Effects of rumen-native microbial feed supplementation on milk yield, composition, and feed efficiency in lactating dairy cows.

The objective of this study was to evaluate the effects of two rumen-native microbial feed supplements (MFS) on milk production, milk composition, and feed efficiency. A total of 90 multiparous cows between 40 and 60 d in milk were enrolled in a randomized block design study. Within each block (baseline milk yield), cows were randomly assigned to: control (no microbial feed supplementation), MFS1 (0.33 g/kg total mixed ration [TMR] of an MFS containing a minimum of Clostridium beijerinckii at 2 × 106 CFU/g and Pichia kudriavzevii at 2 × 107 CFU/g), or MFS2 (0.33 g/kg TMR of a MFS containing a minimum of C. beijerinckii at 2 × 106 CFU/g, P. kudriavzevii at 2 × 107 CFU/g, Ruminococcus bovis at 2 × 107 CFU/g, and Butyrivibrio fibrisolvens at 2 × 107 CFU/g). Cows were housed in a single group and fed the study diets ad libitum for 270 d. Individual milk yield was recorded using electronic milk meters, and milk fat and protein were measured using optical in-line analyzers at each of two daily milkings. Treatment and treatment by time effects were assessed through multiple linear regression analyses. Treatment effects were observed for milk and energy-corrected milk (ECM) yields, milk fat and protein yields and concentrations, dry matter intake (DMI), and feed efficiency; those effects were conditional to time for milk yield, DMI, and feed efficiency. Overall, milk, ECM, fat, and protein yields were higher for MFS2 compared with control cows (+3.0, 3.7, 0.12, and 0.12 kg/d, respectively). Compared with MFS1, milk yield was higher and protein yield tended to be higher for MFS2 cows (+2.9 and 0.09 kg/d, respectively). In contrast, MFS1 cows produced 0.17 and 0.08 units of percentage per day more fat and protein than MFS2 cows, and 0.07 units of percentage per day more protein than control cows. Dry matter intake and feed efficiency were higher for MFS2 cows compared with MFS1 cows (+1.3 kg/d and 0.06, respectively), and feed efficiency was higher for MFS2 cows compared with control cows (+0.04). Where observed, treatment by time effects suggest that the effects of MFS2 were more evident as time progressed after supplementation was initiated. No effects of microbial supplementation were observed on body weight, body condition score, somatic cell count, or clinical mastitis case incidence. In conclusion, the supplementation of MFS2 effectively improved economically important outcomes such as milk yield, solids, and feed efficiency.

This study evaluates the effects of two rumen-native microbial feed supplements (MFS) on milk yield, composition, and feed efficiency in lactating dairy cows. Ninety multiparous Holstein cows between 40 and 60 d in milk were assigned to control (no microbial feed supplementation), MFS1 (Clostridium beijerinckii and Pichia kudriavzevii), or MFS2 (C. beijerinckii, P. kudriavzevii, Ruminococcus bovis, and Butyrivibrio fibrisolvens) total mixed ration supplementation. Overall, MFS2 cows had higher milk and milk component yields than control and MFS1, while MFS1 cows had higher milk component concentrations than control and MFS2. Feed efficiency was higher for MFS2 compared with control and MFS1 cows. Microbial feed supplementation improved economically important outcomes such as milk yield, solids, and feed efficiency.

Cellulosome Localization Patterns Vary across Life Stages of Anaerobic Fungi.

Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores are powerful biomass-degrading organisms that enhance their degradative ability through the formation of cellulosomes, multienzyme complexes that synergistically colocalize enzymes to extract sugars from recalcitrant plant matter. However, a functional understanding of how fungal cellulosomes are deployed in vivo to orchestrate plant matter degradation is lacking, as is knowledge of how cellulosome production and function vary throughout the morphologically diverse life cycle of anaerobic fungi. In this work, we generated antibodies against three major fungal cellulosome protein domains, a dockerin, scaffoldin, and glycoside hydrolase (GH) 48 protein, and used them in conjunction with helium ion and immunofluorescence microscopy to characterize cellulosome localization patterns throughout the life cycle of Piromyces finnis when grown on simple sugars and complex cellulosic carbon sources. Our analyses reveal that fungal cellulosomes are cell-localized entities specifically targeted to the rhizoids of mature fungal cells and bodies of zoospores. Examination of cellulosome localization patterns across life stages also revealed that cellulosome production is independent of growth substrate in zoospores but repressed by simple sugars in mature cells. This suggests that further exploration of gene regulation patterns in zoospores is needed and can inform potential strategies for derepressing cellulosome expression and boosting hydrolytic enzyme yields from fungal cultures. Collectively, these findings underscore how life cycle-dependent cell morphology and regulation of cellulosome production impact biomass degradation by anaerobic fungi, insights that will benefit ongoing efforts to develop these organisms and their cellulosomes into platforms for converting waste biomass into valuable bioproducts. IMPORTANCE Anaerobic fungi (Neocallimastigomycota) isolated from the guts of herbivores excel at degrading ingested plant matter, making them attractive potential platform organisms for converting waste biomass into valuable products, such as chemicals and fuels. Major contributors to their biomass-hydrolyzing power are the multienzyme cellulosome complexes that anaerobic fungi produce, but knowledge gaps in how cellulosome production is controlled by the cellular life cycle and how cells spatially deploy cellulosomes complicate the use of anaerobic fungi and their cellulosomes in industrial bioprocesses. We developed and used imaging tools to observe cellulosome spatial localization patterns across life stages of the anaerobic fungus Piromyces finnis under different environmental conditions. The resulting spatial details of how anaerobic fungi orchestrate biomass degradation and uncovered relationships between life cycle progression and regulation of cellulosome production will benefit ongoing efforts to develop anaerobic fungi and their cellulosomes into useful biomass-upgrading platforms.

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.

Designing chimeric enzymes inspired by fungal cellulosomes.

Cellulosomes are synthesized by anaerobic bacteria and fungi to degrade lignocellulose via synergistic action of multiple enzymes fused to a protein scaffold. Through templating key protein domains (cohesin and dockerin), designer cellulosomes have been engineered from bacterial motifs to alter the activity, stability, and degradation efficiency of enzyme complexes. Recently a parts list for fungal cellulosomes from the anaerobic fungi (Neocallimastigomycota) was determined, which revealed sequence divergent fungal cohesin, dockerin, and scaffoldin domains that could be used to expand the available toolbox to synthesize designer cellulosomes. In this work, multi-domain carbohydrate active enzymes (CAZymes) from 3 cellulosome-producing fungi were analyzed to inform the design of chimeric proteins for synthetic cellulosomes inspired by anaerobic fungi. In particular, Piromyces finnis was used as a structural template for chimeric carbohydrate active enzymes. Recombinant enzymes with retained properties were engineered by combining thermophilic glycosyl hydrolase domains from Thermotoga maritima with dockerin domains from Piromyces finnis. By preserving the protein domain order from P. finnis, chimeric enzymes retained catalytic activity at temperatures over 80 °C and were able to associate with cellulosomes purified from anaerobic fungi. Fungal cellulosomes harbor a wide diversity of glycoside hydrolases, each representing templates for the design of chimeric enzymes. By conserving dockerin domain position within the primary structure of each protein, the activity of both the catalytic domain and dockerin domain was retained in enzyme chimeras. Taken further, the domain positioning inferred from native fungal cellulosome proteins can be used to engineer multi-domain proteins with non-native favorable properties, such as thermostability.

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.

Catabolic repression in early-diverging anaerobic fungi is partially mediated by natural antisense transcripts.

