Engineering of Escherichia coli for Krebs Cycle-Dependent Production of Malic Acid

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.

Since in Escherichia coli, malate is mainly produced during anaerobic fermentation that is redox neutral, cofactor supply may become limiting in biosyntheses that employ additional reduction steps for the conversion of malate into other target molecules. To construct a platform strain in which malate production is increased, and in which sufficient NAD(P)H and ATP cofactors are produced for additional bioconversion steps, we developed a strain that produces malate via the  Krebs cycle and glyoxylate shunt. Following this strategy, the ATP and NAD(P)H output of the malate-producing pathway can be increased at the expense of a decreased maximum theoretical yield which drops to 1.33 mol/mol.

As acetate is the major metabolic by-product during aerobic growth on glucose, we engineered E. coli by inactivating both malate dehydrogenases (Dmdh Dmqo) and malic enzymes (DsfcA DmaeB), and the major acetate-producing pathway (DackA-pta). Significant malate secretion was found to depend on the expression of a malate-insensitive Ppc_K620S mutant. In baffled flask cultures with glucose, the resulting strain MG1655 Dmdh Dmqo DsfcA DmaeB DackA-pta pACT3-ppc_K620Sproduced malate, fumarate, acetate and pyruvate at molar yields of 0.48, 0.11, 0.33 and 0.28, respectively. In controlled bioreactor cultures this strain accumulated 25 g/l malate with a volumetric productivity of 0.53 g/l/h at a yield of 0.5 mol/mol.

Acetate production could not be totally prevented by DackA-pta deletion and strains deprived of the ATP/acetate-producing pathway showed retarded growth. Therefore, we chose to engineer alternative metabolic targets to increase malate production without deleting acetate pathway. Finally, we inactivated the transcriptional repressors, IclR and ArcA, and additionally overexpressed the NADH-insensitive citrate synthase mutant GltA_R164L to increase carbon flux into the TCA cycle and consequently minimized the carbon overflow through the acetate pathway. In baffled flask cultures with glucose as the carbon source, the optimized strain MG1655 Dmdh Dmqo DsfcA DmaeB DiclR DarcA pACT3-ppc_K620S –gltA_R164L produced malate, fumarate, acetate and pyruvate at molar yields of 0.8, 0.11, 0.33 and 0.28, respectively.