Engineering Saccharomyces Cerevisiae For Rapid Cellobiose Utilization

Simultaneous saccharification and fermentation is widely applied in theproduction of cellulosic ethanol. However, there are two bottlenecks in thesimultaneous saccharification and fermentation process: the inhibition of theaccumulated cellobiose on cellulases and the mismatch between the temperatures forenzymatic hydrolysis and fermentation.In this study, genes encoding cellobiose transporter (CDT) and β-glucosidase(BGL) were integrated on the pRS426plasmid and introduced into an industrialSaccharomyces cerevisiae strain SyBE001601. Strain SyBE001602was obtained andevolution engineering was carried out to improve the cellobiose utilization capacity ofthe strain. The evolved strain SyBE001603showed a cellobiose consumption rate of2.35g/L/h and ethanol productivity of1.17g/L/h, which is1.8-fold and3.9-foldhigher than strain SyBE001602. The CDT gene expression level of strainSyBE001603increased5.74-fold and the BGL gene expression level increased0.21-fold compared to SyBE001602, respectively. To investigate whether or not theincreased expression level of the CDT and BGL would influence the overallcellobiose utilization, the genes of CDT and BGL were overexpressed in fivelaboratory chasis strains. The results proved that the improved gene expression levelof CDT and BGL could facilitate the cellobiose consumption and ethanol productivity,but the effects were chassis-dependent.Subsequently, the fermentation performance of strain SyBE001603was studied.The cellobiose consumption rates were almost the same at30°C and38°C, and evenat42°C40g/L cellobiose were consumed completely within30h. The strainexhibited the highest growth rate and cellobiose consumption rate at34°C. Besides,the strain showed a high cellobiose consumption rate of3.67g/L/h at34°C and3.04g/L/h at38°C with an initial cellobiose concentration of80g/L. At pH4.5the strainconsumed80g/L cellobiose completely within42h and showed higher growth rate atpH5.0~6.0. During simultaneous saccharification and fermentation the cellobioseaccumulation was minimized by strain SyBE001603and the inhibition of cellobioseon cellulase was relieved. The ethanol yield increased23%and the ethanolproductivity increased significantly. Meanwhile, strain SyBE001603showed tolerance to the inhibitors inlignocellulosic hydrolysate. The strain can be tolerant to3g/L furfural,1.5g/L phenol,4g/L acetic acid and a multiple inhibitors of2g/L furfural,0.8g/L phenol and1.5g/L acetic acid, which exhibited potential in industrial production.