Study On Key Genetic Factors Effecting High Expression Of 6×mGLP1-M Fusion Protein In Pichia Pastoris

Pichia pastoris（P.pastoris）is widely used heterogeneous protein expression system.Compared with other heterologous protein expression system,P.pastoris expression system has many advantages such as post-translational modification abilities,high cell density fermentation and easy purification of heterologous proteins.However,it was found that not all heterologous proteins could be expressed efficiently in P.pastoris,which seriously restricted the application of P.pastoris expression system.For example,the modified human gut-derived hormone,glucagon-like peptide-1（6×m GLP1-M,GLP1-M）,which has drawn attracted widespread attention because it relies on glucose concentration to promote insulin secretion from pancreatic beta cells to lower blood sugar levels without causing severe hypoglycemia.However GLP1-M has not been produced in yeast due to its extremely low expression level in P.pastoris.The expression level of fusion protein GLP1-M hexamer in P.pastoris could not been effectively improved by the traditional methods such as codon optimization,high copy number,overexpression with PDI（Protein Disulfide Isomerase）and so on.With the rise and development of m RNA sequencing technology,transcriptome sequencing（RNA-Seq）has become an important method to reveal the regulation of protein expression.Transcriptome studies can illustrate the regulation mechanism of host during the expression of heterologous proteins,which helps to explore the mechanism of heterologous protein expression and provide important theoretical guidance for the expression of exogenous proteins.Based on this,some representative engineering strains of P.pastoris were selected which can express and produce different expression levels of heterologous proteins,and then the transcriptome differences of these P.pastoris engineering strains were analyzed and studied by RNA-Seq technology and Log2 fold changes≥1,P≤0.01 were set as significantly differentially expressed genes.Through RNA-Seq data analysis,242 common differentially expressed genes were obtained between high-level and low-level protein expression strains.All the differential genes were divided into gene co-expression modules by WGCNA（Weight gene co-expression network analysis）and correlated with the protein expression trait of P.pastoris.Total 18 modules were obtained,of which the midnightblue module was the strongest correlation and significance contained 68 differentially expressed genes,the black module is the second.We overexpressed these genes of black module,in this study,52 genes were screened,and none of them significantly increased the expression level of GLP1-M fusion protein in P.pastoris.On the other hand,Gene co-expression network nodes was performed on the 68 genes included in the module,and Venn analysis was performed on the common differential genes between high-and low-level protein expression strains.There were 10 genes in this module of midnightblue,they are all located at the gene co-expression node.We knockout differential genes through CRISPR/Cas9 to regulated the recombinant protein 6×m GLP1-M expression level in P.pastoris.Finally,a key gene PAS＿chr1-3＿0003（gene ID:gene0）that affects the high expression of heterologous protein 6×m GLP1-M in P.pastoris was screened.The gene was named GESE（GLP1-M expression in GS115 enhancer）according to its ability to increase the expression level of 6×m GLP1-M in P.pastoris.Through the analysis of experiment yeast Two-Hybrid,it was concluded that the gene GESE regulates the yeast glycolysis metabolism pathway by interacting with triose phosphate isomerase（Triose-phosphate isomerase,TPI）.Without the regulation of GESE after which was knockout through CRISPR/Cas9.TPI tends to converted dihydroxyacetone phosphate into glyceraldehyde-3-phosphate.The accumulation of glyceraldehyde-3-P is catalyzed to produce Glycerate-1,3-P₂ by the enzyme of GAPDH（Glyceraldehyde-3-phosphate dehydrogenase）,the process promoted the transcription of GAPDH with GAP as its promoter,which also promoted the transcription level of recombinant protein 6×m GLP1-M with GAP as its promoter indirectly.At the same time,due to the accelerated of metabolism,we obtained a new engineering strain,which shortens the time of P.pastoris to fermentation and expressing heterologous proteins.In summary,through transcriptome sequencing,gene editing and gene function identification,we have screened a key factor GESE that affecting the efficient expression.When the gene is knocked out,the expression level of recombinant protein 6×m GLP1-M in P.pastoris can be significantly improved,and shorten the fermentation time,which is of great significance for shortening the industrial production time of yeast and has certain reference value for improving the efficient expression of heterologous proteins in P.pastoris.