Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant.
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 Δpta 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.
Tripathi
Shital A
SA
Mascoma Corporation, Lebanon, NH 03755, USA.
Olson
Daniel G
DG
Argyros
D Aaron
DA
Miller
Bethany B
BB
Barrett
Trisha F
TF
Murphy
Daniel M
DM
McCool
Jesse D
JD
Warner
Anne K
AK
Rajgarhia
Vineet B
VB
Lynd
Lee R
LR
Hogsett
David A
DA
Caiazza
Nicky C
NC
D001426:Bacterial Proteins / Q000235:genetics; D002264:Carboxylic Acids / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000235:genetics*; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000633:toxicity; D010709:Phosphate Acetyltransferase / Q000235:genetics; D010957:Plasmids; D012641:Selection, Genetic
D001426:Bacterial Proteins; D002264:Carboxylic Acids; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D010709:Phosphate Acetyltransferase