an emerging field for metabolic engineering of lactic acid bacteria · 2010-08-23 ·...

NUTRACEUTICALS:An emerging field for metabolic

engineering of Lactic Acid BacteriaMALVIKA 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 ofoptimizing genetic and regulatoryprocesses within cells to increase the cells'production of a certain substance

Controlled over expression of desiredgenes

Inactivation of undesired genes

Examples of metabolicengineering of LAB

• Increased production of diacetyl fromglucose 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 industriallyimportant microbes, used in a large variety offood fermentations

• The NICE system for controlled heterologousand homologous gene expression in Lacticacid bacteria has been employed in many ofthe 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 foodgrade 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-upfrom 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 andLactococcus lactis execute de novobiosynthesis of folates to secrete surplusfolate

• Therefore can be used to make starter withincreased folate levels

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

(Smid etal. 2001)

Part of Folate gene cluster L. lactiscloned 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-folatederivatives intracellularly

• Absorbed in human guts as monoglutamyl folatederivatives

• -glutamyl hydrolase cDNA introduced in L.lactis

• Resulted in an inversion of folate spatialdistribution

(Sybesma et al. 2002)

High production of folate by overexpression of whole fol gene cluster

Folate production in engineeredLb. gasseri

Folate level in the organs of animals depletedin 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 riboflavinbiosynthesis has been demonstrated to berate limiting

(Humbelin et al. 1999)

• The gene coding for this enzyme, ribA, hasbeen brought to overexpression in L. lactisusing the NICE-system

• This resulted in a 3-fold overproduction ofriboflavin

Production of non-metabolisable sugars

• Mannitol and sorbitol (polyols) and trehalose couldreplace sucrose, lactose, glucose or fructose infood products

• In colon they are fermented by micro-organisms toshort-chain fatty acids (mainly butyrate) which mayprevent colon cancer

• Trehalose is therapeutic against illnesses, such asthe Creutzfeld-Jakob disease

• Mannitol and sorbitol have stool-bulkingproperties 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 inbiological cells

(Shen et al. 1997)

Activation of Sorbitol production

• Heterofermentative lactic acid bacteria such asLeuconostoc mesenteroides are known toproduce mannitol in the fermentation of fructose

(Soetaert et al. 1995)

• homofermentative lactic acid bacteria can alsoproduce 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 othermetabolites

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

• Similar results were obtained when MPDH wasoverproduced in a strain with decreasedphosphofructokinase (PFK) activity

• Production of mannitol by Lactococcus lactis canbe increased if excretion of this polyol is facilitated,by introducing the mannitol-transporter present inLeuconostoc mesenteroides.

Increasing Mannitol production

Effect of pH on the production of mannitol and sorbitolby

Lb. plantarum VL202

Tagatose production

• A potential sucrose replacement.

• Higher sweetening power than similar componentssuch as mannitol, sorbitol and erythritol

• Much lower caloric value

(Zehner 1988)

• Recently been launched on the food market as lowcalorie sugar, as prebiotic

Calorific values of differentsugars

• 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/orlacD genes resulting in production of eithertagatose-6-P or tagatose-1,6-diphosphate

• Disruption of lacD was accomplished via a twostep procedure

– recombination process, involving integration of anerythromycin-resistance plasmid containing only thelacC and lacF genes via single crossing-over

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

Production of polysaccharides

• Exopolysaccharides (EPS)

– Some polysaccharides produced by lacticacid 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 onlarge plasmids

• Conjugally transferred from onelactococcal strain to the next, therebyintroducing the EPS-producing capacity inthe recipient strain

( van Kranenburg et al. 1997)

Polysaccharide gene cluster in variousLAB

Improving sugar conversion

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

• In liquid fermented dairy products, suchas yoghurt or buttermilk, usually less thanhalf is fermented to lactic acid

• These products are unsuitable for lactoseintolerant persons

• The lactose is converted to galactose andlater to galactitol

• For most lactic acid bacteria, galactose is apoor substrate

• The efficiency lactose utilization by L.lactiscan be increased by metabolic engineering

• Secondly lactose metabolism in L. lactis canbe modified in such a way that the glucosemoiety will end up in the product, whilegalactose will be fully used for growth, in thisway providing a natural sweetening processfor dairy products

Galactose of Lactose being fully utilized andGlucose ends up in the product

• Free galactose is accumulated intracellularly as aresult of the absence of galactokinase activity inthese strains

• Streptococcus thermophilus, gene forgalactokinase is completely intact, but that one ormore point mutations have taken place leading to a‘silent’ phenotype (Vaughan et al. 2001).

• Sometimes these mutations may revert backspontaneously

• To enhance the galactose utilization thesemutations can be reverted deliberately

- Galactosides and their hydrolyticenzymes

Removal of raffinose

• Soy- and pulse-derived food products containhigh levels of -galactosides such as stachyoseand raffinose

• These are not metabolized in human gut due tolack of - galactosidase

• These undigested - galactosides accumulate inthe lower gut and induce gastric problems likeflatulence

• By applying metabolic engineering strategies, lacticacid bacteria can be constructed with high -galactosidase activities

• Starters for removal of -galactosides during soyfermentation

• Possible probiotics to deliver -galactosidaseactivity in the gut for prevention of flatulence

• In Lactobacillus plantarum gene (melA) code for-galactosidase

(Silvestroni et al. 2002)

• For construction of starter and probiotic bacteriawith high -galactosidase activity, the melA iscloned in L. lactis in three different constructionsresulting in

– expression of the enzyme in the cytoplasm formaximum protection of enzyme activity

– expression as a secreted enzyme for maximumexposure to the sugar substrate

– expression on the surface but anchored to thesurface of the cell

Conclusion• Metabolic engineering has provided a powerful

and effective tool for production of nutraceuticals

• Metabolic engineering approach can also beapplied for production of more benificial product.

• With increasing knowledge of the genomicanalysis metabolic engineering can further beexplored for more nutraceutical production.