Reorganization of a synthetic microbial

consortium for one-step vitamin C fermentation En‑Xu Wang1,2, Ming‑Zhu Ding1,2*, Qian Ma1,2, Xiu‑Tao Dong1,2 and Ying‑Jin Yuan1,2 Background: 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 Ketoguloni-cigenium vulgare. Despite its high production efficiency, the long incubation period and an additional second steriliza‑ tion 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. Results: 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 delet‑ ing 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. Conclusions: 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 enhance‑ ment of the mutualism between the microbes and gave us a better understanding of the synthetic consortium