Industrial biotechnology

agustin krisna wardani

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

Technique:

1. Controlled over expression of desired genes

2. 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 (Nisin Controlled gene Expression) 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 production in engineered Lb. gasseri

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. They display equivalent sweetness and taste (Dwivedi 1978)

• In colon they are fermented by micro-organisms to short-chain fatty acids (mainly butyrate) which may prevent colon cancer

• Mannitol and sorbitol have stool-bulking properties and can be used as dietary fibers

• They are active as bifidogenic prebiotic

• Cholesterol lowering , immunomodulant

• 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)

• 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

Increasing Mannitol production

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

Removal of raffinose

• Soy- 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

• 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

(Silvestroni et al. 2002)

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

• 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

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.