MmN

Za

b

a

ARAA

KMEH31O

1

atyanei(hgt(tpap

(

1d

Biochemical Engineering Journal 54 (2011) 151–157

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /be j

etabolic pathway analysis of 1,3-propanediol production with a geneticallyodified Klebsiella pneumoniae by overexpressing an endogenousADPH-dependent alcohol dehydrogenase

hen Chena, Hongjuan Liub,∗, Dehua Liua,∗

Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR ChinaInstitute of Nuclear and New Energy Technology, Tsinghua University, Beijing 100084, PR China

r t i c l e i n f o

rticle history:eceived 30 June 2010ccepted 4 February 2011vailable online 22 February 2011

eywords:etabolic pathway analysis

lementary mode analysis

a b s t r a c t

Coenzyme limitation is one of the most important issues for 1,3-propanediol (PDO) production. Elemen-tary mode analysis indicated that pentose phosphate pathway and TCA cycle were the most efficientpathways for generating reducing equivalent NADPH and NADH. Under the optimal condition for PDOproduction, 0.542 mol NADPH/(mol glycerol), accounting for 61.7% of the total reducing equivalent wouldbe produced, which requires the fast conversion of NADPH for PDO synthesis. Based on the above anal-ysis, an endogenous NADPH-dependent alcohol dehydrogenase (HOR) was cloned and overexpressedfor NADPH usage in Klebsiella pneumoniae ACCC10082. The activities of HOR and total 1,3-propanediol

OR-Hydroxypropionaldehyde,3-Propanediolverxpression

dehydrogenase (PDOR) increased 5.8-fold and 1.1-fold than that of the wild type strain. In the fed-batchfermentation, the PDO concentration and yield of the constructed strain increased 10.4% and 9.4% whilethe highest 3-hydroxypropionaldehyde accumulation reduced 35.1% compared with that of the wild typestrain. Metabolic flux analysis suggested that the increase of PDO yield was due to the enhanced carbonflux flowed to pentose phosphate pathway which provided coenzyme for HOR utilization. This work is

derstducti

helpful for the further unimprovement of PDO pro

. Introduction

The biological conversion of glycerol to 1, 3-propanediol (PDO),promising chemical as the monomer of the novel polymer poly-

rimethylene terephathalate (PTT), has been refocused in recentears as a result of the surplus of glycerol that is being produceds a major by-product in biodiesel industry [1,2]. The mecha-ism for the glycerol conversion by Klebsiella pneumoniae has beenxtensively studied [3,4]. The metabolic pathway can be dividednto two branches: the reductive branch and the oxidative branchFig. 1). In the reductive branch, glycerol is first converted to 3-ydroxypropionaldehyde (3-HPA) by a coenzyme B12-dependentlycerol dehydratase (GDHt) and the latter is further reducedo PDO by a NADH-dependent 1,3-propanediol oxidoreductasePDOR) [5]. On the other hand, glycerol is oxidized to dihydroxyace-

one (DHA) by a NAD+-dependent glycerol dehydrogenase (GDH) orhosphorylated into glycerol-3-phosphate (G3P) by glycerol kinasend both DHA and G3P can be further transferred into DHA phos-hate (DHAP) which will enter glycolysis [6].

∗ Corresponding authors. Tel.: +86 10 62794742; fax: +86 10 62794742.E-mail addresses: dhliu@tsinghua.edu.cn (D. Liu), liuhongjuan@tsinghua.edu.cn

H. Liu).

369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2011.02.005

anding of PDO metabolism in K. pneumoniae and also useful for the strainon.

© 2011 Elsevier B.V. All rights reserved.

It has been generally recognized that the conversion of 3-HPA toPDO is mainly catalyzed by PDOR with the association of coenzymeNADH. Recent years, an NADPH-dependent alcohol dehydrogenasewas found more effective than NADH-dependent 1,3-propanedioldehydrogenase for PDO production. A substitution of dhaT gene toyqhD gene encoding a NADPH-dependent alcohol dehydrogenasein recombinant Escherichia coli resulted in high titers of approxi-mately 130 g/L PDO production, which have never been obtained inthe identical strain utilizing dhaT gene [7]. The proteomic analysisof K. pneumoniae from glycerol fermentation identified an endoge-nous NADPH-dependent oxidoreductase (HOR) in K. pneumoniae[8]. HOR was supposed to play important role in the conversionof 3-HPA to PDO and showed high activity at the late phase of thefermentation [8]. In the latest report, Seo et al. confirmed that HORwas the isoenzyme of PDOR and was responsible for the biotrans-formation of PDO production [9]. However, the effect of HOR on themetabolism of K. pneumoniae was still not clear.

