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Ceriporiopsis subvermispora is a white-rot fungus with great potential for industrial and biotechnological applications, such as the pretreatment of lignocellulose in biorefineries, as it decomposes the lignin in the plant cell wall without causing severe cellulose degradation. A genetic transformation system was recently developed; however, gene-targeting experiments to disrupt or modify the gene(s) of interest remain challenging, and this is a bottleneck for further molecular genetic studies and breeding of C. subvermispora . Herein, we report efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene mutagenesis in this fungus. Two plasmids expressing Cas9 together with a different pyrG -targeting single-guide RNA were separately introduced into the monokaryotic C. subvermispora strain FP-90031-Sp/1, which frequently generated strains that exhibited resistance to 5-fluoroorotic acid and uridine/uracil auxotrophy. Southern blot analyses and genomic polymerase chain reaction followed by DNA sequencing of some mutants revealed that they were pyrG mutants. We also observed that hygromycin resistance of the pyrG mutants was frequently lost after repeated subcultivations, indicating that a maker-free genome editing occurred successfully. It is also suggested that a gene mutation(s) can be introduced via a transient expression of Cas9 and a single-guide RNA; this feature, together with high-frequency gene targeting using the CRISPR/Cas9 system, would be helpful for studies on lignocellulose-degrading systems in C. subvermispora . Key points • Efficient plasmid-based CRISPR/Cas9 was established in C. subvermispora. • The mutations can be introduced via a transient expression of Cas9 and sgRNA. • A maker-free CRISPR/Cas9 is established in this fungus.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted gene targeting is a promising method used in molecular breeding. We recently reported the successful introduction of this method in the monokaryotic Pleurotus ostreatus (oyster mushroom), PC9. However, considering their application in mushroom breeding, dikaryotic strains (with targeted gene mutations in both nuclei) need to be generated. This is laborious and time-consuming because a classical crossing technique is used. Herein, we report a technique that targets both nuclei of dikaryotic P. ostreatus, PC9×#64 in a transformation experiment using plasmid-based CRISPR/Cas9, with the aim of developing a method for efficient and rapid molecular breeding. As an example, we targeted strains with low basidiospore production ability through the meiosis-related genes mer3 or msh4. Four different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting mer3 or msh4 were constructed and separately introduced into PC9×#64. Eight of the 38 dikaryotic transformants analyzed produced no basidiospores. Genomic PCR suggested that msh4 or mer3 mutations were introduced into both nuclei of seven out of eight strains. Thus, in this study, we demonstrated simultaneous gene targeting using our CRISPR/Cas9 system, which may be useful for the molecular breeding of cultivated agaricomycetes.

ABSTRACT Until recently, classical breeding has been used to generate improved commercial mushroom strains; however, classical breeding remains to be laborious and time-consuming. In this study, we performed gene mutagenesis using Cas9 ribonucleoprotein (Cas9 RNP) as a plasmid-free genome editing in Pleurotus ostreatus, which is one of the most economically important cultivated mushrooms. The pre-assembled Cas9/sgRNA targeting pyrG was introduced into protoplasts of a wild-type monokaryotic P. ostreatus strain PC9, which resulted in a generation of strains exhibiting resistance to 5-fluoroorotic acid. Small insertions/deletions at the target site were identified using genomic PCR followed by sequencing. The results showed Cas9 RNP-assisted gene mutagenesis could be applied for the molecular breeding in P. ostreatus and in other edible mushroom strains. Furthermore, gene disruption via split-marker recombination using the Cas9 RNP system was also successfully demonstrated in wild-type P. ostreatus PC9. This method could overcome the disadvantages of NHEJ-deficiency in conventional studies with gene targeting, and also difficulty in gene targeting in various non-model agaricomycetes. Cas9 ribonucleoprotein-assisted gene mutagenesis and gene disruption via split-marker recombination open the door for genome editing in the cultivated edible mushroom Pleurotus ostreatus.

Pleurotus ostreatus is one of the most commercially produced edible mushrooms worldwide. Improved cultivated strains with more useful traits have been obtained using classical breeding, which is laborious and time-consuming. Here, we attempted efficient gene mutagenesis using plasmid-based CRISPR/Cas9 as the first step for non-genetically modified (non-GM) P. ostreatus generation. Plasmids harboring expression cassettes of Cas9 and different single guide RNAs targeting fcy1 and pyrG were individually transferred into fungal protoplasts of the PC9 strain, which generated some strains exhibiting resistance to 5-fluorocytosine and 5-fluoroorotic acid, respectively. Genomic PCR followed by sequencing revealed small insertions/deletions or insertion of a fragment from the plasmid at the target site in some of the drug-resistant strains. The results demonstrated efficient CRISPR/Cas9-assisted genome editing in P. ostreatus, which could contribute to the molecular breeding of non-GM cultivated strains in the future. Furthermore, a mutation in fcy1 via homology-directed repair using this CRISPR/Cas9 system was also efficiently introduced, which could be applied not only for precise gene disruption, but also for insertions leading to heterologous gene expression in this fungus.

