Metabolic Engineering of Gluconobacter Oxydans Wsh-003 for the Direct Production of 2-Keto-L-Gulonic Acid from D-Sorbitol

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 (Gluconobacter oxydans, Bacillus megaterium and Ketogulonicigenium vulgare), making the mix-culture system redundant and complicated. Thus, replacement of the conventional two-step fermentation process with a one-step process could be revolutionary in vitamin C industry. Based on the genomic analysis of Gluconobacter oxydans and Ketogulonicigenium vulgare, a group of dehydrogenases, which use pyrroloquinoline quinine (PQQ) as the cofactor, responsible for the stepwise conversion of D-sorbitol to L-sorbose, L-sorbosone, and finally 2-KLG, were identified. In order to achieve the direct production of 2-keto-L-gulonic acid from D-sorbitol, systematic metabolic engineering were performed on an industrial strain G. oxydans WSH-003. Coordination expression of these dehydrogenases in an industrial strain G. oxydans WSH-003 achieved the one-step production of 2-KLG. Based on a CRISPR-Cas9/Red system established in the strain, the efficiency of 2-KLG production were further significantly enhanced by integrative expression of dehydrogenases and PQQ biosynthesis genes with a series of constitutive promoters with gradient strength, and knockout of a series identified non-essential membrane proteins.