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.

Recent reductions in the size of facial pigmented basal cell carcinoma at diagnosis and the surgical margin: A retrospective and comparative study.

The present, retrospective, single-center study analyzed various factors associated with primary basal cell carcinoma (BCC) in the period before and after the introduction of dermoscopy (BD and AD, respectively). The demographic data of patients with primary BCC between 2001 and 2005 (BD: 84 patients, 90 cases) and 2011 and 2018 (AD: 297 patients, 320 cases) were analyzed. In the pigmented BCC-predominant cohort (94%), the proportion of smaller tumors as well as the total number of tumors significantly increased during AD (median tumor size, 10.0 mm in BD, 8.0 mm in AD; Mann-Whitney U-test, p = 0.011). BCC were excised with a significantly narrower margin during AD (median, 2.0 mm) than during BD (median, 3.0 mm; Mann-Whitney U-test, p < 0.001; odds ratio, 0.30; multivariate logistic regression analysis, p < 0.001); the incomplete excision rate was 1.9%, and the recurrence rate was 0%. The present study suggests that the introduction of dermoscopy might have aided in the early diagnosis of smaller BCC, especially in the face region, and determining the appropriate surgical margin. The smaller pigmented BCC can be excised with a narrower margin than stated in the guidelines (4 mm).

Overexpression and repression of the tyrosinase gene in Lentinula edodes using the pChG vector.

Tyrosinase is an industrially useful enzyme, however, it causes gill browning of Lentinula edodes fruiting bodies during preservation. In this study, we constructed two vectors, pChG-gTs and pChG-gTa, expressing sense and antisense tyrosinase gene of L. edodes, respectively, using promoters derived from the glyceraldehyde-3-phosphate dehydrogenase gene. The host strain SR-1 of L. edodes was selected because of its fast growth, high protoplast yield, and high regeneration rate. Upon transformation of the host strain SR-1 with the pChG-gTs vector, a clone with 3.6-fold and 14.5-fold higher tyrosinase activity in vegetative mycelia and in fresh gills, respectively, than that of the host strain was obtained from nine transformants. Similarly, two clones containing the pChG-gTa vector with effectively repressed tyrosinase gene expression in vegetative mycelia and gills during the late stage of post-harvest preservation of fruiting bodies were obtained from 10 transformants. However, it remained unclear whether repression of the tyrosinase gene prevented gill browning, as the host strain also showed less browning than a commercial strain. Thus, this study highlights the usefulness of the pChG vector in expressing homologous enzyme coding genes in the vegetative mycelia and fruiting bodies of L. edodes.

Heterologous expression of the Pleurotus ostreatus MnP3 gene by the laccase gene promoter in Lentinula edodes.

Lentinula edodes (shiitake), which have a powerful ligninolytic system, is one of the most important edible mushrooms in Asia. In this study, we introduced the manganese peroxidase (MnP, EC 1.11.1.13) gene from Pleurotus ostreatus driven by L. edodes laccase 1 gene promoter into L. edodes for expression. The resulting transformant expressed the recombinant gene and showed a higher level of MnP activity than that of the wild-type strain.

A case of a superficial spreading melanoma in situ diagnosed via digital dermoscopic monitoring with high dynamic range conversion.

A 48-year-old woman presented with a 3 mm, pigmented macule at her first visit to our clinic. The macule, which showed complete symmetry and a typical network, was tentatively diagnosed as a Clark nevus; a 6-month follow-up was recommended, and the patient returned 7 months later. At the second visit, the lesion had enlarged to a diameter of 5 mm, and dermoscopy revealed that it had maintained its typical pigment network. At this point, evidence-based monitoring would have led to excision but the decision was made to continue monitoring. Owing to poor compliance, the patient went another 2 years without follow-up. When we assess small lesions, such as this, the usefulness of dermoscopy is apparent. Additionally, we examined the benefits and drawbacks of high dynamic range (HDR) conversion of the dermoscopy images and their helpfulness for inspecting small lesions. Although the delicate structures present in the lesion can be recognized by a dermoscopy expert and HDR image conversion has a capacity to highlight important structures, there is also a risk that HDR image conversion may mask some of the structural changes. However, a comparison of the original dermoscopy images with the HDR-converted images provides newly trained dermoscopists the opportunity to recognize new findings and to distinguish the differences in the findings between both the types of images. Therefore, such comparisons might be useful for obtaining an accurate diagnosis by using dermoscopy and HDR image conversion.

Effective induction of pblac1 laccase by copper ion in Polyporus brumalis ibrc05015.

Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.

Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence.

