| The Pichia pastoris expression system is a highly effective and versatile system forvarious recombinant heterologous proteins expression, which is also used for analysis ofprotein expression and secretion mechanism.The development of gene sequence technologyhas made the P.pastoris transcriptome as the mainly tool to identify gene expressionregulation mechanism under protein production condition. However, gene expression at thetranscript level may not correlate well with protein synthesis levels due to transcription,translation and posttranslational modification. Proteomics is the systematic study of diverseproperties of proteins in a parallel manner with the aim of providing detailed descriptions ofstructure, function and control of biological systems in different protein expression conditions.Therefore, the proteomics has become an important tool to identify protein regulationmechanism during the protein expression. With the rapid development of mass spectrometryand the complete genome sequencing of P.pastoris, the study of proteomic in P.pastoris isavailable. Herein, the isobaric tag for relative and absolute quantification (iTRAQ) wasutilized to investigate P.pastoris proteomic and try to find the heterologous protein secretionregulation mechanism.Gene dosage is deemed to be one of major bottlenecks for foreign protein expression, andmulti-copy strains could drastically enhance the expression level of recombinant proteins inP.pastoris. Firstly, we designed and synthesized the full length of family10xylanase genefrom Bacillus halodurans C125(xyn10) by optimizing the P.pastoris preferred codons.Secondly, we integrated the multi-copy expression cassette based on vector integration intothe rDNA locus of nontranscribed intergenic space(NTS)to screen multi-copy strains. Astrain harboring4copies (G4) has the highest expression of xyn10after two methodsscreening compared with the other multi-copy strain. The expression level of xyn10in thestrain containing four copies is2.84fold as that in single copy strain. Our result showed thatthe production of a secreted heterologous protein can reach a limit with a certain number ofgene copy whereas a further increase of transcription and translation based on higherabundance of gene copies would not enhance the secretion process any more or even decreasethe secretion. Co-overexpression of the spliced HAC1gene in the four copies strain (G4-H) could further improve the xyn10production approximately1.43fold. Therefore, it isimportant to improve the secretion of heterologous proteins in P. pastoris by adjusting genecopy numbers and overexpressing some helper factors. In order to testify the expression levelof xylanase in harvested G4-H strain in fermenter scale, we cultivated G4-H in a2Lfermentor. The fermentation curve indicated that the highest recombinant xylanase activityreached3361U/mL after methanol induction for about132h. Accordingly, the secretoryprotein yield was up to5.2g/L in the culture supernatant.For iTRAQ proteomic analysis, three recombinant strains（G1，G4and G4-H）aftermethanol induction and G0as control strain were conducted. A total of4613unique peptideswere obtained from the iTRAQ-LC–MS/MS proteomic analysis. After a matching analysis ofpeptides with annotated genome and protein information of P. pastoris strain GS115fromNCBI, a total of1167proteins were matched. Approximately352proteins were detected asdifferentially expressed proteins of low expression strain G1, high expression strain G4andco-overexpressing HAC1strain. Compared with the low expression strain, GO analysis andKEGG metabolic pathway analysis revealed that up-regulated proteins were involved incarbon, energy and amino acid metabolism, indicating enough carbon and energy resourceshould be offered to the expression of xyn10. The high expression of xyn10caused a proteinprocessing stress for recombinant P. pastoris cell factories. Proteins involved in proteinprocessing in ER were up-regulated, however, the proteins involved in amino acid andmethanol metabolism were down-regulated. The unfolded protein response was induced byco-overexpression of the spliced HAC1in the high expression strain G4-H. On the one hand,the proteins involved in ER folding and ER resident chaperones were significantlyup-regulated to improve the protein folding efficiency. The proteins involved in proteintransport were also up-regulated to increase the secretion yield. On the other hand, attenuationof protein synthesis involved in ribosome biogenesis was down-regulated to reduce theunfolded proteins entry to ER. Therefore, the strain G4-H might alleviate the proteinprocessing stress in the ER. Simultaneously, the protein levels involved in energy metabolismand amino acid metabolism were up-regulated. However, the co-overexpression HAC1strainwas growing notably slower in minimal methanol medium. The proteomic data showed that areduced ribosomal translation rate may be the primary cause of a growth rate defect in the G4-H strain. |
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