Pyruvate Production by Escherichia coli by Use of Pyruvate Dehydrogenase Variants

Abstract

Altering metabolic flux at a key branch point in metabolism has commonly been accomplished through gene knockouts or by modulating gene expression. An alternative approach to direct metabolic flux preferentially toward a product is decreasing the activity of a key enzyme through protein engineering. In Escherichia coli, pyruvate can accumulate from glucose when carbon flux through the pyruvate dehydrogenase complex is suppressed. Based on this principle, 16 chromosomally expressed AceE variants were constructed in E. coli C and compared for growth rate and pyruvate accumulation using glucose as the sole carbon source. To prevent conversion of pyruvate to other products, the strains also contained deletions in two nonessential pathways: lactate dehydrogenase (ldhA) and pyruvate oxidase (poxB). The effect of deleting phosphoenolpyruvate synthase (ppsA) on pyruvate assimilation was also examined. The best pyruvate-accumulating strains were examined in controlled batch and continuous processes. In a nitrogen-limited chemostat process at steady-state growth rates of 0.15 to 0.28 h^-1, an engineered strain expressing the AceE[H106V] variant accumulated pyruvate at a yield of 0.59 to 0.66 g pyruvate/g glucose with a specific productivity of 0.78 to 0.92 g pyruvate/g cells·h. These results provide proof of concept that pyruvate dehydrogenase complex variants can effectively shift carbon flux away from central carbon metabolism to allow pyruvate accumulation. This approach can potentially be applied to other key enzymes in metabolism to direct carbon toward a biochemical product. IMPORTANCE Microbial production of biochemicals from renewable resources has become an efficient and cost-effective alternative to traditional chemical synthesis methods. Metabolic engineering tools are important for optimizing a process to perform at an economically feasible level. This study describes an additional tool to modify central metabolism and direct metabolic flux to a product. We have shown that variants of the pyruvate dehydrogenase complex can direct metabolic flux away from cell growth to increase pyruvate production in Escherichia coli. This approach could be paired with existing strategies to optimize metabolism and create industrially relevant and economically feasible processes.

Keywords: batch; chemostat; fermentation; point mutation; pyruvic acid.

FIG 1

Comparison of E. coli ldhA poxB AceE variants grown in shake flasks with 5 g/liter glucose: specific growth rate (gray bars) and pyruvate yield (black bars). WT AceE, wild-type AceE. Error bars indicate standard deviations from three replicates.

FIG 2

Controlled 1.0-liter batch growth of E. coli AceE variants with 15 g/liter glucose. (a) MEC825 (C ldhA poxB aceE::aceE); (b) MEC961 (C ldhA poxB aceE::aceE ppsA); (c) MEC826 (C ldhA poxB aceE::aceE^[H106V]); (d) MEC905 (C ldhA poxB aceE::aceE^[H106V] ppsA). ■, glucose; ▲, pyruvate; ○, OD.

FIG 3

Comparison of glucose uptake rate (circles) and pyruvate yield (grams per gram; triangles) of MEC905 (C ldhA poxB aceE::aceE^[H106V] ppsA; filled symbols) and MEC961(C ldhA poxB aceE::aceE ppsA; empty symbols) during steady-state growth at the indicated dilution rates.

FIG 4

Growth and pyruvate formation in a controlled 1.0-liter repeated batch process with 15 g/liter glucose and 2 g/liter acetate. A 17-ml solution containing 15 g glucose (and no acetate) was added when the initial glucose was depleted. (a) MEC992 (C ldhA poxB aceE ppsA); (b) MEC994 (C ldhA poxB aceE::aceE^{[H106M;E401A]} ppsA). ■, glucose; ▲, pyruvate; ●, acetate; ○, OD.