Elicitation of natural products biosynthesis from endophytic microorganisms´ interactions

Texto

(1)UNIVERSITY OF SÃO PAULO SCHOOL OF PHARMACEUTICAL SCIENCES OF RIBEIRÃO PRETO. Elicitation of natural products biosynthesis from endophytic microorganisms´ interactions. Andrés Mauricio Caraballo Rodríguez. Ribeirão Preto 2016.

(2) UNIVERSITY OF SÃO PAULO SCHOOL OF PHARMACEUTICAL SCIENCES OF RIBEIRÃO PRETO. Elicitation of natural products biosynthesis from endophytic microorganisms´ interactions. Tese de Doutorado apresentada ao Programa de Pós-Graduação em Ciências Farmacêuticas para obtenção do Título de Doutor em Ciências Área de Concentração: Produtos Naturais e Sintêticos Dissertation submitted to the Pharmaceutical Sciences Graduate Program in partial fulfillment of the requirements for the degree of Doctor in Philosophy.. Field of Study: Natural and synthetic products PhD candidate: Andrés Mauricio Caraballo Rodríguez Advisor: Mônica Tallarico Pupo, PhD.. Corrected version of the doctoral dissertation submitted to the Pharmaceutical Sciences Graduate Program 10/02/2017. The original version is available at the School of Pharmaceutical Sciences of Ribeirão Preto/USP. Ribeirão Preto 2016.

(3) i ABSTRACT. CARABALLO-RODRÍGUEZ, A. M. Elicitation of natural products biosynthesis from endophytic microorganisms´ interactions. 2016. 169 p. Doctoral Dissertation. School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo, Ribeirão Preto, 2016. In order to continue with the study of natural products from previously isolated endophytes of the Brazilian medicinal plant Lychnophora ericoides, the main goal of this work focused on the metabolic exchange of endophytic microorganisms from this plant. As it has been demonstrated during the last years, elicitation of different molecules is consequence of microbial interactions, mainly due to the need to compete, survive and establish microbial communities in shared environments. Recent mass spectrometry related approaches, such as imaging and molecular networking, in combination with purification and structural elucidation were applied to the current study of the microbial interactions of endophytes. During this study, several chemical families were identified, such as polyene macrocycles, pyrroloindole alkaloids, leupeptins, angucyclines, siderophores, amongst others. Amongst the polyene macrocycles, the well-known antifungal amphotericin B was identified as a biosynthetic product of the endophytic actinobacteria Streptomyces albospinus RLe7. When S. albospinus RLe7 interacted with an endophytic fungus, Coniochaeta sp. FLe4, a red pigmentation was observed. A new fungal compound was detected from microbial interactions. Isolation and structure elucidation of this new compound enabled to demonstrate the elicitation of fungal secondary metabolites by amphotericin B, an actinobacterial metabolite. It was also demonstrated the elicitation of griseofulvin and its analogue dechlorogriseofulvin from another endophytic fungus, FLe9, as a consequence of exposition to amphotericin B. In addition, investigation of the chemical profile of another endophytic actinobacteria, S. mobaraensis RLe3, led to reveal this strain as angucycline-derivatives producer. Besides that, coculturing of this actinobacteria against Coniochaeta sp. FLe4 also demonstrated elicitation of angucycline analogues. In conclusion, this study demonstrated elicitation of natural products from microbial interactions as well as new compounds from endophytes from L. ericoides and contributed to the identification of microbial metabolites.. Keywords: Endophytes; microbial interactions; natural products; molecular networking; elicitation; amphotericin B..

