Functional Heterologous Expression of an Engineered Full Length CipA from Clostridium thermocellum in Thermoanaerobacterium saccharolyticum

Journal Article · Tue Jan 01 00:00:00 EST 2013 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/1754-6834-6-32· OSTI ID:1091630

Currie, Devin ^[1]; Herring, Christopher ^[2]; Guss, Adam M ^[3]; Olson, Daniel G. ^[4]; Hogsett, David ^[2]; Lynd, Lee R ^[4]

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1. Dartmouth College
2. Mascoma Corporation
3. ORNL
4. Thayer School of Engineering at Dartmouth

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 cipA 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.

• Research Organization: Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• DOE Contract Number: DE-AC05-00OR22725
• OSTI ID: 1091630
• Journal Information: Biotechnology for Biofuels, Vol. 6; ISSN 1754-6834
• Publisher: BioMed Central
• Country of Publication: United States
• Language: English