[Background] Adaptive laboratory evolution is one of directed evolution methods to improve strain fitness. It improves the tolerance to toxic products or rapidly utilizes specific substrates. Due to the rather low mutation rate of native cells caused by the mismatch repair mechanism in the genome, adaptive laboratory evolution usually takes a long period to screen desired evolved strains and more likely to fail because of microbial contamination during a long process. [Objective] To develop controllable hypermutable cells of Bacillus subtilis. [Methods] A marker-free mutation delivery system is used to replace the mismatch repair system operon MutSL with a rigorous xylose-inducible promoter. [Results] The mutation rates of controllable hypermutable B. subtilis cells could be regulated by the concentration of inducer (xylose). The phenethyl alcohol tolerance experiment indicated that the hypermutable B. subtilis cells had better tolerance than the original strain, suggesting that it is more easily to obtain the mutant with desired trait. The adaptive evolution experiment of growth inhibition by substrate crude glycerol showed that the evolved strain showed the shorter lag time, higher biomass, and greater specific growth rate than the starting strain. [Conclusion] The hypermutable B. subtilis cells had better evolvability and adaptability, which could have a wide range of applications.
