Biology Reference
In-Depth Information
E. coli
by removing the genes responsible for beta-oxidation of free fatty acids that had
accumulated as a result of their earlier interventions. This provided a three- to four-fold
increase in fatty acid titer.
65
A caveat for gene deletion attempts is that cellular metabolism can be highly robust to gene
deletions, compensating for knockouts by increasing flux to a metabolite through other
pathways that may affect the biofuel production pathway.
66
Another caveat is that the
deletion of competing pathways may paradoxically decrease the final titer of the desired
product, possibly indicating that the competing pathways may in fact generate energy or
cofactors that are required for cell growth. Deleting pathways necessary for the generation of
an essential metabolite will result in auxotrophy. In this case, rather than deleting the
competing pathway, it may be better to decrease the expression of the pathway.
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One
can imagine a compromise between attenuation and complete elimination of a
competing but necessary pathway, such as engineering competing pathways to reduce
flux or shut off after the growth phase is completed, enabling precursors to flow
entirely into biofuel production. This principle was demonstrated by down-regulating
the first gene in the biosynthesis pathway for ergosterol in a strain of yeast producing
artisemic acid. Ergosterol is required for survival, and decreasing production without
completely eliminating it minimized flux into this competing pathway leading to an
increase in artisemic acid titers.
68,69
INCREASING PRECURSOR CONCENTRATION BY UP-REGULATING
INPUT PATHWAYS
Along with deleting competing pathways, the availability of precursor metabolites
can be increased by up-regulating the native pathways that produce them. Higher levels
of 1-butanol were engineered by overexpressing native
E. coli
genes
ilvA
and
leuABCD
,
increasing the pool of 2-ketobutyrate and directing it towards the synthesis of
2-ketovalerate, precursors of 1-butanol.
29
This method also uncouples enzyme expression
from native transcriptional negative feedback, as highlighted in a production improvement
of 2-methyl-1-butanol.
70
However, overexpressing native genes may not increase flux as
much as desired due to allosteric regulation of the enzymes themselves, as discussed above.
Furthermore, if a pathway enzyme is inhibited by its own substrate, increasing precursor
pools may not increase the flux as quickly as without substrate inhibition.
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218
IN SILICO MODELING TO FIND DELETION AND UP-REGULATION CANDIDATES
Because it can be extremely difficult to predict the effect of knockouts on metabolism, and
even more difficult to experimentally screen a large library of knockout mutants for
production increases, in silico modeling of cellular metabolism can suggest unintuitive
candidates for gene deletion that may increase titers. This approach has been used to
improve the production of succinate in
E. coli
.
72
In silico methods can also be used to not
only pick gene deletion candidates, but also native gene candidates for up-regulation. Choi
et al. used an in silico model to predict gene candidates for overexpression that would result
in an increased titer of the isoprenoid lycopene.
73
Impressively, most of the gene
overexpression candidates selected resulted in increased yield. Combining several of the
overexpression candidates into a single strain resulted in a yield increase of approximately
five-fold over the wild-type strain. Gene deletion candidates were also chosen from an in
silico model and combined with the amplification targets, resulting in further increases in
yield. The authors acknowledge that not all suggested targets resulted in increased yield,
potentially due to poorly-understood feedback regulation mechanisms. As our knowledge of
post-translational regulation of native pathways grows and becomes incorporated into in
silico models, their predictions will likely improve.
