Construction And Performance Analysis Of GPI Protein Deficient Strain Library In Pichia Pastoris

The GPI proteins are a type of proteins that necessary for cell survival and maintenance of normal cell morphology in yeast.In order to study the functions of GPI proteins in Pichia pastoris,the strains library with deleted GPI protein in Pichia pastoris was constructed and analyzed.The possible functions of GPI protein were inferred by phenotypic screening.A methanol-tolerant strain was screened and analyzed to understand the response of the strain and the function of the missing protein under methanol stress by proteomics and transcriptome methods.On the basis that the hydrophobicity of the immobilized carrier can significantly affect the activity of lipase,two methods of improving the surface hydrophobicity,including the screening from the deleted strain library and modification of cell surface with hydrophobins,were used to improve the lipase activity.The specific research content is list below.(1)Using the deleting system called Cre/loxP recombinant enzyme to delete 50 potential GPI proteins in Pichia pastoris,finally a strain library containing 45 strains was constructed.It was found that the knockout GPI proteins were not necessary for cell growth.According to the phenomenon that absence of cell wall protein can cause the remodeling of cell wall structure,five strains with significantly increased methanol tolerance,four strains with higer polysaccharide content in cell wall and four strains with significantly improved surface hydrophobicity were screened from the strain library.They all have the potential application.According to the phenomenon of cell wall structure remodeling that may be caused by the absence of protein,Five strains with significant increase in methanol tolerance were screened from the missing strain bank.4 strains with significantly increased cell wall polysaccharide content and 4 strains with obvious surface hydrophobicity were selected,which have potential application value.(2)The cell wall proteins of Pichia pastoris were analyzed by proteomics,and finally 164 cell wall proteins of were identified.Their bioinformatics analysis has enriched the information of Pichia pastoris cell wall protein.The characteristics of cell wall protein with methanol tolerant strain gcw19? response to methanol stress were studied.A large number of proteins with unknown functions,cell wall,stress response,protein synthesis and fate,metabolism and proteins interacting with environment were found to have changed significantly.The response of these proteins resulted in the formation of a new surface protein system in strain gcw19? under methanol stress,and the strain could adapt to the environment better.(3)Comprehensive analysis the gene expression of methanol tolerant strain gcw19? by transcriptome.It was found that the gene transcription level of most predicted GPI proteins in strain gcw19?,were significantly up-regulated under methanol stress,resulting in the cell surface protein remodeling.In addition,the changes of amino acid metabolism pathway in strain gcw19? were also obvious.It showed that strain gcw19? was favorable to the absorption of extracellular amino acids into the cell,which indicated that the GCW19 protein was an obstacle to the absorption of amino acids.The experiment results also showed that the absorption and utilization of amino acids by strain gcw19? was stronger than that of the control strain GS115.Other pathways such as cell wall synthesis,cell cycle pathway,sterol synthesis,protein biosynthesis,cell stress have also been found differentially altered genes.The co-regulation of these pathways enable the strain gcw19? to better adapt to methanol stress.(4)Strains gcw13?,gcw22?,ku70? gcw30? and ku70? gcw53? were screened from the library of GPI protein deletion because of their strong cell surface hydrophobic properties.Using the four strains as immobilized carriers,the activity of displayed CALB was effectively improved.Especially the enzyme activity of strain gcw22?/CALB-GCW61 increased by 69% after 120 h fermentation.We also introduce two kinds of fungal hydrophobic protein SC3 and HFBI to modify the cell surface,and the hydrophobic properties of the cell surface were also improved.The strain GS115/HFBI-61/CALB-51 showed good surface hydrophobic properties and lipase activity,and the final hydrolysis and synthesis activity were increased by 37% and 109%,respectively.