Abstract: An engineered Escherichia coli constitutively expressing sorbose dehydrogenase (SDH) was previously constructed for producing the vitamin C precursor 2-keto-L-gulonic acid (2-KLG) with L-sorbose as substrate. However, this strain has a poor tolerance to L-sorbose. In order to solve this problem, adaptive laboratory evolution was carried out. Firstly, to enhance the tolerance of L-sorbose, the starting strain was grown and subcultured in the medium with different gradient concentration of L-sorbose by applying Microbial Microdroplet Culture System designed based on microfluidic technology. An evolutionary strain 2-F6 that could tolerate high concentration of L-sorbose was obtained after further verifying on the shaking flasks. Then, the sorbosone dehydrogenase (SNDH) was expressed in 2-F6 to promote the strength of 2-KLG production. The inoculation amount, fermentation temperature, induction time for SNDH, IPTG concentration and L-sorbose addition were optimized in the shaking flasks, and the titer of 2-KLG reached 6.05 g/L under the optimized optimal conditions. Finally, after scaling up in a 5 L fermenter, 2-KLG of 5.70 g/L was obtained. The results obtained could provide references for the production of vitamin C precursor 2-KLG with one-step fermentation process.
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