The Natural Product Domain Seeker version 2 (NaPDoS2) webtool relates ketosynthase phylogeny to biosynthetic function.

The Natural Product Domain Seeker (NaPDoS) webtool detects and classifies ketosynthase (KS) and condensation domains from genomic, metagenomic, and amplicon sequence data. Unlike other tools, a phylogeny-based classification scheme is used to make broader predictions about the polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes in which these domains are found. NaPDoS is particularly useful for the analysis of incomplete biosynthetic genes or gene clusters, as are often observed in poorly assembled genomes and metagenomes, or when loci are not clustered, as in eukaryotic genomes. To help support the growing interest in sequence-based analyses of natural product biosynthetic diversity, here we introduce version 2 of the webtool, NaPDoS2, available at http://napdos.ucsd.edu/napdos2. This update includes the addition of 1417 KS sequences, representing a major expansion of the taxonomic and functional diversity represented in the webtool database. The phylogeny-based KS classification scheme now recognizes 41 class and subclass assignments, including new type II PKS subclasses. Workflow modifications accelerate run times, allowing larger datasets to be analyzed. In addition, default parameters were established using statistical validation tests to maximize KS detection and classification accuracy while minimizing false positives. We further demonstrate the applications of NaPDoS2 to assess PKS biosynthetic potential using genomic, metagenomic, and PCR amplicon datasets. These examples illustrate how NaPDoS2 can be used to predict biosynthetic potential and detect genes involved in the biosynthesis of specific structure classes or new biosynthetic mechanisms.

Structure-function analysis of a new PL17 oligoalginate lyase from the marine bacterium Zobellia galactanivorans DsijT.

Alginate is a major compound of brown macroalgae and as such an important carbon and energy source for heterotrophic marine bacteria. Despite the rather simple composition of alginate only comprising mannuronate and guluronate units, these bacteria feature complex alginolytic systems that can contain up to seven alginate lyases. This reflects the necessity of large enzyme systems for the complete degradation of the abundant substrate. Numerous alginate lyases have been characterized. They belong to different polysaccharide lyase (PL) families, but only one crystal structure of a family 17 (PL17) alginate lyase has been reported to date, namely Alg17c from the gammaproteobacterium Saccharophagus degradans. Biochemical and structural characterizations are helpful to link sequence profiles to function, evolution of functions and niche-specific characteristics. Here, we combined detailed biochemical and crystallographic analysis of AlyA3, a PL17 alginate lyase from the marine flavobacteria Zobellia galactanivorans DsijT, providing the first structure of a PL17 in the Bacteroidetes phylum. AlyA3 is exo-lytic and highly specific of mannuronate stretches. As part of an "alginate utilizing locus", its activity is complementary to that of other characterized alginate lyases from the same bacterium. Structural comparison with Alg17c highlights a common mode of action for exo-lytic cleavage of the substrate, strengthening our understanding of the PL17 catalytic mechanism. We show that unlike Alg17c, AlyA3 contains an inserted flexible loop at the entrance to the catalytic groove, likely involved in substrate recognition, processivity and turn over.

Alginate Degradation: Insights Obtained through Characterization of a Thermophilic Exolytic Alginate Lyase.

Enzymatic depolymerization of seaweed polysaccharides is gaining interest for the production of functional oligosaccharides and fermentable sugars. Herein, we describe a thermostable alginate lyase that belongs to polysaccharide lyase family 17 (PL17) and was derived from an Arctic Mid-Ocean Ridge (AMOR) metagenomics data set. This enzyme, AMOR_PL17A, is a thermostable exolytic oligoalginate lyase (EC 4.2.2.26), which can degrade alginate, poly-ß-d-mannuronate, and poly-α-l-guluronate within a broad range of pHs, temperatures, and salinity conditions. Site-directed mutagenesis showed that tyrosine Y251, previously suggested to act as a catalytic acid, indeed is essential for catalysis, whereas mutation of tyrosine Y446, previously proposed to act as a catalytic base, did not affect enzyme activity. The observed reaction products are protonated and deprotonated forms of the 4,5-unsaturated uronic acid monomer, Δ, two hydrates of DEH (4-deoxy-l-erythro-5-hexulosuronate), which are formed after ring opening, and, finally, two epimers of a 5-member hemiketal called 4-deoxy-d-manno-hexulofuranosidonate (DHF), formed through intramolecular cyclization of hydrated DEH. The detection and nuclear magnetic resonance (NMR) assignment of these hemiketals refine our current understanding of alginate degradation.IMPORTANCE The potential markets for seaweed-derived products and seaweed processing technologies are growing, yet commercial enzyme cocktails for complete conversion of seaweed to fermentable sugars are not available. Such an enzyme cocktail would require the catalytic properties of a variety of different enzymes, where fucoidanases, laminarinases, and cellulases together with endo- and exo-acting alginate lyases would be the key enzymes. Here, we present an exo-acting alginate lyase that efficiently produces monomeric sugars from alginate. Since it is only the second characterized exo-acting alginate lyase capable of degrading alginate at a high industrially relevant temperature (≥60°C), this enzyme may be of great biotechnological and industrial interest. In addition, in-depth NMR-based structural elucidation revealed previously undescribed rearrangement products of the unsaturated monomeric sugars generated from exo-acting lyases. The insight provided by the NMR assignment of these products facilitates future assessment of product formation by alginate lyases.

