Functional Comparison And Preliminary Study Of Applications Of Several Yeast Promoters

The promoter is an important cis-element for regulation of gene expression, the strength of the promoter function is very important for the expression of target genes.In order to find a better promoter for transcriptional initiation, this experiment compared different promoters in the expression of foreign proteins using the green fluorescent protein (GFP) as a reporter. The promoter of glycerol-3-phosphate dehydrogenase gene(Scgpd) which is the key enzyme in glycerol synthesis from Saccharomyces cerevisiae were cloned by PCR, and introduced into pYX212, pGAPZB and pPIC9K to construct pYX212-gfp and pYX-PG, pGAPZB-gfp and pGPDZB-gfp, pPIC9K-gfp and pPIC9K-PG simultaneously. The recombinant plasmids were transformed into S.cerevisiae W303-1A, Pichia pastoris X33 and P.pastoris GS115 by electroporation. In the medium containing glucose or NaCl with different concentrations for culturing the recombinant strains, GFP was detected by fluorescent microscopy. By the expression of GFP, the function of pScgpd and promoter of triosephosphate isomerase (pTPI), promoter of methanol oxidase (pAOX1), promoter of 3-GAPDH (pGAP) were compaired. Fluorescence microscopy assays showed that pScgpd was induced by the performance of hypertonic conditions. The gfp gene was functionally expressed under the control of the promoters in recombinant S.cerevisiae and P.pastoris. Furthermore, the expression of gfp at different level was conducted by the different concentrations of glucose or NaCl within a certain range for the recombinant strains. However, compared with the promoter pTPI, pGAP and pAOX1 corresponding to the host S.cerevisiae W303-1A, P.pastoris X33 and P.pastoris GS115, the expression level under the control of the promoter pScgpd was still weak.Based on the results of function analysis above, pAOX1 was used for the expression of chitinase from S.cerevisiae W303-1A in P.pastoris GS115. Sccts gene was cloned and the recombinant plasmid pPIC9K-Sccts was constructed. Following linearization with Sal I, the plasmid was used to transform into P.pastoris strain GS115 by means of electroporation. One recombinant strain producing chitinase were screened by G418 and shake flask fermentation. Primary grope for fermentation conditions was undertaken on the substrate concentration, the inoculum concentration, the methanol concentration, induced pH and induced time. The optimal conditions were made certain that the substrate concentration was 5%, the inoculum concentration was 3%, the methanol concentration was 2.5%, inducing pH was 6.4 and induced time was 108h. The enzymic activity of chitinase was 46.0U/mL.Analysis of the fermentation process was also performed in a 5L fermenter. Optimization of fermentation parameter such as the initial stirring speed, initial ventilatory capacity, feeding process of methanol adding and the temperature of induction were ascertained. It was determined that the initial stirring speed was 600r/min, initial ventilatory was 3L/min, the coupling value of methanol adding and dissolved oxygen was 30%, the temperature of induction was 28℃. The enzymic activity of chitinase was maximal with 94.6U/mL.