Elementary mode analysis is one of the most powerful tools formetabolic pathway analysis which calculate the solution space that

contains all possible steady-state flux distributions of a networkby considering the stoichiometry of carbon and cofactor [10]. Thestoichiometry of the metabolic network, including carbon as wellas cofactor requirements, is fully considered in elementary modeanalysis. On the other hand, elementary mode analysis also allows

152 Z. Chen et al. / Biochemical Engineering Journal 54 (2011) 151–157

Glycerol

GA-3-P

Fructrose-6-P

Glucose-6-P

Ribose-5-P

Sed-7-P

Erythrose-4-P

2NADPHCO2

NADH

PG

NADH

PEP

ATP

Pyruvate

Oxaloacetate

CO2

Acetyl-CoA

Formate

Ethanol

AcetateATP

Lactate

DHA

DHAP

Ribulose-5-P

Xylulose-5-P

Glycerol-3-P

Fructrose-6-P

Fructrose-1,6-P

GA-3-P

Glycerol_ext

ATP

NADH

NADH

ATP

NADHCO2

2NADH

Citrate

Isocitrate

α-Ketoglutarate

Succinyl-CoA

Succinate

Fumarate

Malate

Glyoxylate

NADPHCO2

NADHCO2

NADH

NADH

Biomass

Pyruvate

PEP

Oxaloacetate

α-Ketoglutarate

Acetyl-CoA

Erythrose-4-P

Ribose-5-P

Glucose-6-P

NADH+2ADP+O2 NAD+2ATP

ATP ADP

NADHNADPH

Succinate_ext

glpF

gldA

glpK

glpD

tpi12

pflpdh

ppc

pyc

pykAF

ldhA

gltA

acnB

icd

sucABsucCD

sdhABCD

fumABC

mdh

aceA

aceB

adhE

pta, ackA

ATP

R1

R2R4

R5

R10

R14

R15

R20

R16

R21

R22

R23

R24

R25

R26

R27

R28

R29

R11

R12

R13

R30

R31 R32

R33

R34

R35

R40

R41R42

R43

R44

R51

R52

R53

R50

R60

R70

R71

R72

R54

R6

NADPH

Glucose-6-P

GA-3-PPG

ATP

dhaKR3

3-HPA1,3-propanediol

NADH

2,3-butanediolNADH

R7

R55

3-HPA_ext

F hydroG uvate.

docfms

ig. 1. The representative metabolic pathway of glycerol. Abbreviations: 3-HPA, 3-A-3-P, glyceraldehyde-3-phosphate; PG, phosphoglycerate; PEP, phosphoenolpyr

etermining the overall capacity, i.e., theoretical maximum yield,

f a cellular system and studying the effects of any genetic modifi-ation [11]. Based on such studies, rational design can be obtainedor the efficient production and genetic modification. Recently, ele-

entary mode analysis have been used for genome scale metabolictudies dealing with, e.g., the rational design of methionine produc-

xypropionaldehyde; DHA, dihydroxyacetone; DHAP, dihydroxyacetone phosphate;

tion in E. coli and Corynebacterium glutamicum [12], the production

of polyhydroxybutanoate in yeast [13], the growth-related aspectsin Saccharomyces cerevisiae [14,15] and E. coli [16,17].

To discuss the function of the endogenous HOR on the PDOsynthesis by K. pneumoniae, an endogenous HOR gene was clonedand overexpressed in K. pneumoniae ACCC10082 in this study.

ineeri

Mypttpf

2

2

TeeP3wGwJgttmPKP

2

omi1rwwwTwdits

2

ut

TS

K

Z. Chen et al. / Biochemical Eng

etabolic pathway analysis including the elementary mode anal-sis and metabolic flux analysis was intensively used for theredicting and analyzing the cellular flux distribution variation byhe perturbation of HOR. This work is considered to be helpful forhe further understanding of the PDO metabolic mechanism in K.neumoniae and also useful for the further optimization of PDOermentation.

