Soil Metagenome Research Articles

Changes in prokaryote communities of southern chernozem under the influence of different farming systems

Soil microbiota or microbiocenosis is profoundly influenced by various physical, chemical, and biological environmental factors. Therefore, it serves as a good indicator of soil condition. With the introduction of new farming systems in agriculture, an intensive study and understanding of soil microbial community will make it possible to comprehensively analyze these systems including determining the degree of their impact on agroecosystems. The present study analyzed the structure of prokaryote communities in soil samples collected from the southern chernozem where different agricultural techniques are used, namely conventional, which involves plowing and a new method to the region, i.e., no-till farming. The outcomes were compared with the virgin land. The changes in soil metagenome were analyzed using high productive sequencing of 16S rRNA gene libraries. The authors found out that farming systems significantly influenced the changes in communities of prokaryotes of the southern chernozems, thereby creating conditions for the more active development of some groups of microorganisms while limiting others. A relative resistance to the effects of farming systems was noted for the genera Bacillus and Paenibacillus.

Antarctic tundra soil metagenome as useful natural resources of cold-active lignocelluolytic enzymes.

Lignocellulose composed of complex carbohydrates and aromatic heteropolymers is one of the principal materials for the production of renewable biofuels. Lignocellulose-degrading genes from cold-adapted bacteria have a potential to increase the productivity of biological treatment of lignocellulose biomass by providing a broad range of treatment temperatures. Antarctic soil metagenomes allow to access novel genes encoding for the cold-active lignocellulose-degrading enzymes, for biotechnological and industrial applications. Here, we investigated the metagenome targeting cold-adapted microbes in Antarctic organic matter-rich soil (KS 2-1) to mine lignolytic and celluloytic enzymes by performing single molecule, real-time metagenomic (SMRT) sequencing. In the assembled Antarctic metagenomic contigs with relative long reads, we found that 162 (1.42%) of total 11,436 genes were annotated as carbohydrate-active enzymes (CAZy). Actinobacteria, the dominant phylum in this soil's metagenome, possessed most of candidates of lignocellulose catabolic genes like glycoside hydrolase families (GH13, GH26, and GH5) and auxiliary activity families (AA7 and AA3). The predicted lignocellulose degradation pathways in Antarctic soil metagenome showed synergistic role of various CAZyme harboring bacterial genera including Streptomyces, Streptosporangium, and Amycolatopsis. From phylogenetic relationships with cellular and environmental enzymes, several genes having potential for participating in overall lignocellulose degradation were also found. The results indicated the presence of lignocellulose-degrading bacteria in Antarctic tundra soil and the potential benefits of the lignocelluolytic enzymes as candidates for cold-active enzymes which will be used for the future biofuel-production industry.

Metagenomic and chemical characterization of soil cobalamin production

Cobalamin (vitamin B12) is an essential enzyme cofactor for most branches of life. Despite the potential importance of this cofactor for soil microbial communities, the producers and consumers of cobalamin in terrestrial environments are still unknown. Here we provide the first metagenome-based assessment of soil cobalamin-producing bacteria and archaea, quantifying and classifying genes encoding proteins for cobalamin biosynthesis, transport, remodeling, and dependency in 155 soil metagenomes with profile hidden Markov models. We also measured several forms of cobalamin (CN-, Me-, OH-, Ado-B12) and the cobalamin lower ligand (5,6-dimethylbenzimidazole; DMB) in 40 diverse soil samples. Metagenomic analysis revealed that less than 10% of soil bacteria and archaea encode the genetic potential for de novo synthesis of this important enzyme cofactor. Predominant soil cobalamin producers were associated with the Proteobacteria, Actinobacteria, Firmicutes, Nitrospirae, and Thaumarchaeota. In contrast, a much larger proportion of abundant soil genera lacked cobalamin synthesis genes and instead were associated with gene sequences encoding cobalamin transport and cobalamin-dependent enzymes. The enrichment of DMB and corresponding DMB synthesis genes, relative to corrin ring synthesis genes, suggests an important role for cobalamin remodelers in terrestrial habitats. Together, our results indicate that microbial cobalamin production and repair serve as keystone functions that are significantly correlated with microbial community size, diversity, and biogeochemistry of terrestrial ecosystems.

