[Development of a method for vector transformation of the methyltrophic yeast Pichia methanolica].

A method for transformation of the methylotrophic yeast Pichia methanolica (formerly P. pinus MH4) was developed. Mutants leu1 were shown to be transformed with different efficiency using 2.2-10.7-kb linear and circular DNA molecules containing the LEU2 gene of the yeast Saccharomyces cerevisiae, which complemented the leu1 mutation of the recipient. Efficiency of transformation with short molecules was higher than with long molecules. Transformation with linear DNA was more efficient than with circular DNA of equal size. Transformants contained both replication-unstable and integration forms in different proportions. Significant rearrangements in the episome and integration forms of transforming DNA were found in transformants obtained using the 10.7-kb circular YEp13 plasmid. Integration of linear DNA was not accompanied by rearrangements in DNA molecules. Certain clones isolated after transformation with linear DNA contained autonomously replicating circular vector molecules formed as a result of a ligase reaction in vivo. The LEU2 gene of S. cerevisiae contains an unknown sequence acting as the ARS replicon in cells of both P. methanolica and of another species of the methylotrophic yeasts, Hansenula polymorpha.