University of Groningen

Engineering of sugar metabolism in Lactococcus lactisPool, Weia Arianne

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ENGINEERING OF SUGAR METABOLISM IN LACTOCOCCUS LACTIS

Wietske Pool

Printed by: PrintPartners Ipskamp B.V. The work described in this thesis was carried out in the Molecular Genetics group of the Groningen Biomolecular Sciences and Biotechnology Institute (Faculty of Mathematics and Natural Sciences, University of Groningen, the Netherlands). The author gratefully acknowledges the Groningen Biomolecular Sciences and Biotechnology Institute for financially supporting the printing of this thesis.

RIJKSUNIVERSITEIT GRONINGEN

ENGINEERING OF SUGAR METABOLISM IN LACTOCOCCUS LACTIS

Proefschrift

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

vrijdag 20 juni 2008 om 14:45 uur

door

Weia Arianne Pool

geboren op 17 september 1977 te Boelenslaan

Promotores: Prof. dr. O.P. Kuipers Prof. dr. J. Kok Beoordelingscommissie: Prof. dr. L. Dijkhuizen Prof. dr. B. Poolman Prof. dr. J. Hugenholtz

CONTENT

Chapter 1 General Introduction 7 Chapter 2 Natural sweetening of food products by engineering Lactococcus lactis for glucose production 31 Chapter 3 Functional characterization of three different glucose uptake routes in Lactococcus lactis 51 Chapter 4 Lactococcus lactis strains engineered to improve galactose removal from dairy products reveal metabolic bottlenecks and alternative catabolic pathways 81 Chapter 5 The α-phosphoglucomutase of Lactococcus lactis is unrelated to the α-D-phosphohexomutase superfamily and encoded by the essential gene pgmH 103 Chapter 6 Summary and general discussion 137 Abbreviations 149 References 155 Nederlandse Samenvatting (voor niet-ingewijden) 171 Nawoord 181

CHAPTER 1

GENERAL INTRODUCTION

Wietske A. Pool

Chapter 1

8

Lactoccoccus lactis

Bacteria represent the oldest, most simple and most diverse life-forms on earth. They can be found everywhere on the planet and they can multiply fast even under the most harsh conditions. Up to now only a relatively low number of species has been identified (218). The species that have been identified are usually the most abundant ones, which are easy to grow in a laboratory setting. The molecular genetic and biochemical work described in this thesis was performed on a strain of the lactic acid bacterium Lactococcus lactis. Lactic acid bacteria (LAB) are bacteria that can ferment a great variety of sugars to predominantly lactate in a process that is called homolactic fermentation. LAB can also be heterofermentative, producing acetate, ethanol, formate and carbon dioxide, apart from lactate. LAB are widely used in food fermentations such as in the production of cheese and yogurt, in sausage fermentation, and in the manufacture of certain wines. Well studied genera of LAB are Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus (72). The species L. lactis, formerly known as lactic or group N Streptococcus, consists of 3 subspecies: L. lactis subsp. cremoris, L. lactis subsp. lactis and L. lactis subsp. hordniae. The first two are used in starter cultures for dairy products including all sorts of cheeses and sour products, where traits like fast acidification, production of food preservatives, production of flavour compounds, and production of exopolysaccharides for texture development are of utmost importance (35, 76). Recently, interest is also growing in some plant-associated non-dairy L. lactis strains, providing possible new flavour-forming traits benificial for dairy fermentations (66). L. lactis is also used extensively as a model organism for facultative anaerobic low-GC Gram-positive bacteria since this food-grade bacterium is easy to culture under laboratory conditions and has a relatively simple metabolism. As a representative of L. lactis subsp. cremoris, strain MG1363 is studied intensively. The model for L. lactis subsp. lactis is strain IL1403. Because of their industrial importance and their role in fundamental research, L. lactis subsp. cremoris and L. lactis subsp. lactis have been studied in great detail. The nucleotide sequence of the whole chromosome has been determined of both model strains IL1403 (15) and MG1363 (215) and also of L. lactis subsp. cremoris strain SK11 (108). This genomic information as well as the in-depth knowledge of the physiology and biochemistry of L. lactis has recently allowed to conduct advanced metabolic engineering strategies e.g. to obtain bacteria with nutraceutical traits. Nutraceuticals represent a wide range of foods and food components with a claimed health benefit. Examples of such beneficial strain engineering in L. lactis are the simultaneous overproduction of

Introduction

9

the vitamins folate and riboflavin (183), and other industrially important features like the production of the buttery flavor diacetyl (64), or the production of the sweet amino acid L-alanine (61). The aim of the work presented in this Thesis is to gain lactococcal strains with diverse sugar uptake and metabolic capacities, focussing on the sugars glucose, galactose and lactose. Sugar transport systems

Most bacteria have the ability to use a great variety of carbon sources as an evolutionary advantage to be able to adapt to their ever-changing habitats. The first step in bacterial carbon metabolism is the uptake of a carbohydrate, which can take place via several mechanistically different uptake routes. First, carbohydrates can be actively imported via primary transport systems. In this type of transport the carbohydrate is imported at the expense of ATP, which is hydrolysed to ADP and inorganic phosphate (Pi). The transporters involved are called ABC-transporters, since they carry an ATP-binding cassette domain. In LAB, this type of transport system is mainly used for the internalization of amino acids and di-, tri- and oligopeptides. Although sugar transport via ABC-transporters seems not to be very common in LAB, some studies show that it is possible. In Lactobacillus acidophilus, the four-component ABC-transport systems of the MsmEFGK-family are involved in the uptake of the sugars raffinose and fructo-oligosaccharides (7, 8). The disaccharide maltose is transported in L. lactis by an ABC-transport system (90). MalEFGK2 is a well-studied ABC-transporter involved in maltose transport in Escherichia coli (16, 129, 168). In archaea, most ABC-transporters characterized to date are involved in the uptake of carbohydrates, for example for the uptake of glucose (GlcSTUV), arabinose (AraSTUV), cellobiose (CbtABCDF), maltose (MalEFGK) and trehalose (TreSTUV) in Sulfolobus solfataricus (2, 46). A second type of sugar transport, so-called secondary transport, is driven by the energy stored in electrochemical gradients (78). Secondary transport includes symport (a sugar is imported together with a coupling-molecule, mostly H+ or Na+), antiport (a sugar is excreted while a coupling-molecule is imported at the same time), and uniport (uptake of only a sugar molecule). Secondary sugar transport in LAB is also rarely described. The best studied example of a secondary transport protein of LAB is the lactose transporter (LacS) of Streptococcus thermophilus (48, 143, 145). LacS can import lactose via two mechanisms: it can internalize lactose together with H+, but in vivo the fastest reaction is the antiport of lactose and

Chapter 1

10

galactose. This antiport reaction is favourable, since galactose (formed by hydrolysis of lactose in the cell) cannot be metabolized by most S. thermophilus strains (48, 212). The third mechanism of carbohydrate transport in bacteria is group translocation via the phosphoenolpyruvate (PEP)-dependent : carbohydrate phosphotransferase system (PTS), which was first discovered more than forty years ago in E. coli (85). The PEP:PTS is a general system used by many organisms for the internalization of a great diversity of carbohydrates (42, 148, 205). The system involves uptake accompanied by phosphorylation of the transported carbohydrate. The phosphate group is donated to the carbohydrate via a phosphate transfer cascade originating from phosphoenolpyruvate (PEP) (Fig. 1).

Glucose-6P

EIIAP

EIIB

EII CD (ptnABCD)

Out

In

Glucose

PEP

HPr(ptsH)

P-HPr

P-EI

(ptsI)EI

Pyruvate

Membrane

Glucose-6P

EIIAP

EIIB

EII CD (ptnABCD)

Out

In

Glucose

PEP

HPr(ptsH)

P-HPr

P-EI

(ptsI)EI

Pyruvate

Membrane

Figure 1: The phosphoenolpyruvate-dependent : carbohydrate phosphotransferase system (PEP:PTS). In this example glucose is the imported molecule, the EII-conformation of a mannose-class EII is expected (148). The phosphate cascade from phosphoenolpyruvate (PEP) via enzyme I (EI, encoded by ptsI), histidine protein (HPr, encoded by ptsH) and enzyme IIA (EIIA, encoded by part of ptnABCD in L. lactis MG1363) to the imported sugar is shown.

First, the phosphate of PEP is transferred to Enzyme I (EI), which concomitantly transfers the phosphate to the histidine residue at position 15 of the histidine protein (HPr), followed by phosphate transfer to Enzyme II (EII). EII transfers the phosphate

Introduction

11

group to the imported carbohydrate. EI and HPr (encoded by ptsI and ptsH, respectively) are used for most of the different carbohydrates that can be imported via PEP:PTSs by a certain organism, while EII is carbohydrate-specific. The functional EII can have different domains or may consist of several separate EII proteins. EII always contains one membrane-bound protein (which may consist of different domains, e.g. IIABC), or two integral membrane-bound proteins (e.g. IIC and IID). Furthermore, one cytosolic protein (formerly called Enzyme III) consisting of one or two domains (e.g. IIA or IIAB) may be present (148). The PEP:PTSs use one PEP molecule for the transport and concomitant phosphorylation of the imported sugar, and is energetically the most favourable sugar uptake system. The PEP molecule used in this reaction is energetically equivalent to one ATP molecule since one ATP is formed in the glycolytic reaction from PEP tot pyruvate. The active transport performed by the primary and secondary transport systems need energy for the transport (one or two ATP-molecule, or one or more proton(s)), and an additional ATP molecule for the phosphorylation of the imported sugar (77, 148). When, by secondary transport, a sugar molecule is antiported with an end-product instead of a proton, this type of transport is energetically comparable to transport via a PEP:PTS, or it can even cost less energy. PEP:PTSs are often used by (facultative) anaerobic bacteria. Under anaerobic conditions, glycolysis is the only way to produce energy in the form of ATP, since respiration is not possible, and these anaerobic bacteria (even more than aerobic bacteria) have to use this energy efficiently (75, 148). Carbohydrate transport systems and further carbohydrate metabolism

in L. lactis

Sugar transport has been studied in L. lactis for more than 30 years. L. lactis can import various sugars (188), of which the most common ones are depicted in Table 1. More recently, as a consequence of the increase in genome information, the genes of several transporters have been annotated, occurring either on the chromosome or on plasmids and some of the encoded proteins have been studied in more detail (15, 122). L. lactis is a facultatively anaerobic bacterium: it tolerates low levels of oxygen. It can use different carbohydrates as carbon source (70), all of which are degraded to pyruvate via the Embden-Meyerhof-Parnas (EMP) pathway (Fig. 2). The pyruvate produced has several alternative fates depending on the environmental and intracellular conditions.

Chapter 1

12

TABLE 1: Carbohydrate transport systems of L. lactis

Transport system Gene(s) involved Reference

PST-transporters Fructose-PTS fruA (9) Sucrose-PTS sacB previously scrA (104, 193, 197) Cellobiose/Glucose-PTS ptcBAC (141) Mannose/Glucose-PTS ptnABCD (195) Trehalose-PTS gene unknown (4) Lactose-PTS lacFE (37, 189) Galactose-PTS gene unknown (190)

Non-PTS transporters Maltose permease malK (5, 90) Galactose permease galP (54, 190) Glucose permease gene unknown (141) Trehalose gene unknown (4)

The first steps in the metabolism of glucose, galactose and lactose all lead to the production of glucose-6-phosphate (G6P), after which the same route through glycolysis follows (Fig. 2). G6P is converted to fructose-1,6-bisphosphate (FBP) by phosphoglucose isomerase and 6-phosphofructo-1-kinase. The hexose FBP is converted to the triose-phosphates dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) by the enzyme fructose-bisphosphate aldolase. The triose-phosphates can be interconverted by triosephosphate isomerase. 3-Phosphoglycerate (3-PGA) is formed from GAP by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). After that, 3-PGA is converted to PEP and subsequently to pyruvate by an enolase and pyruvate kinase (PK) respectively. At the level of pyruvate, the metabolic pathway branches. Although L. lactis is generally growing homofermentatively, under less favourable growth conditions, it can perform a mixed-acid fermentation in which besides lactate also acetate, ethanol, 2,3-butanediol and formate are produced (30, 67, 112). Anaerobic conditions combined with fast growth lead mainly to the end product lactate formed from pyruvate by lactate dehydrogenase (LDH). Under certain circumstances some side-products may be formed (Fig. 2).

Introduction

13

Figure 2: Schematic overview of the fermentation of carbohydrates by L. lactis. Carbohydrates can be imported via PTSs or via non-PTS permeases, after which they are converted to glucose-6-phosphate. Glucose-6-phosphate enters the Embden-Meyerhof-Parnas pathway which results in the conversion to, mainly, lactate. The points of formation or expenditure of ATP and NADH are depicted as well as some of the enzymes involved. Abbreviations: FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 3-

PGA, 3-phosphoglycerate; PEP, phosphoenol-pyruvate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; Pi, inorganic phosphate; NAD+, nicotinamide adenine nucleotide; NADH, dihydronicotinamide adenine dinucleotide; CO2, carbon dioxide; O2, oxygen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PFL, pyruvate formate lyase; PDH, pyruvate dehydrogenase; ALS, α-acetolactate synthase.

Glucose-6PHexose

Out

In

Carbohydrates

FBP

GAP

PEP

Lactate

PK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

Pyruvate

CO2

ADP + PiATP

ADP + Pi

ATP

ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

PEPPyruvate

α-Acetolactate

Acetoin

2,3-Butanediol

CO2

NAD+NADH

NAD+NADH

ATPADP + Pi

Diacetyl

Membrane

CO2

O2

ALS

Glucose-6PHexose

Out

In

Carbohydrates

FBP

GAP

PEP

Lactate

PK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

PyruvatePyruvate

CO2

ADP + PiATP

ADP + Pi

ATP

ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

PEPPyruvate

α-Acetolactate

AcetoinAcetoin

2,3-Butanediol

CO2

NAD+NADH

NAD+NADH

ATPADP + Pi

Diacetyl

Membrane

CO2

O2

ALS

Chapter 1

14

When the growth conditions of the bacterium are not optimal (e.g. when glucose is limited or when the cells are grown on a less favourable sugar), pyruvate can also be used as substrate for pyruvate formate lyase (PFL), which leads to the production of formate, acetate and ethanol. When L. lactis is grown in the presence of (small amounts of) oxygen, pyruvate can be used as substrate for pyruvate dehydrogenase (PDH) and, in that case, carbon dioxide is produced besides acetate and ethanol. When pyruvate accumulates, it can also be converted to 2,3-butanediol or, when small amounts of oxygen are present, to diacetyl, since the affinity of α-acetolactate synthase (ALS, the first enzyme necessary to produce diacetyl or 2,3-butanediol from pyruvate) for pyruvate is very low (176). The control of the flux through glycolysis in L. lactis has been studied extensively over the last decades. Glycolytic intermediates have been determined using different methods and growth conditions, and several glycolytic enzymes and their effectors have been described (122). Almost all sugar substrates added to L. lactis are ultimately recovered as end-products, showing that glycolysis is mainly used to generate energy (75). During fermentation two netto ATP molecules are gained. The ratio of NADH/NAD+ is balanced when lactate is formed. Under anaerobic conditions, glycolysis is the only way to produce energy in the form of ATP, since respiration is not possible. Some L. lactis strains are able to grow very well aerobically in later states of growth, when haem is provided using a respiratory pathway, but this is mainly benificial for strains growing on plants or animals (53, 157). Although the glycolytic pathway for the metabolism of sugars in L. lactis has been studied widely (75, 122, 152, 192), a detailed understanding of glucose transport and the initial steps in glucose degradation is missing. As shown in this thesis, the first steps in glucose metabolism of L. lactis are crucial for the characteristics of fermentation. The first steps in glucose metabolism

Glucose is taken up and phosphorylated by L. lactis via a PEP:PTS, or it is imported via a non-PTS permease, and phophorylated by glucokinase (Fig. 3). The PTS is considered to be the main route for glucose metabolism (36, 148, 191). Recently, we have shown that L. lactis can import glucose via two different PTSs, the mannose/glucose-PTS (EIIman/glc), encoded by ptnABCD, and the cellobiose/glucose-PTS (EIIcel/glc), encoded by ptcBAC (this Thesis, Chapter 2, (141)). EIIman/glc consists

Introduction

15

of a cytosolic EIIAB protein and an integral membrane EIICD protein, while EIIcel/glc

consists of a cytosolic EIIAB protein and the membrane protein EIIC.

GALACTOSE

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Out

InLactose-6P

Glucose-6P

Glucose

LACTOSE

Galactose-6P

Tagatose-6P

Tagatose-1,6diP

Triose-P

GALACTOSEGLUCOSE GLUCOSE

Glucose-6P

GalP

GalM

GalK

GalT

LacFE

LacGLacAB

LacC

LacD

Glk

PgmH

PtnABCD

GLYCOLYSIS

Non-PEP:PTS PEP:PTS

Membrane

GALACTOSE

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Out

InLactose-6P

Glucose-6P

Glucose

LACTOSE

Galactose-6P

Tagatose-6P

Tagatose-1,6diP

Triose-P

GALACTOSEGLUCOSE GLUCOSE

Glucose-6P

GalP

GalM

GalK

GalT

LacFE

LacGLacAB

LacC

LacD

Glk

PgmH

PtnABCD

GLYCOLYSIS

Non-PEP:PTS PEP:PTS

Membrane

Figure 3: The first steps in glucose, galactose and lactose metabolism in L. lactis. On the left, the non-PEP:PTS transporters for galactose and glucose (transporter unknown) are shown. GalPMKT belong to the Leloir pathway. The PEP:PTS transporters are depicted on the right. LacABCD belong to the tagatose-6P-pathway. The capital P in some of the metabolites signifies phosphate. Abbreviations: GalP, Galactose permease; GalM, galactomutarotase; GalK, galactokinase; GalT, galactose-1-phosphate uridylyltransferase; PgmH, α-phosphoglucomutase; Glk, glucokinase; LacFE, EIIlac; LacG, phospho-β-galactosidase; LacAB, galactose-6-phosphate isomerase; LacC, tagatose-6-phosphate kinase; LacD, tagatose-1,6-diphosphate aldolase; PtnABCD, EIIglc/man.

When glucose is imported via a non-PTS permease (of which the encoding gene(s) is/are not known yet), it is phophorylated to G6P by glucokinase (encoded by glk). The glucokinase of L. lactis is an ATP-dependent protein of 33.8 kD (based on the nucleotide sequence of glk), which is able to phosphorylate intracellular glucose (195). The protein has three domains: an ATP-binding site at the N-terminus of the protein, a NagC-domain (regulation of the use of N-acetylglucosamine) spanning almost the complete protein and a ROK-motif (repressor, ORF, kinase) in the middle

Chapter 1

16

of the protein. Proteins belonging to the ROK-family known so far are bacterial sugar kinases, transcriptional repressors, or have as yet uncharacterized functions (196). Proteins having a NagC-domain usually also have a ROK-domain and most of these are also sugar kinases and transcriptional regulators. Glucokinases from the Gram-positive bacteria Streptomyces coelicolor (86), Staphylococcus xylosus (214), Bacillus megaterium (177) and Corynebacterium glutamicum (134), which also contain a ROK-motif, contribute to carbon catabolite repression (CCR), although the precise role in CCR remains unknown (CCR will be discussed later in this Chapter). E. coli NagC and Mlc, which are sugar-specific regulatory proteins (140, 166), belong to the ROK-family of proteins and both have an additional almost identical DNA-binding motif. Nevertheless, NagC coordinates the metabolism of aminosugars while Mlc regulates genes involved in sugar uptake together with the cAMP/CAP complex (139). Glk proteins without a ROK-motif have so far not been shown to fulfil regulatory functions. The main role of glucokinase in L. lactis is thought to be phosphorylation of glucose, derived from glucose uptake or from the intracellular hydrolysis of disaccharides, such as lactose (195). The first steps in galactose metabolism

Galactose is imported in L. lactis by galactose permease (GalP), a secondary transport system (symport) that couples galactose uptake to sodium translocation (54, 142). The imported galactose is converted to glucose-1-phosphate (G1P) via the Leloir pathway (Fig. 3), comprising steps catalyzed by galactose mutarotase (GalM), galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalT) and UDP-galactose-4-epimerase (GalE) (54). The next step is a reversible reaction converting G1P to G6P, which is catalyzed by α-phosphoglucomutase (PgmH). G6P then enters glycolysis (Fig. 2). The L. lactis MG1363 gal genes are clustered in an operon (galPMKTE) (54). The gene encoding α-phosphoglucomutase in L. lactis MG1363 has recently been identified (this Thesis, Chapter 5, (121)). Besides through the Leloir pathway, L. lactis can possibly metabolize galactose via the tagatose-6-phosphate pathway. In this case, galactose uptake is most likely mediated by a PEP:PTS (187). The genes for this putative system have not been identified yet but they might be lacFE, the genes that specify the lactose-PTS (see below). Whether the Leloir pathway or the tagatose-6-phosphate pathway is primarily used, is strain-dependent (187). L. lactis ssp. cremoris MG1363, the laboratory strain used in this study, mainly uses the Leloir pathway for galactose fermentation (54).

Introduction

17

The first steps in lactose metabolism

L. lactis is able to ferment the milk sugar lactose, which consists of a galactose and a glucose moiety. The disaccharide lactose is imported and phosphorylated by a lactose-PTS encoded by lacFE, and is hydrolyzed to glucose and galactose-6-phosphate (Gal6P) by phospho-β-galactosidase (LacG) (Fig. 3). Glucose, the preferred sugar for L. lactis, is subsequently phosphorylated to G6P by glucokinase and further metabolized, while Gal6P is further degraded to the triose-phosphates GAP and DHAP by the tagatose-6-phosphate pathway enzymes galactose-6-phosphate isomerase (LacAB), tagatose-6-phosphate kinase (LacC) and tagatose-1,6-diphosphate aldolase (LacD). The lactose metabolic genes are present in an operon (lacABCDFEG, (210)) on, among others, lactococcal plasmid pMG820 (106), a derivative of the lactose/proteinase plasmid pLP712 of L. lactis NCDO712 (the parental strain of L. lactis MG1363). Besides being metabolized via the tagatose-6-phosphate pathway, some of the Gal6P is probably dephosphorylated and expelled into the medium as a consequence of CCR, a fact that will be discussed later in this chapter, and in Chapter 2 of this Thesis. When the glucose in the cell is depleted, catabolite repression is relieved and galactose can be used. The external galactose is then probably imported by a galactose permease and further metabolized through the Leloir pathway mentioned before. Regulation by HPr

Bacteria will choose a sugar substrate from the medium in a hierarchical manner. They use the substrate that yields maximum benefit of growth first (21, 56). Metabolic pathways for less favourable sugars are downregulated via so-called Carbon Catabolite Repression, CCR, which combines global transcriptional control with inducer exclusion (202). The phosphorylation state of HPr plays a crucial role in CCR in low-GC Gram-positive bacteria like L. lactis, whereas EIIAglc fulfills a similar role in enteric Gram-negative bacteria (202). Here, CCR in Gram-positive bacteria will be further described. HPr can be phosphorylated either at histidine 15 by EI, resulting in HPr-His-P, or at serine 46 by a unique HPr kinase/phosphatase (HPrK/P) (155), resulting in HPr-Ser-P (Fig. 4). HPr-His-P is involved in the phosphate transfer cascade of the PEP:PTS, required for uptake and phosphorylation of sugars, while HPr-Ser-P is involved in the regulation of sugar uptake. HPr-Ser-P is able to exclude uptake of less favourable sugars by inhibition of specific (PTS and non-PTS) sugar transporters while it also activates the global

Chapter 1

18

transcriptional regulator CcpA (carbon catabolite protein A) by binding to it, all inducing CCR (43, 161, 182, 221). HPr-Ser-P is also able to stimulate inducer expulsion (discussed later). The ratio of HPr-His-P and HPr-Ser-P, which is mainly controlled by HPrK/P, depends on the available nutrients and determines which sugar will be used first. When a favourable sugar is used, sugar metabolism in the cell is fast, and high levels of FBP and ATP accumulate, which stimulates the kinase activity of HPrK/P and leads to phosphorylation of HPr to HPr-Ser-P. When a less favourable sugar is used or when glycolysis is progressing, FBP- and ATP-levels go down and inorganic phosphate (Pi) is formed, which inhibits the kinase acitivity of HPrK/P, resulting in a higher availability of HPr for the PEP:PTS (42, 81).

Sugar-P

Sugar-PTS

SugarOut

Membrane

In

PEP

HPr

HPr-His-P

P-EI

EI

Pyruvate

HPr-Ser-P

HPr Kinase / Phosphatase

Inducer expulsionCcpA

FBP +

Pi_

+ ++

Sugar-P

Sugar-PTS

SugarOut

Membrane

In

PEP

HPr

HPr-His-P

P-EI

EI

Pyruvate

HPr-Ser-P

HPr Kinase / Phosphatase

Inducer expulsionCcpA

FBP +

Pi_

+ ++

Figure 4: Schematic overview of the role of the histidine protein (HPr) in Gram-positive bacteria. HPr can be phosphorylated at histidine 15 (HPr-His-P) or at serine 46 (HPr-Ser-P). Abbreviations: EI, Enzyme I; PEP, phosphoenolpyruvate; FBP, fructose-1,6-bisphosphate; Pi, inorganic phosphate; CcpA, carbon catabolite protein A; + , activation; - , inhibition.

Inducer expulsion and inducer exclusion

In some cases uptake and hydrolysis of a disaccharide (e.g. lactose) can result in a favoured hexose and a less favoured hexose-phosphate. A mechanism to verify that only the favoured sugar is metabolized is inducer expulsion, which reduces the amount of intracellular sugar phosphates (10, 141, 198). In L. lactis, a constitutively

Introduction

19

expressed broad-range hexose-6-phosphate hydrolase has been characterized that is held responsible for the dephosphorylation of sugars (194). Furthermore, an inducible hexose-6-phosphate hydrolase has been discovered that can be activated by HPr-Ser-P (220). The genes encoding both enzyme activities have not been identified and, also, the mechanism by which the unphosphorylated sugars are expelled into the medium is as yet unknown. Another regulatory mechanism which ensures that the most favoured sugar is used first is called inducer exclusion (160). HPr-Ser-P is involved in inducer exclusion in LAB. In L. lactis, for example, the uptake of ribose and maltose is strongly inhibited by HPr-Ser-P when glucose is present (116). When ptsH is mutated at serine 46, so that HPr-Ser-P can not be formed, glucose inhibition is relieved, suggesting a role for HPr-Ser-P in the inducer exclusion of ribose and maltose (116). In Gram-negative bacteria, EIIAglc directly binds and inhibits the transporter of the less favoured sugar, e.g. it inhibits the maltose transport system of E. coli by binding to MalK (39). It is assumed that in low-GC Gram-positive bacteria like L. lactis this exclusion mechanism functions in a homologous way to that in Gram-negative bacteria, with HPr-Ser-P being used instead of EIIAglc (42). Regulation by CcpA

The carbon catabolite protein CcpA, first discovered in Bacillus subtilis (59, 63, 114), is also present in L. lactis (105). CcpA is a transcriptional regulator protein that binds to a specific DNA-sequence called a cre box (catabolite responsive element). A cre box can be present within the promoter region or in the coding region of a target gene. The consensus sequence for the cre box in B. subtilis is WWTGNAARCGNWWWCAWW (N, W and R represent any base, A or T, and G or A, respectively) (114). Recently, the functional cre sites in L. lactis were analyzed, resulting in a cre consensus of WWGWAARCGYTWWMA (Y and M stand for bases C or T, and A or C, respectively) for L. lactis (225). This sequence is, thus, very similar to the consensus cre sequence of B. subtilis. In B. subtilis, the binding of CcpA to its DNA-targets depends on the presence of activated cofactors, like HPr-Ser-P and FBP. Glucose triggers phosphorylation of HPr at serine 46 and binding of HPr-Ser-P to CcpA stimulates DNA-binding of CcpA (103). High concentrations of FBP, as a result of high glycolytic flux, also activate CcpA. CcpA is a pleiotropic regulator protein: it can function as a repressor (e.g. of the gal-operon) as well as an activator (as shown for the las-operon) in L. lactis (105). The genes controlled by

Chapter 1

20

CcpA have been identified in B. subtilis. Among the repressed genes were those of the TCA-cycle, while the glycolytic genes were induced (119, 203, 223). The genes involved in the synthesis of glutamate were also upregulated by CcpA, showing that a link exists between carbon and nitrogen metabolism (47). CcpA is also involved in global regulation of sugar metabolism in L. lactis (105). L. lactis CcpA was recently found to intertwine the regulation of carbon and nitrogen metabolism by regulation of pepQ, a gene located in a tail-to-tail arrangement to ccpA (55, 225), as was suggested for the CcpA-like protein PepR1 of Lactobacillus delbrueckii (165). Regulation of glycolysis by metabolites

Besides regulation by HPr-Ser-P and CcpA, control of the glycolytic pathway in L. lactis also takes place at several points by glycolytic metabolites (Fig. 5).

Glucose-6-Phosphate

FBP

Triose-P

PEP

PK LDH

Acetyl-CoA

Acetate Ethanol

PFLPDH

Formate

Pyruvate

CO2

3-PGA

GAPDH

Lactate

+ +

-

Glucose-6-Phosphate

FBP

Triose-P

PEP

PK LDH

Acetyl-CoA

Acetate Ethanol

PFLPDH

Formate

PyruvatePyruvate

CO2

3-PGA

GAPDH

Lactate

+ +

-

Figure 5: Schematic overview of the regulation of the glycolytic flux by metabolites in L. lactis. Abbreviations: FBP, fructose-1,6-bisphosphate; Triose-P, triose-phosphates; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; CO2, carbon dioxide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PFL, pyruvate formate lyase; PDH, pyruvate dehydrogenase; + , activation; - , inhibition.

Introduction

21

FBP is one of those regulating metabolites. A high level of FBP is known to activate LDH and PK, while a high level of inorganic phosphate together with a low FBP-concentration (e.g. when the cells are starved) inhibit PK (109, 153, 186, 191, 199). Inhibition of PK leads to accumulation of PEP and 3PGA (152). The metabolism of pyruvate is also influenced by concentrations of the glycolytic intermediates FBP, GAP, and DHAP. Under anaerobic conditions, LDH and PFL compete for pyruvate. During fast growth, high levels of FBP are present that activate LDH, while the high levels of DHAP and GAP under these conditions inhibit the activity of PFL. As a consequence mainly lactate is formed under these conditions. When the bacterium uses a less favourable sugar, or growth rates are lowered by any other cause, less FBP, DHAP, and GAP accumulate, releasing PFL inactivation and allowing the enzyme to metabolize some of the pyruvate to formate, acetate and ethanol. Regulation of glycolysis by redox- and energy-state has also been described. It has been suggested that the intracellular NADH/NAD+-ratio controls the flux through glycolysis mainly via the activity of GAPDH producing NADH (50), while other studies measuring metabolites in vivo suggested a more important role for the ATP/ADP/Pi pool of the cells (127, 132). Furthermore, which type of regulation plays the major role may be strain-dependent, as was shown for the regulation of LDH enzymes of different strains of L. lactis (209). In vivo NMR spectroscopy

A large part of the results described in this thesis was obtained by in vivo Nuclear Magnetic Resonance (NMR). NMR spectroscopy is a powerful analytical technique that makes use of the intrinsic magnetism of some atomic nuclei. When placed in a stationary magnetic field those nuclei (non-zero spin) become aligned with, or against, the field in two energy states. A magnetic resonance (transitions of the nuclei between the states) is induced when the sample is excited by a pulse of radio-frequency radiation. The transition to the lower energy state results in a resonance that can be detected by spectroscopy at a specific frequency. The frequency at which the resonance is detected depends on the specific microenvironment of the nucleus. The electrons surrounding the nucleus of the atom under study and the electrons of nearby atoms are also influenced by the magnetic field, which in turn affects the magnetic field experienced by the nucleus. This means that the same atom might resonate at a different frequency during NMR spectroscopy depending on its surrounding environment. In this way, different molecules can be identified. The separation of resonance frequencies is termed the

Chapter 1

22

“chemical shift” and is expressed in parts per million (ppm). Thus, an NMR spectrum usually consists of several such signals, positioned according to their chemical shifts. As NMR is a quantitative technique, the area of a particular resonance (calculated by integration) arising from a molecule X is proportional to the concentration of X in the sample. Biologically relevant atoms of which the nuclei have a net spin and, therefore, can be detected by NMR are for example 1H, 13C, 15N and 31P. Not all of these atoms are highly abundant in nature; the natural abundance of 13C for example is only 1.1%. Compounds of interest can be isotopically enriched for 13C at one specific carbon atom. With this technique it is possible to follow the fate of this individual carbon atom through different metabolic pathways in a non-invasive, non-destructive way. NMR is mainly used as an analytical technique by (bio)-chemists to elucidate the structure of molecules. Using NMR in living microorganisms, for example to follow changes in the concentrations of metabolites, became appealing with the development of more powerful magnets. Nevertheless, already in 1972, when the techniques for 13C-enrichment had improved, NMR was used to trace 13C-enriched metabolites in vivo (44). All NMR experiments with living cells described in this Thesis were performed with Ana Rute Neves in the group of Helena Santos (ITQB, Oeiras, Portugal). This team optimized the in vivo NMR technique to study sugar metabolism in L. lactis over the last decade (122, 124, 152). Great focus was given to the regulation of glycolysis and the application of NMR to identify metabolic bottlenecks and direct metabolic engineering strategies. A main drawback of NMR is its low sensitivity, which limits in vivo observations to metabolites present at relatively high concentrations (mM range). Therefore, the majority of NMR experiments are conducted with thick suspensions of non-growing cells. To maintain the cells under specific conditions during the NMR experiment we use a circulating system for on-line NMR. A concentrated cell suspension of non-growing cells in the mid-exponential phase of growth is used in a 50 ml mini-fermenter (Fig. 6).

Introduction

23

pH

SOS

Detection zone

50 mL fermenter

Argon

Waterbath

6.5Pump (35 mL/min) NaOH

30°C

NMR-tube

pH

SOS

Detection zone

50 mL fermenter

Argon

Waterbath

6.5Pump (35 mL/min) NaOH

30°C

NMR-tube

Figure 6: Schematic overview of an in vivo NMR experimental setup. The cells circulate through the system (between the fermenter and the NMR tube) at a speed of 35 ml/min. An ”SOS tube” is present to avoid overflow. The temperature, pH and anaerobicity of the cell suspension are controlled. This figure was adjusted from (120).

The cells are circulated between the mini-fermenter and the NMR tube at a speed of 35 ml/min. The cells are constantly stirred in the fermenter and are kept under anaerobic conditions, by flushing with argon gas. The pH of the culture is automatically controlled between 6.45 and 6.55, by titration with a concentrated solution of NaOH. The cells are kept at a constant temperature of 30°C. Circulation was started, an initial spectrum (30 s) was acquired, and 13C-labeled sugar was supplied at a time designated zero (final concentration 20 mM or 40 mM). The time-course for substrate consumption, product formation, and build-up of the pools of intracellular metabolites was monitored online with a time resolution of 30 s. The fermentation of the substrate is followed in vivo until no further changes in end-products and intracellular metabolites are detected, after which the spectra are analyzed in detail (124).

Chapter 1

24

DNA-microarray analysis

The expanding miniaturization in the laboratory, the use of pipetting-robots and the growing computer power has resulted in an exponential growth of the number of sequenced genomes. The DNA-microarray technique has become a very important tool for whole genome transcription analysis. With DNA-microarrays the transcriptional profile of at least two different strains, or cells under different conditions, or at different points in time can be compared. Amplicons of all genes of L. lactis IL1403 were spotted in duplo on a glass slide, on which labelled cDNA can hybridize. In the studies presented in this Thesis two different L. lactis strains were compared and differentially expressed genes were made visible. Total RNA was isolated from cells growing in mid-exponential phase, after which cDNA was produced from the RNA. The cDNA was labelled with a Cy3 or Cy5 label, and hybridized to the slides. The whole experiment was performed in triplo and the labelled cDNA was hybridized to a total of six slides, using Cy3 and Cy5 dye-swaps (Fig. 7) (83).

RNA isolation, cDNA production, cDNA labelling

6 slides total (including dye-swaps)

Grow 6 separate cultures

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

RNA isolation, cDNA production, cDNA labelling

6 slides total (including dye-swaps)

Grow 6 separate cultures

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

WT mut

WT-Cy3 mut-Cy5

mut-Cy3 WT-Cy5

Figure 7: Experimental setup of a DNA-microarray experiment. Two situations (e.g. wildtype strain (WT) versus mutant (mut)) are compared. The cells are grown in triplo. After RNA isolation, cDNA production and cDNA labelling, the labelled cDNA is hybridized to the DNA-microarray using Cy3 and Cy5 dye-swaps. This methodology results in 6 slides, each containing duplicate spots of each gene.

