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The secretome, the complement of extracellular proteins, is a reflection of the interaction of an organism with its host or substrate, thus a determining factor for the organism’s fitness and competitiveness. Hence, the secretome impacts speciation and organismal evolution. The zoosporic Chytridiomycota , Blastocladiomycota , Neocallimastigomycota , and Cryptomycota represent the earliest diverging lineages of the Fungal Kingdom. The review describes the enzyme compositions of these zoosporic fungi, underscoring the enzymes involved in biomass degradation. The review connects the lifestyle and substrate affinities of the zoosporic fungi to the secretome composition by examining both classical phenotypic investigations and molecular/genomic-based studies. The carbohydrate-active enzyme profiles of 19 genome-sequenced species are summarized. Emphasis is given to recent advances in understanding the functional role of rumen fungi, the basis for the devastating chytridiomycosis, and the structure of fungal cellulosome. The approach taken by the review enables comparison of the secretome enzyme composition of anaerobic versus aerobic early-diverging fungi and comparison of enzyme portfolio of specialized parasites, pathogens, and saprotrophs. Early-diverging fungi digest most major types of biopolymers: cellulose, hemicellulose, pectin, chitin, and keratin. It is thus to be expected that early-diverging fungi in its entirety represents a rich and diverse pool of secreted, metabolic enzymes. The review presents the methods used for enzyme discovery, the diversity of enzymes found, the status and outlook for recombinant production, and the potential for applications. Comparative studies on the composition of secretome enzymes of early-diverging fungi would contribute to unraveling the basal lineages of fungi.

The ubiquity and relevance of extracellular DNA (exDNA) are well-known and increasingly gaining importance in many fields of application such as medicine and environmental microbiology. Although sources and types of exDNA are manifold, ratios of specific DNA-molecules inside and outside of living cells can give reliable information about the activity of entire systems and of specific microbial groups or species. Here, we introduce a method to discriminate between internal (iDNA), as well as bound and free exDNA, and evaluate various DNA fractions and related ratios (ex:iDNA) regarding their applicability to be used as a fast, convenient, and reliable alternative to more tedious RNA-based activity measurements. In order to deal with microbial consortia that can be regulated regarding their activity, we tested and evaluated the proposed method in comparison to sophisticated dehydrogenase- and RNA-based activity measurements with two anaerobic microbial consortia (anaerobic fungi and syntrophic archaea and a microbial rumen consortium) and three levels of resolution (overall activity, total bacteria, methanogenic archaea). Furthermore, we introduce a 28S rRNA gene-specific primer set and qPCR protocol, targeting anaerobic fungi (Neocallimastigomycota). Our findings show that the amount of actively released free exDNA (fDNA) strongly correlates with different activity measurements and is thus suggested to serve as a proxy for microbial activity.

Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process significantly impacts energy production and waste management, influencing greenhouse gas emissions. Traditional research has largely focused on anaerobic bacteria and methanogens for methane production. However, the potential of anaerobic lignocellulolytic fungi for degrading lignocellulosic biomass remains less explored. In this study, buffalo rumen inocula were enriched and acclimatized to improve lignocellulolytic hydrolysis activity. Two consortia were established: the anaerobic fungi consortium (AFC), selectively enriched for fungi, and the anaerobic lignocellulolytic microbial consortium (ALMC). The consortia were utilized to create five distinct microbial cocktails–AF0, AF20, AF50, AF80, and AF100. These cocktails were formulated based on varying of AFC and ALMC by weights (w/w). Methane production from each cocktail of lignocellulosic biomasses (cassava pulp and oil palm residues) was evaluated. The highest methane yields of CP, EFB, and MFB were obtained at 337, 215, and 54 mL/g VS, respectively. Cocktails containing a mix of anaerobic fungi, hydrolytic bacteria ( Sphingobacterium sp.), syntrophic bacteria ( Sphaerochaeta sp.), and hydrogenotrophic methanogens produced 2.1–2.6 times higher methane in cassava pulp and 1.1–1.2 times in oil palm empty fruit bunch compared to AF0. All cocktails effectively produced methane from oil palm empty fruit bunch due to its lipid content. However, methane production ceased after 3 days when oil palm mesocarp fiber was used, due to long-chain fatty acid accumulation. Anaerobic fungi consortia showed effective lignocellulosic and starchy biomass degradation without inhibition due to organic acid accumulation. These findings underscore the potential of tailored microbial cocktails for enhancing methane production from diverse lignocellulosic substrates. Key Points Enhanced methane production from diverse lignocellulosic biomasses using enriched microbial cocktails. Efficient degradation of lignocellulosic biomass and starch by consortia of anaerobic fungi constituent. The number of anaerobic fungi affects the amount of methane production. Importance of tailored microbial cocktails for maximizing methane production.

