Use of a Phosphonate Methyltransferase in the Identification of the Fosfazinomycin Biosynthetic Gene Cluster

Abstract

Natural product discovery has been boosted by genome mining approaches, but compound purification is often still challenging. We report an enzymatic strategy for “stable isotope labeling of phosphonates in extract” (SILPE) that facilitates their purification. We used the phosphonate methyltransferase DhpI involved in dehydrophos biosynthesis to methylate a variety of phosphonate natural products in crude spent medium with a mixture of labeled and unlabeled S‐adenosyl methionine. Mass‐guided fractionation then allowed straightforward purification. We illustrate its utility by purifying a phosphonate that led to the identification of the fosfazinomycin biosynthetic gene cluster. This unusual natural product contains a hydrazide linker between a carboxylic acid and a phosphonic acid. Bioinformatic analysis of the gene cluster provides insights into how such a structure might be assembled.

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

“…Putative ATP-grasp ligase and GCN5-related N-acetyltransferases (GNAT; FemXAB nonribosomal peptidyltransferase) encoded by neighboring genes may catalyze peptide bond formation. Similar enzymes are used in the biosynthesis of the phosphonopeptides rhizocticin (21) (ATP-grasp ligases) and dehydrophos (37) (GNAT), and a combination of both has been suggested to account for the peptide linkages in fosfazinomycin (38). Argolaphos has broad-spectrum antibacterial activity, with the highest efficacy against Salmonella typhimurium, Escherichia coli, and Staphylococcus aureus, and it is weakly inhibitory against Mycobacterium smegmatis (SI Appendix, Fig.…”

“…Including the eight additional molecules we recently reported elsewhere (24,38,40,45) (SI Appendix, Fig. S4), this program has yielded 19 new phosphonate natural products to date.…”

New insights into the biosynthesis of fosfazinomycin

“…1, 2 At present, the mechanism of N-N bond formation is not understood. 3, 4 In recent years, the biosynthetic gene clusters have been reported for several such compounds, including valanimycin, 3, 5 kutzneride, 4, 6 and fosfazinomycin, 7 as well as molecules containing diazo groups such as kinamycin, 8, 9 lomaiviticin, 10, 11 and cremeomycin 12, 13 (Fig. 1a).…”

Discovery of the lomaiviticin biosynthetic gene cluster in Salinispora pacifica

“…Intriguingly, a recent study by van der Donk, Metcalf, and co-workers identified homologs of lom32–35 in a putative fosfazinomycin ( fzm ) biosynthetic gene cluster. 25 Fosfazinomycin contains a hydrazido linkage, and the presence of these lom genes in the fzm cluster provides further evidence for their involvement in N–N bond formation (Figure 3B). We also noted that fznP displays significant sequence homology to hypothetical protein lom29 (30% amino acid identity, 43% similarity).…”