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Title: Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Abstract

Abstract Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycinmore » B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.« less

Authors:
; ; ; ; ; ; ; ; ; ; ; ; ; ; ORCiD logo
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Energy Efficiency and Renewable Energy (EERE), Transportation Office. Bioenergy Technologies Office; German Academic Exchange Service (DAAD)
OSTI Identifier:
1671836
Alternate Identifier(s):
OSTI ID: 1764520
Grant/Contract Number:  
AC02-05CH11231; 57380837
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 13 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
Netherlands
Language:
English
Subject:
09 BIOMASS FUELS; filamentous fungi; thermoascus aurantiacus; agrobacterium tumefaciens; genetic transformation; CRISPR/Cas9 system; sexual crossing; xylanases; enzyme production

Citation Formats

Gabriel, Raphael, Prinz, Julia, Jecmenica, Marina, Romero-Vazquez, Carlos, Chou, Pallas, Harth, Simon, Floerl, Lena, Curran, Laure, Oostlander, Anne, Matz, Linda, Fritsche, Susanne, Gorman, Jennifer, Schuerg, Timo, Fleißner, André, and Singer, Steven W. Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus. Netherlands: N. p., 2020. Web. https://doi.org/10.1186/s13068-020-01804-x.
Gabriel, Raphael, Prinz, Julia, Jecmenica, Marina, Romero-Vazquez, Carlos, Chou, Pallas, Harth, Simon, Floerl, Lena, Curran, Laure, Oostlander, Anne, Matz, Linda, Fritsche, Susanne, Gorman, Jennifer, Schuerg, Timo, Fleißner, André, & Singer, Steven W. Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus. Netherlands. https://doi.org/10.1186/s13068-020-01804-x
Gabriel, Raphael, Prinz, Julia, Jecmenica, Marina, Romero-Vazquez, Carlos, Chou, Pallas, Harth, Simon, Floerl, Lena, Curran, Laure, Oostlander, Anne, Matz, Linda, Fritsche, Susanne, Gorman, Jennifer, Schuerg, Timo, Fleißner, André, and Singer, Steven W. Sat . "Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus". Netherlands. https://doi.org/10.1186/s13068-020-01804-x.
@article{osti_1671836,
title = {Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus},
author = {Gabriel, Raphael and Prinz, Julia and Jecmenica, Marina and Romero-Vazquez, Carlos and Chou, Pallas and Harth, Simon and Floerl, Lena and Curran, Laure and Oostlander, Anne and Matz, Linda and Fritsche, Susanne and Gorman, Jennifer and Schuerg, Timo and Fleißner, André and Singer, Steven W.},
abstractNote = {Abstract Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.},
doi = {10.1186/s13068-020-01804-x},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 13,
place = {Netherlands},
year = {2020},
month = {10}
}

Journal Article:
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https://doi.org/10.1186/s13068-020-01804-x

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Works referenced in this record:

Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism
journal, December 2013

  • Kim, S.; Stahlberg, J.; Sandgren, M.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 1
  • DOI: 10.1073/pnas.1316609111

The new frontier of genome engineering with CRISPR-Cas9
journal, November 2014


Evolution and Diversity of Plant Cell Walls: From Algae to Flowering Plants
journal, June 2011


Xylose induces cellulase production in Thermoascus aurantiacus
journal, November 2017

  • Schuerg, Timo; Prahl, Jan-Philip; Gabriel, Raphael
  • Biotechnology for Biofuels, Vol. 10, Issue 1
  • DOI: 10.1186/s13068-017-0965-z

Regulation of Pyrimidine Biosynthesis in Saccharomyces cerevisiae1
journal, January 1968


Network of nutrient-sensing pathways and a conserved kinase cascade integrate osmolarity and carbon sensing in Neurospora crassa
journal, September 2017

  • Huberman, Lori B.; Coradetti, Samuel T.; Glass, N. Louise
  • Proceedings of the National Academy of Sciences, Vol. 114, Issue 41
  • DOI: 10.1073/pnas.1707713114

