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.

Enhancement of malate production through engineering of the periplasmic rTCA pathway in Escherichia coli

“…The OD 600 was measured using a spectrophotometer. Glucose analysis was quantified by the SBA‐90E biological sensor (Dong et al, ). Pyruvate, PEP, oxaloacetate, malate, fumarate, α‐ketoglutarate, lactate, acetate, and succinate levels were determined by high‐performance liquid chromatography using an Atlantis dC18 column at a flow rate of 0.6 ml/min (Dong et al, ).…”

“…The OD 600 was measured using a spectrophotometer. Glucose analysis was quantified by the SBA-90E biological sensor (Dong et al, 2017).…”

“…Pyruvate, PEP, oxaloacetate, malate, fumarate, α-ketoglutarate, lactate, acetate, and succinate levels were determined by high-performance liquid chromatography using an Atlantis dC18 column at a flow rate of 0.6 ml/min (Dong et al, 2017).…”

“…Recently, Escherichia coli has proven to be an excellent biocatalyst platform for metabolic engineering (Gupta, Reizman, Reisch, & Prather, ; Long, Au, Sandoval, Gebreselassie, & Antoniewicz, ), and much of the work focused on improving malate production has sought to redirect carbon flow into malate. However, as the malate biosynthesis pathway is located in the cytoplasm, these studies have focused on cytoplasmic engineering, including of the one‐step synthesis pathway (Dong et al, ), rTCA pathway (Moon, Hong, Kim, & Lee, ; Zhang, Wang, Shanmugam, & Ingram, ), and noncyclic glyoxylate pathway (Gao et al, ). Thus, one of the challenges in metabolic engineering for the production of malate involves enhancing the transmission efficiency of intermediate metabolites in metabolic or synthetic pathways by limiting metabolic crosstalk.…”

Engineered chromosome-based T7 RNA polymerase in Escherichia coli W3110 for orthogonal T7 promoter circuit as a cell factory

“…A comparative proteomics study between BL21 and W3110 manifested that W3110 maintained growth and metabolism at lower oxygen levels, thus enabling foreign protein to be gradually expressed [23]. Therefore, W3110 strain has also been applied to produce various chemicals, including L-methionine [24][25], L-homoserine [26], and L-malate [27].…”