. Materials and methods

.1. Strains and plasmids construction

Strains and plasmids used in this study are listed in Table 1.he wild type K. pneumoniae ACCC10082 was used as the par-nt strain. The HOR gene from the wild type K. pneumoniae strainncoding a potential alcohol dehydrogenase was amplified byCR using primers P1: 5′- CCGGAATTCATGAATAATTTTGACCTGC-′ and P2: 5′- ACGCGGATCCTTAGCGAGCAGCCTCGTA-3′ (primersere designed according to sequence of yqhD gene undereneBank accession no. NC 011283). The amplified HOR geneas inserted into a vector pMD18-T Simple (Takara Bio Inc.,

apan), resulted in pMD18-T Simple-HOR, from which the HORene was digested by EcoRI and BamHI and introduced intohe EcoRI–BamHI site of the expression vector pKD6 [18] underhe control of tac promotor. The kanamycin resistance transfor-

ants were selected and the insert was confirmed by colonyCR, restriction digestion and sequencing. The confirmed colony. pneumoniae (pDK6-HOR) was designated as K. pneumoniaeD1.

.2. Media and culture conditions

LB medium was used as a rich medium for the routine growthf K. pneumoniae. The composition of the seed and fermentationedia was described as Chen et al. [19]. The seeds were grown

n a 500-mL shake flask containing 100 mL media at 37 ◦C and20 rpm for 12 h. The batch and fed-batch cultivation were car-ied out in a 5L stirring bioreactor (B. Braun Biotech International)ith a working volume of 4 L. Temperature, pH and agitation speedere maintained at 37 ◦C, 6.8 and 250 rpm respectively. The pHas controlled by adding a solution of 50% (mass fraction) NaOH.

he inoculated volume was 10% (v/v). The microaerobic conditionas controlled by aerating 0.5 vv m air. 0.2 mmol/L isopropyl-�--thiogalactoside (IPTG) was added to the culture media at 4 h to

nduce the overexpression of HOR. The glycerol concentration inhe medium was maintained at 10–30 g/L by feeding the glycerololution.

.3. Analytical methods of cell mass and metabolites

The cell concentration was measured as absorbance at 650 nmsing a Beckman DU640 UV/VIS spectrophotometer (Interna-ional MI-SS Inc., CA). The concentration of glycerol, PDO,

able 1trains and plasmids used in this work.

Strains orplasmids

Genotype/phenotype Reference

K. pneumoniaeACCC10082

Parent strain Lab conservation

K. pneumoniaePD1

Recombinant strain, harboringpDK6-HOR

This work

pDK6 Expressing vector, Kmr, 5.1 kb Kleiner et al. [18]pDK6-HOR pDK6 cloned with 1.2 kb HOR

gene, Kmr, 6.3 kbThis work

m: kanamycin.

ng Journal 54 (2011) 151–157 153

ethanol, acetate, lactate, succinate, and 2,3-butanediol were deter-mined by HPLC with a refractive index detector (Shimadzu10AVP HPLC system, Shimadzu, Japan). An Aminex HPX-87H col-umn (300 mm × 7.8 mm) (Bio-Rad, USA) was used with 5 mMH2SO4 as mobile phase at 0.8 mL min−1. The column wascontrolled at 65 ◦C [19,20]. The concentration of 3-HPA wasmeasured according to the method described by Circle et al.[21].

2.4. Enzyme assays of HOR and PDOR

After the strain was induced by IPTG for 4 h, the cells were har-vested. Cell pellets were washed and re-suspended in phosphatebuffer containing 100 mmol/L �-mercaptoethanol (pH 7.5) andthen disrupted ultrasonically at 4 ◦C for 10 min with 3 s pulses and200 W by a cell sonicator (SCIENTZ JY 92-II, China). Cell debris wasremoved by centrifugation (20,000 × g for 20 min at 4 ◦C), resultingin a cell-free extract, which was subsequently used for HOR activityassay.

The enzyme activity of HOR was measured according to themethod of Pérez [22]. The reaction mixture (1.0 mL) contained100 mM potassium carbonate buffer (pH 9.0), 30 mM ammoniumsulfate, 2 mM NADPH, 100 mM 3-HPA and cell lysate. NADPH oxi-dation was determined at 340 nm using Beckman DU640 UV/VISspectrophotometer. The enzyme assays of PDOR was carried outaccording to the methods described by Ahrens et al. [23]. One unitof enzyme activity is defined as the amount of enzyme required toreduce 1 �mol of substrated per minute. Protein concentration wasdetermined according to Bradford’s method [24].

2.5. Elementary mode analysis and metabolic flux analysis

The glycerol metabolic network of K. pneumoniae undermicroaerobic condition was constructed (Fig. 1) based on KEGGdatabase (http://www.genome.jp/kegg/metabolism.html) as wellas the references [5,19,25]. It includes glycerol dissimilation path-ways, glycolysis pathway (EMP), pentose phosphate pathway (PPP),tricarboxylic acid (TCA) cycle, biosynthesis pathway, anaplerosisand respiratory chain as depicted in Fig. 1. For the interconversionof NADH and NADPH, a cytosolic transhydrogenase transferringprotons from NADPH to NAD+ and a membrane-bound transhydro-genase reducing NADP+ by oxidation of NADH was implemented[25]. For ATP production in the respiratory chain, a P/O ratio of 2for NADH was assumed [25]. The precursor demand for biomassformation was taken from the literature [25].

