l-Cysteine production by metabolically engineered Corynebacterium glutamicum

Abstract

l-Cysteine is a commercially important amino acid. Here, we report the construction of l-cysteine-producing Corynebacterium glutamicum using a metabolic engineering approach. l-Serine O-acetyltransferase (SAT), encoded by cysE gene, is a key enzyme of l-cysteine biosynthesis, because of its feedback inhibition by l-cysteine. Therefore, we introduced a mutation into the C. glutamicum cysE gene, which appeared to desensitize SAT against feedback inhibition by l-cysteine. We successfully produced l-cysteine by overexpressing this mutant cysE gene in C. glutamicum, while the wild-type strain scarcely produced l-cysteine. To enhance the biosynthesis of l-serine (a substrate for SAT), a mutant serA gene, encoding D-3-phosphoglycerate dehydrogenase to desensitize it against feedback inhibition by l-serine, was additionally overexpressed in the mutant cysE-overexpressing strain and its l-cysteine production was indeed improved. Moreover, we disrupted the ldh gene encoding l-lactate dehydrogenase and the aecD gene encoding cysteine desulfhydrase to prevent the formation of lactic acid as a by-product and degradation of l-cysteine produced at the stationary phase, respectively, which resulted in enhanced l-cysteine production. However, since the concentration of l-cysteine produced still decreased at the stationary phase despite the aecD disruption, NCgl2463 encoding a possible cystine importer protein was further disrupted to prevent cystine import, because the produced l-cysteine is immediately oxidized to cystine. As a result, the time before the start of the decrease in l-cysteine concentration was successfully prolonged. Approximately 200 mg/L of l-cysteine production was achieved by overexpression of mutant cysE and serA genes and disruption of aecD and NCgl2463 genes in C. glutamicum.