title: Stepwise metabolic engineering of Gluconobacter oxydans WSH-003 for the direct production of 2-keto-L-gulonic acid from D-sorbitol.
abstract: 2-Keto-L-gulonic acid (2-KLG), the direct precursor of vitamin C, is currently produced by a two-step fermentation route from D-sorbitol. However, this route involves three bacteria, making the mix-culture system complicated and redundant. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. In this study, different combinations of five L-sorbose dehydrogenases (SDH) and two L-sorbosone dehydrogenases (SNDH) from Ketogulonicigenium vulgare WSH-001 were introduced into Gluconobacter oxydans WSH-003, an industrial strain used for the conversion of d-sorbitol to L-sorbose. The optimum combination produced 4.9g/L of 2-KLG. In addition, 10 different linker peptides were used for the fusion expression of SDH and SNDH in G. oxydans. The best recombinant strain (G. oxydans/pGUC-k0203-GS-k0095) produced 32.4g/L of 2-KLG after 168h. Furthermore, biosynthesis of pyrroloquinoline quinine (PQQ), a cofactor of those dehydrogenases, was enhanced to improve 2-KLG production. With the stepwise metabolic engineering of G. oxydans, the final 2-KLG production was improved to 39.2g/L, which was 8.0-fold higher than that obtained using independent expression of the dehydrogenases. These results bring us closer to the final one-step industrial-scale production of vitamin C.
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Gao
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Yudong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Jiangsu Jiangshan Pharmaceutical Co., Ltd. Jingjiang 214500, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address: zhoujw1982@jiangnan.edu.cn.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, Jiangsu 214122, China. Electronic address: jchen@jiangnan.edu.cn.
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000096:biosynthesis / Q000235:genetics; D020600:Gluconobacter oxydans / Q000201:enzymology / Q000235:genetics; D060847:Metabolic Engineering; D041902:Rhodobacteraceae / Q000201:enzymology / Q000235:genetics; D013012:Sorbitol / Q000378:metabolism*; D013400:Sugar Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013400:Sugar Acids; D013012:Sorbitol; D002237:Carbohydrate Dehydrogenases; C104557:sorbose dehydrogenase; C011491:provitamin C
keywords: Linker peptides; One-step fermentation; Pyrroloquinoline quinine; Vitamin C; l-sorbose dehydrogenase; l-sorbosone dehydrogenase