HIGH COPY NUMBER INTEGRATION INTO THE RIBOSOMAL DNA OF THE YEAST PHAFFIA-RHODOZYMA

This report describes a transformation system leading to stable high c opy number integration into the ribosomal DNA (rDNA) of the astaxanthi n-producing yeast Phaffia rhodozyma. A plasmid was constructed that co ntains the transposon Tn5 encoded kanamycin resistance gene (Km(R)) fu sed in frame to the 5'-terminal portion of the Phaffia actin gene. Thi s marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that compri ses the 18S rDNA and promotes high copy integration leading to stable Phaffia transformants that maintained the plasmid at high copy number after 15 generations of non-selective growth. Phaffia, strain CBS 6938 , was found to contain the rDNA units in clusters distributed over thr ee chromosomes with a total copy number of 61. Phaffia transformants w ere shown to have over 50 copies of pGB-Ph9 integrated in tandem in ch romosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophores is strongly suggest that Phaffia is haploid.