Molecular cloning and heterologous expression of the dehydrophos biosynthetic gene cluster

Abstract

Dehydrophos is a vinyl phosphonate tripeptide produced by Streptomyces luridus with demonstrated broad-spectrum antibiotic activity. To identify genes necessary for biosynthesis of this unusual compound we screened a fosmid library of S. luridus for the presence of the phosphoenolpyruvate mutase gene, which is required for biosynthesis of most phosphonates. Integration of one such fosmid clone into the chromosome of S. lividans led to heterologous production of dehydrophos. Deletion analysis of this clone allowed identification of the minimal contiguous dehydrophos cluster, which contained 17 open reading frames (ORFs). Bioinformatic analyses of these ORFs are consistent with a proposed biosynthetic pathway that generates dehydrophos from phosphoenolpyruvate. The early steps of this pathway are supported by analysis of intermediates accumulated by blocked mutants and in vitro biochemical experiments.

FIG. 1

(I) Bioassay demonstrating heterologous production of dehydrophos. Production of dehydrophos was assayed against sensitive (a) and resistant (b) strains of E. coli. Filter disks were spotted with supernatant from WM4467, synthetic standard of dehydrophos, or supernatant from wild type S. lividans, as indicated. (II) ³¹P NMR demonstrating heterologous production of dehydrophos. (a) Supernatant collected from WM4467, (b) As (a) but containing synthetic dehydrophos added as an internal standard.

FIG. 2

Gene organization of fosmid 17E11-4. Dark arrows indicate ORFs thought to be involved in dehydrophos biosynthesis. The solid lines indicate DNA fragments whose deletion has no effect on production of dehydrophos, while the dotted lines indicate deletions that abolish production.

FIG. 3

dhpB mutants accumulate DHEP. ³¹P NMR spectra of concentrated culture supernatant from a dhpB mutant (a), and that same sample spiked with a synthetic standard of DHEP (b).

FIG. 4

³¹P NMR spectra of DhpA assay. HEP was incubated with purified DhpA and α-ketoglutarate (c), incubated without enzyme (a), or incubated without α-ketoglutarate(b). The reaction shown in panel c was then spiked with DHEP to verify the product (d).

FIG. 5

dhpC and dhpH mutants accumulate a phosphate ester. ³¹P NMR spectra of concentrated culture supernatants from a dhpC mutant (a), a dhpH mutant,(c) and those same supernatants after treatment with calf intestinal alkaline phosphatase (b and d, respectively).

FIG 6

Proposed pathway for dehydrophos biosynthesis (a), and the portion of the serine biosynthetic pathway that bears structural similarity to step V, VI, and VII of the proposed pathway (b). Broken arrows indicate hypothetical reactions. OP-EP = 1-oxo-2-phosphorylethylphosphonate.