Expression on wood, molecular cloning and characterization of three lignin peroxidase (LiP) encoding genes of the white rot fungus Phlebia radiata

Lignin peroxidase (LiP) is the first enzyme connected to oxidative breakdown of the aromatic plant heteropolymer lignin and related xenobiotics. However, this extracellular enzyme has been described in only a few species of wood-decaying basidiomycetous fungi. The white rot basidiomycete Phlebia radiata 79 readily produces a versatile set of lignin-oxidizing enzymes including lignin and manganese peroxidases (LiPs and MnPs) and laccases. Here we describe genomic and primary structure of two new LiP-encoding genes, Pr-lip1 and Pr-lip4, and genomic characterization for isozyme LiP3/LIII of P. radiata, encoded by the gene depicted Pr-lip3. Pr-lip1 and Pr-lip4 code for 370- and 361-amino-acid long proteins beginning with 26- and 24-amino-acid secretion pre-propeptides, respectively. Translated LiP1 and LiP4 share the highest protein sequence identity (74 and 86%) with P. radiata LiP3, and 70% identity with the one deduced LiP from Bjerkandera adusta. The three P. radiata LiP sequences form a coherent phylogenetic cluster, which is further supported by similarities within gene organization interrupted by 11-introns. To find out the significance of LiP upon fungal growth on natural lignocellulose, such as wood, we studied ligninolytic gene expression on hardwood (milled alder) and softwood (spruce chips). All the LiP-encoding genes were expressed on wood with predominance of Pr-lip3 transcript abundance, in particular on spruce wood chips, where also time-dependent expression of the multiple lip genes was observed.