Document Type

Article

Publication Date

8-6-2010

Publication Title

Applied and Environmental Microbiology

Department

Thayer School of Engineering

Abstract

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 ΔpyrF 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 pta strain did not produce acetate. These results are the first examples of targeted homologous recombination and metabolic engineering in C. thermo-cellum, a microbe that holds an exciting and promising future in the biofuel industry and development of sustainable energy resources.

DOI

10.1128/AEM.01484-10

Original Citation

Tripathi SA, Olson DG, Argyros DA, Miller BB, Barrett TF, Murphy DM, McCool JD, Warner AK, Rajgarhia VB, Lynd LR, Hogsett DA, Caiazza NC. Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant. Appl Environ Microbiol. 2010 Oct;76(19):6591-9. doi: 10.1128/AEM.01484-10. Epub 2010 Aug 6. PMID: 20693441; PMCID: PMC2950449.

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