Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

BACKGROUND The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as beta-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail delta-integration. RESULTS In cocktail delta-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. Although the total integrated gene copy numbers of cocktail delta-integrant strain was about half that of a conventional delta-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). This suggests that optimization of the cellulase expression ratio improves PASC degradation activity more so than overexpression. CONCLUSIONS To our knowledge, this is the first report on the expression of cellulase genes by delta-integration and optimization of various foreign genes by delta-integration in yeast. This method should be very effective and easily applied for other multi-enzymatic systems using recombinant yeast.