Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Butkovich, Lazarina V.; Leggieri, Patrick A.; Lillington, Stephen P.; Navaratna, Tejas A.; Swift, Candice L.; Malinov, Nikola G.; Zalunardo, Thea R.; Vining, Oliver B.; Lipzen, Anna; Wang, Mei; Yan, Juying; Ng, Vivian; Grigoriev, Igor V.; O'Malley, Michelle A.

doi:10.1016/j.fgb.2024.103958

Title: Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

Journal Article · 31 December 2024 · Fungal Genetics and Biology

DOI: https://doi.org/10.1016/j.fgb.2024.103958 · OSTI ID:2544989

Butkovich, Lazarina V. ^[1]; Leggieri, Patrick A. ^[1]; Lillington, Stephen P. ^[1]; Navaratna, Tejas A. ^[1]; Swift, Candice L. ^[1]; Malinov, Nikola G. ^[1]; Zalunardo, Thea R. ^[1]; Vining, Oliver B. ^[1]; Lipzen, Anna ^[2]; Wang, Mei ^[2]; Yan, Juying ^[2]; Ng, Vivian ^[2]; Grigoriev, Igor V. ^[3]; O'Malley, Michelle A. ^[4]

University of California, Santa Barbara, CA (United States)
USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
USDOE Joint Genome Institute (JGI), Berkeley, CA (United States); Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States); University of California, Berkeley, CA (United States)
University of California, Santa Barbara, CA (United States); Joint BioEnergy Institute (JBEI), Emeryville, CA (United States)

Anaerobic gut fungi of the phylum Neocallimastigomycota are microbes proficient in valorizing low-cost but difficult-to-breakdown lignocellulosic plant biomass. Characterization of different fungal life stages and how they contribute to biomass breakdown are critical for biotechnological applications, yet we lack foundational knowledge about the transcriptional, metabolic, and enzyme secretion behavior of different life stages of anaerobic gut fungi: zoospores, germlings, immature thalli, and mature zoosporangia. A Miracloth-based technique was developed to enrich cell pellets with zoospores - the free-swimming, flagellated, young life stage of anaerobic gut fungi. By contrast, fungal mats contained relatively more vegetative, encysted, mature sporangia that form films. Global gene expression profiles were compared from two sample types (zoospore-enriched cell pellets vs. mature mats) harvested from the anaerobic gut fungal strain Neocallimastix californiae G1. Despite cultures being grown on glucose, the fungal zoospore-enriched samples were transcriptionally primed to encounter plant matter substrate, as evidenced by upregulation of catabolic carbohydrate-active enzymes and putative carbohydrate transporters. Furthermore, we report significant differential gene expression for gene annotation groups, including putative secondary metabolites and transcription factors. Understanding global gene expression differences between the fungal zoospore-enriched cells and mature fungi aid in characterizing fungal development, unmasking gene function, and guiding cultivation conditions and engineering targets to promote enzyme secretion.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: National Science Foundation (NSF); US Army Research Office (ARO); USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science (BSS)

Grant/Contract Number:: AC02-05CH11231; SC0020420; SC0022142

OSTI ID:: 2544989

Journal Information:: Fungal Genetics and Biology, Journal Name: Fungal Genetics and Biology Vol. 176; ISSN 1087-1845

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

References (62)

