We developed a methylotrophic yeast, Pichia pastoris, as a host for DNA transformations. The system is based on an auxotrophic mutant host of P. pastoris which is defective in histidinol dehydrogenase. As a selectable marker, we isolated and characterized the P. pastoris HIS4 gene. Plasmid vectors which contained either the P. pastoris or the Saccharomyces cerevisiae HIS4 gene transformed the P. pastoris mutant host. DNA transfer was accomplished by a modified version of the spheroplast generation (CaCI2-polyethylene glycol)-fusion procedure developed for S. cerevisiae. In addition, we report the isolation and characterization of P.pastoris DNA fragments with autonomous replication sequence activity. Two fragments, PARS1 and PARS2, when present on plasmids increased transformation frequencies to 105/,ug and maintained the plasmids as autonomous elements in P. pastonis cells.

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As a first step in a genetic approach towards understanding peroxisome biogenesis and function, we have sought to isolate mutants of the methylotrophic yeast Hansenula polymorpha which are deficient in peroxisomes. A collection of 260 methanol‐utilization‐defective strains was isolated and screened for the ability to utilize a second compound, ethanol, the metabolism of which involves peroxisomes. Electron microscopical investigations of ultrathin sections of selected pleiotropic mutants revealed two strains which were completely devoid of peroxisomes. In both, different peroxisomal matrix enzymes were active but located in the cytosol; these included catalase, alcohol oxidase, malate synthase and isocitrate lyase. Subsequent backcrossing experiments revealed that for all crosses involving both strains, the methanol‐ and ethanol utilizing‐deficient phenotypes segregated independently of each other, indicating that different gene mutations were responsible for these phenotypes. The phenotype of the backcrossed peroxisome‐deficient derivates was identical: defective in the ability to utilize methanol but capable of growth on other carbon sources, including ethanol. The mutations complemented and therefore were recessive mutations in different genes.

Pichia pastoris and Hansenula polymorpha are methylotrophic yeasts capable of utilizing methanol, as a sole source of carbon and energy. Growth of these yeast species on methanol requires the synthesis of cytosolic and peroxisomal enzymes combined with the proliferation of peroxisomes. Peroxisomes are also abundantly present in the alkane-utilizing yeast Yarrowia lipolytica upon growth of cells on oleic acid. This feature has made these yeast species attractive model systems to dissect the molecular mechanisms controlling peroxisome biogenesis. We have found that upon glucose- or ethanol-induced catabolite inactivation, metabolically superfluous peroxisomes are rapidly and selectively degraded within the vacuole by a process called pexophagy, the selective removal of peroxisomes by autophagy-like processes. Utilizing several genetic screens, we have identified a number of genes that are essential for pexophagy. In this review, we will summarize our current knowledge of the molecular events of pexophagy.

We developed the alkane and fatty-acid utilizing yeast Candida tropicalis as a host for DNA transformations. The system is based on an auxotrophic mutant host of C. tropicalis which is defective in orotidine monophosphate decarboxylase (ura3). The ura3 host was isolated by mutagenesis and a double-selection procedure that combined nystatin enrichment selection and 5-fluoro-orotic acid resistance selection. As a selectable marker, we isolated and characterized the C. tropicalis URA3 gene. Plasmid vectors that contained the C. tropicalis URA3 gene transformed the C. tropicalis mutant host at a frequency of 10(3) to 10(4) transformants per micrograms of plasmid DNA. Vectors that contained the Saccharomyces cerevisiae URA3 gene could not transform C. tropicalis. DNA transfer was accomplished by modified versions of either spheroplast generation (CaCl2-polyethylene glycol)-fusion or cation (LiCl) procedures developed for S. cerevisiae. Plasmid vectors that had been cut within the C. tropicalis URA3 fragment integrated by homologous recombination at the URA3 locus.