TRANSFORMATION IN THE METHYLOTROPHIC YEAST PICHIA-METHANOLICA UTILIZING HOMOLOGOUS ADE1 AND HETEROLOGOUS SACCHAROMYCES-CEREVISIAE ADE2 AND LEU2 GENES AS GENETIC-MARKERS

Development of transformation systems for methylotrophic yeasts is the starting point for research aimed at developing molecular genetics of these genera and will be the key to their further successful use in biotechnology. We transformed Pichia methanolica using selector genes ADE2 and LEU2 from Saccharomyces cerevisiae and ADE1 (homologue of S. cerevisiae ADE2 gene) from P. methanolica which was cloned and sequenced in our laboratory (Hiep et al., 1991). Lithium transformation of P. methanolica strains was inefficient with intact plasmids. Linearization of plasmids at a unique restriction site within the ADE1 gene prior to transformation substantially increased its frequency. Transformation with linear ADE1, ADE2 or LEU2 gene fragments was even more effective. Introduced DNA fragments either circularized in vivo, irrespective of the structures of their ends, giving unstable transformants; or integrated at different sites of the host genome. Using this transformation system, we obtained a disruption of the ADE1 gene on the chromosome by inserting the S. cerevisiae LEU2 gene. The disruption mutation ade1=LEU2 was used to study the mechanism of intragenic recombination in P. methanolica.