MIBiG 3.0 : a community-driven effort to annotate experimentally validated biosynthetic gene clusters

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/

antiSMASH v7.0.1 modified for petrobactin BGC detection

A custom version of antiSMASH v7.0.1, modified to add a specific detection rule for homologs of the petrobactin biosynthetic genes asbABCDE. A BGC region meets the “AsbABCDE” rule if it contained matches to existing antiSMASH models for IucA_IucC (asbA/asbB), AMP-binding (asbC), and PP-binding (asbD), as well as the Pfam model for DUF6005 (asbE, PF19468.2)

Genome Mining for Microbial Siderophores: Improving Structural Predictions

Nearly all life requires iron; however, under aerobic conditions, Fe(III) is nearly insoluble. To meet their metabolic needs, many bacteria synthesize siderophores: small-molecule, high-affinity Fe(III) chelators. The discovery of new siderophores has been streamlined by genome mining, where bacterial genomes of interest are scanned for siderophore biosynthetic gene clusters (BGCs). This dissertation is focused on the development of new bioinformatics tools to improve siderophore structural predictions, as well as the application of those tools towards traditional natural product isolation.Some peptidic siderophores contain the chelating ligand β-hydroxyaspartate (β-OHAsp), which provides bidentate OO′ coordination to Fe(III). Current genome mining methods cannot reliably predict which aspartate residues will be hydroxylated, nor the resulting stereochemistry, leaving structural ambiguities which must be rectified experimentally. Through coupling BGCs with verified structures, the origin of the β-OHAsp diastereomers in siderophores is reported. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate–dependent aspartyl β-hydroxylases were identified in siderophore BGCs. Each aspartyl β-hydroxylase subtype effects distinct diastereoselectivity. A previously undescribed, noncanonical member of the nonribosomal peptide synthetase (NRPS) condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized.The DHB-CAA-Ser family of triscatechol siderophores each contain a different cationic amino acid (CAA = D-Arg, D-Lys, or L-Orn), hinting at the existence of their diastereomers with L-Arg, L-Lys, and D-Orn. With no knowledge of which bacteria, if any, are capable of their production, a novel high-throughput genome mining workflow was developed, the Catechol Siderophore Cluster Analysis (CatSCAn). Over 11,000 bacterial genomes were scanned for siderophore BGCs, revealing about 1% potentially capable of the biosynthesis of the combinatoric suite of DHB-CAA-Ser siderophores with L- and D-CAA. The NRPS from Marinomonas sp. TW1 was found to be unusually promiscuous, producing a relatively rare example of a suite of siderophores with D-Arg, D-Orn, and L-Orn amino acids.CatSCAn produced a rich dataset of putative BGCs that may be coupled with structurally- characterized siderophores to study the chemistry and biology of triscatechol siderophores. A detailed analysis of BGCs highlighted key features within siderophore pathways. Mapping known and predicted siderophores onto a phylogenetic tree of NRPS proteins reveals repeated changes in CAA stereochemistry and identity within the DHB-CAA-Ser family. Parsimony suggests that the ancestor of all DHB-CAA-Ser siderophores was trivanchrobactin, (DHB-DArg-Ser)3. A model of DHB-CAA-Ser siderophore evolution is presented where enzyme promiscuity allows for the biosynthesis of new siderophores.Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin, butanochelin, and pentanochelin. This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(III) and may be further modified via thiol–ene or azide–alkyne click chemistry

Precursor-directed biosynthesis of catechol compounds in Acinetobacter bouvetii DSM 14964.

Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin (1), butanochelin (2), and pentanochelin (3). This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(iii) and may be further modified via thiol-ene or azide-alkyne click chemistry

Precursor-directed biosynthesis of catechol compounds in Acinetobacter bouvetii DSM 14964.

Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin (1), butanochelin (2), and pentanochelin (3). This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(iii) and may be further modified via thiol-ene or azide-alkyne click chemistry

Genome mining strategies for metallophore discovery

Many bacteria use small-molecule chelators called metallophores to acquire trace metals from their environment. These molecules play a central role in interactions between bacteria, plants, and animals. Hence, knowing their full diversity is key to combatting infectious diseases as well as harnessing beneficial microbial communities. Metallophore discovery has been streamlined by advances in genome mining, where genomes are scanned for genes involved in metallophore biosynthesis. This review highlights recent trends and advances in predicting the presence and structure of metallophores based solely on genomic information. Recent work suggests new families of metallophores remain hidden from current homology-based approaches. Their discovery will require new genome mining approaches that move beyond biosynthesis to consider metallophore transporters, regulation, and evolution

Genomics-driven discovery of chiral triscatechol siderophores with enantiomeric Fe(iii) coordination.

