Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation

Early-branching gut fungi possess a large, comprehensive array of biomass-degrading enzymes

The fungal kingdom is the source of almost all industrial enzymes in use for lignocellulose bioprocessing. We developed a systems-level approach that integrates transcriptomic sequencing, proteomics, phenotype, and biochemical studies of relatively unexplored basal fungi. Anaerobic gut fungi isolated from herbivores produce a large array of biomass-degrading enzymes that synergistically degrade crude, untreated plant biomass and are competitive with optimized commercial preparations from Aspergillus and Trichoderma. Compared to these model platforms, gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. These enzymes are universally catabolite-repressed and are further regulated by a rich landscape of noncoding regulatory RNAs. Additionally, we identified several promising sequence-divergent enzyme candidates for lignocellulosic bioprocessing

Top-Down enrichment guides in formation of synthetic microbial consortia for biomass degradation

Consortium-based approaches are a promising avenue toward efficient bioprocessing. However, many complex microbial interactions dictate community dynamics and stability that must be replicated in synthetic systems. The rumen and/or hindguts of large mammalian herbivores harbor complex communities of biomass-degrading fungi and bacteria, as well as archaea and protozoa that work collectively to degrade lignocellulose, yet the microbial interactions responsible for stability, resilience, and activity of the community remain largely uncharacterized. In this work, we demonstrate a “top-down” enrichment-based methodology for selecting a minimal but effective lignocellulose-degrading community that produces methane-rich fermentation gas (biogas). The resulting enrichment consortium produced 0.75–1.9-fold more fermentation gas at 1.4–2.1 times the rate compared to a monoculture of fungi from the enrichment. Metagenomic sequencing of the top-down enriched consortium revealed genomes encoding for functional compartmentalization of the community, spread across an anaerobic fungus (Piromyces), a bacterium (Sphaerochaeta), and two methanogenic archaea (Methanosphaera and Methanocorpusculum). Guided by the composition of the top-down enrichment, several synthetic cocultures were formed from the “bottom-up” using previously isolated fungi, Neocallimastix californiae and Anaeromyces robustus paired with the methanogen Methanobacterium bryantii. While cross-feeding occurred in synthetic co-cultures, removal of fungal metabolites by methanogens did not increase the rate of gas production or the rate of substrate deconstruction by the synthetic community relative to fungal monocultures. Metabolomic characterization verified that syntrophy was established within synthetic co-cultures, which generated methane at similar concentrations compared to the enriched consortium but lacked the temporal stability (resilience) seen in the native system. Taken together, deciphering the membership and metabolic potential of an enriched gut consortium enables the design of methanogenic synthetic co-cultures. However, differences in the growth rate and stability of enriched versus synthetic consortia underscore the difficulties in mimicking naturally occurring syntrophy in synthetic systems

Transcriptomic characterization of Caecomyces churrovis: a novel, non-rhizoid-forming lignocellulolytic anaerobic fungus

Anaerobic gut fungi are the primary colonizers of plant material in the rumen microbiome, but are poorly studied due to a lack of characterized isolates. While most genera of gut fungi form extensive rhizoidal networks, which likely participate in mechanical disruption of plant cell walls, fungi within the Caecomyces genus do not possess these rhizoids. Here, we describe a novel fungal isolate, Caecomyces churrovis, which forms spherical sporangia with a limited rhizoidal network yet secretes a diverse set of carbohydrate active enzymes (CAZymes) for plant cell wall hydrolysis. Despite lacking an extensive rhizoidal system, C. churrovis is capable of growth on fibrous substrates like switchgrass, reed canary grass, and corn stover, although faster growth is observed on soluble sugars. Gut fungi have been shown to use enzyme complexes (fungal cellulosomes) in which CAZymes bind to non-catalytic scaffoldins to improve biomass degradation efficiency. However, transcriptomic analysis and enzyme activity assays reveal that C. churrovis relies more on free enzymes compared to other gut fungal isolates. Only 15% of CAZyme transcripts contain non-catalytic dockerin domains in C. churrovis, compared to 30% in rhizoid-forming fungi. Furthermore, C. churrovis is enriched in GH43 enzymes that provide complementary hemicellulose degrading activities, suggesting that a wider variety of these activities are required to degrade plant biomass in the absence of an extensive fungal rhizoid network. Overall, molecular characterization of a non-rhizoid-forming anaerobic fungus fills a gap in understanding the roles of CAZyme abundance and associated degradation mechanisms during lignocellulose breakdown within the rumen microbiome

