Brucella pituitosa strain BU72, a new hydrocarbonoclastic bacterium through exopolysaccharide-based surfactant production.

Hydrocarbon and heavy metal pollution are amongst the most severe and prevalent environmental problems due to their toxicity and persistence. Bioremediation using microorganisms is considered one of the most effective ways to treat polluted sites. In the present study, we unveil the bioremediation potential of Brucella pituitosa strain BU72. Besides its ability to grow on multiple hydrocarbons as the sole carbon source and highly tolerant to several heavy metals, BU72 produces different exopolysaccharide-based surfactants (EBS) when grown with glucose or with crude oil as sole carbon source. These EBS demonstrated particular and specific functional groups as determined by Fourier transform infrared (FTIR) spectral analysis that showed a strong absorption peak at 3250 cm-1 generated by the -OH group for both EBS. The FTIR spectra of the produced EBS revealed major differences in functional groups and protein content. To better understand the EBS production coupled with the degradation of hydrocarbons and heavy metal resistance, the genome of strain BU72 was sequenced. Annotation of the genome revealed multiple genes putatively involved in EBS production pathways coupled with resistance to heavy metals genes such as arsenic tolerance and cobalt-zinc-cadmium resistance. The genome sequence analysis showed the potential of BU72 to synthesise secondary metabolites and the presence of genes involved in plant growth promotion. Here, we describe the physiological, metabolic, and genomic characteristics of Brucella pituitosa strain BU72, indicating its potential as a bioremediation agent.

The effects of synthesis gas feedstocks and oxygen perturbation on hydrogen production by Parageobacillus thermoglucosidasius.

BACKGROUND: The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen gas (H2) through the water-gas shift (WGS) reaction. To date this process has been evaluated under controlled conditions, with gas feedstocks comprising carbon monoxide and variable proportions of air, nitrogen and hydrogen. Ultimately, an economically viable hydrogenogenic system would make use of industrial waste/synthesis gases that contain high levels of carbon monoxide, but which may also contain contaminants such as H2, oxygen (O2) and other impurities, which may be toxic to P. thermoglucosidasius. RESULTS: We evaluated the effects of synthesis gas (syngas) mimetic feedstocks on WGS reaction-driven H2 gas production by P. thermoglucosidasius DSM 6285 in small-scale fermentations. Improved H2 gas production yields and faster onset towards hydrogen production were observed when anaerobic synthetic syngas feedstocks were used, at the expense of biomass accumulation. Furthermore, as the WGS reaction is an anoxygenic process, we evaluated the influence of O2 perturbation on P. thermoglucosidasius hydrogenogenesis. O2 supplementation improved biomass accumulation, but reduced hydrogen yields in accordance with the level of oxygen supplied. However, H2 gas production was observed at low O2 levels. Supplementation also induced rapid acetate consumption, likely to sustain growth. CONCLUSION: The utilisation of anaerobic syngas mimetic gas feedstocks to produce H2 and the relative flexibility of the P. thermoglucosidasius WGS reaction system following O2 perturbation further supports its applicability towards more robust and continuous hydrogenogenic operation.

Time-Course Transcriptome of Parageobacillus thermoglucosidasius DSM 6285 Grown in the Presence of Carbon Monoxide and Air.

Parageobacillus thermoglucosidasius is a metabolically versatile, facultatively anaerobic thermophile belonging to the family Bacillaceae. Previous studies have shown that this bacterium harbours co-localised genes coding for a carbon monoxide (CO) dehydrogenase (CODH) and Ni-Fe hydrogenase (Phc) complex and oxidises CO and produces hydrogen (H2) gas via the water-gas shift (WGS) reaction. To elucidate the genetic events culminating in the WGS reaction, P. thermoglucosidasius DSM 6285 was cultivated under an initial gas atmosphere of 50% CO and 50% air and total RNA was extracted at ~8 (aerobic phase), 20 (anaerobic phase), 27 and 44 (early and late hydrogenogenic phases) hours post inoculation. The rRNA-depleted fraction was sequenced using Illumina NextSeq, v2.5, 1x75bp chemistry. Differential expression revealed that at 8 vs 20, 20 vs 27 and 27 vs 44 hours post inoculation, 2190, 2118 and 231 transcripts were differentially (FDR < 0.05) expressed. Cluster analysis revealed 26 distinct gene expression trajectories across the four time points. Of these, two similar clusters, showing overexpression at 20 relative to 8 hours and depletion at 27 and 44 hours, harboured the CODH and Phc transcripts, suggesting possible regulation by O2. The transition between aerobic respiration and anaerobic growth was marked by initial metabolic deterioration, as reflected by up-regulation of transcripts linked to sporulation and down-regulation of transcripts linked to flagellar assembly and metabolism. However, the transcriptome and growth profiles revealed the reversal of this trend during the hydrogenogenic phase.

