Construction Of A Novel Unmarked Expression System For Pichia Pastoris And Recombinant Expression Of Novel Chitosanases

Pichia pastoris is widely used for industrialized production of recombination proteins,due to the efficiency exocytosis ability and lower culture costs.The main ways for strain modification include for increasing the transgenes copies in hosts or coexpressing the molecular chaperone,but these two methods cannot be performed infinitely due to the limited screening pressures of yeasts?In this study,we established a new way to acquire the high-copies and no-resistance yeast strains,which through the numerous copies transgenes intergraded into the hosts'genomes,and then the resistant genes of plasmids were deleted by the Cre recombinase that were induced by methanol.Next,the non-resistance yeasts could be inserted by another expression plasmids.Finally,the single resistant gene utilized to improve the exogenous gene copies becomes a reality.When the plasmids were constructing,we found that the AOX promotor induced by methanol was distinguished by E.coil which could initiate the Cre recombinase expression.Until the plasmids were recombined,the Cre cassette was not be deleted.We inserted the lacO gene into the plasmids to regulate the Cre recombinase expression in E.coil,and then acquired a new vector expressed in Pichia pastoris.AppA is a phytase comes from E.coil,in the feed industry,it has a potential of industrialization,but the low expression limits the industrial production.In this study,we evaluated the function of pMC vector in construction multi-copy stain and the effect of different promoters using phytase appA as reporting gene.Our results confirmed that more gene copis produced more recombination protein secreted.The secretion level of AppA of multi-copy stain has been raised up to 2.82-fold under the promoter of GAP compared with single copy,and up to 4.45-fold under the promoter of AOX compared with single copy,respectively.When the gene copy number exceeds a certain limit,due to highly occupied molecular chaperone in host,quite a part of the nascent peptide chain were targeted to endoplasmic reticulum associated degradation pathway(ERAD).As gene copy number increase gradually at this time,the secretory enzyme activity will decline.Compared with the expression level of AppA under the condition of same gene dosage but different promoters,the results perform that the expression level of 1,2,and 4 Copies in AOX group are lower than those in GAP group,but 8 and 12 copies in AOX group are higher than those in GAP group.These results confirmed that GAP promoter do have higher efficiency of transcription with single copy foreign gene than AOX promoter,and have lower expression level with multi-copy heterologous gene,suggesting that the differences exist between GAP promoter and AOX promoter in "promoter recognition and regulation".Above,the pMC vector improved the exogenous gene copies,as a result,the protein expression was increased effectively.Yeast are the most common hosts used in industrial,add the low-cost carbon source into culture medium is a effect way to control costs.Molasses is a by-product of the sugar producing industry and its main ingredients is sucrose,which cannot be utilized as sole carbon source for P.pastoris.So invertase-coding genes SUC2 was imported into the GAP-12appA transformants,to attempt make the P.pastoris to utilize sucrose.The results confirmed that SUC2 made the P.pastor is to utilize sucrose,but the enzyme activity was reduced by 41.3%.It is assumed that there is interference between the single peptide of a-Factor and SUC2.Among the enzymes used for industrial of yeast-recombined expression,there are a lot of publications about chitosanases and chitinases,indicating that the two enzymes play a important role in biological control of plant fungal diseases.Aquabacterium sp.A7-Y is a gram-negative bacterium which stocked in our lab.The results of genome sequencing show that there are many gene copies encode chitosanases and chitinases existed in the genome of Aquabacterium sp.A7-Y.Nine of those were heterologously expessed in E.coli.Only two chitosanases,which named as ChoA and ChoB,can be detected the activity.The full length of choA nucleic acid sequence is 1404 bp,encoding 467 amino acids.Its N-terminal 23 amino acids are signal peptide sequence.The homology between ChoA and endogulcanse(Thermobifida fusca)is 61%.The full length of ChoB nucleic acid sequence is 1551 bp,encoding 516 amino acids and there is no signal peptide sequence detected.The homology between ChoB and chitosanase(Streptomyces sp.Sirexaa-e)is 73%.Recombinant proteins ChoA and ChoB were successfully expressed in E.coli BL21(DE3)without signal peptide sequence,data shows that the recombinant ChoB has a very high level of expression,and can be purified by Ni2+-NTA affinity chromatography.On the contrary,the expression level of ChoA is very low.Six-lysine-tag was added to the N-terminal of ChoA,which increased the expression level of ChoA.The biochemical characterizations of ChoA and ChoB were analyzed.The specific activiy of ChoA is 18.53 U·mg-1 and the specific activity of ChoB is 13.18 U·mg-1.For both two enzymes,the optional activity pH is 5.0,and optional activity temperature is 40 ?.ChoA has a higher thermal stability than ChoB,and is less sensitive to chemical reagents.ChoA can hydrolyze chitosan and sodium carboxymethyl cellulose.ChoB can hydrolysis chitosan specifically.While the chitosan hydrolysis product for ChoA is chitotriose,chitotetraose,chitopentaose,chitohexaose,the chitosan hydrolysis product for ChoB is chitobiose and chitotriose.Apparently,there are some functional crosses between the two enzymes in the wild-type bacterium,so the differences in enzyme activity are reasonable for their physiological distribution.Those datas indicated that ChoA and ChoB were both contributed to inhibit the growth of two plant pathogenic fungi.To further study and identify the function of pMC expression vector,ChoA and ChoB encoding gene were used to construct pMC vector,and were integrated into P.pastoris.The tranformants of choB cannot be detected the enzymatic activity,and the activity of recombinant choA is 0.178 U·mL-1.Generation of multi-copy transformants of choA,data shows that maximum activiy(0.324 U·mL-1)was obtained when the copy number of choA is 4,which is 1.82-fold than the transformants that contain only 1 copy of choA.Co-express PDI,EROl,BIP,HAC1,SSA1,YDJ1,SSA4,SSE1,SSO1,SS02 With choA and PDI?ERO1?HAC1?YJD1?SSA4?SSE1?SSO2 have promotion effect on ChoA expression,and can increase 31%,16%,17%,21%,15%,24%and 11%on GAP-1choA transformant,and can increase 80%,30%,38%,31%,21%,36%,and 21 on GAP-4choA respectively.We constructed 6 molecular chaperones in one pMC-GAP vector,and co-expressed with choA,and increase the copy number of choA.This can increase the enzymatic activity lto 2.319 U·mL-1 of activity,which is 13.03-fold of that of the transformant with single copy gene.Our results indicated the "compound" optimization strategy can improve the expression of the target protein;thus codon optimization,the combination of different promoters with signal peptide,construction of multi-copy tranformants and co-expressing molecular chaperone are a complete set of optimization process.pMC expression vector can be rapidly constructed recombinant yeast with high copy number of heterologous gene and extra molecular chaperone,and has a wide application prospect in the future.Above all,the efficiency of protein expression could by improved by the optional codon,the different assemble of promoter and signal peptide,the multi-copies transformants and the coexpression of molecular chaperone.The rapid construction the no resistance of the multi-copies or coexpressed molecular chaperone yeast transformants,makes the pMC vectors had extensive applications.