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| Construction and Preliminary Evaluation of Saccharomyces cerevisiae Strains Co-expressing Three Types of Cellulase Via Cocktail δ-integration |
| LIANG Xiang nan1, ZHANG Kun2, ZOU Shao lan2, WANG Jian jun1, MA Yuan yuan2, HONG Jie fang2 |
1. School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China;
2. Tianjin R & D Center for Petrochemical Technology, Tianjin University, Tianjin 300072, China |
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Abstract 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.
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Received: 13 April 2016
Published: 25 November 2016
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