Construction Of Recombinant Saccharomyces Cerevisiae Applied To Simultaneous Saccharification And Fermentation Of Cellulose Yielding Ethanol

Cellulases can be used in conjunction with yeasts to degrade biomass to ethanol wherein theβ-glucosidase degrades cellobiose to glucose that yeasts can further ferment into ethanol. This production of ethanol from readily available sources of cellulose can provide a stable, renewable fuel source. So simultaneous saccharification and fermentation process(SSF) is regarded as the most effective pathway to convert cellulose to ethanol, in which cellulase is the key factor. However the major rate limiting step to ethanol production from biomass is the insufficient amount ofβ-glucosidase in the cellulase system. Therefore it becomes our research focus to introduce cellobiose metabolic pathway into the Saccharomyces cerevisiae and increase theβ-glucosidase enzyme activity.Primers was designed according to theβ-glucosidase coding sequences of Saccharomycopsis fibuligera and Trichoderma reesei. Then S. fibuligeraβ-glucosidase gene bgl1 was amplified with routine PCR and the T. reeseiβ-glucosidase gene bglⅠwas amplified with cross-over PCR in order to remove the two introns within gene bglⅠ. Recombinant plasmid pYX-sfbgl and pYX-tbgl were successfully constructed by inserting the corresponding gene fragments into the downstream of TPI promoter of shuttle plasmid pYX212. pYX-sfbgl and pYX-tbgl were transformed into S. cerevisiae W303-1A through electroporation respectively. The both genes were expressed actively and enzyme activity reached 0.504(IU/mL extract)and 0.124(IU/mL extract)respectively.In order to achieve high copy numbers integration the rDNA locus with 100-200 copies tandemly presented in S. cerevisiae chromosome was chosen as target integrating site. A rDNA fragment from S. cerevisiae replaced the 2μsequence of the starting vector pYX212 through which not only gave the vector rDNA integrating site but also deprived it of the ability of replicating autonomously. Then the resulting plasmid was modified appropriately which finally gave birth to rDNA integrating vector pYX-MIRY.The enzyme activity and enzymatic character analysis indicated thatβ-glucosidase from S. fibuligera could fit the condition of SSF process, so this enzyme was selected as the subsequent research target. The gene bgl1 was ligated with pYX-MIRY and then the resulting plasmid was co-transformed with pSKsym?Km into the host S. cerevisiae Y. A recombinant S. cerevisiae rRBGL was obtained through screening and applied to the simultaneous saccharification and fermentation of microcrystalline cellulose to ethanol. Compared with the host strain in SSF process, the results demonstrated that when the S. cerevisiae was enabled to hydrolyze cellobiose, it could partly relieve feedback inhibition to cellulase caused by cellobiose accumulation. It concluded that introduction of cellobiose metabolic pathway to yeast strain increased the ethanol yield compared with host strain.