Developing Anaerobic Fungal Tools for Efficient Upgrading of Lignocellulosic Feedstocks
Manish Pareek1* (email@example.com), Radwa Hanafy1, Casey Hooker1,2, Ethan Hillman2, Javier Muñoz Briones2, and Kevin Solomon1, 2
1University of Delaware, and 2Purdue University
This project develops genetic and epigenetic tools for emerging model anaerobic fungi to identify the genomic determinants of their powerful biomass-degrading capabilities, facilitate their study, and enable direct fungal conversion of untreated lignocellulose to bioproducts.
Deconstruction of plant cell walls is a significant bottleneck to the economical production of affordable biofuels and bioproducts from abundant and renewable plant biomass. Anaerobic fungi (Neocallimastigomycota) from the digestive tracts of large herbivores, however, have evolved unique abilities to degrade untreated fiber-rich plant biomass by combining hydrolytic strategies from the bacterial and fungal kingdoms (Haitjema et al. 2017). Anaerobic fungi secrete the largest known diversity of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom, which unaided can degrade up to 60% of the ingested plant material within the animal digestive tract (Seppälä et al. 2017, Youssef et al. 2013). Unlike many other fungal systems, these CAZymes are tightly regulated and assembled in fungal cellulosomes to synergistically degrade plant material, including untreated agricultural residues, bioenergy crops, and woody biomass with comparable efficiency regardless of composition (Haitjema et al. 2017, Solomon et al. 2016, Solomon et al. 2018, Hooker et al. 2018).
The team’s efforts to characterize gut fungal CAZymes reveal industrially relevant properties such as remarkable stability and activity towards untreated plant biomass (Hooker et al. 2018, Hillman et al. 2021). Gut fungal CAZymes liberate sugars from cellulosic substrates for over a week after inoculation with some sugar metabolized to organic acids. However, model bioproduction hosts such as K. marxianus can capture the carbon in these sugars and acids in a two-stage process to efficiently upgrade this carbon to high value solvents, fragrances and advanced fuels derived from esters and aromatic alcohols (e.g., ethyl-acetate and 2-phenylethanol; Hillman et al. 2021). Similarly, anaerobic fungal biosynthetic enzymes possess unique cofactor substrate preferences that support higher catalytic efficiencies, which are easily overlooked via heterologous expression due to the extremely high AT content (~83%) of gut fungal genomes and biased codon preferences (Hillman et al. 2021). Thus, there is an unmet need to build genetic tools and methods to study these enzymes natively in anaerobic fungi.
As a first step to tool development, the team sequenced the genomes of three novel anaerobic fungal isolates to enable part mining. High quality genomic DNA isolations were paired with PacBio long-read sequencing and Hi-C (chromosomal conformation capture) sequencing to achieve the first chromosomally resolved genomes for isolates belonging to the genera Neocallimastix and Piromyces. The team’s assemblies incorporate more than 99% of the genome into 12-25 chromosomes with N50<10. Parts that regulate gene expression (e.g., promotors and terminators) were then identified via sequence homology to assemble a nascent genetic toolbox for heterologous expression in anaerobic fungi.
To introduce these parts, the team optimized delivery of DNA with fluorescently labelled oligonucleotides and circular plasmids. Both Piromyces and Neocallimastix isolates were naturally competent for double-stranded DNA, taking up any DNA supplemented to the growth media and localizing it to the nucleus. Transformation was observed in multiple life stages suggesting that natural competence is a robust property of anaerobic fungi and a facile method to introduce heterologous DNA. Using natural competency, team members then validated heterologous expression of anaerobic fluorescent reporters and codon-optimized antibiotic resistance markers expressed via identified constitutive promoters and terminators synthesized by the DOE Joint Genome Institute’s Biological and Environmental Research Support Science (JGI-BERSS) program. Researchers also identified and validated nuclear localization sequences (NLS) from anaerobic fungal histone proteins, which displayed distinct sequence motifs from conventional NLSs used in model organisms. However, due to a lack of known autonomously replicating sequences, heterologous DNA must be supplemented to growth media daily to achieve stable phenotypes. Efforts are underway to achieve stable transformants via the use of Cas9 ribonucleoproteins (RNPs) together with split-marker cassettes.
In conclusion, anaerobic fungi hold a wealth of potential for biocatalysis from renewable plant substrates. Leveraging natural competency to introduce exogenous DNA, researchers have achieved the first simple methods for targeted heterologous expression in anaerobic fungi. The team’s growing toolbox for anaerobic fungi form foundational tools to generate a deeper systems-level understanding of anaerobic fungal physiology while establishing fundamental knowledge about regulation of gut fungal CAZymes. Ultimately, the team enables predictive biology in anaerobic fungi and derive insight into microbial plant deconstruction to advance the development of economical biofuels and bioproducts.
Haitjema, C. H., et al. 2017. “A Parts List for Fungal Cellulosomes Revealed by Comparative Genomics,” Nature Microbiology 2, 17087.
Hillman, E. T., et al. 2021. “Hydrolysis of Lignocellulose by Anaerobic Fungi Produces Free Sugars and Organic Acids for Two‐Stage Fine Chemical Production with Kluyveromyces marxianus,” Biotechnology Progress 37(5), e3172.
Hooker, C., et al. 2018. “Hydrolysis of Untreated Lignocellulosic Feedstock is Independent of S-Lignin Composition in Newly Classified Anaerobic Fungal Isolate Piromyces sp. UH3-1,” Biotechnology for Biofuels 11:293.
Seppälä, Susanna et al. 2017. “The Importance of Sourcing Enzymes From Non-conventional Fungi for Metabolic Engineering and Biomass Breakdown,” Metabolic Engineering 44, 45–59.
Solomon, K. V., et al. 2016. “Early-Branching Gut Fungi Possess a Large, Comprehensive Array of Biomass-Degrading Enzymes,” Science 351, 1192–1195.
Solomon, K. V., et al. 2018. “Catabolic Repression in Early-Diverging Anaerobic Fungi is Partially Mediated by Natural Antisense Transcripts,” Fungal Genetics & Biology 121:1–9.
Youssef, N.H., et al. 2013. “The Genome of the Anaerobic Fungus Orpinomyces sp. Strain C1A Reveals the Unique Evolutionary History of a Remarkable Plant Biomass Degrader,” Appl Environ Microbiol 79(15), 4620–34.
This project is supported by the Office of Biological and Environmental Research through the DOE Office of Science under Award No DE-SC0022206. A portion of this research was performed under the Facilities Integrating Collaborations for User Science (FICUS) initiative and used resources at the DOE Joint Genome Institute and the Environmental Molecular Sciences Laboratory, which are DOE Office of Science User Facilities. Both facilities are sponsored by the Office of Biological and Environmental Research and operated under Contract Nos. DE-AC02- 05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL).