Enhancement of ergothioneine production in Corynebacterium glutamicum by increasing osmotic pressure

Ergothioneine (EGT), which exhibits strong antioxidant properties, is an amino acid derivative with a betaine structure. Currently, studies have examined EGT import systems and its physiological roles in various organisms. Despite the broad applicability of EGT, industrial production with high productivity has not yet been achieved. In this study, we aimed to develop fermentative production methods for EGT using Corynebacterium glutamicum as a host and successfully achieved the highest yield of EGT (459 mg L^-1) reported to date. A cysteine-producing strain C. glutamicum CYS-2, which was constructed in a previous study, was engineered to enhance the biosynthesis of histidine and S-adenosylmethionine, both of which, along with cysteine, are required for EGT production. Additionally, heterologous metabolic pathways for EGT biosynthesis from Mycolicibacterium smegmatis and Methylobacterium pseudosasicola were introduced into the engineered strain, which was designated CHS2. In batch cultivation, the CHS2 strain produced more EGT than the CYS-2 strain harboring the same EGT biosynthesis pathway. Interestingly, batch cultivation of the CHS2 strain under high osmotic pressure conditions prolonged the time for EGT production and increased the intracellular accumulation of EGT. These results suggest that increasing osmotic pressure together with engineering the biosynthesis of cysteine, histidine, and S-adenosylmethionine is an effective strategy for enhancing EGT production in recombinant C. glutamicum harboring heterologous EGT biosynthesis pathways. KEY POINTS: • Ergothioneine production in C. glutamicum was enhanced by metabolic engineering. • Osmotic pressure affects ergothioneine production in engineered C. glutamicum. • Ergothioneine may function as a compatible solute in C. glutamicum.