The elementary mode analysis was carried out by usingMETATOOL [26,27]. The script files and compiled sharedlibrary of METATOOL 5.1 can be downloaded from the META-TOOL website (http://www.biozentrum.uni-wuerzburg.de/bioinformatik/computing/metatool/). The mathematical details ofthe algorithm are described elsewhere [27].

3. Results

3.1. Characterization and cloning of HOR in K. pneumoniae

To identify and characterize the endogenous NADPH-dependentoxidoreductase (HOR), the chromosomal DNA of K. pneumoniae wascompared through NCBI database and an open reading frames (ORF)located at 703518–704681 bp was identified, which is annotatedas alcohol dehydrogenase (GeneBank accession no. NC 011283).

Based on this template, the HOR gene of K. pneumoniae ACCC 10082was cloned and sequenced. The ORF of HOR gene is 1164 bp whichis consisted of 387 amino acids. The amino acid sequence shared88% identities with the yqhD gene from E. coli K-12 but only show25% identity with the dhaT from K. pneumoniae. The active sites of

154 Z. Chen et al. / Biochemical Engineering Journal 54 (2011) 151–157

roduc

Esto

Fig. 2. The optimal metabolic flux distribution of glycerol for 1,3-propandiol p

. coli yqhD (Asp194, 198His, His267 and His281) [17] were con-erved in the HOR and PDOR from K. pneumoniae which indicatedhat they may share the similar functions. The NADPH binding sitesf E. coli yqhD (residues 37–40, 93–99 and 179–182) [17] were also

tion under microaerobic conditions. The abbreviations are the same as Fig. 1.

conserved in the K. pneumoniae HOR but not conserved in K. pneu-moniae PDOR. The GGGS motif (residues 37–40) of HOR involving ininteraction with the 2′-phosphate group of NADPH was not foundin PDOR. This indicated that they may use different cofactors.

ineering Journal 54 (2011) 151–157 155

3

oamonpotz

PpmN(ee

G

G

wrtppNwntNtKopaPap

3

od0tPtioitHt

3p

fs

Table 2Enzyme activity analysis of PDOR and HOR in K. pneumoniae ACCC10082 and K.pneumoniae PD1.

Strains NADH-dependent NADPH-dependent

10.4% and 10.4% respectively compared with the wild type strain.The PDO yield was 57.1%, which increased 9.4% than that of thewild type strain. The main byproduct 2,3-butanediol concentrationincreased 76.4% and the concentration of lactate decreased 19.6%.

Fig. 3. Cell growth, PDO and 2.3-butanodiol production by wild type K. pneumo-niae ACCC10082 and constructed K. pneumoniae PD1 in the fed-batch fermentation.Symbols: for K. pneumoniae ACCC10082: (�) PDO, (�) OD, and (�) 2.3-butanodiol.For K. pneumoniae PD1: (�) PDO, (�) OD, and (♦) 2.3-butanodiol.

0

5

10

15

20

25

50403020100

Conce

ntr

atio

n o

f ac

etat

e an

d l

acta

te (

g/L

)

Z. Chen et al. / Biochemical Eng

.2. Elementary mode analysis for PDO production

Elementary mode analysis was used to analyze the theoreticalptimum flux distribution for PDO production by K. pneumoniaend the results are shown in Fig. 2. Under optimal condition, theaximum PDO yield could reach 0.875 mol/mol under microaer-

bic condition considering both PPP pathway and TCA cycle, ando cell and by-products such as lactate, acetate and ethanol wereroduced. This value is near to the report of Zhang et al. The yieldf 0.844 mol/mol was obtained in recombinant E. coli by amplifyinghe gene yqhD encoding 1,3-propanediol oxidoreductase isoen-yme and the gene dhaB encoding glycerol dehydratase [28].