Comparative genomic analysis of Parageobacillus thermoglucosidasius strains with distinct hydrogenogenic capacities.

BACKGROUND: The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius produces hydrogen gas (H2) by coupling CO oxidation to proton reduction in the water-gas shift (WGS) reaction via a carbon monoxide dehydrogenase-hydrogenase enzyme complex. Although little is known about the hydrogenogenic capacities of different strains of this species, these organisms offer a potentially viable process for the synthesis of this alternative energy source. RESULTS: The WGS-catalyzed H2 production capacities of four distinct P. thermoglucosidasius strains were determined by cultivation and gas analysis. Three strains (DSM 2542T, DSM 2543 and DSM 6285) were hydrogenogenic, while the fourth strain (DSM 21625) was not. Furthermore, in one strain (DSM 6285) H2 production commenced earlier in the cultivation than the other hydrogenogenic strains. Comparative genomic analysis of the four strains identified extensive differences in the protein complement encoded on the genomes, some of which are postulated to contribute to the different hydrogenogenic capacities of the strains. Furthermore, polymorphisms and deletions in the CODH-NiFe hydrogenase loci may also contribute towards this variable phenotype. CONCLUSIONS: Disparities in the hydrogenogenic capacities of different P. thermoglucosidasius strains were identified, which may be correlated to variability in their global proteomes and genetic differences in their CODH-NiFe hydrogenase loci. The data from this study may contribute towards an improved understanding of WGS-catalysed hydrogenogenesis by P. thermoglucosidasius.

LEA Proteins and the Evolution of the WHy Domain.

The late embryogenesis abundant (LEA) family is composed of a diverse collection of multidomain and multifunctional proteins found in all three domains of the tree of life, but they are particularly common in plants. Most members of the family are known to play an important role in abiotic stress response and stress tolerance in plants but are also part of the plant hypersensitive response to pathogen infection. The mechanistic basis for LEA protein functionality is still poorly understood. The group of LEA 2 proteins harbor one or more copies of a unique domain, the water stress and hypersensitive response (WHy) domain. This domain sequence has recently been identified as a unique open reading frame (ORF) in some bacterial genomes (mostly in the phylum Firmicutes), and the recombinant bacterial WHy protein has been shown to exhibit a stress tolerance phenotype in Escherichia coli and an in vitro protein denaturation protective function. Multidomain phylogenetic analyses suggest that the WHy protein gene sequence may have ancestral origins in the domain Archaea, with subsequent acquisition in Bacteria and eukaryotes via endosymbiont or horizontal gene transfer mechanisms. Here, we review the structure, function, and nomenclature of LEA proteins, with a focus on the WHy domain as an integral component of the LEA constructs and as an independent protein.

In silico characterization of the global Geobacillus and Parageobacillus secretome.

BACKGROUND: Geobacillus and Parageobacillus are two ecologically diverse thermophilic genera within the phylum Firmicutes. These taxa have long been of biotechnological interest due to their ability to secrete thermostable enzymes and other biomolecules that have direct applications in various industrial and clinical fields. Despite the commercial and industrial interest in these microorganisms, the full scope of the secreted protein, i.e. the secretome, of Geobacillus and Parageobacillus species remains largely unexplored, with most studies focusing on single enzymes. A genome-wide exploration of the global secretome can provide a platform for understanding the extracellular functional "protein cloud" and the roles that secreted proteins play in the survival and adaptation of these biotechnologically relevant organisms. RESULTS: In the present study, the global secretion profile of 64 Geobacillus and Parageobacillus strains, comprising 772 distinct proteins, was predicted using comparative genomic approaches. Thirty-one of these proteins are shared across all strains used in this study and function in cell-wall/membrane biogenesis as well as transport and metabolism of carbohydrates, amino acids and inorganic ions. An analysis of the clustering patterns of the secretomes of the 64 strains according to shared functional orthology revealed a correlation between the secreted profiles of different strains and their phylogeny, with Geobacillus and Parageobacillus species forming two distinct functional clades. CONCLUSIONS: The in silico characterization of the global secretome revealed a metabolically diverse set of secreted proteins, which include proteases, glycoside hydrolases, nutrient binding proteins and toxins.

