Multi-scale engineering and modeling of heterologous natural product biosynthesis in Escherichia coli.
engineering of biological systems for industrial applications is a complex process.
Optimization of a cellular phenotype is complicated by the sheer number of genetic and
environmental variables. With regards to heterologous natural product biosynthesis, this
is further complicated by the foreign nature of the metabolic pathways and structurally
complex products involved. To ... read moreaddress this problem, heuristic and systematic approaches
were employed for the engineering of two heterologous natural products (a polyketide and
an isoprenoid) in Escherichia coli. The methods developed and applied herein are
critical to advancing the field of heterologous natural product biosynthesis to the
scale of competitive industrial bioprocesses. Stoichiometric modeling was applied to
survey heterologous hosts for supporting polyketide biosynthesis. Simulations under
different host and environmental conditions revealed multiple gene knockouts that were
capable of improving product titer. Work has shown that multiple pathways exist in
nature for producing the two precursors necessary for polyketide production; however, E.
coli does not possess these. These heterologous pathways were expressed, and with
concurrent substrate feeding experiments, their effects were analyzed on polyketide
production. Native gene over-expressions and deletions also improved polyketide titer.
Due to an inability to thoroughly search genomic space with the aforementioned
computational method, a new algorithm was developed to identify knockout targets based
on network topology and applied to isoprenoid production. By using a genetic algorithm,
this method identified a four knockout strain capable of improved titer, while reducing
computation time by several orders of magnitude. When constructed in the laboratory
using an accelerated genome evolution method, isoprenoid yield improved nearly 3-fold in
some cases. The aforementioned algorithm was reformulated in an attempt to identify
over-expression targets for improving isoprenoid titer. This method identified four
targets, three of which improved titer when implemented genetically, though failed to
meet the predicted levels of improvement. Upon over-expression of the isoprenoid
biosynthetic pathway genes, one gene improved titer to a higher extent than the
predicted targets (almost 4-fold), showing that the rate-limiting step lies within the
pathway itself. Applying heuristics for isoprenoid production, heterologous gene
promoter strength, strain background, and process-related parameters were varied and
allowed for a 240-fold improvement in titer.
Thesis (Ph.D.)--Tufts University, 2011.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Blaine Pfeifer.
Committee: Kyongbum Lee, Abraham Sonenshein, and Gregory Stephanopoulos.
Keyword: Chemical Engineering.read less
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