An efficient gene replacement and deletion system for an extreme thermophile, Thermus thermophilus.

A Thermus thermophilus host strain of which the leuB gene was totally deleted was constructed from a delta pyrE strain by a two step method. First, the leuB gene was replaced with the pyrE gene. Second, the inserted pyrE gene was deleted by using 5-fluoroorotic acid. A plasmid vector with the leuB marker was constructed and the plasmid complemented the leuB deficiency of the host. When the leuB gene from Escherichia coli and its derivative encoding a stabilized enzyme were expressed with the host-vector system, their growth temperature reflected the stability of the enzyme. These results suggest that the gene replacement deletion method using the pyrE gene is useful for the construction of a reliable plasmid vector system and it can be applied to the selection of stabilized enzymes.