title: LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors: Department of Chemical and Biological Engineering, Tufts University, Medford, MA, USA.
authors: J W
authors: JW
authors: Armando
authors: B A
authors: BA
authors: Boghigian
authors: B A
authors: BA
authors: Pfeifer
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism*; D002853:Chromatography, Liquid; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry; D011422:Propionates / Q000378:metabolism; D043368:Ralstonia solanacearum / Q000235:genetics; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D000214:Acyl Coenzyme A; D029968:Escherichia coli Proteins; D011422:Propionates; C015357:methylmalonyl-coenzyme A; C009061:propionyl-coenzyme A; D003066:Coenzyme A Ligases; C068072:propionate - CoA ligase