Phylogeny of the industrial relevant, thermophilic genera Myceliophthora and Corynascus
journal, May 2011


Thermoascus aurantiacus is an Intriguing Host for the Industrial Production of Cellulases
journal, April 2017


A new approach to Cas9-based genome editing in Aspergillus niger that is precise, efficient and selectable
journal, January 2019


Functional genomics of lipid metabolism in the oleaginous yeast Rhodosporidium toruloides
journal, March 2018

  • Coradetti, Samuel T.; Pinel, Dominic; Geiselman, Gina M.
  • eLife, Vol. 7
  • DOI: 10.7554/eLife.32110

Comparison and characterization of α-amylase inducers in Aspergillus nidulans based on nuclear localization of AmyR
journal, January 2012

  • Murakoshi, Yuriko; Makita, Tomohiro; Kato, Masashi
  • Applied Microbiology and Biotechnology, Vol. 94, Issue 6
  • DOI: 10.1007/s00253-012-3874-x

A bacterial pioneer produces cellulase complexes that persist through community succession
journal, November 2017


Thermophilic lignocellulose deconstruction
journal, May 2014

  • Blumer-Schuette, Sara E.; Brown, Steven D.; Sander, Kyle B.
  • FEMS Microbiology Reviews, Vol. 38, Issue 3
  • DOI: 10.1111/1574-6976.12044

Isolation of genomic clones containing the amdS gene of Aspergillus nidulans and their use in the analysis of structural and regulatory mutations.
journal, August 1983

  • Hynes, M. J.; Corrick, C. M.; King, J. A.
  • Molecular and Cellular Biology, Vol. 3, Issue 8, p. 1430-1439
  • DOI: 10.1128/MCB.3.8.1430

The complex transcriptional landscape of the anucleate human platelet
journal, January 2013

  • Bray, Paul F.; McKenzie, Steven E.; Edelstein, Leonard C.
  • BMC Genomics, Vol. 14, Issue 1
  • DOI: 10.1186/1471-2164-14-1

Conservation and diversity of the regulators of cellulolytic enzyme genes in Ascomycete fungi
journal, April 2017


Development of genetic tools for Myceliophthora thermophila
journal, May 2015


Carbon Catabolite Repression of Gene Expression and Conidiation inNeurospora crassa
journal, October 1998


Thermoascus aurantiacus is a promising source of enzymes for biomass deconstruction under thermophilic conditions
journal, January 2012

  • McClendon, Shara D.; Batth, Tanveer; Petzold, Christopher J.
  • Biotechnology for Biofuels, Vol. 5, Issue 1
  • DOI: 10.1186/1754-6834-5-54

Challenges and opportunities in synthetic biology for chemical engineers
journal, November 2013


Redesigning the regulatory pathway to enhance cellulase production in Penicillium oxalicum
journal, April 2015


Xylose Triggers Reversible Phosphorylation of XlnR, the Fungal Transcriptional Activator of Xylanolytic and Cellulolytic Genes in Aspergillus oryzae
journal, May 2011

  • Noguchi, Yuji; Tanaka, Hisaki; Kanamaru, Kyoko
  • Bioscience, Biotechnology, and Biochemistry, Vol. 75, Issue 5
  • DOI: 10.1271/bbb.100923

CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens
journal, May 2018

  • Concordet, Jean-Paul; Haeussler, Maximilian
  • Nucleic Acids Research, Vol. 46, Issue W1
  • DOI: 10.1093/nar/gky354

Strategies for the transformation of filamentous fungi
journal, February 2002


Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains
journal, May 2015


Ultrastructure of thermophilic fungi
journal, June 1981


Xyr1 (Xylanase Regulator 1) Regulates both the Hydrolytic Enzyme System and d-Xylose Metabolism in Hypocrea jecorina
journal, October 2006

  • Stricker, Astrid R.; Grosstessner-Hain, Karin; Würleitner, Elisabeth
  • Eukaryotic Cell, Vol. 5, Issue 12
  • DOI: 10.1128/EC.00211-06

