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.
2009 by Japan Society for Bioscience, Biotechnology, and Agrochemistry