[Enzyme production mechanism of anaerobic fungus Orpinomyces sp. YF3 in yak rumen induced by different carbon source].

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, ß-glucan metabolic process, cellulase activity, endo-1,4-ß-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.

Enzyme production mechanism of anaerobic fungus Orpinomyces sp. YF3 in yak rumen induced by different carbon source / 生物工程学报

In order to investigate the enzyme production mechanism of yak rumen-derived anaerobic fungus Orpinomyces sp. YF3 under the induction of different carbon sources, anaerobic culture tubes were used for in vitro fermentation. 8 g/L of glucose (Glu), filter paper (Flp) and avicel (Avi) were respectively added to 10 mL of basic culture medium as the sole carbon source. The activity of fiber-degrading enzyme and the concentration of volatile fatty acid in the fermentation liquid were detected, and the enzyme producing mechanism of Orpinomyces sp. YF3 was explored by transcriptomics. It was found that, in glucose-induced fermentation solution, the activities of carboxymethyl cellulase, microcrystalline cellulase, filter paper enzyme, xylanase and the proportion of acetate were significantly increased (P < 0.05), the proportion of propionate, butyrate, isobutyrate were significantly decreased (P < 0.05). The results of transcriptome analysis showed that there were 5 949 differentially expressed genes (DEGs) between the Glu group and the Flp group, 10 970 DEGs between the Glu group and the Avi group, and 6 057 DEGs between the Flp group and the Avi group. It was found that the DEGs associated with fiber degrading enzymes were significantly up-regulated in the Glu group. Gene ontology (GO) function enrichment analysis identified that DEGs were mainly associated with the xylan catabolic process, hemicellulose metabolic process, β-glucan metabolic process, cellulase activity, endo-1,4-β-xylanase activity, cell wall polysaccharide metabolic process, carbohydrate catabolic process, glucan catabolic process and carbohydrate metabolic process. Moreover, the differentially expressed pathways associated with fiber degrading enzymes enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were mainly starch and sucrose metabolic pathways and other glycan degradation pathways. In conclusion, Orpinomyces sp. YF3 with glucose as carbon source substrate significantly increased the activity of cellulose degrading enzyme and the proportion of acetate, decreased the proportion of propionate, butyrate and isobutyrate. Furthermore, the degradation ability and energy utilization efficiency of fungus in the presence of glucose were improved by means of regulating the expression of cellulose degrading enzyme gene and participating in starch and sucrose metabolism pathway, and other glycan degradation pathways, which provides a theoretical basis for the application of Orpinomyces sp. YF3 in practical production and facilitates the application of Orpinomyces sp. YF3 in the future.

Beef cattle that respond differently to fescue toxicosis have distinct gastrointestinal tract microbiota.

Tall fescue (Lolium arundinaceum) is a widely used forage grass which shares a symbiosis with the endophytic fungus Epichloë coenophiala. The endophyte produces an alkaloid toxin that provides herbivory, heat and drought resistance to the grass, but can cause fescue toxicosis in grazing livestock. Fescue toxicosis can lead to reduced weight gain and milk yields resulting in significant losses to the livestock industry. The objective of this study was to identify bacterial and fungal communities associated with fescue toxicosis tolerance. In this trial, 149 Angus cows across two farms were continuously exposed to toxic, endophyte-infected, fescue for a total of 13 weeks. Of those 149 cows, 40 were classified into either high (HT) or low (LT) tolerance groups according to their growth performance (weight gain). 20 HT and 20 LT cattle balanced by farm were selected for amplicon sequencing to compare the fecal microbiota of the two tolerance groups. This study reveals significantly (q<0.05) different bacterial and fungal microbiota between HT and LT cattle, and indicates that fungal phylotypes may be important for an animal's response to fescue toxicosis: We found that fungal phylotypes affiliating to the Neocallimastigaceae, which are known to be important fiber-degrading fungi, were consistently more abundant in the HT cattle. Whereas fungal phylotypes related to the genus Thelebolus were more abundant in the LT cattle. This study also found more pronounced shifts in the microbiota in animals receiving higher amounts of the toxin. We identified fungal phylotypes which were consistently more abundant either in HT or LT cattle and may thus be associated with the respective animal's response to fescue toxicosis. Our results thus suggest that some fungal phylotypes might be involved in mitigating fescue toxicosis.

Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficiency of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15-33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

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.

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.

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.

Responses of anaerobic rumen fungal diversity (phylum Neocallimastigomycota) to changes in bovine diet.

AIMS: Anaerobic rumen fungi (Neocallimastigales) play important roles in the breakdown of complex, cellulose-rich material. Subsequent decomposition products are utilized by other microbes, including methanogens. The aim of this study was to determine the effects of dietary changes on anaerobic rumen fungi diversity. METHODS AND RESULTS: Altered diets through increasing concentrate/forage (50 : 50 vs 90 : 10) ratios and/or the addition of 6% soya oil were offered to steers and the Neocallimastigales community was assessed by PCR-based fingerprinting with specific primers within the barcode region. Both a decrease in fibre content and the addition of 6% soya oil affected Neocallimastigales diversity within solid and liquid rumen phases. The addition of 6% soya oil decreased species richness. Assemblages were strongly affected by the addition of 6% soya oil, whereas unexpectedly, the fibre decrease had less effect. Differences in volatile fatty acid contents (acetate, propionate and butyrate) were significantly associated with changes in Neocallimastigales assemblages between the treatments. CONCLUSIONS: Diet clearly influences Neocallimastigales assemblages. The data are interpreted in terms of interactions with other microbial groups involved in fermentation processes within the rumen. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge on the influence of diet on anaerobic fungi is necessary to understand changes in microbial processes occurring within the rumen as this may impact on other rumen processes such as methane production.

Degradation of lignified secondary cell walls of lucerne (Medicago sativa L.) by rumen fungi growing in methanogenic co-culture.

AIMS: To compare the abilities of the monocentric rumen fungi Neocallimastix frontalis, Piromyces communis and Caecomyces communis, growing in coculture with Methanobrevibacter smithii, to colonize and degrade lignified secondary cell walls of lucerne (alfalfa) hay. METHODS AND RESULTS: The cell walls of xylem cylinders isolated from stems of lucerne contained mostly xylans, cellulose and lignin together with a small proportion of pectic polysaccharides. All of these major components were removed during incubation with the three fungi, and differing cell wall polysaccharides were degraded to different extents. The greatest dry weight loss was found with N. frontalis and least with C. communis, and scanning electron microscopy revealed that these extensively colonized different cell types. C. communis specifically colonized secondary xylem fibres and showed much less degradation than N. frontalis and P. communis. CONCLUSIONS: Neocallimastix frontalis and P. communis were efficient degraders of the cell walls of lucerne xylem cylinders. Degradation occurred of pectic polysaccharides, xylan and cellulose. Loss of lignin from the xylem cylinders probably resulted from the cleavage of xylan releasing xylan-lignin complexes. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike rumen bacteria, the rumen fungi N. frontalis, P. communis and C. communis are able to degrade lignified secondary walls in lucerne stems. These fungi could improve forage utilization by ruminants and may have potential in the degradation of lignocellulosic biomass in the production of biofuels.

Polysaccharidase and glycosidase production of avicel grown rumen fungus Orpinomyces sp. GMLF5.