Genomic and functional analyses of fungal and bacterial consortia that enable lignocellulose breakdown in goat gut microbiomes

The herbivore digestive tract is home to a complex community of anaerobic microbes that work together to break down lignocellulose. These microbiota are an untapped resource of strains, pathways and enzymes that could be applied to convert plant waste into sugar substrates for green biotechnology. We carried out more than 400 parallel enrichment experiments from goat faeces to determine how substrate and antibiotic selection influence membership, activity, stability and chemical productivity of herbivore gut communities. We assembled 719 high-quality metagenome-assembled genomes (MAGs) that are unique at the species level. More than 90% of these MAGs are from previously unidentified herbivore gut microorganisms. Microbial consortia dominated by anaerobic fungi outperformed bacterially dominated consortia in terms of both methane production and extent of cellulose degradation, which indicates that fungi have an important role in methane release. Metabolic pathway reconstructions from MAGs of 737 bacteria, archaea and fungi suggest that cross-domain partnerships between fungi and methanogens enabled production of acetate, formate and methane, whereas bacterially dominated consortia mainly produced short-chain fatty acids, including propionate and butyrate. Analyses of carbohydrate-active enzyme domains present in each anaerobic consortium suggest that anaerobic bacteria and fungi employ mostly complementary hydrolytic strategies. The division of labour among herbivore anaerobes to degrade plant biomass could be harnessed for industrial bioprocessing

Experimentally validated reconstruction and analysis of a genome-scale metabolic model of an anaerobic Neocallimastigomycota fungus

Anaerobic gut fungi in the phylum Neocallimastigomycota typically inhabit the digestive tracts of large mammalian herbivores, where they play an integral role in the decomposition of raw lignocellulose into its constitutive sugar monomers. However, quantitative tools to study their physiology are lacking, partially due to their complex and unresolved metabolism that includes the largely uncharacterized fungal hydrogenosome. Modern omics approaches combined with metabolic modeling can be used to establish an understanding of gut fungal metabolism and develop targeted engineering strategies to harness their degradation capabilities for lignocellulosic bioprocessing. Here, we introduce a high-quality genome of the anaerobic fungus Neocallimastix lanati from which we constructed the first genome-scale metabolic model of an anaerobic fungus. Relative to its size (200 Mbp, sequenced at 62× depth), it is the least fragmented publicly available gut fungal genome to date. Of the 1,788 lignocellulolytic enzymes annotated in the genome, 585 are associated with the fungal cellulosome, underscoring the powerful lignocellulolytic potential of N. lanati. The genome-scale metabolic model captures the primary metabolism of N. lanati and accurately predicts experimentally validated substrate utilization requirements. Additionally, metabolic flux predictions are verified by 13C metabolic flux analysis, demonstrating that the model faithfully describes the underlying fungal metabolism. Furthermore, the model clarifies key aspects of the hydrogenosomal metabolism and can be used as a platform to quantitatively study these biotechnologically important yet poorly understood early-branching fungi

Fungal diversity notes 253–366: taxonomic and phylogenetic contributions to fungal taxa

