Influence Of Heterologous Proteins Secretion Yield In Komagataella Phaffii After Separate Knockout Of Twelve Endogenous Genes

The methylotrophic yeast Komagataella phaffii(Pichia pastoris)is one of the most important expression platforms for the production of recombinant proteins.It offers many advantages such as: fast growth;growth at high cell densities;high microbiological safety;easy genetic manipulation and ability to produce heterologous proteins at high levels.In addition,the secretory expression ability of this yeast is important for simplifying the downstream protein purification process.However,secretory expression of heterologous proteins in K.phaffii is often subject to many factors,any one factor is suboptimal in protein secretion,it can create a bottleneck that leads to poor production yields.Thus,many studies on yeast secretion systems have focused on the engineering of the fermentation process,vector systems,and host strains.But finally the drawback of low secretion level can not be completely solved.Recently,strain engineering by genetic modification has showed significant prospect for overcoming the drawbacks.Especially in Saccharomyces cerevisiae and Schizosaccharomyces pombe,it has been made great progress.In order to improve the secretion yield of heterologous proteins in K.phaffii by the way of strain engineering,this study investigated the effect of the respective single knockout of 12 genes on the secretion yield of heterologous proteins in K.phaffii.First,based on homology with their S.cerevisiae and S.pombe counterparts,we identified 12 protein secretion genes ATO2,CZCO,FMA2,FPR3,GCN5,MON2,OMA1,PMR1,PSP3,SBH1,TDA3 and VPS10 in K.phaffii.Then the 12 genes was disrupted via a CRISPR/Cas9 genome editing system,respectively.Finally,the protein secretion level of the resulting strain was assessed by secretion expression an EGFP gene.The results showed that the GCN5 mutant strain could hardly secrete heterologous proteins;moreover both ATO2 and PMR1 mutant strains showed significantly decreased of secretion protein production;the PSP3 and TDA3 mutant strains showed slightly decreased of secretion protein production at fermentation anaphase;and the secretion level of another mutant strains did not exhibit significant change compared with that of the wild type.These results revealed partial function of these gene and offered important reference for the subsequent study about these gene.