Metabolic Engineering of Escherichia coli KO11 with the NADH Regeneration System for Enhancing Ethanol Production

摘要

Cofactor regulation for the production of reduced metabolites, such as ethanol, plays an important role in the metabolic pathways of microorganisms. In this study, the intracellular NADH/NAD~(+) ratio in the ethanologenic strain Escherichia coli KO11 was increased by the overexpression of formate dehydrogenase from Mycobacterium vaccae . To avoid the generation of pyruvate-derived byproducts, genes coding for the lactate- and acetate-producing pathways were successfully deleted using genome editing based on the CRISPR-Cas9 system. In the culture of the constructed strain, the NADH regeneration system increased the intracellular NADH/NAD~(+) ratio in the presence of formate, leading to enhanced ethanol production; the amount of ethanol produced was 36% more than that produced by the original KO11 strain in culture for 24?h. A detailed investigation revealed that the transient pyruvate accumulation plays a key role in the enhanced ethanol production using the NADH regeneration system.