Journal of Microbiology

Full article

Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii: Hui-Gang Han, Rutuja Nandre, Hyerang Eom, Yeon-Jae Choi, Hyeon-Su Ro; J. Microbiol. 2025;63(6):e2502011. Published online June 30, 2025; DOI: https://doi.org/10.71150/jm.2502011

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Abstract PDF Supplementary Material: A thermophilic strain of Paecilomyces variotii (MR1), capable of surviving temperatures above 40°C, was isolated from a paper mill and investigated as a host for heterologous protein production. To prevent environmental dissemination of spores, UV mutagenesis was employed to create a conidia-deficient strain, UM7. This strain underwent gene editing using Cas9-gRNA ribonucleoprotein (RNP) with HR donor DNA fragments, incorporating promoter sequences amplified from the genomic DNA of P. variotii (P_H4, P_P2, P_S8, P_tub, P_tef1, and P_gpdA), along with a signal sequence-tagged eGFP, flanked by 5’-upstream (336 bp) and 3’-downstream (363 bp) regions of pyrG. Co-transformation of HR donor DNA with RNP into protoplasts yielded 48 mutant pyrG transformants capable of surviving in the presence of 5-fluoroorotic acid (5-FOA). Sequence analysis identified 16 of the 48 pyrG-disrupted mutants carrying complete HR donor DNAs with the six different promoter sequences, indicating successful homology-directed repair (HDR). Evaluation of promoter strength revealed that P_gpdA was the most effective for intracellular GFP production; however, it resulted in negligible extracellular GFP signal under all promoter conditions. A newly edited strain with an HDR integration module connecting P_gpdA directly to eGFP, without the signal sequence, exhibited enhanced GFP expression in both mycelial cells and culture broth, suggesting that the signal peptide negatively affect protein expression and secretion. This work represents the first successful RNP-mediated gene editing in P. variotii, contributing to the application of this thermophilic fungus in protein production.

Research Article

Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein: Yeon-Jae Choi, Hyerang Eom, Rutuja Nandre, Minseek Kim, Youn-Lee Oh, Sinil Kim, Hyeon-Su Ro; J. Microbiol. 2025;63(1):e.2409006. Published online January 24, 2025; DOI: https://doi.org/10.71150/jm.2409006

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The presence of multiple nuclei in a common cytoplasm poses a significant challenge to genetic modification in mushrooms. Here, we demonstrate successful gene editing in both nuclei of a dikaryotic strain of Ganoderma lucidum using the Cas9-gRNA ribonucleoprotein complex (RNP). The RNP targeting the pyrG gene was introduced into dikaryotic protoplasts of G. lucidum, resulting in the isolation of 31 mycelial colonies resistant to 5-fluoroorotic acid (5-FOA). Twenty-six of these isolates were confirmed as dikaryotic strains by the presence of two distinct A mating type markers, denoted as A1 and A2. All dikaryons exhibited clamp connections on their mycelial hyphae, while the remaining 5 transformants were monokaryotic. Subsequent sequence analysis of PCR amplicons targeting pyrG revealed that two dikaryons harbored disrupted pyrG in both nuclei (pyrG-/pyrG-), while 10 and 14 displayed pyrG+/pyrG- (A1/A2) and pyrG-/pyrG+ (A1/A2) configurations, respectively. The disruption was achieved through non-homologous end joining repair, involving deletion or insertion of DNA fragments at the site of the double-strand break induced by RNP. Importantly, the nuclei were stable throughout 10 serial transfers over a period of 6 months. These findings highlight the capability of RNP to target genes across multiple nuclei within the same cytoplasm.

Citations

Citations to this article as recorded by

Gene Editing in Ganoderma lucidum: Development, Challenges, and Future Prospects
Shiqi He, Yuanchao Liu, Zhi Zhang, Manjun Cai, Yufan Hao, Huiping Hu
Journal of Fungi.2025; 11(4): 310. CrossRef
Development of a CRISPR/Cas9 RNP-mediated genetic engineering system in Paecilomyces variotii
Hui-Gang Han, Rutuja Nandre, Hyerang Eom, Yeon-Jae Choi, Hyeon-Su Ro
Journal of Microbiology.2025; 63(6): e2502011. CrossRef
CRISPR/Cas9-mediated gene editing in filamentous fungi, focusing on mushrooms and plant-pathogenic fungi
Rutuja Nandre, Hyerang Eom, Yeon-Jae Choi, Yanjiao Zhang, Hyeon-Su Ro
Fungal Biology Reviews.2025; 54: 100446. CrossRef

