Cloning and characterization of lignin degrading enzymes in Polyporus brumalis for biological pretreatment of lignocellulosic biomass

Abstract

The genes encoding lignin degradation-related enzymes including laccase and manganese peroxidase (MnP) were isolated and overexpressed in a white-rot fungus, Polyporus brumalis, followed by functional analysis of the transformants. Two laccase (pblac1 and pblac2) and six MnP (pbmnp1-6) cDNAs were cloned from P. brumalis (KFRI 20912) which was isolated by the Korea Forest Research Institute. The deduced amino acid sequences of the laccase and MnP genes shared 70% and 62-96% identity, respectively. An RT-PCR analysis indicated that the RNAs of these genes were predominantly expressed in shallow stationary culture (SSC) in a liquid medium and increased after treatment of dibutyl phthalate (DBP) and wood chips. Especially, the transcription levels of pblac1 and pbmnp4 were higher than those of other genes and were proportional to the corresponding enzyme specific activity, suggesting that the transciption level of the two genes plays an important role in enzyme activity. Both pblac1 and pbmnp4 genes under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter were overexpressed by genetic transformation in P. brumalis. The enzyme activities of both laccase and MnP in the transformants were significantly higher than those of the wild type. The transformants exhibited more effective decolorization of the dye Remazol Brilliant Blue R than the wild type. When incubating with wood chips from red pine (softwood) and tulip tree (hardwood) for 15 and 45 days, the transformant with enhanced laccase activity showed higher lignin-degrading activity as well as higher wood-chip weight loss than the wild type. When the wood chips treated with the transformant were enzymatically saccharified, the highest sugar yields were found to be 32.5% for red pine wood and 29.5% for tulip tree wood based on the dried wood weights that were about 1.6-fold higher than those for the wild type. These results suggested that overexpression of the laccase gene from P. brumalis significantly contributs to the pretreatment of lignocellulose for increasing sugar yields. Thus, the identification of the genes for laccase and MnP cDNAs is the first step to characterize the molecular events related to the lignin degradation ability of white-rot fungi, which can contribute to the efficient production of lignin degradation enzymes and lead to the utilization of these fungi for the biological pretreatment of lignocellulosic biomass.