Anaerobic fungi and methanogenic archaea are two classes of microorganisms found in the rumen microbiome that metabolically interact during lignocellulose breakdown. Here, stable synthetic co-cultures of the anaerobic fungus Caecomyces churrovis and the methanogen Methanobacterium bryantii (not native to the rumen) were formed, demonstrating that microbes from different environments can be paired based on metabolic ties. Transcriptional and metabolic changes induced by methanogen co-culture were evaluated in C. churrovis across a variety of substrates to identify mechanisms that impact biomass breakdown and sugar uptake. A high-quality genome of C. churrovis was obtained and annotated, which is the first sequenced genome of a non-rhizoid-forming anaerobic fungus. C. churrovis possess an abundance of CAZymes and carbohydrate binding modules and, in agreement with previous studies of early-diverging fungal lineages, N6-methyldeoxyadenine (6mA) was associated with transcriptionally active genes. Co-culture with the methanogen increased overall transcription of CAZymes, carbohydrate binding modules, and dockerin domains in co-cultures grown on both lignocellulose and cellulose and caused upregulation of genes coding associated enzymatic machinery including carbohydrate binding modules in family 18 and dockerin domains across multiple growth substrates relative to C. churrovis monoculture. Two other fungal strains grown on a reed canary grass substrate in co-culture with the same methanogen also exhibited high log2-fold change values for upregulation of genes encoding carbohydrate binding modules in families 1 and 18. Transcriptional upregulation indicated that co-culture of the C. churrovis strain with a methanogen may enhance pyruvate formate lyase (PFL) function for growth on xylan and fructose and production of bottleneck enzymes in sugar utilization pathways, further supporting the hypothesis that co-culture with a methanogen may enhance certain fungal metabolic functions. Upregulation of CBM18 may play a role in fungal–methanogen physical associations and fungal cell wall development and remodeling.

Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome. Anaerobic gut fungi (Neocallimastigomycetes) live in the digestive tract of large herbivores, where they are vastly outnumbered by bacteria. It has been suggested that anaerobic fungi challenge growth of bacteria owing to the wealth of biosynthetic genes in fungal genomes, although this relationship has not been experimentally tested. Here, we cocultivated the rumen bacteria Fibrobacter succinogenes strain UWB7 with the anaerobic gut fungi Anaeromyces robustus or Caecomyces churrovis on a range of carbon substrates and quantified the bacterial and fungal transcriptomic response. Synthetic cocultures were established for at least 24 h, as verified by active fungal and bacterial transcription. A. robustus upregulated components of its secondary metabolism in the presence of Fibrobacter succinogenes strain UWB7, including six nonribosomal peptide synthetases, one polyketide synthase-like enzyme, and five polyketide synthesis O-type methyltransferases. Both A. robustus and C. churrovis cocultures upregulated S -adenosyl- l -methionine (SAM)-dependent methyltransferases, histone methyltransferases, and an acetyltransferase. Fungal histone 3 lysine 27 trimethylation marks were more abundant in coculture, and heterochromatin protein-1 was downregulated. Together, these findings suggest that fungal chromatin remodeling occurs when bacteria are present. F. succinogenes strain UWB7 upregulated four genes in coculture encoding drug efflux pumps, which likely protect the cell against toxins. Furthermore, untargeted nonpolar metabolomics data revealed at least one novel fungal metabolite enriched in coculture, which may be a defense compound. Taken together, these data suggest that A. robustus and C. churrovis produce antimicrobials when exposed to rumen bacteria and, more broadly, that anaerobic gut fungi are a source of novel antibiotics. IMPORTANCE Anaerobic fungi are outnumbered by bacteria by 4 orders of magnitude in the herbivore rumen. Despite their numerical disadvantage, they are resilient members of the rumen microbiome. Previous studies mining the genomes of anaerobic fungi identified genes encoding enzymes to produce natural products, which are small molecules that are often antimicrobials. In this work, we cocultured the anaerobic fungus Anaeromyces robustus or Caecomyes churrovis with rumen bacteria Fibrobacter succinogenes strain UWB7 and sequenced fungal and bacterial active genes via transcriptome sequencing (RNA-seq). Consistent with production of a fungal defense compound, bacteria upregulated genes encoding drug efflux pumps, which often export toxic molecules, and fungi upregulated genes encoding biosynthetic enzymes of natural products. Furthermore, tandem mass spectrometry detected an unknown fungal metabolite enriched in the coculture. Together, these findings point to an antagonistic relationship between anaerobic fungi and rumen bacteria resulting in the production of a fungal compound with potential antimicrobial activity.

Abstract Traditional methods for enumerating and identifying microbial populations within the rumen can be time consuming and cumbersome. Methods that involve culturing and microscopy can also be inconclusive, particularly when studying anaerobic rumen fungi. A real-time PCR SYBR Green assay, using PCR primers to target total rumen fungi and the cellulolytic bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes, is described, including design and validation. The DNA and crude protein contents with respect to the fungal biomass of both polycentric and monocentric fungal isolates were investigated across the fungal growth stages to aid in standard curve generation. The primer sets used were found to be target specific with no detectable cross-reactivity. Subsequently, the real-time PCR assay was employed in a study to detect these populations within cattle rumen. The anaerobic fungal target was observed to increase 3.6-fold from 0 to 12 h after feeding. The results also indicated a 5.4-fold increase in F. succinogenes target between 0 and 12 h after feeding, whereas R. flavefaciens was observed to maintain more or less consistent levels. This is the first report of a real-time PCR assay to estimate the rumen anaerobic fungal population.

Lignocellulose is a promising feedstock for biofuel production as a renewable, carbohydrate-rich and globally abundant source of biomass. However, challenges faced include environmental and/or financial costs associated with typical lignocellulose pretreatments needed to overcome the natural recalcitrance of the material before conversion to biofuel. Anaerobic fungi are a group of underexplored microorganisms belonging to the early diverging phylum Neocallimastigomycota and are native to the intricately evolved digestive system of mammalian herbivores. Anaerobic fungi have promising potential for application in biofuel production processes due to the combination of their highly effective ability to hydrolyse lignocellulose and capability to convert this substrate to H2 and ethanol. Furthermore, they can produce volatile fatty acid precursors for subsequent biological conversion to H2 or CH4 by other microorganisms. The complex biological characteristics of their natural habitat are described, and these features are contextualised towards the development of suitable industrial systems for in vitro growth. Moreover, progress towards achieving that goal is reviewed in terms of process and genetic engineering. In addition, emerging opportunities are presented for the use of anaerobic fungi for lignocellulose pretreatment; dark fermentation; bioethanol production; and the potential for integration with methanogenesis, microbial electrolysis cells and photofermentation.

