Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

Mechanism of high-mannose N-glycan breakdown and metabolism by Bifidobacterium longum.

Bifidobacteria are early colonizers of the human gut and play central roles in human health and metabolism. To thrive in this competitive niche, these bacteria evolved the capacity to use complex carbohydrates, including mammalian N-glycans. Herein, we elucidated pivotal biochemical steps involved in high-mannose N-glycan utilization by Bifidobacterium longum. After N-glycan release by an endo-ß-N-acetylglucosaminidase, the mannosyl arms are trimmed by the cooperative action of three functionally distinct glycoside hydrolase 38 (GH38) α-mannosidases and a specific GH125 α-1,6-mannosidase. High-resolution cryo-electron microscopy structures revealed that bifidobacterial GH38 α-mannosidases form homotetramers, with the N-terminal jelly roll domain contributing to substrate selectivity. Additionally, an α-glucosidase enables the processing of monoglucosylated N-glycans. Notably, the main degradation product, mannose, is isomerized into fructose before phosphorylation, an unconventional metabolic route connecting it to the bifid shunt pathway. These findings shed light on key molecular mechanisms used by bifidobacteria to use high-mannose N-glycans, a perennial carbon and energy source in the intestinal lumen.

Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.

Publisher Correction: Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Hydroxyquinol Degradation Pathway in Rhodococcus jostii RHA1 and Agrobacterium Species Is an Alternative Pathway for Degradation of Protocatechuic Acid and Lignin Fragments.

Deletion of the pcaHG genes, encoding protocatechuate 3,4-dioxygenase in Rhodococcus jostii RHA1, gives a gene deletion strain still able to grow on protocatechuic acid as the sole carbon source, indicating a second degradation pathway for protocatechuic acid. Metabolite analysis of wild-type R. jostii RHA1 grown on medium containing vanillin or protocatechuic acid indicated the formation of hydroxyquinol (benzene-1,2,4-triol) as a downstream product. Gene cluster ro01857-ro01860 in Rhodococcus jostii RHA1 contains genes encoding hydroxyquinol 1,2-dioxygenase and maleylacetate reductase for degradation of hydroxyquinol but also putative mono-oxygenase (ro01860) and putative decarboxylase (ro01859) genes, and a similar gene cluster is found in the genome of lignin-degrading Agrobacterium species. Recombinant R. jostii mono-oxygenase and decarboxylase enzymes in combination were found to convert protocatechuic acid to hydroxyquinol. Hence, an alternative pathway for degradation of protocatechuic acid via oxidative decarboxylation to hydroxyquinol is proposed.IMPORTANCE There is a well-established paradigm for degradation of protocatechuic acid via the ß-ketoadipate pathway in a range of soil bacteria. In this study, we have found the existence of a second pathway for degradation of protocatechuic acid in Rhodococcus jostii RHA1, via hydroxyquinol (benzene-1,2,4-triol), which establishes a metabolic link between protocatechuic acid and hydroxyquinol. The presence of this pathway in a lignin-degrading Agrobacterium sp. strain suggests the involvement of the hydroxyquinol pathway in the metabolism of degraded lignin fragments.

Microbial Communities of the Gut and Nest of the Humus- and Litter-Feeding Termite Procornitermes araujoi (Syntermitinae).

The evolution of the symbiotic association with microbes allowed termites to decompose ingested lignocellulose from plant-derived substrates, including herbivore dung and soil humus. Representatives of the Syntermitinae (Termitidae) range in their feeding habits from wood and litter-feeding to humus-feeding species. However, only limited information is available about their feeding ecology and associated microbial communities. Here we conducted a study of the microbial communities associated to the termite Procornitermes araujoi using Illumina sequencing of the 16S and ITS rRNA genes. This species has been previously included in different feeding guilds. However, most aspects of its feeding ecology are unknown, especially those associated to its symbiotic microbiota. Our results showed that the microbial communities of termite guts and nest substrates of P. araujoi differed significantly for bacteria and fungi. Firmicutes dominated the bacterial gut community of both workers and soldiers, whereas Actinobacteria was found in higher prevalence in the nest walls. Sordariomycetes was the most abundant fungal class in both gut and nest samples and distinguish P. araujoi from the grass/litter feeding Cornitermes cumulans. Our results also showed that diversity of gut bacteria were higher in P. araujoi and Silvestritermes euamignathus than in the grass/litter feeders (C. cumulans and Syntermes dirus), that could indicate an adaptation of the microbial community of polyphagous termites to the higher complexity of their diets.