Biomass-degrading enzymes are catabolite repressed in anaerobic gut fungi Henske, John K.; Gilmore, Sean P.; Haitjema, Charles H. AIChE Journal, Vol. 64, Issue 12 https://doi.org/10.1002/aic.16395	journal	October 2018
Anaerobic gut fungi: Advances in isolation, culture, and cellulolytic enzyme discovery for biofuel production: Anaerobic Gut Fungi Haitjema, Charles H.; Solomon, Kevin V.; Henske, John K. Biotechnology and Bioengineering, Vol. 111, Issue 8 https://doi.org/10.1002/bit.25264	journal	May 2014
Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose Henske, John K.; Wilken, St. Elmo; Solomon, Kevin V. Biotechnology and Bioengineering, Vol. 115, Issue 4 https://doi.org/10.1002/bit.26515	journal	January 2018
Toward understanding the origin and evolution of cellular organisms Kanehisa, Minoru Protein Science, Vol. 28, Issue 11 https://doi.org/10.1002/pro.3715	journal	September 2019
Synthesis and assembly of fungal melanin Eisenman, Helene C.; Casadevall, Arturo Applied Microbiology and Biotechnology, Vol. 93, Issue 3 https://doi.org/10.1007/s00253-011-3777-2	journal	December 2011
Manipulation of fungal development as source of novel secondary metabolites for biotechnology Gerke, Jennifer; Braus, Gerhard H. Applied Microbiology and Biotechnology, Vol. 98, Issue 20 https://doi.org/10.1007/s00253-014-5997-8	journal	August 2014
Transient expression of the β-glucuronidase gene after biolistic transformation of the anaerobic fungus Neocallimastix frontalis Durand, R.; Rascle, C.; Fischer, M. Current Genetics, Vol. 31, Issue 2 https://doi.org/10.1007/s002940050190	journal	February 1997
Ultrastructural description of a new chytrid genus of caecum anaerobe, Caecomyces equi gen. nov., sp. nov., assigned to the Neocallimasticaceae Gold, Julie J.; Brent Heath, I.; Bauchop, Tom Biosystems, Vol. 21, Issue 3-4 https://doi.org/10.1016/0303-2647(88)90039-1	journal	January 1988
The anaerobic fungi in rumen fibre digestion Bauchop, T. Agriculture and Environment, Vol. 6, Issue 2-3 https://doi.org/10.1016/0304-1131(81)90021-7	journal	November 1981
Metabolic energy generation in hydrogenosomes of the anaerobic fungus Neocallimastix: evidence for a functional relationship with mitochondria Marvin-Sikkema, Femke D.; Driessen, Arnold J. M.; Gottschal, Jan C. Mycological Research, Vol. 98, Issue 2 https://doi.org/10.1016/S0953-7562(09)80187-1	journal	February 1994
Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi Solomon, Kevin V.; Henske, John K.; Theodorou, Michael K. Anaerobe, Vol. 38 https://doi.org/10.1016/j.anaerobe.2015.11.008	journal	April 2016
Biofilm disruption enhances growth rate and carbohydrate-active enzyme production in anaerobic fungi Leggieri, Patrick A.; Valentine, Megan T.; O'Malley, Michelle A. Bioresource Technology, Vol. 358 https://doi.org/10.1016/j.biortech.2022.127361	journal	August 2022
Leveraging anaerobic fungi for biotechnology Hooker, Casey A.; Lee, Kok Zhi; Solomon, Kevin V. Current Opinion in Biotechnology, Vol. 59 https://doi.org/10.1016/j.copbio.2019.03.013	journal	October 2019
Functions of fungal melanin beyond virulence Cordero, Radames J. B.; Casadevall, Arturo Fungal Biology Reviews, Vol. 31, Issue 2 https://doi.org/10.1016/j.fbr.2016.12.003	journal	March 2017
SMURF: Genomic mapping of fungal secondary metabolite clusters Khaldi, Nora; Seifuddin, Fayaz T.; Turner, Geoff Fungal Genetics and Biology, Vol. 47, Issue 9 https://doi.org/10.1016/j.fgb.2010.06.003	journal	September 2010
Characterization of the Carbohydrate Binding Module 18 gene family in the amphibian pathogen Batrachochytrium dendrobatidis Liu, Peng; Stajich, Jason E. Fungal Genetics and Biology, Vol. 77 https://doi.org/10.1016/j.fgb.2015.03.003	journal	April 2015