The ruminal microbiota allows ruminants to utilize fibrous feeds and is in the limelight of ruminant nutrition research for many years. However, the overwhelming majority of investigations have focused on bacteria, whereas anaerobic fungi (AF) have been widely neglected by ruminant nutritionists. Anaerobic fungi are not only crucial fiber degraders but also important nutrient sources for the host. This review summarizes the current findings on AF and, most importantly, discusses their new application potentials in modern ruminant nutrition. Available data suggest AF can be applied as direct-fed microbials to enhance ruminal fiber degradation, which is indeed of interest for high-yielding dairy cows that often show depressed ruminal fibrolysis in response to high-grain feeding. Moreover, these microorganisms have relevance for the nutrient supply and reduction of methane emissions. However, to reach AF-related improvements in ruminal fiber breakdown and animal performance, obstacles in large-scale AF cultivation and applicable administration options need to be overcome. At feedstuff level, silage production may benefit from the application of fungal enzymes that cleave lignocellulosic structures and consequently enable higher energy exploitation from forages in the rumen. Concluding, AF hold several potentials in improving ruminant feeding and future research efforts are called for to harness these potentials.

This study investigated silage quality characteristics and ruminal fiber degradability of grass and straw ensiled with either anaerobic fungi (AF) supernatant with active fungal enzymes or mixed ruminal fluid as novel silage additives. Compared to control silages, AF supernatant improved the quality of grass and straw silages as evidenced by decreased pH, acetic acid concentration, and dry matter losses. Likewise, mixed ruminal fluid enhanced lactic acid fermentation, which further resulted in lower pH of the treated grass silage. The ruminal fiber degradability was determined using in situ incubations and, compared to controls, the cellulose degradability was higher for grass silage with AF supernatant, whereas ruminal degradability of straw silage was reduced by this treatment. In contrast, mixed ruminal fluid did not influence fiber degradability of silages in the rumen. Concluding, both novel additives improved silage quality, whereas only AF supernatant enhanced ruminal fiber degradability of grass silage and therefore may represent an approach for improving forage utilization by ruminants. Key points • Enzymes of anaerobic fungi supernatant improve quality of grass and straw silages. • Mixed ruminal fluid enhances lactic acid fermentation when ensiling grass and straw. • Enzymes of anaerobic fungi supernatant increase ruminal grass silage degradability.

Anaerobic fungi found in the guts of large herbivores are prolific biomass degraders whose genomes harbor a wealth of carbohydrate-active enzymes (CAZymes), of which only a handful are structurally or biochemically characterized. Here, we report the structure and kinetic rate parameters for a glycoside hydrolase (GH) family 5 subfamily 4 enzyme (CelD) from Piromyces finnis, a modular, cellulosome-incorporated endoglucanase that possesses three GH5 domains followed by two C-terminal fungal dockerin domains (double dockerin). We present the crystal structures of an apo wild-type CelD GH5 catalytic domain and its inactive E154A mutant in complex with cellotriose at 2.5 and 1.8 Å resolution, respectively, finding the CelD GH5 catalytic domain adopts the (β/α)8-barrel fold common to many GH5 enzymes. Structural superimposition of the apo wild-type structure with the E154A mutant-cellotriose complex supports a catalytic mechanism in which the E154 carboxylate side chain acts as an acid/base and E278 acts as a complementary nucleophile. Further analysis of the cellotriose binding pocket highlights a binding groove lined with conserved aromatic amino acids that when docked with larger cellulose oligomers is capable of binding seven glucose units and accommodating branched glucan substrates. Activity analyses confirm P. finnis CelD can hydrolyze mixed linkage glucan and xyloglucan, as well as carboxymethylcellulose (CMC). Measured kinetic parameters show the P. finnis CelD GH5 catalytic domain has CMC endoglucanase activity comparable to other fungal endoglucanases with kcat = 6.0 ± 0.6 s−1 and Km = 7.6 ± 2.1 g/L CMC. Enzyme kinetics were unperturbed by the addition or removal of the native C-terminal dockerin domains as well as the addition of a non-native N-terminal dockerin, suggesting strict modularity among the domains of CelD.Key points• Anaerobic fungi host a wealth of industrially useful enzymes but are understudied.• P. finnis CelD has endoglucanase activity and structure common to GH5_4 enzymes.• CelD’s kinetics do not change with domain fusion, exhibiting high modularity.