| Proteins(short chain peptides)have been of great interest for applications in disease detection and therapy,enzymes,chemicals and bioenergy.In recent years,extracellular expression of proteins by microorganisms has become a new R&D hotspot due to its unique advantages.Saccharomyces cerevisiae,as a typical eukaryotic model bacterium,has a clear genetic background,fast growth rate,complete post-translational modification system,high robustness and food-grade biosafety,making it a leader in microbial extracellular protein expression research.However,the weak extracellular expression of S.cerevisiae proteins has limited its application.The polarization pathway of S.cerevisiae regulates vesicular transport,which is an important part in protein extracellular expression.And the improvement of vesicular transport can effectively enhance protein extracellular expression.At the same time,moderate defects in the cell wall also promotes the extracellular expression of proteins in S.cerevisiae.To this end,this thesis focuses on these two biological pathways as the starting points,engineering the polarization and cell wall synthesis pathways to improve the extracellular expression of S.cerevisiae,using secretion and surface display as the extracellular expression modes.The main progresses are,I.Engineering the polarization pathway to promote extracellular expression of protein in S.cerevisiae.1.Construction of extracellular expression system of S.cerevisiae proteinUsing S.cerevisiae CEN.PK 113-5D as a chassis strain,knockout TPI1(the encoding gene of triose-phosphate isomerase)to release its glucose utilization and plasmids with the POT1 as a marker for backfilling glucose utilization.POT1 encodes the triose-phosphate isomerase in S.pombe.Glucose utilization was used to establish an extracellular expression system of α-amylase.The INU1 signal peptide from Kluvveromvces lactis was selected and ligated to the N-terminal end of the reporter protein α-amylase and placed in the strong promoter TDH3 to construct the protein secretory expression system.Further,the SED1 anchor protein was attached to the C-terminus of α-amylase to construct the protein surface display expression system in S.cerevisiae.2.Screening of polarization pathway genes that promote protein extracellular expressionThirteen essential genes related to axial budding,bipolar budding and cytoskeleton synthesis during cell polarization were selected.These genes were introduced into the above secretion and surface display expression systems one by one by single-copy plasmids,and the results showed that the enhancement of several genes could enhance the extracellular expression of α-amylase,but the trend of the effect on these two expression systems was not completely consistent,only the RAS protein GTPase gene BUD1 and the cell membrane protein gene BUD10 could enhance the extracellular expression of in both surface display and secretion expression systems.Further,the integration of these two genes into the genome,respectively,showed that the stable overexpression states of BUD1 and BUD10 elevated total α-amylase activity by 56.1% and 37.2%,respectively,in the surface display system;and by 49.7% and20.4%,respectively,in the secretory expression system.Thus,it can be seen that the overexpression of BUD1 has the most significant promotion effect on the extracellular expression of α-amylase in both systems.3.The combination of BUD1 and polarization pathway genes enhanced the promotion of protein extracellular expressionBased on the above genomic integration of BUD1,13 polarization pathway genes were introduced separately by single-copy plasmids one by one,and some genes in both extracellular expression systems could further promote α-amylase extracellular expression.The genes with significant enhancement effect were selected to be integrated into the genome as well.In the protein surface display system,BUD7 and CDC42,in concert with BUD1,elevated total α-amylase activity by 14.1% and 24.6%,respectively.In the secretion expression system,the increase of BUD1’s own copy number increased the total α-amylase activity by 31.2%;while BUD1 synergized with BUD10 also increased the total α-amylase activity by 25.3%.Compared with the parent strain,the best combination of double genes overexpression increased the totalα-amylase activity by about 100.0% in both extracellular expression systems.4.The above best combination of double genes overexpression also promoted the extracellular expression of β-glucosidase in both systems,and the overexpression of the best combination of surface display(BUD1 and CDC42)also promoted the extracellular expression of the protein of the surface display systems constructed with AGA1 and DAN4 anchored structural domains.II.Moderate impairment of cell wall synthesis promotes extracellular expression of proteins in S.cerevisiae.1.Knockout the gene of cell wall synthesis pathway affects protein extracellular expressionSeventy-nine strains that single knocked out gene directly involved in cell wall synthesis were selected from the BY4742 single-knockout library.β-glucosidase and SUC2 signal peptide derived from S.fibuligera were placed under the control of TEF1 promoter and PGK1 terminator to obtain plasmid p JBGL1.The plasmid p JBGL-L-SED1 was obtained from the C-terminal of β-glucosidase by linker binding to SED1 anchor protein,and the above two plasmids were introduced into the extracellular expression system of β-glucosidase respectively.The results of enzyme activity analysis showed that 19 gene singly knockouts promoted the extracellular expression of β-glucosidase(BGL1)in the secretion system,among which Ypk1Δ,Dfg5Δ,Fyv5Δ and Ccw12Δ increased the enzyme activity by 109.2%,89.3%,78.4%and 41.0%,respectively.In the surface display system,26 gene singly knockouts promoted the extracellular expression of β-glucosidase,in which Ypk1Δ,Dfg5Δ,Fyv5Δ,and Kre1Δ elevated the total enzyme activity by 182.1%,167.0%,147.4%,and 62.9%,respectively.The extracellular expression of β-glucosidase was significantly promoted by Ypk1Δ,Dfg5Δ and Fyv5Δ in both extracellular expression systems,and Ypk1Δ had the greatest effect on the extracellular expression of both systems.2.Double knockout genes of cell wall synthesis pathway promotes protein extracellular expressionThe double knockout of YPK1,DFG5,FYV5,CCW12 and KRE1,which were able to promote protein expression in different systems.Compared with Ypk1Δ,in the secretion and surface display system,Dfg5ΔYpk1Δ increased the total β-glucosidase activity by 62.70% and 50.1%,respectively;Compared with Fyv5Δ,Ccw12ΔFyv5Δincreased the total β-glucosidase activity by 203.0% and 148.4%,respectively.Contrary to expectations,Ccw12ΔFyv5Δ,an independent combination of Fyv5Δ,had the most significant effect on extracellular protein expression,and the totalβ-glucosidase activity in secretion and surface display systems increased by 159.0%and 118.0%,to 522.0 and 297.1 U/g DCW,respectively.The above genes deletion had different effects on cell wall integrity Ccw12Δ was unable to grow on Congo red plate,Fyv5Δ was almost the same as the wild type,and the state of Ccw12ΔFyv5Δ grew on the Congo red plate between Ccw12Δ and Fyv5Δ.The results showed that Fyv5Δ had little effect on the cell wall integrity and could repair the cell wall damage caused by.Ccw12Δ some extent.The overall expression of Ccw12ΔFyv5Δ was inhibited,but the average extracellular protein expression capacity of each cell was significantly improved,and the total β-glucosidase activity in the secretion expression system was increased from 101.1 to 522.0 U/g DCW,an increase of 441.7%.The totalβ-glucosidase activity of the corresponding surface display system was increased from48.4 to 297.1 U/g DCW,by 513.6%.Volume enzyme activity increased by 347.4% and441.5%,from 0.3 and 0.2 U/m L to 1.5 and 1.1 U/m L respectively in secretion and surface display systems.These results indicated that moderate cell wall integrity damage was beneficial to the extracellular expression of proteins,and although the cell growth was inhibited,the extracellular expression production of proteins was significantly increased.3.Ccw12ΔFyv5Δ promoted the extracellular expression of the surface display system which constructe with AGA1 and DAN4 anchored structural domains,and Ccw12ΔFyv5Δ also had a significant effect on the extracellular expression ofα-amylase and cellobiohydrolase. |
PDF Full Text Request