Onstruction Of Recombinant Industrial Saccharomyces Cerevisiae By Multi-gene Transformation And Alcoholic Fermentation To Cassava

Under the background of fossil energy is going to be exhausted and cellulose, lignin fuel ethanol production technology is not perfect, cassava will be used as important material for fuel ethanol production in China in the present and a period of future. The main limited factor for the application of cassava ethanol is the high cost of production. Yeast is the "soul" in the ethanol industries. If constructing new metabolic pathways of starch and cellulose in yeast cells to partial substitution the function of exogenous enzymes in cassava ethanol production, then exogenous enzymes would be saved. Starch conversion and ethanol yield would be improved and production costs would be reduced.The strain used in our study was industrial Saccharomyces cerevisiae NY-K The purpose was to improve the utilization ratio of cassava matrix. A multi-gene integrated yeast expression vector with three indivial expression cassette was constructed. Amylase gene, endoglucanase gene and β-glucosidase gene were integrated into the NY-K genome and multi-substrate metabolic activity was added to the transformants. So the transformants could perform post-saccharification to starch in alcoholic fermentation stage and have the function of degrading cellulose. The conversion of starch in cassava matrix was more efficient and cellulose could be degrade in certain degreen. At last ethanol yield was improved.At first, a new integrated yeast expression vector pYES2M was contructed. Primers were designed based on the sequence of promoter (Ppgk) of phosphogly cerate kinase gene, ribosomal gene rDNA, copper resistance gene CUP1in S. cerevisiae and signal peptide a-MF in Pichia pastoris expression vector pPIC9K. PCR was performed and four fragments including Ppgk, rDNA,CUP1and a-MF were obtained. Ppgk sequence cloned has been submitted to GenBank, accession nummber was FJ415226. Episomal plasmid pYES2was used as the parent plasmid and the four amplified fragments were introduced into pYES2one by one. Expression vector pYES2M was contructed. Copper resistance gene was used as a selection marker in pYES2M for avoiding the introduction of a new drug-resistance selection marker. Biological security was improved in the source.A methods of copper resistance as selection marker for screenig industrial yeast transformants was established. And a rapid identification method for industrial Saccharomyces cerevisiae transformants by colony PCR was established.Then glucoarnylase gene gal was amplified by PCR based on Aspergillus awamori genomic DNA. Endolucanase gene eg3and β-glucosidase gene bgll were amplified by PCR based on Trichoderma viride totol RNA.Restriction enzyme Sph I site in gal fragment was deleted. The intron in eg3and bgll was deleted individually. Then gal,eg3and bgll was inserted into the multiple cloning site of the vector pYES2M individually. Three yeast integrated expression vector pYES2M-gal,pYES2M-eg3and pYES2M-bgll were constructed and were introduced into the host strain S. cerevisiae NY-K by electroporation method. Functional identification and SDS-PAGE electrophoresis were performed and the three recombinant yeasts were found to produce functional extracellular recombinant enzyme.Second, the special genes were spiced through overlap extension PCR and inserted into the multiple cloning site of the vector pYES2M. Two yeast integrated expression vector pYES2M-eg3-ga1and pYES2M-eg3-bgl1were constructed. In every vector the gene eg3,bgl1and ga1contained complete expression cassette. The linear pYES2M-eg3-ga1and pYES2M-eg3-bgl1were introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and both two recombinant yeast were found to produce independent (non-fusion) functional extracellular recombinant enzyme.At last, expression cassette PMFgaT was inserted into the vector pYES2M-eg3-bgll and a yeast trivalent integrated expression vector pYES2M-eg3-gal-bgll was constructed. The linear pYES2M-eg3-gal-bgll was introduced into the host strain S.cerevisiae NY-K by electroporation method. Enzyme activity detection and SDS-PAGE electrophoresis were performed and recombinant yeast was found to produce independent (non-fusion) functional extracellular recombinant enzyme.Recombinant yeast integrated gal could performed alcoholic fermentation on soluble starch, ethanol concentration was1.595～1.614g/100mL at the end of fermentation. The value was higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated eg3could performed alcoholic fermentation on CMC. ethanol concentration was0.220～0.259g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K. Recombinant yeast integrated bgll could performed alcoholic fermentation on cellobiose. ethanol concentration was0.323～0.340g/100mL at the end of fermentation. The value was a little higher than the strain NY-K/pYES2M and NY-K.Trivalent recombinant S. cerevisiae NY-K/pYES2M-eg3-gal-bgl1performed alcohol fermentation on cassava matrix directly, ethanol concentration was1.64g/100mL and0.42g/100mL at the end of fermentation for recombinant and parent strain individually. Alcoholic fermentation was performed again after enzyme were added into cassava matrix, ethanol concentration was9.14g/100mL and9.17g/100mL at the end of fermentation for recombinant strain NY-K/pYES2M-eg3-gal-bgl1and NY-K/pYES2M-eg3-ga1individually, ethanol concentration was8.93～9.20g/100mL at the end of fermentation for other recombinants and8.68g/100mL for parent strain.NY-K/pYES2M-eg3-eg3-bgll was transfer in cassava matrix and alcoholic fermentation was performed by SSF and SHF mode, ethanol concentration of the liquor was9.27g/100mL and9.10g/100mL for SSF and SHF. ethanol concentration of SSF was higher than SHF.Based on the results,the conclusion was that ethanol concentration (9.27g/100mL), starch utilization rate (93.79%) and ethanol production rate (53.26%) for recombinant yeast NY-K/pYES2M-eg3-eg3-bgl1arrived the requirement of ethanol industris under SSF model, and glucoamylase and other enzymes can be saved in the process of production. Transgenic yeast strain NY-K/pYES2M-eg3-eg3-bgl1might be useful for cassava ethanol industries.