| Metabolomics 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 states 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 systems. |
