Cloning and homologous expression of novel lignin peroxidase genes in the white-rot fungus Phanerochaete sordida YK-624.
Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.
Sugiura
Tatsuki
T
Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Japan.
Yamagishi
Kenji
K
Kimura
Toshiyuki
T
Nishida
Tomoaki
T
Kawagishi
Hirokazu
H
Hirai
Hirofumi
H
D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal; D015870:Gene Expression; D016681:Genome, Fungal / Q000235:genetics; D008969:Molecular Sequence Data; D010544:Peroxidases / Q000096:biosynthesis / Q000235:genetics*; D020075:Phanerochaete / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
D018076:DNA, Complementary; D004271:DNA, Fungal; D011994:Recombinant Proteins; D010544:Peroxidases; C042858:lignin peroxidase