Journal of Microbiology

Review

Extracellular vesicles in human fungal pathogens: Biogenesis, functions, and translational applications: Catia Mota, Heeyoun Hwang, Hyun Ah Kang; J. Microbiol. 2026;64(7):e2606008. Published online July 31, 2026; DOI: https://doi.org/10.71150/jm.2606008

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Abstract PDF: Fungal extracellular vesicles (EVs) have emerged as critical mediators of fungal physiology, virulence, and host–pathogen interactions. Since their first description in Cryptococcus neoformans, EVs have been identified in several fungal species and shown to transport a broad repertoire of bioactive cargo. Increasing evidence indicates that fungal EVs participate in multiple biological processes, including cell wall remodeling, stress adaptation, biofilm formation, antifungal resistance, and modulation of host immune responses. Recent advances in cryo-electron microscopy, multi-omics approaches, and functional genetics have substantially expanded our understanding of the molecular mechanisms governing EV biogenesis, cargo selection, and extracellular trafficking. These studies have further revealed that EV cargo loading is a highly regulated process linked to intracellular proteostasis, glycosylation, lipid homeostasis, and environmental adaptation. In parallel, the intrinsic immunogenicity and structural stability of fungal EVs have highlighted their translational potential as diagnostic biomarkers, vaccine platforms, therapeutic targets, and nanoscale delivery systems. Given the increasing global burden of invasive fungal infections, this review focuses on EVs derived from clinically relevant human fungal pathogens. We summarize recent advances in EV biogenesis, cargo regulation, their roles in pathogenesis, highlight emerging translational applications, and discuss key unresolved questions and future research directions in the field.

Article

Meiotic prophase roles of Pds5 in recombination and chromosome condensation in budding yeast: Jeong Hwan Joo , Hyun Ah Kang , Keun Pil Kim , Soogil Hong; J. Microbiol. 2022;60(2):177-186. Published online February 1, 2022; DOI: https://doi.org/10.1007/s12275-022-1635-9

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Genetic variation in eukaryotes is mediated during meiosis by the exchange of genetic material between homologous chromosomes to produce recombinant chromosomes. Cohesin is essential to promote proper chromosome segregation, chromosome morphogenesis, and recombination in meiotic cells. Cohesin consists of three main subunits–Smc1, Smc3, and the kleisin subunit Mcd1/Scc1 (Rec8 in meiosis)–and cohesin accessory factors. In Saccharomyces cerevisiae, the cohesin regulatory subunit Pds5 plays a role in homolog pairing, meiotic axis formation, and interhomolog recombination. In this study, we examine the prophase functions of Pds5 by performing physical analysis of recombination and three-dimensional high-resolution microscopy analysis to identify its roles in meiosis-specific recombination and chromosome morphogenesis. To investigate whether Pds5 plays a role in mitoticlike recombination, we inhibited Mek1 kinase activity, which
result
ed in switching to sister template bias by Rad51-dependent recombination. Reductions in double-strand breaks and crossover products and defective interhomolog recombination occurred in the absence of Pds5. Furthermore, recombination intermediates, including single-end invasion and double-Holliday junction, were reduced in the absence of Pds5 with Mek1 kinase inactivation compared to Mek1 kinase inactivation cells. Interestingly, the absence of Pds5
result
ed in increasing numbers of chromosomes with hypercompaction of the chromosome axis. Thus, we suggest that Pds5 plays an essential role in recombination by suppressing the pairing of sister chromatids and abnormal compaction of the chromosome axis.

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Recombination-coupled DNA synthesis facilitates post-invasion steps in meiotic crossover and noncrossover formations
Hyungseok Choi, Jun Seo Lee, Jeong H Joo, Soogene Lee, Keun P Kim
Nucleic Acids Research.2025;[Epub] CrossRef
Multiple Autopolyploid Arabidopsis lyrata Populations Stabilized by Long-Range Adaptive Introgression Across Eurasia
Alison D Scott, Uliana K Kolesnikova, Anna Glushkevich, Laura Steinmann, Nikita P Tikhomirov, Ursula Pfordt, Magdalena Bohutínská, Robin Burns, Alexey P Seregin, Filip Kolar, Roswitha Schmickl, Polina Yu Novikova, Kathryn Hodgins
Molecular Biology and Evolution.2025;[Epub] CrossRef
RPA interacts with Rad52 to promote meiotic crossover and noncrossover recombination
Jeong H Joo, Soogil Hong, Mika T Higashide, Eui-Hwan Choi, Seobin Yoon, Min-Su Lee, Hyun Ah Kang, Akira Shinohara, Nancy Kleckner, Keun P Kim
Nucleic Acids Research.2024; 52(7): 3794. CrossRef
Cohesin is required for meiotic spindle assembly independent of its role in cohesion in C. elegans
Karen P. McNally, Elizabeth A. Beath, Brennan M. Danlasky, Consuelo Barroso, Ting Gong, Wenzhe Li, Enrique Martinez-Perez, Francis J. McNally, Sarit Smolikove
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Yeast polyubiquitin unit regulates synaptonemal complex formation and recombination during meiosis
Min-Kyung Jo, Kiwon Rhee, Keun Pil Kim, Soogil Hong
Journal of Microbiology.2022; 60(7): 705. CrossRef