The impact of chromatin remodelling on cellulase expression in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is used for industry-scale production of plant cell wall-degrading enzymes, in particular cellulases, but also xylanases. The expression of the encoding genes was so far primarily investigated on the level of transcriptional regulation by regulatory proteins. Otherwise, the impact of chromatin remodelling on gene expression received hardly any attention. In this study we aimed to learn if the chromatin status changes in context to the applied conditions (repressing/inducing), and if the presence or absence of the essential transactivator, the Xylanase regulator 1 (Xyr1), influences the chromatin packaging. RESULTS: Comparing the results of chromatin accessibility real-time PCR analyses and gene expression studies of the two prominent cellulase-encoding genes, cbh1 and cbh2, we found that the chromatin opens during sophorose-mediated induction compared to D-glucose-conferred repression. In the strain bearing a xyr1 deletion the sophorose mediated induction of gene expression is lost and the chromatin opening is strongly reduced. In all conditions the chromatin got denser when Xyr1 is absent. In the case of the xylanase-encoding genes, xyn1 and xyn2, the result was similar concerning the condition-specific response of the chromatin compaction. However, the difference in chromatin status provoked by the absence of Xyr1 is less pronounced. A more detailed investigation of the DNA accessibility in the cbh1 promoter showed that the deletion of xyr1 changed the in vivo footprinting pattern. In particular, we detected increased hypersensitivity on Xyr1-sites and stronger protection of Cre1-sites. Looking for the players directly causing the observed chromatin remodelling, a whole transcriptome shotgun sequencing revealed that 15 genes encoding putative chromatin remodelers are differentially expressed in response to the applied condition and two amongst them are differentially expressed in the absence of Xyr1. CONCLUSIONS: The regulation of xylanase and cellulase expression in T. reesei is not only restricted to the action of transcription factors but is clearly related to changes in the chromatin packaging. Both the applied condition and the presence of Xyr1 influence chromatin status.

Transcriptional profiling of Neurospora crassa Δmak-2 reveals that mitogen-activated protein kinase MAK-2 participates in the phosphate signaling pathway.

The filamentous fungus Neurospora crassa is an excellent model system for examining molecular responses to ambient signals in eukaryotic microorganisms. Inorganic phosphate (Pi) is an essential growth-limiting nutrient in nature and is crucial for the synthesis of nucleic acids and the flow of genetic information. The genetic and molecular mechanisms controlling the response to Pi starvation in N. crassa include at least four genes (nuc-2, preg, pogv, and nuc-1), which are involved in a hierarchical regulatory activation network. In a previous work, we identified a number of genes modulated by NUC-2 protein, including the mak-2 gene, which codes for a mitogen-activated protein kinase (MAPK), suggesting its participation in the phosphate signaling pathway. Thus, to identify other genes involved in metabolic responses to exogenous phosphate sensing and the functioning of the MAPK MAK-2, we performed microarray experiments using a mak-2 knockout strain (Δmak-2) grown under phosphate-shortage conditions by comparing its transcription profile to that of a control strain grown in low- and high-phosphate cultures. These experiments revealed 912 unique differentially expressed genes involved in a number of physiological processes related to phosphate transport, metabolism, and regulation as well as posttranslational modification of proteins, and MAPK signaling pathways. Quantitative Real-time PCR gene expression analysis of 18 selected genes, using independent RNA samples, validated our microarray results. A high Pearson correlation between microarray and quantitative Real-time PCR data was observed. The analysis of these differentially expressed genes in the Δmak-2 strain provide evidence that the mak-2 gene participates in the hierarchical phosphate-signaling pathway in N. crassa in addition to its involvement in other metabolic routes such as the isoprenylation pathway, thus revealing novel aspects of the N. crassa phosphorus-sensing network.

