Sponge-Derived Kocuria and Micrococcus spp. as Sources of the New Thiazolyl Peptide Antibiotic Kocurin

Forty four marine actinomycetes of the family Microccocaceae isolated from sponges collected primarily in Florida Keys (USA) were selected from our strain collection to be studied as new sources for the production of bioactive natural products. A 16S rRNA gene based phylogenetic analysis showed that the strains are members of the genera Kocuria and Micrococcus. To assess their biosynthetic potential, the strains were PCR screened for the presence of secondary metabolite genes encoding nonribosomal synthetase (NRPS) and polyketide synthases (PKS). A small extract collection of 528 crude extracts generated from nutritional microfermentation arrays was tested for the production of bioactive secondary metabolites against clinically relevant strains (Bacillus subtilis, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Candida albicans). Three independent isolates were shown to produce a new anti-MRSA bioactive compound that was identified as kocurin, a new member of the thiazolyl peptide family of antibiotics emphasizing the role of this family as a prolific resource for novel drugs.     

Insights into the Variation in Bioactivities of Closely Related Streptomyces Strains from Marine Sediments of the Visayan Sea against ESKAPE and Ovarian Cancer

Marine sediments host diverse actinomycetes that serve as a source of new natural products to combat infectious diseases and cancer. Here, we report the biodiversity, bioactivities against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) and ovarian cancer, and metabolites variation among culturable actinomycetes isolated from the marine sediments of Visayan Sea, Philippines. We identified 15 Streptomyces species based on a 16S rRNA gene sequence analysis. The crude extracts of 10 Streptomyces species have inhibited the growth of ESKAPE pathogens with minimum inhibitory concentration (MIC) values ranging from 0.312 mg/mL to 20 mg/mL depending on the strain and pathogens targeted. Additionally, ten crude extracts have antiproliferative activity against A2780 human ovarian carcinoma at 2 mg/mL. To highlight, we observed that four phylogenetically identical Streptomyces albogriseolus strains demonstrated variation in antibiotic and anticancer activities. These strains harbored type I and II polyketide synthase (PKS) and non-ribosomal synthetase (NRPS) genes in their genomes, implying that their bioactivity is independent of the polymerase chain reaction (PCR)-detected bio-synthetic gene clusters (BGCs) in this study. Metabolite profiling revealed that the taxonomically identical strains produced core and strain-specific metabolites. Thus, the chemical diversity among these strains influences the variation observed in their biological activities. This study expanded our knowledge on the potential of marine-derived Streptomyces residing from the unexplored regions of the Visayan Sea as a source of small molecules against ESKAPE pathogens and cancer. It also highlights that Streptomyces species strains produce unique strain-specific secondary metabolites; thus, offering new chemical space for natural product discovery.     

Bioprospecting from Marine Sediments of New Brunswick,Canada: Exploring the Relationship between Total Bacterial Diversity and Actinobacteria Diversity

Actinomycetes are an important resource for the discovery of natural products with therapeutic properties. Bioprospecting for actinomycetes typically proceeds without a priori knowledge of the bacterial diversity present in sampled habitats. In this study, we endeavored to determine if overall bacterial diversity in marine sediments, as determined by 16S rDNA amplicon pyrosequencing, could be correlated with culturable actinomycete diversity, and thus serve as a powerful tool in guiding future bioprospecting efforts. Overall bacterial diversity was investigated in eight marine sediments from four sites in New Brunswick, Canada, resulting in over 44,000 high quality sequences (x = 5610 per sample). Analysis revealed all sites exhibited significant diversity (H’ = 5.4 to 6.7). Furthermore, statistical analysis of species level bacterial communities (D = 0.03) indicated community composition varied according to site and was strongly influenced by sediment physiochemical composition. In contrast, cultured actinomycetes (n = 466, 98.3% Streptomyces) were ubiquitously distributed among all sites and distribution was not influenced by sediment composition, suggesting that the biogeography of culturable actinomycetes does not correlate with overall bacterial diversity in the samples examined. These actinomycetes provide a resource for future secondary metabolite discovery, as exemplified by the antimicrobial activity observed from preliminary investigation.     

