Biosynthesis of rhizocticins, antifungal phosphonate oligopeptides produced by Bacillus subtilis ATCC6633

Abstract

Rhizocticins are phosphonate oligopeptide antibiotics containing the C-terminal nonproteinogenic amino acid (Z)-l-2-amino-5-phosphono-3-pentenoic acid (APPA). Here we report the identification and characterization of the rhizocticin biosynthetic gene cluster (rhi) in Bacillus subtilis ATCC6633. Rhizocticin B was heterologously produced in the nonproducer strain Bacillus subtilis 168. A biosynthetic pathway is proposed on the basis of bioinformatics analysis of the rhi genes. One of the steps during the biosynthesis of APPA is an unusual aldol reaction between phosphonoacetaldehyde and oxaloacetate catalyzed by an aldolase homolog RhiG. Recombinant RhiG was prepared, and the product of an in vitro enzymatic conversion was characterized. Access to this intermediate allows for biochemical characterization of subsequent steps in the pathway.

Figure 1

Chemical structures of the phosphonate antibiotics rhizocticins (A) and plumbemycins (B). C) The threonine synthase reaction inhibited by APPA. D) Chemical structures of representative phosphonate antibiotics whose biosynthetic pathways have been studied.

Figure 2

Organization of the rhizocticin gene cluster and surrounding genes on the B. subtilis ATCC6633 chromosome. The same locus of B. subtilis 168 genome is also shown for comparison. Genes with a high degree of homology between the two strains (>90% identity) are shown in yellow. The biosynthetic clusters for rhizocticin and sporulation killing factor are shown in blue and green, respectively. The corresponding location of these loci in the other genome is denoted with a star of the same color.

Figure 3

Analysis of rhizocticin B production by B. subtilis MMG272. A) ³¹P NMR spectra of partially purified spent medium of B. subtilis MMG272 and of the same sample supplemented with rhizocticin B authentic standard. The concentrations of components in the sample B. subtilis MMG272 + rhizocticin B are the same as in the individual sample of B. subtilis MMG272. Both spectra were collected for 400 transients and adjusted to the same absolute vertical scale. B) LC-MS analysis of partially purified spent medium of B. subtilis MMG272. The fragmentation of the rhizocticin B parent ion is shown and the peaks corresponding to the characteristic fragments are labeled.

Figure 4

Proposed pathways for the biosynthesis of rhizocticins.

Figure 5

Reaction catalyzed by RhiG-C-His. A) Scheme of reaction catalyzed by RhiG-C-His. B) ³¹P NMR spectrum of the RhiG-C-His assay with unlabeled substrates.

Figure 6

Proposed mechanism for the RhiG catalyzed transformation. Amino acid residues coordinating divalent metal cation (panel A, RhiG numbering, W denotes water) are based on the alignment with the homolog DmpG shown in panel B (conserved residues are in bold, ligands to M²⁺ are labeled with arrows).