Transcriptome Study Of Pichia Pastoris Based On RNA-Seq Technology And Excavations Of Novel Expression Elements

The Pichia pastoris expression system is a highly effective and versatile system for theexpression of various recombinant heterologous proteins, which is widely used as abioengineering platform for producing industrial and biopharmaceutical proteins, studyingprotein expression and secretion mechanisms, and analyzing metabolite synthesis andperoxisome biogenesis. With the development of DNA microarray and mRNA sequencetechnology; the P. pastoris transcriptome has become an important tool to identify thetranscriptional regulation model of the cells under protein production conditions. Due to thelack of a reported P. pastoris genome sequence and related DNA microarrays, thetranscriptome of P. pastoris has not been deeply studied. Herein, the high-throughput RNAsequencing technology (RNS-Seq) was utilized to interrogate P. pastoris transcriptome.RNA samples of P. pastoris were sequenced by RNA-Seq technology. The obtainedsequence fragments (reads) represent1200-fold P. pastoris genome lengths, which wascomplete enough to accurately annotate transcript structures. The P. pastoris genome-widetranscriptome map was depicted according to the reads matched in P. pastoris genome andgenes.4907of the total5313P. pastoris protein-coding genes had transcriptional activitybased on the RPKM value.The transcript structures of P. pastoris genes were analyzed on the genome scale based onRNA-Seq data. The results defined or extended5’UTRs for914transcribed genes and3’UTRsfor924transcribed genes in P. pastoris, and the length distribution of UTRs and theirfunctional categories were studied.291upstream open reading frames (uORF) and15upstreaminitiation codons (uATG) were detected in the identified5’UTRs. The results also identified27novel transcripts and4new exons.Alternative splicing (AS) of P. pastoris gene transcriptswere analyzed on the genome scale based on RNA-Seq data.270AS events were detected in254P. pastoris genes (11.91%of all P. pastoris genes). Based on the comparison of theamount and the length distribution between retained introns (RIs) and cassette exons (CEs), it issuggested that P. pastoris might perform splice site recognition predominantly by the introndefinition (ID) mechanism.Differentially expressed genes were identified based on the negative binomial distributionwith variance and mean linked by local regression. In total,1885genes were differentiallyexpressed under SDEM culture compared with SDEG culture,940up-regulated genes and945down-regulated genes. Compared with the SDEG culture, in the SDEM culture, GO analysis and KEGG metabolic pathway analysis revealed that up-regulated genes were specificallylocated in proteasome, autophagy, phagosome, thiamine metabolism, N-glycan biosynthesis,methane metabolism, protein processing in the endoplasmic reticulum, and protein exportpathways. Genes involved in biosynthesis of unsaturated fatty acids and citrate cycle weredown-regulated. These results described the metabolic characteristics of heterologous proteinproduction of P. pastoris with methanol induction.Based on the RNA-Seq data, we excavate the expression elements of P. pastorisexpression system. Three endogenous signal peptides were identified and the secretionefficiency of them was higher than α-factor. Two IRES elements were also identified in the5’UTR of P. pastoris genes. Besides, three inducible promoters were obtained based on thedifferential expression analyse.