The Microsporum canis genome is organized into five chromosomes based on evidence from electrophoretic karyotyping and chromosome end mapping.

The karyotype of Microsporum canis was analyzed by contoured-clamped homogeneous electric field (CHEF) gel electrophoresis. Four chromosomal bands that correspond to five chromosomes ranging from 3.0-6.2 Mb were identified, adding the total genome size to approximately 24.9 Mb. To confirm the number of chromosomes in M. canis, the number of telomeres was assessed by using a telomeric probe (TTAGGG)(4) in Southern blot analyses of digested genomic DNA. Treatment of M. canis DNA with Bal31 exonuclease revealed progressive shortening of the DNA fragments positive for the (TTAGGG)(4) sequence, supporting location of repeats at the chromosome ends. These results can aid in improving the understanding of the genetic characterization of M. canis and the molecular epidemiology of dermatophytoses caused by this fungus.

rpb2 is a reliable reference gene for quantitative gene expression analysis in the dermatophyte Trichophyton rubrum.

The selection of reference genes used for data normalization to quantify gene expression by real-time PCR amplifications (qRT-PCR) is crucial for the accuracy of this technique. In spite of this, little information regarding such genes for qRT-PCR is available for gene expression analyses in pathogenic fungi. Thus, we investigated the suitability of eight candidate reference genes in isolates of the human dermatophyte Trichophyton rubrum subjected to several environmental challenges, such as drug exposure, interaction with human nail and skin, and heat stress. The stability of these genes was determined by geNorm, NormFinder and Best-Keeper programs. The gene with the most stable expression in the majority of the conditions tested was rpb2 (DNA-dependent RNA polymerase II), which was validated in three T. rubrum strains. Moreover, the combination of rpb2 and chs1 (chitin synthase) genes provided for the most reliable qRT-PCR data normalization in T. rubrum under a broad range of biological conditions. To the best of our knowledge this is the first report on the selection of reference genes for qRT-PCR data normalization in dermatophytes and the results of these studies should permit further analysis of gene expression under several experimental conditions, with improved accuracy and reliability.

Role of pH in the pathogenesis of dermatophytoses.

The secretion of proteolytic enzymes by dermatophytes is a key factor in their invasion and subsequent dissemination through the stratum corneum of the host. During the first stages of infection, dermatophytes respond to the skin by de-repressing a number of genes coding for proteins and enzymes such as adhesins, lipases, phosphatases, DNAses, non-specific proteases, and keratinases. These proteins have their optimal activity at acidic pH values, which matches the acidic pH of human skin, allowing the pathogen to adhere and penetrate the host tissue, scavenge nutrients and overcome host defence mechanisms. The conserved PacC/Rim101p signal transduction pathway mediates diverse metabolic events involved in ambient pH sensing and in the virulence of pathogenic microorganisms. The seven dermatophyte genomes analysed here revealed the presence of the PacC/Rim101p pH-responsive signal transduction pathway, which consists of the six pal genes (palA, B, C, F, H and I) and the transcription factor PacC. The PacC binding site was present in the promoter regions of pacC, palB, palI and palH genes of all dermatophytes, suggesting functional equivalency with the signalling cascade of other fungi. Moreover, the promoter region of pacC gene of the seven dermatophytes had multiple PacC DNA-binding sites, suggesting that these genes, like their homologues in model fungi, are auto-regulated.

Glycoside hydrolase subfamily GH5_57 features a highly redesigned catalytic interface to process complex hetero-ß-mannans.

