Efficient protein expression in a robust Escherichia coli strain and its application for kinetic resolution of racemic glycidyl o-methylphenyl ether in high concentration

A robust Escherichia coli (E. coli) strain, with a dual protection system to fight against microbial contamination and T7 phage infection, is used as a chassis to overexpress various proteins in auxotrophic MOPS medium. Among them, a robust EH (epoxide hydrolase)-producing E. coli was used as a biocatalyst to enantioselectively resolve racemic glycidyl o-methylphenyl ether (rac-o-GMPE) for preparing (R)-o-GMPE with 6416.7 U/g wet cells. The optimal temperature, pH and biocatalyst dosage were 25 °C, 7.0 and 0.2 mg/mL in aqueous buffer, respectively. The maximal yield (23.0 %) and ee (99.2 %) were achieved within 12 min in 20 mM rac-o-GMPE. Additionally, 98.0 % ee and 38.7 % yield were observed by cell-supplemental strategy with substrate concentration of 100 mM. Furthermore, to alleviate substrate inhibition and product toxicity toward the cells, a biphasic system (aqueous buffer: butyl acetate = 0.5:1) was developed to catalyze 3 M rac-o-GMPE with the initial reaction rate of 549.7 mmol/L/h, 91.7 % ee, 17.1 % yield and 3.66 g/L/h space-time yield.