Introduction

25

Since all genes are present in duplo on each slide, for each gene a total of twelve spots can be analyzed. The spots on the slides were processed and normalized using software developed by our laboratory (208). Differentially expressed genes were selected using a variant of the paired t-test on normalized ratio data (101). Nutra Cells

The work described in this thesis was part of an RTD-project sponsored by the European Commission through contract QLK1-CT-2000-01376 (acronym: Nutra Cells). This project was concerned with the development of health-promoting components in food as a result of bacterial activity. The title “Nutra Cells” is based on the term "Nutraceuticals", which was put together from "nutrition" and "pharmaceutical" in 1989 by Stephen DeFelice (20). Nutraceuticals can be defined as a wide range of foods and food components with a claimed medical or health benefit. In the “Nutra Cells” project, ten academic and industrial partners aimed to improve the nutritional value of foods by engineering micro-organisms for nutraceutical production during food fermentation processes or by addition of these nutraceuticals as ingredients (65). In this EU-project the trehalose producing Propionibacterium freudenreichii ssp. shermanii NIZO B365 was shown to produce high levels of trehalose, even when grown in skim milk (27). Trehalose is a low-calorie sugar for humans and could therefore replace high-calorie sugars like sucrose. Another topic of interest was the removal of raffinose and other α-galacto-oligosaccharide sugars from soy-products, since these sugars cannot be metabolized by humans and lead to digestive inconveniences. Lactobacillus fermentum CRL722 was found to produce a thermostable α-galactosidase that is able to remove these raffinose-type sugars when added to the process as a purified protein or when expressed in situ in an L. lactis host (28, 93, 94). Also the α-galactosidase in Lactobacillus plantarum ATCC 8014 was characterized (174). Besides sugar engineering work, several groups have obtained promising results in the production of vitamins by microorganisms. In L. lactis, the pathways for the production of folate (vitamin B11) and riboflavin (vitamin B2) were studied (23-25, 184, 185). Further engineering resulted in a multivitamin-producing L. lactis strain, synthesizing both folate and riboflavin (183). The work described in this Thesis aimed at removing galactose and lactose from dairy fermentation products by engineering of L. lactis in an effort to help individuals suffering from lactose intolerance or galactosemia (see below). Furthermore, an effort was undertaken to make L. lactis produce glucose from lactose, to reduce the

Chapter 1

26

residual lactose in the end-product and to use the glucose produced as a natural sweetener. Fundamentally, we were interested in the first steps of glucose, lactose and galactose metabolism in L. lactis and in the regulatory effect of the use of the different transporters on the rest of sugar metabolism. Galactosemia

In healthy people, galactose is metabolized in a way comparable to that in bacteria like L. lactis and also the names of the human enzymes correspond to their bacterial counterparts (17). Galactose is first imported in human liver cells by a permease, or galactose is formed inside the cell from lactose hydrolysis. The next step is to phosphorylate galactose to Gal1P via galactokinase (GalK). Gal1P is further metabolized to UDP-galactose by galactose-1-phosphate uridylyltransferase (GalT). The interconversion of UDP-galactose and UDP-glucose is catalyzed by uridyl diphosphogalactose-4-epimerase (GalE), after which UDP-glucose can enter glycolysis. Galactosemia is an autosomal recessive inherited enzyme deficiency, with an incidence of about 1 in 35,000 humans in Europe (18, 62, 170). The most common form is called classical galactosemia, which is caused by a deficiency in GalT (17, 98). Other types of galactosemia are rarer and are a result of a deficiency in GalK (113, 133, 164) or GalE (201). Individuals suffering from classical galactosemia are not able to further metabolize Gal1P. Product-inhibition of the kinase results in accumulation of free galactose, which can be converted to either galactitol (206) or galactonate (33), which together with Gal1P can negatively affect different organs. Patients with galactosemia can suffer from cataracts in the eyes, ovarian failure and neurological problems such as cerebral edema, progressive decline in IQ and speech difficulties (158). The severity of the disease can be influenced by the genotype, the hight of galactose intake, and the amount of galactose produced endogeneously. Galactosemia patients have a life-long dietary restriction of dairy products and have to exercise care in consuming fruits and vegetables. If LAB used in dairy industry could be engineered in such a way that the residual galactose and lactose in the end-products could be minimized to close to zero, people suffering from galactosemia could use these dairy products in their diet. Lactose intolerance

About four billion people are unable to digest the milk-sugar lactose properly (26). These lactose intolerant individuals miss the enzyme lactase, which should be

Introduction

27

produced in the small intestine. Lactase deficiency leads to discomfort when milk products are digested. The presence of lactose in the small intestine causes water resorption, leading to nausea, cramps and diarrhoea. The anaerobic bacteria present in the large intestine are able to digest the left-over lactose, which leads to toxin formation and gas production (26). The severity of the symptoms depends on the amount of lactose an individual can tolerate and on the person’s age and ethnicity. After reaching the age of two, humans start to produce less lactase. Lactose intolerance is very common in the Asian and African population, while the condition is least common among the northern European population. Exclusion of milk and dairy products from the diet is a suitable way to overcome the symptoms of lactose intolerance, but this may lead to nutritional problems. Alternatives like using only fully fermented dairy products in the diet, probiotics with bacterial lactase activity (117), or fermented dairy products with low to no residual lactose (this thesis) are currently under investigation.

Outline of the Thesis

In this thesis the first steps in the metabolism of the sugars lactose, glucose and galactose in L. lactis MG1363 are investigated. Using various metabolic engineering strategies combined with state-of-the-art in vivo NMR technology, the in-depth analysis of the metabolism of the sugars in the engineered strains underpins the importance of the first steps in metabolism with respect to the regulation of the glycolytic flux and the formation of end products. In Chapter 2, the engineering of L. lactis for a more efficient use of lactose as a substrate is described. To achieve this, the metabolism of the favoured sugar glucose had to be blocked. This work led to the identification of a glucose-transporting PTS. This system, encoded by ptcBAC was not known to transport glucose. The final strain even produced glucose (“natural sweetening strain”) as an end-product, since it could not metabolize the glucose moiety of lactose. Chapter 3 describes the characterization of the different glucose transport systems in L. lactis MG1363 by making targeted single and multiple deletion strains. Transport activities as well as enzymatic activities of key metabolic enzymes were measured and end products of fermentation were determined. Thus, the new glucose-PTS discovered in Chapter 2 is compared to the glucose-PTS already known (EIIman/glc) and to non-PTS glucose transport. How glucose is metabolized by L. lactis depends on the transport system being used.

Chapter 1

28

Besides glucose and lactose metabolism also galactose metabolism has been studied. Galactose consumption by L. lactis MG1363 can be improved, as is shown in Chapter 4. In this Chapter the bottleneck in the galactose metabolic pathway was shown to be at the level of α-phosphoglucomutase activity. As the lactococcal gene for α-phosphoglucomutase was at the time not known, the gene encoding α-phosphoglucomutase from S. thermophilus was overexpressed in L. lactis. This improved the metabolism of galactose. Chapter 5 describes the isolation of α-phosphoglucomutase activity from L. lactis MG1363. The α-phosphoglucomutase activity of L. lactis is encoded by yfgH (hereafter named pgmH), which is an essential gene for growth, as shown by a conditional knock-out strain. Interestingly, sequence and biochemical analyses showed that PgmH of L. lactis is related to the eukaryotic phosphomannomutases. Finally, Chapter 6 summarizes the most important results of the work described in this Thesis and puts them in perspective. Directions for future experiments are given in order to further improve the knowledge on sugar metabolism and its regulation in L. lactis.

Introduction

29

CHAPTER 2

NATURAL SWEETENING OF FOOD PRODUCTS BY ENGINEERING LACTOCOCCUS LACTIS FOR GLUCOSE

PRODUCTION

Wietske A. Pool, Ana R. Neves, Jan Kok, Helena Santos, Oscar P. Kuipers.

Based on Metab. Eng. 2006, 8:456-464.

Chapter 2

32

Natural sweetening

33

SUMMARY

We show that sweetening of food products by natural fermentation can be achieved by a combined metabolic engineering and transcriptome analysis approach. A Lactococcus lactis ssp. cremoris strain was constructed in which glucose metabolism was completely disrupted by deletion of the genes coding for glucokinase (glk), EIIman/glc (ptnABCD), and the newly discovered glucose-PTS EIIcel (ptcBAC). After introducing the lactose metabolic genes, the deletion strain could solely ferment the galactose moiety of lactose, while the glucose moiety accumulated extracellularly. Additionally, less lactose remained in the medium after fermentation. The resulting strain can be used for in situ production of glucose, circumventing the need to add sweeteners as additional ingredients to dairy products. Moreover, the enhanced removal of lactose achieved by this strain could be very useful in the manufacture of products for lactose intolerant individuals.

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34

INTRODUCTION

Nutraceuticals comprise a wide range of foods or food components, including fermenting bacteria, with a claimed medical or health benefit. Lactococcus lactis is a lactic acid bacterium used in the dairy industry for the production of fermented milk products and, thus, is a good target for the production of nutraceuticals. Additionally, L. lactis is a suitable model organism for metabolic pathway engineering (73), since it has a relatively simple carbon metabolism and many molecular cloning tools are available (84, 95, 96). The metabolism of L. lactis has already been successfully engineered, e.g. for the production of the sweet amino acid L-alanine (61), the production of the buttery flavor diacetyl (64), the production of mannitol (51, 219), and for the simultaneous overproduction of the vitamins folate and riboflavin (183). The aim of the present study was to disrupt glucose uptake and metabolism in L. lactis in such a way that, when growing on lactose it excretes glucose, which can be used as a natural sweetener in dairy products. A second goal was to reduce lactose contents in the final product. Like in many other bacteria, the phosphoenolpyruvate:sugar phosphotransferase system (PEP:PTS), mediating uptake and phosphorylation of carbohydrates (42, 148, 161), is the main sugar transport system in L. lactis. Uptake of glucose in L. lactis can take place either via these PEP:PTS systems or via one or more non-PTS transporter(s), after which the sugar is phosphorylated to glucose-6-phosphate by glucokinase. Subsequently, glucose-6-phosphate enters glycolysis (Fig. 1). PEP:PTS systems for six different (types of) sugars, i.e. fructose, mannose, sucrose, mannitol, β-glucosides and cellobiose, have been annotated in the nucleotide sequence of the genome of L. lactis ssp. lactis IL1403 (15). Homologues of these PTS genes are present in the genome sequence of L. lactis ssp. cremoris MG1363 (215). The mannose/glucose-PTS is considered to be the main uptake system for glucose (195). The milk-sugar lactose is imported by a dedicated PEP:PTSlac. The lactose-phosphate formed during transport is hydrolyzed intracellularly to galactose-6-phosphate, which is metabolized further through the tagatose-6-phosphate pathway, and to glucose, which enters glycolysis after phosphorylation by glucokinase. The genes for lactose usage are located on the lactococcal plasmid pMG820 (106) (Fig. 1). Directed engineering of L. lactis results in stable and clear genotypes, in contrast to genotypes resulting from classical mutagenesis techniques, by which various genetic changes may go undetected (6, 128). The directed engineering approach proposed here, involving deletion of a known and a newly discovered PTS-system

Natural sweetening

35

specific for glucose and the gene encoding glucokinase, as well as the introduction of the lactose metabolic genes, resulted in a strain producing glucose (Fig. 1).

Figure 1: Model of glucose metabolism in L. lactis NZ9000. Lactose is taken up by the lactose-PTS specified by plasmid pMG820 (Lac+). The galactose moiety of lactose is used for metabolism via the tagatose-6-phosphate pathway, also supplied in pMG820. The glucose moiety is phosphorylated by glucokinase before entering glycolysis. Glucose can be imported via either the PTSman/glc (ptnABCD) or the newly discovered PTScel (ptcBAC, encircled), consisting of a cytosolic EIIAB-part and an integral membrane EIICD-part or a cytosolic EIIAB-part and an integral membrane EIIC-part, respectively. Glucose is excreted as observed in L. lactis NZ9000Glc–Lac+. Question-mark: unidentified glucose transporter(s). Dashed crosses: glk, ptnABCD, and ptcBA deletions made in this study.

The fate of carbon in each sugar moiety during lactose fermentation in the engineered strains was investigated using 13C-labelled substrates and in vivo Nuclear Magnetic Resonance (NMR) (124). The production of glucose from lactose could be demonstrated in non-growing cells as well as during cell growth in defined medium and during growth in skim milk.

Chapter 2

36

EXPERIMENTAL PROCEDURES

Microbial strains and growth conditions

Strains and plasmids used in this study are listed in Table 1. The strains were grown in M17 (Difco, Sparks, MD) with 0.5% galactose (w/v) or 2% lactose (w/v) at 30ºC or 37°C, in chemically defined medium (CDM (144)), with 1% glucose (w/v) or 2% lactose (w/v) or in 10% reconstituted skim milk (Oxoid Ltd., Basingstoke, England). The optical density of milk-grown culture was measured using the milk clearing method of Kanasaki (69), in which 100 μl of culture was mixed with 900 μl 0.5 M borate (pH 8.0) containing 10 mM EDTA; after 30 minutes incubation at room temperature the optical density at 600 nm was measured. When necessary, erythromycin and chloramphenicol were used at a final concentration of 5 μg/ml. For growth in a 2 L fermentor (New Brunswick BioFlo, Edison, NJ), the medium was gassed with argon for 10 min prior to inoculation (4% inoculum from a culture grown overnight); the pH was kept at 6.5 by automated addition of 5 N NaOH, and an agitation rate of 70 rpm was used. Growth was monitored by measuring the optical density at 595 or 600 nm. DNA techniques

General DNA techniques were performed essentially as described (162). Plasmid DNA was isolated by the method of Birnboim and Doly (12). Restriction enzymes, T4 DNA ligase, Expand polymerase and Taq polymerase were obtained from Roche Applied Science (Mannheim, Germany) and used according to the supplier’s instructions. PCR was performed in an Eppendorf thermal cycler (Eppendorf, Hamburg, Germany). Specific cloning procedures

Gene deletions were all performed in L. lactis strain NZ9000 and were constructed with the help of L. lactis strains LL108 and LL302 and pORI280-derivatives using a two-step homologous recombination method described before (96). This method does not leave antibiotic resistance markers in the chromosome, and multiple deletions in one strain can be easily realized. Primers used for cloning are listed in Table 1.

Natural sweetening

37

TABLE 1: Lactococcal strains, plasmids, and primers used Strain Description Reference

NZ9000 Derivative of MG1363 carrying pepN::nisRK (84) LL302 RepA+ MG1363, carrying single copy of pWV01 repA in pepX (95) LL108 RepA+ MG1363, Cmr, carrying copies of pWV01 repA in chromosome (95) NZ9000Δglk Derivative of NZ9000 with a 404-bp deletion in glk This work NZ9000ΔptnABCD Derivative of NZ9000 with a 1736-bp deletion in ptnABCD This work NZ9000ΔglkΔptnABCD Derivative of NZ9000Δglk with a 1736-bp deletion in ptnABCD This work NZ9000ΔglkΔptcBA Derivative of NZ9000Δglk with g a 657-bp deletion in ptcBA This work NZ9000Glc− Derivative of NZ9000ΔglkΔptnABCD with a 657-bp deletion in ptcBA This work NZ9000Lac+ NZ9000 carrying pMG820 This work NZ9000Glc−Lac+ NZ9000Glc− carrying pMG820 This work

Plasmid Description Reference

pMG820 Lac mini-plasmid (23.7 kb), with lacFEGABCD, derivative of pLP712 (106) pORI280 Emr, LacZ+, ori+ of pWV01, replicates only in strains providing RepA in trans (96) pORI280-glk’ Emr, derivative of pORI280, for integration in L. lactis glk This work pORI280-ptnABCD’ Emr, derivative of pORI280, for integration in L. lactis ptnABCD This work pORI280-ptcBA’ Emr, derivative of pORI280, for integration in L. lactis ptcBA This work pVE6007 Cmr, temperature-sensitive derivative of pWV01 (107)

Primer Sequence (5' to 3') Restriction-site Location annealing part

Glk5 GCTCTAGACCAGATCGTTTGGATGCG XbaI 196-178 bp up. glk TSS Glk6 CGCGGATCCTTAAGCAGCAACGTTAGCG BamHI 345-360 bp down. glk TSS Glk7 CGCGGATCCGGGTTCTACTTTAAACCCTGC BamHI 765-785 bp down. glk TSS Glk8 GGAAGATCTGGATAGAAAGATTCCATCC BglII 399-417 bp down. glk SC Ptn1 GCTCTAGAGGAGGTTACTCACATTGAG XbaI 14-5 bp down. ptnAB TSS Ptn2 CCATCGATGCTCTTGAGTCTGGAGTCC ClaI 560-578 bp down. ptnAB TSS Ptn3 CCATCGATCTCCACTCTTCTTCTTCATCG ClaI 422-442 bp down. ptnD TSS Ptn4 GAAGATCTTGAGCCACAGATTCTCTCC BglII 132-150 bp down. ptnD SC Ptc1 GCTCTAGAGTCATCTCTGACCCCTTTC XbaI 560-541 bp up. ptcB TSS Ptc2 CGGGATCCTTAGGCTGCACATGCAAGTGC BamHI 19-36 bp down. ptcB TSS Ptc3 CGGGATCCCCTTGCAGTAGAAGTTGTTG BamHI 294-313 down. ptcA TSS Ptc4 CGGAATTCCGGATAAGTTACATCGCTAAATG EcoRI 509-531 bp down. ptcA SC

Abbreviations: TSS, translational start site; up., upstream; down., downstream; SC, stopcodon.

Chapter 2

38

Chromosomal DNA of L. lactis NZ9000 was used as a template in PCR amplifications. An L. lactis NZ9000Δglk strain, in which only the first 360 bp were left of the 969 bp of glk, was engineered using the primers glk5, glk6, glk7, and glk8. L. lactis NZ9000ΔptnABCD, in which ptnAB is disrupted after 578 bp, ptcC is completely deleted and the first 441 bp of ptcD are missing, was made using the primers ptn1, ptn2, ptn3, and ptn4. L. lactis NZ9000ΔptcBA, carrying only the first 36 bp of ptcB and the last 58 bp of ptcA, was made using the primers ptc1, ptc2, ptc3, and ptc4. Glucokinase enzymatic assays

Cells (~109) were harvested at mid-exponential growth-phase, resuspended in 1 ml 10 mM potassium phosphate (KPi) buffer (pH 7.2) and disrupted with 0.5 g glass beads (∅ 50-105 μm, Fischer Scientific BV, Den Bosch, the Netherlands), using a Mini-BeadBeater-8 (Biospec Products, Inc., Bartlesville, OK) with two 1 min pulses of homogenization, and a 1 min interval on ice. Cell debris was pelleted and glucokinase activity in the cell-free extract was assayed spectrophotometrically by the glucose-6-phosphate dehydrogenase (Glc6P-DH) (EC1.1.1.49) : NADPH-coupled assay (147). The assay mixture contained 10 mM KPi (pH 7.2), 5 mM MgCl2, 1 mM NADP+, 1 mM ATP, 1 U Glc6P-DH, 20 mM glucose and cell-free extract (usually 20 μl) in a total volume of 250 μl. Protein was determined by the method of Bradford (19). In vivo NMR

Cells were grown in medium containing 2% lactose (w/v), harvested in the mid-logarithmic phase of growth, and were resuspended in 50 mM KPi buffer (pH 6.5) to a protein concentration of approximately 15 mg protein / ml. In vivo NMR experiments were performed using the on-line system described earlier (124). Lactose specifically labeled on the galactose moiety ([1-13CGal]-lactose, 20 mM) or on the glucose moiety ([1-13CGlc]-lactose, 20 mM) was added to the cell suspension at time-point zero. The time course of lactose consumption, product formation, and changes in the pools of intracellular metabolites were monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, an NMR-sample extract was prepared as described previously (124, 127). Carbon-13 spectra were acquired at 125.77 MHz on a Bruker DRX500 spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). All in vivo

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experiments were run using a quadruple nuclei probe head at 30oC, as described before (124). Lactate was quantified in the NMR-sample extract by 1H-NMR in a Bruker AMX300 (Bruker BioSpin GmbH). The concentration of other metabolites (e.g. glucose) was determined in fully relaxed 13C spectra of the NMR-sample extracts as described (127). Transcriptome analysis

mRNA-levels of L. lactis strains NZ9000 and NZ9000ΔglkΔptnABCD were compared by transcriptome analysis using L. lactis DNA-microarrays (83). The strains were grown independently 3 times with Cy3 and Cy5-dye-swaps of each repetition. Thus, a total of 6 hybridized slides, all containing duplicate spots of each amplicon, resulted in a maximum of 12 measurements for each gene. The experiments were performed essentially as described (83), with the following modifications. RNA-isolation. Macaloid was prepared by resuspending 2 g Macaloid (Bentone MA, Elementis Specialities Inc, Hightstown, NJ) in 100 ml 10 mM Tris / 1 mM EDTA-buffer (T10E1) (pH 8.0), boiling for 5 min, cooling to room temperature, sonicating by burst until the macaloid formed a gel, centrifuging and resuspending in 50 ml T10E1 (pH 8.0). Cells (40 ml; ~2 x 109) were harvested at the mid-exponential growth phase by centrifugation (13,000 x g, 1 min, 20°C) and the cell pellets were immediately frozen in liquid nitrogen. After thawing on ice, the cells were resuspended in 500 μl T10E1 (pH 8.0) (treated with DEPC; Sigma-Aldrich, St. Louis, MO), and placed in a rubber sealed screw-capped tube. After adding 500 mg glass beads (∅ 50-105 μm), 50 µl 10% SDS, 500 µl phenol / chloroform, and 175 μl Macaloid, the cells were disrupted in a Mini-BeadBeater-8 (Biospec Products, Inc.) using two 1 min pulses (homogenize), with a 1 min interval on ice. After centrifugation (20,000 x g, 10 min, 4°C), 500 μl of supernatant was extracted with 500 μl phenol / chloroform, centrifuged as before, followed by an extraction with only 500 μl chloroform. After centrifugation (20,000 x g, 5 min, 4°C), total RNA was isolated from the water phase using the ‘High Pure RNA Isolation Kit’ of Roche Applied Science, according to the manufacturer’s instructions, except that final elution was performed with 50 μl elution buffer. RNA yield was determined spectrophotometrically at 260 nm. RNA quality was examined using the Agilent 2100 Bioanalyzer (Agilent Technologies, Amstelveen, the Netherlands). cDNA labeling and hybridization. Single-strand reverse transcription and indirect labeling of 20 µg of total RNA with Cy3-dCTP or Cy5-dCTP were performed with the CyScribe Post Labelling Kit of Amersham (Roosendaal, the Netherlands) according

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40

to the manufacturer’s instructions. Hybridization of the purified labeled cDNA to an aldehyde-coated glass slide (Cel Associates / Telechem International Inc., Sunnyvale, CA) on which 2108 amplicons of L. lactis strain IL1403 had been spotted in duplicate, was performed as described (83) at 42°C. Bioinformatic analysis. Spot quantitations were processed and normalized using automated grid-based Lowess transformation (f = 0.5) software (208). Differentially expressed genes were selected at a p-value lower than 0.00001 and a ratio of 2 and higher by using a variant of the paired t-test on normalized ratio data (101). Sugar determinations by HPLC

Samples of L. lactis NZ9000Glc⎯Lac+ or L. lactis NZ9000Lac+ grown in CDM containing 2% (w/v) lactose were taken at different stages of growth and centrifuged (2,000 × g, 5 min, 4ºC). Samples of milk-grown cultures of L. lactis NZ9000Glc⎯Lac+ or L. lactis NZ9000Lac+ were taken at different stages of growth and cleared using the method of Kanasaki (69) before centrifugation (2,000 × g, 5 min, 4ºC). Supernatants were filtered over 0.45 μm nylon membranes (Millipore, Bedford, MA) and stored at -20ºC until analysis by high performance liquid chromatography using a refractive index detector (Shodex RI-101, Showa Denko K. K., Japan). Lactose, glucose and galactose in the supernatants of CDM-grown cultures were quantified using an Aminex HPX-87P column (Bio-Rad Laboratories Inc., Hercules, CA) at 80ºC, with H2O as the elution fluid and a flow rate of 0.6 ml / min. Lactose, glucose, and galactose in the supernatants of milk-grown cultures were quantified using an HPX-87H anion exchange column (Bio-Rad Laboratories Inc.) at 60ºC, with 5 mM H2SO4 as the elution fluid and a flow rate of 0.5 ml / min.

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RESULTS

Deletion of glk and ptnABCD is not sufficient to fully block glucose

metabolism

As a first step in the process of blocking glucose metabolism, a deletion was made in the glucokinase gene (glk) in L. lactis ssp. cremoris strain NZ9000 (Fig. 1). NZ9000 showed glucokinase activity (0.14 ± 0.02 U/mg), while no glucose-phosphorylating activity was detected in NZ9000Δglk. The latter strain was still able to grow in chemically defined medium (CDM) with 1% (w/v) glucose, although the growth characteristics were different from those of strain NZ9000 (Fig. 2).

Figure 2: Growth of L. lactis in glucose-containing CDM. L. lactis strains NZ9000 (■), NZ9000Δglk (□), NZ9000ΔglkΔptnABCD (●), NZ9000ΔglkΔptcBA (○) and NZ9000Glc⎯ (▲) were grown in CDM with 1.0% glucose at 30°C in microtiterplates. OD595 was measured at 1 hr intervals. For each strain the μmax is presented in the inset.

NZ9000Δglk showed a lower maximum growth rate and the culture reached a higher final cell density. As NZ9000Δglk is unable to use intracellular glucose, or glucose imported via a non-PTS permease system, growth of this strain on glucose is strictly dependent on uptake via one or more PEP:PTS(s). Removal via double cross-over recombination of the genes encoding the mannose/glucose-PTS (ptnABCD) in L. lactis NZ9000Δglk resulted in a strain (NZ9000ΔglkΔptnABCD) still able to grow on glucose (Fig. 2). Surprisingly, the growth characteristics of L. lactis NZ9000ΔglkΔptnABCD resembled those of NZ9000Δglk in CDM with 1% (w/v)

Chapter 2

42

glucose without pH-control, although the maximum growth rate of the double mutant was slightly higher. These results indicated that, apart from EIIman/glc, another efficient glucose-PTS is functional in L. lactis NZ9000. Transcriptome analysis indicates the additional glucose PTS in L. lactis

To elucidate which PEP:PTS besides EIIman/glc is able to transport glucose in L. lactis NZ9000, mRNA-levels in strains NZ9000 and NZ9000ΔglkΔptnABCD grown in CDM with 1% (w/v) glucose were compared using DNA-microarrays. The assumption was that the gene(s) encoding the glucose-PTS operative in NZ9000ΔglkΔptnABCD would be expressed at a higher level in this mutant than in L. lactis NZ9000. In NZ9000ΔglkΔptnABCD, the genes ptcB and ptcA were both overexpressed more than 5 times, while none of the other PTS-genes were significantly overexpressed (Table 2). TABLE 2: Comparison of PTS-gene expression in L. lactis NZ9000ΔglkΔptnABCD and L. lactis NZ9000 Gene Annotation Ratio a) p-value

ptcB cellobiose EIIB 5.2 3.E–08 ptcA cellobiose EIIA 5.0 7.E–07 ptcC cellobiose EIIC 1.1 8.E–02 celB cellobiose EIIC 0.8 4.E–01 fruA fructose EIIBC 0.9 3.E–01 mtlD mannitol EIIBC 1.1 7.E–01 mtlF mannitol EIIA 0.9 6.E–01 ptbA β-glucoside EIIABC 0.8 1.E–02 yleD sucrose EIIBC 0.9 5.E–01 yleE β-glucoside EIIABC 1.1 5.E–01 yidB cellobiose EIIC 1.1 1.E–01 yedF β-glucoside IIABC 1.0 8.E–01

a) L. lactis NZ9000ΔglkΔptnABCD over L. lactis NZ9000

The sequences of L. lactis ssp. cremoris MG1363 ptcB and ptcA are resp. 93% and 91% identical to the corresponding genes of L. lactis spp. lactis IL1403. L. lactis spp. lactis IL1403 and L. lactis spp. cremoris MG1363 share >85% sequence homology, enabling efficient hybridization of probes (IL1403 amplicons on the slides) and target cDNA (from MG1363), as shown previously (41). The genes ptcB and ptcA have

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been annotated as part of a cellobiose-PTS, encoding the protein complex EIIBAcel (15). The chromosomal organization of the genes encoding PTScel is ptcB-ptcA-yecA-ptcC, in which yecA encodes a putative transcriptional regulator and ptcC encodes EIICcel (15). To examine whether EIIBAcel could use glucose as a substrate, a food-grade strain was constructed in which the glk, ptnABCD, and ptcBA genes were deleted. L. lactis NZ9000ΔglkΔptnABCDΔptcBA was isolated on a medium with galactose as the sole carbon and energy source. The strain could not grow in a medium containing glucose as the sole carbon source (Fig. 2), showing that ptcBA is part of a glucose transporter. Removal of glk, ptnABCD, and ptcBA was sufficient to fully block glucose metabolism in L. lactis NZ9000, and this glucose-negative NZ9000-derivative will be named NZ9000Glc⎯ from here onwards. As L. lactis strains NZ9000ΔglkΔptcBA and NZ9000ΔglkΔptnABCD could metabolize glucose (Fig. 2), both L. lactis EIIcel and EIIman/glc can use glucose as a substrate. Furthermore, the ability to use cellobiose as a substrate was heavily impaired in strain NZ9000ΔptcBA (data not shown), showing that EIIcel also plays a role in cellobiose uptake in L. lactis. NZ9000Glc-Lac+ produces glucose under several conditions

Lactococcal plasmid pMG820, carrying the genes for lactose-PTS and the tagatose-6-phosphate pathway (106), was introduced in L. lactis strains NZ9000 and NZ9000Glc⎯, providing both strains with the ability to use lactose as a substrate (Lac+). Growth of the two resulting strains in CDM with 2% (w/v) lactose was analyzed in batch cultures with and without pH control. Sugar (lactose, glucose, and galactose) concentrations in the medium were determined at several points during growth using High Performance Liquid Chromatography (HPLC). L. lactis NZ9000Glc⎯Lac+ had a lower maximum growth rate and the culture-pH dropped more slowly than that of L. lactis NZ9000Lac+ when the strains were grown as standing cultures without pH-control (Fig. 3A&B). Under these conditions L. lactis NZ9000Glc⎯Lac+ used more lactose from the medium (the concentration was lowered by 16.7 mM, compared to 8.0 mM by NZ9000Lac+) before growth ceased by the lowered pH. Unlike L. lactis NZ9000Lac+, which did not produce glucose, NZ9000Glc⎯Lac+ excreted equivalent amounts of glucose (17.1 mM) compared to the lactose consumed (Fig. 3 A&B).

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44

A

C

B

D

E F

A

C

B

D

E F

Figure 3: Growth of L. lactis in batch cultures with and without pH control in lactose-CDM and skim milk. Culture-pH and extracellular concentrations of lactose, glucose, and galactose measured during growth of L. lactis NZ9000Lac+ (A, C, E) and L. lactis NZ9000Glc⎯Lac+ (B, D, F) in CDM with 2% lactose (w/v) without pH control (A, B), with the pH controlled at 6.5 in a fermentor (C, D), and grown in skim milk without pH-control (E, F). Symbols: growth OD600 (■), pH (●), lactose (○), glucose (□), galactose (▲). Data are from a representative experiment. Standard errors (not shown) never exceeded 10% of the given value.

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When grown in a fermentor with the pH controlled at 6.5 and lactose as the limiting factor, L. lactis NZ9000Glc⎯Lac+ grew to about half of the cell density of NZ9000Lac+ (Fig. 3 C&D). L. lactis NZ9000Glc⎯Lac+ produced equimolar amounts of glucose (52.7 mM) from lactose (52.3 mM) under these conditions, while NZ9000Lac+ metabolized both the galactose and the glucose moiety of lactose (Fig. 3 C&D). When grown under limiting carbon conditions, L. lactis NZ9000Glc⎯Lac+ reached a lower cell density than NZ9000Lac+ because only half of the total carbon supplied (the galactose moiety of lactose) could be metabolized. A slight accumulation of galactose, most probably resulting from dephosphorylation of galactose-6-phosphate, was observed in L. lactis NZ9000Lac+ grown as standing culture and during growth under pH controlled conditions (Fig. 3 A&C). To examine whether the characteristics of the strains in synthetic laboratory media would prevail during milk fermentation, L. lactis NZ9000Lac+ and NZ9000Glc⎯Lac+ were grown in skim milk, in which lactose (~150 mM, which is ~5%) is the main carbon source. Lactose, galactose, and glucose concentrations in the culture supernatants were measured at different growth-stages using HPLC (Fig. 3 e&f). As in synthetic medium, lactose (46 mM) was used to produce glucose (up to a concentration of 38 mM) by L. lactis NZ9000Glc⎯Lac+, while in NZ9000Lac+ the lactose concentration decreased only by 29 mM and no glucose was detected. The final pH reached by milk-grown cultures was 4.2, which is the same as that reached by CDM-grown cultures. Fate of carbon in the glucose and galactose moieties of lactose

To follow the fate of each sugar moiety of lactose during fermentation, lactose metabolism of L. lactis NZ9000Lac+ and L. lactis NZ9000Glc⎯Lac+ was analyzed in vivo by 13C-NMR using non-growing cells under controlled conditions of pH, temperature and gas atmosphere (124). Lactose labeled either on the glucose moiety ([1-13CGlc]-lactose) or on the galactose moiety ([1-13CGal]-lactose) was used as a substrate. L. lactis NZ9000Lac+ metabolized both the galactose moiety and the glucose moiety of lactose to lactate (Fig. 4A). Galactose accumulated transiently to low levels and was used upon lactose depletion. L. lactis NZ9000Glc⎯Lac+ only metabolized the galactose moiety of lactose and did not accumulate galactose (Fig. 4B). Strain NZ9000Glc⎯Lac+ did not metabolize the glucose moiety but, instead, glucose derived from lactose was completely excreted. Consequently, L. lactis NZ9000Glc⎯Lac+ produced half of the amount of lactate compared to NZ9000Lac+, from the same amount of lactose. Also,

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46

a difference in the pattern of intracellular metabolites was detected. In L. lactis NZ9000Lac+ both tagatose-1,6-bisphosphate and fructose-1,6-bisphosphate accumulated up to 24 mM intracellular concentration, whereas in NZ9000Glc⎯Lac+ only tagatose-1,6-bisphosphate (up to 31 mM) was detected (data not shown).

A

B

A

B

Figure 4: In vivo analysis of lactose metabolism using NMR. Kinetics of 20 mM [1-13Cgal]-lactose and 20 mM [1-13Cglc]-lactose consumption and concomitant product formation by non-growing cells of L. lactis, performed under anaerobic conditions at 30ºC and at pH 6.5. A) L. lactis NZ9000Lac+; B) L. lactis NZ9000Glc⎯Lac+. Symbols: [1-13Cgal]-lactose (●), [1-13Cglc]-lactose (○), lactate from [1-13Cgal]-lactose ( ), lactate from [1-13Cglc]-lactose ( ), glucose (□), galactose (▲).

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DISCUSSION

We show that removing glk, ptnABCD and ptcBA results in a complete blockage of glucose metabolism in L. lactis NZ9000. A strain carrying deletions in these three genetic loci cannot use glucose present extracellularly, nor can it metabolize glucose formed intracellularly by lactose-6-phosphate hydrolysis. Glucose derived from lactose-6-phosphate hydrolysis is expelled into the medium. The system responsible for glucose excretion has not yet been identified but it is probable that a non-PTS permease is involved, since passive diffusion of glucose over the cell membrane has not been reported. Previously, a glucokinase- and PTSman/glc-deficient mutant of L. lactis ssp. lactis strain 133, which was blocked in glucose metabolism, was obtained by classical mutagenesis (195). Interestingly, we show now by transcriptome analysis and self-cloning techniques that, in addition to a glucokinase and PTSman/glc deletion, removal of a PTScel is necessary to completely prevent glucose fermentation, at least in L. lactis ssp. cremoris NZ9000. The possibility that L. lactis strain 133 does not have a PTScel with the ability to use glucose cannot be disregarded, but this is unlikely since L. lactis strains 133 and IL1403 belong to the same subspecies and the latter does contain ptcBAC, the genes encoding PTScel. Most probably, the classical mutagenesis approach led to genetic changes that went undetected (128). The decreased maximum growth-rates and higher final cell densities reached by the strains deleted for glucokinase alone or together with a deletion in PTSman/glc or PTScel cannot be fully explained at this point. Obviously, deletion of glucose metabolic genes will decrease the metabolic efficiency and thereby affect the growth-rate. Sugar metabolism is a highly regulated process and disruption of genes involved, especially those encoding PEP:PTS components, might have a big regulatory impact on the overall sugar metabolism. L. lactis strains with mutations in glucose transport systems showed a shift to a mixed-acid fermentation (data not shown) and therefore it is likely that under non-controlled conditions of pH, autoacidification is slower allowing these strains to reach a higher biomass than the wildtype, L. lactis NZ9000. Furthermore, we demonstrate that the PTSman/glc and the PTScel are the only two PTS systems able to use glucose as a substrate in L. lactis NZ9000. PTSs can be specific for more than one sugar (148), although, to our knowledge, a PTS with specificity for both cellobiose and glucose, such as PtcBAC identified here, has not been reported. In addition to using these two glucose PTSs, L. lactis can import glucose via one or more non-PTS permease(s), which remain elusive. Our results

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48

show that the DNA-microarray technique is a useful tool for rational screening, e.g. to pursue new metabolic engineering strategies. Growth of L. lactis without pH control, as in natural milk fermentations, is halted when the pH reaches values below 4.2. Under these conditions, L. lactis NZ9000Glc⎯Lac+ uses more lactose than NZ9000Lac+ before growth ceases, since only the galactose moiety of the available lactose is converted to lactate, with concomitant acidification, leaving the glucose untouched. Therefore, in addition to the production of glucose, the residual level of lactose in the medium is lower than that in the medium of a fully grown culture of NZ9000Lac+. Most importantly, L. lactis NZ9000Glc⎯Lac+ also uses more lactose from the medium and produces glucose during fermentation in skim milk, which is a good indication that this strain is suitable for use in milk fermentations. NZ9000Glc⎯Lac+ produces glucose from lactose under all conditions tested, as shown in Figs. 3 and 4. Furthermore, in vivo NMR coupled to specifically labeled lactose showed that L. lactis NZ9000Glc⎯Lac+ directly uses galactose-6-phosphate, formed by lactose-6-phosphate hydrolysis for metabolism, while the glucose moiety of lactose-6-phosphate is expelled into the medium. L. lactis NZ9000Lac+ accumulates a transient, low level of galactose, which is consumed upon lactose depletion. This transient extracellular accumulation of galactose indicates that the cell has a clear preference for utilization of the glucose moiety of lactose. Most likely, free galactose results from dephosphorylation of galactose 6-phosphate by a phosphatase as the result of an inducer expulsion mechanism. Such a two-step reaction of inducer expulsion caused by glucose has been described before in L. lactis and other Gram-positive bacteria (156, 198, 220). When lactose is depleted, the extracellular galactose is used via the Leloir pathway (54). In summary, L. lactis NZ9000Glc⎯Lac+ is functional in two ways. First, it could be used to produce glucose from lactose, which could serve as a natural sweetener in fermented dairy products. This in situ produced glucose could replace, at least in part, the frequent addition of other sweeteners to dairy products, as the sweetness of glucose is about 60% of that of sucrose (167). The production of glucose in combination with the lower acidification achieved by L. lactis NZ9000Glc⎯Lac+ might give rise to a milder tasting end-product, e.g. when the strain is used as an adjunct starter culture in cheese or buttermilk production. Second, L. lactis NZ9000Glc⎯Lac+ uses only the galactose moiety of lactose for growth, which leads to more effective lactose removal from the medium. Therefore, the strain could be used as a nutraceutical to produce milk fermentation products with lower residual lactose concentrations, which would be suitable in a diet for individuals suffering from lactose intolerance.

