Efficient biosynthesis of ( R )-mandelic acid from styrene oxide by an adaptive evolutionary Gluconobacter oxydans STA

Background ( R )-mandelic acid ( R -MA) is a highly valuable hydroxyl acid in the pharmaceutical industry. However, biosynthesis of optically pure R -MA remains significant challenges, including the lack of suitable catalysts and high toxicity to host strains. Adaptive laboratory evolution (ALE) was a promising and powerful strategy to obtain specially evolved strains. Results Herein, we report a new cell factory of the Gluconobacter oxydans to biocatalytic styrene oxide into R -MA by utilizing the G. oxydans endogenous efficiently incomplete oxidization and the epoxide hydrolase (SpEH) heterologous expressed in G. oxydans . With a new screened strong endogenous promoter P 12780 , the production of R -MA was improved to 10.26 g/L compared to 7.36 g/L of using P lac . As R -MA showed great inhibition for the reaction and toxicity to cell growth, adaptive laboratory evolution (ALE) strategy was introduced to improve the cellular R -MA tolerance. The adapted strain that can tolerate 6 g/L R -MA was isolated (named G. oxydans STA), while the wild-type strain cannot grow under this stress. The conversion rate was increased from 0.366 g/L/h of wild type to 0.703 g/L/h by the recombinant STA, and the final R -MA titer reached 14.06 g/L. Whole-genome sequencing revealed multiple gene-mutations in STA, in combination with transcriptome analysis under R -MA stress condition, we identified five critical genes that were associated with R -MA tolerance, among which AcrA overexpression could further improve R -MA titer to 15.70 g/L, the highest titer reported from bulk styrene oxide substrate. Conclusions The microbial engineering with systematic combination of static regulation, ALE, and transcriptome analysis strategy provides valuable solutions for high-efficient chemical biosynthesis, and our evolved G. oxydans would be better to serve as a chassis cell for hydroxyl acid production.