Journal Article

Diversity of A mating type in Lentinula edodes and mating type preference in the cultivated strains: Byeongsuk Ha , Sinil Kim , Minseek Kim , Yoon Jung Moon , Yelin Song , Jae-San Ryu , Hojin Ryu , Hyeon-Su Ro; J. Microbiol. 2018;56(6):416-425. Published online June 1, 2018; DOI: https://doi.org/10.1007/s12275-018-8030-6

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Abstract

Diversity of A mating type in Lentinula edodes has been assessed by analysis of A mating loci in 127 strains collected from East Asia. It was discovered that hypervariable sequence region with an approximate length of 1 kb in the A mating locus, spanning 5􍿁􀁇region of HD2-intergenic region-5􍿁􀁇region of HD1, could represent individual A mating type as evidenced by comprehensive mating analysis. The sequence analysis revealed 27 A mating type alleles from 96 cultivated strains and 48 alleles from 31 wild strains. Twelve of them commonly appeared, leaving 63 unique A mating type alleles. It was also revealed that only a few A mating type alleles such as A1, A4, A5, and A7 were prevalent in the cultivated strains, accounting for 62.5% of all A mating types. This implies preferred selection of certain A mating types in the process of strain development and suggests potential role of A mating genes in the expression of genes governing mushroom quality. Dominant expression of an A mating gene HD1 was observed from A1 mating locus, the most prevalent A allele, in A1-containing dikaryons. However, connections between HD1 expression and A1 preference in the cultivated strains remain to be verified. The A mating type was highly diverse in the wild strains. Thirty-six unique A alleles were discovered from relatively small and confined area of mountainous region in Korean peninsula. The number will further increase because no A allele has been recurrently observed in the wild strains and thus newly discovered strain will have good chances to contain new A allele. The high diversity in small area also suggests that the A mating locus has evolved rapidly and thus its diversity will further increase.