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ACKNOWLEDGEMENTS

This work was financed by the European Commission through contract QLK1-CT-2000-01376, within the research programme "Quality of Life and Management of Living Resources" under the Key Action "Food, Nutrition & Health" acronym: Nutra Cells. A. R. Neves acknowledges a fellowship of FCT. We would like to thank Thijs Kouwen for expert technical assistance and Sacha A.F.T van Hijum for assisting with the DNA-microarray data analysis.

CHAPTER 3

FUNCTIONAL CHARACTERIZATION OF THREE DIFFERENT GLUCOSE UPTAKE ROUTES IN LACTOCOCCUS LACTIS

Wietske A. Pool, Ana R. Neves, Helena Santos, Oscar P. Kuipers, Jan Kok.

Chapter 3

52

Glucose transport and metabolism

53

SUMMARY

Molecular and physiological characteristics of lactococcal strains harbouring mutations in three different glucose uptake or dissimilation routes were determined, to gain insight into the specific roles of the two glucose-transporting phosphoenolpyruvate-dependent phosphotransfer systems (PEP:PTS) (EIIglc/man and EIIcel/glc) and the non-PTS glucose transport route(s) in Lactococcus lactis. Disruption of any of the pathways resulted in a considerable change in glucose transport. EIIcel/glc and the non-PTS route(s), had a preference for transport of the β-anomer of glucose. The main route for glucose transport was via EIIglc/man. Furthermore, a regulatory role for glucokinase (Glk) is proposed. Disruption of either glk, EIIglc/man, EIIcel/glc or both PTS systems had significant effects on overall glucose metabolism: changes in the pools of intracellular metabolites (fructose-1,6-bisphosphate (FBP), 3-phosphoglycerate (3PGA) and phosphoenolpyruvate (PEP)), in end-products formed (PEP, 3PGA, lactate, acetate, ethanol, 2,3-butanediol), and in key enzyme activities were observed. The lower lactate dehydrogenase (LDH) and pyruvate kinase (PK) activities, which might be caused by a lower FBP pool, together with transcriptional regulation, indicated by the induction of the gene encoding pyruvate formate lyase (pfl) in the glucose-PTS negative strain, resulted in the shifted fermentation pattern. This study demonstrates and specifies the roles that the three sugar uptake systems play in directing glucose uptake and subsequent metabolic processes in L. lactis.

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INTRODUCTION

Lactococcus lactis, a lactic acid bacterium used in the dairy industry for the production of fermented milk products, has been studied intensely over the last decades. Its relatively simple carbon metabolism and the availability of a number of molecular cloning tools make L. lactis an attractive organism for in depth studies on sugar utilization (84, 96). In L. lactis, glucose is taken up either via a phosphoenolpyruvate-dependent phosphotransferase system (PEP:PTS), or it is imported via a non-PTS permease, and phophorylated by glucokinase (Fig. 1). The PTS is considered to be the main route for glucose metabolism (36, 148, 191). PEP:PTSs are group translocators, which import and phosphorylate sugars via a phosphoryl-transfer process. The sugar-specific enzyme II (EII) imports the sugar, while the non-sugar-specific phosphocarriers enzyme I (EI) and histidine protein (HPr) are involved in the phosphate cascade in which the phosphate moiety of PEP is transferred to the incoming sugar (42, 148, 205). Recently, we have shown that L. lactis can import glucose via two different PTS-systems, the mannose/glucose-PTS encoded by ptnABCD, and the cellobiose/glucose-PTS specified by ptcBAC (141). PTSman/glc consists of a cytosolic EIIAB protein and an integral membrane EIICD protein, the PTScel/glc is made up of a cytosolic EIIAB protein and an integral membrane EIIC protein. The glucokinase of L. lactis is an ATP-dependent protein of 33.8 kD (based on the nucleotide sequence of glk), which is able to phosphorylate intracellular glucose (195). The protein has three domains: an ATP-binding site at the N-terminus, a NagC-domain, specifying a possible transcriptional regulatory site, spanning almost the complete protein and a ROK-motif also present in certain sugar kinases and regulator proteins, in the middle of the protein. The main role of glucokinase in L. lactis is so far thought to be the prevention of accumulation of unphosphorylated glucose from the hydrolysis of disaccharides, such as lactose (195). Therefore, a deletion of glucokinase is expected to have no or at most a limited effect on the metabolism of the monosaccharide glucose in L. lactis.. The main end-product of lactococcal sugar fermentation is lactic acid (homolactic fermentation). The control of the flux through glycolysis has been studied extensively over the last decades: glycolytic intermediates have been determined using different conditions, and several glycolytic enzymes and their effectors have been described (122).

Glucose transport and metabolism

55

Figure 1: Schematic overview of glucose metabolism in L. lactis NZ9000. α- And β-glucose can be imported via PTSman/glc (EII man/glc; ptnABCD), PTScel/glc (EII cel/glc; ptcBAC), or by (an) as yet unidentified non-PTS glucose transporter(s). The points of formation or expenditure of ATP and NADH are depicted as well as some of the enzymes involved (bold). Abbreviations: glucose-6P, glucose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; CO2, carbon dioxide; O2, oxygen;

ATP, adenosine triphosphate; ADP, adenosine diphosphate; Pi, inorganic phosphate; NAD+, nicotinamide adenine nucleotide; NADH, dihydronicotinamide adenine dinucleotide; GLK, glucokinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PK, pyruvate kinase; LDH, lactate dehydrogenase; PFL, pyruvate formate lyase; PDH, pyruvate dehydrogenase; ALS, α-acetolactate synthase.

Glucose-6P

Out

In

Glucose

FBP

GAP

PEP

Lactate

PK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

Pyruvate

CO2

ADP + PiATP

ADP + Pi

ATP

ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

PEPPyruvate

α-Acetolactate

Acetoin

2,3-Butanediol

CO2

NAD+NADH

NAD+NADH

ATPADP + Pi

Diacetyl

Membrane

CO2

O2

ALS

Glucose-6PGlucose

FBP

GAP

PEPPK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

Pyruvate

CO2

ADP + PiATP

ADP + Pi ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

α-Acetolactate

Acetoin

2,3-Butanediol

CO2

NAD+NADH

ATPADP + Pi

O2

ALS

GLK

Non-PTS EIIman/glc EIIcel/glc

Glucose-6P

Out

In

Glucose

FBP

GAP

PEP

Lactate

PK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

PyruvatePyruvate

CO2

ADP + PiATP

ADP + Pi

ATP

ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

PEPPyruvate

α-Acetolactate

AcetoinAcetoin

2,3-Butanediol

CO2

NAD+NADH

NAD+NADH

ATPADP + Pi

Diacetyl

Membrane

CO2

O2

ALS

Glucose-6PGlucose

FBP

GAP

PEPPK

LDH

Acetyl-CoA

Acetate Ethanol

PFL PDHFormate

PyruvatePyruvate

CO2

ADP + PiATP

ADP + Pi ADP + PiATP

DHAP

ATPADP + Pi

NAD+

NADH

NADH NAD+

2NADH2NAD+

3-PGA

GAPDH

α-Acetolactate

AcetoinAcetoin

2,3-Butanediol

CO2

NAD+NADH

ATPADP + Pi

O2

ALS

GLK

Non-PTS EIIman/glc EIIcel/glc

Chapter 3

56

Although L. lactis is generally homofermentative, under conditions of limited glucose availability or when it has to metabolize less favourable sugars, it can perform a mixed-acid fermentation in which besides lactate also acetate, ethanol, 2,3-butanediol and formate are produced (30, 67, 112).Regulation takes place at several points in the glycolytic pathway. A high level of fructose-1,6-bisphosphate (FBP) is known to activate lactate dehydrogenase (LDH) and pyruvate kinase (PK), while high levels of inorganic phosphate (Pi) together with low FBP-concentrations (when the cells are starved) inhibit PK (109, 153, 186, 191, 199). Inhibition of PK leads to accumulation of phosphoenolpyruvate (PEP) and 3-phosphoglycerate (3PGA) (152). Pyruvate metabolism is also influenced by concentrations of the glycolytic intermediates FBP, glyceraldehyde-3-phosphate (GAP), and dihydroxyacetone-phosphate (DHAP). Under anaerobic conditions LDH and pyruvate formate lyase (PFL) compete for pyruvate. During fast growth, high FBP-levels are present that activate LDH, while the high levels of DHAP and GAP under these conditions inhibit the activity of PFL (50). When a less favourable sugar is used or growth rates are lowered, less FBP, DHAP, and GAP accumulate, relieving PFL inactivation, enabling the enzyme to metabolize some of the pyruvate. Regulation of glycolysis by redox- and energy-state modulation has also been described (50). The intracellular NADH / NAD+ ratio has been suggested to control the flux through glycolysis mainly via the activity of GAP-dehydrogenase, producing NADH (50), while other studies measuring metabolites in vivo suggest a more important role for the ATP/ADP/Pi content of the cells (127, 132). In addition to regulation by glycolytic intermediates, sugar metabolism in L. lactis is also regulated at the transcriptional level via the transcriptional regulator CcpA (carbon catabolite protein A), which is involved in global metabolic control (105). Recently, L. lactis CcpA was found to intertwine regulation of carbon and nitrogen metabolism by regulating pepQ (225), a gene located tail-to-tail to ccpA (55). Besides being part of the PTS sugar phosphorylation cascade, which ultimately leads to the transfer of a phosphate-group from PEP to the incoming sugar, HPr also plays a role in regulation, since HPr phosphorylated at serine 46 acts as a cofactor for DNA binding of CcpA in Gram-positive bacteria (42, 43). Although the glycolytic pathway for the metabolism of glucose in L. lactis has been studied widely (75, 122, 152, 192), a detailed understanding of glucose transport and the initial steps in glucose degradation is missing. In this paper we analyze the first steps in glucose metabolism of L. lactis and show that the mechanism and efficiency of transport are crucial for the characteristics of fermentation. Mutant strains using different glucose uptake routes (PTSglc/man, PTScel/glc or non-

Glucose transport and metabolism

57

PTS/glucokinase) showed great differences in transport characteristics for the glucose anomers and in the dynamics of the intracellular metabolite pools.

Chapter 3

58

EXPERIMENTAL PROCEDURES

Microbial strains and growth conditions

Strains and plasmids used in this study are listed in Table 1. The strains were grown in M17 (Difco, Sparks, MD) with 0.5% galactose (w/v) or 0.5% glucose (w/v) at 30ºC or 37°C, or in chemically defined medium (CDM (144)), with 1% glucose (w/v). When appropriate, erythromycin or chloramphenicol was used at a final concentration of 5 μg/ml. For growth in a 2 L Biostat® MD fermentor (B. Braun Biotech, Inc., Allentown, PA), the medium was gassed with argon for 60 min prior to inoculation (4% inoculum from a culture grown overnight); the pH was kept at 6.5 by automated addition of 10 M NaOH, and an agitation rate of 70 rpm was used to keep the system homogeneous. Growth was monitored by measuring the optical density at 600 nm. DNA techniques

General DNA techniques were performed essentially as described elsewhere (162). Plasmid DNA was isolated by the method of Birnboim and Doly (12). Restriction enzymes, T4 DNA ligase, Expand polymerase and Taq polymerase were obtained from Roche Applied Science (Mannheim, Germany) and used according to the supplier’s instructions. PCR was performed in an Eppendorf thermal cycler (Eppendorf, Hamburg, Germany). Specific cloning procedures

Gene deletions were all performed in L. lactis NZ9000 and were constructed using a two-step homologous recombination method (96). This method does not leave antibiotic resistance markers in the chromosome, and multiple deletions in one strain can be easily realized. Primers used for cloning are listed in Table 1. Chromosomal DNA of L. lactis NZ9000 was used as a template in PCR amplifications. L. lactis NZ9000ΔptcBA and NZ9000ΔptnABCDΔptcBA, carrying only the first 36 bp of ptcB and the last 58 bp of ptcA, were made using the primerpairs ptc1/ptc2 and ptc3/ptc4 (L. lactis NZ9000 and NZ9000ΔptnABCD were used as parent strains).

Glucose transport and metabolism

59

TABLE 1: Lactococcal strains, plasmids and primers used in this study L. lactis strains Description Reference

NZ9000 MG1363 carrying pepN::nisRK (84) LL302 RepA+ MG1363, carrying a single copy of pWV01 repA in pepXP (95) LL108 RepA+ MG1363, Cmr, with multiple copies of pWV01 repA in the chromosome (95) NZ9000Δglk NZ9000 containing a 404-bp deletion in glk (141) NZ9000ΔptnABCD NZ9000 containing a 1736-bp deletion in ptnABCD (141) NZ9000ΔptcBA NZ9000 containing a 657-bp deletion in ptcBA This work NZ9000ΔglkΔptnABCD NZ9000Δglk with a 1736-bp deletion in ptnABCD (141) NZ9000ΔptnABCDΔptcBA NZ9000ΔptnABCD with a 657-bp deletion in ptcBA This work

Plasmids Description Reference

pORI280 Emr, LacZ+, ori+ of pWV01, replicates only in strains providing RepA in trans (96) pORI280-ptcBA’ Emr, pORI280-derivative specific for integration in L. lactis ptcBA (141) pVE6007 Cmr, temperature-sensitive derivative of pWV01 (107)

Primer Sequence (5' to 3') Restriction site Location annealing part

Ptc1 GCTCTAGAGTCATCTCTGACCCCTTTC XbaI 560-541 bp up. ptcB TSS Ptc2 CGGGATCCTTAGGCTGCACATGCAAGTGC BamHI 19-36 bp down. ptcB TSS Ptc3 CGGGATCCCCTTGCAGTAGAAGTTGTTG BamHI 294-313 down. ptcA TSS Ptc4 CGGAATTCCGGATAAGTTACATCGCTAAATG EcoRI 509-531 bp down. ptcA SC

Abbreviations: TSS, (putative) Translational Start Site; SC, Stop-Codon; up., upstream; down., downstream.

In vivo NMR experiments

Cells were grown in CDM containing 1% glucose (w/v), harvested in the mid-logarithmic phase of growth, washed twice with 5 mM potassium phosphate (KPi) buffer (pH 6.5), and resuspended in 50 mM KPi (pH 6.5), to a protein concentration of approximately 15 mg protein/ml. In vivo NMR experiments were performed using the on-line system described earlier (124). Glucose specifically labeled with 13C on carbon one (20 mM) was added to the cell suspension at time-point zero. The time course of glucose consumption, product formation, and changes in the pools of intracellular metabolites were monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, an NMR-sample extract was prepared as described previously (124, 127). Carbon-13 spectra were acquired at 125.77 MHz on a Bruker DRX500 spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany). All in vivo experiments were run using a

Chapter 3

60

quadruple nuclei probe head at 30oC, as described before (124). Lactate concentration was determined in the NMR-sample extract by 1H-NMR in a Bruker AMX300 (Bruker BioSpin GmbH). The concentrations of other metabolites were determined in fully relaxed 13C spectra of the NMR-sample extracts as described (127). Intracellular NAD+ and NADH were measured in vivo by 13C-NMR using a method described before (127). Enzyme activity assays

Cell suspensions used in the in vivo NMR studies were diluted in 50 mM KPi-buffer pH 6.5, to a final OD600 of 25, and aliquoted in 500 μl quantities. The cells were disrupted with 0.5 g glass beads (∅ 50-105 µm), using a Mini-BeadBeater-8 (Biospec products Inc., Bartlesville, OK) with two 1 min pulses (homogenize), with a 1 min interval on ice. Cell debris was pelleted twice and the cell-free extracts obtained were immediately used for spectrophotometric determination of enzymatic activities using a microtiterplate-reader at 340 nm (GENios, Tecan Group Ltd., Maennedorf, Switzerland). Total protein was determined by the method of Bradford (19). Glucokinase: Glucokinase (Glk) (EC2.7.1.2) activity was assayed spectrophotometrically by the glucose-6-phosphate dehydrogenase (Glc-6P-DH) (EC1.1.1.49) : NADPH-coupled assay as described (147), with minor changes. The assay mixture contained KPi-buffer (10 mM, pH 7.2), MgCl2 (5 mM), NADP+ (1 mM), ATP (1 mM), Glc-6P-DH (1 U). Glucose (20 mM) was used to start the reaction. Pyruvate Kinase: Pyruvate Kinase (PK) (EC2.7.1.40) activity was assayed spectrophotometrically by the lactate dehydrogenase (LDH) (EC1.1.1.27) : NADH-coupled reaction as described earlier (50). Lactate Dehydrogenase: Lactate Dehydrogenase (LDH) activity was assayed spectrophotometrically by NADH measurement, as described previously (50). 14C-Glucose transport assays

Mid-exponential phase cells were washed twice in KPi-buffer (5 mM, pH 6.5) and resuspended in the assay-buffer KPi (50 mM, pH 6.5). Glucose uptake was analyzed at 30ºC in cell suspensions with an appropriate cell density, dependent on the strain tested. The transport reaction (3 ml) was initiated by adding D-[U-14C]-Glucose (100 nM), with a specific activity of 317 mCi/mmol; glucose concentrations were increased up to 10 mM by using cold D-glucose (Merck, Darmstadt, Germany).

Glucose transport and metabolism

61

Time-point samples (0.5 ml cell suspension) were taken from the reaction and filtered using a vacuum pump and nitrocellulose filters with a pore size of 0.45 μm (Millipore, Billerica, MA). The reaction was immediately stopped by washing the cells on the filter with 5 ml KPi-buffer (50 mM, pH 6.5). The filters were placed in a vial, covered with scintillation fluid (Ultima Gold LSC Cocktail, Packard BioScience, Groningen, the Netherlands) and the 14C-activity was determined using a Packard TriCarb 2000 CA liquid scintillation analyzer (Packard Instrument, Meriden, CT). Data from at least two independent experiments were analyzed. The calculations were compared to a Michaelis-Menten model. The glucose uptake data resembled Michaelis-Menten uptake kinetics. Estimation of kinetic properties for α- and β-glucose consumption

A mathematical model was developed that accounted for two different Michaelian uptake kinetics of α- and β-glucose anomers and for the first-order kinetics of the anomers’ interconversion. The first-order rate constants of glucose anomerization were determined by performing 13C-NMR time series of solutions of α-[1-13C]-glucose (20 mM) in KPi (50 mM, pH 6.5), which were allowed to anomerize to equilibrium at 30ºC; at this temperature the relative percentages of α-glucose and β-glucose in equilibrium were 38 and 62%, respectively. The first-order rate constants determined were: for the conversion of α-glucose into β-glucose 0.108±0.001 min-1

and for the conversion of β-glucose into α-glucose 0.063±0.001 min-1. With these constants, the α- and β-glucose consumption kinetics were fitted to the model and the Vmax and Km determined. The model was developed in Matlab v7.3.0 (The MathWorks, Inc., Natick, MA) as a set of ordinary differential equations. The ODE23s function was used to solve the differential equations and the least squares method was used to find the set of parameters that best fitted the experimental data.

[ ] [ ] [ ] [ ][ ]

[ ] [ ] [ ] [ ][ ]⎪

⎪

⎩

⎪⎪

⎨

⎧

⋅+

⋅−⋅−⋅=

∂∂

⋅+

⋅−⋅−⋅=

∂∂

>−−>−−

>−−>−−

ββ

β

αββα

αα

α

βααβ

ϕββ

βαβ

ϕαα

αβα

GlcKGlcV

GlckGlcktGlc

GlcKGlcV

GlckGlcktGlc

m

m

max

max

Where φx is a linear function defined as:

Chapter 3

62

⎪⎪⎩

⎪⎪⎨

⎧

−≥⇐

−<⇔<⋅+⇐⋅+

=

x

x

x

xxxxx

x

ba

t

ba

ttbatba

11

11

ϕ

This function was required in order to obtain a good fit to the initial time-points of the glucose consumption kinetics. Indeed, in some strains the maximal glucose consumption rate was only reached after a lag time of a few minutes. To model this phenomenon, we assumed that initially (at t=0), the transport rate is only a fraction (φx=ax and 0<ax<1) of the expected Michaeliean rate, and that this fraction (φx) increases linearly with time until it reaches the expected Michaeliean rate (when t≥(1-ax)/bx and φx=1). Transcriptome analysis

Levels of mRNA in L. lactis strains NZ9000 and NZ9000ΔptnABCDΔptcBAC were compared by transcriptome analysis using full-genome amplicon-based L. lactis IL1403 DNA-microarrays (83). Cells grown in M17 with 0.5% glucose were harvested at the mid-exponential phase of growth. The strains were grown and analyzed independently in triplicate, with Cy3 and Cy5 dye-swaps of each repetition. This resulted in a total of 6 hybridized slides, all containing duplicate spots of each amplicon. Thus, maximally 12 measurements were obtained for each gene. The experiments were performed essentially as described by van Hijum et al, 2005 (207), with the modifications introduced by Pool et al., 2006 (141).

Glucose transport and metabolism

63

RESULTS

Glucose fermentation patterns differ greatly between L. lactis transport

mutants

Various isogenic L. lactis glucose transport mutants were made that use different glucose uptake systems. The mutant strains have a deletion in either glk (encoding glucokinase), in ptnABCD (encoding EIIman/glc), or in ptcBA (encoding EIIBAcel/glc) or in both ptnABCD and ptcBA. The glk mutation was made to disable the further metabolism of glucose imported by (a) yet unknown non-PTS glucose permease system(s). The chromosomal organization of the genes and the position and extent of the deletions are depicted in Fig 2A.

Strain μmax (h-1)NZ9000 0.72NZ9000Δglk 0.51NZ9000ΔptnABCD 0.59NZ9000ΔptcBA 0.55NZ9000ΔptnABCDΔptcBA 0.61

Δglk

ΔptnABCD

ΔptcBA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 12Time (h)

Gro

wth

(OD

600)

A

B

ptnAB ptnC ptnD

glk yvaB

ptcB ptcA yecA ptcC

Strain μmax (h-1)NZ9000 0.72NZ9000Δglk 0.51NZ9000ΔptnABCD 0.59NZ9000ΔptcBA 0.55NZ9000ΔptnABCDΔptcBA 0.61

Strain μmax (h-1)NZ9000 0.72NZ9000Δglk 0.51NZ9000ΔptnABCD 0.59NZ9000ΔptcBA 0.55NZ9000ΔptnABCDΔptcBA 0.61

Δglk

ΔptnABCD

ΔptcBA

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 12Time (h)

Gro

wth

(OD

600)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 2 4 6 8 10 120 2 4 6 8 10 12Time (h)

Gro

wth

(OD

600)

A

B

ptnAB ptnC ptnD

glk yvaB

ptcB ptcA yecA ptcC

ptnAB ptnC ptnD

glk yvaB

ptcB ptcA yecA ptcC

Figure 2: Genetic structure of L. lactis deletion strains and their growth characteristics. A) Schematic overview of the genes and the deletions studied in this work. Hooked arrow, putative promoter; lollipop, terminator structure; dashed areas, deleted sequence. B) L. lactis strains were grown at 30°C without pH control (standing bottles), under anaerobic conditions in CDM + 1% glucose. The maximum growth rates (μmax) for each strain is given at the right.

Chapter 3

64

The growth curves for each deletion strain studied, grown anaerobically in CDM with 1% glucose, are shown in Figure 2B, together with the maximum growth rates (μmax) for each strain. All the deletion strains show a lower maximum growth rate (between 0.51 and 0.61 h-1) than the parental strain L. lactis NZ9000 (0.72 h-1). Glucose consumption and the dynamics of appearance of glycolytic intermediates in the mutants were compared mutually and with L. lactis NZ9000. The fate of [1-13C]-glucose during metabolism in all of the strains was followed in time using in vivo NMR. (Table 2, Fig. 3). L. lactis NZ9000 displayed a homolactic fermentation with a maximum glucose consumption rate of 0.44 μmol·min-1·mg protein-1. FBP reached a maximum concentration of about 50 mM just prior to glucose depletion, upon which around 8 mM 3-PGA was formed (Fig. 3A). L. lactis strain NZ9000ΔptnABCD showed a heavily disrupted glucose metabolism, which was expected since the EIIman/glc

encoded by ptnABCD has been proposed to be the main sugar transport system in L. lactis. The maximal consumption rate is lowered to 0.24 and the overall glucose consumption rate is lowered to only 0.06 μmol·min-1·mg protein-1 and the strain shifted to a mixed acid fermentation (Fig. 3C). Besides that, high concentrations of 3-PGA and PEP accumulated. Based on these results EIIman/glc appears indeed to be the main glucose transport system. Interestingly, the consumption of glucose appeared to consist of two stages: a first phase with a relatively fast rate followed by a much lower rate. To see if these two phases in glucose consumption might be a consequence of anomeric specificity of the different transport systems, the glucose consumption is split into the consumption of α-glucose and β-glucose in Fig. 3. This showed that the non-PTS transport route and EIIcel/glc probably have a preference for the β-anomer of glucose, which we will later quantify (see below). The strain in which both glucose-transporting PTS systems were disrupted, L. lactis strain NZ9000ΔptnABCDΔptcBA, showed a glucose fermentation pattern comparable to that of NZ9000ΔptnABCD, although the maximum concentration of FBP accumulating during fermentation and the maximal glucose consumption rate was lower.

Glucose transport and metabolism

65

TABLE 2: Glucose fermentation pattern and glycolytic intermediates formed by L. lactis NZ9000 and its isogenic deletion strains analyzed by in vivo NMR.

Strain Functional FT a) β-glc Glycolytic Intermediates (mM) c) transport routes pref.b) FBPmax PEP 3-PGA

NZ9000 EIIman/glc, EIIcel/glc, non-PTS H +/- 49.5 ± 0.5 - 8.3 ± 2.5 Δglk EIIman/glc, EIIcel/glc MA ++ 44.2 ± 0.9 13.2 ± 2.4 31.8 ± 2.7 Δptn d) EIIcel/glc, non-PTS MA ++ 43.5 ± 1.0 13.2 ± 2.4 35.2 ± 2.8 Δptc e) EIIman/glc, non-PTS MA +/- 20.2 ± 2.1 - - ΔptnΔptc d,e) non-PTS MA ++ 33.2 ± 0.7 13.6 ± 2.1 33.6 ± 2.5

a) FT, fermentation type; H, homolactic fermentation; MA, mixed-acid fermentation

b) β-glc pref., qualitative preference for β-glucose shown: +/-, slight preference; ++, preference.

c) FBPmax, maximum fructose-1,6-bisphosphate concentration; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate

d) Δptn, ΔptnABCD e) Δptc, ΔptcBA

Surprisingly, disruption of the non-PTS-route for glucose metabolism in L. lactis strain NZ9000Δglk also led to quite large differences with respect to glucose utilization compared to its parent L. lactis NZ9000 (Table 2, Fig. 3B). This was not expected since the non-PTS uptake system(s) were suggested to play no or only a minor role in glucose consumption (195). The maximal glucose consumption rate was lowered about 4-fold to 0.20 μmol·min-1·mg protein-1 in L. lactis NZ9000Δglk, with a 4-fold increase of the 3-PGA concentration. Also considerable amounts of PEP (13.2 mM) accumulated, which was not detectable in L. lactis NZ9000. FBP accumulated to a slightly lower level in L. lactis NZ9000Δglk compared to L. lactis NZ9000. Furthermore, deletion of glk resulted in a shift to a mixed acid fermentation. In fact, the glucose fermentation pattern of L. lactis NZ9000Δglk resembled that of L. lactis strains NZ9000ΔptnABCD and NZ9000ΔptnABCDΔptcBA. L. lactis NZ9000ΔptcBA displayed a glucose fermentation pattern that differed completely from that of L. lactis NZ9000 and all the other deletion strains studied here (Fig. 3D). L. lactis NZ9000ΔptcBA showed a mixed-acid fermentation, but no PEP nor 3-PGA were formed as final fermentation products. The maximum level of accumulated FBP was less than half of that in L. lactis NZ9000. Furthermore, the consumption of glucose in two stages, as shown in L. lactis strains NZ9000ΔptnABCDΔptcBA, NZ9000ΔptnABCD, and less pronounced in NZ9000Δglk, was not observed in L. lactis NZ9000ΔptcBA.

Chapter 3

66

A

D

E F

0

10

20

30

40

50

Con

cent

ratio

n(m

M)

0 5 10 15 20 25 30 35Time (min)

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0 5 10 15 20 25 30 35Time (min)

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cent

ratio

n(m

M)

0

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NZ9000 ΔptnABCD

ΔptcBA ΔptnABCDΔptcBA

Con

cent

ratio

n (m

M)

B

C

0

10

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30

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50

Con

cent

ratio

n(m

M)

0 5 10 15 20 25 30 35Time (min)

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cent

ratio

n(m

M)

0 5 10 15 20 25 30 35Time (min)

0

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0 5 10 15 20 25 30 35Time (min)

Con

cent

ratio

n(m

M)

Δglk

GCR: 0.44 GCR: 0.20

GCR: 0.24 GCR: 0.13

GCR: 0.15

NZ9000 Δglk

ΔptnABCD ΔptcBA

ΔptnABCD ΔptcBA

A

D

E F

0

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cent

ratio

n(m

M)

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cent

ratio

n(m

M)

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NZ9000 ΔptnABCD

ΔptcBA ΔptnABCDΔptcBA

Con

cent

ratio

n (m

M)

B

C

0

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cent

ratio

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A

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cent

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0 5 10 15 20 25 30 350 5 10 15 20 25 30 35Time (min)

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cent

ratio

n(m

M)

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NZ9000 ΔptnABCD

ΔptcBA ΔptnABCDΔptcBA

Con

cent

ratio

n (m

M)

B

C

0

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cent

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cent

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cent

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0 5 10 15 20 25 30 350 5 10 15 20 25 30 35Time (min)

0

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0 5 10 15 20 25 30 35Time (min)

Con

cent

ratio

n(m

M)

Δglk

GCR: 0.44GCR: 0.44 GCR: 0.20GCR: 0.20

GCR: 0.24GCR: 0.24 GCR: 0.13GCR: 0.13

GCR: 0.15GCR: 0.15

NZ9000 Δglk

ΔptnABCD ΔptcBA

ΔptnABCD ΔptcBA

Figure 3: Kinetics of 20 mM [1-13C]-glucose metabolism, shown by in vivo NMR. A-E) [1-13C]-glucose consumption, product formation and pools of intracellular metabolites of L. lactis under anaerobic conditions at 30°C with pH controlled at 6.5. Results are shown for L. lactis strains NZ9000 (A), NZ9000Δglk (B), NZ9000ΔptnABCD (C), NZ9000ΔptcBA (D) and NZ9000ΔptnABCDΔptcBA (E). Maximal glucose consumption rates (GCR, in μmol·min-1·mg protein-1) are boxed in the upper right corners of the graphs. Symbols used: yellow diamond, α-glucose; black diamond, β-glucose; red triangle, fructose-1,6-bisphosphate; blue circle, 3-phosphoglycerate; green diamond, phosphoenolpyruvate. The lines drawn in the graph are interpolations. F) End-products formed by the indicated strains. Symbols used: White, lactate; black, acetate; stripes, ethanol; grey, 2,3-butanediol.

Glucose transport and metabolism

67

Mixed-acid fermentation is accompanied by a high NADH/NAD+ ratio

Since the genetic deletions in the L. lactis strains studied here led to a shift from a homolactic to a mixed-acid fermentation, the redox balance of one of the deletion strains (L. lactis NZ9000ΔptnABCDΔptcBA) was compared to that of the wildtype, L. lactis NZ9000. Besides the regulation of carbox flux by the FBP concentration, it has been suggested before that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is influenced by a high ratio of NADH/NAD+, controls the flux (50). The kinetics of glucose consumption, accumulation and breakdown of FBP and the levels of nicotinamide adenine nucleotide (NAD+) and dihydronicotinamide adenine dinucleotide (NADH) were measured in vivo in order to investigate the redox-state of the cells of both strains (Fig. 4). A B

0

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/ FB

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)

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+/ N

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Figure 4: Evolution of glucose, FBP, lactate, acetate, NAD+ and NADH in non-growing cells of L. lactis strains NZ9000 (A) and NZ9000ΔptnABCDΔptcBA (B). The cells were grown in CDM with 5 mg/L of [5-13C]-nicotinic acid, before and after a 40 mM [1-13C]-glucose pulse. Glucose was added at time 0 min; dark-grey shaded area is before glucose addition. Light-grey shaded area is the fast phase of glucose consumption in L. lactis NZ9000ΔptnABCDΔptcBA, Conditions were anaerobic, 30°C and pH was controlled at 6.5. Symbols: dark-blue diamond, glucose; red triangle, FBP; green square, lactate; purple triangle, acetate; gold square, NAD+; light-blue circle, NADH. Fitted lines are simple interpolations.

Before addition of glucose, ~ 5 mM of NAD+ was present in the cells of both strains while NADH could not be detected. The addition of 40 mM [1-13C]-glucose at time-point zero resulted in an immediate accumulation of FBP in both strains due to glucose metabolism. When FBP levels decrease at the onset of glucose depletion, NADH was formed at the expense of NAD+. During the formation of the end-

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products, NAD+ is recovered slowly to 5 mM in both strains. The two stages in glucose consumption in L. lactis NZ9000ΔptnABCDΔptcBA as shown by Fig. 3E can also be seen in Fig. 4B. During the first (fast) part of the glucose consumption, in this case during the first 10 min after glucose addition, mainly lactate was formed (data can be obtained from Fig. 3, although in that case only half the amount of glucose (20 mM) was added, and, thus, the fast phase only lasted for about 5 min). This fast phase of glucose consumption led to a mainly homolactic fermentation. During the second (slow) phase of glucose consumption, L. lactis NZ9000ΔptnABCDΔptcBA shifted to a mixed-acid fermentation (see Fig. 3E). The onset of this mixed-acid fermentation is accompanied by a high NADH/NAD+ ratio (above 0.2). L. lactis NZ9000ΔptnABCDΔptcBA recovers the original concentration of NAD+ (~ 5 mM) at the moment the glycolysis stops. PK and LDH activities are lowered in all mutants, while Glk activity

varies

Since LDH and PK are crucial and highly regulated enzymes in glycolysis (186, 191, 199), the activity of these enzymes was analyzed in L. lactis NZ9000 and the mutant strains. Furthermore, the activity of glucokinase was determined, as a glk-deletion had a considerable effect on glucose metabolism. In all 4 mutant strains the PK and LDH activities were lower than those in the wildtype (Table 3). PK and LDH activities were decreased about 1.5 times and 2 times respectively, which is in agreement with the mixed-acid fermentation seen in all mutants. The decrease in LDH-activity, apparently, does not cause a problem in NAD+ recovery, since Fig. 4 shows that NAD+ is formed quickly from NADH after FBP depletion in L. lactis NZ9000ΔptnABCDΔptcBA. The activity of Glk in L. lactis NZ9000 is only 0.14 U·mg protein-1. As expected, L. lactis NZ9000Δglk does not exhibit glucokinase activity. Interestingly, mutations in PTSman/glc (ptnABCD) and PTScel/glc (ptcBA) had opposite effects on the activity of Glk. Deletion of ptnABCD decreased the Glk-activity about 2.5 to 3 times, while deletion of ptcBA increased the Glk-activity about 3.5 to 4 times. When both PTSs are disrupted (NZ9000ΔptnABCDΔptcBA), the Glk-activity was comparable to that of the PTSman/glc single deletion strain.

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TABLE 3: Enzyme activity in L. lactis of glucokinase (Glk), pyruvate kinase (PK) and lactate dehydrogenase (LDH) a).

L. lactis strain Glk PK LDH

NZ9000 0.14 (0.02) 1.90 (0.06) 30.44 (1.65) NZ9000Δglk 0.00 (0.00) 1.15 (0.05) 14.29 (2.06) NZ9000ΔptnABCD 0.05 (0.02) 1.14 (0.02) 14.02 (0.31) NZ9000ΔptcBA 0.52 (0.01) 1.37 (0.04) 15.54 (1.36) NZ9000ΔptnABCDΔptcBA 0.07 (0.02) 1.26 (0.07) 11.70 (0.50)

a) In all cases enzyme activities are given (in U·mg protein-1) and standard deviations determined on at least 3 independent experiments are given between brackets.

