Construction Of Recombinant Saccharomyces Cerevisiae With Direct Utilization Of Cellulose As Sole Carbon Source

Cellulose is one kind of natural polysaccharides that abundantly existed and universal distribution. Its raw material has some characteristics such as cheap, easy to access, renewable and environmental friendly. Industrial production of bioethanol using cellulose as raw material is one kind of effective measure of developing new energy and relieving energy crisis, it is also one of research hotspots currently in the world. How to improve the utilization of cellulose is the current problem to be solved.Based on the previous work of our laboratory, inducible promoter sequence was replaced by constitute promoter sequence, a modified integrated secretion expression vector pHBM368-pgk was constructed. Then the signal peptide coding sequence from pPIC9K was linked with heterologous exoglucanase gene (cbh, GeneBank:AY861348), endoglucanase gene (eg, GeneBank:EU169241) and β-glucosidase gene (bg, GeneBank:EU169241) respectively. The positive clones were obtained respectively by linking with pHBM368-pgk. The auxotroph Saccharomyces cerevisiae were transformed with linearized plasmids DNA by electroporation transformation method respectively.The recombinant S. cerevisiae integrated three cellulase respectively were screened by auxotrophic selection medium (INVSc-P-SE, integrated endoglucanase gene eg; INVSc-P-SC, integrated exoglucanase gene cbh; INVSc-P-SB, integrated β-glucosidase gene bg), the Congo red was used to screen the strains with cellulase activity. The expression patterns were determined by halo around the colony. Due to the fact that carboxymethylcellulose is not the optimum substrate of β-glucosidase, the recombinant S. cerevisiae INVSc-P-SB did not appear halo around the colony obviously. In accordance with determination of DNS method, the enzyme activity of β-glucosidase, exoglucanase and endoglucanase in one milliliter crude enzyme reached45.22U/mL,72.11U/mL and75.45U/mL at50℃, respectively.The microcrystalline cellulose was used as sole carbon source in fermentation aimed at confirming whether recombinant S. cerevisiae could culture in medium which only have cellulose as carbon source, and whether constitutive promoter could enhance efficiency of cellulase hydrolysis. Simultaneous saccharification and fermentation was applied in this work that lasted84hours. The biomass trend of single inoculation showed that INVSc-P-SE strain reached stationary phase in about24hours and lasted a long time with the highest biomass. INVSc-P-SC strain also reached stationary phase in about24hours but lasted a shorter duration, and the biomass was lower than the former. INVSc-P-SB strain reached stationary phase in about48hours and biomass was lower than the former two. The biomass curve of mixed inoculation showed that its maximum biomass was higher than any other biomass of single inoculation. Experimental results showed that the recombinant S. cerevisiae could be cultured in the medium which was added microcrystalline cellulose as sole carbon source in one liter fermentation. Based on the trend of biomass, promotion of cellulase hydrolysis efficiency by mixed inoculation provided an evidence for cellulase synergy. The results in this study compared with former studies in our laboratory, indicated that constitutive promoter was more conducive to high-efficiency expression of exogenous gene than inducible promoter. In summary, the recombinant S. cerevisiae could take advantage of cellulose as sole carbon source effectively, which laid the foundation for converting cellulose into bioethanol efficiently in industrial application.