Citations

Citations to this article as recorded by

Simultaneous gene editing of both nuclei in a dikaryotic strain of Ganoderma lucidum using Cas9-gRNA ribonucleoprotein
Yeon-Jae Choi, Hyerang Eom, Rutuja Nandre, Minseek Kim, Youn-Lee Oh, Sinil Kim, Hyeon-Su Ro
Journal of Microbiology.2025; 63(1): e:2409006. CrossRef
Intraspecies Variation Offers Potential to Improve White Rot Fungi for Increasing Degradability of Lignocellulose for Ruminants
Anton S. M. Sonnenberg, Nazri Nayan, John W. Cone, Arend F. van Peer
Journal of Fungi.2024; 10(12): 858. CrossRef
Effect of a Mating Type Gene Editing in Lentinula edodes Using RNP/Nanoparticle Complex
Minseek Kim, Minji Oh, Ji-Hoon Im, Eun-Ji Lee, Hojin Ryu, Hyeon-Su Ro, Youn-Lee Oh
Journal of Fungi.2024; 10(12): 866. CrossRef
Structural Analysis of the A Mating Type Locus and Development of the Mating Type Marker of Agaricus bisporus var. bisporus
Yeon-Jae Choi, Sujin Jung, Hyerang Eom, Thimen Hoang, Hui-Gang Han, Sinil Kim, Hyeon-Su Ro
Journal of Fungi.2023; 9(3): 284. CrossRef
Determination and Analysis of Hyper-Variable A Mating Types in Wild Strains of Lentinula edodes in Korea
Mi-Jeong Park, Eunjin Kim, Yeun Sug Jeong, Mi-Young Son, Yeongseon Jang, Kang-Hyeon Ka
Mycobiology.2023; 51(1): 26. CrossRef
Haplotype-Resolved Genome Analyses Reveal Genetically Distinct Nuclei within a Commercial Cultivar of Lentinula edodes
Qi Gao, Dong Yan, Shuang Song, Yangyang Fan, Shouxian Wang, Yu Liu, Yu Huang, Chengbo Rong, Yuan Guo, Shuang Zhao, Wentao Qin, Jianping Xu
Journal of Fungi.2022; 8(2): 167. CrossRef
Mitochondrial Effects on the Physiological Characteristics of Lentinula edodes
Minseek Kim, Seong-Hyeok Yang, Hui-Gang Han, Eunbi Kim, Sinil Kim, Youn-Lee Oh, Hyeon-Su Ro
Mycobiology.2022; 50(5): 374. CrossRef
Comparative structural analysis on the mitochondrial DNAs from various strains of Lentinula edodes
Sinil Kim, Hyerang Eom, Rutuja Nandre, Yeon Jae Choi, Hwayong Lee, Hojin Ryu, Hyeon-Su Ro
Frontiers in Microbiology.2022;[Epub] CrossRef
Genetic structure and evolutionary diversity of mating-type (MAT) loci in Hypsizygus marmoreus
Gang Wang, Yuanyuan Wang, Lianfu Chen, Hongbo Wang, Lin Guo, Xuan Zhou, Meijie Dou, Baiyu Wang, Jingxian Lin, Lei Liu, Zhengchao Wang, Youjin Deng, Jisen Zhang
IMA Fungus.2021;[Epub] CrossRef
Macrosynteny analysis between Lentinula edodes and Lentinula novae-zelandiae reveals signals of domestication in Lentinula edodes
Christopher Alan Smith
Scientific Reports.2021;[Epub] CrossRef
Analysis of Genetic Diversity and Population Structure of Wild Strains and Cultivars Using Genomic SSR Markers inLentinula edodes
Hwa-Yong Lee, Suyun Moon, Hyeon-Su Ro, Jong-Wook Chung, Hojin Ryu
Mycobiology.2020; 48(2): 115. CrossRef
Structure analysis of A and B mating type loci in a representative commercial strain of Pleurotus eryngii
Yejin Ju, Sinil Kim, Minseek Kim, Jae San Ryu, Hyeon-Su Ro
Scientia Horticulturae.2020; 274: 109686. CrossRef
Molecular analysis of B mating type diversity in Lentinula edodes
Byeongsuk Ha, Yoon Jung Moon, Yelin Song, Sinil Kim, Minseek Kim, Cheol-Won Yoon, Hyeon-Su Ro
Scientia Horticulturae.2019; 243: 55. CrossRef
Variable Number Tandem Repeats in the Mitochondrial DNA of Lentinula edodes
Sinil Kim, Yelin Song, Byeongsuk Ha, Yoon Jung Moon, Minseek Kim, Hojin Ryu, Hyeon-Su Ro
Genes.2019; 10(7): 542. CrossRef
Activation of the Mating Pheromone Response Pathway ofLentinula edodesby Synthetic Pheromones
Byeongsuk Ha, Sinil Kim, Minseek Kim, Hyeon-Su Ro
Mycobiology.2018; 46(4): 407. CrossRef

Research Support, Non-U.S. Gov'ts

Isolation and Characterization of a Mycovirus in Lentinula edodes: Hyo-Kyoung Won , So-Jung Park , Dong-Kyu Kim , Myeung Ju Shin , Nari Kim , Song-Hee Lee , Young-Chul Kwon , Han Kyu Ko , Hyeon-Su Ro , Hyun-Sook Lee; J. Microbiol. 2013;51(1):118-122. Published online March 2, 2013; DOI: https://doi.org/10.1007/s12275-013-2351-2

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Abstract

A mycovirus was isolated from an edible mushroom, Lentinula edodes, that was suffering from a severe epidemic. Fractionation of the diseased cell extract by isopycnic centrifugation with 50% CsCl revealed that the diseased mushroom was infected by Lentinula edodes spherical virus (LeSV), a new spherical virus with a diameter of 55 nm. The particle of LeSV encapsidated the 12 kb RNA genome by a 120 kDa coat protein. BLAST analysis of the partially sequenced LeSV genome showed 95% sequence identity with a putative RNAdependent RNA polymerase (RdRp) gene of the mycovirus HKB, which was previously reported as being a doublestranded RNA (dsRNA) element. In contrast to HKB, the RNA genome in LeSV is encapsidated by the 120 kDa coat protein. To confirm that the LeSV coat protein is encoded by the viral genome, the N-terminal amino acid sequence of the coat protein was determined. The resulting N-terminal amino acid sequence, N-SALDVAPVVPELYFXXLEV-C, was found to be located in the middle of the HKB ORF1, suggesting that the LeSV coat protein was indeed encoded by the virus. To detect LeSV in L. edodes, a primer set targeting the RdRp gene was designed based on the partial sequence of the LeSV genome. RT-PCR analysis showed that 56 of the 84 commercially available dikaryotic cultivars carry LeSV. The transmission pattern of the virus was determined by analysing basidiospores from LeSV-infected and LeSVfree fruiting bodies. Nine out of 10 basidiospores from the LeSV-infected cultivars contained the virus while the spores from the LeSV-free parent were free of LeSV, suggesting that vertical transmission is the primary mode of LeSV propagation.