Extracellular and cell-associated enzyme preparations were obtained from ruminal anaerobic fungi Orpinomyces sp. GMLF5 grown in culture containing microcrystalline cellulose (avicel) as sole energy source and degradation capacities of the preparations towards several polysaccharides and glycosides were studied. Fungus showed substantial increases in xylanase, carboxymethyl cellulase (CMCase), lichenase, amylase, beta-xylosidase, beta-glucosidase and alpha-L-arabinofuranosidase activities between 72 and 168 hours. High amounts of cell associated beta-xylosidase were noted in 4 and 5 days old cultures. Optimum temperature and pH of the polysaccharidases were found at 50 degrees C and 6.0-6.5, respectively. Xylanase was found to be virtually stable at 50 degrees C, CMCase and lichenase were stable at 40 degrees C for 200 min, however amylase was found more sensitive to heat treatment. The fibrolytic enzymes of the isolate GMLF5 were observed to be capable of hydrolyze the avicel.

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria.

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.

Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2.

An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 beta-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).

In vitro degradation of wheat straw by anaerobic fungi from small ruminants.

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.

In vitro degradation of cell-wall and digestibility of cereal straws treated with anaerobic ruminal fungi.

Ruminal fungal isolates (Orpinomyces sp.; C-14, Piromyces sp.; C-15, Orpinomyces sp.; B-13 and Anaeromyces sp.; B-6), were evaluated under anoxic conditions for their effect on in vitro dry matter digestibility, neutral detergent fibre, acid detergent fibre and acid detergent lignin using rice and wheat straw as substrate. There was no significant effect of the fungal isolates on the disappearance of the substrates along with rumen liquor when compared to control. The doses of 10(6) cfu/ml of the isolate were found to have maximum degradation of straws in comparison to the doses of 10(3) cfu/ml.

Classical and molecular approaches as a powerful tool for the characterization of rumen polycentric fungi.

Ribosomal ITS1 and ITS2 fragments from 8 isolates of polycentric rumen anaerobic fungi were PCR-amplified and sequenced; the sequences obtained were aligned with published data and phylogenetic analyses were performed. Analysis of the ITS1 fragment clearly differentiated between the two polycentric genera Orpinomyces and Anaeromyces and this classification is supported by morphological observation. A multi-order phylogram based on ITS2 sequences proved that anaerobic rumen fungi are separated from aerobic chytrids, which form a well-supported monophylum with the highest possible bootstrap proportion values of 100%. Sequence analysis of ITS regions is a powerful tool for classification of anaerobic fungi but morphological description of strains is still necessary because some genera of rumen fungi display a high genetic heterogeneity.

Properties of a recombinant beta-glucosidase from polycentric anaerobic fungus Orpinomyces PC-2 and its application for cellulose hydrolysis.

A beta-glucosidase (BglA, EC 3.2.1.21) gene from the polycentric anaerobic fungus Orpinomyces PC-2 was cloned and sequenced. The enzyme containing 657 amino acid residues was homologous to certain animal, plant, and bacterial beta-glucosidases but lacked significant similarity to those from aerobic fungi. Neither cellulose- nor protein-binding domains were found in BglA. When expressed in Saccharomyces cerevisiae, the enzyme was secreted in two forms with masses of about 110 kDa and also found in two forms associated with the yeast cells. Km and Vmax values of the secreted BglA were 0.762 mM and 8.20 micromol/(min x mg), respectively, with p-nitrophenyl-beta-D-glucopyranoside (pNPG) as the substrate and 0.310 mM and 6.45 micromol/(min.mg), respectively, for the hydrolysis of cellobiose. Glucose competitively inhibited the hydrolysis of pNPG with a Ki of 3.6 mM. Beta-glucosidase significantly enhanced the conversion of cellulosic materials into glucose by Trichoderma reesei cellulase preparations, demonstrating its potential for use in biofuel and feedstock chemical production.

Production of beta-glucosidase using immobilised Piromyces sp. KSX1 and Orpinomyces sp. 478P1 in repeat-batch culture.

Two anaerobic fungi, one a monocentric strain ( Piromyces sp. KSX1) and the other a polycentric strain ( Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. beta-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, beta-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more beta-glucosidase than strain 478P1, the maximum specific beta-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor.