Biology Reference
In-Depth Information
contributed to designing a strain in which overproduction of BDO was the method to
balance redox and enable anaerobic growth.
An effort to identify gene amplification targets was also considered a meaningful method
for strain optimization, as well as gene deletion targets. Flux scanning based on enforced
objective flux (FSEOF) was able to identify gene amplification targets by scanning all the
metabolic fluxes in the metabolic model and selecting fluxes which increase when flux
toward desired product formation was enforced as an additional constraint.
9
In other words,
gene amplification targets represent the fluxes increasing gradually from their initial value of
the wild-type strain to a value close to the maximum theoretical yield. This algorithm was
applied to overproduction of lycopene in
E. coli
.
9
Although amplification gene targets
through FSEOF algorithm contributed to increase lycopene production, final strain
optimization was further reflected by the FSEOF and MOMA simulation results. Flux
variability scanning based on enforced objective flux (FVSEOF) with grouping reaction (GR)
constraints was also used to identify gene amplification targets.
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FVSEOF scan changes in
the variabilities of metabolic fluxes in response to an artificially enforced objective flux of
product formation so that putrescine production was experimentally increased by
overexpression of each identified amplification gene.
SYNTHETIC BIOLOGY FOR SYSTEMS-LEVEL METABOLIC
ENGINEERING
Development of synthetic biology tools is driven by the applications in which these tools
are employed, ranging from the characterization of genetic circuits to the metabolic
engineering of microorganisms for production of high-value industrial compounds.
However, while synthetic biology tools are powerful in their ability to control and design
cellular functions at the local level, they are limited in determining how the changes will
affect the overall cellular phenotype. Therefore, these tools are combined with other
established tools that would expand their focus from the genetic level to the systems level
(
Fig. 8.2
). One such tool is the genome-scale metabolic models that are widely available for
various host organisms, including widely studied and employed host systems in biology
and biotechnology, such as
E. coli
,
Saccharomyces cerevisiae
, and even
Homo sapiens
.
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By
employing the genome-scale metabolic models, researchers are able to simulate the effect
synthetic biology tools have at the systems level.
152
For instance, systems metabolic engineering of microorganisms for the production of high-
value compounds would seek to achieve high productivity and yield of the target product
while maintaining a sufficiently high growth rate as well as other objectives, including but
not limited to minimal byproduct formation. Strategies employing many of the synthetic
biology tools have been investigated towards this goal. One example is the identification of
novel pathways for the production of 3-hydroxypropionate (3HP) by employing BNICE.
10
Here, all candidate pathways that were thermodynamically feasible were identified and
discussed. However, although the metabolic reactions identified may be thermodynamically
feasible, they would be irrelevant if the pathways failed to achieve high production levels of
3HP for large-scale production. To investigate this, the pathways were incorporated into a
genome-scale metabolic model of
E. coli
and the maximum theoretical yield of 3HP was
examined. Through the use of the genome-scale metabolic model, it was found that the
cofactors of the novel pathways can influence the maximum yield of 3HP (e.g. ATP and
NADH). Furthermore, other tools and methods that have been established in the systems
metabolic engineering of host organisms for the production of a target compound can be
employed to complement the introduction of the novel pathways.
10
Another example of employing systems-level strategies in conjunction with a synthetic
pathway prediction tool is the engineering of
E. coli
to produce 1,4-butanediol (14BDO).
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