Metabolic Engineering of <i>Corynebacterium glutamicum</i> for Fuel Ethanol Production under Oxygen-Deprivation Conditions

TLDR

The study engineered the central metabolic pathway of Corynebacterium glutamicum to produce ethanol. A recombinant C. glutamicum strain expressing Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase under the ldhA promoter was constructed, enabling high‑level ethanol production from glucose under oxygen‑deprived, non‑growth conditions.

Abstract

The central metabolic pathway of <i>Corynebacterium glutamicum</i> was engineered to produce ethanol. A recombinant strain which expressed the <i>Zymomonas mobilis</i> genes coding for pyruvate decarboxylase <i>(pdc)</i> and alcohol dehydrogenase <i>(adhB)</i> was constructed. Both genes placed under the control of the <i>C. glutamicum ldhA</i> promoter were expressed at high levels in <i>C. glutamicum,</i> resulting, under oxygen-deprivation conditions, in a significant yield ofethanol from glucose in a process characterized by the absence of cellular growth. Addition of pyruvate in trace amounts to the reaction mixture induced a 2-fold increase in the ethanol production rate. A similar effect was observed when acetaldehyde was added. Disruption of the lactate dehydrogenase <i>(ldhA)</i> gene led to a 3-fold higher ethanol yield than wild type, with no lactate production. Moreover, inactivation of the phosphoenolpyruvate carboxylase <i>(ppc)</i> and <i>ldhA </i>genes revealed a significant amount of ethanol production and a dramatic decrease in succinate without any lactate production, when pyruvate was added. Since the reaction occurred in the absence of cell growth, the ethanol volumetric productivity increased in proportion to cell density of ethanologenic <i>C. glutamicum</i> in a process under oxygen-deprivation conditions. These observations corroborate the view that intracellular NADH concentrations in <i>C. glutamicum</i> are correlated to oxygen-deprived metabolic flows.