Studies On Promoter And Internal Ribosome Entry Site (IRES) In Pichia Pastoris

Pichia pastoris has become an important industrial microorganism because of its fast growth,high-density fermentation,simple genetic manip?lation and post-translational modification.After the genome sequencing,Pichia pastoris is not limited as the expression system of heterologous proteins,but gradually applied to synthetic biology and metabolic engineering.Whether it is used for heterologous protein expression,synthetic biology or metabolic engineering,the development of gene expression elements is essential for Pichia pastoris system.Based on the idea,this study focused on promoters and internal ribosome entry site(IRES)screening,and we hoped to screen some functional promoters and IRES elements which can used in Pichia pastoris.According to transcript sequencing data of Pichia pastoris,the high transcriptional genes in Pichia pastoris were analyzed,and 18 candidate genes were identified(ACO1,ALD4,ATO2,ATP1,ATP2,CIT1,CTA1,FDH1,HSP12,ICL1,OLE1,PCK1,POR1,QCR7,RGI2,SSA4,TDH3,TMA10).Primers were designed to amplify the promoter regions of the 18 genes,and amplified flagments were cloned into the pPICZA expression vector of Pichia pastoris in which the AOX1 promoter was already removed.Using the Lac Z as a reporter gene,the transcriptional activities of promoter regions were analyzed.The res?lts showed that the ALD4,ATP1,OLE1,PCK1,RGI2,SSA4,TDH3,and TMA10 promoter regions had higher transcriptional activity.Compared with the GAP promoter,the transcriptional activities of the ALD4,ATP1,OLE1,POR1,RGI2,SSA4,TDH3,and TMA10 promoters were higher by 311.7%,104.0%,187.0%,177.4%,266.3%,329.7%,218.2%,169.4%,respectively.To Microbial growth,carbon sources have a large impact on its metabolism.Therefore,the transcriptional activities of the ALD4,ATP1,ATP2,FDH1,OLE1,PCK1,POR1,RGI2,SSA4,TDH3,and TMA10 promoters were analyzed in the medium containing glycerol,glucose,methanol,and sorbitol as a only carbon source,respectively.The res?lts showed that ATP1 had the highest transcription levels in the medium containing glycerol,and ALD4,RGI2,SSA4 and TMA10 had the highest transcription levels in the medium containing glucose,and ATP2,FDH1 and TDH3 had the highest transcription levels in the medium containing methanol,and OLE1,PCK1 and POR1 promoters had the highest transcription levels in the medium containing sorbitol.The promoters(ALD4,ATP1,ATP2,FDH1,OLE1,PCK1,POR1,RGI2,SSA4 and TMA10)with high transcriptional activity was analyzed for secreted expression using EGFP as reporter.The res?lts showed that the promoters of ATP1,ATP2,FDH1,OLE1,RGI2,SSA4,and TMA10 had higher expression levels,and the expression levels of ATP1,ATP2,FDH1,RGI2,SSA4,and TMA10 promoters were 303.3%,283.7%,250.0%,145.7%,233.3%,and 115.3% higher than the commercial GAP promoters,respectively.Analysis based on four carbon sources revealed that the FDH1 promoter can be induced by methanol similar to the commercial promoter AOX1.The activities of FDH1 and AOX1 promoters in both intracell?lar and extracell?lar expression were compared.The res?lts showed that the FDH1 promoter was superior to the AOX1 promoter both for intracell?lar and extracell?lar expression,and the transcription of FDH1 promoter was not inhibited by glycerol.The disadvantage is that FDH1 promoter has a higher background expression before induction compared to the AOX1 promoter.On the IRES study,34 IRESs which function in mammals,insects,plants or Saccharomyces cerevisiae were selected based on published data.The 34 IRESs were analyzed using a bicistronic reporter vector with EGFP and LacZ as reporter genes,respectively.It was found that the LacZ gene can be transcribed in a promoterless expression vector of Pichia pastoris,which co?ld be caused by the crytic promoter in the vector backbone or the LacZ gene.In order to use the LacZ as the reporter in the study,the ?-complement system of the LacZ gene was established in Pichia pastoris based on the ?-complementary property of ?-galactosidase which encoded by LacZ.The 1-92 amino acid of ?-galactosidase in N-terminal was expressed in the bicistronic reporter vector,and the C-terminus(93-1024 aa)of ?-galactosidase was expressed in Pichia pastoris GS115.After screening,a total of 7 IRESs which were functional in Pichia pastoris were obtained,the IRES sequences were TEV,PVY,RhPV,TRV-IGR,KSHV,crTMV viruses and YPA1 gene 5'UTR sequence of Saccharomyces cerevisiae.Among them,the IRESs of TEV and TRV-IGR have the highest translational activity.In this study,the promoters and IRES sequences were developed,which further enriched gene expression elements in the Pichia pastoris system,and provided more reference and selection for the expression of m?ltiple genes in Pichia pastoris.This study is helpf?l to promote the development and improvement of Pichia pastoris system.