Kinase Investigation For Possible Regulators Of Alcohol Oxidase 1 Promoter And Application

As one of the most popular recombinant protein expression systems,more than 5000 types of recombinant proteins have been successfully expressed in Pichia pastoris.In most cases,recombinant protein expression is driven by the outstanding promoter of the alcohol oxidase I gene?AOX1?.AOX1 promoter?PAOX1?is induced only by methanol and repressed by other carbon sources such as glucose,glycerol and ethanol.Since PAOX1 induction requires methanol,this toxic and inflammable material needs special handling and is not suitable for producing edible and medical products.One way to solve the problem is to develop a methanol-free expression system,which does not rely on methanol to induce PAOX1.This requires us to have a clear understanding about the regulatory mechanisms of PAOX1.However,so far our knowledge is mainly focused on single factors,such as carbon sensor,transporter and transcription factor.Kinases play a vital role in signal transduction,but few researches focus on them at present.Therefore,we looked up all the genes with kinase annotations in the database of Pichia pastoris,and constructed a mutant library with each kinase gene knocked out.We screened the growth feature and Aox enzymatic activity in each knockout strain.Based on the screening results,the ?hog1 strain was picked for a further phosphoproteomic analysis to identify any possible substrates.In addition,we constructed two potential noval methanol-free expression systems ?gut1-HpGCY1-Glycerol and ?dak-DHA,and elucidated the recombinant protein expression abilities of these two systems.In the genome of P.pastoris,152 genes were annotated as kinase coding genes.We knocked out 92 of them separately and examined the mutant strain phenotypes under different carbon sources.First of all,we identified 23 kinase genes which affected cell growth under one or more carbon sources.Among them,the kinase genes with more obvious effects are PAS_chr3₀667,PAS_chr2-1₀168 and PAS_chr3₀112?necessary for general cell growth on all three carbon sources?,PAS_chr4₀783?specifically related with glycerol supported cell growth?,PAS_chr1-4₀340,PAS_chr1-4₀498,PAS_chr3₀841,PAS_chr1-3₀213,PAS_chr3₀360,PAS_chr2-1₀211,PAS_chr3₀042,PAS_FragB₀061?specifically involved in methanol supported cell growth?.By measuring the Aox enzymatic activities,we identified 9 kinases possibly participating in PAOX1 regulation,including 5 in glycerol mediated repression?PAS_chr1-3₀232,PAS_chr1-4₀271,PAS_chr1-1₀105,PAS_chr3₀220 and PAS_chr4₀783?and 4 in methanol mediated activation?PAS_chr2-1₀64?,PAS_chr2-1₀028,PAS_chr2-1₀639 and PAS_chr1-4₀498).Based on the kinase screening results,we chose the glycerol cultured mutant APAS_chr1-3₀232?Ahogl?for a phosphoproteome assay.By comparing with glycerol and methanol cultured WT strain,we found similar patterns of the distribution of phosphorylation sites and phosphorylated amino acids species,which only differed a little bit in the absolute quantitiy.Through the analysis of GO and COG,we also found a consistant distribution in all three samples.However,by analyzing different types of phosphoproteins in three samples,we obtained phosphoproteins which were differentially phosphorylated in different strains and carbon soures,and identified 157 possible substrates of Hogl.These results provided a basis for future study.Based on the kinase screening results,we also noticed two knockouts APAS_chr4₀783?Agutl?and APAS_chr3-0841?Adak?.Based on them,we successly constructed two potential methanol-free recombinant protein expression systems:Agut1-HpGCY1-Glycerol and Adak-DHA.Agutl-HpGCYl-Glycerol system could utilize glycerol as the sole carbon source to induce the expression of PAOX1 and Adak-DHA could utilize dihydroxyacetone?DHA?as the sole carbon source to induce the same promoter.PAOX1 in both of the two systems was fully repressed by glucose.Thus,these two noval systems preserved the regulatable nature of PAOX1.Q-PCR results suggested that the the transcriptional levels of the related genes,including those within the methanol utilization pathway and peroxisomes biogenesis,were elevated if compared with glycerol or DHA cultured WT.The de-repression of PAOX1 was also observed at the transcriptional level,together with elevated Aox protein levels at the translational level.In order to test the abilities of the two potential noval systems in recombinant protein production,we expressed GFP under PAOX1 in Agut1-HpGCY1-Glycerol system and Adak-DHA system.We found that the Adak-DHA expression system functioned better than the Agut1-HpGCY1-Glycerol system.In order to further elucidate the potential of the Adak-DHA system,we expressed three more recombinant proteins in this strain.As a result,the expression levels of three recombinant proteins in the Adak-DHA system reached 50?60%of methanol induced WT system,and became comparable or even higher than the constitutive PGAp system in single cell expression ability.