Aspergillus Sydowii Marine Fungal Bloom in Australian Coastal Waters,Its Metabolites and Potential Impact on Symbiodinium Dinoflagellates

Dust has been widely recognised as an important source of nutrients in the marine environment and as a vector for transporting pathogenic microorganisms. Disturbingly, in the wake of a dust storm event along the eastern Australian coast line in 2009, the Continuous Plankton Recorder collected masses of fungal spores and mycelia (~150,000 spores/m³) forming a floating raft that covered a coastal area equivalent to 25 times the surface of England. Cultured A. sydowii strains exhibited varying metabolite profiles, but all produced sydonic acid, a chemotaxonomic marker for A. sydowii. The Australian marine fungal strains share major metabolites and display comparable metabolic diversity to Australian terrestrial strains and to strains pathogenic to Caribbean coral. Secondary colonisation of the rafts by other fungi, including strains of Cladosporium, Penicillium and other Aspergillus species with distinct secondary metabolite profiles, was also encountered. Our bioassays revealed that the dust-derived marine fungal extracts and known A. sydowii metabolites such as sydowic acid, sydowinol and sydowinin A adversely affect photophysiological performance (F_v/F_m) of the coral reef dinoflagellate endosymbiont Symbiodinium. Different Symbiodinium clades exhibited varying sensitivities, mimicking sensitivity to coral bleaching phenomena. The detection of such large amounts of A. sydowii following this dust storm event has potential implications for the health of coral environments such as the Great Barrier Reef.     

Identification of Rosellinia species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus Dematophora as inferred from polythetic taxonomy

Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.     

Secondary Metabolite Production Potential of Mangrove-Derived Streptomyces olivaceus

Mangroves are intertidal extreme environments with rich microbial communities. Actinobacteria are well known for producing antibiotics. The search for biosynthetic potential of Actinobacteria from mangrove environments could provide more possibilities for useful secondary metabolites. In this study, whole genome sequencing and MS/MS analysis were used to explore the secondary metabolite production potential of one actinobacterial strain of Streptomyces olivaceus sp., isolated from a mangrove in Macau, China. The results showed that a total of 105 gene clusters were found in the genome of S. olivaceus sp., and 53 known secondary metabolites, including bioactive compounds, peptides, and other products, were predicted by genome mining. There were 28 secondary metabolites classified as antibiotics, which were not previously known from S. olivaceus. ISP medium 2 was then used to ferment the S. olivaceus sp. to determine which predicted secondary metabolite could be truly produced. The chemical analysis revealed that ectoine, melanin, and the antibiotic of validamycin A could be observed in the fermentation broth. This was the first observation that these three compounds can be produced by a strain of S. olivaceus. Therefore, it can be concluded that Actinobacteria isolated from the mangrove environment have unknown potential to produce bioactive secondary metabolites.     

Towards Commercial Production of Sponge Medicines

Sponges can provide potential drugs against many major world-wide occurring diseases. Despite the high potential of sponge derived drugs no sustainable production method has been developed. Thus far it is not fully understood why, when, where and how these metabolites are produced in sponges. For the near future sea-based sponge culture seems to be the best production method. However, for controlled production in a defined system it is better to develop in vitro production methods, like in vitro sponge culture or even better sponge cell culture, culture methods for symbionts or the transfer of production routes into another host. We still have insufficient information about the background of metabolite production in sponges. Before production methods are developed we should first focus on factors that can induce metabolite production. This could be done in the natural habitat by studying the relation between stress factors (such as predation) and the production of bioactive metabolites. The location of production within the sponge should be identified in order to choose between sponge cell culture and symbiont culture. Alternatively the biosynthetic pathways could be introduced into hosts that can be cultured. For this the biosynthetic pathway of metabolite production should be unraveled, as well as the genes involved. This review discusses the current state of sponge metabolite production and the steps that need to be taken to develop commercial production techniques. The different possible production techniques are also discussed.     

Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus,Aspergillus versicolor ZBY-3

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.     

Harnessing the Potential of Halogenated Natural Product Biosynthesis by Mangrove-Derived Actinomycetes

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH₂-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.     

