Download Nutraceuticals- Emerging Field of Metabolic Engineering of Lactic

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, Meerut250002, U.P., India.
*Email: [email protected]
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
Mannitol
Sorbitol
Erythritol
4.0 cal/gm
1.5 cal/gm
2.5 cal/gm
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
- 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 - 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 αgalactosidase 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.