Metabolomics-Driven Discovery Of Cryptic Natural Products From Marine Microorganisms

Microorganisms such as marine bacteria and fungi possess a remarkable capability for the synthesis of a broad variety of biologically active secondary metabolites.Major challenges in the screening of these microorganisms are to activate the expression of cryptic biosynthetic gene clusters and the development of technologies for efficient dereplication of known compounds.Here we report the efficient mining of cryptic natural products produced by marine hydrothermal vent microorganisms,following strategies based on the activation of silent genetic clusters combined with metabolomics analytical methods.1)We established a strategy based on the theory that_dormant or inert biosynthesis pathways respond to heavy metal stress and metabolomics analysis aiming to locate as well as predict the bioactive structures.Employing this strategy,a novel compound 1 exhibiting antibiotic properties against the Gram-positive bacterium Bacillus sub til is was unveiled from hydrothermal vent Streptomyces sp.WU20.2)We developed a co-culturing device that allows culturing of multispecies microbial strains that are physically separated but can exchange dissolved or colloidal chemical signals and then performed a metabolomics survey of the secondary metabolisms of both a fungal-bacterial community and an actinomycetic-actinomycetic community using a metabolomic approach integrating LC-MS analysis,conventional statistical tools and molecular networking.LC-MS measurements revealed a pronounced influence of the chemical communication on the metabolic profiles of synthetic communities with a group of molecules being newly-induced or upregulated in co-cultures.More specifically,a class of unusual diphenyl ethers with polyhydroxy sidechains including a novel antibiotic 7 was discovered to be induced by chemical communication in the community composed of Cladosporium sp.WUH1 and Bacillus subtilis CMCC(B)63501.Further investigation on the fungal-bacterial cross-talk inferred that the production of these diphenyl ethers might be a defensive response against the growth inhibition resulting from surfactins,a class of antifungal cyclopeptides,secreted by B.subtilis.3)We conducted a MS-based metabolomics survey to investigate the effect of chemical communication on the secondary metabolism of a fungal-bacterial community comprising Penicillium citrinum Y34 and Pseudomona.s aeruginosa CMCC(B)10104.Analysis of the molecular map generated by MS molecular networking techniques revealed new citrinin-related metabolites secreted by Penicillium citrinum while the production of these molecules was limited for f'urther structural elucidation in the stationary co-cultures.Hence,a new co-culture device with external mechanical forces was then developed to accelerate the exchan,ge of chemical information and multivariate data analysis combined with molecular mapping prioritized two cross-talk-induced citrinin analogues which were positively-correlated with the exchange rate in the new co-cultures.MS-guided isolation resulted in the characterization of the previously undescribed antibiotic citrinolide(29).This highly-oxidized citrinin adduct representing a novel skeleton showed significantly enhanced antibiotic property against the partner strain P.aeruginosa than its precursor citrinin,suggesting a role in the microbial competition during the cross-talk.4)We performed a metabolomics survey of the secondary metabolism of a hydrothermal vent flungal-bacterial community comprising Aspergillus sclerotiorurym DX9 and Streptomyces sp.WU20.The fungal strain was found to increase the secretion of notoamides and cyclo(Pro-Trp)produced by the actinomycetes was discovered to be the responsible molecule,which led to the hypothesis that the fungi transformed cyclo(Pro-Trp)synthesized by the actinomycetes as the biosynthetic precursors of notoamides in the cross-talk.Further analysis showed Streptomyces sp.WU20 was efficient in transforming amino acids into cyclo(Pro-Trp)and adding tryptophan as well as proline into the cross-talk could enhance the induction of the notoamide accumulation.