Cloning of several lignin peroxidase (LIP)-encoding genes: sequence analysis of the LIP6 gene from the white-rot basidiomycete, Phanerochaete chrysosporium

Abstract

A Phanerochaete chrysosporium BKMF1767 genomic library, constructed in the BamHI site of vector YRp12, was screened with the lignin peroxidase(LIP)-encoding cDNAs, CLG4 and CLG5, that have been shown to encode LIP2 (previously H2) and LIP6 (previously H10), respectively. Six distinct LIP genomic clones, designated pGLG1, pGLG2, pGLG3, pGLG4, pGLG5, and pGLG6, were isolated in this study. Probe CLG4 hybridized only to pGLG1. Probe CLG5 gave intense hybridization to pGLG2 and weaker hybridization to clones pGLG3 through pGLG6, but showed little or no hybridization to pGLG1. These results, in agreement with previous biochemical results, indicate the existence of LIP gene subfamilies. The limits and transcriptional orientation of the LIP gene in each clone were determined. The sequence data showed that pGLG2 contains the LIP6 gene, which encodes a protein identical in amino acid (aa) composition to that encoded by CLG5. It contains a leader sequence of 27 aa and a mature protein of 344 aa (Mr 36,607). Archetypal TATA-box-like and CAAT-box-like sequences in the 5'-noncoding region are located 51 and 97 nt upstream from the cDNA start point, respectively. S1 nuclease analysis of the 5' region of LIP6 revealed two transcription start points 8 nt apart downstream from the TATA box. Comparison of the sequence of LIP6 with its corresponding cDNA CLG5 showed that the gene contains nine small introns which range in size from 50 to 62 bp. These introns contained consensus splice junction sequences similar to those reported in other fungal and yeast introns.