A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis

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Author(s)Hooker, Casey, A.
Author(s)Hanafy, Radwa
Author(s)Hillman, Ethan T.
Author(s)Muñoz Briones, Javier
Author(s)Solomon, Kevin V.
Date Accessioned2023-05-04T20:31:34Z
Date Available2023-05-04T20:31:34Z
Publication Date2023-04-21
DescriptionThis document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Synthetic Biology, copyright © 2023 American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://doi.org/10.1021/acssynbio.2c00502. This article will be embargoed until 04/21/2024.
AbstractAnaerobic fungi are powerful platforms for biotechnology that remain unexploited due to a lack of genetic tools. These gut fungi encode the largest number of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom, making them attractive for applications in renewable energy and sustainability. However, efforts to genetically modify anaerobic fungi have remained limited due to inefficient methods for DNA uptake and a lack of characterized genetic parts. We demonstrate that anaerobic fungi are naturally competent for DNA and leverage this to develop a nascent genetic toolbox informed by recently acquired genomes for transient transformation of anaerobic fungi. We validate multiple selectable markers (HygR and Neo), an anaerobic reporter protein (iRFP702), enolase and TEF1A promoters, TEF1A terminator, and a nuclear localization tag for protein compartmentalization. This work establishes novel methods to reliably transform the anaerobic fungus Neocallimastix frontalis, thereby paving the way for strain development and various synthetic biology applications.
SponsorC.A.H. was supported by a National Science Foundation Graduate Research Fellowship under DGE-1333468. This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, under award number DESC0022018/DE-SC0022206 and the National Science Foundation under grant no. IOS-2128272. A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) program (proposal DOI: 10.46936/ fics.proj.2018.50397/60000049) and used resources at the DOE Joint Genome Institute (https://ror.org/04xm1d337) and the Environmental Molecular Sciences Laboratory (https://ror.org/04rc0xn13), which are DOE Office of Science User Facilities operated under contract nos. DE-AC02- 05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL). The LSM980 confocal microscope was acquired with a shared instrumentation grant (1S10RR027273-01) from the National Institutes of Health (NIH). Microscopy access was supported by grants from the NIH-NIGMS (P20 GM103446), the NIGMS (P20 GM139760), and the State of Delaware. This publication was made possible by the Delaware INBRE program, supported by a grant from the National Institute of General Medical Sciences−NIGMS (P20 GM103446) from the National Institutes of Health and the State of Delaware. Last, we would like to express our gratitude and pay our respects to the late Dr. Richard West for his considerable help in the Flow Cytometry Core facility.
CitationHooker, Casey A., Radwa Hanafy, Ethan T. Hillman, Javier Muñoz Briones, and Kevin V. Solomon. “A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix Frontalis.” ACS Synthetic Biology 12, no. 4 (April 21, 2023): 1034–45. https://doi.org/10.1021/acssynbio.2c00502.
ISSN2161-5063
URLhttps://udspace.udel.edu/handle/19716/32724
Languageen_US
PublisherACS Synthetic Biology
Keywordsanaerobic gut fungi
Keywordstransformation
Keywordsparts
Keywordsprotein localization
Keywordsgene expression
Keywordsanaerobic reporter
TitleA Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis
TypeArticle
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