Construction of anhydrotetracycline-inducible ClosTron system in Clostridium cellulolyticum

Abstract:

[Background] Clostridium cellulolyticum is a mesophilic model strain for ethanol production from lignocellulose. Constructing the gene targeting technology with controllable induction is an important means to study the genetic regulation of C. cellulolyticum. [Objective] To construct an anhydrotetracycline-inducible ClosTron gene targeting system in C. cellulolyticum.[Methods] Firstly, we determined the sensitivity of C. cellulolyticum to anhydrotetracycline and selected the appropriate inducer concentration, and then tested the inducible effect using β-glucosidase gene as reporter gene. Secondly, we constructed an anhydrotetracycline-induced ClosTron plasmid by combining the anhydrotetracycline-inducible operon with group II intron elements. Finally, the mspI, ldh and ack were selected as targets to determine the targeting efficiency in C. cellulolyticum.[Results] The anhydrotetracycline-inducible system could induce the ClosTron expression in C. cellulolyticum, and the targeting efficiency at mspI, ldh and ack sites were 29.48%±15.51%, 23.61%±7.08% and 28.09%±6.97%, respectively. [Conclusion] The anhydrotetracycline-inducible ClosTron system was successfully constructed in C. cellulolyticum, which laid a foundation for genetic engineering of Clostridium.