Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

A Gene-Editing System for Large-Scale Fungal Phenotyping in a Model Wood Decomposer

Authors:

Jonathan S. Schilling1* (schillin@umn.edu), Jiwei Zhang1* (zhan3437@umn.edu), Igor Grigoriev2, Daniel Cullen3, Hugh D. Mitchell4

Institutions:

1University of Minnesota–Saint Paul; 2DOE Joint Genome Institute; 3U.S. Department of Agriculture Forest Service, Forest Products Laboratory; 4Environmental Molecular Sciences Laboratory

Goals

This project combines CRISPR-Cas9-based genome-editing and network analysis for large-scale phenotyping in a model wood decomposer fungus relevant to the DOE mission area. The overall goal is to develop a high-throughput genetic platform that enables discovery of distinctive genes and genetic features that speed wood degradation by brown rot fungal species. The research endeavors to provide stand-alone tools and resources for discovering novel fungal genetic mechanisms that can be used together to advance relevant plant biomass conversion research in the post-genomic era.

Abstract

This research focuses on a group of unique wood decomposer basidiomycete fungi—brown rot fungi—that harbor industrially relevant pathways for extracting carbohydrates from lignocellulose and have broad relevance to global carbon cycling. Distinct from other fungi, brown rot species use non-enzymatic reactive oxygen species mechanisms to modify lignin and selectively extract sugars. Their degradative mechanisms, from a process efficiency standpoint, represent a pathway upgrade relative to the ancestral approaches in white rot species (Hibbett and Donoghue 2001; Eastwood et al. 2011). Fungi obtained this capacity evolutionarily by shedding rather than gaining carbohydrate-active enzymes repertoire genes (Martinez et al. 2009; Floudas et al. 2012; Riley et al. 2014). This paradox therefore makes brown rot fungi a promising candidate for discovering unknown genetic mechanisms governing plant biomass degradation. Although DOE mission relevance is clear and major genomically informed advances in brown rot have been achieved, progress is limited by an inability to manipulate genes in any brown rot fungal strain.

The potential key roles of fungal genome reshuffling and gene regulation in determining brown rot efficacy are widely recognized (Zhang et al. 2016; Zhang et al. 2017; Zhang et al. 2019). Using functional genomic tools, a staggered two-step (i.e., oxidation-then-hydrolysis) gene regulation model for brown rot was elucidated in the Proceedings of the National Academy of Sciences (Zhang et al. 2016) and mBio (Zhang et al. 2019). Although these genomic studies have greatly advanced understanding of brown rot, its genetic basis remains uncharacterized and unharnessed. For example, (1) gene function in the two-step model remain unverified and ambiguous; (2) the gene regulatory mechanism used to control and consolidate the two steps is unclear; and (3) the functions of most genes identified by multi-omics are either hypothetical or unknown. The existence of these gaps is primarily due to the lack of a robust genome-editing tools for validating and discovering brown rot genetic features.

This project will integrate systems biology, genome-editing, and network modeling to address these key gaps. Three project objectives include:

Objective 1: Create a CRISPR-Cas9-mediated gene-editing system and use it to target genes. To genetically manipulate brown rot fungal species, the research team first created a DNA transformation procedure in a model species—Gloeophyllum trabeum. A series of genetic tools were then developed to test and control gene expression in the fungus, including a collective of promoters, a laccase reporter system for reporting extracellular protein function (Li et al. 2023), and a GFP reporter system for testing intracellular protein function and nuclear localization signals and for localizing the cellular loci of lignocellulolytic enzymes. The strain’s dikaryotic genome was resolved using long-read PacBio sequencing to enable gene editing on both alleles. A pre-assembled Cas9-single guide RNA (sgRNA) ribonucleoprotein method was attempted to target benzoquinone reductase, a key Fenton gene. Mutants with successful disruptions of one or two alleles were obtained. Mutation mechanisms involved in the editing process were studied. Although editing efficacy is low (2% to 5%), the method is acceptable to test brown rot gene functions at a singular gene level (e.g., by targeting crucial candidate genes pinpointed by omics and network analysis).

Objective 2: Model a carbon-utilizing network governing brown rot and use it to mine decay genes. To build a carbon-utilizing gene network for discovering novel brown rot genetic features, transcriptome response to a broad spectrum of lignocellulose derivative carbon sources was measured in two brown rot species, G. trabeum and Rhodonia placenta. This species comparison enabled identification of shared or distinct mechanisms. Different network analyzing tools were tested and compared, and key modules and their “hub” genes associated with lignocellulose polymers or monomers were identified. DNA affinity purification (DAP-seq) was then used to identify the cis- and trans-regulatory elements involved in the carbon signaling pathway, and the key regulatory machinery unique to brown rot was revealed (Zhang et al. 2022). Networks derived from gene co-expression and DAP-seq were overlapped. In the context of the full project, this objective will complement the gene targets for large-scale phenotypic screening.

Objective 3: Develop multiplexed genome editing for large-scale phenotypic screens. This objective aims to develop a pipeline to use the multiplexing sgRNA library for genome-editing and mutant library construction for large-scale phenotypic screens, followed by next-generation sequencing to discover key functional genes. An all-in-one Cas9 and sgRNA expression construct was built and used to target genes. Several candidate genes were selected for disruption experiments to test the method’s editing efficiency. Insertion frequency of the gene constructs was studied. Moving forward, the multiplexed sgRNA library will be expressed in G. trabeum to specifically study the pathways associated with lignin utilization revealed by network analysis as a step toward large-scale phenotypic screening.

This project aims to provide stand-alone tools and resources to elucidate fundamental microbial processes relevant to the DOE mission area, advancing new engineering designs for lignocellulose bioconversion.

References

Eastwood, D. C., et al. 2011. “The Plant Cell Wall-decomposing Machinery Underlies the Functional Diversity of Forest Fungi,” Science 5;333(6043), 762–5. DOI:10.1126/science.1205411.

Floudas, D., et al. 2012. “The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes,” Science 336(6089), 1715–9.

Hibbett, D. S., and M. J. Donoghue. 2001. “Analysis of Character Correlations Among Wood Decay Mechanisms, Mating Systems, and Substrate Ranges in Homobasidiomycetes,” Systems Biology 50(2), 215–42.

Li, W. et al. 2023. “A Laccase Gene Reporting System that Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum,” Microbiology Spectrum 11(1), e04246-22.

Martinez, D., et al. 2009. “Genome, Transcriptome, and Secretome Analysis of Wood Decay Fungus Postia placenta Supports Unique Mechanisms of Lignocellulose Conversion,” Proceedings of the National Academy of Sciences 106(6), 1954–9.

Riley, R., et al. 2014. “Extensive Sampling of Basidiomycete Genomes Demonstrates Inadequacy of the White-Rot/Brown-Rot Paradigm for Wood Decay Fungi,” Proceedings of the National Academy of Sciences 111(27), 9923–8.

Zhang, J., et al. 2016. “Localizing Gene Regulation Reveals a Staggered Wood Decay Mechanism for the Brown Rot Fungus Postia placenta,” Proceedings of the National Academy of Sciences 113(39), 10968–73.

Zhang, J., and J. S. Schilling. 2017. “Role of Carbon Source in the Shift from Oxidative to Hydrolytic Wood Decomposition by Postia placenta,” Fungal Genetics and Biology 106, 1–8. DOI:10.1016/j.fgb.2017.06.003.

Zhang, J., et al. 2019. “Gene Regulation Shifts Shed Light on Fungal Adaption in Plant Biomass Decomposers,” mBio 10(6):e02176-19.

Zhang, J., et al. 2022. “Distinctive Carbon Repression Effects in the Carbohydrate-selective Wood Decay Fungus Rhodonia placenta,” Fungal Genetics and Biology 159, 103673.

Funding Information

This research is supported by the DOE Office of Science, BER Program, grant no. DE-SC0022151.