Allopeptimicins: unique antibacterial metabolites generated by hybrid PKS-NRPS, with original self-defense mechanism in Actinoallomurus

Abstract

In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C₂₀ polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.

Fig. 1. (A) HPLC profile of the extracted fermentation broth from a culture of Actinoallomurus ID145808. The green and red boxes denote the regions of the chromatogram containing fractions with and devoid of antibacterial activity, respectively. (B) MS of the highlighted regions in (A). (C and D) UV-Vis spectra of the fractions at 6.89 and 7.88 minutes, respectively.

Fig. 2. Structure of allopeptimicin congeners. In red stereocenters configuration inferred by bioinformatic analysis are reported.

Fig. 3. (A) The allopeptimicin biosynthetic gene cluster, apt. The predicted functions for the open reading frames within the cluster are color-coordinated with the different panels. (B) Proposed biosynthetic pathway for piperazic acid (Piz) (pink rectangle), l-allo-Ile (light blue rectangle) and the β-amino acid starter unit (red rectangle) with the individual steps catalyzed by apt enzymes. (C) Domain arrangement and predicted biosynthetic intermediate for each PKS and NRPS polypeptide in the cluster, color-coordinated with the chemical structure. The black star indicates the AT domain recognizing a methylmalonate unit.

Fig. 4. Production of allopeptimicins without (A) and with (B) threonine supplementation. Values calculated as means of three replicates with standard deviation. Allopeptimicins A and B are indicated by blue and orange bars, respectively. The grey line indicates the total complex (A + B) observed.