Production of Induced Secondary Metabolites by a Co-Culture of Sponge-Associated Actinomycetes,Actinokineospora sp. EG49 and Nocardiopsis sp. RV163

Two sponge-derived actinomycetes, Actinokineospora sp. EG49 and Nocardiopsis sp. RV163, were grown in co-culture and the presence of induced metabolites monitored by ¹H NMR. Ten known compounds, including angucycline, diketopiperazine and β-carboline derivatives 1–10, were isolated from the EtOAc extracts of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163. Co-cultivation of Actinokineospora sp. EG49 and Nocardiopsis sp. RV163 induced the biosynthesis of three natural products that were not detected in the single culture of either microorganism, namely N-(2-hydroxyphenyl)-acetamide (11), 1,6-dihydroxyphenazine (12) and 5a,6,11a,12-tetrahydro-5a,11a-dimethyl[1,4]benzoxazino[3,2-b][1,4]benzoxazine (13a). When tested for biological activity against a range of bacteria and parasites, only the phenazine 12 was active against Bacillus sp. P25, Trypanosoma brucei and interestingly, against Actinokineospora sp. EG49. These findings highlight the co-cultivation approach as an effective strategy to access the bioactive secondary metabolites hidden in the genomes of marine actinomycetes.     

Potential Anti-Obesity,Anti-Steatosis,and Anti-Inflammatory Properties of Extracts from the Microalgae Chlorella vulgaris and Chlorococcum amblystomatis under Different Growth Conditions

Microalgae are known as a producer of proteins and lipids, but also of valuable compounds for human health benefits (e.g., polyunsaturated fatty acids (PUFAs); minerals, vitamins, or other compounds). The overall objective of this research was to prospect novel products, such as nutraceuticals from microalgae, for application in human health, particularly for metabolic diseases. Chlorella vulgaris and Chlorococcum amblystomatis were grown autotrophically, and C. vulgaris was additionally grown heterotrophically. Microalgae biomass was extracted using organic solvents (dichloromethane, ethanol, ethanol with ultrasound-assisted extraction). Those extracts were evaluated for their bioactivities, toxicity, and metabolite profile. Some of the extracts reduced the neutral lipid content using the zebrafish larvae fat metabolism assay, reduced lipid accumulation in fatty-acid-overloaded HepG2 liver cells, or decreased the LPS-induced inflammation reaction in RAW264.7 macrophages. Toxicity was not observed in the MTT assay in vitro or by the appearance of lethality or malformations in zebrafish larvae in vivo. Differences in metabolite profiles of microalgae extracts obtained by UPLC-LC-MS/MS and GNPS analyses revealed unique compounds in the active extracts, whose majority did not have a match in mass spectrometry databases and could be potentially novel compounds. In conclusion, microalgae extracts demonstrated anti-obesity, anti-steatosis, and anti-inflammatory activities and could be valuable resources for developing future nutraceuticals. In particular, the ultrasound-assisted ethanolic extract of the heterotrophic C. vulgaris significantly enhanced the anti-obesity activity and demonstrated that the alteration of culture conditions is a valuable approach to increase the production of high-value compounds.     

Complementary Analytical Platforms of NMR Spectroscopy and LCMS Analysis in the Metabolite Profiling of Isochrysis galbana

This study was designed to profile the metabolites of Isochrysis galbana, an indigenous and less explored microalgae species. ¹H Nuclear Magnetic Resonance (NMR) spectroscopy and Liquid Chromatography-Mass Spectrometry (LCMS) were used to establish the metabolite profiles of five different extracts of this microalga, which are hexane (Hex), ethyl acetate (EtOAc), absolute ethanol (EtOH), EtOH:water 1:1 (AqE), and 100% water (Aq). Partial least square discriminant analysis (PLS–DA) of the generated profiles revealed that EtOAc and Aq extracts contain a diverse range of metabolites as compared to the other extracts with a total of twenty-one metabolites, comprising carotenoids, polyunsaturated fatty acids, and amino acids, that were putatively identified from the NMR spectra. Meanwhile, thirty-two metabolites were successfully annotated from the LCMS/MS data, ten of which (palmitic acid, oleic acid, α-linolenic acid, arachidic acid, cholesterol, DHA, DPA, fucoxanthin, astaxanthin, and pheophytin) were similar to those present in the NMR profile. Another eleven glycerophospholipids were discovered using MS/MS-based molecular network (MN) platform. The results of this study, besides providing a better understanding of I. galbana’s chemical make-up, will be of importance in exploring this species potential as a feed ingredient in the aquaculture industry.     

