Expression of Plectasin Gene in Clostridium butyricum

Objective:To construct a eukaryotic recombinant expression strain of mycelomycin(Plectasin,Pt) and study its expression level and antibacterial activity in Clostridium tyrobutyricum. A new type of food additive with high efficiency,green and antibacterial function will be provided. Method:The promoter thl was first cloned into the vector pMTL82151 to obtain the expression plasmid pMTL82151-thl,and then the optimized Pt gene was artificially synthesized according to the codon preference of the Clostridium butyricum expression system,and then the bacteria were produced. The silkworm gene Pt was cloned downstream of the promoter thl,and the expression plasmid pMTL82151-Pt was constructed and transformed into Clostridium tyrobutyricum. The positive recombinants were screened by thiamphenicol resistance,PCR identification,SDS-PAGE analysis and agar holes The constitutive expression strain of mycelium was obtained by diffusion method,and the Clostridium tyrobutyricum pMTL82151-Pt fermentation expression of the engineered strain Clostridium tyrobutyricum was studied. Result:The thl gene was ligated with the plasmid pMTL82151 to obtain the expression plasmid pMTL82151-thl. The mycelia gene Pt was cloned downstream of the promoter thl,and the expression plasmid pMTL82151-Pt was successfully constructed. Constitutive expression of the Pt gene in Clostridium tyrobutyricum was achieved. SDS-PAGE analysis showed a clear target band at 4.4 kDa. The supernatant of the engineered bacteria was expressed by fermentation and culture has a significant inhibitory effect on Staphylococcus aureus ATCC25923.The half-inhibitory concentration(IC₅₀) of Staphylococcus aureus ATCC25923 was 12.74 μg/mL. Conclusion:The expression of mycelium in Clostridium butyricum was successfully applied in this study. It has laid the foundation for mass production and promotion of mycelium in the future as a food additive and an antibiotic substitute in meat animal husbandry.