We first de novo sequenced the genome of Saccharothrix sp. ST-888. An assembly of the sequence reads yielded 2822 contigs with 8605814 total base pairs. In the biosynthesis of fosfomycin, bialaphos and FR-900098, the initial reactions begin with a conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate (Supplementary Figure S1).1, 2 This conversion is catalyzed by PEP phosphomutase (PEPPM), which is encoded by the fom1, bcpB or frbD genes, for the biosynthesis of fosfomycin, bialaphos or FR-900098, respectively.1, 2 Therefore, we hypothesized that the initial reaction in phosphonothrixin biosynthesis is also catalyzed by PEPPM. Thus, we retrieved a PEPPM homolog from the genomic DNA sequence of Saccharothrix sp. ST-888 and identified a homologous open reading frame (ORF) with 34%, 40%, and 49% identities to Fom1, BcpB and FrbD, respectively (Supplementary Figure S2). Moreover, an analysis of the regions flanking the PEPPM homologous gene revealed that 14 additional genes were located in these regions in the same direction as the PEPPM homologous gene, suggesting that these 15 genes form a biosynthetic gene cluster for phosphonothrixin.

We constructed a cosmid library for the ST-888 genome using the cosmid vector pOJ4466 and screened the library for cosmids that contained the regions including the PEPPM homologous gene using the gene itself as a probe. Subsequently, three positive cosmids (cos-1, cos-11 and cos-12) were introduced into Streptomyces albus J1074, which has previously been used as a heterologous host.7 The resultant transformants were cultured for heterologous expression under the identical conditions used for ST-888.3 The metabolites, which were partially purified by anion exchange chromatography using AG 1-X8 resin (Cl− form; Bio-Rad, Tokyo, Japan), were analyzed using 31P NMR spectroscopy (Figure 1a). The heterologous production of phosphonothrixin was confirmed in the transformants harboring cos-11 or cos-12. The titers of phosphonothrixin in both the transformants were ~8.7 mg l−1 broth, which were one-half that of the wild-type ST-888 strain (17.3 mg l−1). This result indicates that both cos-11 and cos-12 contain all the genes required for phosphonothrixin biosynthesis. Some unidentified phosphonates were also produced by the transformants. The production of multiple phosphonates upon the heterologous expression of phosphonate biosynthetic gene clusters has been previously reported.8 We suspect that these unknown metabolites may be the biosynthetic intermediates of phosphonothrixin or phosphonates that are detoxified by the heterologous host.

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