Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate l-Val or l-Leu. The amino acid selectivity of PvfC and its homologues likely direct pvf signaling activity. We explored the natural diversity of the active site residues present in 92% of the adenylation domains of PvfC homologues and identified key residues for substrate selection and catalysis. Sequence similarity network (SSN) analysis revealed grouping of PvfC homologues that harbor the same active site residues and activate the same amino acids. Our work identified PvfC as a gatekeeper for the structure and bioactivity of the pvf-produced signaling molecules. The combination of active site residue identification and SSN analysis can improve the prediction of aliphatic amino acid substrates for NRPS adenylation domains.

Targeted Rediscovery and Biosynthesis of the Farnesyl-Transferase Inhibitor Pepticinnamin†E.

The natural product pepticinnamin E potently inhibits protein farnesyl transferases and has potential applications in treating cancer and malaria. Pepticinnamin E contains a rare N-terminal cinnamoyl moiety as well as several nonproteinogenic amino acids, including the unusual 2-chloro-3-hydroxy-4-methoxy-N-methyl-L-phenylalanine. The biosynthesis of pepticinnamin E has remained uncharacterized because its original producing strain is no longer available. Here we identified a gene cluster (pcm) for this natural product in a new producer, Actinobacteria bacterium OK006, by means of a targeted rediscovery strategy. We demonstrated that the pcm cluster is responsible for the biosynthesis of pepticinnamin E, a nonribosomal peptide/polyketide hybrid. We also characterized a key O-methyltransferase that modifies 3,4-dihydroxy-l-phenylalanine. Our work has identified the gene cluster for pepticinnamins for the first time and sets the stage for elucidating the unique chemistry required for biosynthesis.

Identification of the Biosynthetic Pathway for the Antibiotic Bicyclomycin.

Diketopiperazines (DKPs) make up a large group of natural products with diverse structures and biological activities. Bicyclomycin is a broad-spectrum DKP antibiotic with unique structure and function: it contains a highly oxidized bicyclic [4.2.2] ring and is the only known selective inhibitor of the bacterial transcription termination factor, Rho. Here, we identify the biosynthetic gene cluster for bicyclomycin containing six iron-dependent oxidases. We demonstrate that the DKP core is made by a tRNA-dependent cyclodipeptide synthase, and hydroxylations on two unactivated sp3 carbons are performed by two mononuclear iron, α-ketoglutarate-dependent hydroxylases. Using bioinformatics, we also identify a homologous gene cluster prevalent in a human pathogen Pseudomonas aeruginosa. We detect bicyclomycin by overexpressing this gene cluster and establish P. aeruginosa as a new producer of bicyclomycin. Our work uncovers the biosynthetic pathway for bicyclomycin and sheds light on the intriguing oxidation chemistry that converts a simple DKP into a powerful antibiotic.

Large-Scale Bioinformatics Analysis of Bacillus Genomes Uncovers Conserved Roles of Natural Products in Bacterial Physiology.

Bacteria possess an amazing capacity to synthesize a diverse range of structurally complex, bioactive natural products known as specialized (or secondary) metabolites. Many of these specialized metabolites are used as clinical therapeutics, while others have important ecological roles in microbial communities. The biosynthetic gene clusters (BGCs) that generate these metabolites can be identified in bacterial genome sequences using their highly conserved genetic features. We analyzed an unprecedented 1,566 bacterial genomes from Bacillus species and identified nearly 20,000 BGCs. By comparing these BGCs to one another as well as a curated set of known specialized metabolite BGCs, we discovered that the majority of Bacillus natural products are comprised of a small set of highly conserved, well-distributed, known natural product compounds. Most of these metabolites have important roles influencing the physiology and development of Bacillus species. We identified, in addition to these characterized compounds, many unique, weakly conserved BGCs scattered across the genus that are predicted to encode unknown natural products. Many of these "singleton" BGCs appear to have been acquired via horizontal gene transfer. Based on this large-scale characterization of metabolite production in the Bacilli, we go on to connect the alkylpyrones, natural products that are highly conserved but previously biologically uncharacterized, to a role in Bacillus physiology: inhibiting spore development. IMPORTANCEBacilli are capable of producing a diverse array of specialized metabolites, many of which have gained attention for their roles as signals that affect bacterial physiology and development. Up to this point, however, the Bacillus genus's metabolic capacity has been underexplored. We undertook a deep genomic analysis of 1,566 Bacillus genomes to understand the full spectrum of metabolites that this bacterial group can make. We discovered that the majority of the specialized metabolites produced by Bacillus species are highly conserved, known compounds with important signaling roles in the physiology and development of this bacterium. Additionally, there is significant unique biosynthetic machinery distributed across the genus that might lead to new, unknown metabolites with diverse biological functions. Inspired by the findings of our genomic analysis, we speculate that the highly conserved alkylpyrones might have an important biological activity within this genus. We go on to validate this prediction by demonstrating that these natural products are developmental signals in Bacillus and act by inhibiting sporulation.

Direct Capture Technologies for Genomics-Guided Discovery of Natural Products.

Microbes are important producers of natural products, which have played key roles in understanding biology and treating disease. However, the full potential of microbes to produce natural products has yet to be realized; the overwhelming majority of natural product gene clusters encoded in microbial genomes remain "cryptic", and have not been expressed or characterized. In contrast to the fast-growing number of genomic sequences and bioinformatic tools, methods to connect these genes to natural product molecules are still limited, creating a bottleneck in genome-mining efforts to discover novel natural products. Here we review developing technologies that leverage the power of homologous recombination to directly capture natural product gene clusters and express them in model hosts for isolation and structural characterization. Although direct capture is still in its early stages of development, it has been successfully utilized in several different classes of natural products. These early successes will be reviewed, and the methods will be compared and contrasted with existing traditional technologies. Lastly, we will discuss the opportunities for the development of direct capture in other organisms, and possibilities to integrate direct capture with emerging genome-editing techniques to accelerate future study of natural products.