Cliona varians-Derived Actinomycetes as Bioresources of Photoprotection-Related Bioactive End-Products

Sunscreen and sunblock are crucial skincare products to prevent photoaging and photocarcinogenesis through the addition of chemical filters to absorb or block ultraviolet (UV) radiation. However, several sunscreen and sunblock ingredients, mostly UV filters, have been associated with human and environmental safety concerns. Therefore, the exploration and discovery of promising novel sources of efficient and safer compounds with photoprotection-related activities are currently required. Marine invertebrates, particularly their associated microbiota, are promising providers of specialized metabolites with valuable biotechnological applications. Nevertheless, despite Actinobacteria members being a well-known source of bioactive metabolites, their photoprotective potential has been poorly explored so far. Hence, a set of methanolic extracts obtained from Cliona varians-derived actinomycetes was screened regarding their antioxidant and UV-absorbing capacities (i.e., photoprotection-related activities). The active extract-producing strains were identified and classified within genera Streptomyces, Micrococcus, Gordonia, and Promicromonospora. This is the first report of the isolation of these microorganisms from C. varians (an ecologically important Caribbean coral reef-boring sponge). The in vitro cytotoxicity on dermal fibroblasts of oxybenzone and the selected active extracts revealed that oxybenzone exerted a cytotoxic effect, whereas no cytotoxic effect of test extracts was observed. Accordingly, the most active (SPFi > 5, radical scavenging > 50%) and nontoxic (cell viability > 75%) extracts were obtained from Streptomyces strains. Finally, LC-MS-based characterization suggested a broad chemical space within the test strains and agreed with the reported streptomycetes’ chemodiversity. The respective metabolite profiling exposed a strain-specific metabolite occurrence, leading to the recognition of potential hits. These findings suggest that marine Streptomyces produce photoprotectants ought to be further explored in skincare applications.     

Genus Nocardiopsis: A Prolific Producer of Natural Products

Actinomycetes are currently one of the major sources of bioactive secondary metabolites used for medicine development. Accumulating evidence has shown that Nocardiopsis, a key class of actinomycetes, has the ability to produce novel bioactive natural products. This review covers the sources, distribution, bioactivities, biosynthesis, and structural characteristics of compounds isolated from Nocardiopsis in the period between March 2018 and 2021. Our results reveal that 67% of Nocardiopsis-derived natural products are reported for the first time, and 73% of them are isolated from marine Nocardiopsis. The chemical structures of the Nocardiopsis-derived compounds have diverse skeletons, concentrating on the categories of polyketides, peptides, terphenyls, and alkaloids. Almost 50% of the natural products isolated from Nocardiopsis have been discovered to display various bioactivities. These results fully demonstrate the great potential of the genus Nocardiopsis to produce novel bioactive secondary metabolites that may serve as a structural foundation for the development of novel drugs.     

Solid-Phase Extraction Embedded Dialysis (SPEED), an Innovative Procedure for the Investigation of Microbial Specialized Metabolites

Solid-phase extraction embedded dialysis (SPEED technology) is an innovative procedure developed to physically separate in-situ, during the cultivation, the mycelium of filament forming microorganisms, such as actinomycetes and fungi, and the XAD-16 resin used to trap the secreted specialized metabolites. SPEED consists of an external nylon cloth and an internal dialysis tube containing the XAD resin. The dialysis barrier selects the molecular weight of the trapped compounds, and prevents the aggregation of biomass or macromolecules on the XAD beads. The external nylon promotes the formation of a microbial biofilm, making SPEED a biofilm supported cultivation process. SPEED technology was applied to the marine Streptomyces albidoflavus 19-S21, isolated from a core of a submerged Kopara sampled at 20 m from the border of a saltwater pond. The chemical space of this strain was investigated effectively using a dereplication strategy based on molecular networking and in-depth chemical analysis. The results highlight the impact of culture support on the molecular profile of Streptomyces albidoflavus 19-S21 secondary metabolites.     

Isolation and Structure Elucidation of New Cytotoxic Macrolides Halosmysins B and C from the Fungus Halosphaeriaceae sp. Associated with a Marine Alga

Two new cytotoxic metabolites, halosmysins B and C, have been isolated from the fungus Halosphaeriaceae sp. OUPS-135D-4 separated from the marine alga Sargassum thunbergii. These chemical structures have been elucidated by 1D and 2D NMR, and HRFABMS spectral analyses. The new compounds had the same 14-membered macrodiolide skeleton as halosmysin A, which was isolated from this fungal strain previously. As the unique structural feature, a diketopiperazine derivative and a sugar are conjugated to the 14-membered ring of halosmysins B and C, respectively. The absolute stereostructures of them were elucidated by the chemical derivatization such as a hydrolysis, the comparison with the known compounds (6R,11R,12R,14R)-colletodiol and halosmysin A, and a HPLC analysis of sugar. In addition, their cytotoxicities were assessed using murine P388 leukemia, human HL-60 leukemia, and murine L1210 leukemia cell lines. Halosmysin B was shown to be potent against all of them, with IC₅₀ values ranging from 8.2 ± 1.8 to 20.5 ± 3.6 μM, though these values were slightly higher than those of halosmysin A.     

