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Biotechnol Biofuels. 2018 Jun 7;11:157. doi: 10.1186/s13068-018-1151-7. eCollection 2018.

Biomimetic strategy for constructing Clostridium thermocellum cellulosomal operons in Bacillus subtilis.

Chang JJ#1, Anandharaj M#2,3,4, Ho CY5, Tsuge K6, Tsai TY2, Ke HM2,4, Lin YJ2, Ha Tran MD3,4,5, Li WH2,3,4,7,8, Huang CC5,9.

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1Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, 402 Taiwan.
2Biodiversity Research Center, Academia Sinica, Taipei, 11529 Taiwan.
3Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, National Chung Hsing University and Academia Sinica, Taipei, 11529 Taiwan.
4Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, 40227 Taiwan.
5Department of Life Sciences, National Chung Hsing University, Taichung, 40227 Taiwan.
6Institute for Advanced Biosciences, Keio University, 403-1 Nipponkoku, Daihoji, Tsuruoka, Yamagata 997-0017 Japan.
7Biotechnology Center, National Chung Hsing University, Taichung, 40227 Taiwan.
8Department of Ecology and Evolution, University of Chicago, Chicago, IL 60637 USA.
9Innovation and Development Center of Sustainable Agriculture, National Chung Hsing University, Taichung, 40227 Taiwan.
Contributed equally



Enzymatic conversion of lignocellulosic biomass into soluble sugars is a major bottleneck in the plant biomass utilization. Several anaerobic organisms cope these issues via multiple-enzyme complex system so called 'cellulosome'. Hence, we proposed a "biomimic operon" concept for making an artificial cellulosome which can be used as a promising tool for the expression of cellulosomal enzymes in Bacillus subtilis.


According to the proteomic analysis of Clostridium thermocellum ATCC27405 induced by Avicel or cellobiose, we selected eight highly expressed cellulosomal genes including a scaffoldin protein gene (cipA), a cell-surface anchor gene (sdbA), two exoglucanase genes (celK and celS), two endoglucanase genes (celA and celR), and two xylanase genes (xynC and xynZ). Arranging these eight genes in two different orders, we constructed two different polycistronic operons using the ordered gene assembly in Bacillus method. This is the first study to express the whole CipA along with cellulolytic enzymes in B. subtilis. Each operon was successfully expressed in B. subtilis RM125, and the protein complex assembly, cellulose-binding ability, thermostability, and cellulolytic activity were demonstrated. The operon with a higher xylanase activity showed greater saccharification on complex cellulosic substrates such as Napier grass than the other operon.


In this study, a strategy for constructing an efficient cellulosome system was developed and two different artificial cellulosomal operons were constructed. Both operons could efficiently express the cellulosomal enzymes and exhibited cellulose saccharification. This strategy can be applied to different industries with cellulose-containing materials, such as papermaking, biofuel, agricultural compost, mushroom cultivation, and waste processing industries.


Bacillus subtilis; Biomimetic operon; Biomimetic strategy; Cellulosome; Clostridium thermocellum

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