Structure and Candidate Biosynthetic Gene Cluster of a Manumycin-Type Metabolite from Salinispora pacifica

Abstract

A new manumycin-type natural product named pacificamide (1) and its candidate biosynthetic gene cluster (pac) were discovered from the marine actinobacterium Salinispora pacifica CNT-855. The structure of the compound was determined using NMR, electronic circular dichroism, and bioinformatic predictions. The pac gene cluster is unique to S. pacifica and found in only two of the 119 Salinispora genomes analyzed across nine species. Comparative analyses of biosynthetic gene clusters encoding the production of related manumycin-type compounds revealed genetic differences in accordance with the unique pacificamide structure. Further queries of manumycin-type gene clusters from public databases revealed their limited distribution across the phylum Actinobacteria and orphan diversity that suggests additional products remain to be discovered in this compound class. Production of the known metabolite triacsin D is also reported for the first time from the genus Salinispora. This study adds two classes of compounds to the natural product collective isolated from the genus Salinispora, which has proven to be a useful model for natural product research.

Figure 1.

Representative manumycin-type natural products.

Figure 2.

NMR assignments of pacificamide (1). (a) ¹H and ¹³C chemicals shifts (in ppm) based on ¹H NMR and HSQC data. (b) Spin systems observed by COSY. (c) ¹³C chemical shifts (in ppm) and key correlations observed by HMBC. (d) Key NOESY correlations and key spin–spin coupling constants (³J) observed by ¹H NMR and HETLOC.

Figure 3.

Pacificamide BGC and biosynthesis. (a) Candidate pacificamide (pac) and daryamide (dar, two contigs) BGCs. Asukamycin (asu) BGC is shown for reference. Gene numbering for pac in CNT-855, dar, and asu is shown. Genes are color-coded by function [wine: ketosynthases; navy blue: 3,4-AHBA synthesis; purple: oxidoreductases in epoxyquinol synthesis; salmon: biosynthetic (shared); light salmon: biosynthetic (not shared); light green: regulation; light blue: transport; gray: other]. (b) pac gene annotations with asu and dar homologues. (c) Proposed pacificamide biosynthetic pathway (numbers represent pac genes). (d) LCMS extracted ion chromatogram (EIC) for pacificamide [M + Na]⁺ from strains CNT-855 (red) and CNS-960 (blue).

Figure 4.

Manumycin-type BGCs identified in bacterial genome sequences. Colored genes represent pac gene homologues. Related groups of BGCs (I–XI) are shaded gray if they have been linked to a known metabolite or unshaded if they remain orphan (entries 37–39 are not predicted to encode manumycin-type metabolites). Structures linked to the pac, dar, man, esp, asu, and col BGCs are shown with the source species in bold. Colored squares indicate family level taxa (orange: Micromonosporaceae, blue: Streptomycetaceae, and green: Pseudonocardiaceae).