Abstract
The biotechnological production of dicarboxylic acids (C4) from renewable carbon sources represents an attractive approach for the provision of these valuable compounds by green chemistry means. Glycerol has become a waste product of the biodiesel industry that serves as a highly reduced carbon source for some microorganisms. Escherichia coli is capable of consuming glycerol to produce succinate under anaerobic fermentation, but with the deletion of some tricarboxylic acid (TCA) cycle genes, it is also able to produce succinate and malate in aerobiosis. In this study, we investigate possible rate-limiting enzymes by overexpressing the C-feeding anaplerotic enzymes Ppc, MaeA, MaeB, and Pck in a mutant that lacks the succinate dehydrogenase (Sdh) enzyme. The overexpression of the TCA enzyme Mdh and the activation of the glyoxylate shunt was also examined. Using this unbiased approach, we found that phosphoenol pyruvate carboxylase (Ppc) overexpression enhances an oxidative pathway that leads to increasing succinate, while phosphoenol pyruvate carboxykinase (Pck) favors a more efficient reductive branch that produces mainly malate, at 57.5% of the theoretical maximum molar yield. The optimization of the culture medium revealed the importance of bicarbonate and pH in the production of malate. An additional mutation of the ppc gene highlights its central role in growth and C4 production.
Highlights
IntroductionTwo enzymes are potentially capable of driving PEP carboxylation to produce oxaloacetate (OAA); on the one hand, PEP carboxylase (Ppc) is the main component responsible for this reaction in E. coli during glucose fermentation
The Department of Energy of the United States and the European Commission have included the four-carbon (C4) dicarboxylic acids, succinate, malate, and fumarate, among the top 12 selected building blocks [1,2]
The tricarboxylic acid (TCA) cycle was split into two lineal pathways by deletion of the succinate dehydrogenase
Summary
Two enzymes are potentially capable of driving PEP carboxylation to produce oxaloacetate (OAA); on the one hand, PEP carboxylase (Ppc) is the main component responsible for this reaction in E. coli during glucose fermentation. This is, in basic terms, an irreversible reaction that wastes the high-energy phosphate of PEP [6]. Pck gene expression is repressed at high glucose concentration in Escherichia coli and is only activated to operate in a reverse direction for gluconeogenesis during the oxidative metabolism of organic acids [7,8]. The oxidative branch, which diverts OAA and Acetyl-CoA to isocitrate, operates at a low rate to produce α-ketoglutarate for NH4 assimilation [10]
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