Construction And Evaluation Of Saccharomyces Cerevisiae Strains Expressing Cellulolytic Enzyme For Ethanol Production From Cellulose

Consolidated bioprocessing (CBP), which could simplify the process and reducecosts of fermentation and hydrolysis, was considered as the cheapest and the mostefficient ethanol production process. One of the obstacles of CBP is the lack of amicroorganism that can directly and efficiently utilize cellulose to produce ethanol.Saccharomyces cerevisiae is one of the best host strains to construct CBP strains.Strains expressing one or multiple cellulase genes were constructed via-integrationin this thesis. And the relation factors, which affected on cellulase activities, growth,and the fermentation ability, were investigated, and the fermentation performancefrom cellulose was evaluated to estimate the potential application as CBP microbe.Polyploidy series (1N-4N) strains expressing bgl1gene were constructed, usingBGL-a which was-integrated Aspergillus aculeatus bgl1as the parent strain. Andcellulase activities, growth, and the fermentation ability, were investigated. Theresults were as follows:(1) Diploid strain BGL-a had obviously lower activity thanBGL-aa and BGL-. The activity increased with ploidy level increasing.(2) Themaximum OD660decreased with ploidy level, but mating type did not affect thegrowth of strains.(3) Ploidy and mating type showed no significant influence on thefermentation performance in10%glucose and10%cellobiose media, but thefermentation performance of ploidy series strains from acid-and alkali-pretreatedcorncob was greatly dependent on nutrient availability. Regardless of nutritional status,diploid strain BGL-a had the best fermentation ability with the highest ethanol titer.The highest ethanol titer was19.76±1.39g/L and the ethanol yield reached41.92%ofthe theoretical yield.within7days, with only TIJ cellulase (10FPU/g biomass) andurea addition.The six cellulase genes (Trichoderma reesei cbh1, cbh2, egl2and A. aculeatuscbh1、egl1、bgl1) were simultaneously integrated into the W303-1A chromosome, andobtained four strains, named LY-1, LY-2, LY-3, and LY-4. The results of the ethanolproduction from alkali-pretreated corncob and cellulase activities determinationshowed that:(1) Among the strains, the strain LY-3had the best fermentation abilitywith the highest ethanol titer28.08±1.26g/L within7days, with only TIJ cellulase (10FPU/g biomass) addition, the ethanol yield of LY-3reached59.57%of thetheoretical yield. The highest ethanol titer and ethanol yield of the control strainW303-1A were19.54±0.87g/L and41.46%, respectively. So the ethanol yield of LY-3is increased by18%compared with W303-1A.(2) The cellulase activities of strainLY-4is lower than that of LY-3, which coincided with the insignificant improvementof their fermentation ability. Therefore, strain LY-3is the best. It was failed toimprove the fermentation of strain LY-3through hybridization with W3.