Activation of the Silent Secondary Metabolite Production by Introducing Neomycin-Resistance in a Marine-Derived Penicillium purpurogenum G59

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.     

Mining Indonesian Microbial Biodiversity for Novel Natural Compounds by a Combined Genome Mining and Molecular Networking Approach

Indonesia is one of the most biodiverse countries in the world and a promising resource for novel natural compound producers. Actinomycetes produce about two thirds of all clinically used antibiotics. Thus, exploiting Indonesia’s microbial diversity for actinomycetes may lead to the discovery of novel antibiotics. A total of 422 actinomycete strains were isolated from three different unique areas in Indonesia and tested for their antimicrobial activity. Nine potent bioactive strains were prioritized for further drug screening approaches. The nine strains were cultivated in different solid and liquid media, and a combination of genome mining analysis and mass spectrometry (MS)-based molecular networking was employed to identify potential novel compounds. By correlating secondary metabolite gene cluster data with MS-based molecular networking results, we identified several gene cluster-encoded biosynthetic products from the nine strains, including naphthyridinomycin, amicetin, echinomycin, tirandamycin, antimycin, and desferrioxamine B. Moreover, 16 putative ion clusters and numerous gene clusters were detected that could not be associated with any known compound, indicating that the strains can produce novel secondary metabolites. Our results demonstrate that sampling of actinomycetes from unique and biodiversity-rich habitats, such as Indonesia, along with a combination of gene cluster networking and molecular networking approaches, accelerates natural product identification.     

In Vivo Metabolism Study of Xiamenmycin A in Mouse Plasma by UPLC-QTOF-MS and LC-MS/MS

Xiamenmycin A is an antifibrotic leading compound with a benzopyran skeleton that is isolated from mangrove-derived Streptomyces xiamenensis. As a promising small molecule for fibrotic diseases, less information is known about its metabolic characteristics in vivo. In this study, the time-course of xiamenmycin A in mouse plasma was investigated by relative quantification. After two types of administration of xiamenmycin A at a single dose of 10 mg/kg, the plasma concentrations were measured quantitatively by LC-MS/MS. The dynamic changes in the xiamenmycin A concentration showed rapid absorption and quick elimination in plasma post-administration. Four metabolites (M1–M4) were identified in blood by UPLC-QTOF-MS, and xiamenmycin B (M3) is the principal metabolite in vivo, as verified by comparison of the authentic standard sample. The structures of other metabolites were identified based on the characteristics of their MS and MS/MS data. The newly identified metabolites are useful for understanding the metabolism of xiamenmycin A in vivo, aiming at the development of an anti-fibrotic drug candidate for the therapeutic treatment of excessive fibrotic diseases.     

Actinomycetes from Red Sea Sponges: Sources for Chemical and Phylogenetic Diversity

The diversity of actinomycetes associated with marine sponges collected off Fsar Reef (Saudi Arabia) was investigated in the present study. Forty-seven actinomycetes were cultivated and phylogenetically identified based on 16S rRNA gene sequencing and were assigned to 10 different actinomycete genera. Eight putatively novel species belonging to genera Kocuria, Mycobacterium, Nocardia, and Rhodococcus were identified based on sequence similarity values below 98.2% to other 16S rRNA gene sequences available in the NCBI database. PCR-based screening for biosynthetic genes including type I and type II polyketide synthases (PKS-I, PKS-II) as well as nonribosomal peptide synthetases (NRPS) showed that 20 actinomycete isolates encoded each at least one type of biosynthetic gene. The organic extracts of nine isolates displayed bioactivity against at least one of the test pathogens, which were Gram-positive and Gram-negative bacteria, fungi, human parasites, as well as in a West Nile Virus protease enzymatic assay. These results emphasize that marine sponges are a prolific resource for novel bioactive actinomycetes with potential for drug discovery.