Acetyl coenzyme A (acetyl-CoA) is an important molecule of central metabolism because more acetyl-CoA is withdrawn from central metabolism than any other biochemical intermediate on a molar basis, and because many industrially relevant products are derived from acetyl-CoA. We examine the hypothesis that modifying citrate synthase, encoded by gene gltA in Escherichia coli, will limit acetyl-CoA flux into the tricarboxylic acid cycle and lead to increased acetyl-CoA availability for the generation of products derived from this metabolite. A colorimetric method was developed to screen a library of random gltA mutants for increased acetate production in E. coli C ΔpoxB ΔldhA Δacs grown on glucose as the sole carbon source. We generated multiple gltA variant strains with elevated acetate generation compared to the wild-type gltA background. A strong negative correlation was observed between growth rate and acetate formation. We also report on the development of a GFP-based reporter for increased acetyl-CoA availability. These results are discussed in the context of the synthesis of several biochemicals.

Modifying citrate synthase activity to increase acetyl-CoA availability in Escherichia coli

ACS Fall 2021

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