INTRODUCTION OF A FEED BACK RESISTANT ALPHA-ISOPROPYLMALATE SYNTHASE GENE OF SACCHAROMYCES-CEREVISIAE INTO SAKE YEAST

A mutant LEU4 gene (LEU4fbr-2), responsible for both the overproductio n 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 m utant of Saccharomyces cerevisiae F-7. The single copy number of LEU4f br-2 complemented the leucine auxotrophy of S. cerevisiae HB190 (a, le u4, leu5), and also transformed it to TFL-resistant. Leucine-insensiti ve alpha-isopropylmalate synthase activity was detected in the crude e xtract of the Leu+ transformant. Also sake yeast Kyokai no. 7 (K-7) wa s transformed by the LEU4fbr-2 gene to TFL-resistant. The resulting tr ansformants produced 3-30-fold higher levels of iso-amyl alcohol (appr ox. 50-475 ppm) in shaking cultures, while in static cultures the incr ease in productivity was only 2.5-fold compared with that of recipient strain K-7. The isolated LEU4fbr-2 gene may be useful as a positive s electable marker for the transformation of industrial yeast.