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Assembling a cellulase cocktail and a cellodextrin transporter into a yeast host for CBP ethanol production

Jui-Jen Chang12, Feng-Ju Ho1, Cheng-Yu Ho3, Yueh-Chin Wu1, Yu-Han Hou1, Chieh-Chen Huang3*, Ming-Che Shih4* and Wen-Hsiung Li125*

Author Affiliations

1 Biodiversity Research Center, Academia Sinica, 115, Taipei, Taiwan

2 Genomics Research Center, Academia Sinica, 115, Taipei, Taiwan

3 Department of Life Sciences, National Chung Hsing University, 402, Taichung, Taiwan

4 Agricultural Biotechnology Research, Center, Academia Sinica, 115, Taipei, Taiwan

5 Department of Ecology and Evolution, University of Chicago, 60637, Chicago, IL, USA

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Biotechnology for Biofuels 2013, 6:19  doi:10.1186/1754-6834-6-19

Published: 4 February 2013



Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed.


To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1,4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol.


Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.

Cellulosic ethanol; Crystalline cellulose; Cocktail formulation; Synthetic biology; Consolidated bioprocess