L-malate production by metabolically engineered Escherichia coli

Abstract

Escherichia coli strains (KJ060 and KJ073) that were previously developed for succinate production have now been modified for malate production. Many unexpected changes were observed during this investigation. The initial strategy of deleting fumarase isoenzymes was ineffective, and succinate continued to accumulate. Surprisingly, a mutation in fumarate reductase alone was sufficient to redirect carbon flow into malate even in the presence of fumarase. Further deletions were needed to inactivate malic enzymes (typically gluconeogenic) and prevent conversion to pyruvate. However, deletion of these genes (sfcA and maeB) resulted in the unexpected accumulation of D-lactate despite the prior deletion of mgsA and ldhA and the absence of apparent lactate dehydrogenase activity. Although the metabolic source of this D-lactate was not identified, lactate accumulation was increased by supplementation with pyruvate and decreased by the deletion of either pyruvate kinase gene (pykA or pykF) to reduce the supply of pyruvate. Many of the gene deletions adversely affected growth and cell yield in minimal medium under anaerobic conditions, and volumetric rates of malate production remained low. The final strain (XZ658) produced 163 mM malate, with a yield of 1.0 mol (mol glucose(-1)), half of the theoretical maximum. Using a two-stage process (aerobic cell growth and anaerobic malate production), this engineered strain produced 253 mM malate (34 g liter(-1)) within 72 h, with a higher yield (1.42 mol mol(-1)) and productivity (0.47 g liter(-1) h(-1)). This malate yield and productivity are equal to or better than those of other known biocatalysts.

FIG. 1.

Engineering of a pathway for malate production. The central pathway for succinate in K060 and K073 is portrayed, showing the inactivation of fumarase genes predicted to accumulate malate. The stoichiometry of succinate production is shown assuming excess reductant. G6P, glucose-6-phosphate.

FIG. 2.

Pathways concerned with malate metabolism and succinate production. The native fermentation pathway produces malate as an intermediate between OAA and fumarate. Fumarate is subsequently reduced to succinate. Gray arrows represent alternative routes to succinate which do not involve malate. Succinate can be produced from OAA through either an aspartate bypass (aspartate aminotransferase and aspartase) or by using the glyoxylate bypass (citrate synthase, aconitate hydratase, and isocitrate lyase). Although two genes that encode enzymes for PEP carboxylation to OAA are shown, pck was found to be responsible for 97% of this reaction. Intermediate steps in glycolysis have been omitted for clarity. Original gene deletions in the succinate-producing parent (KJ073) are marked by stars. Sites of new deletions described in this paper are also marked (×). Abbreviations: Acetyl-CoA, acetyl-coenzyme A; Acetyl-P, acetylphosphate; α-KG, α-ketoglutarate.