Community structure and functional genes in radionuclide contaminated soils in Chernobyl and Fukushima.

Chernobyl and Fukushima were subjected to radionuclide (RN) contamination that has led to environmental problems. In order to explore the ability of microorganisms to survive in these environments, we used a combined 16S rRNA and metagenomic approach to describe the prokaryotic community structure and metabolic potential over a gradient of RN concentrations (137Cs 1680-0.4 and 90Sr 209.1-1.9 kBq kg-1) in soil samples. The taxonomic results showed that samples with low 137Cs content (37.8-0.4 kBq kg-1) from Fukushima and Chernobyl clustered together. In order to determine the effect of soil chemical parameters such as organic carbon (OC), Cesium-137 (137Cs) and Strontium-90 (90Sr) on the functional potential of microbial communities, multiple predictor model analysis using piecewiseSEM was carried out on Chernobyl soil metagenomes. The model identified 46 genes that were correlated to these parameters of which most have previously been described as mechanisms used by microorganisms under stress conditions. This study provides a baseline taxonomic and metagenomic dataset for Fukushima and Chernobyl, respectively, including physical and chemical characteristics. Our results pave the way for evaluating the possible RN selective pressure that might contribute to shaping microbial community structure and their functions in contaminated soils.

Uncovering the biosynthetic potential of rare metagenomic DNA using co-occurrence network analysis of targeted sequences

Sequencing of DNA extracted from environmental samples can provide key insights into the biosynthetic potential of uncultured bacteria. However, the high complexity of soil metagenomes, which can contain thousands of bacterial species per gram of soil, imposes significant challenges to explore secondary metabolites potentially produced by rare members of the soil microbiome. Here, we develop a targeted sequencing workflow termed CONKAT-seq (co-occurrence network analysis of targeted sequences) that detects physically clustered biosynthetic domains, a hallmark of bacterial secondary metabolism. Following targeted amplification of conserved biosynthetic domains in a highly partitioned metagenomic library, CONKAT-seq evaluates amplicon co-occurrence patterns across library subpools to identify chromosomally clustered domains. We show that a single soil sample can contain more than a thousand uncharacterized biosynthetic gene clusters, most of which originate from low frequency genomes which are practically inaccessible through untargeted sequencing. CONKAT-seq allows scalable exploration of largely untapped biosynthetic diversity across multiple soils, and can guide the discovery of novel secondary metabolites from rare members of the soil microbiome.

High-throughput sequencing approach to access the impact of nanozeolite treatment on species richness and evenness of soil metagenome

Abstract Microbial diversity is a very crucial component for the soil health maintenance. The present study investigated the effects of nanozeolite on bacterial diversity of soil from the agricultural field which was under 4-year field trial with wheat crop. Nanozeolite was amended in the treated plot whereas, the control plot did not receive any treatment. The bacterial population was targeted through the hypervariable (V3) region, which is a part of 16SrDNA. The 16SrDNA region is a conserved region among the bacterial species, but to investigate the diversity among the same species the hypervariable region are the best suited sequences. More than 1 million reads per treatment revealed very high levels of diversity. The majority of the sequences were attribute to the Proteobacteria (about 23–25%), 15% and 30–35% fitted into Actinobacteria and unknown phylum, respectively. Significant higher abundances of bacterial species with NZ treatment encompassed the population associated with nutrient cycling, residue decomposition and xenobiosis. The alpha diversity index also indicated better diversity and evenness within the treated soil than untreated soil. Our findings support the importance of nanozeolite for better survival of soil microorganisms especially bacteria.

