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Combined evolutionary and metabolic engineering improve 2-keto-L-gulonic acid production in Gluconobacter oxydans
Dong Li1, Li Liu1, Zhijie Qin1
1Science Center for Future Foods, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education and School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology, Jiangnan University, Wuxi 214122, China.
Abstract:
The direct fermentation of the precursor of vitamin C, 2-keto-L-gulonic acid (2-KLG), has been a long-pursued goal. Previously, a strain of Gluconobacter oxydans WSH-004 was isolated that produced 2.5 g/L 2-KLG, and through adaptive evolution engineering, the strain G. oxydans MMC3 could tolerate 300 g/L D-sorbitol. This study verified that the sndh-sdh gene cluster encoded two key dehydrogenases for the 2-KLG biosynthesis pathway in this strain. Then G. oxydans MMC3 further evolved through adaptive evolution to G. oxydans 2-KLG5, which can tolerate high concentrations of D-sorbitol and 2-KLG. Finally, by increasing the gene expression levels of the sndh-sdh and terminal oxidase cyoBACD in G. oxydans 2-KLG5, the 2-KLG accumulation in the 5-L fermenter increased to 45.14 g/L by batch fermentation. The results showed that combined evolutionary and metabolic engineering efficiently improved the direct production of 2-KLG from D-sorbitol in G. oxydans.
