Corynebacterium Glutamicum Engineered As a Designer Bug for the Production of Pyruvate and Succinate

Corynebacterium glutamicum is a non-pathogenic, Gram-positive organism that grows on a variety of substrates and is used for the production of amino acids (e.g. L‑glutamate, L‑lysine and L‑valine). The aim of the present work was to engineer C. glutamicum to produce pyruvate aerobically and exploitation of the resulting strain as platform for anaerobic succinate production.

In our study we modified C. glutamicum for the production of pyruvate and decreased formation of byproducts. By stepwise inactivation of the pyruvate dehydrogenase complex, the pyruvate:quinone oxidoreductase, the L‑lactate dehydrogenase and attenuation of the acetohydroxyacid synthase [AHAS], efficient pyruvate production was achieved. The deletion of the genes encoding alanine aminotransferase and pyruvate:valine aminotransferase led to a strong reduction of the side product L‑alanine and together with the attenuation of the AHAS to decreased L‑valine formation. In fed-batch fermentations with adjusted oxygen supply during growth and production (0‑5% DO), the engineered strain produced more than 500 mM pyruvate with a yield of 0.97 mol per mole of glucose and a productivity of 0.92 mmol∙g_(CDW)⁻¹∙h⁻¹ (i.e., 0.08 g∙g_(CDW)⁻¹∙h⁻¹) in the production phase [1].

For production of succinate with this mutant, a tri-phasic fed-batch fermentation process in a single bioreactor was established: Aerobic growth on acetate was followed by a self-induced microaerobic phase at the end of growth by minimal aeration. Subsequently, an anaerobic production phase was realized by CO₂ gassing. Under these conditions a final succinate concentrations above 330 mM from 325 mM glucose was obtained, with a Y_P/S of 1.02 mole succinate per mole of glucose [2].

References:

[1] Wieschalka S, Blombach B, Eikmanns BJ (2012) Engineering Corynebacterium glutamicum for the production of pyruvate. Appl Microbiol Biotechnol. doi: 10.1007/s00253-011-3843-9

[2] Wieschalka S, Blombach B, Bott M, Eikmanns BJ (2013) Bio-based production of organic acids with Corynebacterium glutamicum. Microb Biotechnol 6:87–102. doi: 10.1111/1751-7915.12013