Abstract
A mutant LEU4 gene ( LEU4 fbr-2), responsible for both the overproduction of iso-amyl alcohol in yeast and the phenotype of yeast resistant to 5,5,5-trifluoro- dl-leucine (TFL), was isolated from a TFL-resistant mutant of Saccharomyces cerevisiae F-7. The single copy number of LEU4 fbr-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, leu4, leu5), and also transformed it to TFL-resistant. Leucine-insensitive α-isopropylmalate synthase activity was detected in the crude extract of the Leu + transformant. Also sake yeast Kyokai no. 7 (K-7) was transformed by the LEU4 fbr-2 gene to TFL-resistant. The resulting transformants produced 3∼30-fold higher levels of iso-amyl alcohol (approx. 50∼475 ppm) in shaking cultures, while in static cultures the increase in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4 fbr-2 gene may be useful as a positive selectable marker for the transformation of industrial yeast.
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