Lentinula edodes is one of the most important edible mushrooms, but no method for analyzing its molecular genetics has yet been established. RNA interference (RNAi) is a mechanism that inhibits expression of specific genes at the post-transcriptional stage and has been used to analyze the genetics of several fungal species. RNAi was used to examine the expression of the laccase (EC 1.10.3.2) gene lcc1 of L. edodes, which encodes a lignin-degrading enzyme. Vector pChG'-ivrL1 that expressed a 40 bp homologous inverted repeat sequence from lcc1 was constructed. This was transformed into L. edodes using the restriction enzyme mediated integration method (REMI). Lcc1 protein was not detected in two of 57 transformants (ivrL1#26, ivrL1#32) where the lcc1 transcription levels were suppressed. Thus, a 40 bp inverted repeat sequence expression vector suppressed expression of the target gene in L. edodes. Lcc1 downregulated transformants (ivrL1#32) did not form a thick aerial mycelium mat on agar medium. Electron microscopy showed hyphae of ivrL1#32 had many short branches with low mycelial density, a thin cell wall, and few fibrous layers as compared to the host strain. These morphological phenotypes would be caused by the absence of Lcc1, and that provides some clue to resolve the biological function of Lcc1 in L. edodes. Our results show that RNAi can be used for gene silencing in L. edodes.

Construction of novel vectors for transformation of Lentinula edodes using a chitin synthase gene promoter.

Novel vectors (pLCHS-hph and pChG-bar) containing expression unit driving hph as a selectable marker gene by chitin synthase gene promoter were constructed for Lentinula edodes transformation. Expression of the hph gene in random selected transformants was confirmed by RT-PCR method. Thus, both vectors are useful for L. edodes transformation.

Characterization of an extracellular laccase, PbLac1, purified from Polyporus brumalis.

Polyporus brumalis (strain ibrc05015) secreted high amounts of laccases (Lacs) in liquid medium. With 2,2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) as a substrate, Lac activity was 7.72 U ml⁻¹ and this strain secreted a maximum 0.23 mg ml⁻¹ of total protein. The enzyme, PbLac1 was purified to homogeneity using hydrophobic and anion-exchange chromatography. The purified PbLac1 had a molecular mass of 63.4 kDa as determined by polyacrylamide-gel electrophoresis. PbLac1 oxidized a wide range of substrates such as 3,4-dihydroxy l-phenylalanine (l-DOPA) and catechol, but not tysorine. The activity of PbLac1 was increased by addition of 10.0 mM Cu(2+). PbLac1 could decolorize several industrial dyes, such as Remazol Brilliant Blue R known as model dyes of environmental xenobiotics. In addition, PbLac1 decolorized a wide range of substrates, such as the carcinogen, Poly R-478, in the presence of violuric acid as mediator. The E° value of PbLac1 was 0.80 V±0.01 versus normal hydrogen electrode, which is a very high redox potential compared to those of other basidiomycetous Lacs. These results suggest the potential utility of PbLac1 for industrial applications.

Cloning and characterization of a Lentinula edodes class II chitin synthase gene, LeChs2.

A genomic DNA sequence and cDNA encoding a putative chitin synthase were isolated from the white rot basidiomycete Lentinula edodes. The gene, named LeChs2, consists of a 2,598-bp open reading frame interrupted by 14 introns and encodes a putative protein of 866 amino acid residues. The data obtained in this study suggest that LeChs2 belongs to the class II chitin synthases.

A chimeric laccase with hybrid properties of the parental Lentinula edodes laccases.

We created a chimeric laccase from two different laccases, Lcc1 and Lcc4, from Lentinula edodes. Lcc1 is a secretory lignin-degrading enzyme produced in liquid cultures of L. edodes. Lcc4 is a tissue-accumulating-type enzyme, which is thought to be involved in melanin synthesis in fruiting body after harvesting. Lcc1 and Lcc4 differ in their Km values for some substrates, especially beta-(3,4-dihydroxyphenyl) alanine (L-DOPA) and catechol. The novel chimeric laccase, Lcc4/1, has properties that are a hybrid of those of Lcc1 and Lcc4. Lcc4/1 acts upon both Lcc1 and Lcc4 substrates and most of its Km values are lower than those of Lcc1 and Lcc4. Homology modeling indicates that the deduced shape of the substrate-binding pocket of the chimeric laccase is larger than that of Lcc1 and similar to that of Lcc4. The other biochemical properties, such as temperature and pH dependency, are intermediate between those of Lcc1 and Lcc4.

Characterization of the post-harvest changes in gene transcription in the gill of the Lentinula edodes fruiting body.

