The Construction And Preliminary Evaluation Of Recombinant Saccharomyces Cerevisiae Utilizing Cellulose To Produce Ethanol

Cellulose is the most abundant polysaccharide in nature. Due to the crisis ofglobal energy, environmental pollutions and food shortage, more and more countriesare using cellulose as the raw material to produce ethanol. Enzymes that canhydrolyze cellulose include endoglucanase (EG), exoglucanase (CBH), andβ-glucosidase (BGL), and their joint effort can hydrolyze cellulose intomonosaccharide, which can then be fermented in the yeast to produce alcohol. Thisthesis targets on expressing these three cellulases in yeast through δ-integration inorder to construct engineering strains that can produce alcohol with cellulose.We have built endoglucanase (egl2, from Trichoderma reesei) gene expressioncassette Ptpi-xyn2s-TEGII-Tadh I and β-glucosidase (bgl1, from Aspergillusaculeatus) gene expression cassette Ptpi-xyn2s-ABGL1-Tadh I, so in this study,we firstly isolated and cloned the full-length exoglucanase gene cbh1from A.aculeatus. After sequenced and compared, cbh1was firstly identified to have oneintron and2exons. On this basis, A. aculeatus cbh1gene expression cassette Ptpi-xyn2s-ACBHI-Tadh I was successfully construced, T. reesei cbh1gene expressioncassette Ptpi-xyn2s-TCBHI-Tadh I and T. reesei cbh2gene expression cassettePtpi-xyn2s–TCBHII-Tadh I were also constructed.In order to integrate these gene expression cassettes into S. cerevisiaechromosome with a large number of copies, we constructed a δ-integration platformcontaining4different selection markers (LEU2, HIS3, TRP1, and URA3). A.aculeatus bgl1and T. reesei egl2were as two reporter genes to evaluate differentselection markers’ effects on the expression of exogenous gene in this δ-integrationplatform.The BGL and EG activities (U/ml) on the5thday showed that a selectionmarker can affect the expression of cellulase genes, LEU2made the highestexpression of exogenous gene (2.97/29.01), then HIS3(2.29/12.41) and TRP1(1.78/8.12), while URA3made the lowest (1.52/1.48); Specific activity of each strainwas almost constant during the5days, within the range of0.193~0.207/1.9~2.1,0.087~0.093/0.87~0.95,0.067~0.073/0.5~0.54, and0.028~0.032/0.09~0.11,respectively.Five δ-integration plasmids were transformed into the Saccharomyces cerevisiae chromosome in three steps. By recombinants screening, PCR identification, enzymeassay and fermentation, it could be confirmed that endoglucanase, exoglucanase andβ-glucosidase gene had been introduced into S. cerevisiae, so that the yeast coulddirectly make use of cellulose to produce alcohol. And when adding cellulase (3.3U/gsubstrate), recombinant strains could produce alcohol up to17.82g/l after5days,while the control strain W303-1A and Angel Yeast produced littlel alcohol,1.23g/land2.12g/l respectively.