Structure-Guided Generation of a Redox-Independent Blue Fluorescent Protein from mBFP

Fluorescent proteins, such as the green fluorescent protein, are used for detection of cellular components and events. However, green fluorescent protein and its derivatives have limited usage under anaerobic conditions and require a long maturation time. On the other hand, the NADPH-dependent blue fluorescent protein (BFP) without oxidative modification of residues is instantly functional in both aerobic and anaerobic systems. BFP proteins belong to a short-chain dehydrogenase/reductase (SDR) protein family, and their fluorescent property changes with reaction time in the presence of a substrate. With the aim of developing a better fluorescent reporter independent of redox state, we elucidated the crystal structure of a tetrameric mBFP from soil metagenomes with and without NADPH. Apart from the previously known regions, structure-guided mutational studies have identified several residues that contribute to the fluorescence of mBFP, including two aromatic residues (F97 and Y157) near the nicotinamide moiety of the bound NADPH. A single histidine mutation at Y157 (Y157H) has conferred more stabilized, time-independent fluorescence even in the presence of substrates. Furthermore, we discovered another SDR protein that can also emit blue fluorescence. These results open a new possibility for the development of BFP as a stable cellular reporter for widespread use, independent of subcellular environments.

Effect of Long-Term Farming Practices on Agricultural Soil Microbiome Members Represented by Metagenomically Assembled Genomes (MAGs) and Their Predicted Plant-Beneficial Genes.

To follow the hypothesis that agricultural management practices affect structure and function of the soil microbiome regarding soil health and plant-beneficial traits, high-throughput (HT) metagenome analyses were performed on Chernozem soil samples from a long-term field experiment designated LTE-1 carried out at Bernburg-Strenzfeld (Saxony-Anhalt, Germany). Metagenomic DNA was extracted from soil samples representing the following treatments: (i) plough tillage with standard nitrogen fertilization and use of fungicides and growth regulators, (ii) plough tillage with reduced nitrogen fertilization (50%), (iii) cultivator tillage with standard nitrogen fertilization and use of fungicides and growth regulators, and (iv) cultivator tillage with reduced nitrogen fertilization (50%). Bulk soil (BS), as well as root-affected soil (RS), were considered for all treatments in replicates. HT-sequencing of metagenomic DNA yielded approx. 100 Giga bases (Gb) of sequence information. Taxonomic profiling of soil communities revealed the presence of 70 phyla, whereby Proteobacteria, Actinobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Thaumarchaeota, Firmicutes, Verrucomicrobia and Chloroflexi feature abundances of more than 1%. Functional microbiome profiling uncovered, i.a., numerous potential plant-beneficial, plant-growth-promoting and biocontrol traits predicted to be involved in nutrient provision, phytohormone synthesis, antagonism against pathogens and signal molecule synthesis relevant in microbe–plant interaction. Neither taxonomic nor functional microbiome profiling based on single-read analyses revealed pronounced differences regarding the farming practices applied. Soil metagenome sequences were assembled and taxonomically binned. The ten most reliable and abundant Metagenomically Assembled Genomes (MAGs) were taxonomically classified and metabolically reconstructed. Importance of the phylum Thaumarchaeota for the analyzed microbiome is corroborated by the fact that the four corresponding MAGs were predicted to oxidize ammonia (nitrification), thus contributing to the cycling of nitrogen, and in addition are most probably able to fix carbon dioxide. Moreover, Thaumarchaeota and several bacterial MAGs also possess genes with predicted functions in plant–growth–promotion. Abundances of certain MAGs (species resolution level) responded to the tillage practice, whereas the factors compartment (BS vs. RS) and nitrogen fertilization only marginally shaped MAG abundance profiles. Hence, soil management regimes promoting plant-beneficial microbiome members are very likely advantageous for the respective agrosystem, its health and carbon sequestration and accordingly may enhance plant productivity. Since Chernozem soils are highly fertile, corresponding microbiome data represent a valuable reference resource for agronomy in general.

