Promoter engineering is one method to control cellulase expression levels. reesei degrades cellulose effectively and is known to produce various types of cellulolytic enzymes and control their expression levels simultaneously depending on the environment the ratio of the cellulases and their synergetic effects are important for effective cellulose degradation. This method however requires higher cellulase degradation for cost-effective ethanol production from cellulosic materials. We previously reported direct ethanol production from phosphoric acid swollen cellulose (PASC) using a yeast strain co-displaying Trichoderma reesei EGII, CBHII, and Aspergillus aculeatus BGL1. Although there are many reports concerning cost reduction for cellulosic material degradation using recombinant bacteria, fungi, and yeast, cellulase degradation efficiency has not been improved enough. Therefore, an efficient and cost-effective method for degradation of cellulosic materials into glucose to produce alternative fuels or other fine chemicals is required.Įfficient degradation of cellulose requires a synergistic reaction of the cellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL). Utilization of biomass, especially cellulosic materials, is desirable because it is abundant, inexpensive, renewable, and has favorable environmental properties. Given the eventual exhaustion of fossil fuels and environmental issues such as global warming and acid rain, utilization of biomass as a source of fuels and fine chemicals has recently become an attractive option. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast. To our knowledge, this is the first report on the expression of cellulase genes by δ-integration and optimization of various foreign genes by δ-integration in yeast. This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. Although the total integrated gene copy numbers of cocktail δ-integrant strain was about half that of a conventional δ-integrant strain, the phosphoric acid swollen cellulose (PASC) degradation activity (64.9 mU/g-wet cell) was higher than that of a conventional strain (57.6 mU/g-wet cell). In cocktail δ-integration, several kinds of cellulase expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (i.e., expressing an optimum ratio of cellulases) are easily obtained. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase.
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