| In recent years,Pichia pastoris has gradually become the one of most commonly used protein expression system.The thousands of proteins have been successfully expressed using this system.Increasing the copy numbers of exogenous genes can fast improve protein secretion level,however,the relation between the copy number and secretion level is not proportional.For example,the heterologous proteins were observed that retained in the intracellular in highcopy strains.Therefore,it is important to investigate the mechanism of intracellular retention and improve the secretion of heterologous proteins in high-copy strains.In our previous work,recombinant strains containing 1,2,4,6 and 8 copy were constructed using EGFP as a reporter gene.The result was indicated that the 2-copy strain had the highest secretion level and the 8-copy strain had the lowest secretion level.Detecting intracellular fluorescence showed that the total fluorescence was proportional to the copy number.The 8-copy strain had the highest fluorescence,but most of the fluorescence was derived from intracellular.Therefore,a large amount of recombinant protein was retained in the endoplasmic reticulum and could not be secreted to the extracellular in 8-copy strain.The aim of this study was to investigate the reasons for the recombinant proteins retaining in the endoplasmic reticulum,and to improve the secretion of the 8-copy EGFP strain by co-expressing downregulated genes in the transcriptome,methanol utilization pathway-related genes,endoplasmic reticulum molecular chaperones,transcription factors,cell cycle proteins and other cofactors.The main results are as follows:1.The down-regulated genes which log2 FC were less than minus 3 and associated with secretion were selected for overexpression according to the transcriptome analysis.The highest secreting 2-copy cell and the lowest secreting 8-copy cell were induced with methanol for 3days and sent to the company for transcriptome sequencing and analysis.Then,genes which log2 FC were less than minus 3 and associated with secretion were selected to be overexpressed in the 8-copy strains,and the results showed that overexpressing the down-regulated genes(Mob1,PAS＿chr1-4＿0570,PAS＿chr2-1＿0659,Fet4,Pry2,Ctr1,Dip5,Yfl040 w,Pet123,PAS＿chr2-1＿0659 and PAS＿chr1-1＿0479)was able to promote EGFP secretion in 8-copy strains,meanwhile,overexpression of PAS＿chr2-1＿0659,Fet4,Pry2 and Dip5 could increase EGFP production by 1.5-fold;2.Overexpressing genes related to the methanol utilization pathway or the pentose phosphate pathway.Proteins synthesis and secretion require a constant supply of energy.Therefore,overexpressing heterologous proteins could impose metabolic stress upon host cells.Enhancing the energy supply may be able to promote EGFP secretion in 8-copy strains.Overexpressing genes related to the methanol utilization pathway or pentose phosphate pathway,which could supply ATP and NAD(P)H.Our results showed that overexpressing transcription factor Gcn5,positive regulator Mxr1,Fdh in the methanol dissimilation pathway and Gnd2 in the pentose phosphate pathway had promote EGFP secretion by approximately 1.3-2-fold.3.Overexpressing molecular chaperones or foldases involved in the secretion pathway.To improve EGFP secretion in 8-copy strains,molecular chaperones or foldases that involved in translocation,folding,glycosylation and vesicular transport were selected for overexpressing.The results indicated overexpressing folding-related molecular chaperones(Ssa1,Aha1,Cne1,GII and Sly1)increased EGFP production by 1.2-1.8-fold,overexpressing molecular chaperones or foldases Ydj1,GII and Sed4 associated with translocation,glycosylation and vesicular transport improved EGFP secretion by 1.4-fold;4.Overexpressing the transcription factors.The transcription factors Gcn4,Hsf1 and Aft1 are involved in amino acid synthesis,endoplasmic reticulum protein secretion and energy production.Overexpressing Gcn4,Hsf1 and Aft1 increased EGFP secretion by about 2-fold;In conclusion,we have obtained helper factors that can enhance secretion.And the next step will be to combine the positive helper factors which can promote secretion to overexpress in the 8-copy strain.Although energy enhancement can enhance secretion,the promotion effect is limited in this study.Therefore,the enhanced energy needs to be directed to the endoplasmic reticulum for folding or secreting.The new cofactors obtained in this study provide additional options for optimizing the secretory expression of heterologous proteins in Pichia pastoris. |
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