High copy number integration into the ribosomal DNA of the yeast Phaffia rhodozyma.
This report describes a transformation system leading to stable high copy number integration into the ribosomal DNA (rDNA) of the astaxanthin-producing yeast Phaffia rhodozyma. A plasmid was constructed that contains the transposon Tn5 encoded kanamycin resistance gene (KmR) fused in frame to the 5'-terminal portion of the Phaffia actin gene. This marker, driven by the Phaffia actin promoter, confers resistance to G418 (Geneticin). The plasmid also contains a rDNA portion that comprises 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 three chromosomes with a total copy number of 61. Phaffia transformants were shown to have over 50 copies of pGB-Ph9 integrated in tandem in chromosomes that contain rDNA loci. The chromosomal shifts that occur as a result of these integrations as shown by pulsed field electrophoresis strongly suggest that Phaffia is haploid.
Wery
J
J
Division of Industrial Microbiology, Department of Food Science, Wageningen Agricultural University, The Netherlands. jan.wery@algemeen.im.wau.nl
Gutker
D
D
Renniers
A C
AC
Verdoes
J C
JC
van Ooyen
A J
AJ
D000199:Actins / Q000235:genetics; D015139:Blotting, Southern; D003001:Cloning, Molecular; D015342:DNA Probes; D004275:DNA, Ribosomal / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D016521:Electrophoresis, Gel, Pulsed-Field; D018628:Gene Dosage; D005800:Genes, Fungal / Q000235:genetics*; D005819:Genetic Markers / Q000235:genetics; D005839:Gentamicins / Q000494:pharmacology; D007612:Kanamycin / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014170:Transformation, Genetic / Q000235:genetics
D000199:Actins; D015342:DNA Probes; D004275:DNA, Ribosomal; D005819:Genetic Markers; D005839:Gentamicins; D007612:Kanamycin; C010680:antibiotic G 418