NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria

MALVIKA MALIK1, RAVINDER NAGPAL1, MONICA PUNIYA2, ARTI BHARDWAJ3, SHALINI JAIN4 and HARIOM YADAV4*

1Dairy Microbiology, 2Dairy Cattle Nutrition, 4Animal Biochemistry,

National Dairy Research Institute, Karnal 132001,

Haryana, Meerut Institute of Engineering and Technology, Meerut-250002, U.P., India.

*Email: yadavhariom@gmail.com

Nutraceuticals

• The term ‘Nutraceuticals’, launched by

Stephen De-Felici in the 1980s

• A food or part of a food that may provide

medicinal or health benefits, including the

prevention and treatment of disease.

Metabolic Engineering

�Metabolic engineering is the practice of

optimizing genetic and regulatory

processes within cells to increase the cells'

production of a certain substance

� Controlled over expression of desired

genes

�Inactivation of undesired genes

Examples of metabolic

engineering of LAB

• Increased production of diacetyl from

glucose and lactose

• Efficient production of L-alanine from sugar

• Production of non-metabolisable sugars

• Galactose and/or lactose removal from dairy

products

• Oligosaccharide production

• Vitamin production

Lactic acid bacteria as cell-factories

• Lactic acid bacteria (LAB) are industrially important microbes, used in a large variety of food fermentations

• The NICE system for controlled heterologous and homologous gene expression in Lactic acid bacteria has been employed in many of the metabolic engineering strategies

(Boels et al. 2001; Sybesma et al. 2002)

Why Lactic acid bacteria?

• The bacterium is food grade

• Plasmid selection mechanisms are available that are food

grade and self cloning

• No endotoxins or inclusion bodies are formed and

• Sophisticated genetic tools enable easy genetic handling

• Simple, non-aerated fermentation makes direct scale-up

from 1-L scale to 1000-L scale possible

• Nisin controlled gene expression can be effectively used

NICE

Increased Vitamins Production

• Folate

– Involved in biosynthesis of nucleotides

– Daily recommended intake for an adult is 200 µg

– Known to prevent neural-tube defect in infants

– Protect against some forms of cancer

• Main sources are vegetables and dairy products

• Milk is good source, fermented dairy products

like yoghurt are also important

• Streptococcus thermophilus and Lactococcus lactis execute de novo biosynthesis of folates to secrete surplus folate

• Therefore can be used to make starter with increased folate levels

• In experimental yoghurt up to 150 µg/L folate has been reported

(Smid etal. 2001)

Part of Folate gene cluster L. lactis

cloned behind strong promoter

• The genes involved in folate biosynthesis have been

analysed completely.

• By genetic eng. several of these genes have been over

expressed in L.lactisNZ9000 using the NICE system

• Individual gene can be over expressed or in

combination

• Folate normally synthesis as polyglutamyl-folate

derivatives intracellularly

• Absorbed in human guts as monoglutamyl folate

derivatives

• γ -glutamyl hydrolase cDNA introduced in L.

lactis

• Resulted in an inversion of folate spatial

distribution

(Sybesma et al. 2002)

High production of folate by over

expression of whole fol gene cluster

Folate production in engineered

Lb. gasseri

Folate level in the organs of animals depleted

in folate and supplemented with LAB folate

Riboflavin (B2)

• Riboflavin-deficiency can lead to:-

– Liver(Ross & Klein 1990) and skin-disorders

(Lakshimi 1998)

– Disturbed metabolism of the red blood cells

(Hassan & Thurnham 1977)

– Reduced performance during physical exercise

(Belko et al. 1983; Bates 1987)

• In Bacillus subtilis first reaction in riboflavin

biosynthesis has been demonstrated to be

rate limiting (Humbelin et al. 1999)

• The gene coding for this enzyme, ribA, has

been brought to overexpression in L. lactis

using the NICE-system

• This resulted in a 3-fold overproduction of

riboflavin

Production of non-metabolisable sugars

• Mannitol and sorbitol (polyols) and trehalose could

replace sucrose, lactose, glucose or fructose in

food products

• In colon they are fermented by micro-organisms to

short-chain fatty acids (mainly butyrate) which may

prevent colon cancer

• Trehalose is therapeutic against illnesses, such as

the Creutzfeld-Jakob disease

• Mannitol and sorbitol have stool-bulking

properties and can be used as dietary fibers

• They are active as bifidogenic prebiotic

• Cholesterol lowering , immunomodulant

• They display equivalent sweetness and taste

(Dwivedi 1978)

• Mannitol can also serve as anti-oxidant in

biological cells

(Shen et al. 1997)

Activation of Sorbitol production

• Heterofermentative lactic acid bacteria such as

Leuconostoc mesenteroides are known to

produce mannitol in the fermentation of fructose

(Soetaert et al. 1995)

• homofermentative lactic acid bacteria can also

produce mannitol

• In both Lactobacillus plantarum (Ferain et al.