CRISPR-Cas9 driven structural elucidation of the heteroexopolysaccharides from Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is a Gram-positive soil bacterium known for producing a wide range of exopolysaccharides. However, due to the biopolymer's complexity, structural elucidation has so far been inconclusive. Combinatorial knock-outs of glycosyltransferases were generated in order to separate distinct polysaccharides produced by P. polymyxa. Using a complementary analytical approach consisting of carbohydrate fingerprints, sequence analysis, methylation analysis as well as NMR spectroscopy, the structure of the repeating units of two additional heteroexopolysaccharides termed paenan I and paenan III were elucidated. Results for paenan I identified a trisaccharide backbone consisting of 1➔4-ß-d-Glc, 1➔4-ß-d-Man and a 1,3,4-branching ß-d-Gal residue with a sidechain comprising of a terminal ß-d-Gal3,4-Pyr and 1➔3-ß-d-Glc. For paenan III, results indicated a backbone consisting of 1➔3-ß-d-Glc, 1,3,4-linked α-d-Man and 1,3,4-linked α-d-GlcA. NMR analysis indicated monomeric ß-d-Glc and α-d-Man sidechains for the branching Man and GlcA residues respectively.

Metagenomic Data Reveal Type I Polyketide Synthase Distributions Across Biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically important natural products, yet only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be delineated into different classes and subclasses based on domain organization and structural features of the compounds encoded. Notably, phylogenetic relationships among PKS ketosynthase (KS) domains provide a method to classify the larger and more complex genes in which they occur. Increased access to large metagenomic datasets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through the analysis of KS domain sequences. Here, we used the webtool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse biomes. We found biome-specific separation with soils enriched in modular cis -AT and hybrid cis -AT KSs relative to other biomes and marine sediments enriched in KSs associated with PUFA and enediyne biosynthesis. By extracting full-length KS domains, we linked the phylum Actinobacteria to soil-specific enediyne and cis -AT clades and identified enediyne and monomodular KSs in phyla from which the associated compound classes have not been reported. These sequences were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting novel structures or enzyme functions remain to be discovered. Lastly, we employed our metagenome-extracted KS domains to evaluate commonly used type I KS PCR primers and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. Importance: Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis coupled with the accumulation of metagenomic sequence data provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the webtool NaPDoS2 to assess type I PKS diversity and distributions by detecting and classifying KS domains across 137 metagenomes. We show that biomes are differentially enriched in KS domain classes, providing a roadmap for future biodiscovery strategies. Further, KS phylogenies reveal both biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS dataset allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across earth's major biomes.

Metagenomic data reveals type I polyketide synthase distributions across biomes.

Microbial polyketide synthase (PKS) genes encode the biosynthesis of many biomedically or otherwise commercially important natural products. Despite extensive discovery efforts, metagenomic analyses suggest that only a small fraction of nature's polyketide biosynthetic potential has been realized. Much of this potential originates from type I PKSs (T1PKSs), which can be further delineated based on their domain organization and the structural features of the compounds they encode. Notably, phylogenetic relationships among ketosynthase (KS) domains provide an effective method to classify the larger and more complex T1PKS genes in which they occur. Increased access to large metagenomic data sets from diverse habitats provides opportunities to assess T1PKS biosynthetic diversity and distributions through their smaller and more tractable KS domain sequences. Here, we used the web tool NaPDoS2 to detect and classify over 35,000 type I KS domains from 137 metagenomic data sets reported from eight diverse, globally distributed biomes. We found biome-specific separation with soils enriched in KSs from modular cis-acetyltransferase (AT) and hybrid cis-AT KSs relative to other biomes and marine sediments enriched in KSs associated with polyunsaturated fatty acid and enediyne biosynthesis. We linked the phylum Actinobacteria to soil-derived enediyne and cis-AT KSs while marine-derived KSs associated with enediyne and monomodular PKSs were linked to phyla from which the compounds produced by these biosynthetic enzymes have not been reported. These KSs were phylogenetically distinct from those associated with experimentally characterized PKSs suggesting they may be associated with novel structures or enzyme functions. Finally, we employed our metagenome-extracted KS domains to evaluate the PCR primers commonly used to amplify type I KSs and identified modifications that could increase the KS sequence diversity recovered from amplicon libraries. IMPORTANCE Polyketides are a crucial source of medicines, agrichemicals, and other commercial products. Advances in our understanding of polyketide biosynthesis, coupled with the increased availability of metagenomic sequence data, provide new opportunities to assess polyketide biosynthetic potential across biomes. Here, we used the web tool NaPDoS2 to assess type I polyketide synthase (PKS) diversity and distributions by detecting and classifying ketosynthase (KS) domains across 137 metagenomes. We show that biomes are differentially enriched in type I KS domains, providing a roadmap for future biodiscovery strategies. Furthermore, KS phylogenies reveal biome-specific clades that do not include biochemically characterized PKSs, highlighting the biosynthetic potential of poorly explored environments. The large metagenome-derived KS data set allowed us to identify regions of commonly used type I KS PCR primers that could be modified to capture a larger extent of environmental KS diversity. These results facilitate both the search for novel polyketides and our understanding of the biogeographical distribution of PKSs across Earth's major biomes.

