Enhancement Of Phytase Expression Based On Gene Knockout Of Pichia Pastoris Autophagy And MAPK Pathway

Due to its high efficiency of methanol-induced promoter alcohol oxidase 1(P_AOX1),P_AOX1has a very strict regulatory mechanism and can strictly regulate the secretion and expression of exogenous proteins.Pichia pastoris(P.pastoris)is the popular used expression system that eukaryotic,as an pivotal way to produce exogenous proteins in industrial application.However,due to the large difference in the expression levels of different exogenous proteins,how to reform the P.pastoris expression system and improve the expression efficiency of target proteins in the yeast system is an urgent problem to be solved.Phytase,as an important feed enzyme preparation,can hydrolyze phytate phosphorus in feed,release inorganic phosphorus and inositol,which can be directly used by livestock and poultry.Phytase addition in feed can not only improve the utilization rate of phosphorus to livestock and poultry,promote the growth,metabolism and reproduction of livestock and poultry,but also reduce the pollution of phytase emission in the environment.Therefore,it is extremely important to explore and optimize the expression system of P.pastoris and realize the efficient production and industrial application of phytase by using the expression system of P.pastoris.Based on the transcriptome analysis of high level phytase expression P.pastoris strain GS115/PPIC9K-Phy A with RNA-seq technology,we found that the Autophagy signaling pathway and MAPK signaling pathway genes were significantly upregulated during methanol induction in P.pastoris,which recognized that these two metabolic pathways have important effects on the expression of foreign proteins.Using the efficient CRISPR/Cas9 editing system constructed by our laboratory,key up-regulated genes in Autophagy and MAPK pathway were knocked out,and then the effects of the missing genes on the growth and metabolism of P.pastoris and the secretion and expression of exogenous proteins were investigated.The results of this study are as follows:Autophagy signaling pathway:(1)Using CRISPR/Cas9 editing system,7 autophagy pathway gene deletion strains were successfully constructed:?PAS?chr2-2?0131??PAS?chr2-1?0203??PAS?chr1-1?0348??PAS?chr3?0637??PAS?chr1-1?0096??PAS?chr3?0092??PAS?chr4?0093??PAS?chr2-1?0263??PAS?chr1-3?0159??PAS?chr4?0986??PAS?chr4?0584??PAS?chr1-3?0225??PAS?chr1-3?0307??PAS?chr3?0673??PAS?chr3?0618??PAS?chr3?0861??PAS?chr1-1?0165??PAS?chr1-4?0069??PAS?chr3?0840.(2)The growth of the gene deletion strain was measured in three different carbon sources:glucose(YNA),glycerin(YNG)and methanol(YNM).The results showed that the deletion of PAS?chr3?0964?PAS?chr1-4?0555?PAS?chr1-1?0097 genes promoted the growth of the strain in glycerol.The deletion of PAS?chr2-1?0641?PAS?chr3?0964?PAS?chr1-1?0353 genes promoted cell utilization of methanol at the late stage of methanol metabolism.The deletion of PAS?chr1-1?0097 gene inhibited glucose utilization.(3)Phytase expression was compared between the deletion strain and the wild type strain.The results showed that the deletion of PAS?chr2-1?0641?PAS?chr4?0704?PAS?chr1-1?0353?PAS?chr1-4?0555 genes increased the phytase expression in P.pastoris.The phytase activity of PAS?chr2-1?0641 deletion strain was384.28U/m L,which was 2.09 times higher than that of the wild type.The enzyme activity of PAS?chr1-1?0353 deletion strain was 440.87U/m L,2.4 times higher than that of PAS?chr1-1?0353 deletion strain.(4)The transcription level of P_AOX1promoter gene AOX1 was detected.The results showed that phytase expression was positively correlated with AOX1 gene transcription level.AOX1 expression of PAS?chr2-1?0641 deletion gene was increased at 2h,4h and 42h after methanol induction.The AOX1 expression of PAS?chr1-1?0353 gene was significantly higher than that of the wild type during methanol induction.MAPK signaling pathway:(1)Using CRISPR/Cas9 editing system,19 MAPK pathway gene deletion strains were successfully constructed:?PAS?chr2-2?0131??PAS?chr2-1?0203??PAS?chr1-1?0348??PAS?chr3?0637??PAS?chr1-1?0096??PAS?chr3?0092??PAS?chr4?0093??PAS?chr2-1?0263??PAS?chr1-3?0159??PAS?chr4?0986??PAS?chr4?0584??PAS?chr1-3?0225??PAS?chr1-3?0307??PAS?chr3?0673??PAS?chr3?0618??PAS?chr3?0861??PAS?chr1-1?0165??PAS?chr1-4?0069??PAS?chr3?0840?(2)The growth of gene deletion strain in different carbon sources of glucose,glycerin and methanol was measured.The results showed that the deletion of PAS?chr2-2?0131 gene promoted glucose metabolism most obviously,followed by methanol metabolism.The deletion of PAS?chr1-3?0307?PAS?chr3?0861 genes significantly increased the utilization rate of glycerol.The deletion of gene PAS?chr3?0840 adversely affected the growth of the strain in glucose and methanol.(3)Phytase expression was compared between the deletion strain and the wild type strain.And it turns out,PAS?chr2-2?0131?PAS?chr2-1?0203?PAS?chr1-1?0348?PAS?chr3?0637?PAS?chr4?0093?PAS?chr2-1?0263?PAS?chr4?0986?PAS?chr1-3?0307?PAS?chr3?0673?PAS?chr3?0861?PAS?chr1-1?0165 genes increased the phytase expression.The enzyme activity of PAS?chr3?0637 deletion strain was 316.01 U/m L,which was 2.09 times higher than that of the wild type.The enzyme activity of PAS?chr3?0861 deletion strain was 254.40 U/m L,1.68 times higher than that of PAS?chr3?0861 deletion strain.(4)The transcription level of P_AOX1promoter gene AOX1 was detected.The results showed that the transcription level of AOX1 gene in PAS?chr3?0637?PAS?chr3?0861 deletion genes with increased phytase activity also increased.In conclusion,this study provided theoretical reference for further improving the expression level of exogenous proteins in P.pastoris by modifying key genes of autophagy signaling pathway and MAPK signaling pathway.