1996) and Lactococus lactis (Neves et al. 2000),

disruption of lactate dehydrogenase (LDH)

resulted in production mannitol along with other

metabolites

• Overproduction of the mannitol-P dehydrogenase (MPDH) in a LDH-deficient L. lactis strain has resulted in strong increase in intracellular mannitol production

• Similar results were obtained when MPDH was overproduced in a strain with decreased phosphofructokinase (PFK) activity

• Production of mannitol by Lactococcus lactis can be increased if excretion of this polyol is facilitated, by introducing the mannitol-transporter present in Leuconostoc mesenteroides.

Increasing Mannitol production

Effect of pH on the production of mannitol and sorbitol

by

Lb. plantarum VL202

Tagatose production

• A potential sucrose replacement.

• Higher sweetening power than similar components

such as mannitol, sorbitol and erythritol

• Much lower caloric value

(Zehner 1988)

• Recently been launched on the food market as low

calorie sugar, as prebiotic

Calorific values of different

sugars

• Glucose 4.0 cal/gm

• Mannitol 1.5 cal/gm

• Sorbitol 2.5 cal/gm

• Erythritol 0.2 cal/gm

• Chosen strategy is to disrupt the lacC and/or lacD genes resulting in production of either tagatose-6-P or tagatose-1,6-diphosphate

• Disruption of lacD was accomplished via a two step procedure

– recombination process, involving integration of an erythromycin-resistance plasmid containing only the lacC and lacF genes via single crossing-over

– removal of lacD (or reversion to the wild-type) in a second, spontaneous, recombination event

Production of polysaccharides

• Exopolysaccharides (EPS)

– Some polysaccharides produced by lactic

acid bacteria have prebiotic

(Gibson & Roberfroid 1995)

– Immunostimulatory

(Hosono et al. 1997)

– Antitumoral

(Kitazawa et al. 1991)

– Cholesterol-lowering activity

(Nakajima et al. 1992a)

• The specific eps genes are encoded on

large plasmids

• Conjugally transferred from one

lactococcal strain to the next, thereby

introducing the EPS-producing capacity in

the recipient strain

( van Kranenburg et al. 1997)

Polysaccharide gene cluster in various

LAB

Improving sugar conversion

• In cow’s milk 4–4.5% (w/v) of lactose

present

• In liquid fermented dairy products, such

as yoghurt or buttermilk, usually less than

half is fermented to lactic acid

• These products are unsuitable for lactose

intolerant persons

• The lactose is converted to galactose and

later to galactitol

• For most lactic acid bacteria, galactose is a

poor substrate

• The efficiency lactose utilization by L.lactis

can be increased by metabolic engineering

• Secondly lactose metabolism in L. lactis can

be modified in such a way that the glucose

moiety will end up in the product, while

galactose will be fully used for growth, in this

way providing a natural sweetening process

for dairy products

Galactose of Lactose being fully utilized and

Glucose ends up in the product

• Free galactose is accumulated intracellularly as a

result of the absence of galactokinase activity in

these strains

• Streptococcus thermophilus, gene for

galactokinase is completely intact, but that one or

more point mutations have taken place leading to a

‘silent’ phenotype (Vaughan et al. 2001).

• Sometimes these mutations may revert back

spontaneously

• To enhance the galactose utilization these

mutations can be reverted deliberately

αααα- Galactosides and their hydrolytic

enzymes

Removal of raffinose

• Soy- and pulse-derived food products contain

high levels of α-galactosides such as stachyose

and raffinose

• These are not metabolized in human gut due to

lack of α- galactosidase

• These undigested α- galactosides accumulate in

the lower gut and induce gastric problems like

flatulence

• By applying metabolic engineering strategies, lactic

acid bacteria can be constructed with high α-

galactosidase activities

• Starters for removal of α-galactosides during soy

fermentation

• Possible probiotics to deliver α-galactosidase

activity in the gut for prevention of flatulence

• In Lactobacillus plantarum gene (melA) code for

α-galactosidase

(Silvestroni et al. 2002)

• For construction of starter and probiotic bacteria

with high α-galactosidase activity, the melA is

cloned in L. lactis in three different constructions

resulting in

– expression of the enzyme in the cytoplasm for

maximum protection of enzyme activity

– expression as a secreted enzyme for maximum

exposure to the sugar substrate

– expression on the surface but anchored to the

surface of the cell

Conclusion

• Metabolic engineering has provided a powerful

and effective tool for production of nutraceuticals

• Metabolic engineering approach can also be

applied for production of more benificial product.

• With increasing knowledge of the genomic

analysis metabolic engineering can further be

explored for more nutraceutical production.