Pecoramyces ruminantium, gen. nov., sp. nov., an anaerobic gut fungus from the feces of cattle and sheep.

The anaerobic gut fungi (AGF) inhabit the rumen and alimentary tracts of multiple ruminant and nonruminant herbivores, belong to a distinct phylum-level lineage (Neocallimastigomycota), and play an important role in plant biomass degradation in many herbivores. As part of a wider effort to obtain AGF with high lignocellulolytic capacities, we isolated and characterized four different AGF strains from the feces of cattle and sheep. Microscopically, isolates produced monocentric thalli and monoflagellated zoospores. Phylogenetic analysis revealed that all isolates formed a monophyletic cluster with strong bootstrap support as a sister clade to the genus Orpinomyces and close to Neocallimastix, an unexpected result because these two genera of AGF form polyflagellated zoospores. Isolates displayed a smooth biofilm-like growth in liquid medium and formed small (0.5-1 mm) pinpoint circular colonies on agar roll tubes. Both endogenous and exogenous sporangia were observed with variable shapes and sizes. Zoospores were mainly spherical, with diameters ranging between 3.8 and 12.5 µm, and mostly a single flagellum. All strains exhibited similar substrate utilization patterns and comparable cellulolytic and xylanolytic activities. Similar ITS1 sequences falling within the same distinctive clade were found on GenBank, with all environmental samples obtained from diverse ruminant and pseudoruminant hosts from three continents, but not from any hindgut-fermenting hosts. Given the high level of sequence divergence between our strains and closest cultured representatives and their distinct microscopic/macroscopic features, we propose a new genus, Pecoramyces, from the name of the taxonomic infraorder Pecora ("horned ruminants" or "higher ruminants"; derived from the Latin word for horned livestock), and a new species, P. ruminantium (since occurrence seems to be specific to ruminant/pseudoruminant foregut, but not hindgut-fermenting mammals).

The genome of the anaerobic fungus Orpinomyces sp. strain C1A reveals the unique evolutionary history of a remarkable plant biomass degrader.

Anaerobic gut fungi represent a distinct early-branching fungal phylum (Neocallimastigomycota) and reside in the rumen, hindgut, and feces of ruminant and nonruminant herbivores. The genome of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, was sequenced using a combination of Illumina and PacBio single-molecule real-time (SMRT) technologies. The large genome (100.95 Mb, 16,347 genes) displayed extremely low G+C content (17.0%), large noncoding intergenic regions (73.1%), proliferation of microsatellite repeats (4.9%), and multiple gene duplications. Comparative genomic analysis identified multiple genes and pathways that are absent in Dikarya genomes but present in early-branching fungal lineages and/or nonfungal Opisthokonta. These included genes for posttranslational fucosylation, the production of specific intramembrane proteases and extracellular protease inhibitors, the formation of a complete axoneme and intraflagellar trafficking machinery, and a near-complete focal adhesion machinery. Analysis of the lignocellulolytic machinery in the C1A genome revealed an extremely rich repertoire, with evidence of horizontal gene acquisition from multiple bacterial lineages. Experimental analysis indicated that strain C1A is a remarkable biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple untreated grasses and crop residues examined, with the process significantly enhanced by mild pretreatments. This capability, acquired during its separate evolutionary trajectory in the rumen, along with its resilience and invasiveness compared to prokaryotic anaerobes, renders anaerobic fungi promising agents for consolidated bioprocessing schemes in biofuels production.

Transcriptomic analysis of lignocellulosic biomass degradation by the anaerobic fungal isolate Orpinomyces sp. strain C1A.

BACKGROUND: Anaerobic fungi reside in the rumen and alimentary tract of herbivores where they play an important role in the digestion of ingested plant biomass. The anaerobic fungal isolate Orpinomyces sp. strain C1A is an efficient biomass degrader, capable of simultaneous saccharification and fermentation of the cellulosic and hemicellulosic fractions in multiple types of lignocellulosic biomass. To understand the mechanistic and regulatory basis of biomass deconstruction in anaerobic fungi, we analyzed the transcriptomic profiles of C1A when grown on four different types of lignocellulosic biomass (alfalfa, energy cane, corn stover, and sorghum) versus a soluble sugar monomer (glucose). RESULTS: A total of 468.2 million reads (70.2 Gb) were generated and assembled into 27,506 distinct transcripts. CAZyme transcripts identified included 385, 246, and 44 transcripts belonging to 44, 13, and 8 different glycoside hydrolases (GH), carbohydrate esterases, and polysaccharide lyases families, respectively. Examination of CAZyme transcriptional patterns indicates that strain C1A constitutively transcribes a high baseline level of CAZyme transcripts on glucose. Although growth on lignocellulosic biomass substrates was associated with a significant increase in transcriptional levels in few GH families, including the highly transcribed GH1 ß-glucosidase, GH6 cellobiohydrolase, and GH9 endoglucanase, the transcriptional levels of the majority of CAZyme families and transcripts were not significantly altered in glucose-grown versus lignocellulosic biomass-grown cultures. Further, strain C1A co-transcribes multiple functionally redundant enzymes for cellulose and hemicellulose saccharification that are mechanistically and structurally distinct. Analysis of fungal dockerin domain-containing transcripts strongly suggests that anaerobic fungal cellulosomes represent distinct catalytic units capable of independently attacking and converting intact plant fibers to sugar monomers. CONCLUSIONS: Collectively, these results demonstrate that strain C1A achieves fast, effective biomass degradation by the simultaneous employment of a wide array of constitutively-transcribed cellulosome-bound and free enzymes with considerable functional overlap. We argue that the utilization of this indiscriminate strategy could be justified by the evolutionary history of anaerobic fungi, as well as their functional role within their natural habitat in the herbivorous gut.