In the optimal condition, the reducing power generated fromPP pathway and TCA cycle would be completely used for PDOroduction. The pathway from glycerol to PPP pathway was theost efficient for generating reducing equivalents, in which 1 molADH and 6 mol NADPH would be produced from 1 mol glycerol

Eq. (1.1)). Another pathway, from glycerol to TCA cycle was alsofficient for reducing equivalents generating, in which 1 mol glyc-rol would produce 5 mol NADH and 1 mol NADPH (Eq. (1.2)).

lycerol + NAD+ + 6NADP+ → 3CO2 + NADH + 6NADPH (1.1)

lycerol + 5NAD+ + NADP+ → 3CO2 + 5NADH + NADPH (1.2)

These two pathways were more efficient than any other path-ays e.g., the acetate synthesis pathway which was previously

egarded as the most effective pathway for NAD(P)H regenera-ion [23]. As indicated in Fig. 2, for the theoretical optimal PDOroduction, there would be 8.33% metabolic flux flowed to PPPathway and 4.17% carbon flux flowed to TCA cycle. 0.542 molADPH/mol glycerol would be produced through both PPP path-ay and TCA cycle, which attributed to 61.9% reducing equivalenteeded for PDO production. Since PDOR utilized NADH as cofac-or, these large amount of NADPH needed to be transferred intoADH by transhydrogenase which required the high activity of

ranshydrogenase. However, the activity of transhydrogenase in. pneumoniase was very low, which would limit the productionf PDO [25]. Thus, overexpression the NADPH dependent 1,3-ropanediol oxidoreducatase is considered to be the more directpproach for efficiently utilizing NADPH to increase the yield ofDO and reduce the accumulation of 3-HPA. The optimal in vivoctivities between NADH-dependent and NADPH-dependent 1,3-ropanediol oxidoreducatase was expected to be 39.1:61.9.

.3. Overexpression of HOR in K. pneumoniase ACCC10082

Based on the above analysis, the NADPH-dependent HOR wasverexpressed in K. pneumoniae and the enzyme activity wasetected. For the wild type strain, the HOR activity was observed as.097 U/mg protein, and the activity of PDOR was 0.452 U/mg pro-ein, which was 4.66 fold to that of HOR. The results suggested thatDOR played the main role for the transformation of 3-HPA in wildype K. pneumoniae. This is far from the ideal situation calculatedn the above study. For the constructed strain, the specific activityf NADH-dependent PDOR changed little while the specific activ-ty of NADPH-dependent HOR increased 5.8-fold compared withhe wild type strain (Table 2). The enzyme assay indicated that theOR gene was successfully overexpressed and it would facilitate

he transformation of 3-HPA into PDO in the cooperation of NADPH.

.4. Cell growth and PDO production of the constructed strain K.

neumoniase PD1 in fed-batch fermentation

According to the metabolic pathway analysis, the PDO fed-batchermentation under microaerobic conditions with the constructedtrain K. pneumoniase PD1 was performed in 5L bioreactors. The

PDOR (U/mg) HOR (U/mg)

K. pneumoniae ACCC10082 0.452 ± 0.038 0.097 ± 0.018K. pneumoniae PD1 0.517 ± 0.067 0.662 ± 0.106

wild type K. pneumoniae ACCC10082 was used as the control. Theresults are shown in Figs. 3 and 4.

As indicated in Fig. 3, the cell growth of K. pneumoniase PD1showed a similar growth rate compared to the wild type strain,which suggested that the overexpression of HOR in K. pneumo-niae ACCC10082 did not inhibit the cell growth. The metabolitesproduction is shown in Fig. 4. It was shown that PDO productionwas enhanced by overexpressing HOR. The concentration and pro-ductivity of PDO reached 60.6 g/L and 1.26 g/(L h), which increased

Time (h)

Fig. 4. Acetate and lactate production by wild type K. pneumoniae ACCC10082 andconstructed K. pneumoniae PD1 in the fed-batch fermentation. Symbols: for K. pneu-moniae ACCC10082: (�) lactate, and (�) acetate. For K. pneumoniae PD1: (�) lactate,and (©) acetate.

156 Z. Chen et al. / Biochemical Engineeri

0

1

2

3

4

5

6

7

50403020100

Time (h)

Conce

ntr

atio

n o

f 3

-HP

A (

mm

ol/

L)

FnA

TrteHpatoPtficNf

3p

swcftTptftwsi

accumulation of 3-HPA mainly occurred at the early phase of the

TC

ig. 5. 3-HPA accumulation in the fed-batch fermentation by wild type K. pneumo-iae ACCC10082 and constructed K. pneumoniae PD1. Symbols: (�) K. pneumoniaeCCC10082, and (©) K. pneumoniae PD1.

he acetate production which was produced at the early phase bute-consumed at the later phase was similar to that of the wildype strain. The results demonstrated that PDO production wasnhanced by overexpressing the endogenous NADPH-dependentOR. The PDO yield and productivity were both increased com-ared with the wild type strain and the cell growth was notffected by the HOR overexpression. This phenomenon was iden-ical with the PDOR and GDH co-overexpression [19]. However,verexpressing PDOR singly caused the cell growth and the finalDO concentration decreasing [29]. The reason was regarded ashat PDOR single overexpression led to the coenzyme NADH insuf-ciency, while GDH overexpression provided enough NADH for theell growth and PDO synthesis. For the case of HOR overexpression,ADPH was utilized as reducing equivalent, which was beneficial

or the cell growth and PDO synthesis.