CO-dependent hydrogen production by the facultative anaerobe Parageobacillus thermoglucosidasius.

BACKGROUND: The overreliance on dwindling fossil fuel reserves and the negative climatic effects of using such fuels are driving the development of new clean energy sources. One such alternative source is hydrogen (H2), which can be generated from renewable sources. Parageobacillus thermoglucosidasius is a facultative anaerobic thermophilic bacterium which is frequently isolated from high temperature environments including hot springs and compost. RESULTS: Comparative genomics performed in the present study showed that P. thermoglucosidasius encodes two evolutionary distinct H2-uptake [Ni-Fe]-hydrogenases and one H2-evolving hydrogenases. In addition, genes encoding an anaerobic CO dehydrogenase (CODH) are co-localized with genes encoding a putative H2-evolving hydrogenase. The co-localized of CODH and uptake hydrogenase form an enzyme complex that might potentially be involved in catalyzing the water-gas shift reaction (CO + H2O → CO2 + H2) in P. thermoglucosidasius. Cultivation of P. thermoglucosidasius DSM 2542T with an initial gas atmosphere of 50% CO and 50% air showed it to be capable of growth at elevated CO concentrations (50%). Furthermore, GC analyses showed that it was capable of producing hydrogen at an equimolar conversion with a final yield of 1.08 H2/CO. CONCLUSIONS: This study highlights the potential of the facultative anaerobic P. thermoglucosidasius DSM 2542T for developing new strategies for the biohydrogen production.

RNA-Seq Based Transcriptome Analysis of Aspergillus oryzae DSM 1863 Grown on Glucose, Acetate and an Aqueous Condensate from the Fast Pyrolysis of Wheat Straw.

Due to its acetate content, the pyrolytic aqueous condensate (PAC) formed during the fast pyrolysis of wheat straw could provide an inexpensive substrate for microbial fermentation. However, PAC also contains several inhibitors that make its detoxification inevitable. In our study, we examined the transcriptional response of Aspergillus oryzae to cultivation on 20% detoxified PAC, pure acetate and glucose using RNA-seq analysis. Functional enrichment analysis of 3463 significantly differentially expressed (log2FC >2 & FDR < 0.05) genes revealed similar metabolic tendencies for both acetate and PAC, as upregulated genes in these cultures were mainly associated with ribosomes and RNA processing, whereas transmembrane transport was downregulated. Unsurprisingly, metabolic pathway analysis revealed that glycolysis/gluconeogenesis and starch and sucrose metabolism were upregulated for glucose, whereas glyoxylate and the tricarboxylic acid (TCA) cycle were important carbon utilization pathways for acetate and PAC, respectively. Moreover, genes involved in the biosynthesis of various amino acids such as arginine, serine, cysteine and tryptophan showed higher expression in the acetate-containing cultures. Direct comparison of the transcriptome profiles of acetate and PAC revealed that pyruvate metabolism was the only significantly different metabolic pathway and was overexpressed in the PAC cultures. Upregulated genes included those for methylglyoxal degradation and alcohol dehydrogenases, which thus represent potential targets for the further improvement of fungal PAC tolerance.

Origin and Evolution of Enzymes with MIO Prosthetic Group: Microbial Coevolution After the Mass Extinction Event.

