Acyl-CoA quantification and the effects upon E. coli polyketide substrates through over-expression of native and Ralstonia solanacearum propionyl-CoA synthetases.
has become an increasingly important methodology for analyzing perturbations in
biological systems along with the more established proteomics and genomics tools
currently available today. The study of small molecule metabolites has been described as
"the metabolic complement of functional genomics" (Villas Boas 2005), and can provide a
snapshot of the complex phenotypic... read morestates of cellular systems. Metabolic studies have
been mainly split into two major groups, global metabolite profiling or targeted
metabolite analysis. This study utilizes targeted metabolite analysis to allow direct
quantification of small molecules of interest, which can give a snapshot of dynamic
metabolic flux and help characterize genetic modifications. In particular, the short
chain acyl-CoA class of metabolites, used as building blocks for the production of
polyketides, was studied. The acyl-CoA levels in several engineered Escherichia coli
strains constructed for improved heterologous polyketide production were quantified
using LC-MS/MS. It was observed upon feeding propionate that the engineered E. coli
strains had increases in both propionyl- and methylmalonyl-CoA of ~6- to 30-fold and
~3.7- to 6.8-fold respectively. The observed increases in acyl-CoA levels reflect the
genetic modifications designed for improved polyketide production and correlate with the
previously observed titer improvements in 6-deoxyerythronolide B (the macrolactone
precursor of the potent polyketide antibiotic erythromycin) (Zhang et al. 2010, Pfeifer
et al.2001). To further improve the levels of available acyl-CoA molecules, a flexible
propionyl-CoA synthetase gene from Ralstonia solanacearum (prpE-RS) was cloned and
expressed in the engineered strain BAP1 (Rajashekhara et al 2004, Pfeifer et al 2000).
Rajashekhara et al. demonstrated substrate flexibility of the PrpE-RS enzyme in vitro,
which should produce an increase in propionyl-, acetyl-, and butyryl-CoA when
overexpressed in Escherichia coli. Induction of the prpE-RS gene resulted in ~1.5-, 15-,
and 8.5-fold increases in acetyl-, butyryl-, and propionyl-CoA, respectively, when fed
with corresponding substrates. However when compared to the empty vector control, no
significant increases in acyl-CoA levels were observed, indicating that the substrate
flexibility observed may be a result of the native PrpE enzyme rather than the
heterologously expressed PrpE-RS enzyme. To confirm this observation, further
experiments comparing both the native and heterologous PrpE enzymes were conducted.
Additionally, the propionate transporter AtoAD was expressed with PrpE-RS resulting in a
1.44- and 1.34-fold increase in butyryl- and acetyl-CoA, but no significant increase in
propionyl-CoA. As a result, the introduction of the flexible PrpE-RS and propionate
transporter AtoAD did not significantly improve the acyl-CoA levels in Escherichia coli.
To further test the availability of alternative acyl-CoA substrates observed for
polyketide biosynthesis, attempts were made to quantify the production of 6-dEB,
acetyl-, and butyryl-6dEB analogs (14-nor-6dEB, 15-Me-6dEB), but no significant
improvements in analog production were observed. Overall, it was observed that the
native PrpE in E. coli demonstrates an intrinsic substrate flexibility resulting in
increased acetyl-, propionyl-, and butyryl-CoA levels. This study developed a platform
for acyl-CoA quantification using LC-MS/MS, helping to metabolically characterize
several engineered polyketide producing E. coli strains. The observed improvements in
acyl-CoA levels and apparent flexibility of the native PrpE enzyme provide insight into
the genetic modifications that may optimize polyketide production and help direct future
engineering approaches for polyketide producing E. coli
Thesis (M.S.)--Tufts University, 2012.
Submitted to the Dept. of Chemical and Biological Engineering.
Advisor: Blaine Pfeifer.
Committee: Kyongbum Lee, and Joshua Kritzer.
Keywords: Chemical engineering, and Biology.read less