Efficient biosynthesis of (R)-mandelic acid from styrene oxide by an adaptive evolutionary Gluconobacter oxydans STA
Fei Liu, Junping Zhou, Mengkai Hu, Yan Chen, Han Jin, Xuewei Pan, Jiajia You, Meijuan Xu, Taowei Yang, Minglong Shao, Xian Zhang, Zhiming Rao
Abstract
Abstract 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.