The two-phase kinetics of glucose consumption is explained by the

preferential utilization of glucose anomers

The ability of NMR to distinguish between α- and β-anomers of glucose allowed investigation of the kinetics of glucose utilization in the L. lactis deletion strains in more detail (Fig. 5). A mathematical model accounting for the Michaelian uptake kinetics of each anomer as well as the first-order kinetics of the α- to β-anomer interconversion was developed (for details see Experimental Procedures). The Vmax for the consumption of the α- and β-anomers and the relative fluxes for the uptake of each anomer as well as for the conversion of α- into β-anomer were estimated (Fig. 5). In all strains examined, β-glucose was consumed at a maximal rate higher than that of α-glucose. In L. lactis strains NZ9000 and NZ9000ΔptcBA, exhibiting a single phase of glucose consumption, both anomers were transported as evidenced by their relative uptake fluxes (Fig. 5A and 5D). The lower maximal glucose consumption rate in L. lactis NZ9000ΔptcBA results from a reduction in the uptake of both α- and β-glucose. This assumption is further supported by the ratio between the anomers, similar to that found in L. lactis NZ9000 (Fig. 5E). In L. lactis NZ9000ΔptnABCD, the rate of glucose consumption was moderately high only when β-glucose was available. Upon depletion of β-glucose, glucose consumption is limited by the flux of α- to β-anomer conversion (Fig. 5B). The data strongly suggests that the glucose transporters present in this strain have high specificity for the β-anomer.

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Figure 5: Kinetics of consumption of the α- and β-anomers of glucose. Time course for the consumption of α- and β-glucose (experimental data and simulated line) and estimated fluxes (µmol·min-1·mg protein-1) for the conversion of α- into β-glucose and for uptake of α- and β-glucose in L. lactis strains NZ9000 (A), NZ9000ΔptnABCD (B), NZ9000Δglk (C), NZ9000ΔptcBA (D). Experimental data was obtained during the metabolism of 20 mM glucose (see Fig. 3). The mathematical model assumed Michaelis-Menten uptake kinetics of α- and β-glucose and first-order kinetics of the anomer interconversion (see Experimental Procedures). Vmax for total glucose uptake as well as for the uptake of α- and β-anomers was also estimated (E). Model simulation of the kinetics of α- and β-glucose consumption (full lines) and model-predicted fluxes (dashed lines). Symbols used: open diamond, glucose; black diamond, β-glucose; red diamond, α-glucose; black dotted line, β-glucose; red dotted line, α-glucose; blue dotted line, conversion of α-glucose into β-glucose. Note the different time scales.

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Similar results were obtained for L. lactis strains NZ9000ΔptnABCDΔptcBA and NZ9000ΔglkΔptnABCD (data not shown). In L. lactis NZ9000Δglk, the maximal consumption rates of the glucose anomers were lower, but the decrease was more pronounced for β-glucose, as in the two strains lacking ptnABCD. However, during the slower phase of glucose utilization the flux of α-glucose uptake was greater than the conversion of α-glucose to β-glucose, indicating that the α-anomer is taken up in the glk mutant. Our data show that L. lactis strains with an intact EIIglc/man system transport both anomers of glucose, whereas strains in which ptnABCD is inactivated have a clear preference for β-glucose. The affinity of glucose transport is highly decreased when EIIman/glc is

deleted

To elucidate which of the transport systems has the highest apparent affinity for glucose, glucose transport was analyzed in cell suspensions of L. lactis NZ9000 and its isogenic mutants using 14C-labelled glucose (Table 4). For each strain the assay conditions were optimized seperately, since the Km for glucose transport differed greatly between the strains. L. lactis NZ9000 had a very high affinity for glucose transport (Km = 5.2 μM), but also low affinity transport could be detected using higher glucose concentrations in the assay. The exact kinetic properties of this low affinity transporter could not be determined. Deletion of EIIcel/glc (NZ9000ΔptcBA) decreased both the Vmax and the Km 2-fold. When EIIglc/man was removed (NZ9000ΔptnABCD), a drastic change in glucose transport affinity was observed. The Km increased to around 5.2 mM, showing that EIIman/glc has the highest affinity for the uptake of glucose of all the glucose uptake systems. L. lactis NZ9000ΔptnABCDΔptcBA showed a similar Km for glucose (8.0 mM) as L. lactis NZ9000ΔptnABCD, but the Vmax was slightly lower. Although the non-PTS permease system(s) was/were expected to be of minor importance in glucose transport, L. lactis NZ9000Δglk showed a 4-fold decreased glucose transport affinity (Km = 20 μM), together with an 18-fold reduction of Vmax for one specific transporter. Deletion of glk seems to lower the capacity of EIIman/glc about 20 times, while the affinity for glucose seems to decrease about 4-fold. Besides this, a low-affinity transporter was detected with a Km of 4.8 mM (Table 4).

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TABLE 4. Kinetic parameters obtained for the transport of glucose in whole cells of L. lactis NZ9000 and its glucose transport mutants.

L. lactis strain Functional Vmax a) KmApp a)

transport routes (nmol·min-1·mg protein-1) (uM)

NZ9000 EIIman/glc, EIIcel/glc, non-PTS 188 ± 11 5.2 ± 0.2 > 160 NDb) NZ9000Δglk c) EIIman/glc, EIIcel/glc, non-PTS 10 ± 1 20 ± 2.3 224 ± 30 4809 ± 245 NZ9000ΔptnABCD EIIcel/glc, non-PTS 171 ± 8 5166 ± 372 NZ9000ΔptcBA EIIman/glc, non-PTS 92 ± 6 3.5 ± 0.8 NZ9000ΔptnABCDΔptcBA non-PTS 120 ± 1 7986 ± 674

a) Values of two independent experiments were averaged and are reported ± SD of the two measurements. Vmax and Km

App (apparent Km) were determined using the following glucose concentrations: NZ9000, 0.1 to 500 µM and 500 µM to 25 mM; NZ9000Δglk, 0.1 µM to 10 mM; NZ9000ΔptnABCD and NZ9000ΔptnABCDΔptcBA, 0.5 µM to 10 mM; NZ9000ΔptcBA, 0.1 to 500 µM.

b) Not exactly determined, above 5 mM. c) Experimental data obtained with whole cells of L. lactis NZ9000Δglk were fitted using non-linear least

squares regression analysis to the sum of two independent Michaelis-Menten equations.

EIIman/glc is a high-capacity / high-affinity transporter; EIIcel/glc is also a high-capacity transporter but it has a low affinity for glucose; the non-PTS(s) is/are (a) low-capacity and low-affinity transporter(s). Different types of transport kinetics (different transporters) could be measured using the appropriate glucose concentration ranges. Transcriptome analysis shows regulation at the genetic level in L. lactis NZ9000ΔptnABCDΔptcBA

To analyze the transcriptional effect of deletion of both glucose transporting PTSs, the transcriptomes of L. lactis strains NZ9000 and NZ9000ΔptnABCDΔptcBA were compared using DNA-microarrays of L. lactis. Any possible non-PTS transport system(s) might be overexpressed in NZ9000ΔptnABCDΔptcBA grown on glucose and may, thus, be identified. Transcriptome analysis of cells grown to the mid-exponential phase of growth in GM17 showed that several genes were differentially expressed in L. lactis NZ9000ΔptnABCDΔptcBA relative to L. lactis NZ9000, and these are categorized in Table 5. Additionally, the results from the transcriptome comparison of L. lactis NZ9000ΔptnABCDΔglk with L. lactis NZ9000 are shown in Table 5 (data obtained from previous results (141)). Although we show that glucokinase enzyme activity in L. lactis NZ9000ΔptnABCDΔptcBA was lowered 2-

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fold compared to L. lactis NZ9000 (Table 3), glk expression was not significantly reduced in the mutant strain. Also the enzymatic activities of PK and LDH were lowered resp. 1.5- and 2.6-fold in L. lactis NZ9000ΔptnABCDΔptcBA compared to L. lactis NZ9000 (Table 3), while the expression of the genes of the las-operon (pfk, pyk and ldh) was not significantly different. The expression of the gal-operon genes (galPMKTE) was not significantly different between both strains (data not shown), showing that the growth on galactose during construction of L. lactis NZ9000ΔptnABCDΔptcBA did not have a permanent effect on the gene expression (data not shown). Gene pfl (encoding pyruvate formate lyase) was expressed to a higher level in L. lactis NZ9000ΔptnABCDΔptcBA, which was expected since the protein encoded by this gene is involved in pyruvate metabolism and is responsible for the increased mixed-acid fermentation observed in this mutant strain. Also pdhA and pdhB (encoding part of the pyruvate dehydrogenase complex) were expressed to a higher level in L. lactis NZ9000ΔptnABCDΔptcBA. The cells used for these DNA-microarray studies were grown semi-anaerobically (the Pdh proteins might function slightly). Since this accounts for all strains tested, the overexpression of pdhA and pdhB in L. lactis NZ9000ΔptnABCDΔptcBA is caused by the genotype. The expression pattern of the genes involved in glycolysis and pyruvate metabolism of L. lactis NZ9000ΔptnABCDΔglk resembles that of L. lactis NZ9000ΔptnABCDΔptcBA. Gene ptsH encoding histidine protein (HPr) was not differentially expressed in L. lactis NZ9000ΔptnABCDΔptcBA, but was overexpressed 1.6 times (p-value 0.005) in L. lactis NZ9000ΔptnABCDΔglk, relative to L. lactis NZ9000. The deletion of glk seems to have an effect on the expression of this regulatory gene. The expression of ccpA, encoding carbon catabolite protein A is differentially overexpressed in both L. lactis NZ9000ΔptnABCDΔptcBA (1.5 times, p-value 0.007) and NZ9000ΔptnABCDΔglk (2.3 times). The increased expression of the noxABE genes, encoding NADH dehydrogenases (noxA and noxB) and an NADH-oxidase (noxE) in both deletion strains tested is in agreement with the overexpression of the pdh genes: it is probably required to keep a proper NAD+/NADH redox balance.

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TABLE 5: Gene expression in L. lactis NZ9000ΔptnABCDΔptcBA or NZ9000ΔptnABCDΔglk, both compared to L. lactis NZ9000

Category Gene Ratio a) Ratio b) Product function ΔptnΔptc ΔptnΔglk

Glycolysis glk 0.9 del Glucokinase and pgi 1.0 1.6 Phosphoglucose isomerase pyruvate pfk 0.7 s 0.5 s 6-Phosphofructokinase metabolism fba 0.8 0.7 s Fructose-bisphosphate aldolase tpiA 0.7 1.0 Triose-phosphate isomerase gapA 1.3 1.4 Glyceraldehyde-3P-dehydrogenase pgk 0.9 0.8 s Phosphoglycerate kinase pmg 0.7 s 0.8 Phosphoglycerate mutase enoA 1.1 1.0 Enolase pyk 1.1 0.7 Pyruvate kinase ldh 0.9 0.8 Lactate dehydrogenase (LDH) pfl 5.1 s 4.2 s Pyruvate-formate lyase (PFL) pflA 1.6 s 1.4 s PFL activating enzyme pdhA 3.6 s 1.4 Pyruvate dehydrogenase E1 comp. α pdhB 2.1 s 2.0 s Pyruvate dehydrogenase E1 comp. β-subunit pdhD 1.4 1.8 s Pyruvate dehydrogenase complex E3 comp. als 1.0 0.9 α-Acetolactate synthase aldB 1.0 1.2 Acetolactate decarboxylase butA 1.2 2.1 s Acetoin reductase butB 1.3 2.0 s 2,3-Butanediol dehydrogenase ackA1 1.5 1.6 s Acetate kinase ackA2 1.4 2.5 s Acetate kinase

Regulation ptsH 0.9 1.6 Histidine protein ccpA 1.5 2.3 s Carbon catabolite protein A

Energy atpB 0.8 0.6 ATP synthase F0, A subunit production atpD 0.8 0.6 s ATP synthase F1, beta subunit and atpG 1.0 0.7 s ATP synthase F1, gamma subunit conversion atpH 0.9 0.7 s ATP synthase F1, delta subunit noxA 1.4 1.5 s NADH dehydrogenase noxB 1.6 2.4 s NADH dehydrogenase noxE 2.0 1.7 s NADH oxidase

Other ytgA 11.3 s 3.0 s Unknown, 2 TMS, pI 9.2 genes c) ytgB 2.9 1.2 Unknown, 2 TMS, pI 12 ytgH 6.6 5.3 s Unknown, part of the ytgBAH operon ymgH 3.2 s 1.6 Unknown, 2 TMS, pI 10 chiA 8.0 s 1.9 s Chitinase yucG 3.1 1.9 Putative chitin binding protein

Abbreviations: del, deleted; s, significant differential expression (p-value < 0.001) a) L. lactis NZ9000ΔptnABCDΔptcBA over L. lactis NZ9000 b) L. lactis NZ9000ΔptnABCDΔglk over L. lactis NZ9000; taken from previous data (141) c) Highly overexpressed genes in L. lactis NZ9000ΔptnABCDΔptcBA, coding for putative membrane

proteins or putatively involved in carbohydrate metabolism. Criteria for selection were the presence of putative transmembrane segments (TMS) and the predicted isoelectric point (pI) (169). TMS prediction was performed using the HMMTOP tool (204). Clone Manager Suite (Sci Ed Software, USA) was used to calculate the pI.

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The gene with the highest fold overexpression in L. lactis NZ9000ΔptnABCDΔptcBA compared to L. lactis NZ9000 is ytgA. This gene is part of the putative operon ytgBAH. All three genes of this operon are overexpressed in L. lactis NZ9000ΔptnABCDΔptcBA. ytgBAH encodes three small proteins of 115, 156, and 91 amino-acid residues, respectively, with as yet unknown functions. Both ytgB and ytgA have 2 putative transmembrane segments and both have a very high pI, which make them promising candidates to be membrane proteins potentially involved in glucose transport. For similar reasons, another candidate gene that could be involved in glucose transport is ymgH. Other interesting differentially expressed genes are chiA and yucG, also located in a putative operon, which are overexpressed resp. 8.0 and 3.1 times in L. lactis NZ9000ΔptnABCDΔptcBA relative to L. lactis NZ9000. These genes are (putatively) involved in the breakdown of chitin, which is a polysaccharide of β-1,4-linked N-acetylglucosamine units. In L. lactis NZ9000ΔptnABCDΔglk, ytgAH, ymgH, chiA and yucG are also overexpressed, but to lesser extent than in L. lactis NZ9000ΔptnABCDΔptcBA.

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DISCUSSION

L. lactis NZ9000 has two types of glucose transport systems, the PEP-PTSs represented by PTSman/glc and PTScel/glc, and an unknown number of non-PTS transporters. Glucose fermentation through the latter requires glucokinase for phosphorylation of glucose after its internalization. In L. lactis NZ9000Δglk no glucose phosphorylating activity was detected, suggesting Glk is the only enzyme in L. lactis NZ9000 able to phosphorylate glucose intracellularly. Disruption of glk leaves only the PTSs to transport and phosphorylate glucose. The PTSs are considered to be the main glucose transporters in L. lactis (36, 148, 191). PTScel/glc appears to have the smaller contribution to the overall glucose uptake by the two glucose-transporting PTSs, since deletion of PTScel/glc only decreased Vmax and Km about 2-fold. Deletion of PTSman/glc alone or together with PTScel/glc, resulted in an approximately 1000-fold decreased affinity for glucose, showing that PTSman/glc is the major glucose transporter in L. lactis. Removal of the glk gene led to several changes in the uptake and further metabolism of glucose by the cell, such as a lower glucose consumption rate and a shift to a mixed-acid fermentation. The kinetic parameters of glucose uptake were also affected by deletion of glk. Especially at low glucose concentrations, when PTSman/glc operates, the maximal capacity was lowered almost 20 times, while the affinity decreased about 4 times. Furthermore, deletion of glk together with ptnABCD resulted in transcriptional activation of ptsH and ccpA, which did not occur (ptsH) or was less pronounced (ccpA) in a ptnABCDptcBA deletion strain. All these data suggest a direct or indirect regulatory role for Glk in glucose metabolism of L. lactis. The presence of a ROK-motif in Glk motivates this suggestion. Proteins belonging to the ROK-family known so far are bacterial sugar kinases, transcriptional repressors, or have as yet uncharacterized functions (196). Interestingly, glucokinases from the Gram-positive bacteria Streptomyces coelicolor, Staphylococcus xylosus, Bacillus megaterium and Corynebacterium glutamicum, which also contain a ROK-motif, contribute to carbon catabolite repression (CCR), although their precise roles remain unknown (86, 134, 177, 214). Proteins having a NagC-domain usually also have a ROK-domain. The abbreviation NagC is derived from the regulation of the use of N-acetylglucosamine. E. coli NagC and Mlc are two sugar-specific regulatory proteins (140, 166) that belong to the ROK-family of proteins. They have additional almost identical DNA-binding motifs, although NagC coordinates the metabolism of aminosugars and Mlc regulates genes involved in

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sugar uptake together with the cAMP/CAP complex (139). Glk proteins without a ROK-motif have so far not been shown to fulfil regulatory functions. Although the main role of glucokinase in L. lactis so far was thought to be a catalytic function in the metabolism of glucose, the results obtained here suggest also a possible regulatory role for L. lactis Glk. Besides this, the activity of Glk itself was affected by the disruption of PTSman/glc or PTScel/glc. Interestingly, deletion of either of the two PTSs had opposite effects on Glk. Glucokinase activity was slightly decreased when PTSglc/man was deleted, but deletion of PTScel/glc resulted in a five-fold increased activity. Also, Glk activity was lower in L. lactis NZ9000ΔptnABCDΔptcBA than in L. lactis NZ9000, although glk transcription was not downregulated in the double mutant, indicating that Glk is regulated by means other than transcriptional control, e.g. translational or post-translational regulation. At this point, a full explanation for these results cannot be put forward. It has been described more than a decade ago that uptake of glucose in L. lactis takes place at (at least) two different sites with anomeric specificity (11). Using the transport system mutants in combination with in vivo NMR allowed elucidating the anomeric preferences of the transporters. The glucose transport system(s) operative in L. lactis NZ9000ΔptnABCD displayed clear specificity for β-glucose. As PTScel/glc is the main glucose transporter in this mutant, since the Vmax and the Km are slightly higher than in L. lactis NZ9000ΔptnABCDΔptcBA, we propose that PTScel/glc has a preference for β-glucose. To unequivocally prove this, the anomeric specificity should be tested in an L. lactis strain deleted for ptnABCD together with the gene(s) encoding the as yet unknown non-PTS transporter(s). The preference for β-glucose of L. lactis NZ9000ΔptnABCDΔptcBA, lacking both glucose-transporting PTS systems, indicates that the non-PTS transporter(s) also has/have a preference for β-glucose. PTSglc/man displays no clear anomeric specificity as L. lactis NZ9000ΔptcBA does not have an absolute preference for either anomer; only a slight preference for the β-anomer is seen. Possessing multiple systems for glucose transport, each with its own glucose anomer specificity, may be an evolutionary advantage for L. lactis. Although transcriptional downregulation did not occur significantly for ldh and pyk in L. lactis NZ9000ΔptnABCDΔptcBA compared to L. lactis NZ9000, all deletion strains tested had lower PK and LDH enzyme activities than their parent, L. lactis NZ9000. The in vivo activities of PK and LDH in the mutants during glucose metabolism may be even lower than the activity measured in cell-free extracts, since the concentration of FBP, the positive effector of PK and LDH (191), is lower in the

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deletion strains than in L. lactis NZ9000 (Table 2). Furthermore, PK and LDH are negatively controlled by high levels of inorganic phosphate (Pi) (191). The concentration of Pi is much higher in L. lactis NZ9000ΔptnABCD (and probably similar to that in L. lactis NZ9000ΔptnABCDΔptcBA) than in L. lactis NZ9000 (data not shown). The concentrations of FBP and Pi counterbalance each other as far as regulation of PK- and LDH-activity is concerned. The lower LDH-activity is in accordance with the mixed-acid fermentation taking place in the deletion strains: the flux towards lactate is decreased and other products can be formed by PFL from pyruvate. Indeed, the pfl gene was shown to be transcriptionally overexpressed (five-fold) in L. lactis NZ9000ΔptnABCDΔptcBA relative to L. lactis NZ9000. The levels of the intracellular metabolites FBP, 3-PGA and PEP were closely related to the glucose transporter used. When the main glucose transporter PTSman/glc is removed alone or together with PTScel/glc, high accumulation of PEP and 3-PGA occured. This can be explained by the fact that without the PTSs no PEP is used for glucose uptake and concomitant phospho-transfer reactions. However, when PTSman/glc is the only PTS used, as in L. lactis NZ9000ΔptcBA, PEP and 3-PGA do not accumulate, and FBP accumulates to a lower maximum level. Deletion of PTScel/glc resulted in an approximately 4 times higher Glk activity compared to the wildtype strain. The higher activity of Glk may activate PTSman/glc, which would result in less PEP accumulation. This could also explain why high levels of PEP and 3PGA are formed in the glk deletion strain. In that case no activating Glk is present and PTSman/glc is less active, as shown by the glucose transport assays.The differences in the concentrations of PEP and 3PGA could also be caused by other regulatory effects, like those imposed by the concentrations of FBP and Pi. Furthermore, the pyruvate pool is controlled by the activity of PK, which catalyzes the reaction from PEP to pyruvate. PK was shown to be less active in all deletion strains tested. In L. lactis, sugar consumption rate has been associated with the shift to mixed-acid fermentation (50). Furthermore, modulation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by the NADH/NAD+ ratio was put forward as the main factor controlling the glycolytic flux (50). In line with this view, homolactic metabolism is a consequence of high NADH/NAD+ ratios (0.05 or above), which inhibit GAPDH. Previously, we had shown that L. lactis glyceraldehyde 3-phosphate is able to support a high glycolytic flux despite a high NADH/NAD+ ratio (127). In this study, the glucose transport mutants are characterized by moderately low glucose consumption rates and they display a clear switch to mixed-acid metabolism (Fig. 2). However, production of acetate and ethanol during the slow phase of glucose consumption was associated with a high NADH/NAD+ ratio, whereas the faster

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conversion of glucose to lactate is accompanied by a low ratio of NADH/NAD+ (Fig. 3). These observations reinforce the view that the NADH/NAD+ ratio is not the main factor controlling the glycolytic flux (127). Our data support the hypothesis that glycolytic flux is limited by the transport step. The transcriptome comparison of L. lactis NZ9000ΔptnABCDΔptcBA and L. lactis NZ9000 revealed a higher expression of pfl, pdhA and pdhB in the former strain. This is in accordance with the shift to a mixed-acid fermentation, as shown by the in vivo NMR experiments. Both the anaerobic (pfl) and the aerobic fermentation route (pdh) are overexpressed, since the cells used for the transcriptome analysis were grown under microaerobic conditions. Surprisingly, also chiA and the downstream gene yucG (both putatively involved in the degradation of chitin) were overexpressed, respectively 8 and 3 times, in L. lactis NZ9000ΔptnABCDΔptcBA. The overexpression of these genes cannot be fully explained, but one could speculate that overexpression of chiA and yucG is a stress response of the cell to prepare for metabolism of polysaccharides. Possible new candidates that could be directly or indirectly involved in glucose transport or (the regulation of) glucose metabolism and, thus, are interesting subjects for further investigation are ytgBAH. The function of these genes is yet unknown, but they are highly upregulated in L. lactis NZ9000ΔptnABCDΔptcBA. ytgA and ytgB are potential membrane proteins and could be involved in non-PTS glucose transport. Also ymgH is a potential candidate glucose transporter, since it is also a putative membrane protein. Summarizing, we show that L. lactis strain NZ9000 is able to transport glucose via three different transport systems, via one of the two PTSs (PTSglc/man or PTScel/glc) or via non-PTS transport. Deletion of one or two of these systems gives rise to major changes in glucose metabolism. The main route for glucose transport was shown to be via PTSglc/man, and a regulatory role for Glk in glucose transport was proposed. It remains to be investigated which targets are regulated by Glk, and if regulation is direct or perhaps occurs via the global carbon metabolism regulator CcpA. Each transport system has its own characteristics, and a clear preference for the β-anomer of glucose was displayed by PTScel/glc and the non-PTS transporter(s).

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ACKNOWLEDGEMENTS

This work was sponsored by the European Commission through contract QLK1-CT-2000-01376, within the research programme "Quality of Life and Management of Living Resources" under the Key Action "Food, Nutrition & Health" acronym: Nutra Cells. We would like to thank Thijs Kouwen for expert technical assistance and Luis Fonseca for the Vmax and Km calculations of the β- and α-glucose anomers.

CHAPTER 4

LACTOCOCCUS LACTIS STRAINS ENGINEERED TO IMPROVE

GALACTOSE REMOVAL FROM DAIRY PRODUCTS REVEAL METABOLIC BOTTLENECKS AND ALTERNATIVE CATABOLIC

PATHWAYS

Wietske A. Pool, Ana R. Neves, Jan Kok, Helena Santos, Oscar P. Kuipers.

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Galactose metabolism

83

SUMMARY

This study aimed at engineering Lactococcus lactis to improve its galactose fermenting capacity. Although several galactose-PTS systems have been described in other bacteria, L. lactis NZ9000 consumes galactose mainly via the Leloir pathway using GalP as the permease. α-Phosphoglucomutase appeared to be a key enzyme in controlling the galactose metabolic flux through the Leloir pathway, since galactose-1-phosphate and glucose-1-phosphate, the metabolites upstream of α-phosphoglucomutase in the pathway, accumulate. Overexpression of genes encoding proteins of the Leloir pathway in L. lactis resulted in an even higher accumulation of these metabolic intermediates. Improved galactose consumption was achieved by overexpression of the galactose permease GalP together with α-phosphoglucomutase from Streptococcus thermophilus, which removed one of the bottlenecks hampering efficient galactose consumption. Additionally, this work revealed that galactose can be transported in L. lactis by PTSlac, encoded by the plasmid pMG820-located lacFE, after which galactose is metabolized via the tagatose-6P-pathway, specified by the same plasmid. The overall glucose consumption rate using this route, however, is lower than when GalP and the Leloir pathway are used.

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INTRODUCTION

Lactococcus lactis is a lactic acid bacterium widely used as a starter in the dairy industry for the production of fermented milk products. Because of its economic importance, L. lactis has been studied intensively for the last three decades. The metabolic potential of this organism is known to be relatively simple. Sugar catabolism involves sugar-specific phosphoenolpyruvate-dependent phosphotransferase systems (PEP:PTS), sugar permeases and catabolic enzymes, e.g. for glycolysis. PEP:PTSs catalyze the uptake and concomitant phosphorylation of a sugar. L. lactis is able to ferment the milk sugar lactose, which consists of a galactose and a glucose moiety. Lactose is imported and phosphorylated by a lactose-PTS system, and hydrolyzed to form glucose and galactose-6-phosphate (210). Glucose, the preferred sugar of L. lactis, is subsequently metabolized, while galactose-6-phosphate is dephosphorylated and expelled into the medium (141), a process that is probably caused by catabolite repression (198). When glucose has been metabolized, catabolite repression is relieved and galactose can be used. As a result of incomplete lactose utilisation, some fermented dairy products contain significant residual amounts of galactose, which can pose health problems. Human individuals having a deficiency in one of the enzymes of the Leloir pathway (GalK or GalT deficiencies are most common), are unable to metabolize galactose, which can result in a disease condition called galactosemia (97). This autosomal recessive genetic disorder leads to accumulation of galactose derivatives like galactitol, galactose-1-phosphate and UDP-galactose in the blood, creating serious health problems. Patients with galactosemia can suffer from cataracts in the eyes and ovarian failure, besides neurological problems like cerebral edema, progressive decline in IQ and speech difficulties (158, 172, 178). For these individuals milk fermentation products with lowered galactose contents are desirable, which could be achieved by constructing starter strains tailored to improve galactose utilization. In L. lactis galactose is imported by galactose permease (GalP), a secondary transport system that couples galactose uptake to ion translocation (54, 142). The imported galactose is further converted to glucose-1-phosphate (G1P) via the Leloir pathway (Fig. 1), which comprises steps catalyzed by galactose mutarotase (GalM), galactokinase (GalK), galactose-1-phosphate uridylyltransferase (GalT) and UDP-galactose-4-epimerase (GalE), together converting galactose to G1P (54). The next step in galactose metabolism is a reversible reaction converting G1P to glucose-6-phosphate (G6P), which is catalyzed by α-phosphoglucomutase. G6P then enters glycolysis.

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85

Galactose

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Glucose-6P

Galactose-6P

Tagatose-6P

Tagatose-1,6-BisP

Triose-P

GalP/??

GalM

GalK

GalT

LacAB

LacC

LacD

PgmH

PTSgal

PEP-PTSNon-PTS

UDP-glc

UDP-galGalE

Galactose

Tagatose-6P pathwayLeloir pathway

Glycolysis

Out

Membrane

In

GalP LacFE???

Galactose

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Glucose-6P

Galactose-6P

Tagatose-6P

Tagatose-1,6-BisP

Triose-P

GalP/??

GalM

GalK

GalT

LacAB

LacC

LacD

PgmH

PTSgal

PEP-PTSNon-PTS

UDP-glc

UDP-galGalE

Galactose

Tagatose-6P pathwayLeloir pathway

Glycolysis

Out

Membrane

In

GalP LacFE???

Figure 1: Schematic overview of the first steps in the metabolism of galactose by L. lactis. Galactose can be imported by a non-PTS system (GalP or possibly another non-PTS transporter indicated by the double questionmark) and metabolized to glucose-1-phosphate via the Leloir pathway and converted to glucose-6-phosphate by PgmH. Galactose may also be imported by a PTS (perhaps LacFE?) and further metabolized to triose-phosphates by the tagatose-6P-pathway. Triose-phosphates enter glycolysis. Abbreviations: GalP, galactose permease; GalM, galactose mutarotase; GalK, galactokinase; GalT, galactose-1-phosphate uridylyltransferase; GalE, UDP-galactose-4-epimerase; PgmH, α-phosphoglucomutase; LacFE, PTSlac; LacAB, galactose-6-phosphate isomerase; LacC, tagatose-6-phosphate kinase; LacD, tagatose-1,6-bisphosphate aldolase; UDP-gal/glc, UDP-galactose/glucose. The letter P at the end of a metabolite represents phosphate.

The L. lactis MG1363 gal genes are clustered in an operon (galPMKTE) (54). The gene encoding α-phosphoglucomutase (α-PGM) of L. lactis MG1363 was not known until very recently, after the work described in this chapter was executed.

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Lactococcal α-PGM turned out to be a very specific and unique enzyme in that it does not belong to the known family of prokaryotic α-phosphoglucomutases (this thesis, Chapter 5, (121)). Although the α-PGM from Escherichia coli encoded by pgmU has been successfully cloned in a metabolic engineering strategy to improve exopolysaccharide production in L. lactis (13), in this paper we used the α-PGM of Strestococcus thermophilus instead, since this lactic acid bacterium is generally regarded as save (GRAS) and it is a closer relative to L. lactis. Besides through the Leloir pathway, L. lactis can metabolize galactose via the tagatose-6-phosphate pathway (Fig. 1) (210). In this case, galactose uptake is mediated by a PEP:PTS, at the expense of one PEP molecule, leading to intracellular galactose-6-phosphate (Gal6P) (148, 187, 190). The PTSlac is suggested to be able to transport galactose (92, 135). Whether the Leloir pathway or the tagatose-6-phosphate pathway is primarily used, is strain-dependent (187). In L. lactis ssp. cremoris MG1363, the laboratory strain employed in this study, the Leloir pathway is used (54). This study aimed at engineering L. lactis to improve its galactose fermenting capacity, as a means to reduce the galactose concentration in dairy products to a minimum. Metabolic engineering strategies often entail the overexpression or deletion of genes operating in competing pathways or of regulator genes of the pathways, to produce a specific product or increase the use of a specific substrate (38). Engineering sugar pathways is often not straightforward, since sugar metabolism is a highly regulated process in which changes in the concentration of metabolic enzymes, and thus of one or more metabolite(s), may have unpredictable effects on the cell. For example, to obtain a higher galactose consumption in Saccharomyces cerevisiae, the gene regulatory network had to be engineered instead of the metabolic enzymes (131). To improve the efficiency of galactose metabolism in L. lactis, the different transport systems were investigated separately and the effects of overexpression of several galactose metabolic genes was examined using in vivo NMR. The knowledge thus gained about the bottlenecks in galactose metabolism allowed designing a successful strategy for the overexpression of genes that led to more efficient and more complete galactose utilization.

