Manipulation of Methyl-Directed Mismatch Repair System to Engineer the Robustness of Clostridium Tyrobutyricum

High robustness performance of industrial microbes is crucial for successful bioprocesses. Microbial production of toxic products and tolerance to biomass hydrolysates requires robust cell growth and metabolism under tough conditions. To improve the aero- and acid tolerance of the organic-acid-producing Clostridium tyrobutylicum, a novel adaptive evolutionary strategy based on stress-induced mutagenesis (SIM) was developed using non-dividing cells. First of all, the mutS/L operon essential for methyl-directed mismatch repair (MMR) activity was inactivated from the genome of C. tyrobutylicum to generate hypermutable cells with over 300-fold increased mutation rates. Secondly, a periodic SIM based adaptive evolution procedure, which synchronized the mutagenesis and the selection process in a single plate-incubation step, was developed using the mutL-deleted mutant. C. tyrobutylicum mutants tolerant to high concentrations of butyric acid (100 g/L), NaCl (90 g/L), and high temperature (55 ºC) were obtained. Combined transcriptome and phenome analysis of C. tyrobutylicum with deficient MMR activity was carried out to better understand the global effects on the regulatory networks. Our analysis showed that 95 (2.1%) of all C. tyrobutylicum genes were induced and 178 (4.0%) genes were repressed, including those for trehalose biosynthesis, nucleotides biosynthesis, carbon metabolism, amino acid utilization, except for acid resistance. Also regulated were the master regulators (ArcA, EvgA, H-NS and RpoS) and gene/operon-specific transcription factors (GadX, GadW, AppY, YdeO, KdgR). These results indicate that stress-induced adaptive evolution in non-dividing cells is an effective approach that can improve microbial tolerance against various stresses and generate robust microbial strains suitable for production of chemicals.