Early-diverging anaerobic fungi (order: Neocallimastigomycota), lignocelluolytic chytrid-like fungi central to fiber degradation in the digestive tracts of large herbivores, are attractive sources of cellulases and hemicellulases for biotechnology. Enzyme expression is tightly regulated and coordinated through mechanisms that remain unelucidated to optimize hydrolytic efficiency. Our analysis of anaerobic fungal transcriptomes reveals hundreds of cis-natural antisense transcripts (cis-NATs), which we hypothesize play an integral role in this regulation. Through integrated genomic and transcriptomic sequencing on a range of catabolic substrates, we validate these NATs in three species (Anaeromyces robustus, Neocallimasix californiae, and Piromyces finnis), and analyze their expression patterns and prevalence to gain insight into their function. NAT function was diverse and conserved across the three fungal genomes studied, with 10% of all metabolic process NATs associated with lignocellulose hydrolysis. Despite these similarities, however, only eleven gene targets were conserved orthologs. Several NATs were dynamically regulated by lignocellulosic substrates while their gene targets were unregulated. This observation is consistent with a hypothesized, but untested, regulatory mechanism where selected genes are exclusively regulated at the transcriptional/post-transcriptional level by NATs. However, only genes with high NAT relative expression levels displayed this phenomenon, suggesting a selection mechanism that favors larger dynamic ranges for more precise control of gene expression. In addition to this mode, we observed two other possible regulatory fates: canonical transcriptional regulation with no NAT response, and positive co-regulation of target mRNA and cognate NAT, which we hypothesize is a fine-tuning strategy to locally negate control outputs from global regulators. Our work reveals the complex contributions of antisense RNA to the catabolic response in anaerobic fungi, highlighting its importance in understanding lignocellulolytic activity for bioenergy applications. More importantly, the relative expression of NAT to target may form a critical determinant of transcriptional vs post-transcriptional (NAT) control of gene expression in primitive anaerobic fungi.

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.

PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

Genomic analysis of methanogenic archaea reveals a shift towards energy conservation.

BACKGROUND: The metabolism of archaeal methanogens drives methane release into the environment and is critical to understanding global carbon cycling. Methanogenesis operates at a very low reducing potential compared to other forms of respiration and is therefore critical to many anaerobic environments. Harnessing or altering methanogen metabolism has the potential to mitigate global warming and even be utilized for energy applications. RESULTS: Here, we report draft genome sequences for the isolated methanogens Methanobacterium bryantii, Methanosarcina spelaei, Methanosphaera cuniculi, and Methanocorpusculum parvum. These anaerobic, methane-producing archaea represent a diverse set of isolates, capable of methylotrophic, acetoclastic, and hydrogenotrophic methanogenesis. Assembly and analysis of the genomes allowed for simple and rapid reconstruction of metabolism in the four methanogens. Comparison of the distribution of Clusters of Orthologous Groups (COG) proteins to a sample of genomes from the RefSeq database revealed a trend towards energy conservation in genome composition of all methanogens sequenced. Further analysis of the predicted membrane proteins and transporters distinguished differing energy conservation methods utilized during methanogenesis, such as chemiosmotic coupling in Msar. spelaei and electron bifurcation linked to chemiosmotic coupling in Mbac. bryantii and Msph. cuniculi. CONCLUSIONS: Methanogens occupy a unique ecological niche, acting as the terminal electron acceptors in anaerobic environments, and their genomes display a significant shift towards energy conservation. The genome-enabled reconstructed metabolisms reported here have significance to diverse anaerobic communities and have led to proposed substrate utilization not previously reported in isolation, such as formate and methanol metabolism in Mbac. bryantii and CO2 metabolism in Msph. cuniculi. The newly proposed substrates establish an important foundation with which to decipher how methanogens behave in native communities, as CO2 and formate are common electron carriers in microbial communities.

A parts list for fungal cellulosomes revealed by comparative genomics.

