| The thesis has conducted systematical research to the effect of several factors,such as the strategy of δ-integration, selection marker, ploidy, mating type,recombination, and hybridization, on the expression of single or multiple cellulasegene(s). Based on these, to investigate the potential application as CBP microbe, therecombinant strains were comprehensively evaluated mainly by the fermentationperformance in cellulose meida.Study on the expression of single cellulase gene.1. The δ-integrative platformwere constructed and evaluated, the results showed that selective marker affected theactivity of strains.2. T. reesei cbh1, cbh2, and A. aculeatus cbh1were successfullyexpressed in Saccharomyces cerevisiae, and there was synergy among CBHs.3. Forδ-integrated strains expressing BGL, the activity, growth, and fermentation abilitywere subject to ploidy, mating type, and carbon source. BGL-aα had the bestfermentation ability with the highest ethanol titer18.26±1.34g/L within7days, withonly TIJ cellulase (10FPU/g biomass) and urea addition.4. The potential applicationof spore strains derived from2N to4N strains was studied, the results indicated thattriploid spore strains could endow the strains with the largest improvement in BGLactivity, and the activity of the stable strain A-8was increased by116.84%, comparedwith the parent strain. The results showed that mating type, ploidy, and carbon sourceinfluenced the strains’ activities and fermentation ability.Study on the co-expression of three types of cellulase genes.1. Strains W1, W2,and W3were constructed by sequential integration strategy. The results of cellulaseactivities and fermentation ability showed that there was synergy among EG, BGL,and CBH. With TIJ cellulase addition (10FPU/g biomass), the highest ethanol titer ofstrain W3within7days was28.20±0.84g/L, and the ethanol yield reached59.82%±1.78%of the theoretical yield.2. Strains LA1, LA2, LA3, and LA4wereconstructed by cocktail integration strategy. The results of cellulase activities andfermentation ability showed that LA3was preferable.3.1N~4N strains constructedfrom LA3. The fermentation performance from acid-and alkali-pretreated corncoband cellulase activities of strains were measured, the results showed that mating typeand ploidy did not influence strains’ fermentation ability and cellulose activities. With TJI cellulase addition (10FPU/g biomass), the three strains LA3a, LD3aa, andLT3aaα with the best fermentation ability. The highest ethanol concentration (g/L) ofthem within10days were35.18±1.56,34.51±1.41, and36.75±1.78, and the ethanolyield were74.63%±3.31%,73.21%±2.99%, and77.96%±3.78%of the theoreticalyield, respectively.4. The fermentation conditions of LA3and the hybrid strainMaα-33in cellulose media were optimized. The results showed that:(1) The optimumTIJ cellulase loading was10FPU/g.(2) The BGL activities in the two strains wereenough for enzymatic degradation system and the commercial BGL was not required.(3)The fermentation performance of the two strains was subject to nitrogenavailability. The optimum nitrogen sources were urea and corn steep liquor power forLA3and Maα-33, respectively.(4) The fermentation ability of CBP strains could beimproved by appropriately raising temperature.(5) The inoculum ratio influenced thefermentation performance, and the inoculum ratio of25%was preferable.In summary, the2N strain BGL-aα with high copy number of integrated bgl1andhigh expression, and the1N strain LA3synergisticly co-expressing egl, bgl, and cbhhad the potential application as CBP strains. In this research the systematical researchto the effect of the δ-integration strategy and many other factors on the exogenousgene expression in the δ-integrated strains would contribute to the development andenrichment of the genetic manipulation techniques in S.cerevisiae. |
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