Diversity and abundance of phosphonate biosynthetic genes in nature

Abstract

Phosphonates, molecules containing direct carbon-phosphorus bonds, compose a structurally diverse class of natural products with interesting and useful biological properties. Although their synthesis in protozoa was discovered more than 50 y ago, the extent and diversity of phosphonate production in nature remains poorly characterized. The rearrangement of phosphoenolpyruvate (PEP) to phosphonopyruvate, catalyzed by the enzyme PEP mutase (PepM), is shared by the vast majority of known phosphonate biosynthetic pathways. Thus, the pepM gene can be used as a molecular marker to examine the occurrence and abundance of phosphonate-producing organisms. Based on the presence of this gene, phosphonate biosynthesis is common in microbes, with ∼5% of sequenced bacterial genomes and 7% of genome equivalents in metagenomic datasets carrying pepM homologs. Similarly, we detected the pepM gene in ∼5% of random actinomycete isolates. The pepM-containing gene neighborhoods from 25 of these isolates were cloned, sequenced, and compared with those found in sequenced genomes. PEP mutase sequence conservation is strongly correlated with conservation of other nearby genes, suggesting that the diversity of phosphonate biosynthetic pathways can be predicted by examining PEP mutase diversity. We used this approach to estimate the range of phosphonate biosynthetic pathways in nature, revealing dozens of discrete groups in pepM amplicons from local soils, whereas hundreds were observed in metagenomic datasets. Collectively, our analyses show that phosphonate biosynthesis is both diverse and relatively common in nature, suggesting that the role of phosphonate molecules in the biosphere may be more important than is often recognized.

Discovery of phosphonic acid natural products by mining the genomes of 10,000 actinomycetes

“…A collection of over 10,000 actinomycetes, composed of ca. 2,500 isolated in our laboratory (23) and nearly 7,500 from the Agricultural Research Service (ARS) Culture Collection (NRRL) at the National Center for Agricultural Utilization Research, were screened for the presence of pepM. The NRRL collection houses the largest public repository of Actinobacteria, encompassing species from 117 different genera, important industrial strains, and the retired strain collections of several prominent streptomycete researchers (e.g., S. A. Waksman, D. Gottlieb, E. B. Shirling).…”

“…acute cystitis; FR-900098 and fosmidomycin, antimicrobials undergoing clinical trials for malaria; and phosphinothricin, the active component in several commercial herbicides (Liberty, Basta, and Rely). Second, the methodology needed for gene-based discovery of phosphonate biosynthetic loci has been rigorously established (17)(18)(19)(20)(21)(22)(23). This method relies on the fact that all but two characterized phosphonate biosynthetic pathways begin with the enzyme phosphoenolpyruvate (PEP) mutase (encoded by pepM) (14).…”

“…Thus, amplification of an internal fragment of pepM with degenerate PCR primers is sufficient to identify strains or plasmid clones that encode phosphonate biosynthetic pathways. Third, gene-based surveys have proven that phosphonate biosynthesis is relatively common in microorganisms, with the largest reservoir of unexplored pathways observed within Actinobacteria (23). Finally, the carbon-phosphorus bond endows phosphonates with unique chemical properties that enable their chemispecific detection from complex mixtures of metabolites using MS and NMR spectroscopy, even when the structure of the molecule in question is unknown (24,25).…”

“…Therefore, the genomic data were used to identify, classify, and dereplicate phosphonate biosynthetic pathways within the candidate strains before characterization of the natural products they produce. For this process, we used two established methods for linking genetic data to phosphonate natural product diversity (23). Initially, we examined the relatedness of the PepM proteins encoded by the 278 strains.…”

Purification and Characterization of Phosphonoglycans from Glycomyces sp. Strain NRRL B-16210 and Stackebrandtia nassauensis NRRL B-16338

“…Recent evidence suggests that ca. 5% of all microorganisms have the genetic capacity to produce phosphonates (3). Moreover, it is likely that these molecules play a significant role in the global phosphorus cycle (3)(4)(5).…”