Article

Assessment of Cre-lox and CRISPR-Cas9 as tools for recycling of multiple-integrated selection markers in Saccharomyces cerevisiae: Hye Yun Moon† , Gyu Hun Sim† , Hyeon Jin Kim , Keunpil Kim , Hyun Ah Kang; J. Microbiol. 2022;60(1):18-30. Published online December 29, 2021; DOI: https://doi.org/10.1007/s12275-022-1580-7

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Abstract PDF

We evaluated the Cre-lox and CRISPR-Cas9 systems as markerrecycling tools in Saccharomyces cerevisiae recombinants containing multiple-integrated expression cassettes. As an initial trial, we constructed rDNA-nontranscribed spacer- or Ty4- based multiple integration vectors containing the URA3 marker flanked by the loxP sequence. Integrants harboring multiple copies of tHMG1 and NNV-CP expression cassettes were obtained and subsequently transformed with the Cre plasmid. However, the simultaneous pop-out of the expression cassettes along with the URA3 marker hampered the use of Cre-lox as a marker-recycling tool in multiple integrants. As an alternative, we constructed a set of CRISPR-Cas9-gRNA vectors containing gRNA targeted to auxotrophic marker genes. Transformation of multiple integrants of tHMG1 and NNV-CP cassettes by the Cas9-gRNA vector in the presence of the URA3 (stop) donor DNA fragments generated the Ura- transformants retaining multiple copies of the expression cassettes. CRISPR-Cas9-based inactivation led to the recycling of the other markers, HIS3, LEU2, and TRP1, without loss of expression cassettes in the recombinants containing multiple copies of tHMG1, NNV-CP, and SfBGL1 cassettes, respectively. Reuse of the same selection marker in marker-inactivated S. cerevisiae was validated by multiple integrations of the TrEGL2 cassette into the S. cerevisiae strain expressing SfBGL1. These results demonstrate that introducing stop codons into selection marker genes using the CRISPR-Cas9 system with donor DNA fragments is an efficient strategy for markerrecycling in multiple integrants. In particular, the continual reuse of auxotrophic markers would facilitate the construction of a yeast cell factory containing multiple copies of expression cassettes without antibiotic resistance genes.

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A comparative review of vector insertion techniques in Saccharomyces cerevisiae
Ritika Gopalakrishnan, Kamala Kannan, Ragul Gunasekaran, Priya Ramachandran, Dhanraj Ganapathy, Sivaperumal Pitchiah
Journal of Microbiological Methods.2026; 241: 107378. CrossRef
MAPS: a marker-free genome integration system for Komagataella phaffii enabling co-production of natural pigments and industrial enzymes
Sarah Schmid, Charis Wardell, Anna Maria Hatzl, Tara Steidl, Martina Geier, Katharina Ebner, Ursula Ellmer-Schaumberger, Anton Glieder, Anita Emmerstorfer-Augustin
Metabolic Engineering.2026; 97: 102487. CrossRef
Prophase roles of replication protein A in crossover formation and meiotic progression
Rose M. Lee, Keun Pil Kim, Jeong H. Joo
Journal of Microbiology.2026; 64(6): e2604001. CrossRef
Multiple metabolic engineering of Saccharomyces cerevisiae for the production of lycopene
Jiaheng Liu, Minxia Song, Xianhao Xu, Yaokang Wu, Yanfeng Liu, Guocheng Du, Jianghua Li, Long Liu, Xueqin Lv
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Fermentation.2024; 10(7): 361. CrossRef
CRISPR/Cas9-based toolkit for rapid marker recycling and combinatorial libraries in Komagataella phaffii
Wei Zhou, Yuanyi Li, Guosong Liu, Weichuang Qin, Dongzhi Wei, Fengqing Wang, Bei Gao
Applied Microbiology and Biotechnology.2024;[Epub] CrossRef
Establishment, optimization, and application of genetic technology in Aspergillus spp.
Jing Gao, Huiqing Liu, Zhenzhen Zhang, Zhihong Liang
Frontiers in Microbiology.2023;[Epub] CrossRef
CRISPR-Cas Technology for Bioengineering Conventional and Non-Conventional Yeasts: Progress and New Challenges
Yuanyuan Xia, Yujie Li, Wei Shen, Haiquan Yang, Xianzhong Chen
International Journal of Molecular Sciences.2023; 24(20): 15310. CrossRef
Genomic and functional features of yeast species in Korean traditional fermented alcoholic beverage and soybean products
Da Min Jeong, Hyeon Jin Kim, Min-Seung Jeon, Su Jin Yoo, Hye Yun Moon, Eun-joo Jeon, Che Ok Jeon, Seong-il Eyun, Hyun Ah Kang
FEMS Yeast Research.2023;[Epub] CrossRef
Multiplex genome editing to construct cellulase engineered Saccharomyces cerevisiae for ethanol production from cellulosic biomass
Yatika Dixit, Preeti Yadav, Arun Kumar Sharma, Poornima Pandey, Arindam Kuila
Renewable and Sustainable Energy Reviews.2023; 187: 113772. CrossRef

