An emerging field for metabolic engineering of Lactic Acid...
Transcript of An emerging field for metabolic engineering of Lactic Acid...
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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]
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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.
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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
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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
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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)
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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
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NICE
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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
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• 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)
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Part of Folate gene cluster L. lactis
cloned behind strong promoter
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• 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
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• 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)
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High production of folate by over
expression of whole fol gene cluster
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Folate production in engineered
Lb. gasseri
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Folate level in the organs of animals depleted
in folate and supplemented with LAB folate
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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)
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• 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
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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
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• 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)
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Activation of Sorbitol production
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• 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
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• 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.
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Increasing Mannitol production
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Effect of pH on the production of mannitol and sorbitol
by
Lb. plantarum VL202
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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
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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
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• 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
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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)
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• 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)
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Polysaccharide gene cluster in various
LAB
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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
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• 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
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Galactose of Lactose being fully utilized and
Glucose ends up in the product
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• 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
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αααα- Galactosides and their hydrolytic
enzymes
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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
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• 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)
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• 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
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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.