Investigation of the structural and immunomodulatory properties of alkali-soluble ß-glucans from Pleurotus eryngii fruiting bodies.

Fungal ß-glucans have received a lot of interest due to their proinflammatory activity towards cells of the innate immune system. Although commonly described as (1➔3)-ß-glucans with varying degree of (1➔6)-branching, the fungal ß-glucans constitute a diverse polysaccharide class. In this study, the alkali-soluble ß-glucans from the edible mushroom Pleurotus eryngii were extracted and characterized by GC, GC-MS and 2D NMR analyses. The extracts contain several structurally different polysaccharides, including a (1➔3)-ß-d-glucan with single glucose units attached at O-6, and a (1➔6)-ß-d-glucan, possibly branched at O-3. The immunomodulatory activities of the P. eryngii extracts were assessed by investigating their ability to bind to the receptor dectin-1, and their ability to induce production of the proinflammatory cytokines TNF-α, IL-6 and IL-1ß in LPS-differentiated THP-1 cells. Although the samples were able to bind to the dectin-1a receptor, they did not induce production of significant levels of cytokines in the THP-1 cells. Positive controls of yeast-derived (1➔3)-ß-d-glucans with branches at O-6 induced cytokine production in the cells. Thus, it appears that the P. eryngii ß-glucans are unable to induce production of proinflammatory cytokines in LPS-differentiated THP-1 cells, despite being able to activate the human dectin-1a receptor.

Visible light-exposed lignin facilitates cellulose solubilization by lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) catalyze oxidative cleavage of crystalline polysaccharides such as cellulose and are crucial for the conversion of plant biomass in Nature and in industrial applications. Sunlight promotes microbial conversion of plant litter; this effect has been attributed to photochemical degradation of lignin, a major redox-active component of secondary plant cell walls that limits enzyme access to the cell wall carbohydrates. Here, we show that exposing lignin to visible light facilitates cellulose solubilization by promoting formation of H2O2 that fuels LPMO catalysis. Light-driven H2O2 formation is accompanied by oxidation of ring-conjugated olefins in the lignin, while LPMO-catalyzed oxidation of phenolic hydroxyls leads to the required priming reduction of the enzyme. The discovery that light-driven abiotic reactions in Nature can fuel H2O2-dependent redox enzymes involved in deconstructing lignocellulose may offer opportunities for bioprocessing and provides an enzymatic explanation for the known effect of visible light on biomass conversion.

Structural elucidation of the fucose containing polysaccharide of Paenibacillus polymyxa DSM 365.

Paenibacillus polymyxa is an avid producer of exopolysaccharides of industrial interest. However, due to the complexity of the polymer composition, structural elucidation of the polysaccharide remained unfeasible for a long time. By using a CRISPR-Cas9 mediated knock-out strategy, all single glycosyltransferases as well as the Wzy polymerases were individually deleted in the corresponding gene cluster for the first time. Thereby, it was observed that the main polymer fraction was completely suppressed (or deleted) and a pure minor fucose containing polysaccharide could be isolated, which was named paenan II. Applying this combinatorial approach, the monosaccharide composition, sequence and linkage pattern of this novel polymer was determined via HPLC-MS, GC-MS and NMR. Furthermore, we demonstrated that the knock-out of the glycosyltransferases PepQ, PepT, PepU and PepV as well as of the Wzy polymerase PepG led to the absence of paenan II, attributing those enzymes to the assembly of the repeating unit.