Abstract
BackgroundMalate 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.ResultsWe 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 for a pivotal role of this permease in malate export.ConclusionsSince 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 malate-derived metabolites such as 2,4-dihydroxybutyric acid.
Highlights
Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry
We show that significant malate production through the Krebs cycle could not be achieved by solely deleting all malate dehydrogenases and malic enzymes, but required the over-expression of a malate-insensitive PEP carboxylase mutant
It was previously demonstrated that the aerobic production of the Krebs cycle intermediates succinate and fumarate could be improved by increasing expression of the anaplerotic enzymes phosphoenolpyruvate (PEP) carboxylase (Ppc) [23, 24], or pyruvate carboxylase [25]
Summary
Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. The four-carbon dicarboxylic acid malate is a Krebs cycle intermediate and a compound of considerable economic interest. It is mainly used as an acidulant in the food and beverage industry, or as a precursor for specialty chemicals [1, 2]. The filamentous fungus Aspergillus flavus was the first identified natural malate-producing organism [4]. During the search for alternative natural malate-producing organisms, the yeast Zygosaccharomyces rouxii and the filamentous fungi Aspergillus oryzae and Penicillium
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