The Construction And Evaluation Of Recombinant Saccharomyces Cerevisiae Strains Expressing Cellulase Genes

Global fossil fuel reserves dwindling, and the various energy, resources andenvironmental problems facing humanity makes the significance of the developmentof renewable alternative fuels, especially cellulosic ethanol increasingly important.Using the traditional microorganism Saccharomyces cerevisiae in the ethanolfermentation industry to express cellulase and produce alcohol from cellulose is animportant direction of research and development of cellulosic ethanol. Endoglucanase(EG) is one of the major components of the cellulase system, and also the key enzymecomposition to reduce the viscosity of the cellulose substrate.In this study, the factors that affect EG gene expression level were firstinvestigated and then the optimal expression cassette was determined. The full-lengthegll gene of Aspergillus aculeatus was cloned by PCR first. On the basis ofsequencing and identifying that it contains two introns and three exons, theappropriate regulatory elements S. cerevisiae tpi promoter, Trichoderma reeseixylanase II secretion peptide xyn2s, and S. cerevisiae adhI terminator were added,constructing two gene cassettes one with anchor peptide while the other without.Similarly, the two types of gene cassette were constructed for Trichoderma reeseiegl2. The above four gene cassettes were connected to the low copy shuttle plasmidvector YCplac33and the high copy shuttle plasmid vector YEplac195, thentransformed into S. cerevisiae W303a, achieving8recombinant strains possessingendoglucanase activity. The growth curves when growing in medium with glucose asthe carbon source indicated the growth conditions between the8recombinant strainswere not significantly different from the control strain. The enzymatic activities whengrowing in glucose medium and the capacity for degradation of sodiumcarboxymethylcellulose (CMC) showed that the strain YE-TrEGII’ containing EGIIgene ORF from T. reesei, YEplac195vector and without anchor peptide obtained thebest effects, which had activity of6.0U/ml at48h and the viscosities at12h wasdecreased to4.7%of the initial values; the strain YC-TrEII containing EGII geneORF from T. reesei, YCplac33vector and with anchor peptide showed the lowestenzyme activity (0.6U/ml at48h) and viscosity change (26.2%of the initial controlviscosity at12h). In order to produce alcohol directly from the cellulose substrate, the S. cerevisiaechromosome repeated sequence-sequence was used as multi-copy integration sitesand the integration vector pGEM-T easy-delta-LEU-TrEGII’ containing T. reeseiEGII gene and without anchor peptide, was constructed. Then it was transformed intovarious S. cerevisiae host with or without the integration vector for expressing A.aculeatus BGL1gene, pGEM-T easy-delta-URA-Aa BGL1’, which had beenconstructed in our laboratory previously. Finally, an integrated strain with the highestactivity and the ability to produce alcohol from CMC was screened and named delta1,whose host was W303a (delta::URA-Aa BGL1’). Its EG and BG activities were1.76U/ml and0.83U/ml when growing in glucose medium at24h, respectively, andproduced1.1g/l ethanol from1%CMC after6days’ high-density fermentation.