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NOTE] The Activity of Phosphoinositide-Specific Phospholipase C Is Required for Vegetative Growth and Cell Wall Regeneration in Coprinopsis cinerea: Young Taek Oh , Chun-Seob Ahn , Kyung-Jin Lee , Jeong-Geun Kim , Hyeon-Su Ro , Jae Won Kim , Chang-Won Lee; J. Microbiol. 2012;50(4):689-692. Published online August 25, 2012; DOI: https://doi.org/10.1007/s12275-012-2004-x

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Abstract

Three isotypes of phosphoinositide-specific phospholipase C designated CcPLC1, CcPLC2, and CcPLC3 were identified in Coprinopsis cinerea, through a search of the genome sequence database. The functional role of the PI-PLCs were studied by using U73122, which specifically inhibits the activity of PI-PLC. The specificity of the inhibitor effect was confirmed by using an inactive structural analog U73433. The inhibition of PI-PLCs activity resulted in severely retarded germination of basidiospores and oidia, reduced hyphal growth, knobbly hyphal tips with many irregular side branches, and aberrant (branch-like structure) clamp cells. Furthermore, U73122 definitely inhibited cell wall formation. Here we report that PI-PLCs play important roles in various aspects of C. cinerea biology.

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Phospholipase C: Diverse functions in plant biotic stress resistance and fungal pathogenicity
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Genetic Introduction of Foreign Genes to Pleurotus eryngii by Restriction Enzyme-Mediated Integration: Won Noh , Sang-Woo Kim , Dong-Won Bae , Jae-Yean Kim , Hyeon-Su Ro; J. Microbiol. 2010;48(2):253-256. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-9278-7

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Abstract: Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 106 mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10-40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.

Analysis of Microbial Communities Using Culture-dependent and Culture-independent Approaches in an Anaerobic/Aerobic SBR Reactor: Shipeng Lu , Minjeong Park , Hyeon-Su Ro , Dae Sung Lee , Woojun Park , Che Ok Jeon; J. Microbiol. 2006;44(2):155-161.; DOI: https://doi.org/2370 [pii]

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Abstract: Comparative analysis of microbial communities in a sequencing batch reactor which performed enhanced biological phosphorus removal (EBPR) was carried out using a cultivation-based technique and 16S rRNA gene clone libraries. A standard PCR protocol and a modified PCR protocol with low PCR cycle was applied to the two clone libraries of the 16S rRNA gene sequences obtained from EBPR sludge, respectively, and the resulting 424 clones were analyzed using restriction fragment length polymorphisms (RFLPs) on 16S rRNA gene inserts. Comparison of two clone libraries showed that the modified PCR protocol decreased the incidence of distinct fragment patterns from about 63% (137 of 217) in the standard PCR method to about 34% (70 of 207) under the modified protocol, suggesting that just a low level of PCR cycling (5 cycles after 15 cycles) can significantly reduce the formation of chimeric DNA in the final PCR products. Phylogenetic analysis of 81 groups with distinct RFLP patterns that were obtained using the modified PCR method revealed that the clones were affiliated with at least 11 phyla or classes of the domain Bacteria. However, the analyses of 327 colonies, which were grouped into just 41 distinct types by RFLP analysis, showed that they could be classified into five major bacterial lineages: α, β, γ- Proteobacteria, Actinobacteria, and the phylum Bacteroidetes, which indicated that the microbial community yielded from the cultivationbased method was still much simpler than that yielded from the PCR-based molecular method. In this study, the discrepancy observed between the communities obtained from PCR-based and cultivation-based methods seems to result from low culturabilities of bacteria or PCR bias even though modified culture and PCR methods were used. Therefore, continuous development of PCR protocol and cultivation techniques is needed to reduce this discrepancy.

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