Glycoside hydrolase family 5 (GH5) harbors diverse substrate specificities and modes of action, exhibiting notable molecular adaptations to cope with the stereochemical complexity imposed by glycosides and carbohydrates such as cellulose, xyloglucan, mixed-linkage ß-glucan, laminarin, (hetero)xylan, (hetero)mannan, galactan, chitosan, N-glycan, rutin and hesperidin. GH5 has been divided into subfamilies, many with higher functional specificity, several of which have not been characterized to date and some that have yet to be discovered with the exploration of sequence/taxonomic diversity. In this work, the current GH5 subfamily inventory is expanded with the discovery of the GH5_57 subfamily by describing an endo-ß-mannanase (CapGH5_57) from an uncultured Bacteroidales bacterium recovered from the capybara gut microbiota. Biochemical characterization showed that CapGH5_57 is active on glucomannan, releasing oligosaccharides with a degree of polymerization from 2 to 6, indicating it to be an endo-ß-mannanase. The crystal structure, which was solved using single-wavelength anomalous diffraction, revealed a massively redesigned catalytic interface compared with GH5 mannanases. The typical aromatic platforms and the characteristic α-helix-containing ß6-α6 loop in the positive-subsite region of GH5_7 mannanases are absent in CapGH5_57, generating a large and open catalytic interface that might favor the binding of branched substrates. Supporting this, CapGH5_57 contains a tryptophan residue adjacent and perpendicular to the cleavage site, indicative of an anchoring site for a substrate with a substitution at the -1 glycosyl moiety. Taken together, these results suggest that despite presenting endo activity on glucomannan, CapGH5_57 may have a new type of substituted heteromannan as its natural substrate. This work demonstrates the still great potential for discoveries regarding the mechanistic and functional diversity of this large and polyspecific GH family by unveiling a novel catalytic interface sculpted to recognize complex heteromannans, which led to the establishment of the GH5_57 subfamily.

Gut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharides.

The largest living rodent, capybara, can efficiently depolymerize and utilize lignocellulosic biomass through microbial symbiotic mechanisms yet elusive. Herein, we elucidate the microbial community composition, enzymatic systems and metabolic pathways involved in the conversion of dietary fibers into short-chain fatty acids, a main energy source for the host. In this microbiota, the unconventional enzymatic machinery from Fibrobacteres seems to drive cellulose degradation, whereas a diverse set of carbohydrate-active enzymes from Bacteroidetes, organized in polysaccharide utilization loci, are accounted to tackle complex hemicelluloses typically found in gramineous and aquatic plants. Exploring the genetic potential of this community, we discover a glycoside hydrolase family of ß-galactosidases (named as GH173), and a carbohydrate-binding module family (named as CBM89) involved in xylan binding that establishes an unprecedented three-dimensional fold among associated modules to carbohydrate-active enzymes. Together, these results demonstrate how the capybara gut microbiota orchestrates the depolymerization and utilization of plant fibers, representing an untapped reservoir of enzymatic mechanisms to overcome the lignocellulose recalcitrance, a central challenge toward a sustainable and bio-based economy.

Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds.

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway.

Filamentous fungi are important cell factories for large-scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR-inducing chemicals and differentially expressed genes were identified by RNA-seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co-chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.

Novel redox-active enzymes for ligninolytic applications revealed from multiomics analyses of Peniophora sp. CBMAI 1063, a laccase hyper-producer strain.

The repertoire of redox-active enzymes produced by the marine fungus Peniophora sp. CBMAI 1063, a laccase hyper-producer strain, was characterized by omics analyses. The genome revealed 309 Carbohydrate-Active Enzymes (CAZymes) genes, including 48 predicted genes related to the modification and degradation of lignin, whith 303 being transcribed under cultivation in optimized saline conditions for laccase production. The secretome confirmed that the fungus can produce a versatile ligninolytic enzyme cocktail. It secretes 56 CAZymes, including 11 oxidative enzymes classified as members of auxiliary activity families (AAs), comprising two laccases, Pnh_Lac1 and Pnh_Lac2, the first is the major secretory protein of the fungi. The Pnh_Lac1-mediator system was able to promote the depolymerization of lignin fragments and polymeric lignin removal from pretreated sugarcane bagasse, confirming viability of this fungus enzymatic system for lignocellulose-based bioproducts applications.

Genome Sequence of Lysinibacillus sphaericus, a Lignin-Degrading Bacterium Isolated from Municipal Solid Waste Soil.

We report here the draft genome sequence of Lysinibacillus sphaericus strain A1, a potential lignin-degrading bacterium isolated from municipal solid waste (MSW) soil and capable of enhancing gas release from lignocellulose-containing soil.

Microbiota modification by probiotic supplementation reduces colitis associated colon cancer in mice.