Article

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans: Jong-Myeong Kim , Hye Yun Moon , Dong Wook Lee , Hyun Ah Kang , Jeong-Yoon Kim; J. Microbiol. 2021;59(10):911-919. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1326-y

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Abstract PDF

Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24,
result
ing in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hyphainducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hyphainducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

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Biocontrol effects and mechanisms of two Streptomyces strains against root rot of Codonopsis pilosula caused by Fusarium oxysporum
Wei Wang, Lulu Zhang, Zhanfeng Cao, Weijing Yang, Xingzheng Wang, Yufen Yang, Ziyi Hu, Shufeng Wei, Ning Yang
Physiological and Molecular Plant Pathology.2026; 145: 103378. CrossRef
Cas5 Regulates the Exposure of β-Glucan, the Cell Surface Hydrophobicity, and the Expression of Cell Wall Proteins to Remodel the Candida albicans Cell Wall and Participates in the Recruitment of Neutrophils
Qiyue Zhang, Guanglin Li, Yanmei Wang, Chen Yang, Wenhui Bai, Qingqing Li, Jiye Zhang, Peipei Zhang
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Che-Kang Chang, Min-Chi Yang, Hsueh-Fen Chen, Yi-Ling Liao, Chung-Yu Lan
Journal of Fungi.2022; 8(11): 1196. CrossRef

Article

Molecular characterization of the Saccharomycopsis fibuligera ATF genes, encoding alcohol acetyltransferase for volatile acetate ester formation: Hye Yun Moon , Hyeon Jin Kim , Ki Seung Kim , Su Jin Yoo , Dong Wook Lee , Hee Je Shin , Jeong Ah Seo , Hyun Ah Kang; J. Microbiol. 2021;59(6):598-608. Published online May 29, 2021; DOI: https://doi.org/10.1007/s12275-021-1159-8

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Abstract PDF

Aroma ester components produced by fermenting yeast cells via alcohol acetyltransferase (AATase)-catalyzed intracellular reactions are responsible for the fruity character of fermented alcoholic beverages, such as beer and wine. Acetate esters are reportedly produced at relatively high concentrations by non-Saccharomyces species. Here, we identified 12 ATF orthologues (SfATFs) encoding putative AATases, in the diploid genome of Saccharomycopsis fibuligera KJJ81, an isolate from wheat-based Nuruk in Korea. The identified SfATF proteins (SfAtfp) display low sequence identities with S. cerevisiae Atf1p (between 13.3 and 27.0%). All SfAtfp identified, except SfAtf(A)4p and SfAtf(B)4p, contained the activation domain (HXXXD) conserved in other Atf proteins. Culture supernatant analysis using headspace gas chromatography mass spectrometry confirmed that the recombinant S. cerevisiae strains expressing SfAtf(A)2p, SfAtf(B)2p, and SfAtf(B)6p produced high levels of isoamyl and phenethyl acetates. The volatile aroma profiles generated by the SfAtf proteins were distinctive from that of S. cerevisiae Atf1p, implying difference in the substrate preference. Cellular localization analysis using GFP fusion revealed the localization of SfAtf proteins proximal to the lipid particles, consistent with the presence of amphipathic helices at their N- and C-termini. This is the first report that systematically characterizes the S. fibuligera ATF genes encoding functional AATases responsible for acetate ester formation using higher alcohols as substrate, demonstrating their biotechnological potential for volatile ester production.

