Cloning and Homologous Expression of Novel Lignin Peroxidase Genes in the White-Rot Fungus Phanerochaete sordida YK-624

Department of Bioscience, Graduate School of Science and Technology, Shizuoka University, Japan.
Bioscience Biotechnology and Biochemistry (Impact Factor: 1.06). 09/2009; 73(8):1793-8. DOI: 10.1271/bbb.90152
Source: PubMed


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.

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Available from: Hirofumi Hirai
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    • "In addition, lignin peroxidase (LiP) and manganese peroxidase (MnP) are considered to be the most effective enzymes for recycling carbon sources fixed as lignin [3]. As genes encoding LiP are quite limited to white rot fungi, including Phanerochaete chrysosporium[4,5], P. sordida[6], Trametes versicolor[7], Phlebia radiata[8,9], P. tremellosa[10], and Bjerkandera sp. [11], genes encoding MnP have drawn attention as an alternative ligninolytic peroxidase due to their wider distribution among basidiomycetes compared to those encoding LiP. "
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    • "UV-64 protoplasts were prepared and then transformed with pPsURA5 and pBUNA2pro-mnp4 using standard techniques. The cotransformed clones were selected by PCR, as described previously (Sugiura et al., 2009), with the following modifications: primers bee2proF4 and mnp4R3 were designed to amplify the mnp4 gene fused with the bee2 promoter. "
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    • "LiPs are secreted as sets of multiple isozymes and isoforms, by P. chrysosporium and a few other corticioid and polyporous white-rot fungi, e.g., Trametes versicolor (Johansson and Nyman 1993), Phlebia radiata (Lundell 1993; Moilanen et al. 1996), Phlebia tremellosa (Vares et al. 1994), Bjerkandera spp. (ten Have et al. 1998), and Phanerochaete sordida (Sugiura et al. 2009). LiP is catalytically the most powerful class II fungal peroxidase with the ability to directly oxidize dimeric lignin model compounds such as the nonphenolic β-O-4 linkagetype arylglycerol-aryl ethers (Scheme 1, reaction 2, substrate A). "
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    Full-text · Article · Jul 2010 · Applied Microbiology and Biotechnology
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