Transformation by complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete Phanerochaete sordida YK-624.
Phanerochaete sordida YK-624 is a hyper lignin-degrading basidiomycete possessing greater ligninolytic selectivity than either P. chrysosporium or Trametes versicolor. To construct a gene transformation system for P. sordida YK-624, uracil auxotrophic mutants were generated using a combination of ultraviolet (UV) radiation and 5-fluoroorotate resistance as a selection scheme. An uracil auxotrophic strain (UV-64) was transformed into a uracil prototroph using the marker plasmid pPsURA5 containing the orotate phosphoribosyltransferase gene from P. sordida YK-624. This system generated approximately 50 stable transformants using 2 x 10(7) protoplasts. Southern blot analysis demonstrated that the transformed pPsURA5 was ectopically integrated into the chromosomal DNA of all transformants. The enhanced green fluorescent protein (EGFP) gene was also introduced into UV-64. The transformed EGFP was expressed in the co-transformants driven by P. sordida glyceraldehyde-3-phosphate dehydrogenase gene promoter and terminator regions.
Yamagishi
Kenji
K
National Agricultural Research Center for the Tohoku Region, National Agriculture and Food Research Organization, Arai, Fukushima 960-2156, Japan. yamagisi@affrc.go.jp
Kimura
Toshiyuki
T
Oita
Sigeru
S
Sugiura
Tatsuki
T
Hirai
Hirofumi
H
D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007218:Industrial Microbiology / Q000379:methods; D008031:Lignin / Q000378:metabolism*; D009154:Mutation; D009962:Orotate Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism; D009963:Orotic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D020075:Phanerochaete / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism / Q000528:radiation effects; D014170:Transformation, Genetic; D014498:Uracil / Q000378:metabolism*
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014498:Uracil; D009963:Orotic Acid; C001242:5-fluoroorotic acid; D008031:Lignin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D009962:Orotate Phosphoribosyltransferase