We compared the gene expression patterns of Lentinula edodes fresh fruiting bodies and fruiting bodies 3 days after harvest, by suppression subtractive hybridization, to characterize the physiologic changes that occur after harvest, such as gill browning and cell wall lysis of the fruiting body, which are responsible for the loss of food quality and value. We found increase of transcription levels of several enzyme encoding genes, such as, two phenol oxidases encoding genes (tyr tyrosinase, lcc4 laccase), and several cell wall degradation-related enzyme-encoding genes, such as mixed-linked glucanase (mlg1), chitinases (chi1, chi2), chitin deacetylase (chd1), and chitosanase (cho1), after harvesting. We isolated a putative transcription factor-encoding gene (L. edodes exp1) with high similarity to exp1 from Coprinopsis cinerea, which is involved in autolysis of the cap during spore diffusion. Transcription of L. edodes exp1 increased post-harvest, which suggests that its target genes are up-regulated after harvesting. These enzymes and the transcription factor may be involved in L. edodes fruiting body senescence.

The tyrosinase-encoding gene of Lentinula edodes, Letyr, is abundantly expressed in the gills of the fruit-body during post-harvest preservation.

The gill browning of Lentinula edodes fruit-bodies during preservation is thought to be due to melanin biosynthesis catalyzed by tyrosinase. We isolated a genomic DNA sequence and cDNA encoding a putative tyrosinase from the white rot basidiomycete Lentinula edodes (shiitake mushroom). The gene, named Letyr, consists of a 1,854-bp open reading frame interrupted by eight introns, and encodes a putative protein of 618 amino acid residues with an estimated molecular mass of 68 kDa. Amino acid residues known to be involved in copper-binding domains were conserved in the deduced amino acid residues of LeTyr. Transcriptional and translational expression of Letyr in the gills of the fruit-body increased during preservation after harvest. This correlation between Letyr expression and fruit-body preservation suggests that tyrosinase gene expression contributes to gill browning.

The basidiomycetous mushroom Lentinula edodes white collar-2 homolog PHRB, a partner of putative blue-light photoreceptor PHRA, binds to a specific site in the promoter region of the L. edodes tyrosinase gene.

We isolated a cDNA homolog of Neurospora crassa wc-2 from the basidiomycetous mushroom Lentinula edodes and termed it phrB cDNA. The deduced PHRB (313 amino acid residues) contained a PAS domain and a zinc-finger motif. Random binding-site selection analysis of the PHRB produced in Escherichia coli revealed that it bound to a 7-bp sequence with the consensus sequence 5'GATA/TTG/T/AC3'. Electrophoretic mobility-shift assay showed that it also bound to the consensus sequence 5'GATATTC3' in the promoter region of the L. edodes tyrosinase gene (Le.tyr). In vitro GST-pulldown immunoblot analysis disclosed that PHRB interacts with a putative blue-light photoreceptor of L. edodes (PHRA), the homolog of N. crassa WC-1, through the PAS B- and/or PAS C domain of PHRA. The expression of phrB and Le.tyr genes in pre-primordial mycelia of L. edodes is induced by light exposure, suggesting that PHRB can regulate the expression of the Le.tyr gene in a light-dependent manner.

Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae.

The shiitake mushroom, Lentinula edodes, has an extracelluar secretory-type laccase, Lcc1, and a fruiting-body-accumulation-type laccase, Lcc4. We previously reported the production of Lcc1 by plant cells, but had difficulty producing Lcc4. Here, we report the production of Lcc1 and Lcc4 by Aspergillus oryzae and the extracellular secretory production of Lcc4 using a modified secretion signal peptide (SP) from Lcc1. Sp-Lcc4 produced by A. oryzae had biochemical activities similar to Lcc4 produced by L. edodes. Lcc1 did not react with beta-(3,4-dihydroxyphenol) alanine (DOPA), but Lcc4 from L. edodes and A. oryzae could oxidize DOPA. K(M) values for the substrates 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate), 2,6-dimethoxyphenol, guaiacol, pyrogallol, and catechol were similar for Lcc4 and Sp-Lcc4. In conclusion, a non-secretory-type fungal laccase is secreted into the culture media with its original enzymatic properties by exploiting modified secretory signal peptide.

Isolation of industrial strains of Aspergillus oryzae lacking ferrichrysin by disruption of the dffA gene.

Based on studies using laboratory strains, the efficiency of gene disruption in Aspergillus oryzae, commonly known as koji mold, is low; thus, gene disruption has rarely been applied to the breeding of koji mold. To evaluate the efficiency of gene disruption in industrial strains of A. oryzae, we produced ferrichrysin biosynthesis gene (dffA) disruptants using three different industrial strains as hosts. PCR analysis of 438 pyrithiamine-resistant transformants showed dffA gene disruption efficiency of 42.9%-64.1%, which is much higher than previously reported. Analysis of the physiological characteristics of the disruptants indicated that dffA gene disruption results in hypersensitivity to hydrogen peroxide. To investigate the industrial characteristics of dffA gene disruptants, two strains were used to make rice koji and their properties were compared to those of the host strains. No differences were found between the dffA gene disruptants and the host strains, except that the disruptants did not produce ferrichrysin. Thus, this gene disruption technique is much more effective than conventional mutagenesis for A. oryzae breeding.