Efficient production of 2-keto-L-gulonic acid from D-glucose in Gluconobacter oxydans ATCC9937 by mining key enzyme and transporter

[Display omitted] •Gluconobacter oxydans ATCC9937 can synthesize 2-KLG from D-glucose.•A 2,5-DKG reductase with stronger activity has been obtained from G. oxydans.•A functional 2,5-DKG transporter KgtpA has been identified from Tatumella citrea.•The highest 2-KLG titer synthesized from D-glucose has been achieved. Direct production of 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) from D-glucose through 2,5-diketo-D-gluconic acid (2,5-DKG) is a promising alternative route. To explore the pathway of producing 2-KLG from D-glucose, Gluconobacter oxydans ATCC9937 was selected as a chassis strain. It was found that the chassis strain naturally has the ability to synthesize 2-KLG from D-glucose, and a new 2,5-DKG reductase (DKGR) was found on its genome. Several major issues limiting production were identified, including the insufficient catalytic capacity of DKGR, poor transmembrane movement of 2,5-DKG and imbalanced D-glucose consumption flux inside and outside of the host strain cells. By identifying novel DKGR and 2,5-DKG transporter, the whole 2-KLG biosynthesis pathway was systematically enhanced by balancing intracellular and extracellular D-glucose metabolic flux. The engineered strain produced 30.5 g/L 2-KLG with a conversion ratio of 39.0%. The results pave the way for a more economical large-scale fermentation process for vitamin C.