Response of Hypogean Actinobacterial Genera Secondary Metabolism to Chemical and Biological Stimuli

Microorganisms within microbial communities respond to environmental challenges by producing biologically active secondary metabolites, yet the majority of these small molecules remain unidentified. We have previously demonstrated that secondary metabolite biosynthesis in actinomycetes can be activated by model environmental chemical and biological stimuli, and metabolites can be identified by comparative metabolomics analyses between stimuli conditions. Herein, we survey the secondary metabolite productivity of a group of 20 phylogenetically diverse actinobacteria isolated from hypogean (cave) environments by applying a battery of stimuli consisting of exposure to antibiotics, metals, and mixed microbial culture. Comparative metabolomics was used to reveal secondary metabolite responses from stimuli. These analyses revealed substantial changes in global metabolomic dynamics with over 30 % of metabolomic features increasing more than 10-fold under at least one stimulus condition. Selected features were isolated and identified via NMR, revealing several known secondary metabolite families including the tetarimycins, aloesaponarins, hypogeamicins, actinomycins, and propeptins. One prioritized metabolite was identified to be a previously unreported aminopolyol polyketide, funisamine, produced by a cave isolate Streptosporangium when exposed to mixed culture. The production of funisamine was most significantly increased in mixed culture with Bacillus sp. The biosynthetic gene cluster responsible for the production of funisamine was identified via genomic sequencing of the producing strain, Streptosporangium sp. KDCAGE35, which facilitated deduction of its biosynthesis. Together, these data demonstrate that comparative metabolomics can reveal the stimulus-induced production of natural products from diverse microbial phylogenies. IMPORTANCE Microbial secondary metabolites are an important source of biologically active and therapeutically relevant small molecules. However, much of this active molecular diversity is challenging to access due to low production levels or difficulty in discerning secondary metabolites within complex microbial extracts prior to isolation. Herein we demonstrate that ecological stimuli increase secondary metabolite production in phylogenetically diverse actinobacteria isolated from understudied hypogean environments. Additionally, we show that comparative metabolomics linking stimuli to metabolite response data can effectively reveal secondary metabolites within complex biological extracts. This approach highlighted secondary metabolites in almost all observed natural product classes, including low abundance analogs of biologically relevant metabolites, as well as a new linear aminopolyol polyketide, funisamine. This study demonstrates the generality of activating stimuli to potentiate secondary metabolite production across diverse actinobacterial genera.