Common biosynthetic origins for polycyclic tetramate macrolactams from phylogenetically diverse bacteria

Abstract

A combination of small molecule chemistry, biosynthetic analysis, and genome mining has revealed the unexpected conservation of polycyclic tetramate macrolactam biosynthetic loci in diverse bacteria. Initially our chemical analysis of a Streptomyces strain associated with the southern pine beetle led to the discovery of frontalamides A and B, two previously undescribed members of this antibiotic family. Genome analyses and genetic manipulation of the producing organism led to the identification of the frontalamide biosynthetic gene cluster and several biosynthetic intermediates. The biosynthetic locus for the frontalamides' mixed polyketide/amino acid structure encodes a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), which resembles iterative enzymes known in fungi. No such mixed iterative PKS-NRPS enzymes have been characterized in bacteria. Genome-mining efforts revealed strikingly conserved frontalamide-like biosynthetic clusters in the genomes of phylogenetically diverse bacteria ranging from proteobacteria to actinomycetes. Screens for environmental actinomycete isolates carrying frontalamide-like biosynthetic loci led to the isolation of a number of positive strains, the majority of which produced candidate frontalamide-like compounds under suitable growth conditions. These results establish the prevalence of frontalamide-like gene clusters in diverse bacterial types, with medicinally important Streptomyces species being particularly enriched.

Fig. 1.

Bacterial polycyclic tetramate macrolactams. (A) Structures of the newly isolated frontalamides. (B) Structures of the frontalamide biosynthetic intermediates FI-1, FI-2, FI-3. (C) Known bacterial polycyclic tetramate macrolactams with structures similar to the frontalamides.

Fig. 2.

LC/MS assays for frontalamide production. UV chromatographs were recorded at 280 nm. (A) LC/MS detection of frontalamides produced by a Streptomyces sp. SPB78 rpsL mutant. #s denote compounds with masses and UV spectra similar to frontalamides A and B. (B) Detection of frontalamide biosynthetic intermediates FI-1, FI-2 and FI-3 in a SPB78 rpsL ftdA double mutant. (C) Spectrum indicating the loss of frontalamide production in a SPB78 rpsL ftdB double mutant.

Fig. 3.

Open reading frame (ORF) map of the frontalamide biosynthetic (ftd) locus from Streptomyces sp. SPB78 compared to ftd-like clusters sourced from sequenced bacterial genomes. Each ORF is color-coded to designate ftd orthologs. Those shown in gray encode for alkane hydroxylases, red for hybrid PKS-NRPS synthetases, green for phytoene dehydrogenases, blue for zinc-dependent alcohol dehydrogenases, and yellow for cytochrome P450s. ORFs shown in black do not have homologs conserved between different species. The S. clavuligerus and S. sp. Mg1 clusters are shown as fragments because their draft genome sequences are incomplete in this region. The bracketed ORFS at the right flank of the L. enzymogenes cluster indicate those not analyzed during the initial characterization of this locus (8). These genes were released directly to GenBank without accompanying analysis.

Fig. 4.

Hypothetical model for frontalamide biosynthesis using a fungal-type hybrid PKS-NRPS mechanism. (A) Domain structure of the iterative type I PKS-NRPS hybrid enzyme TenS from the fungus Beuveria bassiana. Also shown is the mixed polyketide-amino acid tetramate product of TenS, prototenellin A. Note TenS iteratively condenses one acetyl-CoA and four malonyl-CoA substrates before incorporating a single tyrosine residue during the formation of protenellin A. (B) Homology-based biosynthetic model for the production of the mixed polyketide-amino acid tetramate backbone of the frontalamides by FtdB. Using an iterative mechanism to similar to that employed by the fungal enzymes, FtdB would catalyze two rounds of polyketide synthesis, and the two chains are subsequently condensed to the two free amines of ornithine. A Dieckmann cyclization could form the tetramic-acid moiety during release of the mixed polyketide-amino acid product from the FtdB synthetase. The roles of FtdC-FtdF await experimental elucidation but could plausibly be involved in redox tailoring of the FtdB product or for modification of the polyketide chains to direct the cyclizations that produce the 5, 5, 6 polycycle. This scheme represents a possible pathway for frontalamide biosyntheses; future studies are required. Our genetic data establish the hydroxylase role for FtdA, but we cannot ascertain the timing of this reaction during biosynthesis.