Development of a gene-targeting system using CRISPR/Cas9 and utilization of pyrG as a novel selectable marker in Lentinula edodes.

First, we attempted to recombine the Shiitake (Lentinula edodes) pyrG (ura3) gene homologously by introducing a donor vector containing a carboxin resistance gene (lecbxR) flanked by homologous sequences of pyrG into protoplasts of the fungus. However, all the carboxin-resistant transformants only contained ectopic insertions of the exogenous gene and no homologous insertions. Agaricomycetes are generally known for their low efficiency of homologous recombination, and a similar result was shown for L. edodes. We then co-introduced a Cas9 plasmid vector containing a CRISPR/Cas9 expression cassette targeting pyrG and donor plasmid vector. As a result, ∆pyrG strains containing the expected homologous recombination were obtained. However, only two of the seven ∆pyrG strains had the Cas9 sequence; the others did not. Our results suggest that genome editing occurred via the transient expression of the CRISPR/Cas9 cassette in the Cas9 plasmid vector introduced into the fungal cell. Transforming pyrG into a ∆pyrG strain (strain I8) resulted in prototrophic strains with an efficiency of 6.5 strains/experiment.

Effects of overexpression of PKAc genes on expressions of lignin-modifying enzymes by Pleurotus ostreatus.

We studied the role of genes encoding the cAMP-dependent protein kinase A catalytic subunit (PKAc) in the ligninolytic system in Pleurotus ostreatus. The wild-type P. ostreatus strain PC9 has two PKAc-encoding genes: PKAc1 and PKAc2 (protein ID 114122 and 85056). In the current study, PKAc1 and PKAc2 were fused with a ß-tubulin promoter and introduced into strain PC9 to produce the overexpression strains PKAc1-97 and PKAc2-69. These strains showed significantly higher transcription levels of isozyme genes encoding lignin-modifying enzymes than strain PC9, but the specific gene expression patterns differed between the two recombinant strains. Both recombinants showed 2.05-2.10-fold faster degradation of beechwood lignin than strain PC9. These results indicate that PKAc plays an important role in inducing the wood degradation system in P. ostreatus.

Effects of calmodulin on expression of lignin-modifying enzymes in Pleurotus ostreatus.

Previously, we suppressed the expression of genes encoding isozymes of lignin peroxidase (LiP) and manganese peroxidase (MnP) using a calmodulin (CaM) inhibitor, W7, in the white-rot fungus Phanerochaete chrysosporium; this suggested that CaM positively regulates their expression. Here, we studied the role of CaM in another white-rot fungus, Pleurotus ostreatus, which produces MnP and versatile peroxidase (VP), but not LiP. W7 upregulated Mn(2+)-dependent oxidation of guaiacol, suggesting that CaM negatively regulates the production of the enzymes. Suppression of CaM in P. ostreatus using RNAi also led to upregulation of enzyme activity, whereas overexpression of CaM in P. ostreatus caused downregulation. Real-time RT-PCR showed that MnP1-6 and VP3 levels in the CaM-knockdown strain were higher than those in the wild-type strain, while MnP-5 and -6 and VP1 and 2 levels in the CaM-overexpressing strain were lower than in the wild type. Moreover, we also found that another ligninolytic enzyme, laccase, which is not produced by P. chrysosporium, was negatively regulated by CaM in P. ostreatus similar to MnP and VP. Although overexpression of CaM did not reduce the ability of P. ostreatus to digest beech wood powder, the percentage of lignin remaining in the digest was slightly higher than in the wild-type strain digest.

Long serial analysis of gene expression for transcriptome profiling during the initiation of ligninolytic enzymes production in Phanerochaete chrysosporium.

To analyze the transcriptome profile during the initiation of manganese peroxidase (MnP) and lignin peroxidase (LiP) production in Phanerochaete chrysosporium, we constructed long serial analysis of gene expression (LongSAGE) libraries. A total of 13,666 tags (the number of cumulative counted tags) that included 6,945 unique tags (the number of distinct tags) were isolated from the day-3 culture, which just started the enzymes production and was 24 h after veratryl alcohol addition and oxygen-purge into the culture (day-2 culture). A total of 12,402 tags that included 6,396 unique tags were isolated from the day-2 culture, in which the activity of enzymes is not detected. The comparison of the two libraries suggested that 38 genes showed significant (p < or = 0.01) fourfold or greater upregulation; this included the MnP gene (mnp2, mnp3) and LiP H8 gene. On the other hand, 43 genes showed significant (p < or = 0.01) fourfold or greater downregulation. This LongSAGE analysis found many new candidate genes regulating the enzymes production.

Mn(2+) is dispensable for the production of active MnP2 by Pleurotus ostreatus.

The regulation mechanism for expression of versatile peroxidase MnP2 by the basidiomycete fungus Pleurotus ostreatus was examined using chemically defined synthetic media. Expression of MnP2 was down-regulated at the transcription level by nutrient nitrogen, e.g., NH(4)(+), arginine or urea. As is often the case with other fungal manganese peroxidases, active MnP2 was not detected when Mn(2+) was omitted from the culture, while mnp2 transcription was barely affected by Mn(2+). However, Mn(2+) can be substituted by an MnP2 substrate, Poly R-478, since active MnP2 was detected extracellularly when the compound was added to the culture without Mn(2+). Enzyme stability assays with the purified MnP2 indicated an indispensable requirement for a substrate that can be used to complete the catalytic cycle, and avoid inactivation resulting from an excess H(2)O(2). This report is the first of the Mn(2+)-independent production of an active versatile peroxidase by P. ostreatus.

Direct oxidation of polymeric substrates by multifunctional manganese peroxidase isoenzyme from Pleurotus ostreatus without redox mediators.

VPs (versatile peroxidases) sharing the functions of LiP (lignin peroxidase) and MnP (manganese peroxidase) have been described in basidiomycetous fungi Pleurotus and Bjerkandera. Despite the importance of this enzyme in polymer degradation, its reactivity with polymeric substrates remains poorly understood. In the present study, we first report that, unlike LiP, VP from Pleurotus ostreatus directly oxidized two polymeric substrates, bovine pancreatic RNase and Poly R-478, through a long-range electron pathway without redox mediators. P. ostreatus produces several MnP isoenzymes, including the multifunctional enzyme MnP2 (VP) and a typical MnP isoenzyme MnP3. MnP2 (VP) depolymerized a polymeric azo dye, Poly R-478, to complete its catalytic cycle. Reduction of the oxidized intermediates of MnP2 (VP) to its resting state was also observed for RNase. RNase inhibited the oxidation of VA (veratryl alcohol) in a competitive manner. Blocking of the exposed tryptophan by N-bromosuccinimide inhibited the oxidation of RNase and VA by MnP2 (VP), but its Mn2+-oxidizing activity was retained, suggesting that Trp-170 exposed on an enzyme surface is a substrate-binding site both for VA and the polymeric substrates. The direct oxidation of RNase and Poly R by MnP2 (VP) is in sharp contrast with redox mediator-dependent oxidation of these polymers by LiP from Phanerochaete chrysosporium. Molecular modelling of MnP2 (VP) revealed that the differences in the dependence on redox mediators in polymer oxidation by MnP2 (VP) and LiP were explained by the anionic microenvironment surrounding the exposed tryptophan.