A global survey of arsenic-related genes in soil microbiomes

BackgroundEnvironmental resistomes include transferable microbial genes. One important resistome component is resistance to arsenic, a ubiquitous and toxic metalloid that can have negative and chronic consequences for human and animal health. The distribution of arsenic resistance and metabolism genes in the environment is not well understood. However, microbial communities and their resistomes mediate key transformations of arsenic that are expected to impact both biogeochemistry and local toxicity.ResultsWe examined the phylogenetic diversity, genomic location (chromosome or plasmid), and biogeography of arsenic resistance and metabolism genes in 922 soil genomes and 38 metagenomes. To do so, we developed a bioinformatic toolkit that includes BLAST databases, hidden Markov models and resources for gene-targeted assembly of nine arsenic resistance and metabolism genes: acr3, aioA, arsB, arsC (grx), arsC (trx), arsD, arsM, arrA, and arxA. Though arsenic-related genes were common, they were not universally detected, contradicting the common conjecture that all organisms have them. From major clades of arsenic-related genes, we inferred their potential for horizontal and vertical transfer. Different types and proportions of genes were detected across soils, suggesting microbial community composition will, in part, determine local arsenic toxicity and biogeochemistry. While arsenic-related genes were globally distributed, particular sequence variants were highly endemic (e.g., acr3), suggesting dispersal limitation. The gene encoding arsenic methylase arsM was unexpectedly abundant in soil metagenomes (median 48%), suggesting that it plays a prominent role in global arsenic biogeochemistry.ConclusionsOur analysis advances understanding of arsenic resistance, metabolism, and biogeochemistry, and our approach provides a roadmap for the ecological investigation of environmental resistomes.

Molecular cloning, expression and characterization of a novel feruloyl esterase from a soil metagenomic library with phthalate-degrading activity.

To discover novel feruloyl esterases (FAEs) by the function-driven screening procedure from soil metagenome. A novel FAE gene bds4 was isolated from a soil metagenomic library and over-expressed in Escherichia coli. The recombinant enzyme BDS4 was purified to homogeneity with a predicted molecular weight of 38.8kDa. BDS4 exhibited strong activity (57.05 U/mg) toward methyl ferulate under the optimum pH and temperature of 8.0 and 37°C. Based on its amino acid sequence and model substrates specificity, BDS4 was classified as a type-C FAE. The quantity of the releasing ferulic acid can be enhanced significantly in the presence of xylanase compared with BDS4 alone from de-starched wheat bran. In addition, BDS4 can also hydrolyze several phthalates such as diethyl phthalate, dimethyl phthalate and dibutyl phthalate. The current investigation discovered a novel FAE with phthalate-degrading activity and highlighted the usefulness of metagenomic approaches as a powerful tool for discovery of novel FAEs.

Methanethiol and Dimethylsulfide Cycling in Stiffkey Saltmarsh.

Methanethiol (MeSH) and dimethylsulfide (DMS) are volatile organic sulfur compounds (VOSCs) with important roles in sulfur cycling, signaling and atmospheric chemistry. DMS can be produced from MeSH through a reaction mediated by the methyltransferase MddA. The mddA gene is present in terrestrial and marine metagenomes, being most abundant in soil environments. The substrate for MddA, MeSH, can also be oxidized by bacteria with the MeSH oxidase (MTO) enzyme, encoded by the mtoX gene, found in marine, freshwater and soil metagenomes. Methanethiol-dependent DMS production (Mdd) pathways have been shown to function in soil and marine sediments, but have not been characterized in detail in the latter environments. In addition, few molecular studies have been conducted on MeSH consumption in the environment. Here, we performed process measurements to confirm that Mdd-dependent and Mdd-independent MeSH consumption pathways are active in tested surface saltmarsh sediment when MeSH is available. We noted that appreciable natural Mdd-independent MeSH and DMS consumption processes masked Mdd activity. 16S rRNA gene amplicon sequencing and metagenomics data showed that Methylophaga, a bacterial genus known to catabolise DMS and MeSH, was enriched by the presence of MeSH. Moreover, some MeSH and/or DMS-degrading bacteria isolated from this marine environment lacked known DMS and/or MeSH cycling genes and can be used as model organisms to potentially identify novel genes in these pathways. Thus, we are likely vastly underestimating the abundance of MeSH and DMS degraders in these marine sediment environments. The future discovery and characterization of novel enzymes involved in MeSH and/or DMS cycling is essential to better assess the role and contribution of microbes to global organosulfur cycling.