The roles of the zinc finger transcription factors XlnR, ClrA and ClrB in the breakdown of lignocellulose by Aspergillus niger
journal, January 2016


Neurospora v. a Synthetic Medium Favoring Sexual Reproduction
journal, December 1947


Sexual development in the industrial workhorse Trichoderma reesei
journal, August 2009

  • Seidl, V.; Seibel, C.; Kubicek, C. P.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 33
  • DOI: 10.1073/pnas.0904936106

Regulation of the lignocellulolytic response in filamentous fungi
journal, July 2016


Agrobacterium-mediated transformation as a tool for functional genomics in fungi
journal, May 2005

  • Michielse, Caroline B.; Hooykaas, Paul J. J.; van den Hondel, Cees A. M. J. J.
  • Current Genetics, Vol. 48, Issue 1
  • DOI: 10.1007/s00294-005-0578-0

Genetic engineering of filamentous fungi — Progress, obstacles and future trends
journal, March 2008


Conserved and essential transcription factors for cellulase gene expression in ascomycete fungi
journal, April 2012

  • Coradetti, S. T.; Craig, J. P.; Xiong, Y.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 19
  • DOI: 10.1073/pnas.1200785109

Identification of the CRE-1 Cellulolytic Regulon in Neurospora crassa
journal, September 2011


Plant Cell Wall Deconstruction by Ascomycete Fungi
journal, September 2013


Trichoderma reesei RUT-C30 - thirty years of strain improvement
journal, October 2011


Sordaria macrospora: 25 years as a model organism for studying the molecular mechanisms of fruiting body development
journal, March 2020

  • Teichert, Ines; Pöggeler, Stefanie; Nowrousian, Minou
  • Applied Microbiology and Biotechnology, Vol. 104, Issue 9
  • DOI: 10.1007/s00253-020-10504-3

Comparative genomic analysis of the thermophilic biomass-degrading fungi Myceliophthora thermophila and Thielavia terrestris
journal, October 2011

  • Berka, Randy M.; Grigoriev, Igor V.; Otillar, Robert
  • Nature Biotechnology, Vol. 29, Issue 10, p. 922-927
  • DOI: 10.1038/nbt.1976

Sexual development and cryptic sexuality in fungi: insights from Aspergillus species
journal, January 2012


The influence of Aspergillus niger transcription factors AraR and XlnR in the gene expression during growth in d-xylose, l-arabinose and steam-exploded sugarcane bagasse
journal, November 2013

  • de Souza, Wagner Rodrigo; Maitan-Alfenas, Gabriela Piccolo; de Gouvêa, Paula Fagundes
  • Fungal Genetics and Biology, Vol. 60
  • DOI: 10.1016/j.fgb.2013.07.007

A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi
journal, July 2015


Binary Agrobacterium vectors for plant transformation
journal, January 1984


Sexual reproduction in Aspergillus species of medical or economical importance: why so fastidious?
journal, November 2009


Systems analysis of plant cell wall degradation by the model filamentous fungus Neurospora crassa
journal, December 2009

  • Tian, Chaoguang; Beeson, William T.; Iavarone, Anthony T.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 52
  • DOI: 10.1073/pnas.0906810106

A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola
journal, January 2017

  • Pigné, Sandrine; Zykwinska, Agata; Janod, Etienne
  • Fungal Biology and Biotechnology, Vol. 4, Issue 1
  • DOI: 10.1186/s40694-016-0029-3

Transformation of Aspergillus niger using the homologous orotidine-5′-phosphate-decarboxylase gene
journal, March 1987

  • Goosen, Theo; Bloemheuvel, Gerda; Gysler, Christoph
  • Current Genetics, Vol. 11, Issue 6-7
  • DOI: 10.1007/BF00384612

Sexual crossing of thermophilic fungus Myceliophthora heterothallica improved enzymatic degradation of sugar beet pulp
journal, February 2016

  • Aguilar-Pontes, Maria Victoria; Zhou, Miaomiao; van der Horst, Sjors
  • Biotechnology for Biofuels, Vol. 9, Issue 1
  • DOI: 10.1186/s13068-016-0460-y