Discovery of 3-Formyl-Tyrosine Metabolites from Pseudoalteromonas tunicata through Heterologous Expression

Genome mining and identification of natural product gene clusters typically relies on the presence of canonical nonribosomal polypeptide synthetase (NRPS) or polyketide synthase (PKS) domains. Recently, other condensation enzymes, such as the ATP-grasp ligases, have been recognized as important players in natural product biosynthesis. In this study, sequence based searching for homologues of DdaF, the ATP-grasp amide ligase from dapdiamide biosynthesis, led to the identification of a previously unannotated biosynthetic gene cluster in Pseudoalteromonas tunicata. Heterologous expression of the cluster in Escherichia coli allowed for the production and structure determination of two new 3-formyl tyrosine metabolites.Molecular and Cellular Biolog

Dapdiamides, Tripeptide Antibiotics Formed by Unconventional Amide Ligases†

Construction of a genomic DNA library from Pantoea agglomerans strain CU0119 and screening against the plant pathogen Erwinia amylovora yielded a new family of antibiotics, dapdiamides A-E (1-5). The structures were established through 2D-NMR experiments and mass spectrometry, as well as the synthesis of dapdiamide A (1). Transposon mutagenesis of the active cosmid allowed identification of the biosynthetic gene cluster. The dapdiamide family's promiscuous biosynthetic pathway contains two unconventional amide ligases that are predicted to couple its constituent monomers

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

Microbial Biotechnolog

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/

Investigations into the biosynthesis, regulation, and self-resistance of toxoflavin in Pseudomonas protegens Pf-5

Pseudomonas spp. are prolific producers of natural products from many structural classes. Here we show that the soil bacterium Pseudomonas protegens Pf-5 is capable of producing trace levels of the triazine natural product toxoflavin (1) under microaerobic conditions. We evaluated toxoflavin production by derivatives of Pf-5 with deletions in specific biosynthesis genes, which led us to propose a revised biosynthetic pathway for toxoflavin that shares the first two steps with riboflavin biosynthesis. We also report that toxM, which is not present in the well-characterized cluster of Burkholderia glumae, encodes a monooxygenase that degrades toxoflavin. The toxoflavin degradation product of ToxM is identical to that of TflA, the toxoflavin lyase from Paenibacillus polymyxa. Toxoflavin production by P. protegens causes inhibition of several plant-pathogenic bacteria, and introduction of toxM into the toxoflavin-sensitive strain Pseudomonas syringae DC3000 results in resistance to toxoflavin

Genetic Variation of Adenylation Domains of the Anabaenopeptin Synthesis Operon and Evolution of Substrate Promiscuity▿†

Anabaenopeptins (AP) are bioactive cyclic hexapeptides synthesized nonribosomally in cyanobacteria. APs are characterized by several conserved motifs, including the ureido bond, N-methylation in position 5, and d-Lys in position 2. All other positions of the AP molecule are variable, resulting in numerous structural variants. We have identified a nonribosomal peptide synthetase (NRPS) operon from Planktothrix agardhii strain CYA126/8 consisting of five genes (apnA to apnE) encoding six NRPS modules and have confirmed its role in AP synthesis by the generation of a mutant via insertional inactivation of apnC. In order to correlate the genetic diversity among adenylation domains (A domains) with AP structure variation, we sequenced the A domains of all six NRPS modules from seven Planktothrix strains differing in the production of AP congeners. It is remarkable that single strains coproduce APs bearing either of the chemically divergent amino acids Arg and Tyr in exocyclic position 1. Since the A domain of the initiation module (the ApnA A1 domain) has been proposed to activate the amino acid incorporated into exocyclic position 1, we decided to analyze this domain both biochemically and phylogenetically. Only ApnA A1 enzymes from strains producing AP molecules containing Arg or Tyr in position 1 were found to activate these two chemically divergent amino acids in vitro. Phylogenetic analysis of apn A domain sequences revealed that strains with a promiscuous ApnA A1 domain are derived from an ancestor that activates only Arg. Surprisingly, positive selection appears to affect only three codons within the apnA A1 gene, suggesting that this remarkable promiscuity has evolved from point mutations only