DEVELOPMENT OF A TRANSFORMATION SYSTEM FOR THE YEAST YAMADAZYMA (PICHIA) OHMERI

This communication describes the development of genetic tools for the yeast Yamadazyma ohmeri. Nystatin enrichment proved highly effective for isolating various auxotrophic strains, which were classified by complementation analysis. Biosynthetic genes encoding known biochemical functions were isolated by polymerase chain reaction, including YoLEU2 and YoURA3 that were sequenced. Using these homologous genes as selective markers, DNA transformation was accomplished by electroporation. Transformation with pBR322-based plasmids, cut within the coding region of the homologous marker gene, yielded 20 to 50 stable transformants per mu g of DNA. In about 80% of the cases, integration of plasmid DNA sequence occurred by homologous recombination of a single plasmid into the chromosome. Excision of the plasmid permitted gene replacement, as illustrated by the substitution of a wild-type URA3 gene by an in vitro generated deletion. Sequences conferring extrachromosomal replication were isolated from Y. ohmeri DNA. Plasmids based on pBR322 carrying such an ARS and either selective markers transformed at 10(4)/mu g and were shown to replicate freely in Y. ohmeri at an approximate copy number of 40. Unexpectedly, we observed that BS-SKR derivatives carrying either YoLEU2 or YoURA3 but no Y. ohmeri ARS also replicated extrachromosomally. Linearization of transforming plasmids within regions homologous or not to chromosomal sequences stimulated transformation frequencies up to four-fold. The sequences are available for consultation under EMBL accession number Z35101 for YoLEU2 and Z35100 for YOURA3.