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EXPERIMENTAL PROCEDURES

Microbial strains used and growth conditions

Strains and plasmids used in this study are listed in Table 1. The strains were grown at 30ºC in M17 broth (Difco, Sparks, MD) containing 1% (w/v) galactose or in chemically defined medium (CDM) (144) with 1% (w/v) galactose. When appropriate, erythromycin or chloramphenicol was used at 5 μg/ml. Nisin-induction of gene expression was performed by addition of supernatant (dilution 1:10,000) from a fully grown culture of the nisin producer L. lactis NZ9700 (82), when the cells to be tested reached an optical density at 600 nm of 0.5. For growth in a 2 L Biostat® MD fermentor (B. Braun Biotech, Inc., Allentown, PA), the medium was gassed with argon for 1 h prior to inoculation (4% inoculum from a culture grown overnight in the same medium); the pH was kept at 6.5 by automated addition of 5 M NaOH, while an agitation rate of 70 rpm was used. Growth was monitored by measuring the optical density at 600 nm in a Novaspec II spectrophotometer (Amersham Pharmacia Biotech., Uppsala, Sweden). TABLE 1: Strains, plasmids and primers used Strains Description Reference

L. lactis MG1363 L. lactis subsp. cremoris, plasmid free derivative of NCDO712 (52) NZ9000 MG1363 carrying pepN::nisRK (84) NZ9700 Nisin producing transconjugant with the nisin-sucrose transposon Tn5276 (82) LL302 RepA+ MG1363, carrying a copy of pWV01 repA in pepX (95) NZ9000ΔgalP NZ9000 with deletion of galP This work NZ9000ΔgalPMK NZ9000 with deletion of galPMK This work

S. thermophilus ST11 Gal- strain (115)

Plasmids Description Reference

pNZ8048 CmR, nisin inducible PnisA (34) pNG8048e CmR, EmR, derivative of pNZ8048 Lab stock pORI280 EmR, LacZ+, RepA-, ori+ (96) pVE6007 CmR, Ts-ori, derivative of pWV01 (107) pMG820 23.7 kb derivative of pLP712, containing lacFEGABCD (106) pgalP pNG8048e, galP in the NcoI / XbaI site This work pgalPMKT pNG8048e, galPMKT in the NcoI / XbaI site This work ppgmA-ST pNG8048e, pgmA of S. thermophilus in the NcoI / XbaI site This work pgalP-pgmA-ST pgalP, pgmA of S. thermophilus in the XbaI-site This work pgalPMKT-pgmA-ST pgalPMKT, pgmA of S. thermophilus in the XbaI-site This work

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General DNA manipulations

General DNA techniques were performed essentially as described (162). Plasmid DNA was isolated by the method of Birnboim and Doly (12). Restriction enzymes, T4 DNA ligase, Expand polymerase and Taq DNA-polymerase were obtained from Roche Diagnostics GmbH (Mannheim, Germany) and used according to the supplier’s instructions. PCR amplifications were performed in an Eppendorf thermal cycler (Eppendorf, Hamburg, Germany) with L. lactis MG1363 chromosomal DNA as the template, unless described otherwise, using appropriate conditions. Primers used in this study are described in Table 2. Construction of galP and galPMKT overexpression vectors

The PCR products obtained with primer pairs GalA1-fw / GalA-rev and GalA1-fw / GalT-rev were cloned as Eco31I / XbaI restriction fragments in NcoI / XbaI restricted pNG8048e, resulting in the plasmids pGalP (galP cloned downstream of the nisin-inducible promoter PnisA) and pGalPMKT (galPMKT downstream of PnisA), respectively. The plasmids were introduced by electrotransformation (60) in L. lactis NZ9000 and L. lactis NZ9000[pMG820]. TABLE 2: Primers used in this study Name Sequence (5’ to 3’), restrictionsite underlined Restriction sites

GalA1-fw CGGTCTCCCATGAAAGAGGGAAAAATGAAACAACG Eco31I GalA-rev CTAGTCTAGATTATTTCAAACGTTCTTC XbaI GalT-rev CTAGTCTAGATTATTGATTCACAAAATC XbaI PgmA-ST-fw1 CATGCCATGGTAGTTGTGATACAATGTAAGCG NcoI PgmA-ST-fw2 GCTCTAGATAGTTGTGATACAATGTAAGCG XbaI PgmA-ST-rev GCTCTAGATTGGTGTAGCAGCGAAAG XbaI GalA-KO1 GCTCTAGACTTTCGGGAGAAACCGTGG XbaI GalA-KO2 CGGGATCCCCCTCTTTCATGGGAATCC BamHI GalA-KO3 CGGGATCCCCTTTGTAGTCCCAGCGG BamHI GalA-KO4 CGGAATTCGAATGCTATCTTCTCCACC EcoRI GalAMK-KO5 CGGGATCCGATGATTACGAAGTCACTGG BamHI GalAMK-KO6 CGGAATTCAATCGCCAGAAGTTGGTCC EcoRI

Construction of galP and galPMK deletion strains

The PCR products obtained with primer pairs GalA-KO1 / GalA-KO2 and GalA-KO3 / GalA-KO4 were cloned together as XbaI / BamHI and BamHI / EcoRI restriction

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89

fragments in XbaI / EcoRI restricted pORI280 (96), resulting in pORI280-galP’. This plasmid was obtained using L. lactis LL302 (95) as the cloning host. PCR products obtained with primer pairs GalA-KO1 / GalA-KO2 and GalAMK-KO5 / GalAMK-KO6 were cloned as XbaI / BamHI and BamHI / EcoRI restriction fragments in XbaI / EcoRI restricted pORI280, resulting in pORI280-galPMK’, which was obtained and maintained in L. lactis LL302. Introducing pORI280-galP’ or pORI280-galPMK’ together with helper plasmid pVE6007 in L. lactis NZ9000, followed by a two-step homologous recombination event (96), yielded strains NZ9000ΔgalP and NZ9000ΔgalPMK, respectively, the chromosomal structures of which were confirmed by PCR analysis and Southern blotting using Enhanced Chemiluminescence (ECL) detection (Amersham Pharmacia Biotech) with a PCR fragment obtained with primer pair galA-KO1 / galA-KO2 as a probe. Overexpression of pgmA

To clone the pgmA gene of Streptococcus thermophilus ST11, primers were designed on the sequence of S. thermophilus LY03 (IMDST01, culture collection at VU, Brussels, Belgium). The PCR product obtained with primer pairs PgmA-ST-fw1 / PgmA-ST-rev was cloned as an NcoI/XbaI restriction fragment in NcoI/XbaI-restricted pNG8048e, to obtain ppgmA-ST. The PCR product obtained with primer pairs PgmA-ST-fw2 / PgmA-ST-rev was cloned as an XbaI restriction fragment in XbaI-restricted pgalP or pgalPMKT, to obtain pgalP-pgmA-ST and pgalPMKT-pgmA-ST, respectively. Variants of both plasmids that carried pgmA downstream and in the same orientation as the gal gene(s) were selected in L. lactis NZ9000. Plasmid constructions were checked by EcoRI restriction analysis. Determination of α-phosphoglucomutase activity in cell extracts

Cell cultures grown to an OD600 of 0.5 were induced with nisin as described above. After 2 h of induction, a 20 ml sample was centrifuged (6000 rpm, 7 min), washed twice with potassium phosphate (KPi) buffer (5 mM, pH 7.2) and resuspended in 1 ml KPi solution (10 mM, pH 7.2). Cells were disrupted using 0.5 g glass beads (∅ 50-105 μm, Fischer Scientific BV, Den Bosch, the Netherlands), using a Mini-BeadBeater-8 (Biospec products, Inc., Bartlesville, OK) with two 1 min pulses of homogenization, and a 1 min interval on ice. Cell debris was pelleted and α-phosphoglucomutase specific activity was assayed as described before (150). The reaction mixture (250 μl) consisted of: TEA buffer (50 mM; pH, 7.2), 5 mM MgCl2,

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0.5 mM NADP+, 0.5 U glucose-6-phosphate dehydrogenase, 50 μM glucose-1,6-bisphosphate and cell extract (10-20 μl). After 2 min of incubation at 30ºC, the reaction was started by addition of 1.4 mM α-G1P. Enzymes were assayed at 30ºC in a 96 well microtiterplate using the GENios microtiterplate reader and Magelan software (Tecan, Grödig, Austria). One unit of enzyme is defined as the amount of enzyme catalyzing the conversion of 1 μmol of substrate (NADP+) per min under the assay conditions used. Specific activity was expressed as units (μmol·min-1) per milligram of protein. Measurements were done at 340 nm (NADPH absorbance peak). Protein concentrations were determined by the method of Bradford (19). In vivo NMR spectroscopy

Cells were grown in a 2 L fermenter in CDM with 1% w/v galactose and harvested during mid-logarithmic phase (OD600 = 2.2), centrifuged, washed twice and resuspended to a protein concentration of approximately 15 mg/ml in 50 mM KPi buffer (pH 6.5). In vivo NMR experiments were performed using the on-line system described earlier (124). Specifically labeled galactose ([1-13C]-galactose) was added to the cell suspension to a final concentration of 20 mM at time-point zero. The time course of galactose consumption, product formation, and changes in the pools of intracellular metabolites were monitored in vivo. When the substrate was exhausted and no changes in the resonances of intracellular metabolites were observed, a total NMR-sample extract was prepared and used for the quantification of end-products and other metabolites (124, 127). The concentration of labeled lactate determined by 1H-NMR was used as a standard to calculate the concentration of the other metabolites in the sample (124). Due to the fast pulsing conditions used for acquiring in vivo 13C-spectra, correction factors were determined to convert peak intensities into concentrations (124). The correction factors for Gal1P (0.73), α-G1P (0.73), UDP-Gal (0.67) and UDP-Glc (0.67) were determined as follows: an NMR-sample extract supplemented with the pure compounds was circulated through the NMR tube at a rate similar to that used for cell suspensions and 13C-NMR spectra were acquired with a 60º flip angle and a recycle delay of 1.5 s (saturating conditions) or 60.5 s (relaxed conditions). The quantitative kinetic data for intracellular metabolites were calculated as described elsewhere (125, 127). The lower limit for in vivo NMR detection of intracellular metabolites under these conditions was 3-4 mM. Intracellular metabolite concentrations were calculated using a value of 2.9 µl/mg of protein for the intracellular volume of L. lactis (146). Carbon-13 spectra were acquired at 125.77

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MHz on a Bruker DRX500 spectrometer (Karlsruhe, Germany). All in vivo experiments were run using a quadruple nuclei probe head at 30°C, as described before (124). Acquisition of 13C-NMR spectra was performed as described by Neves et al (127). Although individual experiments are illustrated in each figure, each experiment was repeated at least twice and the results were highly reproducible. The values reported are averages of two experiments and the accuracy varied from ±2% (extracellular products) to ±10% in the case of intracellular metabolites with concentrations below 5 mM.

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RESULTS

Galactose can be imported by L. lactis by more than one non-PTS

permease and by a PTS system

The first step in the utilization of substrates by L. lactis, i.e. transport across the cell membrane, is likely to have an important contribution to the overall sugar utilization rate. The first aim was to get an overview of which galactose transport systems are present in L. lactis and to determine the galactose consumption rate in strains using different galactose transport systems. The uptake of galactose in L. lactis is mainly mediated by the galP-encoded galactose permease (54). Since previous work had shown that galactose transport in an L. lactis MG1363 galP mutant was strongly reduced (54), a galP deletion was made in the isogenic strain NZ9000 (MG1363pepN::nisRK) in order to block the Leloir pathway (Fig. 2) and study galactose consumption via a possible PTS by introducing pMG820 in the galP-negative strain. Surprisingly, L. lactis NZ9000ΔgalP was still able to grow in a medium with galactose as the sole carbon source in a way comparable to wildtype L. lactis NZ9000. The maximum growth rate was only slightly decreased relative to that of NZ9000 (Fig. 2). Subsequently, a mutant strain of L. lactis NZ9000 was made in which, apart from galP, also the downstream genes of the operon, galM and galK, were deleted (Fig. 2). Deletion of galPMK resulted in total loss of the capacity to grow in a medium with galactose as the sole source of carbon (Fig. 2), demonstrating that in the galP deletion strain another non-PTS system takes over the role of GalP. Besides non-PTS galactose transport, galactose can be imported via a PEP:PTS. Introduction of plasmid pMG820 (106) with genes lacABCDFEG encoding the lactose-PTS system and the tagatose-6-phosphate pathway enzymes (210) in L. lactis NZ9000ΔgalPMK, re-established the capability of the strain to use galactose. The lactose-PTS encoded by lacFE on pMG820 is a good candidate for a transport system able to import galactose, next to its usual substrate lactose (1, 148). It is unlikely that another galactose-transporting PTS is encoded on pMG820. Recently, sequence analysis of pLP712 ((52), of which pMG820 is a 23,7-kb deletion derivative) indeed revealed that no other sugar transporter is encoded by pMG820 (personal communication: U. Wegmann, Institute of Food Research, Norwich Research Park, Norwich, UK). To be sure that LacFE was the galactose-PTS transporter, we furthermore had to rule out the possibility that another galactose-PTS gene encoded by the chromosome of L. lactis MG1363, of which the activity only visualizes after introduction of the tagatose-6-phosphate pathway genes

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lacABCD located on pMG820. Based on genome mining efforts there is no additional candidate gene encoding a galactose-specific PTS (215). This is the more unlikely since galactose imported via such a PTS would result in intracellular accumulation of Gal6P, which is highly toxic when it is not further metabolized, because a functional tagatose-6P pathway is not encoded by the L. lactis MG1363 chromosome, but by pMG820. From recent results we can conclude that galactose is indeed transported via LacFE, since L. lactis NZ9000ΔgalPMK overexpressing the genes lacABCDFE could metabolize galactose, while NZ9000ΔgalPMK overexpressing only lacFE or lacABCD could not (A.R. Neves, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal, unpublished results).

ΔgalP

ΔgalPMK

galP galM galK galT galE

galP galM galK galT galE

Strain

0.38

0.16

0.36

NZ9000

NZ9000ΔgalP

NZ9000ΔgalPMK

μmax (h-1)

0,0

0,1

1,0

10,0

0 3 6 9 12 15 18 21 24 27

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Figure 2: L. lactis deletion strains used in this study and their growth characteristics. Top: Schematic overview of the genetic make up of the galP and galPMK deletion strains. Hooked arrow, promoter; lollipop, possible terminator structure. Genes are indicated by black arrows; grey regions, parts of the chromosome removed by the double cross-over deletion events; dashed parts, out of frame in the deletion strain. Bottom: Growth curve of L. lactis strains NZ9000, NZ9000ΔgalP and NZ9000ΔgalPMK in CDM with 1.0% galactose; corresponding maximum growth rates are given on the right. For L. lactis NZ9000ΔgalP the first μmax is calculated for the first 3 hours of growth, the second is calculated for the growth after 3 hours. The average of 2 experiments is shown. Differences did not exceed 5%.

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Taken the above into account, galactose uptake in L. lactis NZ9000ΔgalPMK[pMG820] takes place via the PTS encoded by lacFE. Gal6P is subsequently further metabolized via the tagatose-6-phosphate pathway, encoded by lacABCD. Indeed, in vivo NMR on this strain shows accumulation of the typical tagatose-6-phosphate pathway intermediate tagatose-1,6-bisphosphate (TBP) (see below). Galactose consumption via the PEP:PTS (LacFE) was analyzed in L. lactis NZ9000ΔgalPMK[pMG820] without background effects of the Leloir pathway. Galactose consumption rate via the Leloir pathway is higher than via

the PTS-uptake and subsequent tagatose-6-phosphate pathway in L. lactis

Galactose consumption, end-product formation and the dynamics of intracellular metabolites derived from galactose in L. lactis strains NZ9000, NZ9000ΔgalP, and NZ9000ΔgalPMK[pMG820] were monitored by in vivo NMR (Fig. 3), to determine the galactose metabolic pathway used in each strain and their galactose consumption rates. L. lactis NZ9000 accumulated Gal1P and G1P, showing that galactose was consumed via the Leloir pathway and that galactose was imported via a non-PTS permease. The galactose consumption rate in L. lactis NZ9000 was 0.16 μmol·min-

1·mg protein-1. As also shown above, L. lactis NZ9000ΔgalP could still metabolize galactose, although the galactose consumption rate of this strain was much lower than when galP was present, i.e. only 0.09 μmol·min-1·mg protein-1. Slight accumulation of the intracellular metabolites Gal1P and G1P was detected in L. lactis NZ9000ΔgalP cells in vivo by NMR, indicating that the Leloir pathway was used for galactose metabolism. As galactose cannot be transported via GalP in this strain another, yet unknown, galactose non-PTS permease has to be active in L. lactis NZ9000ΔgalP to import galactose. Mining of the L. lactis MG1363 genome revealed no clear candidate gene coding for this non-PTS galactose transport system besides galP.

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galactose

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Figure 3: Analysis of L. lactis galactose consumption. Galactose consumption and analysis of extracellular (A, C, E) en intracellular (B, D, F) metabolites by in vivo NMR of L. lactis strains NZ9000 (A, B), NZ9000ΔgalP (C, D) and NZ9000ΔgalPMK[pMG820] (E, F). For each strain the galactose consumption rate (GalCR) is depicted in μmol·min-1·mg protein-1.

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L. lactis NZ9000ΔgalPMK[pMG820] could metabolize galactose, although no Gal1P nor G1P was detected in this strain. The strain accumulated TBP labelled at C1 already 2 min. after galactose addition (Fig. 4), indicating that galactose is metabolized via the tagatose-6-phosphate pathway and that galactose is internalized via a PTS. After about 6 min. some FBP labelled at C6 is detected, caused by scrambling of the labelled carbon atom at the level of triose phosphate isomerase. This FBP arises from the triose phosphates (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate) by the reverse reaction of fructose-bisphosphate aldolase. The galactose consumption rate in L. lactis NZ9000ΔgalPMK[pMG820], which only uses a galactose PTS for galactose transport, was 0.05 μmol·min-1·mg protein-1. This is considerably lower than the galactose consumption rates in the strains that use the Leloir pathway.

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Overexpression of Leloir genes in L. lactis results in accumulation of

the intermediates Gal1P and G1P

Since galactose metabolism via GalP and the Leloir pathway displayed the highest galactose consumption rate, overexpression of several combinations of Leloir pathway genes was examined to try to increase the galactose consumption rate. The galP gene alone and galP together with galMKT were cloned in pNG8048e, downstream of the nisin-inducible promoter, and the resulting vectors were introduced in L. lactis NZ9000. Overexpression of the Gal proteins was confirmed by SDS-PAGE and Coomassie Brilliant Blue staining (data not shown). Overexpression of galP or galPMKT resulted in lower growth rates on galactose than the empty-vector control strain L. lactis NZ9000[pNZ8048]. Addition of more nisin to the cells with the galP or galPMKT overexpression plasmids decreased the growth rates even more (data not shown), an effect that was not observed with the control plasmid without the gal genes. Further analysis by in vivo NMR revealed, especially in L. lactis NZ9000 overexpressing galPMKT, a high accumulation of the Leloir pathway intermediates Gal1P (up to 24 mM) and G1P (up to 34 mM) (Fig. 5A & 5B). High intracellular concentrations of these phosphorylated intermediates could be toxic and suggests that a bottleneck exists in galactose metabolism at the level of α-PGM (see Fig. 1). Overexpression of α-phosphoglucomutase from S. thermophilus in L. lactis leads to faster galactose consumption

The following step was to overexpress the gene encoding α-PGM, together with the genes of the Leloir pathway, to obtain better galactose consumption by a “push-and-pull” strategy. Since the gene encoding α-PGM in L. lactis was not known at the time, the gene encoding α-PGM of S. thermophilus strain ST11 (pgmA-ST) was used as an alternative to increase the α-PGM activity in L. lactis. The pgmA-ST gene was amplified by PCR and overexpressed alone or downstream of galP or galPMKT in nisin-inducible plasmids in L. lactis NZ9000. Overexpression of S. thermophilus pgmA alone resulted in an approximately forty-fold increased α-phosphoglucomutase enzymatic activity in L. lactis (Table 3), showing that the S. thermophilus enzyme is functional in L. lactis. Overexpression of S. thermophilus pgmA transcriptionally fused downstream of galPMKT led to a ten-fold increased α-PGM activity, compared to the empty vector control L. lactis NZ9000[pNZ8048] (Table 3).

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Figure 5: Galactose consumption and analysis of the intracellular intermediates Gal1P and G1P in different L.lactis strains analyzed by in vivo NMR. L. lactis strains examined: A) NZ9000[pgalP], B) NZ9000[pgalPMKT], C) NZ9000[ppgmA-ST], D) NZ9000[pgalP-pgmA-ST], E) NZ9000[pgalPMKT-pgmA-ST]. Galactose, galactose-1-phosphate (Gal1P) and glucose-1-phosphate (G1P) concentrations are depicted for each strain. Other intracellular metabolites like fructose-1,6-bisphosphate, phosphoenolpyruvate and 2-phosphoglycerate and the external end-products lactate, ethanol, acetate and 2,3-butanediol were detected but are omitted from the graphs for reasons of clarity. Galactose consumption rates (GalCR, in μmol·min-1·mg protein-1) are indicated for each strain in the table at the bottom right.

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TABLE 3: Nisin-inducible α-phosphoglucomutase activity in L. lactis strains used in this study

α-Phosphoglucomutase activity (U/mg protein) Strain Not induced Nisin-induced Overexpressiona) NZ9000[pNZ8048] 0.3 0.2 no NZ9000[ppgmA-ST] 0.4 15.9 ~ 40 x NZ9000[pgalPMKT-pgmA-ST] 0.3 2.9 ~ 10 x

a) Nisin-induced overexpression, calculated from: value “not induced” / value “nisin-induced”

The metabolism of galactose in L. lactis strains overexpressing pgmA-ST alone or together with galP or galPMKT was analyzed in vivo by NMR. All strains showed a mixed-acid fermentation, in which besides lactate, millimolar amounts of ethanol, acetate and 2,3-butanediol were formed (data not shown). A temporary accumulation of FBP was observed and 3PGA and PEP accumulated at the end of metabolism (data not shown). In Fig. 5 (panels C, D & E) galactose consumption and the concentrations of Gal1P and G1P in the three different pgmA-ST overexpressing strains are depicted. Overexpression of pgmA-ST alone or together with galP or galPMKT decreased the accumulation of Gal1P and G1P, previously observed in strains over-expressing galP(MKT) alone (Fig. 5). The galactose consumption rates in the strains overexpressing pgmA-ST together with galP(MKT) increased to 0.23 μmol·min-1·mg protein-1.

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DISCUSSION

To improve galactose utilization in L. lactis, the first steps in galactose utilization were analyzed, to see which of the metabolic routes, the Leloir pathway or the plasmid-introduced tagatose-6-phosphate pathway, was the most efficient. Gene galP, encoding a galactose non-PTS transporter (the first step of the Leloir pathway), was removed from the chromosome in order to be able to examine the plasmid pMG820-encoded tagatose-6-phosphate pathway. Surprisingly, and in conflict with previous results (54), a strain in which the known galactose permease GalP had been removed was still able to grow on galactose. We suspect that the galP deletion previously described (54) had a downstream negative effect, although significant GalK activity was detected, disturbing the maximal activity of the Leloir enzymes, leading to the strongly reduced galactose uptake and metabolism detected. Our strain L. lactis NZ9000ΔgalP used galactose via the Leloir pathway, as the typical Leloir intermediates Gal1P and G1P were detected in L. lactis NZ9000ΔgalP. This could not be caused by residual activity of GalP, as an out-of-frame deletion was made in which only the first four amino acids of the original protein are left. Based on these results we conclude that, next to GalP, at least one more efficient non-PTS galactose transporter has to be encoded by the genome of L. lactis NZ9000. Inspection of the genome sequence of L. lactis MG1363, the parent of strain NZ9000, however, did not yield an obvious candidate gene. Since deletion of galP resulted in a galactose consumption rate that was almost half of that of the L. lactis NZ9000, galactose consumption is most efficient when GalP is used for galactose transport. In L. lactis NZ9000ΔgalP, the maximum galactose consumption rate is decreased to 0.09 μmol·min-1·mg protein-1 from 0.16 μmol·min-

1·mg protein-1 for L. lactis NZ9000. A complete block of galactose metabolism was obtained by deletion of chromosomal galPMK. Although galactose may still be imported via the unidentified putative galactose permease, further conversions should be hindered, since galactose mutarotase (GalM) and galactose kinase (GalK) are not present in the triple gal mutant. The data allows concluding that at least one of these two enzymes (GalM or GalK) has no homologue in L. lactis. With the introduction of pMG820 in L. lactis NZ9000ΔgalPMK, the ability of the strain to metabolize galactose was re-established. In L. lactis NZ9000ΔgalPMK[pMG820] galactose is imported via the plasmid-encoded PTS (LacFE), resulting in Gal6P, which is further metabolized through the tagatose-6P-pathway (LacABCD). Indeed, during galactose fermentation a slight accumulation of the pathway’s intermediate

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TBP was observed by in vivo NMR. Although sugar transport via a PTS is bioenergetically favourable for L. lactis, since only one ATP is spent on transport and phoshorylation of the sugar, the in vivo analyses of galactose metabolism presented here reveal that L. lactis using a PTS in combination with the tagatose-6P-pathway did not reach the same galactose consumption rate as did an L. lactis strain employing a non-PTS galactose permease followed by the Leloir pathway. Of course this may be largely caused by the possibility that the PTS encoded by pMG820 has a low affinity for galactose. We conclude that improvement of L. lactis galactose metabolism via engineering of the Leloir pathway using GalP seems the most viable route, when self-cloning techniques are preferred over heterologous expression of genes. Since accumulation of Gal1P and G1P occurred in the galP(MKT) overexpressing strains, the bottleneck in galactose metabolism via the Leloir pathway was shown to be at the level of α-PGM. Heterologous overexpression of the pgmA gene of S. thermophilus in L. lactis NZ9000 increased the α-PGM activity. Overexpression of this true α-PGM of S. thermophilus together with the L. lactis Leloir genes relieved the galactose metabolic bottleneck and improved the galactose consumption rate significantly. In summary, we can conclude that galactose is consumed in L. lactis most efficiently via the Leloir pathway using GalP as a permease, and that galactose transport by lacFE followed by tagatose-6-phosphate pathway activity is less efficient. Moreover, α-PGM appears to be a key enzyme in controlling the galactose metabolic flux, especially when the first steps in the Leloir pathway are overexpressed. Recently, α-PGM of L. lactis MG1363 was identified and interestingly shown to be related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily (121). Overexpression of galP(MKT) together with pgmA-ST in L. lactis NZ9000 increased the galactose consumption rate significantly to 0.23 μmol·min-1·mg protein-1 compared to 0.16 μmol·min-1·mg protein-1 in wildtype NZ9000. At this point, we do not know whether the strain described in this chapter will leave less residual galactose during a milk fermentation. Further improvement of galactose consumption may require even more sophisticated strategies like engineering of regulatory genes, as was done for Saccharomyces cerevisiae (131). It has been shown that the galactose genes in L. lactis are repressed by the carbon catabolite control protein CcpA when a more favourable sugar like glucose is present (105, 225). To improve galactose utilization independent of environmental conditions, engineering could be focussed on these regulatory pathways, to possibly engineer a strain preferring galactose over glucose. It would also be interesting to introduce pgalP(MKT)-pgmA-ST (the plasmid carrying galP(MKT) together with pgmA of S.

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thermophilus behind the nisin promoter) in the glucose-negative strain (L. lactis NZ9000ΔglkΔptnABCDΔptcBA, Chapter 2, (141)). This strain, with its combination of genetically engineered traits (it is blocked in glucose metabolism and improved for galactose metabolism), would in theory be a very interesting candidate for analysis of residual galactose, especially in an experimental dairy fermentation using a combination of sugar substrates.

CHAPTER 5

THE α-PHOSPHOGLUCOMUTASE OF LACTOCOCCUS LACTIS IS UNRELATED TO THE α-D-PHOSPHOHEXOMUTASE

SUPERFAMILY AND ENCODED BY THE ESSENTIAL GENE pgmH

Ana R. Neves*, Wietske A. Pool*, Rute Castro, Ana Mingote, Filipe Santos, Jan Kok, Oscar P. Kuipers and Helena Santos

Based on J. Biol. Chem. 2006, 281:36864-36873.

* Both authors contributed equally to the results described in this chapter.

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A novel α-phosphoglucomutase

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SUMMARY

α-Phosphoglucomutase (α-PGM) plays an important role in carbohydrate metabolism, by catalyzing the reversible conversion of α-glucose-1-phosphate to glucose-6-phosphate. Isolation of α-PGM activity from cell extracts of Lactococcus lactis strain MG1363 led to the conclusion that this activity is encoded by yfgH, herein renamed to pgmH. Its gene product has no sequence homology to proteins in the α-D-phosphohexomutase superfamily and instead is related to the eukaryotic phosphomannomutases within the haloacid dehalogenase superfamily. In contrast to known bacterial α-PGMs, this 28 kDa enzyme is highly specific for α-glucose-1-phosphate and glucose-6-phosphate and showed no activity with mannose-phosphate. To elucidate the function of pgmH, the metabolism of glucose and galactose was characterized in mutants overproducing or with deficiency of α-PGM activity. Overproduction of α-PGM led to increased glycolytic flux and growth rate on galactose. Additionally, the intracellular concentration of UDP-glucose was decreased. Despite several attempts, we failed to obtain a deletion mutant of pgmH. The essentiality of this gene was proven by using a conditional knock-out strain, in which a native copy of the gene was provided in trans under the control of the nisin promoter. Growth of this strain was severely impaired when α-PGM activity was below the control level. Moreover, the growth-rate and biomass production were directly related to α-PGM activity. We show that the novel L. lactis α-PGM is the only enzyme mediating the interconversion of α-glucose-1-phosphate to glucose-6-phosphate in L. lactis and is essential for growth.

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INTRODUCTION

Phosphoglucomutase (E.C. 5.4.2.2) is widespread in living organisms from bacteria to humans (217). It plays various roles in carbohydrate metabolism, by catalyzing the reversible conversion of α-glucose-1-phosphate (α-G1P) to glucose-6-phosphate (G6P). In higher organisms, its major function is mediating the mobilization of sugar moieties from energy reserves (e.g. glycogen, trehalose, starch). Also, α-PGM activity is essential for the synthesis of UDP-glucose, a sugar donor for the production of glucose-containing polysaccharides. Therefore, PGM is a crucial link between catabolic and anabolic processes. The lactic acid bacterium Lactococcus lactis is used worldwide in the industrial manufacture of fermented milk products. The organism converts sugars primarily into lactic acid, thus providing an efficient means of food conservation. In L. lactis, α-phosphoglucomutase is assumed to be essential for the utilization of galactose via the Leloir pathway (105) and also for the synthesis of precursors of cell wall polysaccharides and exopolysaccharides (40, 74). In a number of Gram positive bacteria, namely Bacillus subtilis and Streptococcus pneumoniae, pgm mutants showed altered cell wall morphology and altered polysaccharide production as well as growth defects on glucose (58, 91, 100). Despite the key metabolic role of α-PGM and the wealth of knowledge on sugar metabolism of L. lactis (122), genes coding for this activity have not been identified in this organism. More than a decade ago the presence of two distinct phosphoglucomutase activities in L. lactis subsp. lactis with specificity for α- and β-anomers of phosphoglucose has been reported (150). A 28 kDa protein, designated β-PGM, was shown to catalyze the reversible conversion of β-G1P to G6P. The L. lactis β-PGM belongs to the haloacid dehalogenase (HAD) enzyme superfamily; its X-ray structure has been determined and the catalytic mechanism investigated (87, 88, 224). β-PGM is a catabolic enzyme in the pathway for maltose and trehalose degradation (99), which is encoded by a gene (pgmB) in the trehalose operon (4, 15). A larger protein (around 65 kDa) showing affinity for the α-anomer of glucose-1-phosphate was partially purified (150). The α-specificity and the protein size, matching the analogous parameters of bacterial α-PGMs, led the authors to conclude that it was the lactococcal α-PGM. It is pertinent noting that all α-PGMs described thus far belong to the α-D-phosphohexomutase superfamily of proteins (171, 217). Intriguingly, a BLASTp search of the available L. lactis genome sequences (15, 215) using members of the α-D-phosphohexomutase superfamily as query, or a domain

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search using the highly conserved regions of proteins in this family, retrieved only FemD, a protein tentatively annotated as a phosphoglucosamine mutase. In this work we report the identification, purification, expression and characterization of L. lactis α-PGM and its encoding gene pgmH (previously yfgH), and show that this activity is essential for growth. To our knowledge the lactococcal α-PGM is the first member of the HAD superfamily of proteins with strict specificity for α-G1P.

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EXPERIMENTAL PROCEDURES Microbial strains and growth conditions

Strains and plasmids used throughout this study are listed in Table 1. For molecular biology procedures, L. lactis strains were cultivated as batch cultures (flasks) without aeration in M17 medium (Difco, Sparks, MD) containing 0.5% glucose (w/v) at 30°C or 38ºC. For physiological studies, L. lactis NZ9000 (84) derivatives, NZ9000[pNZ8048] (control strain), NZ9000[pNZ8048-pgmH] (hereafter designated NZ9000[pgmH+]) and NZ9000ΔpgmH[pgmH+], were grown in Chemically Defined Medium (CDM) (144) at 30ºC in 2 or 5-liter vessels (B. Braun Biostat®, MD), under anaerobic conditions and pH 6.5 as previously described (123). Glucose or galactose was added to a final concentration of 1% (w/v). Plasmid selection was achieved by addition of chloramphenicol (5 mg/l) or erythromycin (5 mg/l) to the growth medium. For overproduction of α-PGM, nisin (1 μg/l) was added to the medium when an optical density at 600 nm (OD600) of 0.5 was reached. For studies in which the nisin-inducible conditional mutant NZ9000ΔpgmH[pgmH+] was used, cells were grown overnight in M17 containing different levels of nisin and washed once with fresh M17 lacking nisin. The washed cultures were subsequently subcultured for 15 h (initial OD600 around 0.025 or 0.05 for growth on glucose or galactose, respectively) in fresh medium, either with or without nisin (0.01 to 1 µg/l). Growth was monitored by measuring the optical density at 600 nm. General DNA techniques

General molecular techniques were performed as described by Sambrook et al. (162). Chromosomal and plasmid DNA were isolated from L. lactis according to Johansen and Kibenich (68) and Birnboim (12), respectively. L. lactis was transformed with plasmid DNA by electroporation as described by Holo and Nes (60) using a Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, CA). All DNA modification enzymes were purchased from Roche Molecular Biochemicals (Mannheim, Germany), and used according to the manufacturer’s directions. Polymerase chain reactions (PCRs) were performed using Expand DNA polymerase (Roche Molecular Biochemicals) and purified with the Roche PCR purification kit (Roche Molecular Biochemicals). Primers (listed in Table 1) were purchased from Biolegio BV (Malden, the Netherlands).

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TABLE 1: Strains, plasmids and primers used in this study Strains Description Reference

MG1363 Plasmid free and prophage cured derivative of NCDO712 (52) NZ9000 Derivative of MG1363 carrying pepN::nisRK (84) LL302 RepA+ MG1363, carrying single copy of pWV01 repA in pepX (95) NZ9000::pORI280-ΔpgmH Derivative of NZ9000 containing pORI280ΔpgmH integrated in the chromosome in pgmH This work NZ9000ΔpgmH[pgmH+] Derivative of NZ9000 containing a 711-bp deletion in pgmH and carrying pNZ8048-pgmH This work

Plasmids Description Reference

pNZ8048 Cmr; inducible expression vector carrying PnisA (84) pNZ8048-pgmH Cmr, derivative of pNZ8048 carrying a copy of pgmH This work pORI280 Emr, LacZ+, ori+ of pWV01, replicates only in strains providing repA in trans (96) pORI280-ΔpgmH Emr; derivative of pORI280 specific for integration in the L. lactis pgmH gene This work pORI13 Emr; integration vector; ori+ repA- derivative of pWV01; promoterless lacZ (163) pORI13-pgmH’ Emr; derivative of pORI13 specific for integration in the L. lactis pgmH gene This work pVE6007 Cmr, temperature-sensitive derivative of pWV01 (107) pNZ8048-femD Cmr, derivative of pNZ8048 carrying a copy of femD This work pNZ8048-galE Cmr, derivative of pNZ8048 carrying a copy of galE This work

Primers Sequence (5’ to 3’) Restriction-site

yfgH-fw CATGTCATGAAAAAAATATTAAGTTTCGACATTG RcaI yfgH-rev GCTCTAGAGAAAATTAAGCTTCTTCCATCGC XbaI yfgH-KO1 CGGAATTCCCAACGCTTCTACATCTTC EcoRI yfgH-KO2 CGGGATCCCTTATCAACGCTTACATTATAAC BamHI yfgH-KO3 CGGGATCCGAAACTGCTGCTATCCTCAAAGC BamHI yfgH-KO4 GCTCTAGACTTCACGCGTTTGGGC XbaI yfgH-fw1 GGAATTCGAAAAAAATATTAAGTTTCG EcoRI yfgH-rev1 GCTCTAGAGCTTGTTTAGCTTCAACC XbaI femD-fw CATGCCATGGGTAAATATTTTGGAACAG NcoI femD-rev GCTCTAGATTATTTCACACCAATTTCCTC XbaI galE-fw CATGTCATGACAGTTTTAGTACTTGGTGG RcaI galE-rev GGACTAGTTCAGTAGCCTTTTGGATGAC SpeI

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Construction of strains and plasmids

The genes pgmH and galE were cloned and overexpressed in L. lactis strain NZ9000 as follows. The coding regions of pgmH and galE were amplified by PCR using primers yfgH-fw/yfgH-rev and galE-fw/galE-rev (Table 1), respectively. The 0.76-kb RcaI/XbaI and the 0.98-kb RcaI/SpeI PCR-products were digested with the indicated enzymes and fragments were cloned into NcoI/XbaI or NcoI/SpeI digested pNZ8048 (84), yielding constructs pNZ8048-pgmH and pNZ8048-galE, respectively. The femD gene was amplified by PCR using primer pair femD-fw/femD-rev (Table 1). The overexpression plasmid pNZ8048-femD was constructed by cloning the 1.36-kb NcoI-XbaI resticted PCR product into similarly digested pNZ8048. The resulting construct was transformed into L. lactis strain NZ9000. L. lactis MG1363 DNA was used as template for all PCR reactions. Several strategies were employed to construct an L. lactis ΔpgmH strain. In the first strategy, a complete deletion of the pgmH gene was tried by using a two-step homologous recombination method as follows: the upstream and downstream flanking regions of pgmH were obtained by PCR using primer pairs yfgH-KO1/yfgH-KO2 and yfgH KO3/yfgH-KO4, and cloned as EcoRI/BamHI and BamHI/XbaI restriction fragments in pORI280 (96), resulting in plasmid pORI280ΔpgmH. The plasmid was obtained and maintained in L. lactis LL302 (95). pORI280ΔpgmH and pVE6007 (107) were co-transformed into L. lactis NZ9000 and this strain was taken through the temperature shift protocol for single and double-crossovers (96). No double-crossover transformants were obtained in M17 supplemented with 0.5% of the following sugars: glucose, maltose, trehalose or a mixture of trehalose (0.5%) and galactose (0.05%). In a second approach, which was used in an attempt to knock out the pgmH by single-crossover plasmid integration, a 0.46-bp fragment, containing the 5’ end of pgmH except two base pairs, was amplified by PCR using primers yfgH-fw1 and yfgH-rev1, double-digested with EcoRI/XbaI, and cloned into the similarly digested pORI13 (163). The resulting plasmid was transformed into NZ9000, but despite several attempts, no erythromycin resistant colonies were obtained. Subsequently, pNZ8048-pgmH was introduced in NZ9000::pORI280-ΔpgmH, obtained as explained above. The resulting chloramphenicol and erythromycin resistant strain was subjected to an excision strategy (96) in M17 medium with 0.5% w/v glucose and containing nisin (0.1 µg/l), yielding NZ9000ΔpgmH[pgmH+]. The addition of nisin was required to induce expression of pgmH from pNZ8048-pgmH. Integration of plasmids in the chromosome and deletions were confirmed by PCR and by Southern blotting. Probe labeling, hybridization, and detection were

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performed using the ECL direct nucleic acid labeling system, according to the specifications of the manufacturer (Amersham Pharmacia Biotech, Little Chalfont, UK). Enzyme assays

Enzymes were assayed at 30ºC immediately after mechanical disruption of a cell suspension by passage through a French press (twice at 120 MPa), and centrifugation for 15 min at 30,000 × g to remove cell debris. Protein concentration was determined by the method of Bradford (19). Routinely and during enzyme purification, α- and β-phosphoglucomutase activities were assayed as described by Qian et al. (150). The 1 ml assay mixture contained 50 mM TEA-HCl (pH 7.2), 5 mM MgCl2, 0.5 mM NADP+, 50 µM glucose-1,6-bisphosphate and 1.75 U glucose-6-phosphate dehydrogenase. Reactions were started by the addition of 1.5 mM α-G1P or β-G1P, respectively. Purification of native α-PGM from L. lactis

The native α-PGM was purified by fast protein liquid chromatography (Amersham Biosciences). All steps were performed in the presence of 0.5 mM EDTA and 5 mM 2-mercaptoethanol, at 4ºC; 15% glycerol (w/v) was added during chromatography. Cell extracts were prepared from 135 g (wet weight) of galactose-grown MG1363 cells that were suspended in 50 mM TEA buffer (50 mM, pH 7.2), containing 5 mM MgCl2. Precipitation steps with protamine sulfate (0.25% w/v) and with solid ammonium sulfate were essentially performed as described by Qian et al. (150). The precipitate collected in the range of 45-85% (NH4)2SO4 saturation was dissolved in TEA buffer (50 mM, pH 7.2) containing 30 mM KCl, and dialyzed against the same buffer. The protein preparation was applied onto a gel filtration Superdex 200 (20 mM Bis-TrisPropane, pH 6.9, containing 45 mM KCl), and α-PGM activity was detected in the flow-through. Active samples were loaded onto a Resource Q column (same buffer as Superdex 200), and elution was carried out with a linear gradient of KCl (45-500 mM). Active fractions, eluted at around 240 mM KCl, were dialyzed against Tris-HCl (10 mM, pH 7.3) containing 4 mM MgCl2 and 1.6 M (NH4)2SO4. The sample was applied to a phenyl-Sepharose column, and elution was carried out with a linear gradient of (NH4)2SO4 (1.6 – 0 M). α-PGM activity was detected at 0.5 M of (NH4)2SO4. The positive fractions were dialyzed against TEA buffer (50 mM, pH 7.5, 30 mM KCl), loaded onto a gel filtration

