Abstract
Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species–specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, gbl kaufen we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.
INTRODUCTION
Actinobacteria are a rich source of specialized metabolites that have been developed into life-saving drugs. Recent advances in genome sequencing and mining have revealed that Actinobacteria have far greater potential for secondary metabolite production than previously realized. Yet, much of this potential remains cryptic, as many biosynthetic genes are poorly expressed in normal laboratory incubation conditions. The manipulation of the signaling mechanisms for gene expression can be a key to activating the expression of dormant biosynthetic genes. However, the regulation of biosynthetic pathways, and the autoregulators themselves, remains poorly understood.
Among the known signaling molecules, gamma-butyrolactones (GBLs) are recognized to be involved in the regulation of morphological development and secondary metabolism in actinomycete bacteria, especially in the genus of Streptomyces. In contrast, among the metabolically prolific marine actinomycete genus Salinispora, far less is known about the signaling pathways regulating the biosynthesis of their rich repertoire of natural products. However, the recently reported potent and selective antimalarial compounds, salinipostins salinipostin A (1)) from Salinispora sp. RL08-036-SPS-B, may shed some light on this. Salinipostins possess a GBL ring analogous to A-factor, as well as a highly unusual phosphotriester ring. Two compounds, cyclipostins and cyclophostin , from Streptomyces have this same structural motif and have been reported as hormone sensitive lipase and acetylcholinesterase inhibitors, respectively. These compounds are estimated to be derived from a similar pathway as the GBL biosynthetic pathway based on their structural similarity.
The most studied GBL compound is A-factor from Streptomyces griseus. The GBL structure is formed from the condensation of beta-ketoacyl acyl carrier protein (ACP) and dihydroxyacetone phosphate by the A-factor synthase AfsA followed by successive reduction by BprA and dephosphorylation. AfsA homologues are common in streptomycetes where they have been shown to be involved not just in GBL biosynthesis but also in the production of gamma-butenolides and furans.
Recently, we showed that the AfsA homologue Spt9 in Salinispora bacteria is responsible for the construction of the salinipostins and its volatile byproducts, salinilactones. The spt9 gene is the terminal gene of a nine-gene biosynthetic locus that is broadly conserved in Salinispora species (spt was annotated as butyrolactone 1 in this reference). Herein, we report the functional characterization of spt genes, spt6 and spt9, toward salinipostin biosynthesis and show that the spt gene cluster is also responsible for the synthesis of novel, natural GBLs as coproducts of salinipostin biosynthesis. Furthermore, we revealed that spt-like biosynthetic gene clusters are broadly distributed among actinomycete genera, suggesting its important role in bacterial signaling.
RESULTS AND DISCUSSION
Discovery of Gamma-Butyrolactone Compounds from Salinispora Species.
We previously reported the global transcriptional activity of biosynthesis gene clusters in Salinispora species gbl kaufen and showed that the spt locus was involved with salinipostin biosynthesis through genetic inactivation of spt9 and a concomitant loss of salinipostin. Upon reexamination of the S. tropica CNB-440 spt9-deletion mutant in comparison with the native CNB-440 strain by liquid chromatography–mass spectrometry (LC-MS), we identified compounds in addition to the salinipostins that were also abolished in the mutant. Inspection of the molecular formulas of compounds 2 (C11H19O4, m/z 215.1280, [M + H]+ calcd. 215.1278) and 3 (C10H17O4, m/z 201.1126, [M + H]+ calcd. 201.1121) suggested that they could be simple, A-factor-like GBL compounds.