AIM: To investigate the effect of probiotic supplementation during the development of an experimental model of colitis associated colon cancer (CAC). METHODS: C57BL/6 mice received an intraperitoneal injection of azoxymethane (10 mg/kg), followed by three cycles of sodium dextran sulphate diluted in water (5% w/v). Probiotic group received daily a mixture of Lactobacillus acidophilus, Lactobacillus rhamnosus and Bifidobacterium bifidum. Microbiota composition was assessed by 16S rRNA Illumina HiSeq sequencing. Colon samples were collected for histological analysis. Tumor cytokines was assessed by Real Time-PCR (Polymerase Chain Reaction); and serum cytokines by Multiplex assay. All tests were two-sided. The level of significance was set at P < 0.05. Graphs were generated and statistical analysis performed using the software GraphPad Prism 5.0. The project was approved by the institutional review board committee. RESULTS: At day 60 after azoxymethane injection, the mean number of tumours in the probiotic group was 40% lower than that in the control group, and the probiotic group exhibited tumours of smaller size (< 2 mm) (P < 0.05). There was no difference in richness and diversity between groups. However, there was a significant difference in beta diversity in the multidimensional scaling analysis. The abundance of the genera Lactobacillus, Bifidobacterium, Allobaculum, Clostridium XI and Clostridium XVIII increased in the probiotic group (P < 0.05). The microbial change was accompanied by reduced colitis, demonstrated by a 46% reduction in the colon inflammatory index; reduced expression of the serum chemokines RANTES and Eotaxin; decreased p-IKK and TNF-α and increased IL-10 expression in the colon. CONCLUSION: Our results suggest a potential chemopreventive effect of probiotic on CAC. Probiotic supplementation changes microbiota structure and regulates the inflammatory response, reducing colitis and preventing CAC.

Lignolytic-consortium omics analyses reveal novel genomes and pathways involved in lignin modification and valorization.

BACKGROUND: Lignin is a heterogeneous polymer representing a renewable source of aromatic and phenolic bio-derived products for the chemical industry. However, the inherent structural complexity and recalcitrance of lignin makes its conversion into valuable chemicals a challenge. Natural microbial communities produce biocatalysts derived from a large number of microorganisms, including those considered unculturable, which operate synergistically to perform a variety of bioconversion processes. Thus, metagenomic approaches are a powerful tool to reveal novel optimized metabolic pathways for lignin conversion and valorization. RESULTS: The lignin-degrading consortium (LigMet) was obtained from a sugarcane plantation soil sample. The LigMet taxonomical analyses (based on 16S rRNA) indicated prevalence of Proteobacteria, Actinobacteria and Firmicutes members, including the Alcaligenaceae and Micrococcaceae families, which were enriched in the LigMet compared to sugarcane soil. Analysis of global DNA sequencing revealed around 240,000 gene models, and 65 draft bacterial genomes were predicted. Along with depicting several peroxidases, dye-decolorizing peroxidases, laccases, carbohydrate esterases, and lignocellulosic auxiliary (redox) activities, the major pathways related to aromatic degradation were identified, including benzoate (or methylbenzoate) degradation to catechol (or methylcatechol), catechol ortho-cleavage, catechol meta-cleavage, and phthalate degradation. A novel Paenarthrobacter strain harboring eight gene clusters related to aromatic degradation was isolated from LigMet and was able to grow on lignin as major carbon source. Furthermore, a recombinant pathway for vanillin production was designed based on novel gene sequences coding for a feruloyl-CoA synthetase and an enoyl-CoA hydratase/aldolase retrieved from the metagenomic data set. CONCLUSION: The enrichment protocol described in the present study was successful for a microbial consortium establishment towards the lignin and aromatic metabolism, providing pathways and enzyme sets for synthetic biology engineering approaches. This work represents a pioneering study on lignin conversion and valorization strategies based on metagenomics, revealing several novel lignin conversion enzymes, aromatic-degrading bacterial genomes, and a novel bacterial strain of potential biotechnological interest. The validation of a biosynthetic route for vanillin synthesis confirmed the applicability of the targeted metagenome discovery approach for lignin valorization strategies.