After major mass extinction events, ancient plants and terrestrial vertebrates were faced with various challenges, especially ultraviolet (UV) light. These stresses probably resulted in changes in the biosynthetic pathways, which employed the MIO (3,5-dihydro-5-methylidene-4H-imidazole-4-one)-dependent enzymes (ammonia-lyase and aminomutase), leading to enhanced accumulation of metabolites for defense against UV radiation, pathogens, and microorganisms. Up to now, the origin and evolution of genes from this superfamily have not been extensively studied. In this report, we perform an analysis of the phylogenetic relations between the members of the aromatic amino acid MIO-dependent enzymes (AAM), which demonstrate that they most probably have a common evolutionary origin from ancient bacteria. In early soil environments, numerous bacterial species with tyrosine ammonia-lyase genes (TAL; EC 4.3.1.23) developed tyrosine aminomutase (TAM; EC 5.4.3.6) activity as a side reaction for competing with their neighbors in the community. These genes also evolved into other TAL-like enzymes, such as histidine ammonia-lyase (HAL, EC 4.3.1.3) and phenylalanine ammonia-lyase (PAL; EC 4.3.1.24), in different bacterial species for metabolite production and accumulation for adaptation to adverse terrestrial environmental conditions. On the other hand, the existence of phenylalanine aminomutase (PAM; EC 5.4.3.10) and phenylalanine/tyrosine ammonia-lyase (PTAL; EC 4.3.1.25) strongly indicates the horizontal gene transfer (HGT) between bacteria, fungi, and plants in symbiotic association after acquiring the PAL gene from their ancestor.

Metabolic shift towards increased biohydrogen production during dark fermentation in the anaerobic fungus Neocallimastix cameroonii G341.

BACKGROUND: Anaerobic fungi of the phylum Neocallimastigomycota have a high biotechnological potential due to their robust lignocellulose degrading capabilities and the production of several valuable metabolites like hydrogen, acetate, formate, lactate, and ethanol. The metabolism of these fungi, however, remains poorly understood due to limitations of the current cultivation strategies in still-standing bottles, thereby restricting the comprehensive evaluation of cultivation conditions. RESULTS: We describe the analysis of growth conditions and their influence on the metabolism of the previously isolated fungus Neocallimastix cameroonii G341. We established a bioreactor process in a stirred tank, enabling cultivation under defined conditions. The optimal growth temperature for the fungus was between 38.5 °C and 41.5 °C, while the optimal pH was 6.6-6.8. Like other dark fermentation systems, hydrogen production is dependent on the hydrogen partial pressure and pH. Shaking the bottles or stirring the fermenters led to an increase in hydrogen and a decrease in lactate and ethanol production. Regulation of the pH to 6.8 in the fermenter nearly doubled the amount of produced hydrogen. CONCLUSIONS: Novel insights into the metabolism of Neocallimastix cameroonii were gained, with hydrogen being the preferred way of electron disposal over lactate and ethanol. In addition, our study highlights the potential application of the fungus for hydrogen production from un-pretreated biomass. Finally, we established the first cultivation of an anaerobic fungus in a stirred tank reactor system.

Bacillales: From Taxonomy to Biotechnological and Industrial Perspectives.

For a long time, the genus Bacillus has been known and considered among the most applicable genera in several fields. Recent taxonomical developments resulted in the identification of more species in Bacillus-related genera, particularly in the order Bacillales (earlier heterotypic synonym: Caryophanales), with potential application for biotechnological and industrial purposes such as biofuels, bioactive agents, biopolymers, and enzymes. Therefore, a thorough understanding of the taxonomy, growth requirements and physiology, genomics, and metabolic pathways in the highly diverse bacterial order, Bacillales, will facilitate a more robust designing and sustainable production of strain lines relevant to a circular economy. This paper is focused principally on less-known genera and their potential in the order Bacillales for promising applications in the industry and addresses the taxonomical complexities of this order. Moreover, it emphasizes the biotechnological usage of some engineered strains of the order Bacillales. The elucidation of novel taxa, their metabolic pathways, and growth conditions would make it possible to drive industrial processes toward an upgraded functionality based on the microbial nature.

Global Transcriptome Profile of the Oleaginous Yeast Saitozyma podzolica DSM 27192 Cultivated in Glucose and Xylose.

