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.
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.
Authors: Matthew Harger; Lei Zheng; Austin Moon; Casey Ager; Ju Hye An; Chris Choe; Yi-Ling Lai; Benjamin Mo; David Zong; Matthew D Smith; Robert G Egbert; Jeremy H Mills; David Baker; Ingrid Swanson Pultz; Justin B Siegel Journal: ACS Synth Biol Date: 2012-09-26 Impact factor: 5.110
Authors: Marta Riera-Borrull; Esther Rodríguez-Gallego; Anna Hernández-Aguilera; Fedra Luciano; Rosa Ras; Elisabet Cuyàs; Jordi Camps; Antonio Segura-Carretero; Javier A Menendez; Jorge Joven; Salvador Fernández-Arroyo Journal: J Am Soc Mass Spectrom Date: 2015-09-17 Impact factor: 3.109
Authors: Stefan Marcus Gaida; Andrea Liedtke; Andreas Heinz Wilhelm Jentges; Benedikt Engels; Stefan Jennewein Journal: Microb Cell Fact Date: 2016-01-13 Impact factor: 5.328