.5. 3-HPA accumulation of the constructed strain K.neumoniase PD1 in fed-batch fermentation

As the important middle metabolites, 3-HPA is the precur-or of PDO, also a toxic substance, the accumulation of whichould cause an irreversible cessation of the fermentation pro-

ess. So, the accumulation of 3-HPA also was analyzed in theermentation. The results are indicated as Fig. 5. For the con-rol, 3-HPA reached two peaks during the whole fermentation.he first peak (6.31 mmol/L) appeared at 12 h and the secondeak (4.23 mmol/L) appeared at 22 h. Both peaks appeared athe early phase of the fermentation when the cells were inast growing. Compared with the control, the 3-HPA accumula-

ion obviously declined and the highest concentration of 3-HPAas only 4.10 mmol/L which decreased 35.1% for the constructed

train K. pneumoniase PD1. The decrease of 3-HPA accumulationmplied that HOR played an important role even in the early

able 3omparation of glycerol metabolism flux distribution in K. pneumoniae ACCC10082 and K

Strains Flux to

PDO (R7) PPP pathway (R11) 2,3-Butanediol (R52) Lactate (R51)

K. pneumoniaeACCC10082

0.522 0.061 0.042 0.215

K. pneumoniaePD1

0.571 0.074 0.072 0.188

ng Journal 54 (2011) 151–157

phase of PDO synthesis by the constructed strain K. pneumoniasePD1.

3.6. Metabolic flux analysis of constructed strain K. pneumoniasePD1

Metabolic flux distribution was compared between the wildtype K. pneumoniase and the constructed strain K. pneumoniase PD1.The results are shown in Table 3. From Table 3, we can see thatthe carbon flux flowed to PPP pathway, the most efficient pathwayfor NADPH regeneration, increased 21.3%. On the other hand, theflux flowed to PDO increased 9.4%, both of which were attributedto the enhancement of PDO production. At the metabolic node ofpyruvate, the carbon flux flowed to 2,3-butanediol increased 72.2%while flowed to lactate and acetyl-CoA decreased 12.6% and 46.6%,respectively. At the Acetyl-CoA node, the carbon flux flowed toTCA cycle, ethanol and acetate decreased 23.5%, 87.5% and 34.3%,respectively. The flux redistribution to the overproduction of thealcohol could be attributed to wide range of substrates specificityof HOR. It has been reported that yqhD could catalyze the trans-formation of C3–C6 aldehydes [30]. The enzyme activity assay alsoconfirmed that HOR could catalyze the transformation of acetointo 2,3-butanediol (data not shown). Thus, the metabolic pathwaysto PDO and 2,3-butanediol could be both strengthened by theHOR overexpression. On the other hand, the PPP pathway, pro-vided more NADPH which was favourable for the HOR utilization.The fully use of both NADH and NADPH also enhanced the 3-HPAtransformation and PDO production in the constructed strain K.pneumoniase PD1.

4. Discussion

According to the elementary mode analysis, an endogenousNADPH-dependent alcohol dehydrogenase was cloned and over-expressed for NADPH usage in K. pneumoniae ACCC10082. Themetabolic pathway analysis of glycerol metabolism indicated thatthe pathway to PPP was the most effective way for generatingreducing equivalent, which was crucial for PDO production. Thismay attribute to the high titer of yqhD-carring recombinant E. coli.What is more, since the PDOR catalyzed the reversible reactionbetween 3-HPA and PDO, the reverse reaction was more and moresignificant with the PDO accumulation, which resulted in the re-consumption of PDO at the later phase of fermentation [31]. Thereversibility of reaction from 3-HPA to PDO catalyzed by HORwas much lower than that catalyzed by PDOR. In our enzymeassays, the reaction from PDO to 3-HPA catalyzed by HOR wasnot detected. This phenomenon was also observed in Pérez’s study[22].

HOR has been identified to be an inherent enzyme in K. pneu-moniae by the previous proteomic study and it mainly played thefunction at the later phase of the fermentation [8]. However, the

fermentation when the HOR was not highly expressed [19]. In thisstudy, the constructed plasmid pKD6-HOR was induced at the earlyphase of the fermentation which consequently accelerated the con-version of 3-HPA to PDO. Thus, the accumulation of 3-HPA at the

. pneumoniae PD1 (unit: mmol mmol−1 glycerol).

