Download Food Biotechnology Dr. Tarek Elbashiti

Food Biotechnology
Dr. Tarek Elbashiti
5. Metabolic Engineering of Bacteria
for Food Ingredients
• Metabolic engineering has been defined as the
directed improvement of product formation or
cellular properties through the modification of
specific biochemical reactions or the introduction
of new ones with the use of recombinant DNA
technology.
• ME can rationally improve the properties of the
microorganisms and their efficiency for producing
different products.
• This illustrates the tremendous potential of ME for
improving the bacteria used by the food industry.
• Food additives produced by bacteria can be
incorporated into foods as nutritional supplements,
flavor enhancers, texturizers, acidulants,
preservatives, emulsifiers, surfactants, thickeners,
or functional food ingredients.
• Applying ME principles and tools to the
production of food ingredients by bacteria has
resulted in the efficient production of both native
and totally novel products by several cultures,
including strains of lactic acid bacteria (LAB),
Escherichia coli, Bacillus subtilis, and
Corynebacterium glutamicum.
• The examples in this chapter (summarized in
Table 5.2) will cover the analysis and
modification of central metabolic pathways,
biosynthetic pathways, and transport systems
involved in producing amino acids, organic
acids, vitamins, carbohydrates, bacteriocins, low
calorie sugars, and aroma compounds.
AMINO ACIDS
• Amino acids have a great variety of current and
potential uses as food, pharmaceuticals, and
animal feed.
• Their main application field is food, where about
50% of the product is applied.
• They can be used as nutritional supplements,
flavor enhancers, sweeteners, and in pre and
postoperative nutrition therapy.
• This section will discuss using ME to produce
some of these amino acids.
• Recent progress in this field can be divided
into three categories:
(1) modification of central metabolic pathways,
(2) modification of biosynthetic pathways, and
(3) modification of transport systems.
• Figures 5.1, 5.2, and 5.3 summarize some of
the examples discussed below.
Modification of Central Metabolic
Pathways
• From reasoning based on metabolic pathways
structure, rerouting a carbon source to produce
a desired amino acid should start by increasing
the availability of precursor metabolites, energy,
and reducing equivalents used in its synthesis.
• Central metabolic pathways meet these criteria,
and therefore engineering central metabolism is
essential for the efficient production of amino
acids.
• The analytical tools included in the ME toolbox
have played an essential role in elucidating the
function of different central pathways and
suggesting useful strategies for redirecting carbon
flow toward the biosynthesis of amino acids.
• For example, it has been shown that the pentose
phosphate pathway (PPP) supports higher fluxes
during the production of L-lysine compared to the
production of L-glutamic acid in C. glutamicum.
• This was attributed to the higher requirements of
reducing power (NADPH) in the production of Llysine.
• Another example is improving aromatic amino
acids and L-histidine production in C. glutamicum
by increasing the availability of their precursor
metabolites, erythrose 4-phosphate and ribose 5phosphate, respectively, as well as NADPH.
• This can be done by modifying the flux through
the PPP, either by increasing the activity of
transketolase (and providing more erythrose 4phosphate for aromatic amino acids
biosynthesis) or by decreasing the activity of
transketolase (and providing more ribose 5phosphate for L-histidine biosynthesis) as shown
in Figure 5.1, strategy 1.
• Both approaches have produced C. glutamicum
strains with an increased capacity for making
aromatic amino acids and L-histidine.
• Figure 5.1 shows additional examples of
engineering central metabolic pathways to
increase the availability of precursor metabolites
used to synthesize aromatic amino acids in C.
glutamicum.
• In general, these strategies are based on
increasing the availability of erythrose 4phosphate (strategy 1, Figure 5.1),
phosphoenolpyruvate (strategies 2, 3, and
4, Figure 5.1), and ribose-5-P (strategy 1
Figure 5.1) by inactivating the enzymes
involved in their consumption and/or
amplifying the enzymes involved in their
production.
Modification of Biosynthetic
Pathways
• After engineering central metabolic pathways, a
sufficient supply of precursor metabolites, energy,
and reducing power is ensured, and efforts then need
to be focused on engineering biosynthetic pathways
that convert precursor metabolites into amino acids.
• Several strategies have been used to achieve this
goal, and some of them are illustrated in Figure 5.2.
• For example, the gene that encodes a rate-limiting
enzyme can be amplified, resulting in the release of a
bottleneck.
• Ozaki et al. and Ikeda et al. used this strategy to
improve the production of L-phenylalanine in C.
glutamicum.
• Overexpression of the gene that encodes
chorismate mutase in C. glutamicum K38 resulted
in a 50% increase in the yield of L-phenylalanine.
• On the other hand, introducing heterologous
enzymes subject to different regulatory
mechanisms can also result in the release of a
bottleneck.
• For example, overexpressing a mutated
(insensitive to L-phenylalanine) E. coli gene that
encoded the bifunctional enzyme chorismate
mutase–prephenate dehydratase in C.
glutamicum KY10694 led to a 35% increase in the
production of L-phenylalanine.
• In addition, expressing the E. coli catabolic
threonine dehydratase (insensitive to L-isoleucine)
in C. glutamicum resulted in increased isoleucine
production.
• A second strategy could be the redirection of
metabolic flux in a branch point.
• Ikeda and Katsumata engineered a tryptophanproducing mutant of C. glutamicum to produce
L-tyrosine or L-phenylalanine in abundance (26
and 28 g/L, respectively) by overexpressing the
branch-point enzymes (chorismate mutase and
prephenate dehydratase), catalyzing the
conversion of the common intermediate
chorismate into tyrosine and phenylalanine.
• Using a similar approach, Katsumata et al.
produced threonine using a lysine-producing C.
glutamicum strain, by amplifying a threonine
biosynthetic operon.
• Other strategies could include introducing
heterologous enzymes that use different
cofactors than those used by the native enzyme
as well as amplifying the enzyme that catalyzes
the steps linking central metabolism and the
biosynthetic pathway.
• Ikeda et al. achieved a 61% increase in
tryptophan yield (50 g/L of tryptophan) in a
tryptophan-producing C. glutamicum KY10894
by coexpressing two enzymes catalyzing the
initial steps in the biosynthesis of aromatic
amino acids (3-deoxy-D-arabinoheptulosonate
7-phosphate synthase) and serine (3phosphoglycerate dehydrogenase) together with
tryptophan biosynthetic enzymes.
Modification of Transport Systems:
• By altering amino acid transport systems, one
could expect to decrease their intracellular
concentration and avoid feedback inhibition.
• The following two examples illustrate that show
the significance of this strategy.
• During the production of L-tryptophan by C.
glutamicum, the accumulation of this product in
the extracellular medium resulted in a backflow
into the cells, which produced severe feedback
inhibition in the biosynthesis of tryptophan.
• Ikeda and Katsumata solved this problem by
creating mutants with lower levels of tryptophan
uptake, which resulted in an accumulation of 10
to 20% more tryptophan than in their parent.
• Another example of transport engineering is the
increase in the fermentation yield of cysteine by
overexpressing multidrug efflux genes (mar
genes) in a cysteine-producing strain of E. coli.