Developing controllable hypermutable Clostridium cells through manipulating its methyl-directed mismatch repair system

Development of controllable hypermutable cells can greatly benefit understanding and harnessing microbial evolution.However,there have not been any similar sys-tems developed for Clostridium,an important bacterial genus.Here we report a novel two-step strategy for de-veloping controllable hypermutable cells of Clostridium acetobutylicum,an important and representative indus-trial strain.Firstly,the mutS/L operon essential for methyl-directed mismatch repair(MMR)activity was inactivated from the genome of C.acetobutylicum to generate hy-permutable cells with over 250-fold increased mutation rates.Secondly,a proofreading control system carrying an inducibly expressed mutS/L operon was constructed.The hypermutable cells and the proofreading control system were integrated to form a controllable hypermut-able system SMBMutC,of which the mutation rates can be regulated by the concentration of anhydrotetracycline(aTc).Duplication of the miniPthl-tetR module of the proof-reading control system further signifi cantly expanded the regulatory space of the mutation rates,demonstrating hypermutable Clostridium cells with controllable mutation rates are generated.The developed C.acetobutylicum strain SMBMutC2 showed higher survival capacities than the control strain facing butanol-stress,indicating greatly increased evolvability and adaptability of the controllable hypermutable cells under environmental challenges.