The Functional Analysis Of Pichia Pastoris Transcription Factor Fhl1p And Its Mechanism On Improvement Of Foreign Protein Production

Pichia pastoris has been widely used as the cell factory for the efficient foreign proteins from fungi,bacteria,plants,mammalian cells and viruses.More than 5000 proteins have been generated in Pichia pastoris.Current research focuses on the effect of gene transcription,post-translational modification,transport and secretion on the foreign protein production.However,no related research was reported to illustrate the connection between protein translation and foreign protein production.As we all know,protein can be biosynthesized by ribosome and the translation efficiency of m RNA significantly affects the final yield.However,overwhelming m RNA level will result in pressure for translation and hamper the process of protein production.Hence,it is promising to enhance the protein yield by improved translation efficiency.Forkhead transcription factor Fhl1p from Saccharomyces cerevisiae regulated the expression of ribosomal proteins and r RNA,impacting the ribosome biosynthesis and translation state.A novel Fhl1p in Pichia pastoris can be a positive candidate to regulate the foreign protein expression.Therefore,it is critical to evaluate the function of Pichia pastoris Fhl1p(encoded by PAS＿chr4＿0980),which was a transcription factor mined by transcriptome data.To learn how the Fhl1p worked on foreign protein synthesis and its function in system,a mutant constructed by deleting whole FHL1 gene was investigated on cell growth,ribosomal protein expression,r RNA biosynthesis and translational state.When Fhl1p was overexpressed,the foreign protein was tested,and the role of Fhl1p on ribosome biosynthesis and protein translation was explored by transcriptome and ribosome profiling.To dissect the function of Fhl1p,its target genes were identified by chromosome immunoprecipitation high-throughput sequencing technology(ChIP-Seq)to help in-depth research and make it realistic to apply Fhl1p in protein expression.Details are as follows:(1)The transcription factor Fhl1p containing canonical forkhead domain in 3D structure was a member of FOX family.The biomass of mutant with FHL1 deleted was 34%,14% and32% of that of the wild-type strain in glucose,glycerol and methanol,respectively.The contents of 25 S r RNA,18 S r RNA and 5.8S r RNA decreased by 31%,43% and 93%,respectively,in mutant.The synthesis of ribosome-related components decreased significantly when FHL1 was knocked out.Meanwhile,the mutant was sensitive to cycloheximide,paromomycin,and anisomycin,showing the structure of ribosome was impaired and the translation activity was low.When red fluorescent protein m RFP was expressed,the production level decreased by 28%.In conclusion,Fhl1p deletion affected cell growth and foreign protein production by destroying ribosome structure and biosynthesis,which is similar to that of Saccharomyces cerevisiae ribosomal protein Fhl1p.(2)The growth and translation can be improved by overexpressing Fhl1p.When reporter proteins such as pectinase,phytase and m RFP expressed in host cells with Fhl1p overexpression,all proteins increased by 20-35%.Further transcriptome analysis showed that significantly changed pathways of pectinase strain with Fhl1p overexpression mainly involved ribosomal biosynthesis,m RNA surveillance pathway,RNA polymerase,endocytosis,cell cycle,regulation of mitophagy-yeast,pyrimidine metabolism,propanoate metabolism and TCA cycle,etc.,among which the rich factor of ribosomal biosynthesis pathway was the highest.Meanwhile,the bioinformatics tool RSAT was used to predict and obtain 2245 genes with GACGC motif sequence in promoter.The 101 biological processes collected were same in transcriptome,proving that up and down regulation of genes were due to Fhl1p overexpression.Ribosome profiling was used to assess the translation efficiency of pectinase strain,demonstrating that Fhl1p overexpression can significantly change the quantity of polyribosomes.Thus,overexpression of Fhl1p help regulate translation-related pathways,improve ribosome biosynthesis,enhance the translation efficiency.(3)ChIP-Seq was applied to gain target genes of Fhl1p.A total of 63 potential genes were found and CIT1 and BRN1 genes were up-regulated in transcriptome.EMSA confirmed that CIT1 and BRN1 were the real target of Fhl1p,indicating Fhl1p regulated TCA cycle and cell cycle pathways.When BRN1 and CIT1 overexpressed respectively in mutant,the growth and protein production defect can be restored,respectively,showing poor growth originated from cell cycle disbalance and CIT1 played an important role in protein generation.Moreover,CIT1 made TCA cycle indispensable in recombinant proteins expression.Above information demonstrated that BRN1 and CIT1 can regulate cell division and metabolism to influence cell growth and foreign proteins expression.In summary,the mechanism of foreign protein improvement with Fhl1p overexpression was analyzed.It was found that overexpression of Fhl1p can promote the expression of genes in translation-related pathways,increase ribosome biosynthesis and boost the translation efficiency.Meanwhile,it can directly up-regulate BRN1 and CIT1 genes and accelerate the generation of foreign proteins.This study provided a theoretical basis for improving foreign protein production by translation regulation in Pichia pastoris or other hosts.