Disruption of seven hypothetical aryl alcohol dehydrogenase genes from Saccharomyces cerevisiae and construction of a multiple knock-out strain

By in silicio analysis, we have discovered that there are seven open readin g frames (ORFs) in Saccharomyces cerevisiae whose protein products show a h igh degree of amino acid sequence similarity to the aryl alcohol dehydrogen ase (AAD) of the lignin-degrading fungus Phanerochaete chrysosporium. Yeast cultures grown to stationary phase display a significant aryl alcohol dehy drogenase activity by degrading aromatic aldehydes to the corresponding alc ohols. To study the biochemical and the biological role of each of the AAD genes, a series of mutant strains carrying deletion of one or more of the A AD-coding sequences was constructed by PCR-mediated gene replacement, using the readily selectable marker kanMX. The correct targeting of the PCR-gene rated disruption cassette into the genomic locus was verified by analytical PCR and by pulse-field gel electrophoresis (PFGE) followed by Southern blo t analysis. Double, triple and quadruple mutant strains were obtained by cl assical genetic methods, while the construction of the quintuple, sextuple and septuple mutants was achieved by using the marker URA3 from Kluyveromyc es lactis, HIS3 from Schizosaccharomyces pombe and TRP1 from S. cerevisiae. None of the knock-out strains revealed any mutant phenotype when tested fo r the degradation of aromatic aldehydes using both spectrophotometry and hi gh performance liquid chromatography (HPLC). Specific tests for changes in the ergosterol and phospholipids profiles did not reveal any mutant phenoty pe and mating and sporulation efficiencies were not affected in the septupl e deletant. Compared to the wild-type strain, the septuple deletant showed an increased resistance to the anisaldehyde, but there is a possibility tha t the nutritional markers used for gene replacement are causing this effect . Copyright (C) 1999 John Wiley & Sons, Ltd.