期刊
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
卷 76, 期 19, 页码 6591-6599出版社
AMER SOC MICROBIOLOGY
DOI: 10.1128/AEM.01484-10
关键词
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资金
- Mascoma Corporation
- BioEnergy Science Center (BESC)
- 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
We report development of a genetic system for making targeted gene knockouts in Clostridium thermocellum, a thermophilic anaerobic bacterium that rapidly solubilizes cellulose. A toxic uracil analog, 5-fluoroorotic acid (5-FOA), was used to select for deletion of the pyrF gene. The Delta pyrF strain is a uracil auxotroph that could be restored to a prototroph via ectopic expression of pyrF from a plasmid, providing a positive genetic selection. Furthermore, 5-FOA was used to select against plasmid-expressed pyrF, creating a negative selection for plasmid loss. This technology was used to delete a gene involved in organic acid production, namely pta, which encodes the enzyme phosphotransacetylase. The C. thermocellum Delta pta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermocellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.
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