Structure and function of fungal zoospores: ecological implications Gleason, Frank H.; Lilje, Osu Fungal Ecology, Vol. 2, Issue 2 https://doi.org/10.1016/j.funeco.2008.12.002	journal	May 2009
A fast and reliable procedure for spore collection from anaerobic fungi: Application for RNA uptake and long-term storage of isolates Calkins, Shelby; Elledge, Nicole C.; Hanafy, Radwa A. Journal of Microbiological Methods, Vol. 127 https://doi.org/10.1016/j.mimet.2016.05.019	journal	August 2016
Designing chimeric enzymes inspired by fungal cellulosomes Gilmore, Sean P.; Lillington, Stephen P.; Haitjema, Charles H. Synthetic and Systems Biotechnology, Vol. 5, Issue 1 https://doi.org/10.1016/j.synbio.2020.01.003	journal	March 2020
Formulation of secondary compounds as additives of biopolymer-based food packaging: A review Moeini, Arash; Germann, Natalie; Malinconico, Mario Trends in Food Science & Technology, Vol. 114 https://doi.org/10.1016/j.tifs.2021.05.040	journal	August 2021
A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast Podolsky, Igor A.; Seppälä, Susanna; Xu, Haiqing Metabolic Engineering, Vol. 66 https://doi.org/10.1016/j.ymben.2021.04.009	journal	July 2021
A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis Hooker, Casey A.; Hanafy, Radwa; Hillman, Ethan T. ACS Synthetic Biology, Vol. 12, Issue 4 https://doi.org/10.1021/acssynbio.2c00502	journal	March 2023
Gene Ontology: tool for the unification of biology Ashburner, Michael; Ball, Catherine A.; Blake, Judith A. Nature Genetics, Vol. 25, Issue 1 https://doi.org/10.1038/75556	journal	May 2000
Phylogenetic diversity and community structure of anaerobic gut fungi (phylum Neocallimastigomycota) in ruminant and non-ruminant herbivores Liggenstoffer, Audra S.; Youssef, Noha H.; Couger, M. B. The ISME Journal, Vol. 4, Issue 10 https://doi.org/10.1038/ismej.2010.49	journal	April 2010
HISAT: a fast spliced aligner with low memory requirements Kim, Daehwan; Langmead, Ben; Salzberg, Steven L. Nature Methods, Vol. 12, Issue 4 https://doi.org/10.1038/nmeth.3317	journal	March 2015
A parts list for fungal cellulosomes revealed by comparative genomics Haitjema, Charles H.; Gilmore, Sean P.; Henske, John K. Nature Microbiology, Vol. 2, Issue 8 https://doi.org/10.1038/nmicrobiol.2017.87	journal	May 2017
Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides Artzi, Lior; Bayer, Edward A.; Moraïs, Sarah Nature Reviews Microbiology, Vol. 15, Issue 2 https://doi.org/10.1038/nrmicro.2016.164	journal	December 2016
Hydrogenosomes in the rumen fungus Neocallimastix patriciarum Yarlett, N.; Orpin, C. G.; Munn, E. A. Biochemical Journal, Vol. 236, Issue 3 https://doi.org/10.1042/bj2360729	journal	June 1986
The anaerobic chytridiomycete fungus Piromyces sp. E2 produces ethanol via pyruvate:formate lyase and an alcohol dehydrogenase E: Alcohol dehydrogenase E in the fungus Piromyces sp. E2 Boxma, Brigitte; Voncken, Frank; Jannink, Sander Molecular Microbiology, Vol. 51, Issue 5 https://doi.org/10.1046/j.1365-2958.2003.03912.x	journal	January 2004
Anaerobic gut fungi are an untapped reservoir of natural products Swift, Candice L.; Louie, Katherine B.; Bowen, Benjamin P. Proceedings of the National Academy of Sciences, Vol. 118, Issue 18 https://doi.org/10.1073/pnas.2019855118	journal	April 2021
Resistant Body Formation in Neocallimastix Sp., An Anaerobic Fungus from the Rumen of a Cow Wubah, D. A.; Fuller, M. S.; Akin, D. E. Mycologia, Vol. 83, Issue 1 https://doi.org/10.1080/00275514.1991.12025977	journal	January 1991
featureCounts: an efficient general purpose program for assigning sequence reads to genomic features Liao, Y.; Smyth, G. K.; Shi, W. Bioinformatics, Vol. 30, Issue 7 https://doi.org/10.1093/bioinformatics/btt656	journal	November 2013
