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Dual Induction of New Microbial Secondary Metabolites by Fungal Bacterial Co-cultivation
Frontiers in microbiology, 2017
The frequent re-isolation of known compounds is one of the major challenges in drug discovery. Many biosynthetic genes are not expressed under standard culture conditions, thus limiting the chemical diversity of microbial compounds that can be obtained through fermentation. On the other hand, the competition during co-cultivation of two or more different microorganisms in most cases leads to an enhanced production of constitutively present compounds or an accumulation of cryptic compounds that are not detected in axenic cultures of the producing strain under different fermentation conditions. Herein, we report the dual induction of newly detected bacterial and fungal metabolites by the co-cultivation of the marine-derived fungal isolate Aspergillus fumigatus MR2012 and two hyper-arid desert bacterial isolates Streptomyces leeuwenhoekii strain C34 and strain C58. Co-cultivation of the fungal isolate MR2012 with the bacterial strain C34 led to the production of luteoride D, a new lute...
Scientific Reports, 2015
Actinomycetes and filamentous fungi produce a wide range of bioactive compounds, with applications as antimicrobials, anticancer agents or agrochemicals. Their genomes contain a far larger number of gene clusters for natural products than originally anticipated, and novel approaches are required to exploit this potential reservoir of new drugs. Here, we show that co-cultivation of the filamentous model microbes Streptomyces coelicolor and Aspergillus niger has a major impact on their secondary metabolism. NMR-based metabolomics combined with multivariate data analysis revealed several compounds that correlated specifically to co-cultures, including the cyclic dipeptide cyclo(Phe-Phe) and 2-hydroxyphenylacetic acid, both of which were produced by A. niger in response to S. coelicolor. Furthermore, biotransformation studies with o-coumaric acid and caffeic acid resulted in the production of the novel compounds (E)-2-(3-hydroxyprop-1-en-1-yl)-phenol and (2E,4E)-3-(2carboxy-1-hydroxyethyl)-2,4-hexadienedioxic acid, respectively. This highlights the utility of microbial co-cultivation combined with NMR-based metabolomics as an efficient pipeline for the discovery of novel natural products.
Recent Advances in the Heterologous Biosynthesis of Natural Products from Streptomyces
Applied Sciences, 2021
Streptomyces is a significant source of natural products that are used as therapeutic antibiotics, anticancer and antitumor agents, pesticides, and dyes. Recently, with the advances in metabolite analysis, many new secondary metabolites have been characterized. Moreover, genome mining approaches demonstrate that many silent and cryptic biosynthetic gene clusters (BGCs) and many secondary metabolites are produced in very low amounts under laboratory conditions. One strain many compounds (OSMAC), overexpression/deletion of regulatory genes, ribosome engineering, and promoter replacement have been utilized to activate or enhance the production titer of target compounds. Hence, the heterologous expression of BGCs by transferring to a suitable production platform has been successfully employed for the detection, characterization, and yield quantity production of many secondary metabolites. In this review, we introduce the systematic approach for the heterologous production of secondary m...
Fungal–fungal co-culture: a primer for generating chemical diversity
Natural Product Reports, 2022
In their natural environment, fungi must compete for resources. It has been hypothesized that this competition likely induces the biosynthesis of secondary metabolites for defence. In a quest to discover new chemical diversity from fungal cultures, a growing trend has been to recapitulate this competitive environment in the laboratory, essentially growing fungi in co-culture. This review covers fungal-fungal co-culture studies beginning with the first literature report in 2002. Since then, there has been a growing number of new secondary metabolites reported as a result of fungal co-culture studies. Specifically, this review discusses and provides insights into (1) rationale for pairing fungal strains, (2) ways to grow fungi for co-culture, (3) different approaches to screening fungal co-cultures for chemical diversity, (4) determining the secondary metabolite-producing strain, and (5) final thoughts regarding the fungalfungal co-culture approach. Our goal is to provide a set of practical strategies for fungal co-culture studies to generate unique chemical diversity that the natural products research community can utilize. 1 Introduction and background 1.1 Fungal secondary metabolites are an untapped resource for new drug leads, but do we need new ways to 'turn on' their biosynthesis? 1.2 Interaction driven secondary metabolite discovery: mimicking the competitive environment of nature in the lab 1.3 Genomics and biosynthetic gene clusters 2 Rationale for pairing fungal strains for co-culturing 2.1 Pairings from endophytic environments 2.1.1 Insights 2.2 Pairings from marine environments 2.2.1 Insights 2.3 Pairings from unique environments 2.3.1 Insights 2.4 Pairings with and/or between pathogens 2.4.1 Insights and examples 2.5 Pairings due to the biosynthesis of antagonistic secondary metabolites 2.5.1 Insights and examples 3 Ways to grow fungi for co-culturing 4 Screening of co-cultures for chemical diversity 4.1 Mass spectrometry 4.2 Nuclear magnetic resonance 4.3 Bioassays 5 Determining the secondary metabolite producer strain 5.1 Structural similarities and monoculture presence 5.2 Spatial location during co-culture 5.3 Different pairing/growing in presence of an extract 5.4 Media doping 5.5 Genomics 6 Final thoughts 7 Author contributions 8 Conicts of interest 9 Acknowledgements 10 Notes and references
A chromatogram-simplified Streptomyces albus host for heterologous production of natural products
2019
Cloning natural product biosynthetic gene clusters from cultured or uncultured sources and their subsequent expression by genetically tractable heterologous hosts is an essential strategy for the elucidation and characterisation of novel microbial natural products. The availability of suitable expression hosts is a critical aspect of this workflow. In this work, we mutagenised five endogenous biosynthetic gene clusters from Streptomyces albus S4, which reduced the complexity of chemical extracts generated from the strain and eliminated antifungal and antibacterial bioactivity. We showed that the resulting quintuple mutant can express foreign BGCs by heterologously producing actinorhodin, cinnamycin and prunustatin. We envisage that our strain will be a useful addition to the growing suite of heterologous expression hosts available for exploring microbial secondary metabolism.