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Kerui Su, Feihang Wang, Anqi Zhao, Beixiao Zhang, Zhihao Fan, Qi Wang, Xuejing Duan, Hanyu Dong, A.E. Solovchenko, Ayesha Shahid, Jingliang Xu
Food Microbiology.2026; 137: 105043. CrossRef
Two Metschnikowia nectar yeast species have similar volatile profiles but elicit differential foraging in bee pollinators
M. Elizabeth Moore, Lindsey Wilson, Nathan Brandt, Ayako Wada‐Katsumata, Ahmed M. Saveer, Coby Schal, Robert R. Dunn, Rebecca E. Irwin, Caiti Smukowski Heil
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Qi Peng, Huihui Zhou, Huajun Zheng, Guangfa Xie
Food Microbiology.2025; 129: 104765. CrossRef
Molecular functional mechanisms of two alcohol acetyltransferases in Lavandula x intermedia (lavandin)
Dafeng Liu, Yanyan Du, Ablikim Abdiriyim, Lvxia Zhang, Daoqi Song, Huashui Deng, Xiongying Wen, Yanyan Zhang, Bingwang Sun
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Anastasia E.C. Rumpl, Joshua R. Goodhew, Paul F. Kelly, Mika Hirano, Michael E. Pyne
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Genome-based exploration of volatile flavor diversity from food yeast species
Su Jin Yoo, Da Eun Kim, Lintang Ignatius Satyawan, Seong-Il Eyun, Che Ok Jeon, Hyun Ah Kang, Zbigniew Lazar
FEMS Yeast Research.2025;[Epub] CrossRef
Genome-Wide Identification and Biochemical Characterization of Alcohol Acyltransferases for Aroma Generation in Wickerhamomyces subpelliculosus Isolates from Fermented Food
Su Jin Yoo, Hyeon Jin Kim, Hye Yun Moon, Min-Seung Jeon, Yong Uk Cho, Che Ok Jeon, Seong-Il Eyun, Hyun Ah Kang
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Yue Deng, Guangjiu Chen, Xuedong Bao, Jie He
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Foods.2024; 13(10): 1546. CrossRef
Genomic and functional features of yeast species in Korean traditional fermented alcoholic beverage and soybean products
Da Min Jeong, Hyeon Jin Kim, Min-Seung Jeon, Su Jin Yoo, Hye Yun Moon, Eun-joo Jeon, Che Ok Jeon, Seong-il Eyun, Hyun Ah Kang
FEMS Yeast Research.2023;[Epub] CrossRef
Beer fermentation performance and sugar uptake of Saccharomycopsis fibuligera–A novel option for low-alcohol beer
Yvonne Methner, Frederico Magalhães, Luis Raihofer, Martin Zarnkow, Fritz Jacob, Mathias Hutzler
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Article

Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha: Jin Ho Choo , Su-Bin Lee , Hye Yun Moon , Kun Hwa Lee , Su Jin Yoo , Keun Pil Kim , Hyun Ah Kang; J. Microbiol. 2021;59(2):151-163. Published online February 1, 2021; DOI: https://doi.org/10.1007/s12275-021-0646-2

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Abstract PDF

Ogataea parapolymorpha (Hansenula polymorpha DL-1) is a thermotolerant methylotrophic yeast with biotechnological applications. Here, O. parapolymorpha genes whose expression is induced in response to heat shock were identified by transcriptome analysis and shown to possess heat shock elements (HSEs) in their promoters. The function of O. parapolymorpha HSF1 encoding a putative heat shock transcription factor 1 (OpHsf1) was characterized in the context of heat stress response. Despite exhibiting low sequence identity (26%) to its Saccharomyces cerevisiae homolog, OpHsf1 harbors conserved domains including a DNA binding domain (DBD), domains involved in trimerization (TRI), transcriptional activation (AR1, AR2), transcriptional repression (CE2), and a C-terminal modulator (CTM) domain. OpHSF1 could complement the temperature sensitive (Ts) phenotype of a S. cerevisiae hsf1 mutant. An O. parapolymorpha strain with an H221R mutation in the DBD domain of OpHsf1 exhibited significantly retarded growth and a Ts phenotype. Intriguingly, the expression of heat-shock-protein‒coding genes harboring HSEs was significantly decreased in the H221R mutant strain, even under non-stress conditions, indicating the importance of the DBD for the basal growth of O. parapolymorpha. Notably, even though the deletion of C-terminal domains (ΔCE2, ΔAR2, ΔCTM) of OpHsf1 destroyed complementation of the growth defect of the S. cerevisiae hsf1 strain, the C-terminal domains were shown to be dispensable in O. parapolymorpha. Overexpression of OpHsf1 in S. cerevisiae increased resistance to transient heat shock, supporting the idea that OpHsf1 could be useful in the development of heatshock‒ resistant yeast host strains.

