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

Developing Anaerobic Fungal Tools for Efficient Upgrading of Lignocellulosic Feedstocks

Authors:

Kunwoo Khim1* (kwkhim@udel.edu), Manish Pareek1, Radwa Hanafy1, Casey Hooker1,2, Kevin Solomon1, 2

Institutions:

1Department of Chemical and Biomolecular Engineering, University of Delaware–Newark; 2Agricultural and Biological Engineering, Purdue University–West Lafayette

Goals

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.

Abstract

Anaerobic fungi (Neocallimastigomycota) from the digestive tracts of large herbivores are emerging model species for the efficient deconstruction of untreated renewable plant biomass due to their integration of hydrolytic strategies from the bacterial and fungal kingdoms (Solomon et al. 2016). 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; Hooker et al. 2018; Solomon et al. 2016; Solomon et al. 2018). Past efforts have characterized remarkable resistance to lignin composition (Hooker et al. 2018), revealed more efficient enzymes for biosynthesis (Hillman et al. 2021a), and created bioprocesses for efficient conversion of agricultural residues to high value products (Hillman et al. 2021b). More recently, researchers have created the first tools for transient and compartmentalized heterologous gene expression in these species (Hooker et al. 2023).

Engineering efforts exploit the natural competency of anaerobic fungi to uptake exogenous nucleic acids. While daily dosing with exogenous DNA enables transient transformation, this requires significant DNA input. To evaluate whether genomic integration is possible, the team created linear codon-optimized hph cassettes that confer hygromycin resistance. A single dose and selection on hygromycin produced several resistant colonies of which 30% had a stable resistance phenotype. Subsequent molecular characterization confirmed integration via non- homologous end joining in various loci including LTR retrotransposons serving as the first example of such in this family.

Researcheres have also sought to identify an autonomous replicating sequence (ARS) for anaerobic fungal plasmid replication and develop self-replicating plasmids. The team is pursuing a number of strategies, including screening shotgun genomic libraries and plasmids from other species for broad host activity. One plasmid that has shown promise is the yeast 2 μm plasmid, a relatively small multi-copy selfish DNA element that resides in the yeast nucleus at a copy number of 40 to 60. Combining AGF-optimized expression cassettes producing eGFP and hygromycin resistance marker, researchers were able to see a steady level of eGFP expression above background levels even approximately 12 days after a single plasmid dose. However, researchers are still unable to maintain a selectable phenotype for long periods with this, implying that the steady state copy number and/or promoter expression is too low. To address this, researchers are currently expanding the promoter library and evaluating the impact of plasmid features (e.g. GC content, size, etc.) on stability.

Leveraging natural competency to introduce exogenous DNA, researchers have achieved the first simple methods for targeted heterologous expression in anaerobic fungi and are optimizing approaches for stable phenotypes. This growing toolbox for anaerobic fungi forms foundational tools to generate a deeper systems-level understanding of anaerobic fungal physiology while establishing fundamental knowledge about regulation of gut fungal CAZymes. Ultimately, this research enables predictive biology in anaerobic fungi and derive insight into microbial plant deconstruction to advance the development of economical biofuels and bioproducts.

References

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. 2021a. “Anaerobic Fungal Mevalonate Pathway Genomic Biases Lead to Heterologous Toxicity Underpredicted by Codon Adaptation Indices,” Microorganisms 9, 1986.

Hillman, E. T., et al. 2021b. “Hydrolysis of Lignocellulose by Anaerobic Fungi Produces Free Sugars and Organic Acids for Two-Stage Fine Chemical Production with Kluyveromyces marxianus,” Biotechnology Progress 37, e3172.

Hooker, C. A., 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.

Hooker, C. A.. et al. 2023. “A Genetic Engineering Toolbox for the Lignocellulolytic Anaerobic Gut Fungus Neocallimastix frontalis,” ACS Synthetic Biology 12, 1034–45.

Seppälä, S., 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, 1192–95.

Solomon, K. V., et al. 2018. “Catabolic Repression in Early-Diverging Anaerobic Fungi is Partially Mediated by Natural Antisense Transcripts,” Fungal Genetics and 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,” Applied and Environmental Microbiology 79, 4620–34.

Funding Information

This project is supported by the BER program 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 (JGI) and the Environmental Molecular Sciences Laboratory (EMSL), which are DOE Office of Science user facilities. Both facilities are sponsored by the BER program and operated under Contract Nos. DE-AC02-05CH11231 (JGI) and DE-AC05-76RL01830 (EMSL).