Abstract
A novel high-throughput screening method is proposed for the directed evolution of exoglucanase facilitated by the co-expression of β-glucosidase, using the glucose released from filter paper as the screening indicator. Three transformants (B1, D6 and G10) with improved activity were selected from 4,000 colonies. The specific activities of B1, D6 and G10 for releasing glucose were, respectively, 1.4-, 1.3- and 1.6-fold higher than that of the wild type. The engineered exoglucanase gene was inserted into an expression vector carrying the previously engineered endoglucanase and β-glucosidase genes, and transformed into Escherichia coli to form a completely engineered cellulase system that showed 8.2-fold increase in glucose production (relative activity) compared to the cells equipped with wild-type enzymes. To our knowledge, this is the first report for directed evolution of an exoglucanase using insoluble cellulose as the screening substrate.
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Abbreviations
- HTS:
-
High-throughput screening
- RBS:
-
Ribosomal-binding site
- IRBS:
-
Internal ribosomal-binding site
- WT:
-
Wild type
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Acknowledgments
This work was financially supported by the Natural Science Foundation of China (Grant No. 21176215 and 21176102), the Outstanding Young Scholar of Zhejiang Province (Grant No. R4110092), the Program for Zhejiang Leading Team of S&T Innovation (Grant No. 2011R50007) and the Fundamental Research Funds for the Central Universities (Grant No. 2014QNA4025).
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Liu, M., Xie, W., Xu, H. et al. Directed evolution of an exoglucanase facilitated by a co-expressed β-glucosidase and construction of a whole engineered cellulase system in Escherichia coli . Biotechnol Lett 36, 1801–1807 (2014). https://doi.org/10.1007/s10529-014-1541-2
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DOI: https://doi.org/10.1007/s10529-014-1541-2