(623bm) Effect of Propionyl-CoA Synthetase From Ralstonia Solanacearum by LC-MS/MS Quantification of Acyl-CoA Thioesters In Escherichia Coli

Metabolomics has become an increasingly important methodology for analyzing perturbations in cellular systems along with the more established proteomics and genomics tools currently available today. More specifically, targeted metabolite analysis allows direct quantification of small molecules of interest, and can give a snapshot of dynamic metabolic flux. This paper utilized quantitative LC-MS/MS to profile the short chain acyl-CoA levels of several engineered Escherichia coli constructed for heterologous polyketide production. It was observed upon feeding propionate, 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. The observed increases in acyl-CoA levels reflect the genetic modifications utilized for improved polyketide production. To further improve the levels of available acyl-CoA molecules, a flexible propionyl-CoA synthetase gene from Ralstonia solanacearum (prpE-RS) was subcloned and expressed in the engineered strain BAP1 (Watanabe et al 2004, Pfeifer et al 2000)). Watanabe et al. demonstrated substrate flexibility of prpE-RS in vitro, which should produce an increase in propionyl-, acetyl-, and butyryl-CoA when expressed in Escherichia coli. Induction of prpE-RS resulted in ~1.5, 15, and 8.5 fold increases in acetyl-, butyryl-, and propionyl-CoA respectively, when fed with the corresponding substrate. 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 T7 controlled PrpE rather than the heterologously expressed PrpE-RS. 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. No significant increase in propionyl-CoA was observed when ATOAD was expressed with PrpE-RS.  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, but it was observed that the native prpE overexpressed in our engineered strains may have an intrinsic substrate flexibility resulting in increased acetyl-, propionyl-, and butyryl-CoA levels upon over-expression providing a more robust platform for polyketide production.