High Efficiency Expression Strategy Of Recombinant Bovine Prochymosin In Pichia Pastoris

Chymosin?EC 3.4.23.4?is an aspartic protease that is a key enzyme in cheese processing.Rennet is traditionally extracted from the calf's stomach,which can no longer meet the increasing needs of cheese production.Genetically engineered strains,as efficient hosts for target protein production,are expected to solve shortage of bovine rennet.In this study,the bovine prorennin gene was heterologously expressed in Pichia pastoris.The expression level of recombinant bovine prorennin in Pichia pastoris was enhanced via codon optimization,fusion expression,promoter selection,and fermentation process optimization.According to the codon bias of Pichia pastoris,the bovine prorennin gene pcw was redesigned to pcm14 gene through OptimumGene^TM algorithm.Then the clpcm14 gene was obtained by insert cherrym ahead of pcm14.The two genes,pcm14 and clpcm14,were then expressed in P.pastoris,respectively.The recombinant strains 2-70 and clp2-91 with the high enzyme activities,2188 SU/mL and 3705 SU/mL,respectively,were selected.The transcription level and copy number of the target genes in the two strains were equal estimated by quantitative PCR.After that,the methanol-induced promoter P_AOX1 was replaced with the constitutive promoter P_GAP in the clpcm14 gene expression cassette,High-expression strain GH-1 was obtained by enzyme activity screening.Furthermore,fermentation processes related to GH-1 strain were investigated and opimized,including pH,feed mode and carbon source.The optimum fermentation conditions in fed batch mode were as follows,pH 4.0 during fermentation and glucose as carbon source.The enzyme activity of the GH-1 strain was up to 12,000 SU/mL,and the protein concentration was 0.28 mg/mL in 3 L fermentor.The production intensity of the constitutive strain GH-1 was 3 times as that of the inducible strain clp2-91.In summary,several optimization strategies were combined for increasing expression level of heterologous proteins in P.pastoris.The selected high-yield strain GH-1 was able to produce prochymosin efficiently.Its production intensity meets demand of enzyme enterprises,and exhibits a potential application in large-scale production.