Abstract Hydrophobins, which are small-secreted proteins with both hydrophobic and hydrophilic parts, can self-assemble into an amphiphilic film at the air-water interface, helping the fungus to form aerial hyphae. In the agaricomycete Pleurotus ostreatus, more than 20 putative hydrophobin genes have been predicted. Of these, two hydrophobin genes, vmh2 and vmh3, are predominantly expressed in the vegetative mycelium. In this study, we focused on the functions of Vmh2 and Vmh3 in vegetative mycelia. Based on the observation of the mycelial cross-section by transmission electron microscopy and the disappearance time of water droplets on the mycelial surface, Vmh2 and Vmh3 were considered essential for the maintenance of the surface hydrophobicity of the mycelium. The Δvmh3 and Δvmh2Δvmh3 strains exhibited relatively slower aerial mycelia formation on a liquid medium, and no significant alteration was observed in Δvmh2 strains. Only the Δvmh3 and Δvmh2Δvmh3 strains grew slower than the wild-type strain under stress conditions involving SDS and H2O2 on agar plates. This study revealed possible distinct roles for these hydrophobins in stress resistance. These results suggest that Agaricomycetes, including P. ostreatus, have evolved to possess multiple different hydrophobins as a means of adapting to various environments. The first report of functional differences between hydrophobins Vmh2 and Vmh3 in Pleurotus ostreatus.

Cis -acting elements play a vital role in regulation of transcription initiation. Several cis -acting elements have been identified in filamentous fungi; however, the fundamental requirements for basic promoter function in basidiomycetes are obscure. In this study, core elements in β 1 -tubulin promoters of basidiomycetes were functionally characterized. Using transient transformation in Ceriporiopsis subvermispora as a promoter assay, we found that a 14-bp region ( β 1 - tubulin core promoter element, BCE), as well as CT-rich stretch, in the β 1 - tubulin promoter of the species played a critical role in the expression of a recombinant hph as a reporter gene. In addition, in silico analysis revealed other members of basidiomycetes also harboured the BCE motif as well as CT-rich stretch in the β 1 - tubulin promoter region, suggesting their functional conservation among the species of basidiomycetes. To confirm the function of BCE, we investigated the effects of BCE motif deletion in the Pleurotus ostreatus β 1 - tubulin promoter on expression levels of a recombinant luminous shrimp luciferase reporter gene, which was targeted into the Pofcy1 locus . Intriguingly, luciferase activity was abolished when the BCE motif was deleted in the β 1 - tubulin promoter, strongly demonstrating its essential function in transcription from this promoter on the chromosome. This study clearly demonstrates the crucial role of the BCE as well as the CT-rich stretch regions in the β 1 - tubulin promoter among basidiomycetes and provides new insights into the fundamental mechanism of transcription initiation in this group.

The global trend toward carbon neutrality and sustainability calls for collaborative efforts in both the basic and applied research sectors to utilize mushroom mycelia as environmentally friendly and sustainable materials. Fungi, along with animals and plants, are one of the major eukaryotic life forms. They have long been utilized in traditional biotechnology sectors, such as food fermentation, antibiotic production, and industrial enzyme production. Some fungi have also been consumed as major food crops, such as the fruiting bodies of various mushrooms. Recently, new trends have emerged, shifting from traditional applications towards the innovative use of mushroom mycelium as eco-friendly bioresources. This approach has gained attention in the development of alternative meats, mycofabrication of biocomposites, and production of mycelial leather and fabrics. These applications aim to replace animal husbandry and recycle agricultural waste for use in construction and electrical materials. This paper reviews current research trends on industrial applications of mushroom mycelia, covering strain improvements and molecular breeding as well as mycelial products and the production processes. Key findings, practical considerations, and valorization are also discussed. Graphical Abstract

Abstract 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. Using the pyrG strain obtained via CRISPR/Cas9-mediated homologous recombination as a host, we evaluated the potential of pyrG gene as a novel selectable marker gene.

Mycelium-based materials derived from white-rot fungi have attracted increasing attention as sustainable and eco-friendly alternatives to conventional products. However, their mechanical strength and durability remain relatively inferior. Molecular breeding of white-rot fungi offers a promising strategy to address these limitations. Recent studies have suggested that mycelial density and cell wall structure play key roles in determining the physical properties of mycelium-based materials. In this study, we disrupted mbp1 , which encodes a transcription factor required for normal mycelial growth and cell wall synthesis, in the white-rot fungus Pleurotus ostreatus and investigated the effects on mycelium mats and mycelium-based composites. The mycelium mats of the dikaryotic mbp1 disruptants exhibited higher Young’s moduli and ultimate tensile strengths than those of the control strain (20b×#61), indicating that mbp1 disruption resulted in stiffer mycelium mats. This may be because of the increased mycelial density in the dikaryotic mbp1 disruptants. In addition, the dikaryotic mbp1 disruptants produced harder mycelium-based composites than the strain 20b×#61. These findings indicate that mbp1 disruption improved the characteristics of mycelium-based composites. To our knowledge, this is the first study demonstrating that molecular breeding can enhance the performance of mycelium-based composites, thereby paving the way for the development of efficient strategies to advance mycelium-based materials. Key points • mbp1 disruption resulted in stiffer mycelium mats. • Dikaryotic mbp1 disruptants formed thinner but denser mycelium mats. • mbp1 disruption improved the mechanical strength of mycelium-based composites.

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.