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 B-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.Peer reviewedMicrobiology and Molecular Genetic

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.Peer reviewedMicrobiology and Molecular GeneticsBiosystems and Agricultural Engineerin

Pecoramyces ruminantium, gen. no., 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 ?m, 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).Peer reviewe

Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi

Six DNA regions were evaluated as potential DNA barcodes for Fungi, the second largest kingdom of eukaryotic life, by a multinational, multilaboratory consortium. The region of the mitochondrial cytochrome c oxidase subunit 1 used as the animal barcode was excluded as a potential marker, because it is difficult to amplify in fungi, often includes large introns, and can be insufficiently variable. Three subunits from the nuclear ribosomal RNA cistron were compared together with regions of three representative protein-coding genes (largest subunit of RNA polymerase II, second largest subunit of RNA polymerase II, and minichromosome maintenance protein). Although the protein-coding gene regions often had a higher percent of correct identification compared with ribosomal markers, low PCR amplification and sequencing success eliminated them as candidates for a universal fungal barcode. Among the regions of the ribosomal cistron, the internal transcribed spacer (ITS) region has the highest probability of successful identification for the broadest range of fungi, with the most clearly defined barcode gap between inter- and intraspecific variation. The nuclear ribosomal large subunit, a popular phylogenetic marker in certain groups, had superior species resolution in some taxonomic groups, such as the early diverging lineages and the ascomycete yeasts, but was otherwise slightly inferior to the ITS. The nuclear ribosomal small subunit has poor species-level resolution in fungi. ITS will be formally proposed for adoption as the primary fungal barcode marker to the Consortium for the Barcode of Life, with the possibility that supplementary barcodes may be developed for particular narrowly circumscribed taxonomic groups