Gene Mining And Function Analysis Of GPI-modified Cell Wall Proteins In Pichia Pastoris

The Pichia pastoris cell surface display system is a kind of rapidly growing andextensive applied eukaryotic cell expression system. Various enzymes were immobilized onPichia pastoris cell surface as whole-cell biocataysts. The whole-cell biocataysts possess thecharacteristic and advantage of immobilized enzyme. Furthermore the preparation method issimple and they are easy to recycle and reuse. By far, most of the anchoring proteins of P.pastoris cell surface display were from Saccharomyces cerevisiae which were covalently ornon-covalently linked to the glucan layer of the cell wall. There are two types cell wallproteins which were covalently linked to the cell wall, Pir-CWPs (proteins with internalrepeats) and GPI-CWPs (glycosylphosphatidylinositol-modified cell wall proteins), and theGPI-CWPs attracted more attention. The genome sequencing results of Pichia pastoris havebeen published in2009, and the gene annotations have not been completed. Very fewendogenous GPI-CWPs of P. pastoris have been confirmed and used in P. pastoris cellsurface display. In this study, GPI-CWPs of P. pastoris were screened for and confirmed andthese proteins were used as anchoring proteins to display CALB on the cell surface. Then,co-displaying systems with various anchoring proteins were constructed and the function ofGCW14were studied preliminarily. The specific research content is list below.(1) Screening for and confirmation GPI-CWPs in Pichia pastorisAccording to the characteristics of the yeast GPI-anchored proteins,50potentialGPI-anchored proteins of Pichia pastoris were obtained. Then they were used as anchoringproteins to display CALB (Candida antarctic lipase B) on the cell surface.13GPI-CWPswere confirmed by flow cytometry and western blot analysis. The CALB hydrolytic activityby each recombinant was measured. The results showed that the hydrolytic activity of CALBcould be detected on the cell surface of the13recombinants and the level of CALB activityvaried with the anchored proteins fused. The highest hydrolytic activity of CALB displayedon the cell surface were found in the recombinants based on Gcw12p, Gcw21p, Gcw51p andGcw61p proteins, reaching1538U/g,1828U/g,1889U/g and2234U/g cell dry weight,respectively. (2) Construction and application of co-displaying systemspPICZαA is used to generate multiple expression cassette copies in a single vector priorto transformation into Pichia pastoris. Then two different selection marker were used to selectrecombinants with1-4copy numbers. A strain harboring8copies based on Gcw61p wasobtained. All recombinants were induced by methanol to144h and the growth curve showedthat the growth of recombinants were inhibited with the increase of copy numbers. Genedosage 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. In this study, a strain based on same anchoring proteins harboring2copies(GS115/CALB-GCW (61+61)) has the highest expression of CALB, reaching4451U/g celldry weight. A strain based on different anchoring proteins harboring3copies(GS115/CALB-GCW (12+51+61)) has the best efficiency of CALB, reaching4493U/g celldry weight. The hydrolytic activity of the co-displaying recombinant depends on thedisplaying efficiency of anchoring protein and Gcw61p has best efficiency to display CALBThe amount of fusion proteins displayed on cell surface of recombinants and the activities ofdisplayed enzymes are not completely corresponding. The space of the cell surface is limitedand not all of the displayed enzyme molecules can show catalytic activity. The cryo-electronmicroscopy was used to observe the structure of the cell wall and the staining method wasused to analyze the change of the cell wall components. The results indicated that the glucanlayer of recombinant displaying fusion protein CALB-Gcw51p has no significant change. Butthe mannoproteins layer made very large changes in the content and structure. Thesephenomena suggesting that the fusion protein CALB-Gcw51p was displayed on P. pastorissurface efficiently and the displaying expression changed the content and structure of cell wallmannoproteins layer.(3) A preliminary study of Gcw14p functionIn the laboratory previous transcriptome data, Gcw14p has the highest transcriptionlevel with glycerin or glucose as carbon source. Therefore Gcw14p was first selected to studyits function by gene knock out and overexpression foreign proteins. When the GCW14wasknocked out (GS115/ΔGCW14), the growth of strain was inhibited. Staining method and measuring of cell wall components revealed that the glucan and the chitin content wereincreased. Three different foreign proteins (CALB, s7-XYN and EGFP) were expressed inGS115/ΔGCW14and the results showed that the activity or fluorescence intensity decreasedcompared with the control strain GS115. These phenomena suggesting that the cell wallglucan layer were incrassated after knock out GCW14, which caused certain obstacles to thesecretion of heterologous proteins. So GCW14gene may participate in controlling the contentof cell wall inner components.