Functional heterologous expression of an engineered full length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

Biotechnology for Biofuels

Lee R Lynd³  David A Hogsett³  Daniel G Olson¹  Adam M Guss²  Christopher D Herring¹  Devin H Currie³ 

[1]Thayer School of Engineering, Dartmouth College, Hanover, NH 03755, USA

[2]Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA

[3]Mascoma Corporation, Lebanon, NH 03766, USA

关键词: Consolidated bioprocessing;    Ethanol;    Anaerobe;    Thermophile;    Cellulosome;    Clostridium thermocellum;    Thermoanaerobacterium saccharolyticum;   

Others  :  798136
DOI  :  10.1186/1754-6834-6-32

 received in 2012-11-06, accepted in 2013-02-08,  发布年份 2013

【 摘 要 】

Background

Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes.

Results

We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipA from C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome.

Conclusion

This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA. In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose.

【 授权许可】

   
2013 Currie et al; licensee BioMed Central Ltd.

【 图 表 】

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