Changes in microbial functional genes within the soil metagenome during forest ecosystem restoration

As important mediators of numerous ecological processes, soil microorganisms play essential roles in the recovery of ecosystems after disturbance. Using next-generation sequencing techniques, microbial taxonomic changes during ecosystem restoration have been widely studied, but data describing microbial community structure alone can be difficult to link to ecosystem processes mediated by microorganisms. Shotgun metagenome sequencing provides a chance to examine changes among thousands of functional genes during the recovery of microbial communities and ecological function. We analyzed 15 soil metagenomes from a chronosequence of mine soils spanning 6–31 years since reforestation along with unmined reference soils. Taxonomic and functional changes indicate a shift from copiotrophic to oligotrophic groups, increasing metabolism of recalcitrant carbon sources and the influence of vegetation. Increases in genes involved in transposable elements, virulence, defense, and stress response suggest more cooperative and competitive interactions among microorganisms with chronosequence age. Within N cycling groups, ammonia and nitrite oxidizing bacteria increased significantly during restoration, but few significant changes were observed in key N-cycle functional genes. The low relative abundances of methanotrophs and methane monooxygenase genes in all reforested soils explains previous observations that methane consumption has not recovered at these sites 31 years after reforestation. This work helps identify possible mechanisms linking the soil microbiome to ecosystem recovery, with a specific focus on N cycling and greenhouse gas emission, to better understand the roles soil microorganisms play in the restoration of ecosystem functions.

Modified Rice Straw Enhanced Cadmium (II) Immobilization in Soil and Promoted the Degradation of Phenanthrene in Co-Contaminated Soil

Very limited information is available about heavy metal-polycyclic aromatic hydrocarbons (PAHs) depollution involving the modified natural material in soil. Using phenanthrene and cadmium (Cd) as model, this study investigated the effect(s) of modified rice straw by a NaOH solution and on PAHs, heavy metal availability, and their interactions. Treatment included chemical contaminant with/without modified/unmodified rice straw. Fourier Transform Infrared (FTIR) analysis revealed that certain functional groups including anionic matters groups, which can a complex with Cd2+, were exposed on the modified rice straw surfaces. Therefore, Cd concentration was significantly reduced by about 60%, 57%, 62.5 %, and, 64% in the root, shoot, CaCl2, diethylenetriaminepentaacetic acid (DTPA), and extractable Cd, respectively. Subsequently, the prediction of the functional profile of the soil metagenome using Clusters Orthologous Groups (COGs) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that the significantly changed individual COGs belonged to the carbohydrate metabolism, ion transports, and signaling (including cytochrome P450s) categories. This indicated that ion transports might be involved in Cd management, while carbohydrate metabolism, including bisphenol, benzoate, ethylbenzene degradation, and cytochrome P450s, were rather involved in phenanthrene metabolism. The exposed functional group might serve as an external substrate, and P450s might serve as a catalyst to activate and initiate phenanthrene metabolism process. These finding offer confirmation that modified straw could promote the reduction of heavy metal and the degradation of PAHs in soil.

