Coexpressing Accessory Proteins Facilitate The Heterologous Protein Secretion Of Saccharomyces Cerevisiae

Saccharo,myces cerevisiae is widely applied in food industry and biological medicine industry due to it is a kind of edible safe fungi.Saccharomyces cerevisiae has a complete system of protein processing to gurantee proper protein folding and posttranslational modification,so that many drug proteins could be produced in this way.However,the heterologous protein secretion level of Saccharomyces cerevisiae is in general quite low,which severely constrict its industrial application.Approaches to improve the secretion level of Saccharomyces erevisiae are mainly from two perspectives,one is to optimize the fermentation conditions,and the other is to adjust the heterologous protein secretion level genetically.Nowadays,strategies to optimize the heterologous protein secretion level of Saccharomyces cerevisiae genetically are mainly targeted at small molecule protein,and rarely at macromolecular proteins over 30KDa.In this research,we try to find a series of secretion and expression strategies suitable for macromolecular proteins,with a case study of laccase(53KDa).In this thesis,the promoter,signal peptide and accessory proteins related to the synthesis and secrection of proteins in Saccharomyces cerevisiae are optimized to improve laccase extracellular secretion level.Besides,we find that co-expression of accessory proteins can also increase the secretion levels of a-amylase and EGFP,indicating that this method has some universality.The details of study are as follows:1?Intracellular enzyme-producing strains with promoter TEF1 and GAL1 is constructed to study intracellular expression of laccase in Saccharomyces cerevisae.With this bases,a-factor signal peptide is added to construct extracellular enzyme-producing strains.The supernatant and intracellular laccase activity is investigated to assess their laccase expression and secretion level.Results show that this laccase gene can be expressed and secreted in Saccharomyces cerevisiae.Strain with GAL 1 promoter has a higher supernatant laccase activity compared with TEF1 promoter,with a value of 129.73 ?2.57U/L and 30.52 ± 2.56 U/L respectively.Therefore,we choose GAL1 for later experiment.2.Four kinds of signal peptide,including Saccharomyces cerevisiae natural a-factor signal peptide,inulase natural signal peptide,synthetic signal peptide and laccase natural signal peptide,is compared The extracellular enzyme activity of laccase mediated by synthetic signal peptide is highest increasing by 139%and its secretion ratio increased by 99%,compared with a-factor signal peptide.While the extracellular enzyme activity of laccase mediated by inulase natural signal peptide and laccase natural signal peptide cannot be detected.3?Eight accessory proteins related to the synthesis or secretion of heterologous proteins are selected and overexpressed to investigate their effect on the laccase secretion.The results shows that strains overexpressing PDI,BIP,SS02 and RPPO show improved extracellular/intracellular enzyme activity and secretion rate.Among them,Strain B/pGSLC-yPRP overexpressing RPPO shows an extracellular enzyme activity of 444.24 ± 2.82 U/L and secretion rate of 25.73 ± 0.37%,which is increased by 69%and 46%compared with Control B/pGSLC-YEpla181 respectively.4?The synergistic effect of PDI,BIP,SS02 and RPPO,which shows positive effect on secretion level is studied.The results show that RPP0/SSO2 pair,RPPO/BIP pair and RPPO/SecI pair have synergistic action to facilitate the intracellular expression and extracellular secretion,the synergistic action of RPP0/SSO2 pair is most significant on the laccase secretion,with an extracellular enzyme activity of 585.21 ± 1.28 U/L and secretion rate of 30.63±0.39%,which is increased by 31%and 18%of that of Control,respectively.5.The universality of the effect of coexpressing RPPO and SSO2 on heterologous protein secretion is studied.The result shows that in the condition of coexpressing RPPO and SS02,the extracellular enzyme activity of a-amylase and extracellular fluorescence intensity of EGFP increased by 73%and 8%relative to those of Control,respectively.In general,coexpressing RPPO and SS02 show a more significant positive effects on the secretion of a-amylase,a macromolecular protein.Finally,by optimizing promoter,signal peptide and overexpressing accessory proteins,the extracellular enzyme activity of laccase increased by 18.17-fold from 30.52 ± 2.56 U/L to 585.21 ± 1.28 U/L.The effects of the synergistic action of accessory proteins also show some universality to heterologous proteins.The purports of this research may reflect in two aspects:on the one hand,the secretion of macromolecule protein-laccase in Sacchaomyceserevisiae is increased,on the other hand,it is first time to find that the accessory proteins RPPO and SS02 working together can increase the secretion level of heterologous proteins.