It would be difficult to overestimate the contribution of genetic manipulation to the study of any biological system and it is an essential tool for the metabolic engineering of microbial biosynthetic and substrate utilization pathways. Hyperthermophilic anaerobes are of particular interest because of their environmental and commercial importance as well as their unique physiology but their extreme growth requirements present unique challenges for developing genetic methods. We have recently developed high efficiency methods for the genetic manipulation of Pyrococcus furiosus and several members of the genus Caldicellulosiruptor. P. furiosus grows optimally at 100 0C, is an obligate anaerobe and produces hydrogen in the absence of sulfur. Caldicellulosiruptor species grow optimally at 80 0C, are obligate anaerobes and have the unique ability to grow on unprocessed, unpretreated lignocellulosic biomass producing ethanol and hydrogen as fermentation products. The combined transformation and recombination frequencies of these strains allowed marker replacement by direct selection using either linear or plasmid DNA. In addition, methods for selection of gene deletions by marker replacement allows the construction of virtually any gene deletion no matter how severe the phenotype as long as the gene is not essential for viability. We suggest that these methods will provide the basis for the development of more sophisticated genetic tools allowing the study and metabolic engineering of these important hyperthermophiles.
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