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Superose 6 column (same buffer as dialysis). α-PGM activity was measured in the flow-through and active fractions were evaluated by SDS-PAGE. Two putative target proteins of 28 and 37 kDa were excised from Coomassie-stained SDS-PAGE gels and the amino acid sequences of their N-termini were determined (45). ORFs encoding the two proteins were identified by BLASTp searches using the genome sequence of L. lactis subsp. lactis IL1403. Purification and characterization of recombinant α-PGM

The α-PGM protein was purified to electrophoretic homogeneity from approximately 65 grams (wet weight) of glucose-grown nisin-induced NZ9000[pNZ8048-pgmH] cells. Cell extract preparation and chromatographic steps were essentially as described above for purification of the native enzyme, except that the gel filtration Superdex 200 (first chromatographic step) was replaced by an anion-exchange Q-Sepharose (20 mM Bis-TrisPropane, pH 6.9; 45-500 mM KCl gradient), and the last chromatographic step (gel filtration) was not required. Also, the hydrophobic step (phenyl-Sepharose) preceded the anion exchange step (Resource Q). The purified protein was stored at -20ºC in 50 mM TEA buffer containing 5 mM MgCl2, 0.5 mM EDTA, 5 mM mercaptoethanol and 15% glycerol until further use. The pH profile of α-PGM was determined in TEA-HCl buffer in the pH range 4-9. The effect of 1 or 5 mM alternative cations (Ni2+, Zn2+, Ca2+, Mn2+, and Li+) was examined in the presence of 50 µM Mg2+. ATP and FBP were examined as potential inhibitors of α-PGM activity. Kinetic constants of the purified α-PGM were determined in the reaction direction α-G1P G6P. The Vmax for the reaction direction G6P α-G1P and substrate specificity were determined by 31P-NMR spectroscopy. The 3 ml reaction mixture contained 50 mM TEA-HCl buffer (pH 7.2), 5 mM MgCl2, 50 µM glucose-1,6-bisphosphate, 3% 2H2O (v/v) and 20 µg of pure enzyme. For substrate specificity, spectra were acquired before and after three hours of incubation at 30ºC in the presence of putative substrates (7.5 mM). The following compounds were examined: α-G1P, G6P, β-G1P, glucosamine-1P, glucosamine-6P, α-mannose-1P, fructose-1P, fructose-6P, FBP, FBP/α-G1P, α-galactose-1P, galactose-6P, ribose-5P, ribulose-5P, 6-phosphogluconate, UDP-galactose and UDP-glucose. For Vmax determinations, the reactions were started by the addition of G6P (50 mM) and the time course for its consumption was monitored. The rate of G6P consumption was calculated by comparison of the area of the G6P resonance to that of a known amount of methylphosphonate added as an internal

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standard. One unit of enzyme activity was defined as the amount of enzyme catalyzing the conversion of 1 µmol of G6P per minute under the experimental conditions used. Molecular mass determination was performed by gel filtration on Superose 12 10/300 GL using 100 mM sodium acetate, pH 7.0. Determination of extra- and intracellular metabolites during growth

Samples (2 ml) of L. lactis NZ9000[pNZ8048] or L. lactis NZ9000[pgmH+] cultures growing in CDM containing either glucose or galactose were collected at different points during growth, centrifuged (2000 × g, 5 min, 4ºC), and supernatant solutions were stored at –20ºC until analysis by high performance liquid chromatography. Fermentation substrates and products were quantified as described before (126). Ethanol extracts for analysis by 31P-NMR and quantification of phosphorylated metabolites in NZ9000[pgmH+] and control strains at mid-exponential growth phase were prepared as described elsewhere (151). The dried extracts were dissolved in 4 ml 2H2O containing 5 mM EDTA (final pH approximately 6.8). Assignment of resonances and quantification of phosphorylated metabolites was based on previous studies (151) or by spiking the NMR-sample extracts with the suspected, pure compounds. The reported values for intracellular phosphorylated compounds are averages of two independent growth experiments and the accuracy was around 15%. NMR experiments and quantification of metabolites

Cells were harvested during mid-logarithmic growth phase (OD600 = 2.2), centrifuged, washed twice and re-suspended to a protein concentration of 16.5 mg/ml in 50 mM KPi buffer, pH 6.5. In vivo NMR experiments were performed as described before (123). Spectra were acquired sequentially prior to and after addition of [1-13C]-glucose or [1-13C]-galactose. After substrate exhaustion, and when no changes in the resonances due to end products and intracellular metabolites were observed, a total NMR-sample extract was prepared and used for quantification of end-products and other metabolites (125, 127). The concentration of labeled lactate determined by 1H-NMR was used as a standard to calculate the concentration of the other metabolites in the sample (124). Due to the fast pulsing conditions used for acquiring in vivo 13C-spectra, correction factors were determined to convert peak intensities into concentrations (124). The correction factors for Gal1P (0.73), α-G1P (0.73), UDP-Gal (0.67) and UDP-Glc

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(0.67) were determined as follows: an NMR-sample extract supplemented with the pure compounds was circulated through the NMR tube at a similar rate to that used for cell suspensions and 13C-NMR spectra were acquired with a 60º flip angle and a recycle delay of 1.5 s (saturating conditions) or 60.5 s (relaxed conditions). The quantitative kinetic data for intracellular metabolites were calculated as described elsewhere (125, 127). The lower limit for in vivo NMR detection of intracellular metabolites under these conditions was 3-4 mM. Intracellular metabolite concentrations were calculated using a value of 2.9 μl/mg of protein for the intracellular volume of L. lactis (146). NMR spectroscopy

NMR-spectra of living cells were run at 30°C using a quadruple-nucleus probe head on a Bruker DRX500 spectrometer (Karlsruhe, Germany). Acquisition of 31P-NMR and 13C-NMR spectra was performed as described by Neves et al. (127). Although individual experiments are illustrated in each figure, each experiment was repeated at least twice and the results were highly reproducible. The values reported are averages of two to four experiments and the accuracy varied from ±2% (extracellular products) to ±10% in the case of intracellular metabolites with concentrations below 5 mM. The quantification of phosphorylated metabolites and the measurement of α-PGM activity were performed as described by Ramos et al. (151). Carbon and phosphorus chemical shifts are referenced to external methanol or H3PO4 (85%) designated at 49.3 ppm and 0.0 ppm, respectively.

Chemicals

[1-13C]Glucose (99% enrichment) and [1-13C]-galactose (99% enrichment) were obtained from Campro Scientific (Veenendaal, The Netherlands) and Cambridge Isotope Laboratories (Andover, MA, USA), respectively. Formic acid (sodium salt) was purchased from Merck Sharp & Dohme (Paço de Arcos, Portugal). All other chemicals were reagent grade and obtained from Sigma-Aldrich (St. Louis, MO).

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RESULTS

Purification of α-PGM activity and identification of the coding gene

A BLASTp search of L. lactis MG1363 and IL1403 genomes using sequences of proteins in the α-D-phosphohexomutase superfamily identified femD as the best hit. FemD, annotated as a putative phosphoglucosamine mutase (15), is a protein with a calculated molecular weight of 48 kDa. To ascertain whether femD encodes a protein with α-PGM activity, the gene (1356 bp) was cloned into pNZ8048, under the control of the nisin inducible promoter, and introduced in NZ9000. Cell extracts of NZ9000[pNZ8048-femD] grown on glucose-M17 and induced with nisin (1 µg/l) contained a clearly overexpressed protein with a size of 48 kDa, as evidenced by SDS-PAGE (not shown). Moreover, the activity of α-PGM in the induced strain was identical to that of control cells (0.15 U/mg protein) indicating that femD does not encode an α-PGM. A PCR strategy to clone the lactococcal α-PGM gene using degenerate primers based on highly conserved regions of phosphohexomutases (171) and the complementation of an E. coli pgm::tet mutant (102) with a genomic library of L. lactis were also attempted without success. Consequently, purification of α-PGM activity was pursued to obtain protein sequence information and identify the gene.

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Figure 1: Purification of the α-PGM activity and the identification of the coding gene. A) Protein samples loaded on SDS-PAGE; different fractions of the flow-through after elution from a Superose6 matrix are shown. Lanes from left to right: protein marker, fraction 18, 20, 22, 23, 24, 26 and 28. At the bottom the α-PGM activity of the samples in each lane is depicted. 2 potential candidates for α-PGM activity are marked with an arrow. B) Protein samples loaded on SDS-PAGE. Lanes from left to right: protein marker, L. lactis NZ9000[pgmH+] uninduced (-) and nisin-induced (+),L. lactis NZ9000[galE+] uninduced (-) and nisin-induced (+). At the bottom the α-PGM activity of the samples in each lane is depicted.

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Extracts from L. lactis MG1363 grown on M17 with galactose exhibited 2-fold higher α-PGM activity (0.3 U/mg protein), than glucose-grown cells and were used for protein isolation. α-PGM was partially purified (100 fold) (as described in the Materials and Methods section). Fractions from the last column contained several bands, as visualized by SDS-PAGE, but only two proteins (28 and 37 kDa) co-eluted with the phosphoglucomutase activity in all purification steps (Fig. 1A). These two protein bands were excised and their N-terminal amino acid sequences determined to be MKKILSFD and MTVLVLG, respectively. The corresponding open reading frames in the L. lactis IL1403 genome sequence were yfgH (NP_266726; hypothetical protein) and galE (NP_268136, UDP-glucose 4-epimerase), respectively. The product of galE was shown to be UDP-glucose 4-epimerase by others (54, 211), and cloning and overexpression of galE in NZ9000 did not lead to enhancement of α-PGM activity (Fig. 1B). The yfgH (herein renamed as pgmH) gene contains 759 bp and codes for a protein with 252 amino acids and a calculated protein mass of 28,276 kDa. The gene was amplified by PCR using MG1363 chromosomal DNA as template, cloned in pNZ8048 and expressed in strain NZ9000. Induction with nisin (1 μg/l) of a glucose-M17 growing NZ9000[pgmH+] culture resulted in a 30-fold increase of α-PGM activity (Fig. 1B), thus proving that pgmH encodes the α-PGM of L. lactis. In the chromosome of L. lactis MG1363, pgmH is flanked by yfgL and yfgG (Fig. 2), both of unknown function (215). All three genes are preceded by Shine-Dalgarno sequences (5’-AAATAGGAGA-3’, for pgmH). A putative promoter region containing an extended -10 (5’-TGTTATAAT-3’) sequence precedes the pgmH coding sequence. Inverted repeat sequences, (5’-AAAAAGCAATCTATTTTGATTAGATTGTTTTT-3’) and (5’-AAAAAGTTGTCATTAATGACAGCTTTTT-3’) followed by AT-rich regions downstream of the yfgL and pgmH stop codons, respectively, could function as transcriptional terminators. Therefore, it is unlikely that the three genes are organized in an operon-like structure and possibly they have unrelated functions. A similar genomic organization is observed for strain L. lactis subps. lactis IL1403 (15).

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yfgF yfgG pgmH yfgL dfpAyfgF yfgG pgmH yfgL dfpAyfgF yfgG pgmH yfgL dfpA

Figure 2: Chromosomal organization of pgmH in L. lactis MG1363. Gene pgmH is located between yfgG and yfgL. Hooked arrow: putative promoter region. Lollipop: putative terminator region.

Interestingly, the product of pgmH (Fig. 3; DQ778336) had no sequence homology with proteins from the α-D-phosphohexomutase family. It showed around 37% identity with hypothetical proteins from human-colonizing Gram-positive bacteria (Bifidofacterium longum ZP_00121741 and Propionibacterium acnes YP_056695), and around 25% identity with eukaryotic phosphomannomutases (e.g. Mus musculus PMM2 NP_058577, Homo sapiens PMM2 AAH08310 and PMM1 AAC51117, and Saccharomyces cerevesiae Sec53 NP_116609) (Fig. 3). These proteins have been classified as members of the HAD superfamily. Despite the low overall identity, the α-PGM sequence contains the four conserved motifs that characterize the HAD superfamily: motif I (DXDX(T/V)), motif II (S/TXX) and motif III and IV (K (X)18-30(G/S)(D/S)) (79, 159).

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M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

-----MAT-----------LCLFDMDGTLTAPRQKITEEMDGFLQKLRQKTKIGVVGGSDFEKLQEQLG--NDV 56-----MAAPGPA-------LCLFDVDGTLTAPRQKITKEMDDFLQKLRQKIKIGVVGGSDFEKVQEQLG--NDV 60-----MAVTAQAARRRERVLCLFDVDGTLTPARQKIDPEVAAFLQKLRSRVQIGVVGGSDYCKIAEQLGDGDEV 69-----MSIAEFAYKEKPETLVLFDVDGTLTPARLTVSEEVRKTLAKLRNKCCIGFVGGSDLSKQLEQLG--PNV 67---------------------------------------MARIIVRLLDRTSVCVISGGQFGQFRTQVVEALVD 35MVVRSWSELDFDNVCSNAKVFGFDLDNTLASSKQPMKPAMIERFCALLDHTVVALISGGGMAVATSQVLDVLTP 74----------------MKKILSFDIDNTLNEPKMPIFPEMAELLATLSQKYIIAPISGQKYDQFLIQIINNLPE 58

: **:*.** . : : : : * .: : :.*· *: ----------------MFKAVLFDLDGVITDT----AEYHFRAWKALAEEIGINGVDR-QFNEQLKGVSREDSL 53

**:*. : * .. : :. :

VEKYD--YVFPENGLVAYKDGKLLCKQNIQGHLGEDVIQDLINYCLSYIANIKLPKKR--GT---FIEFRNGML 123VEKYD--YVFPENGLVAYKDGKLLCRQNIQSHLGEALIQDLINYCLSYIAKIKLPKKR--GT---FIEFRNGML 127IEKFD--YVFAENGTVQYKHGRLLSKQTIQNHLGEELLQDLINFCLSYMALLRLPKKR--GT---FIEFRNGML 136LDEFD--YSFSENGLTAYRLGKELASQSFINWLGEEKYNKLAVFILRYLSEIDLPKRR--GT---FLEFRNGMI 134APRLDRLHLLPACGTQYYRCVDGEWQRIYVEALTDDEKSRAMEAVETCARDLGLWEEHTWGP---VLEDRESQI 106NARRGNLHVMPTSGSRYYRWDGTQWALVYAHDLSEATVAAVSESLERHARELGLWEQQVWGP---RIENRGSQI 145SANLDNFHLFVAQGTQYYAHKAGEWKQVFNYALTDEQANAIMGALEKAAKELGHWDESVLLPGDEINENRESMI 132. . : : * * * : : .. .. * * . :

QKILD--LADKKVSAEEFKELAKRKNDNYVKMIQDVSPADVYPGILQLLKDLRSNKIKIALA----SASKNGPF 121. . : : : : . . : . :

NVSPIGRSCSQEERIEFYELDKKEHIRQKFVADLRKEFAGKGLTFSIGGQISIDVFPEGWDKRYCLRHLEHAG- 196NVSPIGRSCSQEERIEFYELDKKENIRQKFVADLRKEFAGKGLTFSIGGQISFDVFPDGWDKRYCLRHVENDG- 200NISPIGRSCTLEERIEFSELDKKEKIREKFVEDLKTEFAGKGLRFSRGGMISFDVFPEGWDKRYCLDSLDQDS- 209NVSPIGRNASTEERNEFERYDKEHQIRAKFVEALKKEFPDYGLTFSIGGQISFDVFPAGWDKTYCLQHVEKDG- 207TFSALGQQAPVDAKKAWDPSGDK---KLKLREAVAGKLL--DLEVRAGGSTSVDITRVGRDKSFGIAKLLEMTG 175TFSALGQFAPVAAKQAWDRDNTK---KQALVEAVKADLP--HMRVRAGGYTSVDVSECGIDKAYAVRKLTQTLG 214AYSAIGQKAGVEAKQAWDPDMTK---RNEIAKLASQYAP--EFEFEVAGTTTINGFVPGQNKEFGMNHLMEELN 201*.:*: . : : : : : : . .* :.: * :* : : : .

LLEKMNLTGYFDAIADPAEVAASKPAPDIFIAAAH------AVGVAPSESIGLEDSQAG------IQAIKDSG- 182. :. . : . . . .: * : : .

--YKTIYFFGDKTMPGGNDHEIFTDPRTVGYTVTAPEDTRRIC-EGLFP------ 242--YKTIYFFGDKTMPGGNDHEIFTDPRTMGYSVTAPEDTRRIC-ELLFS------ 246--FDTIHFFGNETSPGGNDFEIFADPRTVGHSVVSPQDTVQRCREIFFPETAHEA 262--FKEIHFFGDKTMVGGNDYEIFVDERTIGHSVQSPDDTVKILTELFNL------ 254LSKADVLFYGDRLDEHGNDYPVKAMG-IPCVAVDDWHDTLVKLEDLLSQA----- 224IRADEMVFVGDRMTPTGNDYPAVEAG-AIGVRVENPQDTVQLLDALLARFDTPAR 268VTKEEILYFGDMTQPGGNDYPVVQMG-IETITVRDWKETAAILKAIIAMEEA--- 252

: : *: ***. * .:* : ----ALPIGVGRPEDLGDDIVIVPDT---------SYYTLEFLKEVWLQKQK--- 221

: . *:* *

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

M. musculusH. sapiens PMM2H. sapiens PMM1S. cerevisiaeP. acnesB. longumL. lactis α-PGM

L. lactis β-PGM

-----MAT-----------LCLFDMDGTLTAPRQKITEEMDGFLQKLRQKTKIGVVGGSDFEKLQEQLG--NDV 56-----MAAPGPA-------LCLFDVDGTLTAPRQKITKEMDDFLQKLRQKIKIGVVGGSDFEKVQEQLG--NDV 60-----MAVTAQAARRRERVLCLFDVDGTLTPARQKIDPEVAAFLQKLRSRVQIGVVGGSDYCKIAEQLGDGDEV 69-----MSIAEFAYKEKPETLVLFDVDGTLTPARLTVSEEVRKTLAKLRNKCCIGFVGGSDLSKQLEQLG--PNV 67---------------------------------------MARIIVRLLDRTSVCVISGGQFGQFRTQVVEALVD 35MVVRSWSELDFDNVCSNAKVFGFDLDNTLASSKQPMKPAMIERFCALLDHTVVALISGGGMAVATSQVLDVLTP 74----------------MKKILSFDIDNTLNEPKMPIFPEMAELLATLSQKYIIAPISGQKYDQFLIQIINNLPE 58

: **:*.** . : : : : * .: : :.*· *: ----------------MFKAVLFDLDGVITDT----AEYHFRAWKALAEEIGINGVDR-QFNEQLKGVSREDSL 53

**:*. : * .. : :. :

VEKYD--YVFPENGLVAYKDGKLLCKQNIQGHLGEDVIQDLINYCLSYIANIKLPKKR--GT---FIEFRNGML 123VEKYD--YVFPENGLVAYKDGKLLCRQNIQSHLGEALIQDLINYCLSYIAKIKLPKKR--GT---FIEFRNGML 127IEKFD--YVFAENGTVQYKHGRLLSKQTIQNHLGEELLQDLINFCLSYMALLRLPKKR--GT---FIEFRNGML 136LDEFD--YSFSENGLTAYRLGKELASQSFINWLGEEKYNKLAVFILRYLSEIDLPKRR--GT---FLEFRNGMI 134APRLDRLHLLPACGTQYYRCVDGEWQRIYVEALTDDEKSRAMEAVETCARDLGLWEEHTWGP---VLEDRESQI 106NARRGNLHVMPTSGSRYYRWDGTQWALVYAHDLSEATVAAVSESLERHARELGLWEQQVWGP---RIENRGSQI 145SANLDNFHLFVAQGTQYYAHKAGEWKQVFNYALTDEQANAIMGALEKAAKELGHWDESVLLPGDEINENRESMI 132. . : : * * * : : .. .. * * . :

QKILD--LADKKVSAEEFKELAKRKNDNYVKMIQDVSPADVYPGILQLLKDLRSNKIKIALA----SASKNGPF 121. . : : : : . . : . :

NVSPIGRSCSQEERIEFYELDKKEHIRQKFVADLRKEFAGKGLTFSIGGQISIDVFPEGWDKRYCLRHLEHAG- 196NVSPIGRSCSQEERIEFYELDKKENIRQKFVADLRKEFAGKGLTFSIGGQISFDVFPDGWDKRYCLRHVENDG- 200NISPIGRSCTLEERIEFSELDKKEKIREKFVEDLKTEFAGKGLRFSRGGMISFDVFPEGWDKRYCLDSLDQDS- 209NVSPIGRNASTEERNEFERYDKEHQIRAKFVEALKKEFPDYGLTFSIGGQISFDVFPAGWDKTYCLQHVEKDG- 207TFSALGQQAPVDAKKAWDPSGDK---KLKLREAVAGKLL--DLEVRAGGSTSVDITRVGRDKSFGIAKLLEMTG 175TFSALGQFAPVAAKQAWDRDNTK---KQALVEAVKADLP--HMRVRAGGYTSVDVSECGIDKAYAVRKLTQTLG 214AYSAIGQKAGVEAKQAWDPDMTK---RNEIAKLASQYAP--EFEFEVAGTTTINGFVPGQNKEFGMNHLMEELN 201*.:*: . : : : : : : . .* :.: * :* : : : .

LLEKMNLTGYFDAIADPAEVAASKPAPDIFIAAAH------AVGVAPSESIGLEDSQAG------IQAIKDSG- 182. :. . : . . . .: * : : .

--YKTIYFFGDKTMPGGNDHEIFTDPRTVGYTVTAPEDTRRIC-EGLFP------ 242--YKTIYFFGDKTMPGGNDHEIFTDPRTMGYSVTAPEDTRRIC-ELLFS------ 246--FDTIHFFGNETSPGGNDFEIFADPRTVGHSVVSPQDTVQRCREIFFPETAHEA 262--FKEIHFFGDKTMVGGNDYEIFVDERTIGHSVQSPDDTVKILTELFNL------ 254LSKADVLFYGDRLDEHGNDYPVKAMG-IPCVAVDDWHDTLVKLEDLLSQA----- 224IRADEMVFVGDRMTPTGNDYPAVEAG-AIGVRVENPQDTVQLLDALLARFDTPAR 268VTKEEILYFGDMTQPGGNDYPVVQMG-IETITVRDWKETAAILKAIIAMEEA--- 252

: : *: ***. * .:* : ----ALPIGVGRPEDLGDDIVIVPDT---------SYYTLEFLKEVWLQKQK--- 221

: . *:* *

Figure 3: Multiple sequence alignment of amino acid sequences of the α-PGM from L. lactis subsp. cremoris MG1363 and its putative homologs from Mus musculus, Homo sapiens (PMM1 and PMM2), Saccharomyces cerevisiae, Propionibacterium acnes and Bifidobacterium longum (for accession numbers, see text). The alignment was generated with ClustalW. The eukaryotic α-PMMs belong to the subfamily II of the HAD superfamily. The lactococcal β-PGM, a subfamily I member, is shown in grey. A signature pattern for motif I of the HAD superfamily phosphomutases -DXDXT- (metal binding and nucleophile) is highlighted by a dark grey box (31). The invariant aspartate residue is printed in yellow. The other signature patterns of the HAD superfamily subfamily II (motifs II, III and IV) are highlighted by blue dark boxes (79, 173). Light blue and light green boxes highlight the core motifs 1, 2, 3, 4 and cap loop 5, respectively, of β-PGM (3). Shaded yellow boxes indicate hinge regions connecting core and cap domains. Residues proposed to be important for substrate binding or catalysis are printed in red. The conserved serine residue in motif II is printed in light-blue (residue inversion in α-PMM and lactococcal α-PGM and its homologs).

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Biochemical characterization of recombinant α-PGM

The protein was purified about 13-fold to a specific activity of 65 U/mg protein. Biochemical and kinetic properties are listed in Table 2. TABLE 2: Biochemical and kinetic properties of α-PGM from L. lactis Parameter Value

Apparent mol mass of enzyme (kDa) Native 84.3 Subunit 28 Calculated 28.3 Oligomeric structure α3 pH optimum 6.5 ±0.5 Km (µM) α-G1P 71.4 ±2.8 Kact (µM)a) Glucose-1,6-bisphosphate 16.8 ±1.3 Mg2+ 52.6 ±5.1 Vmax (U/mg protein) α-G1P 65.3 ±1.8 G6P 15.8 ±1.3 Substrate specificity α-G1P 7.5 mM 100%b) G6P 50 mM 23% β-G1P 7.5 mM 0%c) Effect of cations Mg2+ 0 mM < 2%d) Mg2+ 0.05 mM 49% Zn2+ 5 mM 2% Zn2+ 1 mM 28% Ca2+ 5 mM 0.3% Ca2+ 1 mM 1.6% Mn2+ 5 mM 17% Mn2+ 1 mM 21% Ni2+ 5 mM 43% Ni2+ 1 mM 41% Li+ 5 mM 42% Li+ 1 mM 39% Potential inhibitors (I50) FBP 48±1.1 mM ATP No effect

a) Kact is the concentration of activator at which the rate of the reaction is half of Vmax. b) Activities are relative to the value determined for the conversion of α-G1P to G6P, which is

set to 100%, as measured using 31P-NMR. c) The following phosphosugars were also examined but no activity was detected:

glucosamine-1P, glucosamine-6P, α-mannose-1P, fructose-1P, fructose-6P, FBP, α-galactose-1P, galactose-6P, ribose-5P, ribulose-5P, 6-phosphogluconate, UDP-galactose and UDP-glucose; when a mixture of FBP and α-G1P was used, α-G1P was fully converted to G6P, but FBP was not used.

d) Activities are relative to the value determined for the conversion of α-G1P to G6P, which is set to 100%, as measured using the standard coupling assay and 5 mM Mg2+.

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The purified protein was electrophoretically homogenous and SDS-PAGE revealed a single subunit with an apparent molecular mass of 28 kDa. A value of 84.3 kDa was determined by gel filtration (Superose 12 10/300 GL) suggesting a trimeric structure for α-PGM. The pH for maximal activity was 6.5; 50% of the activity was found at pHs 5.6 and 7.5. The rate dependence on α-G1P concentration followed Michaelis-Menten kinetics. Several phosphosugars were examined as putative substrates using the 31P-NMR direct assay. The enzyme catalyzes only the interconversion of α-G1P into G6P. Furthermore, the apparent Vmax value for the reverse direction (G6P α-G1P) was 3-fold lower than that of the forward direction, and the apparent Km value for G6P was in the mM range. Neither α-mannose-1P nor β-G1P were substrates for the enzyme, despite the sequence similarity of L. lactis α-PGM to the eukaryotic phosphomannomutases (around 25%) and β-PGM/G1P phosphodismutases (around 10%). For maximal activity, α-PGM required Mg2+ and glucose-1,6-bisphosphate. In the absence of Mg2+, the activity was reduced below 2% and no activity was detected when glucose-1,6-bisphosphate was omitted from the assay. Zn2+ and Ca2+ strongly inhibited the activity, whereas Ni2+ and Li+ had only a slight inhibitory effect. Partial inhibition was also observed with Mn2+. ATP showed no regulatory effect on α-PGM activity, while FBP moderately inhibited the activity (50% was lost at 48 mM FBP). Pools of glycolytic intermediates in non-growing cells

L. lactis NZ9000 harbouring pNZ8048-pgmH (hereafter named NZ9000[pgmH+]) or pNZ8048 (control) were grown in CDM containing glucose or galactose and induced with nisin for α-PGM production. The effect of α-PGM overproduction on the metabolism of glucose and galactose was studied by in vivo 13C-NMR in non-growing cell suspensions under an argon atmosphere and at pH 6.5 (Fig. 4). (i) [1-13C]-Glucose. The conversion of glucose was homofermentative in NZ9000[pgmH+] and NZ9000[pNZ8048] (lactate production above 91%), the glucose consumption rates being 0.37±0.02 and 0.39±0.02 μmol·min·mg protein-1, respectively. Minor end-products, acetate, ethanol and 2,3-butanediol, were detected in identical amounts in both strains(data not shown). NZ9000[pNZ8048] metabolized glucose like its parent MG1363 (123), showing that glucose metabolism in non-growing NZ9000 cells was not affected by the presence of pNZ8048. The dynamics of intracellular metabolite pools was not appreciably affected by the substantial increase in α-PGM activity from 0.07 U·mg protein-1 in the control to 2.6 U·mg protein-1 in NZ9000[pgmH+]. The maximal concentration of FBP decreased by

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about 10% and 3-PGA increased slightly. Moreover, the UDP-glucose pool decreased from 6.7±0.5 mM in the control to 4.2±0.3 mM in NZ9000[pgmH+] (Fig. 4A & 4B).

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Figure 4: Metabolism of glucose or galactose in non-growing cell suspensions of L. lactis NZ9000[pNZ8048] and NZ9000[pgmH+]. Time course for substrate consumption and pools of intracellular metabolites in non-growing cultures of strains NZ9000[pNZ8048] (A, C) and NZ9000[pgmH+] (B, D) during the metabolism of [1-13C]-glucose (A, B) or [1-13C]-galactose (C, D) as monitored by 13C-NMR. Cells were grown in a bioreactor vessel on glucose (A, B) or galactose (C, D) in de-aerated CDM at pH 6.5, and induced with nisin (1 µg/l) when the OD600 was 0.5. Lactate, ethanol, acetate and 2,3-butanediol were also detected, but their time courses were omitted from the graphs for the sake of simplicity. The glucose or galactose consumption rate (GCR or GalCR respectively) in μmol·min-1·mg protein-1 is depicted in the top-right corner of each graph. Symbols: glucose ( ); galactose ( ); FBP ( ); α-G1P ( ); Gal1P ( ); 3-PGA ( ); PEP ( ); UDP-Glc ( ); UDP-Gal ( ). Fitted lines are simple interpolations.

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(ii) [1-13C]-Galactose. As expected for galactose, the metabolism of pyruvate was shifted to products other than lactate, namely ethanol (2.2±0.2 mM) and acetate (2.4±0.4 mM), but their concentrations were about 35% lower in NZ9000[pgmH+] compared to the control strain NZ9000[pNZ8048] (data not shown). Induction with nisin resulted in a 7-fold higher (0.3 to 2.1 U·mg protein-1) α-PGM activity in the strain harbouring the pgmH construct, a modest change when compared to the 37-fold increase observed in glucose grown cells. Curiously, the galactose consumption rate was 25% greater (from 0.16±0.1 to 0.21±0.1 μmol·min·mg protein-1) in the strain overproducing α-PGM, compared to the control strain. Addition of galactose to a “starved” cell suspension of the control strain resulted in the build-up of the Leloir pathway phosphorylated intermediates, Gal1P and α-G1P, reaching maximal concentrations of 18.0±0.5 and 18.7±0.7 mM, respectively (Fig. 4C). Accumulation of FBP, the predominant metabolite during glucose metabolism, was slightly delayed and reached a maximal concentration of 24.0±1.5 mM. At the onset of galactose depletion, 3-PGA and PEP pools rose to 28.6±2.1 and 11.0±2.2 mM, respectively. These high levels of 3-PGA and PEP denote the utilization of an uptake system for galactose other than a PEP:PTS. Overproduction of α-PGM had a considerable impact on the concentrations of intracellular metabolite pools. As in the control strain, the accumulation of FBP was slightly delayed, but in NZ9000[pgmH+] its maximal concentration was clearly higher. A remarkable reduction in the size of Gal1P and α-G1P pools to 2.4 and 2.2 mM, respectively, revealed α-PGM as the main bottleneck during galactose metabolism in L. lactis. UDP-glc (3.9±0.4 mM) and UDP-gal (3.2±0.3 mM) were detected and these pools were 1.4-fold lower in the strain with increased α-PGM activity (Fig. 4C & 4D). In summary, the overproduction of α-PGM caused notable changes in the dynamics and levels of glycolytic intermediates derived from galactose, whereas no major differences were observed when glucose was used, reflecting the central role of α-PGM in the degradation of galactose. The observations in resting cells raised the question as to how a growing culture would respond to an increase in α-PGM activity. Therefore, we investigated the effect of overexpression of pgmH in growing cells. Impact of α-PGM overproduction in growing cells

(i) Growth characteristics. The effect of overproduction of α-PGM on the growth properties of L. lactis was evaluated using either glucose or galactose as carbon source (Table 3 and Fig. 5).

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The specific activity of α-PGM measured in cells before addition of nisin was consistently higher in the strain carrying pNZ8048-pgmH, most likely due to low basal PnisA expression. Galactose per se induced α-PGM activity 2- and 4-fold in NZ9000[pgmH+] and the control strain, respectively (Table 3). Up-regulation of pgmH expression on galactose was also apparent when the strains were grown on galactose without nisin induction. TABLE 3: Effect of pgmH overexpression on some growth properties during glucose or galactose fermentation by L. lactis. NZ9000[pgmH+] and NZ9000[pNZ8048] were grown in CDM supplemented with chloramphenicol (5 mg/l) and 1% (w/v) glucose or galactose. Nisin (1 µg/l) was added when the culture OD600 reached a value of 0.5. Growth rate constants for the entire growth phase were calculated using linear regressions. α-PGM activity was measured before (OD600, 0.5) and after induction (OD600, 0.5).

Glucose Galactose pNZ8048 pgmH+ pNZ8048 pgmH+

Carbon balance (%) 95 ± 0.5 95 ± 0.5 94 ± 0.3 94 ± 0.1 Biomass yield (g/mol) 29.9 ± 0.3 29.1 ± 0.3 26.5 ± 0.2 26.5 ± 0.4 ATP yield (mol/mol subs.) a) 1.9 ± 0.01 1.9 ± 0.01 2.1 ± 0.01 2.1 ± 0.01

YATP b) 15.6 ± 0.2 15.2 ± 0.2 12.5 ± 0.2 12.9 ± 0.2

Growth rate (µ1) c) 0.80 ± 0.01 0.82 ± 0.01 0.36 ± 0.01 0.45 ± 0.01 Growth rate (µ2) d) 0.58 ± 0.01 0.56 ± 0.02 0.37 ± 0.01 0.50 ± 0.01 α-PGM activity (U/mg)1c) 0.07 ± 0.01 0.16 ± 0.01 0.29 ± 0.01 0.36 ± 0.02 α-PGM activity (U/mg)1d) 0.07 ± 0.01 2.63 ± 0.20 0.34 ± 0.02 2.05 ± 0.16 Lactate/substrate (%) 92 ± 0.4 91 ± 0.4 71 ± 1.0 79 ± 0.8 Other products/substrate (%) 2 ± 0.5 3 ± 0.7 23 ± 0.3 19 ± 0.3

a) The global ATP yield was calculated from the fermentation products assuming that all ATP was synthesized by substrate-level phosphorylation

b) YATP, biomass yield relative to ATP production c) before addition of nisin d) after addition of nisin (1 µg/l)

The growth rate was affected by nisin addition, but the magnitude and the sign of the effect was sugar-dependent (Table 3). In the absence of nisin, the growth rate on glucose was about twice as high as that on galactose. Furthermore, the growth rate on glucose decreased considerably upon addition of nisin, but this negative effect was unrelated to α-PGM overproduction (16-fold increase). Only a 7 fold higher α-PGM activity was achieved during growth on galactose; however, the growth rate of NZ9000[pgmH+] was substantially greater than in the control strain (0.50 versus 0.36

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h-1), reaching a value close to that on glucose (0.56 h-1). The results show that α-PGM activity in the control strain is limiting during growth on galactose, and that this bottleneck was overcome by pgmH overexpression. In glucose-grown cells, increased α-PGM activity had no impact on product formation, with lactate accounting for over 90% of the end-products. When galactose was used as carbon source, the lactate yield increased slightly in the strain overproducing α-PGM (Table 3, Fig. 5), in line with the increased growth rate of strain NZ9000[pgmH+]. Biomass yield, ATP yield, and biomass yield on ATP were dependent on the sugar used and not affected by overproduction of α-PGM (Table 3).

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Figure 5: Growth of L. lactis NZ9000[pNZ8048] and NZ9000[pgmH+] on glucose or galactose in CDM at 30°C and pH 6.5. A) Biomass formation was spectrophotometrically followed during growth on glucose (open symbols) or galactose (closed symbols). Symbols: squares (open and closed) NZ9000[pNZ8048]; triangles (open and closed), NZ9000[pgmH+]. B) End products from the fermentation of glucose (59.3±0.2 mM) and galactose (57.7±0.2 mM). To induce expression of pgmH, nisin (1 µg/l) was added at an OD600 of 0.5. Symbols: lactate ( ); acetate ( ); ethanol ( ). Data are from a representative experiment where the error in each point ≤ 10%.