DeepLoc: prediction of protein subcellular localization using deep learning Almagro Armenteros, José Juan; Sønderby, Casper Kaae; Sønderby, Søren Kaae Bioinformatics, Vol. 33, Issue 21 https://doi.org/10.1093/bioinformatics/btx431	journal	July 2017
KEGG: Kyoto Encyclopedia of Genes and Genomes Kanehisa, Minoru; Goto, Susumu Nucleic Acids Research, Vol. 28, Issue 1, p. 27-30 https://doi.org/10.1093/nar/28.1.27	journal	January 2000
The Gene Ontology resource: enriching a GOld mine Carbon, Seth; Douglass, Eric; Good, Benjamin M. Nucleic Acids Research, Vol. 49, Issue D1 https://doi.org/10.1093/nar/gkaa1113	journal	December 2020
The carbohydrate-active enzyme database: functions and literature Drula, Elodie; Garron, Marie-Line; Dogan, Suzan Nucleic Acids Research, Vol. 50, Issue D1 https://doi.org/10.1093/nar/gkab1045	journal	November 2021
KEGG for taxonomy-based analysis of pathways and genomes Kanehisa, Minoru; Furumichi, Miho; Sato, Yoko Nucleic Acids Research, Vol. 51, Issue D1 https://doi.org/10.1093/nar/gkac963	journal	October 2022
InterPro in 2022 Paysan-Lafosse, Typhaine; Blum, Matthias; Chuguransky, Sara Nucleic Acids Research, Vol. 51, Issue D1 https://doi.org/10.1093/nar/gkac993	journal	November 2022
MycoCosm portal: gearing up for 1000 fungal genomes Grigoriev, Igor V.; Nikitin, Roman; Haridas, Sajeet Nucleic Acids Research, Vol. 42, Issue D1 https://doi.org/10.1093/nar/gkt1183	journal	December 2013
deepTools: a flexible platform for exploring deep-sequencing data Ramírez, Fidel; Dündar, Friederike; Diehl, Sarah Nucleic Acids Research, Vol. 42, Issue W1 https://doi.org/10.1093/nar/gku365	journal	May 2014
dbCAN2: a meta server for automated carbohydrate-active enzyme annotation Zhang, Han; Yohe, Tanner; Huang, Le Nucleic Acids Research, Vol. 46, Issue W1 https://doi.org/10.1093/nar/gky418	journal	May 2018
Characterization and rank assignment criteria for the anaerobic fungi (Neocallimastigomycota) Elshahed, Mostafa S.; Hanafy, Radwa A.; Cheng, Yanfen International Journal of Systematic and Evolutionary Microbiology, Vol. 72, Issue 7 https://doi.org/10.1099/ijsem.0.005449	journal	July 2022
Anaerobic fungi (phylum Neocallimastigomycota ): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential Gruninger, Robert J.; Puniya, Anil K.; Callaghan, Tony M. FEMS Microbiology Ecology, Vol. 90, Issue 1 https://doi.org/10.1111/1574-6941.12383	journal	August 2014
Regulation of glycogen metabolism in yeast and bacteria Wilson, Wayne A.; Roach, Peter J.; Montero, Manuel FEMS Microbiology Reviews, Vol. 34, Issue 6 https://doi.org/10.1111/j.1574-6976.2010.00220.x	journal	November 2010
Relationship between Secondary Metabolism and Fungal Development Calvo, A. M.; Wilson, R. A.; Bok, J. W. Microbiology and Molecular Biology Reviews, Vol. 66, Issue 3 https://doi.org/10.1128/MMBR.66.3.447-459.2002	journal	September 2002
Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii Silva, A. M.; Maia, J. C.; Juliani, M. H. Journal of Bacteriology, Vol. 169, Issue 5 https://doi.org/10.1128/jb.169.5.2069-2078.1987	journal	May 1987
Experimentally Validated Reconstruction and Analysis of a Genome-Scale Metabolic Model of an Anaerobic Neocallimastigomycota Fungus Wilken, St. Elmo; Monk, Jonathan M.; Leggieri, Patrick A. mSystems, Vol. 6, Issue 1 https://doi.org/10.1128/mSystems.00002-21	journal	February 2021
The phylogenetic relationships of the anaerobic chytridiomycetous gut fungi (Neocallimasticaceae) and the Chytridiomycota. II. Cladistic analysis of structural data and description of Neocallimasticales ord.nov. Li, Jinliang; Heath, I. Brent; Packer, Laurence Canadian Journal of Botany, Vol. 71, Issue 3 https://doi.org/10.1139/b93-044	journal	March 1993