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Thermotolerant yeasts and their biotechnological applications
Roksolana Vasylyshyn, Justyna Ruchala, Kostyantyn Dmytruk, Andriy Sibirny
Trends in Biotechnology.2026;[Epub] CrossRef
Lignocellulosic biomass fermentation: a roadmap for Candida famata and Ogataea polymorpha
Dominik Wojdyła, Roksolana Vasylyshyn, Alicja Najdecka, Justyna Ruchala
FEMS Yeast Research.2025;[Epub] CrossRef
Sucrose‐Induced Transcriptomic Response in Ogataea polymorpha TBRC 4839 Reveals its Potential for Recombinant Protein Production
Somsak Likhitrattanapisal, Chitwadee Phithakrotchanakoon, Aekkachai Puseenam, Paopit Siriarchawatana, Natta Wiriyakun, Jiraprapa Nirapun, Warasirin Sornlek, Supawadee Ingsriswang, Niran Roongsawang
Yeast.2025; 42(8-10): 181. CrossRef
A comprehensive review and comparison of L-tryptophan biosynthesis in Saccharomyces cerevisiae and Escherichia coli
Xinru Ren, Yue Wei, Honglu Zhao, Juanjuan Shao, Fanli Zeng, Zhen Wang, Li Li
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Heat shock in Cronobacter sakazakii induces direct protection and cross-protection against simulated gastric fluid stress
Hongmei Niu, MingzheYang, Yonghua Qi, Yangtai Liu, Xiang Wang, Qingli Dong
Food Microbiology.2022; 103: 103948. CrossRef
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Dongxu Shen, Xiaoli He, Peifang Weng, Yanan Liu, Zufang Wu
FEMS Yeast Research.2022;[Epub] CrossRef

Article

Evolutionary analysis and protein family classification of chitin deacetylases in Cryptococcus neoformans: Seungsue Lee , Hyun Ah Kang , Seong-il Eyun; J. Microbiol. 2020;58(9):805-811. Published online September 1, 2020; DOI: https://doi.org/10.1007/s12275-020-0288-9

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Abstract PDF

Cryptococcus neoformans is an opportunistic fungal pathogen causing cryptococcal meningoencephalitis. Interestingly, the cell wall of C. neoformans contains chitosan, which is critical for its virulence and persistence in the mammalian host. C. neoformans (H99) has three chitin deacetylases (CDAs), which convert chitin to chitosan. Herein, the classification of the chitin-related protein (CRP) family focused on cryptococcal CDAs was analyzed by phylogenetics, evolutionary pressure (dN/dS), and 3D modeling. A phylogenetic tree of 110 CRPs revealed that they can be divided into two clades, CRP I and II with bootstrap values (> 99%). CRP I clade comprises five groups (Groups 1–5) with a total of 20 genes, while CRP II clade comprises sixteen groups (Groups 6–21) with a total of 90 genes. CRP I comprises only fungal CDAs, including all three C. neoformans CDAs, whereas CRP II comprises diverse CDAs from fungi, bacteria, and amoeba, along with other carbohydrate esterase 4 family proteins. All CDAs have the signal peptide, except those from group 11. Notably, CDAs with the putative O-glycosylation site possess either the glycosylphosphatidylinositol (GPI)-anchor motif for CRP I or the chitin-binding domain (CBD) for CRP II, respectively. This evolutionary conservation strongly indicates that the O-glycosylation modification and the presence of either the GPI-anchor motif or the chitin-binding domain is important for fungal CDAs to function efficiently at the cell surface. This study reveals that C. neoformans CDAs carrying GPI anchors have evolved divergently from fungal and bacterial CDAs, providing new insights into evolution and classification of CRP family.

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Characterization of novel cold-active chitin deacetylase for green production of bioactive chitosan
Mohamed N. Abd El-Ghany, Salwa A. Hamdi, Ahmed K. Zahran, Mustafa A. Abou-Taleb, Abdallah M. Heikel, Muhammed T. Abou El-Kheir, Mohamed G. Farahat
AMB Express.2025;[Epub] CrossRef
Structural features and roles of O-glycans in the secretion, stability, and immunogenicity of Cryptococcus neoformans Cda1 and MP88 mannoproteins
Eun Jung Thak, Seon Woo Song, Young-Jin Seo, J Andrew Alspaugh, Hyun Ah Kang
International Journal of Biological Macromolecules.2025; 321: 146290. CrossRef
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Effects of altered N-glycan structures of Cryptococcus neoformans mannoproteins, MP98 (Cda2) and MP84 (Cda3), on interaction with host cells
Su-Bin Lee, Catia Mota, Eun Jung Thak, Jungho Kim, Ye Ji Son, Doo-Byoung Oh, Hyun Ah Kang
Scientific Reports.2023;[Epub] CrossRef
Novel Chitin Deacetylase from Thalassiosira weissflogii Highlights the Potential for Chitin Derivative Production
Mengzhen Cheng, Zhanru Shao, Xin Wang, Chang Lu, Shuang Li, Delin Duan
Metabolites.2023; 13(3): 429. CrossRef
Identification and Phylogenetic Analysis of Chitin Synthase Genes from the Deep-Sea Polychaete Branchipolynoe onnuriensis Genome
Hyeongwoo Choi, Sang Lyeol Kim, Man-Ki Jeong, Ok Hwan Yu, Seongil Eyun
Journal of Marine Science and Engineering.2022; 10(5): 598. CrossRef

