LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity
Article first published online: 20 DEC 2011
© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology
Letters in Applied Microbiology
Volume 54, Issue 2, pages 140–148, February 2012
How to Cite
Armando, J.W., Boghigian, B.A. and Pfeifer, B.A. (2012), LC-MS/MS quantification of short-chain acyl-CoA’s in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity. Letters in Applied Microbiology, 54: 140–148. doi: 10.1111/j.1472-765X.2011.03184.x
- Issue published online: 11 JAN 2012
- Article first published online: 20 DEC 2011
- Accepted manuscript online: 26 NOV 2011 04:48AM EST
- 2011/1621: received 23 September 2011, revised 11 November 2011 and accepted 16 November 2011
- Escherichia coli;
Aims: This paper utilized quantitative LC-MS/MS to profile the short-chain acyl-CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl-CoA molecules, a propionyl-CoA synthetase gene from Ralstonia solanacearum (prpE-RS) was synthesized and expressed in the engineered strain BAP1.
Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl- and methylmalonyl-CoA of 6- to 30-fold and 3·7- to 6·8-fold, respectively. Expression of prpE-RS resulted in no significant increases in acetyl-, butyryl- and propionyl-CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl-CoA formation.
Conclusions: The increased acyl-CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE.
Significance and Impact of the Study: The results provide direct evidence of enhanced acyl-CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl-CoA precursor supply for additional heterologous biosynthetic attempts.