Evaluating the utility of hydrothermolysis pretreatment approaches in enhancing lignocellulosic biomass degradation by the anaerobic fungus Orpinomyces sp. strain C1A.

Members of the anaerobic fungi (Phylum Neocallimastigomycota) are efficient biomass degraders and represent promising agents for fuel and chemical production from lignocellulosic biomass. Pretreatment of lignocellulosic biomass is considered an unavoidable first step in enzyme-based saccharification schemes, but its necessity in any proposed anaerobic fungi-based schemes is still unclear. Here, we evaluated the effect of hydrothermal pretreatments on the extent of corn stover and switchgrass degradation by an anaerobic fungal isolate, Orpinomyces sp. strain C1A. Using a factorial experimental design, we evaluated the effect of three different temperatures (180, 190, and 200°C) and three hold times (5, 10, and 15min). Pretreated corn stover and switchgrass were more amenable to degradation by strain C1A when compared to untreated biomass, as evident by the higher proportion of plant biomass degraded compared to untreated controls. However, when factoring in the proportion of biomass lost during the pretreatment process (ranging between 25.78 and 58.92% in corn stover and 28.34 and 38.22% in switchgrass), hydrothermolysis provided negligible or negative improvements to the extent of corn stover and switchgrass degradation by strain C1A. Product analysis demonstrated a shift towards higher ethanol and lactate production and lower acetate production associated with increase in pretreatment severity, especially in switchgrass incubations. The results are in stark contrast to the requirement of pretreatment in enzyme-based schemes for biomass saccharification, and their implications on the potential utility of anaerobic fungi in biofuel and biochemical production are discussed.

Survival of the anaerobic fungus Orpinomyces sp. strain C1A after prolonged air exposure.

Anaerobic fungi are efficient plant biomass degraders and represent promising agents for a variety of biotechnological applications. We evaluated the tolerance of an anaerobic fungal isolate, Orpinomyces sp. strain C1A, to air exposure in liquid media using soluble (cellobiose) and insoluble (dried switchgrass) substrates. Strain C1A grown on cellobiose survived for 11, and 13.5 hours following air exposure when grown under planktonic, and immobilized conditions, respectively. When grown on switchgrass media, strain C1A exhibited significantly enhanced air tolerance and survived for 168 hours. The genome of strain C1A lacked a catalase gene, but contained superoxide dismutase and glutathione peroxidase genes. Real time PCR analysis indicated that superoxide dismutase, but not glutathione peroxidase, exhibits a transient increase in expression level post aeration. Interestingly, the C1A superoxide dismutase gene of strain C1A appears to be most closely related to bacterial SODs, which implies its acquisition from a bacterial donor via cross kingdom horizontal gene transfer during Neocallimastigomycota evolution. We conclude that strain C1A utilizes multiple mechanisms to minimize the deleterious effects of air exposure such as physical protection and the production of oxidative stress enzymes.

Phylogenetic diversity and community structure of anaerobic gut fungi (phylum Neocallimastigomycota) in ruminant and non-ruminant herbivores.

The phylogenetic diversity and community structure of members of the gut anaerobic fungi (AF) (phylum Neocallimastigomycota) were investigated in 30 different herbivore species that belong to 10 different mammalian and reptilian families using the internal transcribed spacer region-1 (ITS-1) ribosomal RNA (rRNA) region as a phylogenetic marker. A total of 267 287 sequences representing all known anaerobic fungal genera were obtained in this study. Sequences affiliated with the genus Piromyces were the most abundant, being encountered in 28 different samples, and representing 36% of the sequences obtained. On the other hand, sequences affiliated with the genera Cyllamyces and Orpinomyces were the least abundant, being encountered in 2, and 8 samples, and representing 0.7%, and 1.1% of the total sequences obtained, respectively. Further, 38.3% of the sequences obtained did not cluster with previously identified genera and formed eight phylogenetically distinct novel anaerobic fungal lineages. Some of these novel lineages were widely distributed (for example NG1 and NG3), whereas others were animal specific, being encountered in only one or two animals (for example NG4, NG6, NG7, and NG8). The impact of various physiological and environmental factors on the diversity and community structure of AF was examined. The results suggest that animal host phylogeny exerts the most significant role on shaping anaerobic fungal diversity and community composition. These results greatly expand the documented global phylogenetic diversity of members of this poorly studied group of fungi that has an important function in initiating plant fiber degradation during fermentative digestion in ruminant and non-ruminant herbivores.

Biodegradation of vinyl chloride and cis-dichloroethene by a Ralstonia sp. strain TRW-1.

An aerobic bacterium, Ralstonia sp. strain TRW-1, that assimilates vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. Phylogenetic analysis of 16S rDNA sequence of the isolate revealed almost 99% sequence similarity to Ralstonia pickettii. To our knowledge, this is the first report describing the isolation of a member of Ralstonia that can degrade VC as the growth substrate. The measured growth yield values for VC and ETH were 11.27 and 18.90 g protein/mole, respectively. The estimated half-velocity constant K (m) values for VC and ETH were 9.09+/-2.97 and 5.73+/-2.96 muM, respectively. These values are almost three- to tenfold higher than for other VC-assimilating Mycobacterium sp. The strain also degrades cis-dichloroethene (cis-DCE) in mineral salts medium containing yeast-extract, beef-extract, casamino acids, or peptone. This ability of the strain TRW-1 to degrade cis-DCE in the presence of a nontoxic, water-soluble substrate is relevant to in-situ remediation of cis-DCE-contaminated aquifers.