Secondary Metabolites Produced during the Germination of Streptomyces coelicolor
Frontiers in Microbiology
Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra-and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments.
PloS one, 2018
Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.
The current work was carried out under a screening program targeted at isolation of bioactive Streptomyces species from soil samples. A total of 54 Streptomyces species were isolated from soil samples, out of which 4 isolates were found to be promising. These isolates were identified as Streptomyces spectabilis, Streptomyces purpurascens, Streptomyces coeruleorubidus and Streptomyces lavendofoliae and their sequences have been deposited in the GenBank. The influence of culture conditions including, incubation time, incubation temperature, initial pH and different carbon and nitrogen sources on growth and bioactive compound formation was investigated. Isolate R1, identified as Streptomyces spectabilis, showed maximum bioactive metabolite production with cellobiose and peptone as the carbon and nitrogen sources, on the 5th day at pH 5 at 30℃. The optimum conditions for production by isolate R3, identified as Streptomyces purpurascens, were observed to be starch and casein as the carbon and nitrogen sources, pH 7, temperature 30℃ and an incubation period of eight days. For isolate R5, identified as Streptomyces coeruleorubidus, maximal production resulted on the sixth day at pH 6 and temperature of 35℃ with mannitol and JBM. Isolate Y8, identified as Streptomyces lavendofoliae, was found to produce high levels of bioactive metabolites in the medium supplemented with starch and peptone on the 10th day at pH 7 and at an incubation temperature of 30℃. The four strains tested here behaved differently, each one requiring specific conditions for maximum growth as well as bioactive metabolite production.
OPTIMIZATION OF ANTIMICROBIAL METABOLITES PRODUCTION BY STREPTOMYCES FRADIAE Original Article
2015
Objectives: The aim of the present study to optimize the culture conditions for the production of secondary metabolites of a sponge-derived actinomycetes Streptomyces fradiae. Methods: Effects of the incubation period, temperature and pH on biomass and antimicrobial metabolite production by Streptomyces fradiae was examined. Moreover, Effects of various carbon, nitrogen and minerals on biomass and antimicrobial metabolite production by Streptomyces fradiae were also assessed. Results: Secondary metabolites production was started after 24 h of incubation of culture broth and reached its maximum levels after 96 h and thereafter gradually declined. The culture medium adjusted to pH 7.0 supported the production of antimicrobial metabolites as compared to other pH levels and optimum temperature for secondary metabolite production was found to be 35 °C. Basal medium amended with arabinose and soybean meal as carbon and nitrogen sources respectively was proved to be the best for the production of bioactive metabolites. Among different minerals tested, only K2HPO4 showed positive influence on secondary metabolite production by the strain. Conclusion: In the present study, optimum levels of culture conditions were determined for the production of antibiotic by Streptomyces fradiae.
PeerJ, 2023
Background. Bioactive compounds (BCs) from natural resources have been extensively studied because of their use as models in the development of novel and important medical and biopreservative agents. One important source of BCs is microorganisms, particularly terrestrial bacteria of the order Actinomycetales. Methods. We characterized Streptomyces sp. KB1 by observing its morphology, physiology, and growth on different media using biochemical tests, optimizing cultural conditions by changing one independent variable at a time. Results. Streptomyces sp. KB1 (TISTR 2304) is a gram-positive and long filamentous bacteria that forms straight to flexuous (rectiflexibile) chains of globose-shaped and smooth-surfaced spores. It can grow under aerobic condition s only at a temperature range of 25-37 • C and initial pH range of 5-10 in the presence of sodium chloride 4% (w/v). Therefore, it is considered an obligate aerobe, mesophilic, neutralophilic, and moderately halophilic bacteria. The isolate grew well on peptone-yeast extract iron, Luria Bertani (LB), and a half-formula of LB (LB/2), but could not grow on MacConkey agar. It utilized fructose, mannose, glucose, and lactose as its carbon source along with acid production and showed positive reactions to casein hydrolysis, gelatin liquefaction, nitrate reduction, urease, and catalase production. Streptomyces sp. KB1 (TISTR 2304) could produce the maximum number of BCs when 1% of its starter was cultivated in a 1,000 ml baffled flask containing 200 ml of LB/2 broth with its initial pH adjusted to 7 with no supplemental carbon source, nitrogen source, NaCl, or trace element at 30 • C, shaken at 200 rpm in an incubator for 4 days.