鸡尾酒δ整合策略构建表达三类纤维素酶的酿酒酵母工程菌株及初步评价

木质纤维素乙醇具有替代化石燃料的潜力，其生产过程包括生物质预处理、纤维素酶生产、水解和发酵等多个步骤。将纤维素酶生产、水解和发酵组合在一起的统合生物加工过程（consolidated bioprocessing，CBP）由于能降低水解和发酵成本而具有应用于纤维素乙醇生产的潜力，该技术的关键是构建能有效降解纤维素的工程菌株，而构建表达纤维素酶的酿酒酵母即是其中一种选择。采用鸡尾酒多拷贝δ整合的策略将7种纤维素酶基因（Trichoderma reesei cbh1、cbh2和egl2，Aspergillus aculeatus cbh1、egl1和bgl1）表达盒整合至酿酒酵母W303-1A染色体上，经4轮整合筛选得到菌株LA1、LA2、LA3和LA4。对这4个菌株进行纤维素酶活性测定，结果表明从LA1到LA3各种纤维素酶活性呈递增趋势，而LA4的酶活性与LA3的酶活水平相当。对菌株LA3进行酸碱预处理玉米芯料的发酵评价，结果表明：①在外加商品化纤维素酶的情况下，与对照菌株W303-1A和AADY相比，LA3能有效利用纤维素料发酵产醇；②与分步整合的菌株W3相比，发酵性能更优；③培养基中的营养成分影响菌株发酵性能。这些结果表明，鸡尾酒δ整合是一种有效的构建酿酒酵母CBP菌株的方法。

Construction and Preliminary Evaluation of Saccharomyces cerevisiae Strains Co-expressing Three Types of Cellulase Via Cocktail δ-integration

Lignocellulosic ethanol has the potential to replace fossil fuels, and its production requires multiple steps including pretreatment, cellulase production, saccharification, and fermentation. By combining enzyme production, saccharification and fermentation into a single step, consolidated bioprocessing (CBP), which could reduce costs of hydrolysis and fermentation, is a promising technology for cellulosic ethanol production. And the key to CBP is the engineering of a microorganism that can efficiently utilize cellulose. Development of cellulolytic Saccharomyces cerevisiae strains is one of strategies. Six cellulase-encoding genes (cbh1, cbh2 and egl2 from Trichoderma reesei, cbh1, egl1, and bgl1 from Aspergillus aculeatus) were integrated into the Saccharomyces cerevisiae W303-1A chromosome via repeated cocktail δ-integration, generating strains LA1, LA2, LA3, and LA4. The aforementioned strains were evaluated by enzyme assay, and the results indicated that the BGL, CMCase, FPase, and PASC degradation activity of LA1, LA2, and LA3 ascended, however, the corresponding cellulase activities of the strain LA4 were similar to that of LA3. The fermentation performance of strain LA3 was evaluated using acid-and alkali-pretreated corncob as substrate, the results showed that:①Compared with the control strains W303-1A and AADY, strain LA3 could efficiently ferment pretreated corncob to ethanol with commercial cellulase addition. ②LA3 exhibited higher fermentation ability than W3 which was constructed via cellulase genes sequentially integration. ③ The nutrition used in the media affected the fermentation performance of S. cerevisiae strains. These results indicated that cocktail δ-integration was an efficient approach to construct CBP-enabling S. cerevisiae strains.