Mechanism Of Glycerol Repression On P_AOX1 In Pichia Pastoris

As one of the most important foreign protein expression systems,Pichia pastoris（P.pastoris）has been extensively used in expression of heterologous protein from various organism such as Clostridium Botulinum F,Trameta versicolor.The successful of this system is due to its strong and inducible promoter of alcohol oxidase 1(P_AOX1),which is activated by methanol,but repressed by some non-fermentation substrates,such as glycerol,glucose,ethanol,etc.Baed on the above reasons,a two-stage fermentation strategy was developed,where in the first stage,glycerol is used to grow biomass,and in the second stage,methanol is fed for induction of protein expression.However,there are some disadvantages for this strategy;for example,it required long fermentation cycle,large energy input,and massive amounts of O₂ for metabolizing methanol.Moreover,in this process,many harmful metabolites such as formaldehyde,hydrogen peroxide,dihydroxyacetone were produced as by-products.All the above reasons limites the application of P_AOX1.Nevertheless,the mechanism of glycerol inhibiting P_AOX1 is not clear,so there is no perfect solution to these defects.In this paper,some methods such as bioinformatics and genetic engineering were adopted to study the mechanism glycerol bringing to P_AOX1.Some major conclusions were summarized below:（1）The first glycerol transporter was confirmed in P.pastoris,and its function in glycerol transportion was confirmed.According to SGD database（Saccharomyces Genome Database,SGD）,a homologous sequence with sugar transporter 1（STL1）in P.pastoris was found（67.8%）,named glycerol transporter 1（GLT1）.Amino acid sequence alignment suggests both STL1and Glt1p belong to major facilitator superfamily（MFS）,a big family existing in many transporter proteins.The above results indicated that Stl1p and Glt1p might share the same function in glycerol transportation.According to transmembrane analysis,Glt1p is a transmembrane protein.Subcellular localization results indicate that Glt1p protein located in cytomembrane in P.pastoris and Schizosaccharomyces pombe（S.pombe）;Moreover,compared with S.pombe（wild-type）,S.pombe（containing GLT1）grows on the medium with glycerol as sole carbon source,which indicates that GLT1 has the capacity to transport glycerol from extracellular into intracellular to support cell growth.The above results indicated that Glt1p is a glycerol transporter in P.pastoris.（2）According to qRT-PCR and other experiments,it was confirmed that Mxr1p protein could bind on P_GLT1 to inhibit its transcription,which will result to glycerol concentration decreased and finally lead to AOX1 expression level increaeds.Detection the relative expression of GLT1 in（35）mx1 mutant and MXR1 over-expression strains.The results show that mRNA expression level of GLT1 in（35）mx1 mutant is 4times than that expressed in wild-type,but the mRNA expression level of GLT1 in MXR1 over-expression strain is 20 percentage of that in wild-type;Aox1p activity in（35）mx1 mutant and MXR1 over-expression strains cultured in different medium（glycerol medium,glycerol plus methanol,methanol medium）were detected,results show that the expression level and activity of Aox1p in（35）mx1 mutant(0.06 U?OD600^-1)（cultured in methanol medium）is approximately 15 percentage of that in wild-type(0.40 U?OD600^-1),but the expression level and activity of Aox1p in MXR1over-expression strains（cultured in methanol medium）(1.15 U?OD600^-1)is 3 times than that in wild-type;Electrophoretic mobility shifts assay（EMSA）results showed that Mxr1 protein binds on P_GLT1(promoter of glycerol transporter 1,P_GLT1)（binding sequence 5’-CYCC-3’）.The above results indicate that Mxr1p inhibits the expression of GLT1 by binding on P_GLT1 and promote the expression of AOX1.（3）GLT1 represses MXR1 transcription via increasing glycerol concentration in cells.Detection the relative expression level of MXR1 in（35）glt1 and GLT1 over-expression strains.In glycerol medium,GLT1 deletion promotes MXR1 transcription.Glt1over-expression inhibits MXR1 transcription;According to the results of Aox1p expression level and enzyme activity detection,it could be found that in glycerol medium,deletion GLT1 would promote Aox1p expression and enzyme activity(0.34U?OD600^-1).Glt1 over-expression would inhibit Aox1p expression and enzyme activity(0.04 U?OD600^-1);Moreover,intracellular glycerol content detection showed that deletion GLT1 would decrease glycerol content in cells,GLT1 over-expression would increase glycerol content in cells.The above results indicated that GLT1 would increase glycerol content in cells,which would inhibit AOX1 expression via inhibit MXR1 expression.（4）New protein（Tkl1p）interacted with Mxr1p in methanol medium was found and it could accelerate formaldehyde degradation via promoting D-Xylulose-5-phosphate（Xu5P）formation.Through pull-down assay and mass spectrometry analysis,Mxr1p protein interacts with certain proteins in Pichia pastoris.Mxr1p protein can interact with UDP-glucose pyrpphosphorylasegene,translationelongationfactor1-alphagene,imidazoleglycerol-phosphate dehydratase gene,and transketolase（TKL1）gene with methanol as the sole carbon source;Via yeast two-hybrid experiment,it was found that Mxr400AA protein in Saccharomyces cerevisiae（S.cerevisiae）interacts with Tkl1p protein in vivo but Mxr150AA can’t,which indicate that the main domain Tkl1p interacting with Mxr1p located from 150AA to 400AA;According to detect xylulose content at different formaldehyde concentrations by High performance liquid chromatography（HPLC）revealed that the xylulose content in Tkl1p overexpression system（added Mxr1p protein）was significantly higher than that in other control groups,the reason may be that the complex formed by Tkl1p and Mxr1p could promoter Fructose-6phosphate（F6P）and Glycaraldehyde-3-phosphate（GAP）go into NOG（no oxidized glycolysis）pathway to form D-Xylulose-5-phosphate（Xu5P）,and excess of Xu5P will be converted to xylulose by some phosphatase.（5）The repression of glycerol on P_AOX1OX1 was decreased in new P.pastoris expression system,which will promote hetelogous protein expression.To evaluate the new P.pichia expression system,strains（wild-type and（35）glt1）expressing enhanced green fluorescent protein（Egfpp）or Human Serum Albumin（Hsap）were constructed,the expression level of Egfpp and Hsap were approximately7.3 times in（35）glt1 mutant than that in wild-type cultured in shake flask level（2.5 to3.5 times）;At 5-liter,the expression of Hsap in（35）glt1 was 4 times than that in wild-type.The quantity expression of Egfpp in（35）glt1 mutant（cultured in glycerol plus methanol）is like that in（35）glt1 mutant（cultured in methanol）.However,compared with biomass in methanol medium,strains cultured in glycerol and methanol is higher.The above results indicate that in glycerol plus methanol medium,the expression level of foreign in（35）glt1 mutant is like that in methanol,but biomass in medium containing glycerol is higher than that in methanol medium,which could promote the overall expression level of heterologous proteins.