Construction And Transcriptome Analysis Of Non-methanol-utilizing Pichia Pastoris With High Expression Of Recombinant Protein

Pichia pastoris has been widely used in the expression of recombinant proteins,mainly due to its highly efficient P_AOX1,which is strictly induced by methanol and inhibited by other carbon sources,such as glycerol,glucose.At present,the regulation mechanism of P_AOX1 under glycerol or glucose has been studied.It was found that the activating transcription factors of regulating P_AOX1 expression were Mxr1,Prm1 and Mit1,and the inhibitory transcription factors were Nrg1 and Mig.A non-methanol dependent Pichia pastoris expression system was obtained by engineering these transcription factors.Methanol is flammable,explosive and toxic,which limits the application of Pichia pastoris in large-scale fermentation,food and pharmaceutical products.When Pichia pastoris metabolizes methanol,it takes a lot of energy to synthesize alcohol oxidase,which accounts for 30%of the total soluble protein.In this study,a non-methanol-utilized high-yield recombinant protein strain was obtained by transforming the genes involved in the methanol metabolism of Pichia pastoris,and the gene expression of the strain was analyzed by transcriptomics.The specific research contents and results are as follows:?1?Construction of high expression recombinant protein strain of non-methanol-utilized Pichia pastorisThrough the Cre/lox gene editing system,the genes related to methanol metabolism regulation were successively knocked out,and?AOX1-WT,?AOX1/2-WT,?AOX1/2-?Mig1-WT,?AOX1/2-?Mig1/2-WT,?AOX1/2-?Mig1/2-?Nrg1-WT were obtained.The strain of?AOX1/2-WT had the best expression ability of recombinant protein in BMGYM medium mixed with 0.4%glycerol and 0.5%methanol.The expression of EGFP was 53.6%higher than WT in BMMY.It showed that after knocking out the AOX gene,it was beneficial to improve the ability of synthesize recombinant protein in Pichia pastoris.?2?Transcriptomic analysis of high expression recombinant protein strains of non-methanol-utilized Pichia pastorisThe expression of genes involved in the metabolic pathway of?AOX1/2-WT were analyzed by transcriptomics.According to the results of differential expression gene enrichment analysis,the most significant changes in gene expression levels were the cellular metabolic pathways and gene information processing pathways,and the expression levels of most of these genes were up-regulated.The expression levels of glycerol transporter GT1 and glycerol degradation pathway-related genes were down-regulated in microbial cells,for alleviating the inhibitory effect of glycerol on P_AOX1,and achieving the expression of P_AOX1 in the presence of glycerol.In the cellular metabolic pathway,precursors,cofactors and energy substances were sufficiently provided by up-regulating the glycolytic pathway and the non-oxidative pathway in the PPP pathway,as well as down-regulating the TCA cycle.The expression level of the protein also was increased by up-regulating the expression level of ribosomal proteins,the genes involved in protein processing and secretion in the endoplasmic reticulum of the protein translation stage.