Abstract(#br) Background(#br)In the industry, the conventional two-step fermentation method was used to produce 2-keto- l -gulonic acid (2-KGA), the precursor of vitamin C, by three strains, namely, Gluconobacter oxydans , Bacillus spp. and Ketogulonicigenium vulgare . Despite its high production efficiency, the long incubation period and an additional second sterilization process inhibit the further development. Therefore, we aimed to reorganize a synthetic consortium of G. oxydans and K. vulgare for one-step fermentation of 2-KGA and enhance the symbiotic interaction between microorganisms to perform better.(#br) Results(#br)During the fermentation, competition for sorbose of G. oxydans arose when co-cultured with K. vulgare . In this study, the competition between the two microbes was... alleviated and their mutualism was enhanced by deleting genes involved in sorbose metabolism of G. oxydans . In the engineered synthetic consortium (H6 + Kv), the yield of 2-KGA (mol/mol) against d -sorbitol reached 89.7 % within 36 h, increased by 29.6 %. Furthermore, metabolomic analysis was used to verify the enhancement of the symbiotic relationship and to provide us potential strategies for improving the synthetic consortium. Additionally, a significant redistribution of metabolism occurred by co-culturing the K. vulgare with the engineered G. oxydans , mainly reflected in the increased TCA cycle, purine, and fatty acid metabolism.(#br) Conclusions(#br)We reorganized and optimized a synthetic consortium of G. oxydans and K. vulgare to produce 2-KGA directly from d -sorbitol. The yield of 2-KGA was comparable to that of the conventional two-step fermentation. The metabolic interaction between the strains was further investigated by metabolomics, which verified the enhancement of the mutualism between the microbes and gave us a better understanding of the synthetic consortium.