Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid

Microbial Cell Factories
Debora TrichezThomas Walther

Abstract

Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using 13C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δmqo ΔmaeAB ΔiclR ΔarcA which expressed malate-insensitive PEP carboxylase PpcK620S and NADH-insensitive citrate synthase GltAR164L. We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing...Continue Reading

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Citations

Jun 25, 2019·Journal of Cellular Biochemistry·Muhammad S NadeemAbdul R Shakoori
Apr 17, 2020·Biotechnology Journal·Ying ZhuChun Li
Aug 20, 2020·Microbial Cell Factories·Youming JiangMin Jiang
Aug 11, 2019·Scientific Reports·Cláudio J R FrazãoJean Marie François
Aug 2, 2021·Metabolic Engineering·Ji Yeon LeeGyoo Yeol Jung

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Methods Mentioned

BETA
PCR
genetic modifications
phosphotransferase

Software Mentioned

Influx
IsoCor

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