Unlike conventional yeasts, several oleaginous yeasts, including Saitozyma podzolica DSM 27192, possess the innate ability to grow and produce biochemicals from plant-derived lignocellulosic components such as hexose and pentose sugars. To elucidate the genetic basis of S. podzolica growth and lipid production on glucose and xylose, we performed comparative temporal transcriptome analysis using RNA-seq method. Approximately 3.4 and 22.2% of the 10,670 expressed genes were differentially (FDR < 0.05, and log2FC > 1.5) expressed under batch and fed batch modes, respectively. Our analysis revealed that a higher number of sugar transporter genes were significantly overrepresented in xylose relative to glucose-grown cultures. Given the low homology between proteins encoded by most of these genes and those of the well-characterised transporters, it is plausible to conclude that S. podzolica possesses a cache of putatively novel sugar transporters. The analysis also suggests that S. podzolica potentially channels carbon flux from xylose via both the non-oxidative pentose phosphate and potentially via the first steps of the Weimberg pathways to yield xylonic acid. However, only the ATP citrate lyase (ACL) gene showed significant upregulation among the essential oleaginous pathway genes under nitrogen limitation in xylose compared to glucose cultivation. Combined, these findings pave the way toward the design of strategies or the engineering of efficient biomass hydrolysate utilization in S. podzolica for the production of various biochemicals.

Not All That Glitters Is Gold: The Paradox of CO-dependent Hydrogenogenesis in Parageobacillus thermoglucosidasius.

The thermophilic bacterium Parageobacillus thermoglucosidasius has recently gained interest due to its ability to catalyze the water gas shift reaction, where the oxidation of carbon monoxide (CO) is linked to the evolution of hydrogen (H2) gas. This phenotype is largely predictable based on the presence of a genomic region coding for a carbon monoxide dehydrogenase (CODH-Coo) and hydrogen evolving hydrogenase (Phc). In this work, seven previously uncharacterized strains were cultivated under 50% CO and 50% air atmosphere. Despite the presence of the coo-phc genes in all seven strains, only one strain, Kp1013, oxidizes CO and yields H2. The genomes of the H2 producing strains contain unique genomic regions that code for proteins involved in nickel transport and the detoxification of catechol, a by-product of a siderophore-mediated iron acquisition system. Combined, the presence of these genomic regions could potentially drive biological water gas shift (WGS) reaction in P. thermoglucosidasius.

Carbon Monoxide Induced Metabolic Shift in the Carboxydotrophic Parageobacillus thermoglucosidasius DSM 6285.

Parageobacillus thermoglucosidasius is known to catalyse the biological water gas shift (WGS) reaction, a pathway that serves as a source of alternative energy and carbon to a wide variety of bacteria. Despite increasing interest in this bacterium due to its ability to produce biological hydrogen through carbon monoxide (CO) oxidation, there are no data on the effect of toxic CO gas on its physiology. Due to its general requirement of O2, the organism is often grown aerobically to generate biomass. Here, we show that carbon monoxide (CO) induces metabolic changes linked to distortion of redox balance, evidenced by increased accumulation of organic acids such as acetate and lactate. This suggests that P. thermoglucosidasius survives by expressing several alternative pathways, including conversion of pyruvate to lactate, which balances reducing equivalents (oxidation of NADH to NAD+), and acetyl-CoA to acetate, which directly generates energy, while CO is binding terminal oxidases. The data also revealed clearly that P. thermoglucosidasius gained energy and grew during the WGS reaction. Combined, the data provide critical information essential for further development of the biotechnological potential of P. thermoglucosidasius.

Characterization and Phylogenetic Analysis of a Novel GH43 ß-Xylosidase From Neocallimastix californiae.

Degradation of lignocellulosic materials to release fermentable mono- and disaccharides is a decisive step toward a sustainable bio-based economy, thereby increasing the demand of robust and highly active lignocellulolytic enzymes. Anaerobic fungi of the phylum Neocallimastigomycota are potent biomass degraders harboring a huge variety of such enzymes. Compared to cellulose, hemicellulose degradation has received much less attention; therefore, the focus of this study has been the enzymatic xylan degradation of anaerobic fungi as these organisms produce some of the most effective known hydrolytic enzymes. We report the heterologous expression of a GH43 xylosidase, Xyl43Nc, and a GH11 endoxylanase, X11Nc, from the anaerobic fungus Neocallimastix californiae in Escherichia coli. The enzymes were identified by screening of the putative proteome. Xyl43Nc was highly active against 4-Nitrophenol-xylopyranosides with a Km of 0.72 mM, a kcat of 29.28 s-1, a temperature optimum of 32°C and a pH optimum of 6. When combined, Xyl43Nc and X11Nc released xylose from beechwood xylan and arabinoxylan from wheat. Phylogenetic analysis revealed that Xyl43Nc shares common ancestry with enzymes from Spirochaetes and groups separately from Ascomycete sequences in our phylogeny, highlighting the importance of horizontal gene transfer in the evolution of the anaerobic fungi.