TCA cycle (R22) Acetyl-CoA (R20) Lactate (R51) Acetate (R53) Ethanol (R50)

0.068 14.8 21.5 3.2 4.8

0.052 7.9 18.8 2.1 0.6

ineeri

erp

sflflctcHg2a

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

Z. Chen et al. / Biochemical Eng

arly phase in the constructed strain was obviously reduced, whichesulted in the risk of fermentation cessation decreasing and PDOroduction enhancing.

According to the metabolic flux analysis, in the constructedtrain, the carbon flux flowed to PPP pathway increased 21.3% andowed to PDO increased 9.4% correspondingly. Additionally, theux flowed to 2,3-butanediol increased 72.2% suggested that HORould catalyze both the reactions from 3-HPA to PDO and from ace-oin to 2,3-butanenol [22,25]. Since 2,3-butanediol is also a valuedhemical which has been received more interest, combination ofOR overexpresssion and other metabolic engineering technolo-ies could be explored for the overproduction of both PDO and,3-butanenol to make the biological fermentation process morettractive.

eferences

[1] A.P. Zeng, H. Biebl, Bulk chemicals from biotechnology: the case of 1,3-propanediol production and the new trends, Adv. Biochem. Eng. Biotechnol.74 (2002) 239–259.

[2] K.K. Cheng, D.H. Liu, Y. Sun, W.B. Liu, 1,3-propanediol production by Kleblsiellapneumoniae under different aeration strategies, Biotechnol. Lett. 26 (2004)911–915.

[3] A.P. Zeng, A. Ross, H. Hibel, C. Tag, B. Gunzel, W.D. Deckwer, Multipleproduct inhibition and growth modeling of Clostridium butyricum and Kleb-siella pneumoniae in glycerol fermentation, Biotechnol. Bioeng. 44 (1994)902–911.

[4] J. Sun, J. Heuvel, P. Soucaille, Y. Qu, A.P. Zeng, Comparative genomic analysis ofdha regulon and related genes for anaerobic glycerol metabolism in bacteria,Biotechnol. Prog. 19 (2003) 263–272.

[5] Z. Chen, H.J. Liu, D.H. Liu, Cell physiology and metabolic flux response ofKlebsiella pneumoniae to aerobic conditions, Process Biochem. 44 (2009)862–868.

[6] R.G. Forage, E.C. Lin, DHA system mediating aerobic and anaerobic dissimilationof glycerol in Klebsiella pneumoniae NCIB 418, J. Bacteriol. 151 (1982) 591–599.

[7] C. Nakamura, G. Whited, Metabolic engineering for the microbial productionof 1,3-propanediol, Curr. Opin. Biotechnol. 14 (2003) 454–459.

[8] W. Wang, J. Sun, M. Hartlep, W.D. Deckwer, A.P. Zeng, Combined use of pro-teomic analysis and enzyme activity assay for metabolic pathway analysis ofglycerol fermentation by Klebsiella pneumoniae, Biotechnol. Bioeng. 83 (2003)525–536.

[9] J.W. Seo, M.Y. Seo, B.R. Oh, S.Y. Heo, J.O. Baek, D. Rairakhwada, L.H. Luo,W.K. Hong, C.H. Kim, Identification and utilization of a 1,3-propanedioloxidoreductase isoenzyme for production of 1,3-propanediol from glyc-erol in Klebsiella pneumoniae, Appl. Genet. Mol. Biotechnol. 85 (2010)659–666.

10] S. Schuster, T.D. Andekar, D.A. Fell, Detection of elementary flux modes inbiochemical networks: a promising tool for pathway analysis and metabolicengineering, Trends Biotechnol. 172 (1999) 53–60.

11] C.H. Schilling, D. Letscher, B.O. Palsson, Theory for the systemic definition ofmetabolic pathways and their use in interpreting metabolic function from apathway-oriented perspective, J. Theor. Biol. 203 (2000) 229–248.

[

ng Journal 54 (2011) 151–157 157

12] J.O. Kromer, C. Wittmann, H. Schroder, E. Heinzle, Metabolic pathway analysisfor rational design of l-methionine, Metab. Eng. 8 (2006) 353–369.

13] R. Carlson, D. Fell, F. Srienc, Metabolic pathway analysis of a recombinant yeastfor rational strain development, Biotechnol. Bioeng. 79 (2002) 121–134.