Article

Functional analysis of Mpk1-mediated cell wall integrity signaling pathway in the thermotolerant methylotrophic yeast Hansenula polymorpha: Hyunah Kim , Eun Jung Thak , Ji Yoon Yeon , Min Jeong Sohn , Jin Ho Choo , Jeong-Yoon Kim , Hyun Ah Kang; J. Microbiol. 2018;56(1):72-82. Published online January 4, 2018; DOI: https://doi.org/10.1007/s12275-018-7508-6

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Abstract PDF

Understanding the characteristics and regulation mechanisms of cell wall integrity (CWI) in yeast is important not only for basic research but also in biotechnological applications. We found significantly different CWIs in two representative strains of the thermotolerant methylotrophic yeast Hansenula polymorpha. Compared to the A16 strain (classified as Ogataea polymorpha), the DL1-L strain (classified as Ogataea parapolymorpha) has a thinner cell wall that was found to be more fragile following long-term cultivation and more sensitive to zymolyase. To gain a deeper insight into this difference, we compared the characteristics of the Mpk1pmediated CWI signaling pathway in the two strains. While a DL1-L mutant deficient in Mpk1p (mpk1Δ) showed severe growth retardation at both normal and high growth temperatures and in the presence of cell-wall disrupting agents, the A16 mpk1Δ mutant displayed only a mild defect in cell growth. Sorbitol effect on rescuing growth retardation was different in the two mpk1Δ strains, which could partly be ascribed to subtle differences in the activation of HOG pathway. Among the cell wall disruptors evaluated, only caffeine clearly increased phosphorylation of Mpk1p in DL1-L, but not in A16. A transcriptome analysis of the DL1-L strain revealed that caffeine significantly increased the expression of a subset of cell-wall related genes in an Mpk1p-dependent manner, but not the expected Rlm1-target genes. Taken together, our data support an essential role for Mpk1p in maintaining CWI in H. polymorpha, although the requirement for Mpk1p and its regulation under diverse stress conditions varies depending on the strain background.

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Phytosphingosine promotes intracellular microbial lipid accumulation and cell structure remodeling during adaptive evolution combined with cell fractionation in wild-type Yarrowia lipolytica DSM 3286
Shaokai Li, Lixia Pan, Bin Zhang, Jie Bao
Biochemical Engineering Journal.2026; 234: 110293. CrossRef
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Roghayeh Shirvani, Maryam Babaei, Motahare Baladi, Matthias G Steiger, Mohammad Barshan-tashnizi
FEMS Yeast Research.2025;[Epub] CrossRef
Optimization, characterization, comparison of self-assembly VLP of capsid protein L1 in yeast and reverse vaccinology design against human papillomavirus type 52
Moh Egy Rahman Firdaus, Apon Zaenal Mustopa, Nurlaili Ekawati, Sheila Chairunnisa, Rosyida Khusniatul Arifah, Ai Hertati, Shasmita Irawan, Anika Prastyowati, Arizah Kusumawati, Maritsa Nurfatwa
Journal of Genetic Engineering and Biotechnology.2023; 21(1): 68. CrossRef
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Tong-tong Zhu, Sheng-han Xiang, Lei Yang, Fang Tang, Wei Li, Ying-bao Liu, Wen-xiu Sun
European Journal of Plant Pathology.2022; 164(4): 525. CrossRef
The cell wall and the response and tolerance to stresses of biotechnological relevance in yeasts
Ricardo A. Ribeiro, Nuno Bourbon-Melo, Isabel Sá-Correia
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The damage and tolerance mechanisms of Phaffia rhodozyma mutant strain MK19 grown at 28 °C
Li-Li Miao, Shuang Chi, Ting-Ting Hou, Zhi-Pei Liu, Ying Li
Microbial Cell Factories.2021;[Epub] CrossRef
Molecular characterization of Hsf1 as a master regulator of heat shock response in the thermotolerant methylotrophic yeast Ogataea parapolymorpha
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Protocol

Structural analysis of N-/O-glycans assembled on proteins in yeasts: Eun Jung Thak , Jungho Kim , Dong-Jik Lee , Jeong Yoon Kim , Hyun Ah Kang; J. Microbiol. 2018;56(1):11-23. Published online January 4, 2018; DOI: https://doi.org/10.1007/s12275-018-7468-x

