Comparative Study On HEGF Expression And Lipidomics Profile In Different Chassis Cells Of Pichia Pastoris

Pichia pastoris is a widely used foreign protein expression system,and P.pastoris GS115 is also the most widely used P.pastoris chassis cell.However,at present,there are few reports on the modification of P.pastoris GS115,which restricts the improvement of the expression efficiency of foreign proteins.Based on this,this study compared the expression efficiency of human epidermal growth factor in ten kinds of P.pastoris chassis cells for the first time,and compared the differences of their lipidomics.First of all,in this study,CRISPR/Cas9 technique was used to knock out the genes of proteins required for the biosynthesis ofβ-1,6-D-glucan（PAS＿chr4＿0508,PAS＿chr1-3＿0114）,aspartate protease genes（PAS＿chr1-1＿0379,PAS＿chr3＿1087）and serine protease genes（PAS＿chr1-4＿0548,PAS＿chr1-1＿0226）in P.pastoris GS115,and six single gene deletion strains were constructed.In addition,there were 4 gene deleted strains（ΔPAS＿chr1-3＿0225,ΔPAS＿chr2-1＿0661,ΔPAS＿chr3＿0370,ΔPAS＿chr3＿0370ΔPAS＿chr2-1＿0661）in the laboratory,its growth characteristics and carbon source yield were compared and screened.at the same time,the content of glucan was compared and observed by transmission electron microscope.Then,according to the better growth strains,the expression efficiency of green fluorescent protein GFP was evaluated by using two different promoters(constitutive P_GAPand inducible P_AOX1).Four excellent strains were obtained,and their hEGF expression efficiency was compared.Finally,the expression level of hEGF protein was compared and analyzed,and it was found that the change of antioxidant capacity of chassis cells during fermentation was one of the important reasons affecting cell viability and protein expression.In order to further explore the reasons,the differences of liposome were compared,the mass spectrometry data were collected by Lipid Search 4.1,and the lipid composition and relative content of membrane lipids were compared after pretreatment.The specific conclusions are as follows:（1）The study of candidate chassis cells showed that:the growth rate of five strains of chassis cells in YPD was higher,and the highest OD₆₀₀ofΔPAS＿chr3＿0370ΔPAS＿chr2-1＿0661 was 29.78,which was 1.32 times higher than that of GS115;the highest carbon source yield ofΔPAS＿chr2-1＿0661 was 0.53 g·g^-1,which was 0.14 g·g^-1higher than that of GS115.Further study on the growth rate of BMGY showed that four strains of chassis cells grew well,includingΔPAS＿chr1-3＿0225,ΔPAS＿chr2-1＿0661,ΔPAS＿chr3＿0370ΔPAS＿chr2-1＿0661,ΔPAS＿chr1-1＿0379,and the glucan content decreased by 2.14%,28.57%,25.00%and 23.57%compared with GS115.Among them,the highest OD₆₀₀ofΔPAS＿chr2-1＿0661 was 35.20,which was 1.53 times higher than that of GS115,and the carbon source yield was 0.65 g·g^-1,which was 0.08 g·g^-1higher than that of GS115.In addition,the outer wall of the chassis cells of the above four strains became loose.（2）The protein expression results of the above four chassis cells showed that:The fluorescence intensity ofΔPAS＿chr2-1＿0661/p GAPZ A-gfp andΔPAS＿chr2-1＿0661/p PICZ A-gfp was the highest,which was 1.57 and 1.35 times higher than that of GS115,respectively.The expression efficiency of hEGF in the four chassis cells was better than that of GS115;and the expression levels ofΔPAS＿chr2-1＿0661/p GAPZ A-hegf andΔPAS＿chr2-1＿0661/p PICZ A-hegf were the highest,which were 40.37 mg·g^-1and 75.26 mg·g^-1,which were 41.40%and66.76%higher than that of GS115,respectively.After 2L fermentation ofΔPAS＿chr2-1＿0661and GS115,the hEGF expression amount ofΔPAS＿chr2-1＿0661/p GAPZ A-hegf was 0.58mg·g^-1,which was 11.53%higher than that of GS115;and the hEGF expression quantity ofΔPAS＿chr2-1＿0661/p PICZ A-hegf was 0.97 mg·g^-1,which was 14.12%higher than that of GS115.（3）The MDA analysis of the markers of peroxidation damage in four chassis cells showed that:The degree of oxidative damage in the later stage of fermentation was lower than that of GS115,which had an important advantage in the fermentation process.The lipidomics analysis to further explore its mechanism showed that 991 kinds of lipids were detected in P.pastoris GS115,including glycerolipids,glycerols and sphingolipids;and the increase of PC content in four strains of chassis cells was related to the decrease of glucan content,while unsaturated glycerolipids also increased,which improved the ability to cope with oxidative stress and oxidative damage,which was one of the reasons for its lower oxidative damage.This paper reveals for the first timeΔPAS＿chr1-3＿0225、ΔPAS＿chr2-1＿0661、ΔPAS＿chr3＿0370、ΔPAS＿chr1-1＿0379 were chassis cells with a higher expression efficiency of hEGF than GS115,and its oxidative damage level became lower in the later stage of fermentation,which is likely due to to an increase in its unsaturated glycerol phospholipids.