Notes on 113 fungal taxa are compiled in this paper, including 11 new genera, 89 new species, one new subspecies, three new combinations and seven reference specimens. A wide geographic and taxonomic range of fungal taxa are detailed. In the Ascomycota the new genera Angustospora (Testudinaceae), Camporesia (Xylariaceae), Clematidis, Crassiparies (Pleosporales genera incertae sedis), Farasanispora, Longiostiolum (Pleosporales genera incertae sedis), Multilocularia (Parabambusicolaceae), Neophaeocryptopus (Dothideaceae), Parameliola (Pleosporales genera incertae sedis), and Towyspora (Lentitheciaceae) are introduced. Newly introduced species are Angustospora nilensis, Aniptodera aquibella, Annulohypoxylon albidiscum, Astrocystis thailandica, Camporesia sambuci, Clematidis italica, Colletotrichum menispermi, C. quinquefoliae, Comoclathris pimpinellae, Crassiparies quadrisporus, Cytospora salicicola, Diatrype thailandica, Dothiorella rhamni, Durotheca macrostroma, Farasanispora avicenniae, Halorosellinia rhizophorae, Humicola koreana, Hypoxylon lilloi, Kirschsteiniothelia tectonae, Lindgomyces okinawaensis, Longiostiolum tectonae, Lophiostoma pseudoarmatisporum, Moelleriella phukhiaoensis, M. pongdueatensis, Mucoharknessia anthoxanthi, Multilocularia bambusae, Multiseptospora thysanolaenae, Neophaeocryptopus cytisi, Ocellularia arachchigei, O. ratnapurensis, Ochronectria thailandica, Ophiocordyceps karstii, Parameliola acaciae, P. dimocarpi, Parastagonospora cumpignensis, Pseudodidymosphaeria phlei, Polyplosphaeria thailandica, Pseudolachnella brevifusiformis, Psiloglonium macrosporum, Rhabdodiscus albodenticulatus, Rosellinia chiangmaiensis, Saccothecium rubi, Seimatosporium pseudocornii, S. pseudorosae, Sigarispora ononidis and Towyspora aestuari. New combinations are provided for Eutiarosporella dactylidis (sexual morph described and illustrated) and Pseudocamarosporium pini. Descriptions, illustrations and / or reference specimens are designated for Aposphaeria corallinolutea, Cryptovalsa ampelina, Dothiorella vidmadera, Ophiocordyceps formosana, Petrakia echinata, Phragmoporthe conformis and Pseudocamarosporium pini. The new species of Basidiomycota are Agaricus coccyginus, A. luteofibrillosus, Amanita atrobrunnea, A. digitosa, A. gleocystidiosa, A. pyriformis, A. strobilipes, Bondarzewia tibetica, Cortinarius albosericeus, C. badioflavidus, C. dentigratus, C. duboisensis, C. fragrantissimus, C. roseobasilis, C. vinaceobrunneus, C. vinaceogrisescens, C. wahkiacus, Cyanoboletus hymenoglutinosus, Fomitiporia atlantica, F. subtilissima, Ganoderma wuzhishanensis, Inonotus shoreicola, Lactifluus armeniacus, L. ramipilosus, Leccinum indoaurantiacum, Musumecia alpina, M. sardoa, Russula amethystina subp. tengii and R. wangii are introduced. Descriptions, illustrations, notes and / or reference specimens are designated for Clarkeinda trachodes, Dentocorticium ussuricum, Galzinia longibasidia, Lentinus stuppeus and Leptocorticium tenellum. The other new genera, species new combinations are Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis from Neocallimastigomycota, Phytophthora estuarina, P. rhizophorae, Salispina, S. intermedia, S. lobata and S. spinosa from Oomycota, and Absidia stercoraria, Gongronella orasabula, Mortierella calciphila, Mucor caatinguensis, M. koreanus, M. merdicola and Rhizopus koreanus in Zygomycota

Robust and effective methodologies for cryopreservation and DNA extraction from anaerobic gut fungi

Cell storage and DNA isolation are essential to developing an expanded suite of microorganisms for biotechnology. However, many features of non-model microbes, such as an anaerobic lifestyle and rigid cell wall, present formidable challenges to creating strain repositories and extracting high quality genomic DNA. Here, we establish accessible, high efficiency, and robust techniques to store lignocellulolytic anaerobic gut fungi long term without specialized equipment. Using glycerol as a cryoprotectant, gut fungal isolates were preserved for a minimum of 23 months at −80 °C. Unlike previously reported approaches, this improved protocol is non-toxic and rapid, with samples surviving twice as long with negligible growth impact. Genomic DNA extraction for these isolates was optimized to yield samples compatible with next generation sequencing platforms (e.g. Illumina, PacBio). Popular DNA isolation kits and precipitation protocols yielded preps that were unsuitable for sequencing due to carbohydrate contaminants from the chitin-rich cell wall and extensive energy reserves of gut fungi. To address this, we identified a proprietary method optimized for hardy plant samples that rapidly yielded DNA fragments in excess of 10 kb with minimal RNA, protein or carbohydrate contamination. Collectively, these techniques serve as fundamental tools to manipulate powerful biomass-degrading gut fungi and improve their accessibility among researchers