Evaluation of synthetic gene encoding α-galactosidase through metagenomic sequencing of paddy soil

Many genes of industrial relevance can be found in soil. In this study, metagenome sequencing of paddy soil was performed with 55.68Gb sequences and 1,787,113 putative open reading frames (ORFs). The functional profiles and metabolic pathway of soil metagenomes were examined using Gene Ontology, Metagenomics RAST, and Kyoto Encyclopedia of Genes and Genomes. To verify the protein function and assembly of ORFs, a putative gene encoding α-galactosidase, namely GalR, which shares 65% identity with an unpublished glycoside hydrolase (GH) 27 family protein, was synthesized using its optimal codon for overexpression in Escherichia coli. GalR was successfully obtained and characterized. The optimal temperature and pH for GalR activity were 30°C and pH 9, respectively. Enzymatic activity indicated that GalR was alkaliphilic and different from acidophilic α-galactosidase in the GH 27 family. Furthermore, 50% of the relative activity of GalR can be attained for 1.7 and 0.7h preincubation at 40°C and 50°C, respectively. Significant inhibition of GalR was observed in the presence of ethylenediaminetetraacetic acid (EDTA), MgCl2, sodium dodecyl sulfate (SDS), and H2O2; however, it was resistant to 0.1% methanol and ethanol and was slightly activated with NaCl and KCl. The specific activity of GalR was achieved at 11.6 and 0.59μmol/min/mg of protein using p-nitrophenyl-α-d-galactopyranoside and raffinose as substrates, respectively. Consequently, the metagenomic sequencing-based strategy can provide information for mining novel genes.

The distribution of novel bacterial laccases in alpine paleosols is directly related to soil stratigraphy

Bacterial laccases are now known to be abundant in soil and to function outside of the cell facilitating the bacterial degradation of lignin. In this study we wanted to test the hypotheses that: i) Such enzymes can be identified readily in stratified paleosols using metagenomics approaches, ii) The distribution of these genes as potential ‘public good’ proteins in soil is a function of the soil environment, iii) Such laccase genes can be readily retrieved and expressed in E. coli cloning systems to demonstrate that de novo assembly processes can be used to obtain similar metagenome-derived enzyme activities. To test these hypotheses, in silico gene-targeted assembly was employed to identify genes encoding novel type B two-domain bacterial laccases from alpine soil metagenomes sequenced on an Illumina MiSeq sequencer. The genes obtained from different strata were heterologously cloned, expressed and the gene products were shown to be active against two classical laccase substrates. The use of a metagenome-driven pipeline to obtain such active biocatalysts has demonstrated the potential for gene mining to be applied systematically for the discovery of such enzymes. These data ultimately further demonstrate the application of soil pedology methods to environmental enzyme discovery. As an interdisciplinary effort, we can now establish that paleosols can serve as a useful source of novel biocatalytic enzymes for various applications. We also, for the first time, link soil stratigraphy to enzyme profiling for widespread functional gene activity in paleosols.

Municipal solid wastes as a resource for environmental recovery: Impact of water treatment residuals and compost on the microbial and biochemical features of As and trace metal-polluted soils

In this study we evaluated the microbiological and biochemical impact of iron-based water treatment residuals (Fe-WTRs) and municipal solid waste compost (MSWC), alone and combined, on three different soils co-contaminated with arsenic (As) and trace-metals (TM), i.e. Pb, Cu and Zn. Overall, all the amendments considered significantly increased the abundance of culturable heterotrophic bacteria, with MSWC showing the greatest impact across all soils (up to a 24% increase). In most of treated soils this was accompanied by a significant reduction of both the (culturable) fungal/bacterial ratio, and the proportion of culturable As(V)- and As(III)-resistant bacteria with respect to total bacterial population. The catabolic potential and versatility of the resident microbial communities (assessed by community level physiological profile) was highly soil-dependent and substantial increases of both parameters were observed in the amended soils with the higher total As concentration (from approx. 749 to 22,600 mg kg−1). Moreover, both carbon source utilisation profile and 16S rRNA soil metagenome sequencing indicated a significant impact of MSWC and Fe-WTRs on the structure and diversity of soil microbial communities, with Proteobacteria, Actinobacteria and Firmicutes being the most affected taxa. The assessment of selected soil enzyme activities (dehydrogenase, urease and β-glucosidase) indicated an increase of metabolic functioning especially in soils treated with MSWC (e.g. dehydrogenase activity increased up to 19.5-fold in the most contaminated soil treated with MSWC). Finally, the microbial and biochemical features of treated (and untreated) contaminated soils (i.e. total bacterial counts, catabolic potential and versatility and soil enzyme activities) were highly correlated with the concentrations of labile As and TM in these latter soils and supported a clear role of the tested amendments (especially MSWC) as As- and TM-immobilising agents.