Glycoside hydrolase enzymes present in the zoospore and vegetative growth stages of the rumen fungi Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate, grown on a range of carbohydrates Williams, Alan G.; Orpin, Colin G. Canadian Journal of Microbiology, Vol. 33, Issue 5 https://doi.org/10.1139/m87-072	journal	May 1987
A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes Koonin, Eugene V.; Fedorova, Natalie D.; Jackson, John D. Genome Biology, Vol. 5, Issue 2 https://doi.org/10.1186/gb-2004-5-2-r7	journal	January 2004
Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters Seppälä, Susanna; Solomon, Kevin V.; Gilmore, Sean P. Microbial Cell Factories, Vol. 15, Issue 1 https://doi.org/10.1186/s12934-016-0611-7	journal	December 2016
Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Love, Michael I.; Huber, Wolfgang; Anders, Simon Genome Biology, Vol. 15, Issue 12 https://doi.org/10.1186/s13059-014-0550-8	journal	December 2014
OrthoFinder: phylogenetic orthology inference for comparative genomics Emms, David M.; Kelly, Steven Genome Biology, Vol. 20, Issue 1 https://doi.org/10.1186/s13059-019-1832-y	journal	November 2019
Transcriptomic characterization of Caecomyces churrovis: a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus Henske, John K.; Gilmore, Sean P.; Knop, Doriv Biotechnology for Biofuels, Vol. 10, Issue 1 https://doi.org/10.1186/s13068-017-0997-4	journal	December 2017
Basal body loss during fungal zoospore encystment: evidence against centriole autonomy Heath, I. Brent; Kaminskyj, Susan G. W.; Bauchop, Tom Journal of Cell Science, Vol. 83, Issue 1 https://doi.org/10.1242/jcs.83.1.135	journal	July 1986
High-quality RNA extraction and the regulation of genes encoding cellulosomes are correlated with growth stage in anaerobic fungi Brown, Jennifer L.; Gierke, Taylor; Butkovich, Lazarina V. Frontiers in Fungal Biology, Vol. 4 https://doi.org/10.3389/ffunb.2023.1171100	journal	July 2023
Glycoside Hydrolase Family 48 Cellulase: A Key Player in Cellulolytic Bacteria for Lignocellulose Biorefinery You, Cai; Liu, Ya-Jun; Cui, Qiu Fermentation, Vol. 9, Issue 3 https://doi.org/10.3390/fermentation9030204	journal	February 2023
Involvement of LaeA and Velvet Proteins in Regulating the Production of Mycotoxins and Other Fungal Secondary Metabolites Hou, Xuwen; Liu, Liyao; Xu, Dan Journal of Fungi, Vol. 10, Issue 8 https://doi.org/10.3390/jof10080561	journal	August 2024
Genetic Regulators and Physiological Significance of Glycogen Storage in Candida albicans Zeitz, Marcus A.; Tanveer, Zainab; Openshaw, Anatole T. Journal of Fungi, Vol. 5, Issue 4 https://doi.org/10.3390/jof5040102	journal	October 2019
Isolation and Biochemical Characterization of Six Anaerobic Fungal Strains from Zoo Animal Feces Stabel, Marcus; Schweitzer, Tabea; Haack, Karoline Microorganisms, Vol. 9, Issue 8 https://doi.org/10.3390/microorganisms9081655	journal	August 2021
Enzyme-based DNA extraction from zoospores of ruminal fungi Tsai, K. -P.; Calza, R. E. Fungal Genetics Reports, Vol. 39, Issue 1 https://doi.org/10.4148/1941-4765.1443	journal	January 1992
A cellular and molecular atlas reveals the basis of chytrid development Laundon, Davis; Chrismas, Nathan; Bird, Kimberley eLife, Vol. 11 https://doi.org/10.7554/eLife.73933	journal	March 2022

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Title: Separation of life stages within anaerobic fungi (Neocallimastigomycota) highlights differences in global transcription and metabolism

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