Isolation and Biochemical Characterization of Six Anaerobic Fungal Strains from Zoo Animal Feces.

Anaerobic fungi are prime candidates for the conversion of agricultural waste products to biofuels. Despite the increasing interest in these organisms, their growth requirements and metabolism remain largely unknown. The isolation of five strains of anaerobic fungi and their identification as Neocallimastix cameroonii, Caecomyces spec., Orpinomyces joyonii, Pecoramyces ruminantium, and Khoyollomyces ramosus, is described. The phylogeny supports the reassignment of Neocallimastix californiae and Neocallimastix lanati to Neocallimastix cameroonii and points towards the redesignation of Cyllamyces as a species of Caecomyces. All isolated strains including strain A252, which was described previously as Aestipascuomyces dubliciliberans, were further grown on different carbon sources and the produced metabolites were analyzed; hydrogen, acetate, formate, lactate, and succinate were the main products. Orpinomyces joyonii was lacking succinate production and Khoyollomyces ramosus was not able to produce lactate under the studied conditions. The results further suggested a sequential production of metabolites with a preference for hydrogen, acetate, and formate. By comparing fungal growth on monosaccharides or on the straw, a higher hydrogen production was noticed on the latter. Possible reactions to elevated sugar concentrations by anaerobic fungi are discussed.

Genomic characterization of a polyvalent hydrocarbonoclastic bacterium Pseudomonas sp. strain BUN14.

Bioremediation offers a viable alternative for the reduction of contaminants from the environment, particularly petroleum and its recalcitrant derivatives. In this study, the ability of a strain of Pseudomonas BUN14 to degrade crude oil, pristane and dioxin compounds, and to produce biosurfactants, was investigated. BUN14 is a halotolerant strain isolated from polluted sediment recovered from the refinery harbor on the Bizerte coast, north Tunisia and capable of producing surfactants. The strain BUN14 was assembled into 22 contigs of 4,898,053 bp with a mean GC content of 62.4%. Whole genome phylogeny and comparative genome analyses showed that strain BUN14 could be affiliated with two validly described Pseudomonas Type Strains, P. kunmingensis DSM 25974T and P. chloritidismutans AW-1T. The current study, however, revealed that the two Type Strains are probably conspecific and, given the priority of the latter, we proposed that P. kunmingensis DSM 25974 is a heteronym of P. chloritidismutans AW-1T. Using GC-FID analysis, we determined that BUN14 was able to use a range of hydrocarbons (crude oil, pristane, dibenzofuran, dibenzothiophene, naphthalene) as a sole carbon source. Genome analysis of BUN14 revealed the presence of a large repertoire of proteins (154) related to xenobiotic biodegradation and metabolism. Thus, 44 proteins were linked to the pathways for complete degradation of benzoate and naphthalene. The annotation of conserved functional domains led to the detection of putative genes encoding enzymes of the rhamnolipid biosynthesis pathway. Overall, the polyvalent hydrocarbon degradation capacity of BUN14 makes it a promising candidate for application in the bioremediation of polluted saline environments.

In silico Proteomic Analysis Provides Insights Into Phylogenomics and Plant Biomass Deconstruction Potentials of the Tremelalles.

