Construction of yeast xylulokinase mutant by recombinant DNA techniques

A Saccharomyces cerevisiae xylulokinase mutant was constructed by using the cloned yeast xylulokinase gene, XYK-Sc, and the gene disruption technique. The S. cerevisiae LEU2 gene was used to disrupt the XYK-Sc gene cloned on pLSK4 by insertion into the unique HIndIII site of the gene. The disrupted gene was liberated from the remainder of the plasmid with XhoI digestion, yielding a 4.4 kb DNA fragment. Transformation of a S. cerevisiae leu2 mutant with this fragment and selection for Leu super(+) complementation resulted in the isolation of transformants that were unable to grow in pure xylulose medium. The ability to grow in xylulose medium and increased xylulokinase activity were obtained by transforming the mutant with a plasmid-borne wild-type XYK-Sc gene. Insertional inactivation of the chromosomal XYK-Sc gene was also demonstrated by xylulokinase assays.