A Novel Bacteriophage of the Order Caudovirales from the Soil Metagenome in the Area of an Underground Coal Seam Fire

A Novel Bacteriophage of the Order Caudovirales from the Soil Metagenome in the Area of an Underground Coal Seam Fire

Author Correction: Identification of a Novel Feruloyl Esterase by Functional Screening of a Soil Metagenomic Library.

The original version of this article unfortunately contained a mistake in the image and caption of Fig. 6. The corrected version of the image and caption is shown here.

Biochemical profiles of two thermostable and organic solvent-tolerant esterases derived from a compost metagenome.

Owing to the functional versatility and potential applications in industry, interest in lipolytic enzymes tolerant to organic solvents is increasing. In this study, functional screening of a compost soil metagenome resulted in identification of two lipolytic genes, est1 and est2, encoding 270 and 389 amino acids, respectively. The two genes were heterologously expressed and characterized. Est1 and Est2 are thermostable enzymes with optimal enzyme activities at 80 and 70°C, respectively. A second-order rotatable design, which allows establishing the relationship between multiple variables with the obtained responses, was used to explore the combined effects of temperature and pH on esterase stability. The response curve indicated that Est1, and particularly Est2, retained high stability within a broad range of temperature and pH values. Furthermore, the effects of organic solvents on Est1 and Est2 activities and stabilities were assessed. Notably, Est2 activity was significantly enhanced (two- to tenfold) in the presence of ethanol, methanol, isopropanol, and 1-propanol over a concentration range between 6 and 30% (v/v). For the short-term stability (2h of incubation), Est2 exhibited high tolerance against 60% (v/v) of ethanol, methanol, isopropanol, DMSO, and acetone, while Est1 activity resisted these solvents only at lower concentrations (below 30%, v/v). Est2 also displayed high stability towards some water-immiscible organic solvents, such as ethyl acetate, diethyl ether, and toluene. With respect to long-term stability, Est2 retained most of its activity after 26days of incubation in the presence of 30% (v/v) ethanol, methanol, isopropanol, DMSO, or acetone. All of these features indicate that Est1 and Est2 possess application potential.

An alkaline active feruloyl-CoA synthetase from soil metagenome as a potential key enzyme for lignin valorization strategies.

Ferulic acid (FA), a low-molecular weight aromatic compound derived from lignin, represents a high-value molecule, used for applications in the cosmetic and pharmaceutical industries. FA can be further enzymatically converted in other commercially interesting molecules, such as vanillin and bioplastics. In several organisms, these transformations often start with a common step of FA activation via CoA-thioesterification, catalyzed by feruloyl-CoA synthetases (Fcs). In this context, these enzymes are of biotechnological interest for conversion of lignin-derived FA into high value chemicals. In this study, we describe the first structural characterization of a prokaryotic Fcs, named FCS1, isolated from a lignin-degrading microbial consortium. The FCS1 optimum pH and temperature were 9 and 37°C, respectively, with Km of 0.12 mM and Vmax of 36.82 U/mg. The circular dichroism spectra indicated a notable secondary structure stability at alkaline pH values and high temperatures. This secondary structure stability corroborates the activity data, which remains high until pH 9. The Small Angle X-Ray Scattering analyses resulted on the tertiary/quaternary structure and the low-resolution envelope in solution of FCS1, which was modeled as a homodimer using the hyperthermophilic nucleoside diphosphate-forming acetyl-CoA synthetase from Candidatus Korachaeum cryptofilum. This study contributes to the field of research by establishing the first biophysical and structural characterization for Fcs, and our data may be used for comparison against novel enzymes of this class that to be studied in the future.