Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi.
Phanerochaete chrysosporium
simultaneously degrades lignin and cellulose, whereas the closely related species,
Ceriporiopsis subvermispora,
also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of
C. subvermispora
and
P. chrysosporium
. Genes encoding manganese peroxidase numbered 13 and five in
C. subvermispora
and
P. chrysosporium
, respectively. In addition, the
C. subvermispora
genome contains at least seven genes predicted to encode laccases, whereas the
P. chrysosporium
genome contains none. We also observed expansion of the number of
C. subvermispora
desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in
C. subvermispora
culture filtrates, but none in
P. chrysosporium
cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two
C. subvermispora
genes were predicted to encode peroxidases structurally similar to
P. chrysosporium
lignin peroxidase and, following heterologous expression in
Escherichia coli
, the enzymes were shown to oxidize high redox potential substrates, but not Mn
2+
. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the
C. subvermispora
genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to
P. chrysosporium
.
The A mating type factor of the fungus Coprinus cinereus regulates essential steps in sexual development. Here we describe features of one of the four specificity genes of the A42 factor. By transformation we show that the gene regulates not only sexual development but also asexual sporulation. DNA sequence analysis shows that the gene beta 1–1, encodes a protein with a DNA binding motif and is thus likely to be a transcription factor. The DNA binding domain is an unusual homeodomain with D replacing the normally invariant N in the recognition helix and apparent absence of helix II. The homeodomain is linked to a helical region related to the POUs domain, which is part of a bipartite DNA binding domain of certain animal transcription factors. Like POU factors, the beta 1–1 protein has regions rich in serine, threonine and proline which are possible transactivation domains. Putative dimerization domains and sites for post‐translational modification are described.
White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood decaying activity within the Basidiomycota genus Pycnoporus. We observed strong conservation in the genome structures and the repertoires of protein coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analyzed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2 producing enzymes with H2O2 consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 LPMO gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood decaying process.
Engineering of fungal laccases with optimum catalytic activity at alkaline pH has been a long-lasting challenge. In this study, a mutant library containing 3000 clones was obtained by error-prone PCR to adapt the optimum pH of a fungal laccase Lcc9 from the basidiomycete Coprinopsis cinerea. After three rounds of functional screening, a mutant with three amino acid changes (E116K, N229D, I393T) named PIE5 was selected. PIE5 showed an optimum pH of 8.5 and 8.0 against guaiacol and 2,6-DMP when expressed in Pichia pastoris, representing the first fungal laccase that possesses an optimum pH at an alkaline condition. Site directed mutagenesis disclosed that N229D contributed the most to the optimum pH increment. A single N229D mutation caused an increase in optimum pH by 1.5 units. When used in indigo dye decolorization, PIE5 efficiently decolorized 87.1 ± 1.1% and 90.9 ± 0.3% indigo dye at the optimum conditions of pH 7.0–7.5 and 60 °C, and with either methyl 3,5-dimethoxy-4-hydroxybenzoate or 2,2′-azino-bis(3-ethylbenzothazoline-6-sulfonate) as the mediator. In comparison, the commercially available fungal laccase TvLac from Trametes villosa decolorized 84.3 ± 1.8% of indigo dye under its optimum conditions (opt. pH 5.0 and 60 °C). The properties of an alkaline-dependent activity and the high indigo dye decolorization ability (1.3-fold better than the parental Lcc9) make the new fungal laccase PIE5 an alternative for specific industrial applications.
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