Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid
2018
Trichez, Débora | Auriol, Clément | Baylac, Audrey | Irague, Romain | Dressaire, Clémentine | Carnicer Heras, Marc | Heux, Stephanie | François, Jean marie | Walther, Thomas
Background: 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 coil for Krebs cycle-dependent production of malic acid that can satisfy these requirements.Results: 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 C-13-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 Delta mdh Delta mqo Delta maeAB Delta iclR Delta arcA which expressed malate-insensitive PEP carboxylase Ppc(K6)(205) and NADH-insensitive citrate synthase GltA(R1641) . We also showed that inactivation of the dicarboxylic acid transporter DcuA strongly reduced malate production arguing for a pivotal role of this permease in malate export.Conclusions: Since more NAD(P)H and ATP cofactors are generated in the Krebs cycle-dependent malate production when compared to pathways which depend on the function of anaplerotic PEP carboxylase or PEP carboxykinase enzymes, the engineered strain developed in this study can serve as a platform to increase biosynthesis of malatederived metabolites such as 2,4-dihydroxybutyric acid.
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تم تزويد هذا السجل من قبل National Institute for Agricultural Research