Rapid Method To Estimate the Presence of Secondary Metabolites in Microbial Extracts

Abstract

Screening microbial secondary metabolites is an established method to identify novel biologically active molecules. Preparation of biological screening samples from microbial fermentation extracts requires growth conditions that promote synthesis of secondary metabolites and extraction procedures that capture the secondary metabolites produced. High-performance liquid chromatography (HPLC) analysis of fermentation extracts can be used to estimate the number of secondary metabolites produced by microorganisms under various growth conditions but is slow. In this study we report on a rapid (approximately 1 min per assay) surrogate measure of secondary metabolite production based on a metabolite productivity index computed from the electrospray mass spectra of samples injected directly into a spectrometer. This surrogate measure of productivity was shown to correlate with an HPLC measure of productivity with a coefficient of 0.78 for a test set of extracts from 43 actinomycetes. This rapid measure of secondary metabolite productivity may be used to identify improved cultivation and extraction conditions by analyzing and ranking large sets of extracts. The same methods may also be used to survey large collections of extracts to identify subsets of highly productive organisms for biological screening or additional study.Microbial extracts have been and continue to be a productive source of new biologically active molecules for drug discovery (2, 13). It is estimated that more than 30% of worldwide human pharmaceutical sales have compounds from natural sources as their origin (12). With advances in genomics and high-throughput screening (HTS) technology, many new therapeutic targets are accessible for identifying pharmaceutical agents. In addition to the historical practice of screening microbial fermentation extracts for antibiotic activity, extracts can now routinely be screened with a variety of new functional, receptor binding, enzyme inhibition, and protein-protein interaction assays. HTS formats used at many large pharmaceutical research organizations are, however, generally incompatible with complex fermentation extracts. The reasons for the incompatibility include nonspecific interference with assay systems, cost in dollars and time to identify and dereplicate active components from a complex mixture, and adverse physical properties for automated liquid-handling equipment. Therefore, additional investment in selection and preparation of fermentation extracts is one strategy to align natural product drug discovery with today's automated HTS assay systems. A typical approach to improve the compatibility of fermentation extracts with HTS was recently reported by Schmid et al., who described a multistage automated solid-phase extraction (SPE) system (12).To make the best use of finite resources in natural product discovery organizations, it is important to identify collections of organisms that produce secondary metabolites, culture conditions that generally support secondary metabolite synthesis, and sample prepar...

EndophyticCephalotheca sulfureaAGH07 reprograms soybean to higher growth

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Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

“…Chemical analyses of filtered ethanol extracts or of SPE eluates were performed by HPLC-ES-MS as described by Julian et al (23) or by ES-MS as described by Higgs et al (20). A standard mixture consisting of caffeine, m-cresol purple, Spinosad (a mixture of isomeric spinosyn factors A and D; Dow Agrosciences, Indianapolis, Ind.…”

“…), narasin A, and tylosin (100 g/ml each) was injected at the beginning, at the end, and after every 10th sample to assess instrument stability and performance during the analyses. Data from the ES-MS studies were evaluated with a UNIX workstation by using the productivity index algorithms described by Higgs and coworkers (20). Background corrections were completed for all treatments by subtracting the chemical productivity score for the uninoculated treatment (20).…”

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HPLC Studio: A Novel Software Utility to Perform HPLC Chromatogram Comparison for Screening Purposes

“…In natural products, fermentation samples can be improved by preselecting strains fermented in most productive media by chemical dereplication of secondary metabolite compounds. [1][2][3][4][5][6][7] Theoretically, increasing the number and quantity of different chemical compounds tested in different targets should increase the chances of finding new leads for therapeutic agents.…”