(ii) Pools of phosphorylated metabolites. Table 4 shows pool sizes for glycolytic intermediates and sugar-nucleotides determined by 31P-NMR in cell extracts derived from mid-exponential phase cultures. FBP was the major metabolite on glucose, whereas on galactose 3-PGA and PEP were also present in high amounts. The size of the FBP pool varied to a small extent and in a sugar-dependent way in response to increased α-PGM activity. As expected, the Leloir pathway intermediate Gal1P was below the detection limit on glucose; on galactose its concentration was slightly

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lower (20%) in the α-PGM overproducing strain. The concentration of α-G1P decreased notably in NZ9000[pgmH+] regardless the sugar used. It is thought that in L. lactis UDP-sugars and UDP-aminosugars are derived from α-G1P and F6P, respectively, hence their concentrations could respond to changes in α-PGM activity. Overproduction of α-PGM resulted in reduction or constancy of UDP-sugars on galactose or glucose grown cells, respectively. Concentrations of UDP-aminosugars and 5-phosphorylribose 1-pyrophosphate in mid-exponential phase cells of L. lactis were not significantly affected by pgmH overexpression, except for UDP-N-acetylglucosamine, the first cytoplasmatic precursor of peptidoglycan, which level responded inversely to an increase in the activity of α-PGM (Table 4). TABLE 4: Effect of pgmH overexpression on the pools of phosphorylated metabolites and UDP-sugars during growth on glucose or galactose. NZ9000[pgmH+] and NZ9000[pNZ8048] were grown in CDM supplemented with choramphenicol (5 mg/l) and 1% (w/v) glucose or galactose. Nisin (1 µg/l) was added when the culture OD600 reached a value of 0.5. Phosphorylated metabolites (mM) were measured in cell extracts obtained during mid-exponential growth phase (OD600 2.2). The average accuracy ±15%.

Glucose Galactose pNZ8048 pgmH+ pNZ8048 pgmH+

α-G1P (α-glucose-1-phosphate) 0.4 0.2 2.4 1.0 Gal1P (galactose-1-phosphate) 0.0 0.0 3.7 3.0 FBP (fructose-1,6-bisphosphate) 13.9 11.8 9.7 11.0 G6P (glucose-6-phosphate) 3.7 3.8 3.3 1.8 3-PGA (3-phosphoglycerate) 2.0 2.8 9.2 7.5 PEP (phosphoenolpyruvate) 1.0 1.6 3.3 3.6 2-PGA (2-phosphoglycerate) 0.1 0.2 0.9 0.6

UDP-Gal (UDP-galactose) 0.5 0.4 1.4 1.0 UDP-Glc (UDP-glucose) 2.4 2.4 4.0 2.8 UDP-GalNa) 1.1 1.2 0.6 0.8 UDP-GlcNAc 3.2 2.6 2.0 2.4 UDP-N-AcMur-pPep 1.2 1.3 1.8 1.9 UDP-GlcN 0.4 0.6 0.7 0.6 PRPP 0.9 0.7 1.6 1.4

α-PGM activity (U/mg) 0.07 2.63 0.34 2.05

a) Resonance at -11.98 tentatively assigned to UDP-galactosamine (136, 151). Abbreviations: UDP-GlcNAc, UDP-N-acetylglucosamine; UDP-N-AcMur-pPep, UDP-N-acetylmuromoyl-pentapeptide; UDP-GlcN, UDP-glucosamine; PRPP, 5-phosphorylribose 1-pyrophosphate.

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Inactivation of the chromosomal pgmH gene and its effect on L. lactis

growth

To investigate whether the α-PGM activity is essential for growth of L. lactis on glucose and galactose we decided to inactivate the pgmH gene (see Experimental Procedures). Several attempts to disrupt pgmH by single-crossover plasmid integration with pORI13-pgmH’ or by a two-step homologous recombination method (96) failed. These results suggest that pgmH plays an essential role in L. lactis. Integration of pORI280ΔpgmH in NZ9000 resulted in erythromycin resistant colonies harboring a disrupted as well as an integral copy of pgmH. Only when pgmH was expressed in trans (under nisin control in pNZ8048) in NZ9000::pORI280ΔpgmH, was it possible to delete the chromosomal copy of pgmH, as confirmed by PCR and Southern analysis (not shown). The resulting strain, NZ9000ΔpgmH[pgmH+] was constructed and maintained in the presence of 0.1 µg/l nisin. To ascertain whether α-PGM was limiting during growth on glucose-M17, low nisin (0.01 µg/l) overnight grown-cultures (α-PGM activity 0.11 U·mg protein-1) were subcultured in fresh medium with increasing concentrations of nisin (0 to 1 µg/l). In the absence of nisin, the mutant strain showed poor growth with an average growth constant of 0.20 h-1 (Fig. 6A); at time-point 12 h, α-PGM activity had decreased to 0.025 U·mg protein-1. Under the same conditions, the control strain NZ9000[pNZ8048] showing a growth rate of 0.55 h-1, reached a biomass concentration five times higher (OD600 of 3.2 as compared to 0.6) and a steady α-PGM activity of 0.15 U·mg protein-1. Modulation of expression of pgmH by varying the nisin concentration in the range 0.01 to 1 µg/l resulted in a series of cultures with α-PGM activity between 63 and 4000% of the control level (Fig. 6B). Nisin concentrations as low as 0.05 µg/l already resulted in overexpression of pgmH (Fig. 6B). A reduction of the growth rate to 70% of that of the control was observed at the lowest α-PGM activity corresponding to 63% of the control level (nisin 0.01 µg/l). The data show that α-PGM activities below the control level do not sustain maximal growth of L. lactis on glucose-M17.

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Figure 6: Effect of α-PGM activity on the growth kinetics of L. lactis in glucose-M17. A) Overnight cultures of NZ9000ΔpgmH[pgmH+] grown in the presence of 0.01 µg/l nisin were subcultured in fresh glucose-M17 containing 0 µg/l ( ), 0.01 µg/l ( ), 0.05 µg/l ( ) and 1 µg/l ( ) of nisin; NZ9000[pNZ8048] was used as the control strain ( ). Growth was monitored for 15 h by measuring the optical density at 600 nm (OD600). B) For each of the growth curves, α-PGM specific activity at time 12 h of culturing is shown. α-PGM specific activity was measured in cell-free extracts as described in Experimental Procedures. Activity in the control strain NZ9000[pNZ8048] is shown as a dashed bar. Data is shown from a representative experiment where the error in each point ≤ 10%.

The effect of controlled limitation of pgmH expression was evaluated during growth on glucose and galactose in M17 medium. Strain NZ9000ΔpgmH[pgmH+] was grown overnight in medium containing different concentrations of nisin and subsequently subcultured in nisin-free medium (Fig. 7A & 7B). α-PGM activity in the overnight cultures at the time of subculturing (17 h after inoculation) showed a similar pattern as in Fig. 6B. In glucose-grown cells, nisin concentrations of 0.1, 0.01 and 0 µg/l in the overnight cultures resulted in 16, 65 and 74% reduction in the growth rates, respectively. In contrast, nisin concentrations of 0.5 and 1 µg/l in the inocula supported growth constants similar to those of the control strain. The effect on growth rates correlated well with the decline of α-PGM activity (Fig. 7A). Moreover, the stepwise reduction in the final optical density was consistent with the nisin amounts used in the overnight cultures. These results show that pgmH is essential for growth of L. lactis on glucose.

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Figure 7: Growth dependence of NZ9000ΔpgmH[pgmH+] on α-PGM activity. Overnight cultures of NZ9000ΔpgmH[pgmH+] grown on glucose-M17 (A) or galactose-M17 (C) containing nisin 0 µg/l ( ), 0.01 µg/l ( ), 0.1 µg/l ( ), 0.5 µg/l ( ) and 1 µg/l ( ) were subcultured in fresh M17 medium without nisin on glucose (A) or galactose (C). NZ9000[pNZ8048] was used as the control strain ( ). Growth was monitored for 15 h by measuring the optical density at 600 nm (OD600). For each of the growth curves in graphs A and C, α-PGM specific activity at time 12 h of culturing are shown in diagrams (B) and (D), respectively. α-PGM specific activity was measured in cell-free extracts as described in the Experimental Procedures. Activity in the control strain NZ9000[pNZ8048] is shown as a dashed bar. The dashed line indicates the wild-type level α-PGM activity. Data is shown from a representative experiment where the error in each point ≤ 10%.

When galactose was used as sole carbon source, the effect of limiting the expression of pgmH on the growth rates and final optical density was more pronounced than on glucose (Fig. 7C). Regardless of the nisin concentration used (0 to 1 µg/l), final biomass and growth rate constants were below 60% of the respective parameters in the control strain (OD600, 3.5 and µ, 0.41 h-1) (Fig. 7C). An α-PGM

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activity of 1.38 U·mg protein-1 was measured at time-point 12 h in cells grown overnight and subcultured in fresh medium with nisin at 0.5 µg/l, but the growth parameters did not improve. Although no final explanation for this intriguing behavior can be put forward, it is possible that complex regulatory mechanisms resulting from cross-reactions involving galactose metabolism and nisin-induction are implicated (29, 80). The severe growth defects shown here for L. lactis NZ9000ΔpgmH[pgmH+] validate the conclusion that pgmH is essential for growth of L. lactis on galactose.

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DISCUSSION

In this report we describe the identification and functional analysis of L. lactis α-phosphoglucomutase, which represents the first characterized member of a novel α-PGM family. Sequence comparison and properties

All α-PGMs characterized so far belong to the α-D-phosphohexomutase superfamily of proteins, comprising α-PGMs (mostly eukaryotic), bacterial and archaeal phosphomannomutases/phosphoglucomutases (PMM/PGMs), phosphoglucosamine mutases (mostly bacterial) and the strictly eukaryotic phosphoacetylglucosamine mutases (171). The α-PGM encoded by L. lactis pgmH did not show any similarity to the members of the α-D phosphohexomutase superfamily nor did it contain the family’s consensus motifs. Instead, it showed sequence homology to eukaryotic phosphomannomutases (Fig. 3 and Fig. 8), an unrelated group of proteins that despite their phosphohexomutase activity belong to the HAD superfamily (PF03332, http://www.sanger.ac.uk/cgi-bin/Pfam). Saccharomyces cerevisiae SEC53 was the first member studied (71), but thereafter several other enzymes have been characterized to some extent (130, 137, 179). These proteins have about 260 amino acids and feature the conserved sequence motifs characteristic of the HAD superfamily (3, 31, 79, 159), which are all present in the pgmH product (Fig. 3). The HAD superfamily comprises two branches that have acquired phosphohexomutase function, the eukaryotic α-phosphomannomutases (PF03332) and β-phosphoglucomutases (PF00702) (87). As all known α-PGMs fall into the α-D-phosphohexomutase superfamily, we propose that the L. lactis α-PGM represents a novel line of α-D-phosphohexomutase evolution (Fig. 8). Unlike the eukaryotic phosphomannomutases, which in general use both mannose-1-phosphate and glucose-1-phosphate, L. lactis α-PGM shows strict specificity for α-G1P. Narrow substrate specificity has been described for the eukaryotic α-PGMs that fall into the α-D-phosphohexomutase superfamily (154), in contrast to the bacterial α-PGMs (PMM/PGM), which have a rather broad substrate preference (222). Within the α-D-phosphohexomutase superfamily, substrate specificity has been related to subtle residue variance in the catalytic domains (171). In the HAD superfamily, like in the α-D-phosphohexomutase superfamily, the catalytic cycle proceeds via a bisphosphorylated sugar intermediate to the reversible conversion of 1-phospho to 6-phosphosugars and requires Mg2+ as cofactor (Table 2, (137, 138, 150, 171)). However, kinetic studies showed that a different reaction mechanism

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operates, in which the active residue is phosphoaspartyl (110, 138). The recent publication of the X-ray structures of HAD superfamily phosphohexomutases, lactococcal β-PGM and human PMM1 sheds some light on the mechanism used (88, 89, 173, 224). In addition to the hexose C1 configuration (α- or β-anomer) specificity, the position and fold of the cap domain allow distinguishing eukaryotic α-PMMs from bacterial β-PGMs (Fig. 3, (3)). The position and fold of the cap domain (Fig. 3) places the L. lactis α-PGM in the HAD superfamily subclass II, whereas β-PGM is a subclass I protein. Sequence identities between L. lactis α-PGM (query sequence) and human α-PMM1 or L. lactis β-PGM are 25% and 10%, respectively. Taken together the structural and sequence similarities and the anomer specificity suggest that the L. lactis α-PGM is mechanistically closer to the α-PMMs than to the β-PGMs. This hypothesis is strengthened by the presence in the α-PGM sequence of the residues that are involved in the catalytic process of α-PMM1, in particular the nucleophile Asp8, the acid/base Asp10 and the Gln51 (Asp19, Asp21 and Gln62 in human α PMM1). However, only some of the conserved positively charged residues at the interface of the cap and core domains in α-PMMs are present in L. lactis α-PGM (Fig. 3), suggesting a mechanism of action different from that of the electrostatic wedge proposed for the α-PMM (173). To analyze the phylogenetic relationship of proteins with phosphohexomutase activity, of which the L. lactis α-PGM was characterized here, the neighbour-joining tree construction method was used (200). A phylogram including both characterized and putative phosphohexomutases from eukaryotic and bacterial sources is given in Fig. 8. The depicted topology clearly separates members of the α-D-phosphohexomutase and HAD superfamilies despite their similar function. Topological organization within the α-D-phosphohexomutase superfamily is identical to that observed by others and has been thoroughly discussed elsewhere (171, 217), but the topology within the HAD branch is intriguing. The reconstruction presented here reflects the family division in the HAD superfamily, with bacterial β-PGMs and eukaryotic α-PMMs divided into two distinct groups that are supported by good bootstrap values. The L. lactis α-PGM is included in a cluster comprising proteins with unknown function of the human-associated organisms Bifidobacterium longum and Propionibacterium acnes and a plant pathogen, Gibberella zeae, that branches from the line leading to the eukaryotic α-PMMs. This suggests a common origin of these proteins and the eukaryotic α-PMMs, which is also supported by the common α-anomeric specificity. A possible explanation relies on independent lateral gene transfer from eukarya to their commensal or pathogenic organisms. Surprisingly, of all the bacteria with available genome sequences, L. lactis appears

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to be unique insofar as it lacks an α-PGM of the α-D-phosphohexomutase superfamily.

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Figure 8: Unrooted phylogenetic tree based on available amino acid sequences of phosphomutases. The ClustalX program (200) was used for sequence alignments and to generate the phylogenetic tree. The significance of the branching order was evaluated by bootstrap analysis of 1,000 computer-generated trees. The bootstrap values are indicated. Bar, 0.1 changes/site. Abbreviations and GenBank accession numbers: Ocun, Oryctolagus cuniculus PGM P00949; Scer, Saccharomyces cerevisiae: PGM NP_013823, PMM NP_116609; Hsap, Homo sapiens: PGM AAH67763, PAGM AAD55097, PMM1 AAC51117, PMM2 AAH08310; Llac, Lactococcus lactis: FemD NP_266580, α-PGM (pgmH) DQ778336 , PGMB NP_266585; YhfA NP_266899; Spne, Streptococcus pneumoniae: PGNM AAL00221, PMM|PGM AAD56627; Spyo, Streptococcus pyogenes: PGNM YP_280210, PMM|PGM YP_282301, PGMB YP_281891; Bsub, Bacillus subtilis: PGNM NP_388058, PMM/PGM CAH04980, PGMB NP_391335; Ecol, Escherichia coli: PNGM AAC76208, PMM O85343, PGM AAC73782; Mmusc, Mus musculus: PAGM NP_082628, PMM NP_058577; Dmel, Drosophila

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melanogaster: PAGM NP_648588; Pacn, Propionibacterium acnes; HP YP_056695; Blon, Bifidobacterium longum: HP ZP_00121741, PGM NP_696782; Gzea, Gibberella zeae: PMM EAA71459; Orys, Oryza sativa: PMM XP_474395; Cpar, Cryptosporidium parvum: PMM EAK87737; Ecun, Encephalitozoon cuniculi: PMM CAD26542; Calb; Candida albicans: PMM EAL02637; Paer, Pseudomonas aeruginosa: PMM/PMG NP_254009; Sthe, Steptococcus thermophilus: PMM/PGM AAV62380; Gxyl, Gluconacetobacter xylinus: PGM P38569; Cglu, Corynebacterium glutamicum: PMM/PGM CAF21203; Vfis, Vibrio fischeri: PGM AAM77720; Nmen, Neisseria meningitides: PGMB CAB85309; Efae, Enterococcus faecalis: PGM NP_814693; Lplan, Lactobacillus plantarum: PGM NP_783891. PGM, phosphoglucomutase; PGNM; phosphoglucosamine mutase; PAGM; phosphoacetylglucosamine mutase; PMM, phosphomannomutase; HP, hypothetical protein; PGMB; beta-phosphoglucomutase. α-D phosphohexomutase and haloacid dehalogenase superfamilies are highlighted by light and dark grey boxes, respectively.

Physiological function of the L. lactis α-PGM

In bacteria, the interconversion of α-G1P to G6P catalyzed by α-D-phosphohexomutases is a key step in the production of UDP-Glc, the glucosyl donor for the synthesis of glucose-containing polysaccharides (cell wall polysaccharides, capsules and exopolysaccharides) and in the catabolism of galactose. We demonstrated that the product of femD, the only gene in the L. lactis chromosome with homology to known α-D-phosphohexomutases, does not display α-PGM activity. Thus far, pgmB was the only gene identified in L. lactis encoding phosphoglucomutase activity (15, 149), but the enzyme is specific for the β-anomer of phosphoglucose, an intermediate in the catabolism of the disaccharides maltose and trehalose (224). In this work we show that the highly specific α-PGM encoded by the pgmH gene mediates the reversible conversion of α-G1P into G6P in L. lactis. Remarkably, all strategies attempted to disrupt pgmH failed. In bacteria, inactivation of α-PGM generally results in altered or defective lipopolysaccharides (49, 111, 118, 216) and/or reduced or abolished polysaccharide production (32, 213) mainly due to a deficient UDP-Glc supply. However, in some Gram-positives growth defects and altered cell morphology have also been associated with disruption of PGM genes (22, 57, 91, 100). Our experiments with varying nisin concentration or progressive decrease of α-PGM activity by using the pgmH conditional knock-out showed dramatic effects on growth rate and final biomass when the activity was below the control level. In the absence of nisin, a residual α-PGM activity, probably due to leakage of PnisA in a high copy system, could still sustain modest growth. Under these conditions, cell division and morphology were affected as denoted by the appearance of long chains comprising cells that had lost their typical lactococcal shape (data not shown). L. lactis strains deficient in UDP-galactose-4-epimerase

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(galE) (14), the major autolysin (acmA) (180) or the lipoteichoic acids d-alanylation (181) showed a similar behaviour, suggesting altered or deficient biosynthesis of cell wall polysaccharides. Earlier attempts to inactivate L. lactis MG1363 galU, encoding the enzyme that catalyzes the conversion of α-G1P to UDP-Glc, were also unsuccessful (14), and as for α-PGM, no other genes coding for a galactose uridyl transferase were found in the available genome sequences (15, 215). Therefore, it is reasonable to conclude that UDP-Glc synthesis in L. lactis relies entirely on pgmH and galU gene products. Moreover, we can speculate that α-PGM exerts considerable flux control in the synthesis of glucose-containing polysaccharides, since we showed that the size of the UDP-Glc pool responds to variations in the level of this enzyme. Under an applied point of view, this knowledge could be exploited to develop strains with improved exopolysaccharide production or altered lytic capacity, both industrially relevant traits. The involvement of pgmH in galactose metabolism became clear from the observed changes in the dynamics of intracellular metabolites in cells overproducing α-PGM. The lower concentration of α-G1P and Gal1P in the strain overexpressing pgmH identifies the step catalyzed by α-PGM as a bottleneck in galactose metabolism. This view is further substantiated by the improved growth and galactose consumption rates in the strain overexpressing pgmH as well as the growth impairment in mutants with reduced α-PGM activity. Overproduction of α-PGM affected the levels of glycolytic metabolites and the glycolytic flux from galactose, but not from glucose, reflecting the different role of pgmH in the metabolism of these two sugars: α-PGM is required for galactose degradation, whereas its main function is providing precursors for biosynthetic pathways during growth on glucose. Altogether, the results presented here support the conclusion that pgmH is the only gene in the L. lactis genome coding for α-PGM activity. Therefore, the purification of a 65 kDa protein with α-PGM activity reported earlier (150) can only be explained on the basis of the different genetic backgrounds used. This work revealed a novel α-D-phoshoglucomutase that falls into the haloacid dehalogenase family, unlike all other known α-PGM. The demonstration of the essentiality of pgmH is a final confirmation of the crucial role played by the enzyme in the physiology of L. lactis. To further understand the unique features unraveled by the present study, the structural determination of this new α-D-phoshoglucomutase is in progress.

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ACKNOWLEDGEMENTS

This work was supported by contract QLK1-CT-2000-01376 of the Commission of the European Communities and contract POCTI/BIO/48333/2002 of Fundação para a Ciência e a Tecnologia (FCT). A. R. Neves acknowledges a post-doctoral fellowship of FCT. We thank Claudia Sanchéz for technical assistance with the HPLC measurements and Thijs Kouwen for cloning the pgmH gene.

CHAPTER 6

SUMMARY AND GENERAL DISCUSSION

Wietske A. Pool

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The work presented in this thesis focuses on the metabolism of glucose, galactose and lactose in L. lactis. In this concluding chapter, the obtained results will be summarized and future prospects will be discussed. The research performed was part of an EU-funded project called ‘Nutracells’, which aimed at proof the concept of gaining ‘healthy’ strains of lactic acid bacteria, useful for the dairy industry. The aim of the work presented in this thesis was to shift the preferences of L. lactis for the sugar glucose to lactose and/or galactose. The resulting end-products obtained by fermentation with these strains would contain less residual lactose and/or galactose, a favourable product characteristic for individuals suffering from lactose intolerance and/or galactosemia. Furthermore, by completely blocking glucose metabolism, glucose would be left over as an end-product and could, thus, function as a natural sweetener. Apart from the relevance of proving this concept for the industrial partners that are part of the nutracells programme, there is a major fundamental interest in the first steps of sugar metabolism and its regulation. At the start of this work it was known that L. lactis could import glucose via PTSman/glc, encoded by ptnABCD and via (a) non-PTS transporter(s). To fully disable glucose metabolism, first the non-PTS route for glucose breakdown was blocked by deletion of glk, the gene encoding glucokinase. This enzyme catalyzes the phophorylation of glucose to glucose-6-phophate, which is the first step after non-PTS glucose transport. Subsequently, ptnABCD was disrupted in L. lactis NZ9000Δglk resulting in L. lactis NZ9000ΔglkΔptnABCD. Surprisingly, the latter strain could still use glucose as a substrate. Import of glucose had to be via a PTS, since the non-PTS-route was blocked by deletion of glk. Using DNA-microarrays, in which the expression of genes in the wildtype strain L. lactis NZ9000 was compared to that in L. lactis NZ9000ΔglkΔptnABCD, revealed several differentially expressed genes, of which genes of putative PTSs overexpressed in L. lactis NZ9000ΔglkΔptnABCD were, of course, most interesting as they potentially encode proteins involved in glucose transport. The genes ptcB and ptcA were five times more expressed in L. lactis NZ9000ΔglkΔptnABCD than in L. lactis NZ9000, which made them promising candidates. Interestingly, ptcBA together with ptcC were annotated as a cellobiose-transporting PTS (PTScel). Removing glk, ptnABCD and ptcBA in one strain resulted in a complete blockage of glucose metabolism, showing that the PTScel is actually a PTScel/glc (see Chapter 2). We confirmed that ptcBAC encodes a PTS transporting both glucose and cellobiose by growing L. lactis NZ9000, L. lactis NZ9000ΔptcBA and L. lactis NZ9000ΔglkΔptnABCDΔptcBA (hereafter named L. lactis NZ9000Glc–) in media containing galactose, glucose or cellobiose (data not shown). All three strains grew perfectly well on galactose. On

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glucose, L. lactis NZ9000 and L. lactis NZ9000ΔptcBA grew, but L. lactis NZ9000Glc– did not. On cellobiose, only L. lactis NZ9000 grew happily, while both L. lactis NZ9000ΔptcBA and L. lactis NZ9000Glc– did not. These results further imply that cellobiose can only be transported via PTScel/glc. The DNA-microarray technology was also employed to find the as yet unknown non-PTS glucose transporter(s), assuming that such transporters would be upregulated in L. lactis NZ9000ΔptnABCDΔptcBA, compared to strain NZ9000, when both strains would be growing on glucose. DNA-microarray results showed that the operon ytgBAH was highly upregulated in NZ9000ΔptnABCDΔptcBA (ytgA 11.3 times, ytgH 6.6 times, ytgB 2.9 times). Previous experiments had shown that ytgH (3.7 times) and ytgA (2.8 times) were also upregulated in NZ9000ΔglkΔptnABCD compared to NZ9000, both growing in the presence of glucose. The function of the proteins encoded by the small genes ytgBAH is as yet unknown, but since ytgB and ytgA both have 2 putative transmembrane domains and a high pI, they might be located in the membrane and involved in transport. To unravel whether ytgBAH indeed have a role in glucose uptake, a ytgBAH deletion strain in an L. lactis NZ9000 background as well as an NZ9000ΔptnABCDΔptcBA background, should be engineered and analyzed for differences in glucose transport. The same strategy could be followed for ymgH, which was overexpressed 3.2-fold in NZ9000ΔptnABCDΔptcBA compared to NZ90000, and also has 2 potential transmembrane domains and a high pI. Once a glucose-negative strain of L. lactis was made that was unable to grow on glucose, the next step was to determine which lactose metabolic pathway is the most efficient to introduce lactose metabolic capacity in L. lactis. The first strategy was to introduce pMG820 (106), a plasmid containing the lac-PTS and tagatose-6-phosphate pathway in L. lactis NZ9000. L. lactis NZ9000[pMG820] grew well on medium supplemented with lactose. The second strategy was to introduce lacSZ from Streptococcus thermophilus ST11 in L. lactis NZ9000 with its normal cre-site and with a single mutation in cre, using the high copy number vector pIL253 (175). LacSZ-producing strains of L. lactis were able to grow in medium supplemented with lactose (whether or not the cre-box upstream of lacSZ was intact), but different clones did not show the same growth pattern. Sequencing data showed that the lacSZ constructs were correct. Possibly, this was caused by variations in the copy-number of the plasmid in the different clones, leading to different amounts of the membrane protein LacS. Unfortunately the same lacSZ-constructs cloned in the low-copy number vector pIL252 failed. The maximum growth rate of the strains

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employing LacSZ for lactose metabolism was much lower than that of strains using lac-PTS together with the tagatose-6-phosphate pathway. On the basis of the results obtained with pMG820 and lacSZ, we decided to focus on the PEP:PTS route for improving lactose metabolism in L. lactis. Plasmid pMG820 was introduced in L. lactis NZ9000 and L. lactis NZ9000Glc– leading to, resp., L. lactis NZ9000Lac+ and L. lactis NZ9000Glc–Lac+. Since the latter strain can only use the galactose moiety of the disaccharide lactose for fermentation, it uses about twice as much lactose from the medium as L. lactis NZ9000Lac+ before growth ceases (due to the acidification of the medium). Besides the lower residual lactose concentration, also the relatively sweet glucose was produced as an end product by L. lactis NZ9000Glc–Lac+. Similar results were obtained in small-scale skim milk fermentations using this strain, which is promising for its application as an adjunct starter culture for industrial dairy applications. During the process of making L. lactis NZ9000Glc–Lac+, several interesting strains were produced, which were investigated in more detail to study glucose import in L. lactis. Questions that still remained unanswered are: which glucose transporter is the most efficient for glucose import, and what effect does the use of a specific transport system have on the further (regulation of) metabolism of glucose? Transport assays showed that PTSman/glc is the major glucose transporter in L. lactis. These studies also revealed that removal of glk resulted in a lowered affinity of PTSman/glc for glucose, suggesting that the presence of glucokinase is important for maximal functioning of this PTS. A direct or indirect regulatory role for glucokinase was suggested. The enzymatic activity of Glk itself is regulated by metabolic components, other glycolytic enzymes or regulator proteins. The possible regulatory role of Glk in sugar metabolism still remains to be investigated along the following lines. L. lactis NZ9000Δglk should be complemented with a functional Glk, to examine whether the Glk-effect is diminished. Glk could also be overexpressed to see if the effect shown on the PTSman/glc by the glk-deletion can be abolished. An L. lactis strain has already been constructed in which glk can be overproduced by addition of nisin, NZ9000[pNZ8048-glk]. After induction with nisin, L. lactis NZ9000[pNZ8048-glk] functionally overexpressed Glk approximately 150 times compared to the strain that was not treated with nisin. An in vivo NMR experiment showed that the glucose fermentation pattern of L. lactis NZ9000[pNZ8048-glk] is comparable to that of L. lactis NZ9000; both have comparable glucose consumption rates and produce similar amounts of intracellular and extracellular metabolites. No further experiments have been performed with L. lactis NZ9000[pNZ8048-glk] so far.

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It would be interesting to perform transport assays with NZ9000[pNZ8048-glk] and compare the results with previous experiments described in Chapter 3, to see if overexpression of Glk has a positive effect on the glucose transport efficiency of PTSman/glc. Another approach to show a link between Glk and PTSman/glc is to make a glk-GFP fusion protein and localize Glk-GFP in the cell. If Glk directly binds to PTSman/glc, it will be visible on the inside of the membrane instead of all throughout the cell, indicating a possible interaction of PTSman/glc with Glk. To find functional sites or amino acids in Glk, the effect of different mutations in the glk-sequence, for example in the ROK-motif could be studied. To investigate if the regulation by Glk is direct or perhaps occurs via the global carbon metabolism regulator CcpA, protein-protein interaction studies could be performed. A more genetic approach would be to use a ccpA-deletion strain of L. lactis and a strain in which both glk and ccpA are deleted. An L. lactis MG1363ΔccpA deletion strain already present in our laboratory collection (225) was used to make an MG1363ΔglkΔccpA double mutant. MG1363 is the parental strain of NZ9000 (MG1363pepN::nisRK). Using DNA-microarrays, the transcriptomes of the different strains should be compared (NZ9000Δglk to MG1363, MG1363ΔccpAΔglk to MG1363 and MG1363ΔccpAΔglk to NZ9000Δglk) to unravel possible clues of the transcriptomes. A comparison of MG1363ΔccpA and MG1363 has already been published (225). Glucose metabolism of L. lactis NZ9000, NZ9000Δglk, MG1363ΔccpA and MG1363ΔccpAΔglk was also analyzed by in vivo NMR. L. lactis MG1363ΔccpAΔglk showed a glucose fermentation pattern comparable to that of MG1363ΔccpA. The glucose consumption rates and the formation of intracellular and extracellular metabolites of both strains were similar, but completely different from those of L. lactis NZ9000 and NZ9000Δglk. NZ9000Δglk showed by far the most drastic change in glucose fermentation, thus it seems that the deletion of ccpA overcomes the problems NZ9000Δglk has. Another interesting result during this work was the discovery of the anomeric specificities of the different glucose transport systems in L. lactis NZ9000 (Chapter 3). PTScel/glc and the non-PTS glucose transporter(s) have a preference for β-glucose, while PTSglc/man displays no clear anomeric specificity and can use both the α-anomer and the β-anomer of glucose. Having multiple systems for glucose transport, with different specificities for the two glucose anomers could improve survival under natural conditions when the cells need to compete with other microorganisms for glucose as sugar substrate. It would be interesting to gain insight in the exact mechanisms of transport of the different glucose transporters and to examine what determines the anomeric specificity.

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All L. lactis glucose deletion strains described in Chapter 3 (NZ9000Δglk, NZ9000ΔptnABCD, NZ9000ΔptcBA and NZ9000ΔptnABCDΔptcBA) display a mixed acid fermentation pattern, while their parent strain L. lactis NZ9000 shows a homolactic fermentation mainly producing lactate. This shift to mixed acid fermentation is caused by key metabolic enzymes like pyruvate kinase (PK) and lactate dehydrogenase (LDH). The deletion strains had lower PK and LDH enzyme activities than L. lactis NZ9000. This is most probably not caused by transcriptional regulation of the genes for these enzymes, since they were not downregulated in a DNA-microarray experiment comparing the transcriptomes of L. lactis NZ9000ΔptnABCDΔptcBA and L. lactis NZ9000. The lower PK and LDH activities are caused by the concentrations of FBP and Pi in the cells (109, 153, 186, 191, 199), which are dependent on the rate of glucose metabolism, while the latter in turn depends highly on the glucose import system used. Lower activity of LDH results in a decreased lactate production, while production of other end products from pyruvate increases due to a higher activity of pyruvate formate lyase (PFL), shifting pyruvate metabolism to mixed acid fermentation. In addition to regulation of PFL at the enzyme level, the pfl gene was also transcriptionally activated in L. lactis NZ9000ΔptnABCDΔptcBA. The genes encoding PTScel/glc are located on the L. lactis chromosome in an operon together with yecA in the order ptcB-ptcA-yecA-ptcC. The yecA gene encodes a possible regulator protein. The PTScel/glc deletion studies decribed in this thesis were all performed with a strain deleted in ptcBA. Since not the complete PTScel/glc was disrupted, it would be interesting to engineer a ptcC deletion mutant and a ptcBAptcC double deletion mutant, to see whether these strains display the same characteristics as NZ9000ΔptcBA. Furthermore, since yecA is in the middle of the PTScel/glc operon, it would be interesting to study the function of YecA. Chapter 4 describes the work performed on the improvement of galactose utilization by L. lactis. It was shown that L. lactis NZ9000 preferably uses the galactose non-PTS permease GalP followed by the Leloir pathway (GalMKT) for the metabolism of galactose. Surprisingly, a galP deletion strain still used galactose via the Leloir pathway, suggesting the presence of another non-PTS galactose permease. Deletion of galPMK resulted in total loss of the ability of the strain to metabolize galactose. To further investigate which other permease(s) transports galactose besides GalP, a DNA-microarray experiment should be performed in which the transcription profiles of L. lactis strains NZ9000 and NZ9000ΔgalP are compared, as

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a gene encoding the putative additional galactose permease might be overexpressed in NZ9000ΔgalP to compensate for the loss of GalP. The lactose-PTS has been suggested to be able to use galactose as well as a substrate (92, 135), but so far this has not been proven. To analyze this possibility we introduced pMG820 (106), a 23,7 kb deletion derivative of pLP712, encoding PTSlac and the tagatose-6P-pathway in L. lactis NZ9000ΔgalPMK. The resulting strain indeed regained the capacity to use galactose as a substrate and produced tagatose-1,6-bisphosphate as an intermediate metabolite. Thus, this strain indeed uses the tagatose-6-phosphate pathway and not the Leloir pathway. This finding suggested that galactose was imported by the lactose-PTS encoded by lacFE. Recent sequencing studies discovered that no other sugar transporter is encoded by pMG820 (personal communication: U. Wegmann, Institute of Food Research, Norwich Research Park, Norwich, UK). Furthermore, we had to rule out the possibility that a(nother) galactose-PTS gene was encoded by the chromosome of L. lactis MG1363, of which the activity only became visible when the tagatose-6-phosphate pathway genes (lacABCD) were introduced on a plasmid (pMG820). Different combinations of lac-genes were introduced in L. lactis NZ9000ΔgalPMK by cloning them behind the nisin inducible promoter on pNZ8048, and galactose metabolic capacity was monitored after induction with nisin. The following preliminary results were obtained using L. lactis NZ9000ΔgalPMK as a negative control and NZ9000pMG820 as a positive control. L. lactis NZ9000ΔgalPMK[pNZ-lacFE] and NZ9000ΔgalPMK[pNZ-lacABCD] could not metabolize galactose, while NZ9000ΔgalPMK[pNZ-lacABCDFE] and NZ9000ΔgalPMK[pNZ-lacABCDFEG] were able to use galactose (A.R. Neves, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal, unpublished results). These data suggest that lacFE is necessary for galactose import and, thus, that LacFE is indeed a galactose/lactose-PTS. It also suggests that there is no other galactose-transporting PTS present on the L. lactis NZ9000 chromosome. The introduction of the β-phosphogalactosidase encoded by lacG is not required for the degradation of galactose. It would be interesting to introduce the plasmid with the nisin inducible lacABCDFEG genes in L. lactis NZ9000Glc– discussed above, to examine whether the growth on lactose would improve when the lac-PTS and the tagatose-6P-pathway are overexpressed instead of normally expressed, when using pMG820. In an effort to improve galactose metabolism in L. lactis NZ9000 we tried to overexpress its favoured route of galactose degradation, the Leloir pathway (Chapter 4). Overexpression of galP(MKT) led to accumulation of Gal1P and G1P, as shown by in vivo NMR. Apparently, the bottleneck in galactose metabolism via

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the Leloir pathway was at the level of α-phosphoglucomutase. The next step was to overexpress the gene encoding α-phosphoglucomutase alone and in combination with galP(MKT). Since a lactococcal gene for α-phosphoglucomutase turned out to be wrongly annotated in the L. lactis genome (femD), the pgmA gene of S. thermophilus was overexpressed in L. lactis NZ9000. This indeed increased the α-phosphoglucomutase activity in the latter strain. Overexpression of S. thermophilus pgmA together with L. lactis galP(MKT) relieved the bottleneck in galactose metabolism and improved the galactose consumption rate significantly. Apparently, the bottleneck in galactose metabolism is the reaction from glucose-1-phosphate to glucose-6-phosphate catalyzed by α-phosphoglucomutase. In this reaction UDP-glucose is produced, which is the glucosyl donor for the synthesis of glucose-containing polysaccharides. A next interesting step was to identify and characterize the α-phosphoglucomutase of L. lactis. In Chapter five of this thesis we described the identification of the genuine gene encoding L. lactis α-phosphoglucomutase. Thus far, all α-PGMs described belong to the α-D-phosphohexomutase superfamily of proteins (171, 217). First, we overexpressed femD, the only gene in the L. lactis chromosome of which the product shows homology to known α-D-phosphohexomutases. FemD, however, did not display α-PGM activity. Purification of α-PGM activity from L. lactis in combination with reverse genetics pinpointed the gene yfgH as specifying α-PGM activity. Consequently, yfgH was renamed pgmH. The α-PGM (pgmH) of L. lactis was characterized biochemically and the determination of the 3D-structure of this novel α-phosphoglucomutase is in progress. Functional analysis of L. lactis α-phosphoglucomutase showed it to be the first characterized member of a novel α-PGM family. At the amino acid (aa) sequence level, L. lactis α-phosphoglucomutase does not show any homology to the members of the α-D-phosphohexomutase superfamily. Instead, it has aa sequence homology with eukaryotic phosphomannomutases. Unlike the eukaryotic phosphomannomutases, which in general use both mannose-1-phosphate and glucose-1-phosphate, and bacterial α-PGMs belonging to the α-D-phosphohexomutase superfamily, which have a rather broad substrate preference, L. lactis α-PGM shows strict specificity for α-G1P. In Chapter 5 (Fig. 8), L. lactis α-PGM is placed in a phylogram with both characterized and putative phosphohexomutases from eukaryotic and bacterial sources.

