Journal
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Volume 77, Issue 23, Pages 8288-8294Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.00646-11
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Funding
- Mascoma Corporation
- BioEnergy Science Center
- Oak Ridge National Laboratory
- U.S. Department of Energy (DOE) Bioenergy Research Center
- Office of Biological and Environmental Research in the DOE Office of Science
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This work describes novel genetic tools for use in Clostridium thermocellum that allow creation of unmarked mutations while using a replicating plasmid. The strategy employed counter-selections developed from the native C. thermocellum hpt gene and the Thermoanaerobacterium saccharolyticum tdk gene and was used to delete the genes for both lactate dehydrogenase (Ldh) and phosphotransacetylase (Pta). The Delta ldh Delta pta mutant was evolved for 2,000 h, resulting in a stable strain with 40:1 ethanol selectivity and a 4.2-fold increase in ethanol yield over the wild-type strain. Ethanol production from cellulose was investigated with an engineered coculture of organic acid-deficient engineered strains of both C. thermocellum and T. saccharolyticum. Fermentation of 92 g/liter Avicel by this coculture resulted in 38 g/liter ethanol, with acetic and lactic acids below detection limits, in 146 h. These results demonstrate that ethanol production by thermophilic, cellulolytic microbes is amenable to substantial improvement by metabolic engineering.
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