PICHIA-STIPITIS GENES FOR ALCOHOL-DEHYDROGENASE WITH FERMENTATIVE ANDRESPIRATORY FUNCTIONS

Two genes coding for isozymes of alcohol dehydrogenase (ADH); designat ed PsADH1 and PsADH2, have been identified and isolated from Pichia st ipitis CBS 6054 genomic DNA by Southern hybridization to Saccharomyces cerevisiae ADH genes, and their physiological roles have been charact erized through disruption, The amino acid sequences of the PsADH1 and PsADH2 isozymes are 80.5% identical to one another and are 71.9 and 74 .7% identical to the S. cerevisiae ADH1 protein. They also show a high level identity with the group I ADH proteins from Kluyveromyces lacti s, The PsADH isozymes are presumably localized in the cytoplasm, as th ey do not possess the amino-terminal extension of mitochondrion-target ed ADHs, Gene disruption studies suggest that PsADH1 plays a major rol e in xylose fermentation because PsADH1 disruption results in a low er growth rate and profoundly greater accumulation of xylitol. Disruptio n of PsADH2 does not significantly affect ethanol production or aerobi c growth on ethanol as long as PsADH1 is present, The PsADH1 and PsADH 2 isozymes appear to be equivalent in the ability to convert ethanol t o acetaldehyde, and either is sufficient to allow cell growth on ethan ol. However, disruption of both genes blocks growth am ethanol. P. sti pitis strains disrupted in either PsADH1 or PsADH2 still accumulate et hanol, although in different amounts, when grown on xylose under oxyge n-limited conditions, The PsADH double disruptant, which is unable to grow on ethanol still produces ethanol from xylose at about 13% of the rate seen in the parental strain, Thus, deletion of both PsADH1 and P sADH2 blocks ethanol respiration but not production, implying a separa te path for fermentation.