NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1,...
-
Upload
joshua-rogers -
Category
Documents
-
view
215 -
download
0
Transcript of NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1,...
![Page 1: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/1.jpg)
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: [email protected]
![Page 2: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/2.jpg)
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.
![Page 3: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/3.jpg)
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
![Page 4: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/4.jpg)
![Page 5: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/5.jpg)
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
![Page 6: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/6.jpg)
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)
![Page 7: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/7.jpg)
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
![Page 8: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/8.jpg)
NICE
![Page 9: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/9.jpg)
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
![Page 10: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/10.jpg)
• 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)
![Page 11: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/11.jpg)
![Page 12: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/12.jpg)
Part of Folate gene cluster L. lactis cloned behind strong promoter
![Page 13: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/13.jpg)
• 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
![Page 14: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/14.jpg)
• 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)
![Page 15: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/15.jpg)
![Page 16: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/16.jpg)
High production of folate by over expression of whole fol gene cluster
![Page 17: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/17.jpg)
Folate production in engineered Lb. gasseri
![Page 18: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/18.jpg)
Folate level in the organs of animals depleted in folate and supplemented with LAB folate
![Page 19: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/19.jpg)
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)
![Page 20: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/20.jpg)
![Page 21: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/21.jpg)
• 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
![Page 22: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/22.jpg)
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
![Page 23: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/23.jpg)
• 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)
![Page 24: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/24.jpg)
![Page 25: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/25.jpg)
Activation of Sorbitol production
![Page 26: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/26.jpg)
• 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
![Page 27: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/27.jpg)
• 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.
![Page 28: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/28.jpg)
Increasing Mannitol production
![Page 29: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/29.jpg)
Effect of pH on the production of mannitol and sorbitol by
Lb. plantarum VL202
![Page 30: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/30.jpg)
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
![Page 31: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/31.jpg)
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
![Page 32: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/32.jpg)
![Page 33: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/33.jpg)
• 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
![Page 34: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/34.jpg)
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)
![Page 35: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/35.jpg)
• 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)
![Page 36: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/36.jpg)
Polysaccharide gene cluster in various LAB
![Page 37: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/37.jpg)
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
![Page 38: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/38.jpg)
• 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
![Page 39: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/39.jpg)
Galactose of Lactose being fully utilized and Glucose ends up in the product
![Page 40: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/40.jpg)
![Page 41: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/41.jpg)
• 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
![Page 42: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/42.jpg)
- Galactosides and their hydrolytic enzymes
![Page 43: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/43.jpg)
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 - galactosidase
• These undigested - galactosides accumulate in the lower gut and induce gastric problems like flatulence
![Page 44: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/44.jpg)
• 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)
![Page 45: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/45.jpg)
• 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
![Page 46: NUTRACEUTICALS: An emerging field for metabolic engineering of Lactic Acid Bacteria MALVIKA MALIK 1, RAVINDER NAGPAL 1, MONICA PUNIYA 2, ARTI BHARDWAJ.](https://reader036.fdocuments.net/reader036/viewer/2022062716/56649dc85503460f94abd9c2/html5/thumbnails/46.jpg)
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.