Ferric complexes of triscatechol siderophores may assume one of two enantiomeric configurations at the iron site. Chirality is known to be important in the iron uptake process, however an understanding of the molecular features directing stereospecific coordination remains ambiguous. Synthesis of the full suite of (DHBL/DLysL/DSer)3 macrolactone diastereomers, which includes the siderophore cyclic trichrysobactin (CTC), enables the effects that the chirality of Lys and Ser residues exert on the configuration of the Fe(iii) complex to be defined. Computationally optimized geometries indicate that the Λ/Δ configurational preferences are set by steric interactions between the Lys sidechains and the peptide backbone. The ability of each (DHBL/DLysL/DSer)3 diastereomer to form a stable Fe(iii) complex prompted a genomic search for biosynthetic gene clusters (BGCs) encoding the synthesis of these diastereomers in microbes. The genome of the plant pathogen Dickeya chrysanthemi EC16 was sequenced and the genes responsible for the biosynthesis of CTC were identified. A related but distinct BGC was identified in the genome of the opportunistic pathogen Yersinia frederiksenii ATCC 33641; isolation of the siderophore from Y. frederiksenii ATCC 33641, named frederiksenibactin (FSB), revealed the triscatechol oligoester, linear-(DHBLLysLSer)3. Circular dichroism (CD) spectroscopy establishes that Fe(iii)-CTC and Fe(iii)-FSB are formed in opposite enantiomeric configuration, consistent with the results of the ferric complexes of the cyclic (DHBL/DLysL/DSer)3 diastereomers

Genomics-driven discovery of chiral triscatechol siderophores with enantiomeric Fe(iii) coordination.

Ferric complexes of triscatechol siderophores may assume one of two enantiomeric configurations at the iron site. Chirality is known to be important in the iron uptake process, however an understanding of the molecular features directing stereospecific coordination remains ambiguous. Synthesis of the full suite of (DHBL/DLysL/DSer)3 macrolactone diastereomers, which includes the siderophore cyclic trichrysobactin (CTC), enables the effects that the chirality of Lys and Ser residues exert on the configuration of the Fe(iii) complex to be defined. Computationally optimized geometries indicate that the Λ/Δ configurational preferences are set by steric interactions between the Lys sidechains and the peptide backbone. The ability of each (DHBL/DLysL/DSer)3 diastereomer to form a stable Fe(iii) complex prompted a genomic search for biosynthetic gene clusters (BGCs) encoding the synthesis of these diastereomers in microbes. The genome of the plant pathogen Dickeya chrysanthemi EC16 was sequenced and the genes responsible for the biosynthesis of CTC were identified. A related but distinct BGC was identified in the genome of the opportunistic pathogen Yersinia frederiksenii ATCC 33641; isolation of the siderophore from Y. frederiksenii ATCC 33641, named frederiksenibactin (FSB), revealed the triscatechol oligoester, linear-(DHBLLysLSer)3. Circular dichroism (CD) spectroscopy establishes that Fe(iii)-CTC and Fe(iii)-FSB are formed in opposite enantiomeric configuration, consistent with the results of the ferric complexes of the cyclic (DHBL/DLysL/DSer)3 diastereomers

Genomic analysis of siderophore β-hydroxylases reveals divergent stereocontrol and expands the condensation domain family

Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the β-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate-dependent aspartyl β-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization-existing either as fused domains (IβHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TβHAsp)-and each directs opposite stereoselectivity of Asp β-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of β-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) β-OHAsp residues of alterobactin arise from hydroxylation by the β-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) β-OHAsp in delftibactin arises from the stand-alone β-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro β-OHAsp, consistent with the presence of both types of β-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate-dependent enzymes (IβHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the β-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized β-OHAsp diastereomers to the phylogenetic tree of siderophore β-hydroxylases, methods to predict β-OHAsp stereochemistry in silico are realized