Basidiomycetes populate a wide range of ecological niches but unlike ascomycetes, their capabilities to decay plant polymers and their potential for biotechnological approaches receive less attention. Particularly, identification and isolation of CAZymes is of biotechnological relevance and has the potential to improve the cache of currently available commercial enzyme cocktails toward enhanced plant biomass utilization. The order Tremellales comprises phylogenetically diverse fungi living as human pathogens, mycoparasites, saprophytes or associated with insects. Here, we have employed comparative genomics approaches to highlight the phylogenomic relationships among thirty-five Tremellales and to identify putative enzymes of biotechnological interest encoded on their genomes. Evaluation of the predicted proteomes of the thirty-five Tremellales revealed 6,918 putative carbohydrate-active enzymes (CAZYmes) and 7,066 peptidases. Two soil isolates, Saitozyma podzolica DSM 27192 and Cryptococcus sp. JCM 24511, show higher numbers harboring an average of 317 compared to a range of 267-121 CAZYmes for the rest of the strains. Similarly, the proteomes of the two soil isolates along with two plant associated strains contain higher number of peptidases sharing an average of 234 peptidases compared to a range of 226-167 for the rest of the strains. Despite these huge differences and the apparent enrichment of these enzymes among the soil isolates, the data revealed a diversity of the various enzyme families that does not reflect specific habitat type. Growth experiment on various carbohydrates to validate the predictions provides support for this view. Overall, the data indicates that the Tremellales could serve as a rich source of both CAZYmes and peptidases with wide range of potential biotechnological relevance.

Genomic insights into the lifestyles, functional capacities and oleagenicity of members of the fungal family Trichosporonaceae.

Trichosporonaceae incorporates six genera of physiologically and ecologically diverse fungi including both human pathogenic taxa as well as yeasts of biotechnological interest, especially those oleagenic taxa that accumulate large amounts of single cell oils (SCOs). Here, we have undertaken comparative genomic analysis of thirty-three members of the family with a view to gain insight into the molecular determinants underlying their lifestyles and niche specializations. Phylogenomic analysis revealed potential misidentification of three strains which could impact subsequent analyses. Evaluation of the predicted proteins coding sequences showed that the free-living members of the family harbour greater numbers of carbohydrate active enzymes (CAZYmes), metallo- and serine peptidases compared to their host-associated counterparts. Phylogenies of selected lipid biosynthetic enzymes encoded in the genomes of the studied strains revealed disparate evolutionary histories for some proteins inconsistent with the core genome phylogeny. However, the documented oleagenic members distinctly cluster based on the constitution of the upstream regulatory regions of genes encoding acetyl-CoA carboxylase (ACC), ATP-citrate synthase (ACS) and isocitrate dehydrogenase [NADP] (ICDH), which are among the major proteins in the lipid biosynthetic pathway of these yeasts, suggesting a possible pattern in the regulation of these genes.

Aestipascuomyces dupliciliberans gen. nov, sp. nov., the First Cultured Representative of the Uncultured SK4 Clade from Aoudad Sheep and Alpaca.

We report on the isolation of the previously-uncultured Neocallimastigomycota SK4 lineage, by two independent research groups, from a wild aoudad sheep rumen sample (Texas, USA) and an alpaca fecal sample (Baden-Württemberg, Germany). Isolates from both locations showed near-identical morphological and microscopic features, forming medium-sized (2-5 mm) white filamentous colonies with a white center of sporangia, on agar roll tubes and a heavy biofilm in liquid media. Microscopic analysis revealed monocentric thalli, and spherical polyflagellated zoospores with 7-20 flagella. Zoospore release occurred through an apical pore as well as by sporangial wall rupturing, a duality that is unique amongst described anaerobic gut fungal strains. Isolates were capable of growing on a wide range of mono-, oligo-, and polysaccharide substrates as the sole carbon source. Phylogenetic assessment based on the D1-D2 28S large rRNA gene subunit (D1-D2 LSU) and internal transcribed spacer-1 (ITS-1) regions demonstrated high sequence identity (minimum identity of 99.07% and 96.96%, respectively) between all isolates; but low sequence identity (92.4% and 86.7%, respectively) to their closest cultured relatives. D1-D2 LSU phylogenetic trees grouped the isolates as a new monophyletic clade within the Orpinomyces-Neocallimastix-Pecoramyces-Feramyces-Ghazallamyces supragenus group. D1-D2 LSU and ITS-1 sequences recovered from the obtained isolates were either identical or displayed extremely high sequence similarity to sequences recovered from the same aoudad sheep sample on which isolation was conducted, as well as several sequences recovered from domestic sheep and few other herbivores. Interestingly, members of the SK4 clade seem to be encountered preferably in animals grazing on summer pasture. We hence propose accommodating these novel isolates in a new genus, Aestipascuomyces (derived from the Latin word for "summer pasture"), and a new species, A. dupliciliberans. The type strain is Aestipascuomycesdupliciliberans strain R4.