A Gene-Editing System for Large-Scale Fungal Phenotyping in a Model Wood Decomposer
Jiwei Zhang1*(email@example.com), Jonathan S Schilling1*(firstname.lastname@example.org), Igor Grigoriev2, Daniel Cullen3, and Hugh D Mitchell4
1University of Minnesota–Saint Paul; 2Joint Genome Institute; 3USDA Forest Products Laboratory and University of Wisconsin–Madison; and 4Environmental Molecular Sciences Laboratory at Pacific Northwest National Laboratory
The team 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 can allow the discoveries of distinctive genes and genetic features enabling the fast wood degradation in brown rot fungal species. Through this research, the team hopes to provide stand-alone tools and resources for discovering novel fungal genetic mechanisms that can be used in combination to advance relevant plant biomass conversion research in the post-genomic era.
This research focuses on a group of unique wood decomposer basidiomycete fungi–brown rot fungi, which harbor the industrially relevant pathways to extract carbohydrates from lignocellulose and have broad relevance to global carbon cycling. Distinct from other fungi, brown rot species use nonenzymatic reactive oxygen species (ROS) 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 (CAZys) repertoire genes (Martinez et al. 2009, Floudas et al. 2012, and 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 the team has made major genomically informed advances in brown rot, progress is limited by an inability to manipulate genes in any brown rot fungal strain.
The team has known that reshuffling of fungal genome and gene regulation might play key roles in determining the brown rot efficacy (Zhang et al. 2016, Zhang et al. 2019, and Zhang et al. 2017). Using functional genomic tools, recently team members elucidated a staggered two-step (i.e., oxidation-then-hydrolysis) gene regulation model for brown rot in PNAS in 2016 and in mBio in 2019 (Zhang et al. 2016, Zhang et al. 2019). Although these genomic studies have greatly advanced understanding of brown rot, its genetic basis remains uncharacterized and unharnessed. Targeted gaps are still remaining for understanding its genetic mechanism. For example, (1) the functions of genes involved the two-step model remain unverified and ambiguous, (2) the gene regulatory mechanism used to control and consolidate two steps is unclear, and (3) the functions of majority of genes identified by multiomics are either hypothetical or unknown and are waiting for interrogation. The existence of these gaps is, to a large extent, due to the lack of a robust genome-editing tool that can allow the validation and discovery of brown rot genetic features.
In this project, team members plan to integrate systems biology, genome-editing, and network modeling to address these key gaps. Three objectives were included, and progresses were made recently towards accomplishing the project goals:
Obj. 1: Create the CRISPR/Cas9-based editing system to validate brown rot gene functions.
To make the genetic manipulation available in brown rot fungal species, researchers first built a genetic transformation platform in a model species: Gloeophyllum trabeum. Based on this, researchers then developed a gene reporting system that relies on a laccase reporter gene and its rapid, colorimetric detection method for optimizing expression elements and transformation procedures (Li et al. 2023). With this, a codon-optimized eSpCas9 gene originally retrieved from Streptococcus pyogenes, was fused to eGFP and expressed in the G. trabeum nucleus led by a nucleoplasmin nucleus localization signal. The sgRNAs targeting a cellulase gene Cel5A (Gene ID 57704) were then expressed in the G. trabeum-Cas9 mutant for the plasmid-based CRISPR-Cas9 gene-editing. Three U6 promoters from Aspergillus niger, Trametes versicolor, and G. trabeum were tested for their efficiencies in driving the sgRNA’s expression, respectively. Parallelly, a preassembled Cas9-sgRNA ribonucleoprotein method was also tried in the same brown rot species for the scarless gene disruption. Going forward, once the gene-editing tool is fully built-up, researchers will continue to work on the genes, which were not functionally validated yet, towards generating a first-ever single-gene mutant library for brown rot phenotypic studies.
Obj. 2: Build a carbon utilization network to discover key genetic features used by brown rot.
To build a gene co-expression network for discovering novel brown rot genetic features, the team measured the transcriptomes of brown rot species in response to a broad spectrum of lignocellulose derivative carbon sources. Two brown rot species, G. trabeum and Rhodonia placenta, were used for cross-species comparisons for discovering the shared or distinct mechanisms. By a genome-wide co-expression modeling, key modules and its “hub” genes associated with lignocellulose polymers or monomers were discovered. DAP-seq (DNA affinity purification) was then used to identify the cis- and trans-regulatory elements involved in the carbon signaling pathway and revealed the regulatory machineries unique to brown rot (papers in prep.). Connecting to the whole project, this objective will complement the gene pool, aiming for large-scale phenotypic screening.
Obj. 3: Integrate network analysis with CRISPR/Cas9 library 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 NGS to discover key functional genes. Firstly, tens of plasmid constructs were used for simultaneously delivery into G. trabeum and were combined with the laccase reporter to optimize a high-throughput transformation procedure. Once this ongoing trial succeed, the team will continue to integrate it with the plasmid-based CRISPR/Cas9 method for large-scale gene editing.
Collectively, by this project researchers anticipate providing stand-alone tools and resources to elucidate fundamental microbial processes relevant to DOE mission area, advancing new engineering designs for lignocellulose bioconversion.
Eastwood, D. C., et al. 2011. “The Plant Cell Wall-Decomposing Machinery Underlies the Functional Diversity of Forest Fungi,” Science 333(762), 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),1715–19. DOI:10.1126/science.1221748.
Li, W., et al. 2023. “A Laccase Gene Reporting System That Enables Genetic Manipulations in a Brown Rot Wood Decomposer Fungus Gloeophyllum trabeum,” Microbiol Spectr 11(1): e04246-22. DOI:10.1128/spectrum.04246-22.
Martinez, D., et al. 2009. “Genome, Transcriptome, and Secretome Analysis of Wood Decay Fungus Postia placenta Supports Unique Mechanisms of Lignocellulose Conversion,” PNAS 106(6), 1954–59. DOI:10.1073/pnas.0809575106.
Riley, R., et al. 2014. “Extensive Sampling of Basidiomycete Genomes Demonstrates Inadequacy of the White-Rot/Brown-Rot Paradigm for Wood Decay Fungi,” PNAS 111(27), 9923–28. DOI:10.1073/pnas.1400592111.
Zhang, J., et al. 2016. “Localizing Gene Regulation Reveals a Staggered Wood Decay Mechanism for the Brown Rot Fungus Postia placenta,” PNAS 113(39), 10968–73. DOI:10.1073/pnas.1608454113.
Zhang, J., et al. 2017. “Role of Carbon Source in the Shift from Oxidative to Hydrolytic Wood Decomposition by Postia placenta,” Fungal Genet Biol 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. DOI:10.1128/mBio.02176-19.
This research is supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research (BER), grant no. DE-SC0022151.