Metabolic engineering of Escherichia coli W3110 to produce L‐malate

ABSTRACT A four‐carbon dicarboxylic acid L‐malate has recently attracted attention due to its potential applications in the fields of medicine and agriculture. In this study, Escherichia coli W3110 was engineered and optimized for L‐malate production via one‐step L‐malate synthesis pathway. First, deletion of the genes encoding lactate dehydrogenase ( ldhA ), pyruvate oxidase ( poxB ), pyruvate formate lyase ( pflB ), phosphotransacetylase ( pta ), and acetate kinase A ( ackA ) in pta‐ackA pathway led to accumulate 20.9 g/L pyruvate. Then, overexpression of NADP + ‐dependent malic enzyme C490S mutant in this multi‐deletion mutant resulted in the direct conversion of pyruvate into L‐malate (3.62 g/L). Next, deletion of the genes responsible for succinate biosynthesis further enhanced L‐malate production up to 7.78 g/L. Finally, L‐malate production was elevated to 21.65 g/L with the L‐malate yield to 0.36 g/g in a 5 L bioreactor by overexpressing the pos5 gene encoding NADH kinase in the engineered E. coli F0931 strain. This study demonstrates the potential utility of one‐step pathway for efficient L‐malate production. Biotechnol. Bioeng. 2017;114: 656–664. © 2016 Wiley Periodicals, Inc.