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Galactose

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Out

In

Lactose-6P

G6P

Glucose

Lactose

Galactose-6P

Tagatose-6P

TBP

Galactose

β-Glucose

Glk

PgmH

Membrane

In

Out

Membrane

Glucose

FBP

DHAP

GAP

3PGA

PEP

GalP? ? LacFE LacFE

Pyruvate Lactate

Mixed acid products

EI~PEI

HPr-His~P HPr

HPr-Ser~P

A

P

B

PtnCD

HPr-Ser~PCcpA

Transcriptional regulationA

P

B

PtcC

G6P

α/β-Glucose β-Glucose

G6P

PTSman/glc PTScel/glc?

+

Other regulatory

roles?

Regulation via CcpA?

PTSlac/gal

Galactose

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Out

In

Lactose-6P

G6P

Glucose

Lactose

Galactose-6P

Tagatose-6P

TBP

Galactose

β-Glucose

Glk

PgmH

Membrane

In

Out

Membrane

Glucose

FBP

DHAP

GAP

3PGA

PEP

GalP? ? LacFE LacFE

Pyruvate Lactate

Mixed acid products

EI~PEI

HPr-His~P HPr

HPr-Ser~P

A

P

B

PtnCD

HPr-Ser~PCcpA

Transcriptional regulationA

P

B

PtcC

G6P

α/β-Glucose β-Glucose

G6P

PTSman/glc PTScel/glc?

+

Other regulatory

roles?

Regulation via CcpA?

Galactose

β-Galactose

α-Galactose

Galactose-1P

Glucose-1P

Out

In

Lactose-6P

G6P

Glucose

Lactose

Galactose-6P

Tagatose-6P

TBP

Galactose

β-Glucose

Glk

PgmH

Membrane

In

Out

Membrane

Glucose

FBP

DHAP

GAP

3PGA

PEP

GalP? ? LacFE LacFE

Pyruvate Lactate

Mixed acid products

EI~PEI

HPr-His~P HPr

HPr-Ser~P

A

P

B

PtnCD

HPr-Ser~PCcpA

Transcriptional regulationA

P

B

PtcC

G6P

α/β-Glucose β-Glucose

G6P

PTSman/glc PTScel/glc?

+

Other regulatory

roles?

Regulation via CcpA?

PTSlac/gal

General discussion

147

Figure 1: Schematic overview of glucose, galactose, and lactose metabolism in L. lactis including all new metabolic components described in this thesis. For details about the known glucose metabolic reactions see Chapter 3, Fig. 1. For details about the known galactose and lactose metabolic reactions see Chapter 1, Fig. 3. For details about the role of the histidine protein (HPr), see Chapter 1, Fig. 4. Besides donating phosphate to glucose via the PTSs, HPr also delivers the phosphate group for uptake and phosphorylation of lactose and galactose imported by the PTSlac/gal, but this detail is omitted from the figure for the sake of simplycity. All newly discovered metabolic proteins or their possible new roles in sugar metabolism are written in bold and/or encircled in dark-grey. PTScel/glc (encoded by ptcBAC) can transport and phosphorylate glucose and is specific for β-glucose. The PTSman/glc can use both α- and β-glucose. The non-PTS glucose transporter(s) is/are specific for β-glucose. Glucokinase (Glk), besides catalyzing the phosphorylation reaction of glucose, shows regulatory effects. PgmH was discovered to be the L. lactis α-phosphoglucomutase. Galactose can be transported by another non-PTS transporter apart from GalP, and it can be transported and phosphorylated by PTSlac/gal encoded by lacFE. Abbreviations: PTSman/glc, phosphoenolpyruvate-dependant phosphotransferase system (PEP:PTS) specific for mannose and glucose (encoded by ptnABCD); PTScel/glc, PEP:PTS specific for cellobiose and glucose (encoded by ptcBAC); PTSlac/gal, PEP:PTS specific for lactose and galactose (encoded by lacFE); G6P, glucose-6-phophate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde-3-phosphate; 3-PGA, 3-phosphoglycerate; PEP, phosphoenolpyruvate; TBP, tagatose-1,6-bisphosphate; Glk, glucokinase; PgmH, α-phosphoglucomutase; GalP, galactose permease; LacFE, EIIlac; PtnABCD, EIIglc/man; PtcBAC, EIIlac/gal; EI, Enzyme I; HPr, Histidine protein; HPr-His-P, HPr phosphorylated at histidine 15; HPr-Ser-P , HPr phosphosrylated at serine 46; CcpA, carbon catabolite protein A; + , activation; The letter P stands for a phosphate-group; ?, as yet unknown non-PTS glucose transporter; ??, newly discovered non-PTS galactose transporter(s) of which the gene(s) is/are as yet unknown.

L. lactis α-PGM is clustered with proteins with unknown function of the human-associated organisms Bifidobacterium longum and Propionibacterium acnes and with a protein with unknown function of the plant pathogen Gibberella zeae. Since L. lactis α-PGM is not homologous to α-PGMs of evolutionary closely related bacteria, we suggest that L. lactis might have obtained the gene for α-PGM by horizontal gene transfer. This might also be the case for the proteins with unknown function of the other organisms in the L. lactis α-PGM cluster. Of course it would be interesting to determine the function of these proteins with as yet unknown functions. L. lactis α-PGM showed around 25% identity with 2 human proteins. Since more and more genome sequences are becoming available, the near future might shed some more light on (the origin of) this intruiging new protein family. L. lactis lacks an α-PGM of the α-D-phosphohexomutase superfamily, a property that seems to be unique among bacteria. The α-PGM encoded by pgmH was

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demonstrated to be essential for L. lactis, which can be explained by the crucial role the enzyme fulfils in the physiology of the organism. α-PGM is not only required during galactose metabolism, but also during glucose metabolism, where its main function is to provide precursors for biosynthetic pathways e.g., for the production of exopolysaccharides. The UDP-Glc pool responds to variations in the level of α-PGM. Since L. lactis is an industrially relevant organism, the discovery of L. lactis α-PGM is interesting from the perspective of application. For example, exopolysaccharide production could be improved through manipulation of α-PGM (activity) in order to alter (improve) texture of food products or strains could be engineered with altered lytic capacity, which could lyse at a favourable time during the fermentation process. Overall, the work described in this thesis yielded very interesting results at both the fundamental and more applied levels. All new metabolic components described in this thesis are depicted in Fig. 1. The applied goals of the Nutracells project were achieved. A strain with improved lactose metabolic capacity was produced by metabolic engineering. This strain produces glucose as end product, which can be used as a natural sweetener. Furthermore, galactose metabolism was improved once the metabolic bottleneck had been revealed and resolved. The next step for application would be to make all the strains food-grade and test these strains in industrial settings for the same traits. Application of the engineered strains is expected to be initiated in the USA rather than in Europe due to the more strict regulations and the lower consumer acceptance of GMO’s in the latter continent. With respect to the fundamental research objectives, this project gained a further and deeper insight in sugar uptake and degradation, mainly in the first steps of metabolism. The presence of an additional galactose non-PTS transporter in L. lactis was determined, the cellobiose-transporting PTS (ptcBAC) was shown to also have affinity for glucose, and the gene encoding a novel α-PGM activity was discovered and characterized. Although metabolic engineering strategies devised on the basis of theoretical know-how do not always lead to the expected results in vivo, along the way interesting fundamental metabolic insights are always gained.

General discussion

149

Abbreviations

151

LIST OF ABBREVIATIONS (in alphabetical order)

3-PGA, 3-phosphoglycerate α-G1P, α-glucose-1-phosphate ADP, adenosine diphosphate ALS, α-acetolactate synthase ATP, adenosine triphosphate CcpA, carbon catabolite protein A CCR, carbon catabolite repression cre, catabolite responsive element DHAP, dihydroxyacetone phosphate EI, Enzyme I EII, Enzyme II EMP, Embden-Meyerhof-Parnas FBP, fructose-1,6-bisphosphate G1P, glucose-1-phosphate G6P, glucose-6-phosphate Gal, galactose Gal1P, galactose-1-phosphate Gal6P, galactose-6-phosphate GalE, UDP-galactose-4-epimerase GalK, galactokinase GalM, galactose mutarotase GalT, galactose-1-phosphate uridylyltransferase GAP, glyceraldehyde-3-phosphate GAPDH, glyceraldehyde-3-phosphate dehydrogenase Glc, glucose HAD, haloacid dehalogenase HPr, histidine protein HPr-His-P, HPr phosphorylated at histidine 15 HPr-Ser-P, HPr phosphorylated at serine 46 HPrK/P, HPr kinase/phosphatase LAB, Lactic Acid Bacteria LDH, lactate dehydrogenase NAD+, nicotinamide adenine nucleotide NADH, dihydronicotinamide adenine dinucleotide NMR, Nuclear Magnetic Resonance

Abbreviations

152

PDH, pyruvate dehydrogenase PEP, phosphoenolpyruvate PEP:PTS, phosphoenolpyruvate-dependent phosphotransferase system PFL, pyruvate formate lyase PGM, phosphoglucomutase Pi, inorganic phosphate PK, pyruvate kinase PMM, phosphomannomutase ROK, repressor-ORF-kinase UDP-Gal, UDP-galactose UDP-Glc, UDP-glucose

153

References

155

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NEDERLANDSE SAMENVATTING VOOR NIET-INGEWIJDEN Dit hoofdstuk is bedoeld voor iedereen die geen kaas heeft gegeten van moleculaire biologie. In dit hoofdstuk wil ik duidelijk maken wat het doel was van mijn laboratorium werk en wat de behaalde resultaten zijn. Bacteriën

Tijdens mijn promotieonderzoek bij de vakgroep moleculaire genetica in Haren heb ik gewerkt met bacteriën. Bacteriën zijn de oudste levensvormen op aarde. De evolutietheorie start met het ontstaan van deze eencellige organismen, waaruit vervolgens over een tijd van miljarden jaren al het andere leven is ontstaan. Bacteriecellen zijn bijna altijd tussen de 1 en 20 micrometer (1 micrometer is een duizendste millimeter) klein en ze zijn met het blote oog dus niet te zien. Hierdoor zijn ze pas ontdekt in 1676 door de Nederlandse bioloog Antonie van Leeuwenhoek toen hij door zijn zelf ontworpen microscoop keek. Inmiddels zijn er vele soorten bacteriën bestudeerd, hoewel het grootste deel van de bacteriesoorten nog niet bekend is, omdat deze onder laboratoriumcondities niet te kweken zijn. Er zijn heel veel verschillende soorten bacteriën en elke soort heeft zich aangepast aan de specifieke leefomgeving waarin ze leeft. Er zijn bacteriën die kunnen leven onder zeer extreme condities zoals bij hoge temperaturen in heet water bronnen, onder hoge druk diep in de oceaan, of bij hoge zoutconcentraties. Maar er zijn ook bacteriën die prima groeien onder (voor ons) normalere omstandigheden, bijvoorbeeld in tuinaarde, in ons maagdarm systeem, of in het vaatdoekje op het aanrecht. Er zijn bacteriën die gevaarlijk kunnen zijn voor ons mensen, zoals Streptococcus pneumoniae die tot een longontsteking kan leiden of Salmonella soorten die een dunne darm infectie kunnen veroorzaken. Maar er zijn ook vele onschadelijke bacteriën, die zelfs door ons gebruikt kunnen worden bijvoorbeeld in de voedingsmiddelenindustrie. Gebruik micro-organismen in de voedingsmiddelenindustrie

In de voedingsmiddelenindustrie wordt al eeuwenlang op grote schaal gebruik gemaakt van bacteriën en andere micro-organismen zoals gist. De eencellige gist (Saccharomyces cerevisiae) zorgt al eeuwen voor de productie van alcohol tijdens het vergisten van bieren en wijnen. Tevens produceert gist het gas koolstofdioxide (CO2) waar gebruik van wordt gemaakt bij het maken van een luchtig brood. Diverse Lactobacillus soorten worden gebruikt voor het produceren van gefermenteerde

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producten zoals salami en zuurkool. De bacteriën Streptococcus thermophilus en Lactobacillus bulgaricus worden samen gebruikt voor de productie van yoghurt uit melk. Deze bacteriën worden toegevoegd aan de melk waar ze melkzuur (lactaat) produceren uit melksuiker (lactose). Deze bacteriën worden melkzuurbacteriën genoemd. De productie van melkzuur resulteert in een snelle verzuring van het product en dat draagt bij aan de smaak. Door deze snelle verzuring wordt tevens de groei van andere (mogelijk schadelijke) bacteriën voorkomen. Voor de productie van kaas worden ook melkzuurbacteriën gebruikt. Na pasteurisatie van de melk wordt er zuursel en stremsel aan de melk toegevoegd. Het zuursel bestaat uit een combinatie van melkzuurbacteriën, en zorgt voor een specifieke smaak en textuur van de kaas. Bij gebruik van een bacterie die CO2 produceert ontstaan er bijvoorbeeld gaten in de kaas. Tevens zorgt het zuursel voor een snelle verzuring van de melk, waardoor de kaas langer houdbaar wordt. Naast het gebruik van melkzuurbacteriën voor het produceren van bepaalde voedingsmiddelen, worden ze tegenwoordig ook toegevoegd aan producten om te dienen als probiotica. Een probioticum is een levend microbiologisch voedingssupplement, dat de gezondheid van de gastheer bevordert door het microbiële evenwicht in de darmen te verbeteren. Van deze probiotische (zuivel)producten zijn tegenwoordig vele varianten op de markt, waarvan de meeste een verbeterde stoelgang of een verhoogde weerstand claimen. Lactococcus lactis als melkzuurfabriekje

De bacterie die voornamelijk is gebruikt voor het werk beschreven in dit proefschrift draagt de naam Lactococcus lactis. L. lactis is een melkzuurbacterie en wordt gebruikt voor de productie van kaas en andere zuivelproducten. Deze bacterie groeit het liefst bij een temperatuur van 30°C in een omgeving met weinig zuurstof. Hiernaast wordt L. lactis tevens veelvuldig gebruikt voor wetenschappelijke vraagstukken omdat L. lactis gemakkelijk en snel te kweken is onder laboratoriumcondities, en omdat de bacterie een relatief simpel metabolisme heeft. Metabolisme is het totaal aan chemische omzettingen dat plaatsvindt in de cel, ook wel de stofwisseling genoemd. L. lactis is een soort melkzuurfabriekje. De bacterie breekt suiker af tot melkzuur waarbij energie vrij komt voor de bacterie om te groeien. Er zijn verschillende soorten suikers (sacchariden), bijvoorbeeld glucose, galactose en lactose. Glucose en galactose bestaan uit één koolstofring en heten ook wel monosaccharides. Lactose is een disaccharide en bestaat uit twee koolstofringen, namelijk een glucose en een galactose deel. Bij de afbraak van lactose ontstaan dus glucose en galactose. In aanwezigheid van diverse soorten

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suiker zal L. lactis eerst zijn meest favoriete suiker afbreken, voordat een andere suiker aan de beurt komt. Glucose is de meest favoriete suiker van L. lactis, en heeft de voorkeur boven galactose, lactose en alle andere suikers. De afbraak van suikers verloopt via diverse stappen. Elke stap wordt uitgevoerd met behulp van een enzym dat een bepaalde reactie katalyseert. Elk organisme produceert een bepaald aantal specifieke enzymen. Welke enzymen kunnen worden geproduceerd is afhankelijk van het erfelijke materiaal (DNA) van het organisme. Van DNA naar eiwit

In het DNA staat aan de hand van een vier-letterige code alle genetische informatie van een organisme beschreven, en vormt dus een soort blauwdruk. Het totale DNA van L. lactis bestaat uit 2.500.000 lettertjes (nucleotiden), en hierin worden bijna 2500 genen beschreven. Elk gen kan worden afgeschreven tot RNA. Dit RNA kan vervolgens worden afgelezen tot een eiwit, zoals een enzym. Niet alle genen in het DNA worden even vaak afgeschreven. Dit is afhankelijk van de enzymen die het organisme op dat moment nodig heeft en is dus een sterk gereguleerd proces. Op het DNA ligt voor elk gen een zogenaamde promoter. Hier kunnen vaak verschillende moleculen aan binden, wat kan leiden tot activatie of inhibitie van het gen. Dit promoter-gebied fungeert dus als een soort aan/uit-knop voor het afschrijven van het gen. Als glucose aanwezig is, worden de enzymen voor de afbraak van glucose door L. lactis aangemaakt en tegelijkertijd wordt de productie van enzymen voor de afbraak van andere suikers geremd, zodat geen energie onnodig door de bacterie wordt verspeeld. ‘Metabolic engineering’

Het doel van dit onderzoek was om inzicht te verkrijgen in een aantal metabole processen van L. lactis. Er werd specifiek gekeken naar het metabolisme van glucose, galactose en lactose. Welke genen spelen hierbij een rol en wat zijn de eigenschappen van de betrokken enzymen? Hiernaast wilden we het metabolisme van de bacterie aanpassen naar onze wensen, door de bacterie genetisch te bewerken, ook wel ‘metabolic engineering’ genoemd. Door middel van moleculair biologische technieken zijn genen in L. lactis te manipuleren. Het is bijvoorbeeld mogelijk specifieke genen uit te schakelen door aan de genetische code te sleutelen. De code kan worden veranderd door slechts één lettertje te veranderen, of door een heel stuk van het gen weg te halen, waardoor er geen functioneel genproduct (eiwit) wordt gemaakt. Het is ook mogelijk om genetische informatie toe te voegen, waardoor de bacterie een extra genproduct gaat maken. Hiernaast kan

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ook de promoter van een gen worden veranderd, waardoor het gen op een andere manier gereguleerd kan worden. Wat waren onze wensen en waarom

De wensen die we hadden zijn beschreven in een project genaamd ‘Nutracells’, gesubsidieerd door de Europese Unie. De term Nutracells is afgeleid van Nutraceuticals, wat een samenvoeging is van ‘nutritional’ en ‘pharmaceutical’. De definitie van een ‘nutraceutical’ is: voeding of een voedingssupplement dat gezondheidsbevorderende eigenschappen bezit, en kan helpen bij het behandelen van een ziekte. De gezondheidsproblemen waar dit onderzoek aan is ontleend zijn de stofwisselingsziektes lactose intolerantie en galactosemie. Personen die last hebben van lactose intolerantie kunnen de melksuiker lactose niet verteren. Het gen dat codeert voor het enzym lactase, nodig voor de afbraak van lactose tot galactose en glucose, is niet functioneel. Lactose intolerante individuen kunnen voedsel waar lactose in zit niet eten, anders krijgen ze last van winderigheid en diarree. Galactosemie is een stofwisselingsziekte waarbij een enzym nodig voor het verteren van galactose ontbreekt. Hierdoor stapelt het toxische stofje galactose-fosfaat zich op in het lichaam, wat o.a. leidt tot problemen in de hersenen, nieren en darmen. Personen die lijden aan galactosemie zijn gebonden aan een levenslang dieet zonder lactose en galactose. Gedurende dit onderzoek is geprobeerd om de melkzuurbacterie L. lactis genetisch zo te bewerken dat de afbraak van glucose metabolisme wordt geblokkeerd en dat het metabolisme van galactose en lactose wordt opgeschroefd. De kennis die op deze manier werd verkregen kan in de zuivelindustrie worden gebruikt voor het uiteindelijk produceren van zuivelproducten zonder of in ieder geval met minder lactose en/of galactose. In Nederland mogen de door ons genetisch gemodificeerde bacteriën (nog) niet in de voedingsmiddelenindustrie worden gebruikt op dit moment. We kunnen met behulp van deze genetische experimenten wel laten zien wat er in de bacterie metabolisch gezien mogelijk is, zodat industriële partners kunnen proberen bacteriën met dezelfde eigenschappen te verkrijgen door ze gericht te kweken zonder genetische modificatie toe te passen. Behaalde resultaten

Glucose, galactose en lactose worden opgenomen in de bacterie-cel met behulp van specifieke transport eiwitten. Daarna worden bijna alle suikers omgezet tot glucose-6-fosfaat. Vanaf glucose-6-fosfaat verloopt de afbraak voor alle suikers via dezelfde route, de zogenaamde glycolyse. Om specifiek de afbraak van glucose te

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blokkeren, hebben we in L. lactis gekeken naar de eerste stappen in het metabolisme van glucose. Allereerst moet glucose de cel in worden getransporteerd en dit kan op twee manieren. Het kan worden opgenomen door een zogenaamde PTS transporter, die de glucose de cel importeert en er gelijk een fosfaatgroep aan vast zet, of door een non-PTS transporter die glucose importeert waarna het nog moet worden gefosforyleerd door een enzym genaamd glucokinase. Voor een overzicht, zie figuur 1. Het gen dat codeert voor de non-PTS transporter was niet bekend, dus om deze glucose afbraak route te blokkeren hebben we het gen dat codeert voor glucokinase (glk) in L. lactis uitgeschakeld. Om de PTS-route te blokkeren hebben we de bekende PTS transporter (PTSman/glc) weggehaald. Op basis van reeds bekende literatuur dachten we dat L. lactis zonder glucokinase en PTSman/glc (glk-, PTSman/glc-) geen glucose meer zou kunnen afbreken. Tot onze verbazing kon L. lactis (glk-, PTSman/glc-) wel degelijk op glucose groeien, wat betekende dat er nog een andere PTS transporter aanwezig moest zijn in L. lactis die glucose kon importeren. De volgende stap was om uit te zoeken welk gen codeert voor deze transporter. We gingen er vanuit dat de nog onbekende glucose-specifieke PTS transporter opgereguleerd zou worden in L. lactis (glk-, PTSman/glc-), om in de afbraak van glucose te kunnen voorzien. Door het totale RNA van L. lactis (glk-, PTSman/glc-) en de moederstam te vergelijken, en te kijken welke genen in L. lactis (glk-, PTSman/glc-) hoger tot expressie kwamen, verkregen we een aantal potentiële genen die betrokken zouden kunnen zijn bij glucose transport. Eén van deze genen stond bekend als een PTS transporter specifiek voor de suiker cellobiose (PTScel). Dit was een goede kandidaat om mogelijk ook glucose te transporteren. Vervolgens werd een L. lactis stam gekloneerd waarin de genen coderend voor glucokinase, PTSman/glc-, en PTScel uitgeschakeld waren. Deze stam bleek niet meer in staat om glucose af te breken en werd daarom glucose-negatief (Glc⎯⎯) genoemd. Hieruit werd geconcludeerd dat PTScel glucose kon transporteren, en deze transporter werd hernoemd tot PTScel/glc. Verschillende mutanten werden gemaakt waarbij de verschillende transport systemen afzonderlijk werden uitgeschakeld. De drie verschillende glucose transport systemen zelf (non-PTS, PTSman/glc, PTScel/glc) en het effect van het gebruik van deze verschillende systemen op het metabolisme van glucose werd vervolgens uitgebreid gekarakteriseerd, om meer te weten te komen over glucose metabolisme in L. lactis. Hiervoor werden verschillende technieken gebruikt waaronder Nucleaire Magnetische Resonantie (NMR), waarmee een specifiek gelabeld koolstof atoom (het kleinst mogelijke deeltje) in de loop van de tijd gevolgd werd. De vakgroep “cel fysiologie en NMR” onder leiding van Prof. Dr. Helena Santos, gelokaliseerd in Oeiras, Portugal, heeft deze techniek in de afgelopen tien jaar geoptimaliseerd voor het volgen van

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suikermetabolisme in L. lactis. Eén glucose molecuul bestaat uit 6 koolstof (C), 6 zuurstof (O), en 12 waterstof (H) atomen. Glucose wordt afgebroken zoals in figuur 1 is beschreven. Door gebruik te maken van een gelabeld C-atoom in glucose kan met behulp van NMR worden bepaald via welke stoffen dit C-atoom wordt overgedragen en wat de eindproducten van de afbraak van glucose uiteindelijk zijn.

Galactose

Galactose-1-fosfaat

Glucose-1-fosfaat

Buiten

Binnen

Lactose-6-fosfaat

Glucose

Lactose

Galactose-6-fosfaat

Glk

α-Pgm

Glucose

Lactaat &

Anderebijproducten

Glucose-6-fosfaat

Glucose

PTSman/glc PTScel/glc

PTSlac/gal

GlucoseGlucose

non-PTS

Galactose

Galactose-1-fosfaat

Glucose-1-fosfaat

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Lactose-6-fosfaat

Glucose

Lactose

Galactose-6-fosfaat

Glk

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Glucose

Lactaat &

Anderebijproducten

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Glucose

PTSman/glc PTScel/glc

PTSlac/gal

GlucoseGlucose

non-PTS

Figuur 1: overzicht glucose, galactose en lactose opname en metabolisme door een L. lactis cel. Glucose kan worden opgenomen en gefosforyleerd tot glucose-6-fosfaat door twee verschillende PTS-transporters (PTSman/glc en PTScel/glc), of het wordt opgenomen door een non-PTS transporter gevolgd door fosforylatie door glucokinase (Glk) in de cel. Galactose wordt voornamelijk opgenomen door een galactose transporter, waarna het wordt afgebroken tot glucose-6-fosfaat via galactose-1-fosfaat en glucose-1-fosfaat. Het enzym α-phosphoglucomutase (α-Pgm) speelt hierbij een belangrijke rol. Lactose wordt opgenomen door PTSlac/galen in de cel gesplitst in glucose en galactose-6-fosfaat. Uiteindelijk wordt alles afgebroken tot lactaat en mogelijk enkele andere bijproducten, afhankelijk van de groeicondities.

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De genen nodig voor de afbraak van lactose werden toegevoegd aan de glucose-negatieve stam van L. lactis. Deze stam (L. lactis Glc⎯⎯Lac+) kon groeien in een omgeving met lactose als enige suikerbron, waarbij alleen het galactose-deel van lactose werd verteerd. Het glucose-deel van lactose kon niet door deze stam worden afgebroken en werd door de cel uitgescheiden. De uitgescheiden glucose zou kunnen worden gebruikt als natuurlijke suiker in fermentaties, waardoor er minder of geen extra suiker aan het product hoeft te worden toegevoegd. Als L. lactis wordt gebruikt voor fermentaties is de zuurgraad meestal de limiterende factor voor de groei. De fermentatie begint vaak met een neutrale pH van ongeveer 6,5. L. lactis stopt met groeien als de pH in zijn omgeving beneden de 4,2 komt. Zoals al eerder gezegd zorgt L. lactis zelf voor de verzuring van zijn omgeving door de productie van melkzuur. Doordat L. lactis Glc⎯⎯Lac+ alleen het galactose-deel van lactose kan afbreken tot lactaat, kan deze stam dus twee keer zoveel lactose afbreken dan de moederstam van L. lactis voordat de groei wordt geremd door de verlaagde pH. Dit verhoogde lactose verbruik leidt dus tot een verlaagde lactose concentratie aan het eind van de fermentatie. Dit hebben we aangetoond voor L. lactis Glc⎯⎯Lac+ onder laboratorium condities. L. lactis Glc⎯⎯Lac+ is naast in laboratorium medium ook gekweekt in magere melk en hierin werden dezelfde resultaten behaald. L. lactis Glc⎯⎯Lac+ is dus een zeer bruikbare stam om gefermenteerde melkproducten met minder lactose te verkrijgen. Hiernaast hebben we geprobeerd een L. lactis stam te “engineeren” waarbij de afbraak van galactose zou worden gestimuleerd. Galactose wordt geïmporteerd in de cel door een galactose transporter, en vervolgens via een reeds bekende route afgebroken (zie figuur 1) tot glucose-6-fosfaat. De genen die coderen voor de betrokken enzymen worden gereguleerd. Als er glucose aanwezig is, worden de genen voor de afbraak van galactose niet afgeschreven. Wij hebben een extra kopie van een aantal van deze genen in L. lactis gezet, met een aangepaste promoter. Deze alternatieve promoter konden we zelf aanzetten, door het stofje nisine toe te voegen aan het groeimedium. Echter, toen we de galactose genen activeerden, nam de bacteriegroei af en er werd juist minder snel galactose afgebroken. De reden hiervoor bleek een opeenhoping van galactose-fosfaat en glucose-fosfaat te zijn; deze stoffen zijn in hoge concentraties toxisch voor de bacterie. In de route voor galactose-afbraak zat dus ergens een enzymatische stap die de bottleneck in de route vormde waardoor deze suiker-fosfaten accumuleerden. De bottleneck bleek te liggen bij het enzym alfa-phosphoglucomutase (α-pgm), die de omzetting van glucose-1-fosfaat naar glucose-6-fosfaat katalyseert.

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Om deze bottleneck te verhelpen werd geprobeerd om naast de activatie van de galactose genen, α-pgm hoger tot expressie te brengen in L. lactis. Omdat het gen dat codeert voor α-pgm in L. lactis niet bekend was, werd het gen voor α-pgm van een andere melkzuurbacterie (Streptococcus thermophilus) gebruikt. Overexpressie van de α-pgm van S. thermophilus samen met de galactose genen van L .lactis in L. lactis leidde tot een snellere galactose opname vergeleken met de moederstam van L. lactis zonder gen-overexpressies. Omdat het voor kloneringen in de toekomst handig was om de α-pgm van L. lactis zelf te gebruiken, en tevens uit pure fundamentele interesse, werd geprobeerd het gen dat codeert voor α-pgm in L. lactis op te sporen. Hiervoor werden diverse strategieën gebruikt. Uiteindelijk bleek de biochemische methode waarbij de activiteit van α-pgm werd geïsoleerd succesvol. Gen yfgH (een gen-naam beginnend met een y staat voor een gen waarvan de functie nog niet bekend is) bleek te coderen voor het enzym α-pgm en gen yfgH werd daarom hernoemd tot gen pgmH. Gen pgmH is het enige gen in L. lactis dat codeert voor een α-pgm, aangezien we hebben aangetoond dat een L. lactis stam waarin pgmH is uitgeschakeld niet meer kan groeien op galactose. Vervolgens werd de α-pgm van L. lactis uitgebreid gekarakteriseerd. De optimale pH en de specificiteit van het enzym werden bepaald, evenals mogelijke factoren die van invloed zouden kunnen zijn op de activiteit van het enzym, zoals de aanwezigheid van een aantal mineralen. De α-pgm van L. lactis bleek een uniek enzym te zijn, niet vergelijkbaar met enzymen met dezelfde functie in homologe bacteriën, en er wordt daarom nog verder onderzoek naar gedaan. Al met al zijn er veel fundamentele vragen betreffende glucose, galactose en lactose metabolisme in L. lactis beantwoord door dit onderzoek. Hiernaast zijn er een aantal nieuwe vraagstukken bijgekomen, die door vervolgonderzoek zullen moeten worden opgelost. Er zijn een aantal zeer interessante L. lactis stammen gekloneerd, die gebruikt zouden kunnen worden voor het produceren van zuivelproducten met minder lactose en galactose en voor het gebruik van glucose als natuurlijke zoetstof in zuivelproducten. De resultaten zijn in dit hoofdstuk niet in detail besproken. Bent u geïnteresseerd geraakt dan verwijs ik u voor de details door naar de hoofdstukken hiervoor.

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Nawoord

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NAWOORD

Zo, daar ligt hij dan, lang verwacht, toch gekomen, mijn boekje. Een nawoord biedt de ruimte om even terug te kijken. In oktober 2000 begon ik als AIO bij de vakgroep moleculaire genetica op het biologische centrum in Haren. Voor mij een bekende plek, omdat ik er tijdens mijn studie ook al één van mijn stages had gedaan. Het knutselen aan Lactococcus lactis en de sfeer in de groep beviel mij wel, dus ik was erg blij dat ik als AIO werd aangenomen op het EU-project ‘Nutracells’. Het project heeft veel leuke resultaten opgeleverd, die hopelijk in de toekomst verder zullen worden uitgebreid. Uiteindelijk, na 4 jaar pipetteren, moesten alle vergaarde resultaten alleen nog ‘even’ op papier gezet worden. Dat dit schrijfwerk gemakkelijk naast mijn nieuwe baan te doen zou zijn, bleek voor mij een illusie. Toch is het gelukt, en hiervoor wil ik graag een aantal mensen bedanken. Als eerste wil ik mijn promotores bedanken. Oscar, jouw enthousiasme heeft mij sterk gemotiveerd tijdens het pipetteren en zeker tijdens het schrijven. Je hield het paadje naar het lab warm, en bleef in een goede afloop geloven. Jan, bedankt voor het uitvoerig lezen van mijn hoofdstukken. Door jou is het boekje een stuk leerbaarder geworden en daarnaast heb ik van je suggesties veel geleerd. Een deel van mijn werk heb ik verricht in Oeiras, Portugal in het lab van Helena Santos. Helena, agradeco o tempo em que trabalhei no seu laboratόrio. A possibilidade de ter trabalhado com o vosso equipamento de RMN para estudos “em vivo” foi muito importante na minha investigação. Rute, a minha SPSS, queria tambem agradecer-te pelo nosso trabalho conjunto. Alem de trabalharmos juntas, tambem conseguimos motivar-nos uma a outra. Gostei de conviver contigo tanto no trabalho (em Groningen e Lisboa) como nas viagens de congressos que fizemos juntas. Sem a tua ajuda o resultado da minha tese seria diferente. Espero continuar a relacionar-me contigo no futuro. Ook wil ik alle ‘MolGenners’ bedanken voor de gezellige sfeer op het lab. Ik heb 4 jaar lang met veel plezier bij moleculaire genetica gewerkt. Naast het feit dat de groep over veel kennis en faciliteiten beschikt, is het een ontzettend gezellige groep. De vele gesprekken, borrels, uitstapjes en feestjes zal ik nooit vergeten. Geen enkele AIO strijdt alleen, en daarom wil ik een aantal van mijn medestrijders bij MolGen speciaal bedanken. Anton, Naomi en Olivera, voor het tikken van vele eitjes en voor het delen van onze AIO-dipjes. Robèr, multiplex labgenootje, immer positief. Harma, het rustpunt in ons werkvertrek. α-Chris, lotgenoot, je mocht altijd

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even uithuilen, maar liever niet met Mariah. Richard (wel wakker blijven!), Nathalie (eh be…), Rasmus (how is your portuguese nowadays), Aldert, Girbe, Anne H., Anne de J., Siger en Sacha. Ook wil ik Thijs bedanken, dat hij heeft meegewerkt aan dit onderzoek tijdens zijn stage. Naast het onderzoekend personeel wil ik Arie (zeg maar in wezen), Mozes en Peter bedanken voor alle bestellingen, media en glaswerk, en Emma en Mirelle voor alle secretariële hulp. Na 4 jaar labwerk bij MolGen in Haren ben ik als docent gaan werken aan de Hanzehogeschool in Groningen voor het instituut voor Life Science & Technology. Hier probeer ik goede nieuwe laboranten te kweken. Het schrijfwerk voor dit proefschrift is verricht naast mijn werkzaamheden als docent. Mede dankzij de stimulerende woorden van vele collega’s is dit boekje toch nog afgekomen. Peter, ben jij de volgende? Ida, bedankt voor de tijd die ik kreeg om het laatste schrijfwerk te verrichten. Alexandra, bedankt voor je Portugese vertaling. De nodige ontspanning naast het werk was minstens zo belangrijk. Daarbij hebben alle familie en (korfbal)vrienden een grote rol gespeeld. Binnenkort weer eens een caneitje drinken in de Jister? En Durkje, ik kom binnenkort echt weer te spinnen hoor! Ria, samen gestudeerd, samen naar Wales (saampjes he). Hoewel je AIO werd bij een hele andere vakgroep, konden we toch alle facetten van het AIO-schap met elkaar delen en nu vele andere zaken natuurlijk. Hebben we bijna alle restaurants in Groningen gehad? Een promotie begint natuurlijk niet zomaar. Hierbij wil mijn ouders bedanken voor de mogelijkheid om te gaan studeren. Het moet niet gemakkelijk zijn geweest om je 17-jarige dochter op een kamertje in Groningen achter te laten. Anton, ik was paranimf bij jouw promotie, ik vind het erg leuk dat jij nu mijn paranimf wil zijn. Johan, ik was getuige bij jouw bruiloft, jij bent ‘ceremoniemeester’ bij mijn promotie (druk met de voorbereidingen?). Hartstikke leuk, we gaan er een mooie dag van maken. Lieve Peter, jij hebt mijn promotie van A tot Z meegemaakt. Bedankt voor alle steun, geduld en vooral het hebben van een groot luisterend oor. Dit feestje wordt van ons samen. En ja, binnenkort mag je Vista op je computer zetten. Tot slot, lieve kleine Amarins, bedankt voor je glimlach. Je hebt er nu nog geen weet van, maar jij bent mijn laatste ‘stok achter de deur’ geweest om dit boekje af te ronden.

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Notes

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