Regulation Networks Of Transcription And Metabolism In A Novel Methanol Free Expression System Of Pichia Pastoris

Pichia pastoris is one of the most widely used hosts for heterologous expression of various recombinant proteins.In P.pastoris,the AOX1 promoter is frequently used for protein expression because it leads to high protein productivity in response to methanol.However,the use of methanol in industrial fermentation is not welcome.It is easily flammable and its catabolism causes abundant heat-releasing,which requires the design of an explosion-proof plant and the supply of increased oxygen.Our lab is working to elucidate the regulation mechanism of AOX1 promoter,and has developed several methanol free expression systems in P.pastoris.Nevertheless,lack of full data of regulation network in P.pastoris,the research results of other yeast species are tried and the large-scale mutation screening method is used to develop AOX1 promoter methanol free expression system so far.These approaches are time-consuming and laborious,and there are some limitations.With the development of mRNA sequencing and metabolites high-throughput analysis technologies,transcriptomics and metabolomics become important tools in studies of the transcriptional and metabolic regulatory networks,strain improvement,and reconstruction of expression system.Although there are some studies in P.pastoris involving DNA chip,mRNA sequencing,metabolism and metabolic flow,the regulatory networks of AOX1 promoter used by system biology has not been reported in P.pastoris.In this work,transcriptional and metabolic regulatory networks of a novel methanol free expression system of P.pastoris were studied by RNA-seq technology and LC/MS-GC/MS platform.By comparative analysis of transcriptome and metabolome between the wild-type strain and new methanol free strain,a number of genes associated with the regulation of AOX1 promoter were identified,and the transcriptional and metabolic regulatory networks were thoroughly understood in this new strain.Based on the basic research of two catabolite repressors Mig1 and Mig2 in P.pastoris,RNA-seq is used to compare the map of transcriptome at the whole genome level between the wild-type strain and carbon derepressed strain ?mig1?mig2.RNA-seq results showed that the expressions of 357 genes were significantly up-regulated in ?mig1?mig2,accounting for 7 percent of the total 5,276 expression genes.The up-regulations of AOX1 and AOX1 expression levels were the highest,as more as 32-fold and 33-fold,respectively.Metabolic pathway analysis revealed that up-regulated genes were specifically located in methanol metabolism and peroxisome biogenesis pathway.The expressions of 1,197 genes were significantly down-regulated,accounting for 23 percent of the total expression genes,which were mainly involved in the mitochondria-related metabolic pathways.The expression of a transcriptional regulation factor,Mit1,was significantly improved in ?mig1?mig2.Moreover,the expression levels of AOX1 and MIT1 in different time points showed that their expression patterns were highly relevant,suggesting that Mit1 might be a regulatory factor of AOX1 promoter.The transcriptome and metabolome of the wild-type strain and the mutant strain?mig1?mig2?nrg 1-Mitl were analyzed using RNA-seq and LC/MS-GC/MS platform.From the high-throughput transcriptome results,9 types of alternative splicing events were detected and 62 novel transcripts identified.The expression levels of the genes related to methanol utilization and peroxisome biosynthesis were significantly up-regulated in the mutant strain which was in response to glucose or glycerol.The cluster analysis of transcriptome data suggested that a number of genes were closely related with the expression of AOX1,and especially P.pastoris could not grow in methanol with the deletion of the gene PAS_chr2-1₀748.Metabolomics data showed that the amino acid and energy metabolism pathways in the mutant strain grown in glucose,glycerol or methanol increased significantly in comparison with the wild-type strain.Although multiple genes were modified in the mutant,the metabolic pathways of P.pastoris were not diminished,and the expression of foreign proteins improved.In this study,the regulation mechanism of AOX1 promoter in P.pastoris was studied by systems biotechnology.This approach provided a wealth of valuable information about the regulation mechanism of AOX1 promoter.With the guidance from the information,strain improvement will be followed up.