14] N.C. Duarte, B.B. Palsson, P. Fu, Integrated analysis of metabolic phenotypes inSaccharomyces cerevisiae, BMC Genomics 5 (2004) 63.

15] J.C. Liao, M.K. Oh, Toward predicting metabolic fluxes in metabolically engi-neered strains, Metab. Eng. 1 (1999) 214–223.

16] R. Carlson, F. Srienc, Fundamental E. coli biochemical pathways for biomass andenergy production: creation of overall flux states, Biotechnol. Bioeng. 86 (2004)149–162.

17] N. Vijayasankaran, R. Carlson, F. Srienc, Metabolic pathway structures forrecombinant protein synthesis in E. coli, Appl. Microbiol. Biotechnol. 68 (2005)737–746.

18] D. Kleiner, W. Paul, M.J. Merrick, Construction of multicopy expression vectorsfor regulated over-production of proteins in Klebsiella pneumoniae and otherenteric bacteria, J. Genet. Microbiol. 134 (1988) 1779–1784.

19] Z. Chen, H.J. Liu, D.H. Liu, Regulation of 3-hydroxypropionaldehyde accumu-lation in Klebsiella pneumoniae by overexpression of dhaT and dhaD genes,Enzyme Microb. Technol. 45 (2009) 305–309.

20] H.J. Liu, D.H. Liu, J.J. Zhong, Quantitive response of Candida krusei in differentgrowth stages to hyperosmotic stress, Process Biochem. 41 (2006) 473–476.

21] S.J. Circle, L. Stone, C.S. Boruff, Acrolein determination by means of tryptophane,Ind. Eng. Chem. Anal. Ed. 17 (1945) 259–262.

22] J.M. Pérez, F.A. Arenas, G.A. Pradenas, J.M. Sandoval, C.C. Vásquez, Escherichiacoli yqhD exhibits aldehyde reductase activity and protects from the harm-ful effect of lipid peroxidation-derived aldehydes, J. Biol. Chem. 283 (2008)7346–7353.

23] K. Ahrens, K. Menzel, A.P. Zeng, W.D. Deckwer, Kinetic, dynamic, and path-way studies of glycerol metabolism by Klebsiella pneumoniae in anaerobiccontinuous culture: III. Enzymes and fluxes of glycerol dissimilation and 1,3-propanediol formation, Biotechnol. Bioeng. 59 (1998) 544–552.

24] M.M. Bradford, A rapid and sensitive method for the quantitation of micro-gram quantities of protein utilizing the principle of protein dye building, Anal.Biochem. 72 (1976) 248–254.

25] Q.R. Zhang, Z.L. Xiu, Metabolic pathway analysis of glycerol metabolism in Kleb-silla pneumonia incorporating oxygen regulatory system, Biotechnol. Prog. 25(2009) 103–115.

26] A. Kamp, S. Schuster, Metatool 5.0: fast and flexible elementary modes analysis,Bioinformatics 22 (2006) 1930–1931.

27] T. Pfeiffer, I. Sánchez-Valdenebro, J.C. Nuno, F. Montero, S. Schuster, METATOOL:for studying metabolic networks, Bioinformatics 15 (1999) 251–257.

28] X.M. Zhang, Y. Li, B. Zhuge, X.M. Tang, W. Shen, Z.M. Rao, H.Y. Fang, J.Zhuge, Optimization of 1,3-propanediol production by novel recombinantEscherichia coli using response surface methodology, J. Chem. Technol. Biotech-nol. 81 (2006) 1075–1078.

29] J. Hao, W. Wang, J.S. Tian, J.L. Li, D.H. Liu, Decrease of 3-hydroxypropionaldehyde accumulation in 1,3-propanediol productionby over-expressing dhaT gene in Klebsiella pneumoniae TUAC01, J. Ind.Microbiol. Biotechnol. 35 (2008) 735–741.

30] G. Sulzenbacher, K. Alvarez, R.H. Van Den Heuvel, C. Versluis, S. Spinelli, V.Campanacci, C. Valencia, C. Cambillau, H. Eklund, M. Tegoni, Crystal structure

of E. coli alcohol dehydrogenase yqhD: evidence of a covalently modified NADPcoenzyme, J. Mol. Biol. 10 (2004) 489–502.

31] P. Zheng, K. Wereath, J. Sun, J. van den Heuvel, A.P. Zeng, Overexpression ofgenes of the dha regulon and its effects on cell growth, glycerol fermenta-tion to 1,3-propanediol and plasmid stability in Klebsiella pneumoniae, ProcessBiochem. 41 (2006) 2160–2169.