Engineering of Rhodococcus cell catalysts for tolerance improvement by sigma factor mutation and active plasmid partition.

Tolerance to various stresses is a key phenotype for cell catalysts, which are used widely in bioproduction of diverse valuable chemicals. Using the Rhodococcus ruber TH strain, which exhibits high nitrile hydratase activity, as the target cell catalyst for acrylamide production, we established a method to improve cell tolerance by stably introducing global transcription perturbation. The σ(70) gene (sigA) of R. ruber was cloned and randomly mutated. An R. ruber TH3/pNV-sigA(M) library containing additional sigA mutants was constructed and used for survival selection. The TH3/M4N1-59 mutant was selected by acrylonitrile/acrylamide double stress and exhibited a 160 % extension of the half-life of nitrile hydratase upon exposure to 40 % acrylamide. A redesigned parDE(M) gene was introduced to Rhodococcus to accomplish stable inheritance of plasmids. A two-batch acrylonitrile hydration reaction was performed using the engineered cells as a catalyst. Compared to TH3, the acrylamide productivity of TH3/M4N1-59DE(M) catalysis increased by 27.8 and 37.5 % in the first and second bioreaction batches, respectively. These data suggest a novel method for increasing the bioconversion productivity of target chemicals through sigA mutation of the cell catalyst.