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Abstract PDF

Protein glycosylation, the most universal and diverse posttranslational modification, can affect protein secretion, stability, and immunogenicity. The structures of glycans attached to proteins are quite diverse among different organisms and even within yeast species. In yeast, protein glycosylation plays key roles in the quality control of secretory proteins, and particularly in maintaining cell wall integrity. Moreover, in pathogenic yeasts, glycans assembled on cell-surface glycoproteins can mediate their interactions with host cells. Thus, a comprehensive understanding of protein glycosylation in various yeast species and defining glycan structure characteristics can provide useful information for their biotechnological and clinical implications. Yeast-specific glycans are a target for glyco-engineering; implementing human-type glycosylation pathways in yeast can aid the production of recombinant glycoproteins with therapeutic potential. The virulenceassociated glycans of pathogenic yeasts could be exploited as novel targets for antifungal agents. Nowadays, several glycomics techniques facilitate the generation of species- and strain-specific glycome profiles and the delineation of modified glycan structures in mutant and engineered yeast cells. Here, we present the protocols employed in our laboratory to investigate the N- and O-glycan chains released from purified glycoproteins or cell wall mannoproteins in several yeast species.

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Article

Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus: Van-Trinh Luu , Hye Yun Moon , Jee Youn Hwang , Bo-Kyu Kang , Hyun Ah Kang; J. Microbiol. 2017;55(8):655-664. Published online July 28, 2017; DOI: https://doi.org/10.1007/s12275-017-7218-5

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Abstract PDF

Nervous necrosis virus (NNV) causes viral encephalopathy and retinopathy, a devastating disease of many species of cultured marine fish worldwide. In this study, we used the dimorphic non-pathogenic yeast Yarrowia lipolytica as a host to express the capsid protein of red-spotted grouper nervous necrosis virus (RGNNV-CP) and evaluated its potential as a platform for vaccine production. An initial attempt was made to express the codon-optimized synthetic genes encoding intact and N-terminal truncated forms of RGNNV-CP under the strong constitutive TEF1 promoter using autonomously replicating sequence (ARS)-based vectors. The full-length recombinant capsid proteins expressed in Y. lipolytica were detected not only as monomers and but also as trimers, which is a basic unit for formation of NNV virus-like particles (VLPs). Oral immunization of mice with whole recombinant Y. lipolytica harboring the ARSbased plasmids was shown to efficiently induce the formation of IgG against RGNNV-CP. To increase the number of integrated copies of the RGNNV-CP expression cassette, a set of 26S ribosomal DNA-based multiple integrative vectors was constructed in combination with a series of defective Ylura3 with truncated promoters as selection markers, resulting in integrants harboring up to eight copies of the RGNNVCP cassette. Sucrose gradient centrifugation and transmission electron microscopy of this high-copy integrant were carried out to confirm the expression of RGNNV-CPs as VLPs. This is the first report on efficient expression of viral capsid proteins as VLPs in Y. lipolytica, demonstrating high potential for the Y. lipolytica expression system as a platform for recombinant vaccine production based on VLPs.

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Article

Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae: Hye Ji Oh , Yun Moon , Seon Ah Cheon , Yoonsoo Hahn , Hyun Ah Kang; J. Microbiol. 2016;54(10):667-674. Published online September 30, 2016; DOI: https://doi.org/10.1007/s12275-016-6401-4

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Abstract PDF

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.

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GREB1: An evolutionarily conserved protein with a glycosyltransferase domain links ERα glycosylation and stability to cancer
Eun Myoung Shin, Vinh Thang Huynh, Sultan Abda Neja, Chia Yi Liu, Anandhkumar Raju, Kelly Tan, Nguan Soon Tan, Jayantha Gunaratne, Xuezhi Bi, Lakshminarayan M. Iyer, L. Aravind, Vinay Tergaonkar
Science Advances.2021;[Epub] CrossRef
Contribution of yeast models to virus research
R Sahaya Glingston, Jyoti Yadav, Jitika Rajpoot, Neha Joshi, Shirisha Nagotu
Applied Microbiology and Biotechnology.2021; 105(12): 4855. CrossRef
A Sweet Embrace: Control of Protein–Protein Interactions by O-Linked β-N-Acetylglucosamine
Heather J. Tarbet, Clifford A. Toleman, Michael Boyce
Biochemistry.2018; 57(1): 13. CrossRef

Research Support, Non-U.S. Gov't

Functional Characterization of Extracellular Chitinase Encoded by the YlCTS1 Gene in a Dimorphic Yeast Yarrowia lipolytica: Jeong-Nam Park , Chang Pyo Han , Dong-Jik Lee , Seon Ah Cheon , Hyun Ah Kang; J. Microbiol. 2014;52(4):284-291. Published online March 29, 2014; DOI: https://doi.org/10.1007/s12275-014-4070-8

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Abstract PDF

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by Omannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.

