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.

Keywords: antibiotics; biosynthesis; hydrazine; natural products.

Figure 1

(A) ³¹P NMR spectrum of fosfomycin (top) and after treatment with DhpI and a mixture of SAM and d₃-SAM (1:1) (bottom). (B) LC-MS analysis of the sample in the bottom spectrum of panel A.

Figure 2

(A) ³¹P NMR spectrum of spent medium of Streptomyces sp. WM 6372. Only the chemical shift region of interest with respect to phosphonates is depicted. (B) ³¹P NMR spectrum after treatment with DhpI and SAM. (C) Structures of the phosphonates giving rise to the signals in panel A and the structures of fosfazinomycin A and B.

Figure 3

(A) ³¹P NMR spectrum of spent medium of Streptomyces sp. XY332. (B) ³¹P NMR spectrum of spent medium of the same strain when grown on ¹⁵NH₄SO₄. The chemical shift variation is the result of small changes in pH as these compounds have pK_a values near 7. (C) DNA fragment of the genome from Streptomyces sp. WM 6372 that contains the pepM gene. For color coding, see Figure S6.

Figure 4

Proposed biosynthetic pathway of fosfazinomycin A. Putative steps are indicated with dashed arrows. FzmFIN are proposed to be involved in formation of the three magenta bonds, whereas the FzmAMNOQR proteins are proposed to be involved in forming and installing the hydrazine core.