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

Abstract

Although natural products have been a particularly rich source of human medicines, activity-based screening results in a very high rate of rediscovery of known molecules. Based on the large number of natural product biosynthetic genes in microbial genomes, many have proposed "genome mining" as an alternative approach for discovery efforts; however, this idea has yet to be performed experimentally on a large scale. Here, we demonstrate the feasibility of large-scale, high-throughput genome mining by screening a collection of over 10,000 actinomycetes for the genetic potential to make phosphonic acids, a class of natural products with diverse and useful bioactivities. Genome sequencing identified a diverse collection of phosphonate biosynthetic gene clusters within 278 strains. These clusters were classified into 64 distinct groups, of which 55 are likely to direct the synthesis of unknown compounds. Characterization of strains within five of these groups resulted in the discovery of a new archetypical pathway for phosphonate biosynthesis, the first (to our knowledge) dedicated pathway for H-phosphinates, and 11 previously undescribed phosphonic acid natural products. Among these compounds are argolaphos, a broad-spectrum antibacterial phosphonopeptide composed of aminomethylphosphonate in peptide linkage to a rare amino acid N(5)-hydroxyarginine; valinophos, an N-acetyl l-Val ester of 2,3-dihydroxypropylphosphonate; and phosphonocystoximate, an unusual thiohydroximate-containing molecule representing a new chemotype of sulfur-containing phosphonate natural products. Analysis of the genome sequences from the remaining strains suggests that the majority of the phosphonate biosynthetic repertoire of Actinobacteria has been captured at the gene level. This dereplicated strain collection now provides a reservoir of numerous, as yet undiscovered, phosphonate natural products.

Keywords: antibiotic; genome mining; natural products; phosphonic acid.

Fig. 1.

Diversity of phosphonate biosynthesis in Actinobacteria. (A) Maximum-likelihood phylogeny of actinomycete PepMs using methylisocitrate lyase as the outgroup (*). Complete labeling of strain names is provided in SI Appendix, Fig. S1. (B) Network diagram of phosphonate gene clusters. In both diagrams, highlighting indicates clades and strains containing pathways for previously described phosphonates (blue) and those phosphonates newly isolated in this study (red). Groups encoding known phosphonate natural products are as follows: a, hydroxynitrilaphos; b, phosphonocystoximate; c, phosphonothrixin; d, valinophos; e, argolaphos; f, dehydrophos; g, fosfazinomycin; h, fosmidomycin; i, plumbemycins and phosacetamycin; j, FR-900098; k, phosphinothricin peptides; l, DMPT; m, fosfomycin; n, 2-hydroxyethylphosphonate polyglycans. Phosphonate gene clusters are available (www.igb.illinois.edu/labs/metcalf/gcf/Phosphonates.html).

Fig. 2.

S. monomycini B-24309. (A) Isolated compounds. (B) Putative argolaphos gene cluster. Core genes encoding for enzymes predicted for AmPn biosynthesis are shown in dark blue (pepM in red). (C) Growth inhibition of E. coli WM6242 by 40 nmol of AmPn and argolaphos in a disk diffusion assay, with (Top) and without (Bottom) isopropyl-β-d-thiogalactopyranoside (IPTG) induction of phosphonate-specific transporters.

Fig. 3.

Comparisons between phosphonocystoximic acid and hydroxynitrilaphos biosynthesis. (A) Putative gene clusters. The majority of genes are shared between the two pathways, with some notable differences. Genes for α-ketoglutarate–dependent dioxygenase (magenta) and PnPy decarboxylase (purple) are absent from the phosphonocystoximate gene cluster, which instead contains a gene for pyridoxamine-phosphate oxidase (yellow). Half of the shared genes encoding the two pathways are inverted relative to each other. Other labeled genes putatively encode for PepM (red), mycothiol transferase (blue, outlined in red), N-hydroxylase (green), and peptidases (brown). (B) Proposed biosynthetic pathways for phosphonocystoximic acids and cyanomethylphosphonic acids. (C) Proposed biosynthetic pathway for glucosinolates, adapted from Halkier and Gershenzon (41). Several intermediates in glucosinolate biosynthesis and metabolism share similarities to the compounds from these pathways. Glutathione is the source of Cys (orange) in glucosinolate biosynthesis, whereas mycothiol (orange and blue) may contribute N-acetylcysteine in phosphonocystoximic acid biosynthesis.

Fig. 4.

S. durhamensis B-3309. (A) Isolated compounds and proposed pathway for valinophos biosynthesis. (B) Putative Vlp gene cluster, with the genetic cassette conserved in phosphonothrixin biosynthesis indicated by brackets. HO-PPA, 2-hydroxy-3-oxopropylphosphonic acid; HOP-PPA, 2-hydroxy-3-oxo-3-phosphoxypropylphosphonic acid; 3-PG, 3-phosphoglycerate.

Fig. 5.

Nonomuraea B-24552. (A) Putative H-phosphinate biosynthetic genes. Core genes encoding for predicted enzymes in DMPT biosynthesis are shown in dark blue (pepM is shown in red). (B) Isolated compounds. (C) ¹H-decoupled ³¹P NMR analysis of spent medium revealing several compounds with chemical shifts characteristic of H-phosphinates. (D) ¹H-coupled ³¹P NMR spectra of concentrated extract. The four largest signals (i–iv) exhibit distinctive splitting patterns and coupling constants consistent with H-phosphinates (i, J_H-P = 505 Hz; ii, J_H-P = 512 Hz; iii, J_H-P = 518 Hz; iv, J_H-P = 519 Hz).

Fig. 6.

Extrapolation of unique phosphonate natural product gene cluster families. Calculations were based on amino acid (blue) or gene cluster family (red) data. The current extent of sampling is shown with filled circles. Extrapolation is based on discovery of pepM⁺ strains, equivalent to sampling of an additional 40,000 actinomycetes in the same manner as the 10,000 screened for this work. Shaded areas show the 95% confidence intervals for the analyses.