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Carmen-Lisset Flores, Joaquín Ariño, Carlos Gancedo
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Davide Gottardi, Lorenzo Siroli, Lucia Vannini, Francesca Patrignani, Rosalba Lanciotti
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Functional analysis of recombinant human and Yarrowia lipolytica O-GlcNAc transferases expressed in Saccharomyces cerevisiae
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Journal of Microbiology.2016; 54(10): 667. CrossRef

Research Support, Non-U.S. Gov't

Cell-Surface Expression of Aspergillus saitoi-Derived Functional α-1,2-Mannosidase on Yarrowia lipolytica for Glycan Remodeling: Hye Yun Moon , Trinh Luu Van , Seon Ah Cheon , Jinho Choo , Jeong-Yoon Kim , Hyun Ah Kang; J. Microbiol. 2013;51(4):506-514. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3344-x

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Abstract PDF

Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man8GlcNAc2 to Man5GlcNAc2 efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.

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Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus
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Characterization of putative glycosylphosphatidylinositol-anchoring motifs for surface display in the methylotrophic yeast Hansenula polymorpha
Seon Ah Cheon, Jinhee Jung, Jin Ho Choo, Doo-Byoung Oh, Hyun Ah Kang
Biotechnology Letters.2014; 36(10): 2085. CrossRef

Research Support, Non-U.S. Gov't

Remodeling of the Glycosylation Pathway in the Methylotrophic Yeast Hansenula polymorpha to Produce Human Hybrid-Type N-Glycans: Seon Ah Cheon , Hyunah Kim , Doo-Byoung Oh , Ohsuk Kwon , Hyun Ah Kang; J. Microbiol. 2012;50(2):341-348. Published online April 27, 2012; DOI: https://doi.org/10.1007/s12275-012-2097-2

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Abstract PDF

As a step forward to achieve the generation of human complex- type N-glycans in the methylotrophic yeast Hansenula polymorpha, we here report the modification of the yeast glycosylation pathway by heterologous expression of the human gene encoding β-1,2-N-acetylglucosaminyltransferase I (GnTI). For the optimal expression of human GnTI in the yeast Golgi compartment, the catalytic domain of the GnTI was fused to various N-terminal leader sequences derived from the yeast type II membrane proteins. The vectors containing GnTI fusion constructs were introduced into the H. polymorpha och1Δ single and och1Δalg3Δ double mutant strains expressing the ER-targeted Aspergillus saitoi α-1,2 mannosidase, respectively. Both of the glycoengineered Hpoch1Δ and Hpoch1ΔHpalg3Δ strains were shown to produce successfully the hybrid-type glycans with a monoantennary N-acetylglucosamine (GlcNAc1Man5GlcNAc2 and GlcNAc1Man3GlcNAc2, respectively) by N-glycan profile analysis of cell wall proteins. Furthermore, by comparative analysis of byproduct formation and the glycosylation site occupancy, we propose that the Hpoch1Δ strain would be more suitable than the Hpoch1ΔHpalg3Δ strain as a host for the production of recombinant proteins with humanized glycans.

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Research Support, Non-U.S. Gov't

Functional Analysis of a Hansenula polymorpha MNN2-2 Homologue Encoding a Putative UDP-N-acetylglucosamine Transporter Localized in the Endoplasmic Reticulum: Jeong-Nam Park , Jinho Choo , Hyun Ah Kang; J. Microbiol. 2011;49(6):1012-1017. Published online December 28, 2011; DOI: https://doi.org/10.1007/s12275-011-1520-4

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The Kluyveromyces lactis UDP-GlcNAc transporter (KlMnn2-2p) is responsible for the biosynthesis of N-glycans containing N-acetylglucosamine. A putative gene of Hansenula polymorpha encoding a KlMnn2-2p homologue, HpMNN2-2, was identified and investigated for its function. The deletion mutant strain of HpMNN2-2 (Hpmnn2-2Δ) showed increased sensitivity to geneticin, hygromycin B, and tunicamycin. However, the Hpmnn2-2Δ strain exhibited increased resistance to Calcofluor white, an inhibitor of chitin biosynthesis, along with a reduced chitin content. The localization of HpMnn2-2p at the endoplasmic reticulum-enriched membrane, different from the Golgi localization of a K. lactis homologue, further supports the involvement of HpMnn2-2p in cell wall chitin biosynthesis.

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Erratum

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans: Jong-Myeong Kim , Hye Yun Moon , Dong Wook Lee , Hyun Ah Kang , Jeong-Yoon Kim; J. Microbiol. 2021;59(11):1063-1063.; DOI: https://doi.org/10.1007/s12275-021-0326-2

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