Multicopy integrative expression and optimization of cecropin AD expression in Pichia pastoris

Abstract:

[Background] Cecropin is extensively studied and which is an effective antimicrobial peptide. It will lay the foundation for the practical application of cecropin in aquaculture and agriculture to realize the commercial production of cecropin. [Objective] To obtain a genetically engineered strain for efficient production of cecropin AD. [Methods] The recombinant vector pGAPZαA-CAD was firstly constructed and then transformed to P. pastoris X33 strain by electric shock. Cecropin AD gene was successfully expressed and X33/GCAD strain was obtained. Secondly, the recombinant vector pUCGAP-CAD was constructed and transformed into X33/GCAD strain. The pGAPZαA-CAD plasmid was integrated into the GAPDH promoter region of P. pastoris X33 with Zeocin as resistance screening label, while pUCGAP-CAD plasmid was integrated into the non-translated rDNA region of P. pastoris X33 with Geneticin as resistance screening label. Finally, a recombinant X33/GUCAD strain with an efficiently expressed cecropin AD gene was obtained. [Results] The antimicrobial compound of X33/GUCAD was cecropin AD which was identified by mass spectrometry. Afterward optimizing its fermentation conditions, we found that the X33/GUCAD strain having higher potential to express cecropin AD by consuming glycerol as a sole carbon source while peptone and yeast extract for organic nitrogen source. [Conclusion] The higher copy number is more beneficial to increase the yield of cecropin AD, and the engineering strain is more stable in the later fermentation process and suitable for industrial production.