A single workflow for multi-species blood transcriptomics.

BACKGROUND: Blood transcriptomic analysis is widely used to provide a detailed picture of a physiological state with potential outcomes for applications in diagnostics and monitoring of the immune response to vaccines. However, multi-species transcriptomic analysis is still a challenge from a technological point of view and a standardized workflow is urgently needed to allow interspecies comparisons. RESULTS: Here, we propose a single and complete total RNA-Seq workflow to generate reliable transcriptomic data from blood samples from humans and from animals typically used in preclinical models. Blood samples from a maximum of six individuals and four different species (rabbit, non-human primate, mouse and human) were extracted and sequenced in triplicates. The workflow was evaluated using different wet-lab and dry-lab criteria, including RNA quality and quantity, the library molarity, the number of raw sequencing reads, the Phred-score quality, the GC content, the performance of ribosomal-RNA and globin depletion, the presence of residual DNA, the strandness, the percentage of coding genes, the number of genes expressed, and the presence of saturation plateau in rarefaction curves. We identified key criteria and their associated thresholds to be achieved for validating the transcriptomic workflow. In this study, we also generated an automated analysis of the transcriptomic data that streamlines the validation of the dataset generated. CONCLUSIONS: Our study has developed an end-to-end workflow that should improve the standardization and the inter-species comparison in blood transcriptomics studies. In the context of vaccines and drug development, RNA sequencing data from preclinical models can be directly compared with clinical data and used to identify potential biomarkers of value to monitor safety and efficacy.

Comparison of DNA extraction methods for 16S rRNA gene sequencing in the analysis of the human gut microbiome.

The gut microbiome is widely analyzed using high-throughput sequencing, such as 16S rRNA gene amplicon sequencing and shotgun metagenomic sequencing (SMS). DNA extraction is known to have a large impact on the metagenomic analyses. The aim of this study was to compare DNA extraction protocols for 16S sequencing. In that context, four commonly used DNA extraction methods were compared for the analysis of the gut microbiota. Commercial versions were evaluated against modified protocols using a stool preprocessing device (SPD, bioMérieux) upstream DNA extraction. Stool samples from nine healthy volunteers and nine patients with a Clostridium difficile infection were extracted with all protocols and 16S sequenced. Protocols were ranked using wet- and dry-lab criteria, including quality controls of the extracted genomic DNA, alpha-diversity, accuracy using a mock community of known composition and repeatability across technical replicates. SPD improved overall efficiency of three of the four tested protocols compared with their commercial version, in terms of DNA extraction yield, sample alpha-diversity, and recovery of Gram-positive bacteria. The best overall performance was obtained for the S-DQ protocol, SPD combined with the DNeasy PowerLyser PowerSoil protocol from QIAGEN. Based on this evaluation, we strongly believe that the use of such stool preprocessing device improves both the standardization and the quality of the DNA extraction in the human gut microbiome studies.

An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi.

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC.

Tandem metalloenzymes gate plant cell entry by pathogenic fungi.

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase-peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection.

In vitro Applications of the Terpene Mini-Path 2.0.

In 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes.

Evolution of Fungal Carbohydrate-Active Enzyme Portfolios and Adaptation to Plant Cell-Wall Polymers.

The postindustrial era is currently facing two ecological challenges. First, the rise in global temperature, mostly caused by the accumulation of carbon dioxide (CO2) in the atmosphere, and second, the inability of the environment to absorb the waste of human activities. Fungi are valuable levers for both a reduction in CO2 emissions, and the improvement of a circular economy with the optimized valorization of plant waste and biomass. Soil fungi may promote plant growth and thereby increase CO2 assimilation via photosynthesis or, conversely, they may prompt the decomposition of dead organic matter, and thereby contribute to CO2 emissions. The strategies that fungi use to cope with plant-cell-wall polymers and access the saccharides that they use as a carbon source largely rely on the secretion of carbohydrate-active enzymes (CAZymes). In the past few years, comparative genomics and phylogenomics coupled with the functional characterization of CAZymes significantly improved the understanding of their evolution in fungal genomes, providing a framework for the design of nature-inspired enzymatic catalysts. Here, we provide an overview of the diversity of CAZyme enzymatic systems employed by fungi that exhibit different substrate preferences, different ecologies, or belong to different taxonomical groups for lignocellulose degradation.

Distribution of methionine sulfoxide reductases in fungi and conservation of the free-methionine-R-sulfoxide reductase in multicellular eukaryotes.

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi.

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus.

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.

Integrative visual omics of the white-rot fungus Polyporus brumalis exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

BACKGROUND: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus Polyporus brumalis efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of P. brumalis for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide. RESULTS: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners. CONCLUSION: As a peculiar feature of P. brumalis, we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.