Discovery of a Phosphonoacetic Acid Derived Natural Product by Pathway Refactoring

The activation of silent natural product gene clusters is a synthetic biology problem of great interest. As the rate at which gene clusters are identified outpaces the discovery rate of new molecules, this unknown chemical space is rapidly growing, as too are the rewards for developing technologies to exploit it. One class of natural products that has been underrepresented is phosphonic acids, which have important medical and agricultural uses. Hundreds of phosphonic acid biosynthetic gene clusters have been identified encoding for unknown molecules. Although methods exist to elicit secondary metabolite gene clusters in native hosts, they require the strain to be amenable to genetic manipulation. One method to circumvent this is pathway refactoring, which we implemented in an effort to discover new phosphonic acids from a gene cluster from <i>Streptomyces</i> sp. strain NRRL F-525. By reengineering this cluster for expression in the production host <i>Streptomyces lividans</i>, utility of refactoring is demonstrated with the isolation of a novel phosphonic acid, <i>O</i>-phosphonoacetic acid serine, and the characterization of its biosynthesis. In addition, a new biosynthetic branch point is identified with a phosphonoacetaldehyde dehydrogenase, which was used to identify additional phosphonic acid gene clusters that share phosphonoacetic acid as an intermediate

Cyanohydrin Phosphonate Natural Product from <i>Streptomyces regensis</i>

<i>Streptomyces regensis</i> strain WC-3744 was identified as a potential phosphonic acid producer in a large-scale screen of microorganisms for the presence of the <i>pepM</i> gene, which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate phosphonomutase. ³¹P NMR revealed the presence of several unidentified phosphonates in spent medium after growth of <i>S. regensis</i>. These compounds were purified and structurally characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric analyses. Three new phosphonic acid metabolites, whose structures were confirmed by comparison to chemically synthesized standards, were observed: (2-acetamidoethyl)­phosphonic acid (<b>1</b>), (2-acetamido-1-hydroxyethyl)­phosphonic (<b>3</b>), and a novel cyanohydrin-containing phosphonate, (cyano­(hydroxy)­methyl)­phosphonic acid (<b>4</b>). The gene cluster responsible for synthesis of these molecules was also identified from the draft genome sequence of <i>S. regensis</i>, laying the groundwork for future investigations into the metabolic pathway leading to this unusual natural product

Cyanohydrin Phosphonate Natural Product from <i>Streptomyces regensis</i>

<i>Streptomyces regensis</i> strain WC-3744 was identified as a potential phosphonic acid producer in a large-scale screen of microorganisms for the presence of the <i>pepM</i> gene, which encodes the key phosphonate biosynthetic enzyme phosphoenolpyruvate phosphonomutase. ³¹P NMR revealed the presence of several unidentified phosphonates in spent medium after growth of <i>S. regensis</i>. These compounds were purified and structurally characterized via extensive 1D and 2D NMR spectroscopic and mass spectrometric analyses. Three new phosphonic acid metabolites, whose structures were confirmed by comparison to chemically synthesized standards, were observed: (2-acetamidoethyl)­phosphonic acid (<b>1</b>), (2-acetamido-1-hydroxyethyl)­phosphonic (<b>3</b>), and a novel cyanohydrin-containing phosphonate, (cyano­(hydroxy)­methyl)­phosphonic acid (<b>4</b>). The gene cluster responsible for synthesis of these molecules was also identified from the draft genome sequence of <i>S. regensis</i>, laying the groundwork for future investigations into the metabolic pathway leading to this unusual natural product

Elucidating the Rimosamide-Detoxin Natural Product Families and Their Biosynthesis Using Metabolite/Gene Cluster Correlations

As microbial genome sequencing becomes more widespread, the capacity of microorganisms to produce an immense number of metabolites has come into better view. Utilizing a metabolite/gene cluster correlation platform, the biosynthetic origins of a new family of natural products, the rimosamides, were discovered. The rimosamides were identified in <i>Streptomyces rimosus</i> and associated with their NRPS/PKS-type gene cluster based upon their high frequency of co-occurrence across 179 strains of actinobacteria. This also led to the discovery of the related detoxin gene cluster. The core of each of these families of natural products contains a depsipeptide bond at the point of bifurcation in their unusual branched structures, the origins of which are definitively assigned to nonlinear biosynthetic pathways <i>via</i> heterologous expression in <i>Streptomyces lividans</i>. The rimosamides were found to antagonize the antibiotic activity of blasticidin S against <i>Bacillus cereus</i>

Discovery of the Antibiotic Phosacetamycin via a New Mass Spectrometry-Based Method for Phosphonic Acid Detection

Naturally occurring phosphonates such as phosphinothricin (Glufosinate, a commercially used herbicide) and fosfomycin (Monurol, a clinically used antibiotic) have proved to be potent and useful biocides. Yet this class of natural products is still an under explored family of secondary metabolites. Discovery of the biosynthetic pathways responsible for the production of these compounds has been simplified by using gene based screening approaches, but detection and identification of the natural products the genes produce have been hampered by a lack of high-throughput methods for screening potential producers under various culture conditions. Here, we present an efficient mass-spectrometric method for the selective detection of natural products containing phosphonate and phosphinate functional groups. We have used this method to identify a new phosphonate metabolite, phosacetamycin, whose structure, biological activity, and biosynthetic gene cluster are reported

A Proteomic Survey of Nonribosomal Peptide and Polyketide Biosynthesis in Actinobacteria

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a “protein-first” method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951−956), for example, improved <i>de novo</i> peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously “orphan” gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2–4 Mbp genomes) used previously

Discovery of the Tyrobetaine Natural Products and Their Biosynthetic Gene Cluster <i>via</i> Metabologenomics

Natural products (NPs) are a rich source of medicines, but traditional discovery methods are often unsuccessful due to high rates of rediscovery. Genetic approaches for NP discovery are promising, but progress has been slow due to the difficulty of identifying unique biosynthetic gene clusters (BGCs) and poor gene expression. We previously developed the metabologenomics method, which combines genomic and metabolomic data to discover new NPs and their BGCs. Here, we utilize metabologenomics in combination with molecular networking to discover a novel class of NPs, the tyrobetaines: nonribosomal peptides with an unusual trimethylammonium tyrosine residue. The BGC for this unusual class of compounds was identified using metabologenomics and computational structure prediction data. Heterologous expression confirmed the BGC and suggests an unusual mechanism for trimethylammonium formation. Overall, the discovery of the tyrobetaines shows the great potential of metabologenomics combined with molecular networking and computational structure prediction for identifying interesting biosynthetic reactions and novel NPs