Effects Of PAS＿chr2-1＿0661 And XP＿002492593 Gene Deletion On Constitutive Expression Of Foreign Proteins In Pichia Pastoris

Pichia pastoris is a widely used eukaryotic host for commercial heterologous protein production.P.pastoris has the advantages of efficient aerobic respiration mode,protein modification ability of advanced eukaryotic cells,easy gene manipulation and less naturally secreted proteins,but there are few reports on the modification of its chassis cell GS115.The main components of yeast cell wall areβ-glucan and mannan,which high content plays an important role in the stress response of yeast in the natural environment.In the industrial fermentation environment,the high content of polysaccharides is likely to be redundant and cause a lot of waste of carbon sources.In recent years,chassis cell design has become an important strategy for optimizing the fermentation performance of strains.Simplifying the cell wall structure is one of the ways.However,there is no report on the modification of P.pastoris cell wall components.The effect of exogenous protein expression in P.pastoris was verified by expressing human epidermal growth factor（h EGF）and S-adenosylmethionine synthase 2（SAM2）.Since cell wall modification may aggravate lipid oxidation due to continuous oxidative stress during fermentation,this study analyzed lipid peroxidation and changes in lipid molecules during fermentation of P.pastoris.The main research contents and conclusions are as follows:（1）The genes encodingβ-1,3-glucan,β-1,6-glucan and mannan in the cell wall of P.pastoris GS115 were knocked out and validated.Based on the p Pp T4-p HTX1-hs Cas9 plasmid,the catalytic subunit genes ofβ-1,3-glucan synthase（PAS＿chr2-1＿0661,PAS＿chr2-1＿0263）,genes required forβ-1,6 glucan biosynthesis（PAS＿chr4＿0508,PAS＿chr3＿0929）,andα-1,2-mannosyltransferase-related genes（XP＿002492593,PAS＿chr1-3＿0138,PAS＿chr3＿0882）were knocked out.A total of 5 single-gene deletion strains and 1 double-gene deletion strain were successfully constructed.Growth ability test results showed that GS115-?PAS＿chr2-1＿0661,GS115-?XP＿002492593,and GS115-?XP＿002492593-?PAS＿chr2-1＿0661 outperformed GS115;The maximum OD₆₀₀ of GS115-?XP＿002492593-?PAS＿chr2-1＿0661 reaches 27.64,which is 21.1%higher than GS115.Compared with GS115,the content ofβ-glucan in the cell wall of GS115-?PAS＿chr2-1＿0661 was decreased by 19.3%,and the content of mannan in the cell wall of GS115-?XP＿002492593 was decreased by 14.3%.The content ofβ-glucan and mannan in the cell wall of GS115-?XP＿002492593-?PAS＿chr2-1＿0661 was 5.5%and 8.4%lower than that of GS115,respectively.（2）The carbon source yield and reporter gene expression of gene deletion strains were compared.The results showed that GS115-?XP＿002492593-?PAS＿chr2-1＿0661 had the highest carbon source yield,reaching 0.53 g?g^-1.Using p GAPZ A-gfp to express GFP,GS115-?PAS＿chr2-1＿0661 and GS115-?XP＿002492593-?PAS＿chr2-1＿0661 expressed 16.6%and21.0%higher fluorescence intensity than GS115 respectively.It shows that the strains with deletion of XP＿002492593 and PAS＿chr2-1＿0661 genes have the potential to express foreign proteins efficiently.（3）The effects of deletion of XP＿002492593 and PAS＿chr2-1＿0661 on the expression and yield of foreign proteins were analyzed.Using hegf and sam2 as the target product genes,constitutive plasmids p GAPZαA-hegf and p GAPZ A-sam2 were constructed,and their expressions were compared at the level of shake flask and fermenter.At the level of 250 m L shake flasks,GS115-?PAS＿chr2-1＿0661/p GAPZαA-hegf and GS115-?XP＿002492593-?PAS＿chr2-1＿0661/p GAPZαA-hegf had the highest exocrine proteins of 50.51 mg?L^-1 and53.12 mg?L^-1,which were higher than GS115 increased by 11.1%and 16.8%;GS115-?XP＿002492593-?PAS＿chr2-1＿0661/p GAPZ A-sam2 expressed SAM2 enzyme activity up to515 U?m L^-1,9.8%higher than GS115;The SAM yield of GS115-?XP＿002492593-?PAS＿chr2-1＿0661 was 19.7%higher than that of GS115,reaching 2.31 g?L^-1.（4）Further analysis of GS115-?XP＿002492593-?PAS＿chr2-1＿0661 affected other cell properties.To compare the differences in oxidative stress during culture,the content of lipid peroxidation product malondialdehyde（MDA）was determined.The MDA content was the highest in the stationary phase and death phase.The MDA content of GS115-?XP＿002492593-?PAS＿chr2-1＿0661 reached the highest level of 0.41μmol?g^-1 dry cells,which was 15.8%higher than that of GS115.For lipid changes and lipid oxidation,the first whole-cell lipidome of P.pastoris was carried out,which laid the foundation for further transformation of chassis.This study showed that the simultaneous deletion of XP＿002492593 and PAS＿chr2-1＿0661 genes in P.pastoris GS115 strain improved the cell growth rate and carbon source yield,and facilitated the expression of exogenous gene hegf and sam2.Therefore,GS115-?XP＿002492593-?PAS＿chr2-1＿0661 is a highly potential chassis cell.