Cellulosomes are large, multiprotein complexes that tether plant biomass-degrading enzymes together for improved hydrolysis1. These complexes were first described in anaerobic bacteria, where species-specific dockerin domains mediate the assembly of enzymes onto cohesin motifs interspersed within protein scaffolds1. The versatile protein assembly mechanism conferred by the bacterial cohesin-dockerin interaction is now a standard design principle for synthetic biology2,3. For decades, analogous structures have been reported in anaerobic fungi, which are known to assemble by sequence-divergent non-catalytic dockerin domains (NCDDs)4. However, the components, modular assembly mechanism and functional role of fungal cellulosomes remain unknown5,6. Here, we describe a comprehensive set of proteins critical to fungal cellulosome assembly, including conserved scaffolding proteins unique to the Neocallimastigomycota. High-quality genomes of the anaerobic fungi Anaeromyces robustus, Neocallimastix californiae and Piromyces finnis were assembled with long-read, single-molecule technology. Genomic analysis coupled with proteomic validation revealed an average of 312 NCDD-containing proteins per fungal strain, which were overwhelmingly carbohydrate active enzymes (CAZymes), with 95 large fungal scaffoldins identified across four genera that bind to NCDDs. Fungal dockerin and scaffoldin domains have no similarity to their bacterial counterparts, yet several catalytic domains originated via horizontal gene transfer with gut bacteria. However, the biocatalytic activity of anaerobic fungal cellulosomes is expanded by the inclusion of GH3, GH6 and GH45 enzymes. These findings suggest that the fungal cellulosome is an evolutionarily chimaeric structure-an independently evolved fungal complex that co-opted useful activities from bacterial neighbours within the gut microbiome.

Mapping the membrane proteome of anaerobic gut fungi identifies a wealth of carbohydrate binding proteins and transporters.

BACKGROUND: Engineered cell factories that convert biomass into value-added compounds are emerging as a timely alternative to petroleum-based industries. Although often overlooked, integral membrane proteins such as solute transporters are pivotal for engineering efficient microbial chassis. Anaerobic gut fungi, adapted to degrade raw plant biomass in the intestines of herbivores, are a potential source of valuable transporters for biotechnology, yet very little is known about the membrane constituents of these non-conventional organisms. Here, we mined the transcriptome of three recently isolated strains of anaerobic fungi to identify membrane proteins responsible for sensing and transporting biomass hydrolysates within a competitive and rather extreme environment. RESULTS: Using sequence analyses and homology, we identified membrane protein-coding sequences from assembled transcriptomes from three strains of anaerobic gut fungi: Neocallimastix californiae, Anaeromyces robustus, and Piromyces finnis. We identified nearly 2000 transporter components: about half of these are involved in the general secretory pathway and intracellular sorting of proteins; the rest are predicted to be small-solute transporters. Unexpectedly, we found a number of putative sugar binding proteins that are associated with prokaryotic uptake systems; and approximately 100 class C G-protein coupled receptors (GPCRs) with non-canonical putative sugar binding domains. CONCLUSIONS: We report the first comprehensive characterization of the membrane protein machinery of biotechnologically relevant anaerobic gut fungi. Apart from identifying conserved machinery for protein sorting and secretion, we identify a large number of putative solute transporters that are of interest for biotechnological applications. Notably, our data suggests that the fungi display a plethora of carbohydrate binding domains at their surface, perhaps as a means to sense and sequester some of the sugars that their biomass degrading, extracellular enzymes produce.

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.

Driving biomass breakdown through engineered cellulosomes.

Extraction of sugar is the rate-limiting step in converting unpretreated biomass into value-added products through microbial fermentation. Both anaerobic fungi and anaerobic bacteria have evolved to produce large multi-cellulase complexes referred to as cellulosomes, which are powerful machines for biomass deconstruction. Characterization of bacterial cellulosomes has inspired synthetic "designer" cellulosomes, consisting of parts discovered from the native system that have proven useful for cellulose depolymerization. By contrast, the multi-cellulase complexes produced by anaerobic fungi are much more poorly understood, and to date their composition, architecture, and enzyme tethering mechanism remain unknown and heavily debated. Here, we compare current knowledge pertaining to the cellulosomes produced by both bacteria and fungi, including their application to synthetic enzyme-tethered systems for tunneled biocatalysis. We highlight gaps in knowledge and opportunities for discovery, especially pertaining to the potential of fungal cellulosome-inspired systems.