(4) ii. RESUMO. CARABALLO-RODRÍGUEZ, A. M. Eliciação da biossíntese de produtos naturais a partir da interação de microorganismos endofíticos. 2016. 169 f. Tese de doutorado, Faculdade de Ciências Farmacêuticas de Ribeirão Preto - Universidade de São Paulo, Ribeirão Preto, 2016. Com o propósito de continuar com os estudos de produtos naturais de microorganismos endofíticos da planta medicinal Brasileira Lychnophora ericoides, o principal objetivo desse trabalho focou-se no estudo da troca metabólica de microorganismos endofíticos dessa planta. Como tem sido demonstrado durante os últimos anos, a elicitação de diferentes compostos é consequência das interações microbianas, principalmente devido à necessidade de competir, sobreviver e estabelecer comunidades microbianas em diversos ambientes naturais. Abordagens recentes de espectrometria de massas, tais como imageamento e molecular networking, junto com purificação e elucidação estrutural foram aplicadas durante o estudo de interações microbianas de endofíticos. Durante este estudo, várias classes químicas foram identificadas, tais como polienos macrocíclicos, alcaloides pirroloindólicos, leupeptinas, anguciclinas, sideróforos, entre outras. Da classe química de polienos macrocíclicos, foi identificada a anfotericina B como produto da biossíntese da actinobactéria endofítica Streptomyces albospinus RLe7. Durante a interação da S. albospinus RLe7 com o fungo endofítico Coniochaeta sp. FLe4, uma pigmentação vermelha foi observada. Um novo composto de origem fúngica foi detectado a partir de interações microbianas. O isolamento e posterior elucidação estrutural do novo composto permitiu demonstrar a eliciação de metabólitos secundários fúngicos pela anfotericina B, metabólito da actinobactéria S. albospinus RLe7. Foi também demonstrada a eliciação de griseofulvina e desclorogriseofulvina a partir de outro fungo endofítico, FLe9, como consequência da exposição à anfotericina B. Adicionalmente, a investigação do perfil químico de outra actinobactéria endofítica, S. mobaraensis RLe3, evidenciou essa linhagem como produtora de compostos da classe das anguciclinas. Além disso, o cocultivo dessa actinobacteria com o fungo endofítico Coniochaeta sp. FLe4 também eliciou análogos de anguciclinas. Em conclusão, neste estudo demonstrou-se a elicitação de produtos naturais a partir das interações microbianas assim como de novos compostos de endofíticos de L. ericoides e contribuiu-se com a identificação de metabólitos microbianos.. Palavras-chave: Endofíticos. Networking. Elicitação.. Interações. microbianas.. Produtos. naturais.. Molecular.

(5) 1. 1.. INTRODUCTION. 1.1.. Relevance of endophytic microorganisms. The first report of an endophytic fungus was published more than a century ago (Freeman, 1904), describing the presence of a non-spore forming fungus in the seeds of Lolium temulentum and suggesting a possible symbiotic relationship between the fungus and the host. The term “endophyte” was later clarified in the biological context by considering that many biological terms evolve over time (Wilson, 1995). The proposed definition considered not just the location but also the infection strategy by the microorganism. Therefore, endophytes were defined as fungi or bacteria that during all or part of their life cycle invade tissues of living plants without causing symptoms of disease (Wilson, 1995). Interestingly, in that paper was emphasized that, instead of being slaves of rules and definitions, the priority should be placed on the biological context of the situation. As the question regarding to why infection by endophytes does not trigger a defense response by the plant host (Wilson, 1995), a balanced antagonism was proposed as a consequence of endophyte-host interaction (Schulz & Boyle, 2005; Schulz, Rommert, Dammann, Aust, & Strack, 1999). By investigating endophytes and plant pathogens, it was found that a higher proportion of endophytes produced herbicidal metabolites when compared to microorganisms isolated from other sources (Schulz et al., 1999). Additionally, it was suggested that secondary metabolites produced by pathogens and endophytes inhabiting the same host may be directed against each other in order to decrease their competition (Schulz et al., 1999). This is a clear example of microbial metabolites as mediators of biological responses in ecological contexts, such as plant-microorganisms systems. Endophytes became interesting as bioactive compounds sources since the first report of an endophytic fungus, Taxomyces andreanae, producing the anticancer compound paclitaxel (Stierle, Strobel, & Stierle, 1993). Paclitaxel (Figure 1) was further investigated and still remains amongst one of the most effective chemotherapeutics currently used against cancer (Cragg, Grothaus, & Newman, 2014; Newman & Cragg, 2016). Paclitaxel has been reported from several endophytes (Somjaipeng, Medina, & Magan, 2016), including actinobacteria such as Kitasatospora sp. (Caruso et al., 2000), as part of the efforts to find sustainable sources for.

(6) 2. this compound (Liu, Gong, & Zhu, 2016). After several years of research focused on obtaining more amounts of this compound, the question about why endophytic microorganisms produced the same compound as their hosts kept unanswered (Talbot, 2015). A relatively recent study provided some answers and gave new insights into the fungi-host interaction, with paclitaxel as one of the mediators of this relationship (Soliman et al., 2015; Soliman & Raizada, 2013). Briefly, as paclitaxel acts as fungicide (Soliman, Trobacher, Tsao, Greenwood, & Raizada, 2013), yew tree formed a symbiotic relationship with taxol-producing endophytes to protect itself against pathogen invasion (Soliman, Tsao, & Raizada, 2011). The endophyte protects diving cells from paclitaxel phytotoxicity by storage in hydrophobic bodies. As a response to pathogen elicitors, the hydrophobic bodies containing the antifungal are released by exocytosis at the pathogen entry points. So, while plant-produced paclitaxel assists general immunity, endophytic-produced paclitaxel might target immunity where plant cells cannot, finally explaining why both organisms produce paclitaxel (Soliman et al., 2015). Maytansine (Figure 1), another important anticancer and cytotoxic compound, was discovered in 1970 from Putterlickia species and in other Celastraceae plants of the genus Maytenus (Kupchan et al., 1977; Kupchan et al., 1972). Interestingly, it was demonstrated a different localization of maytansine in plant tissues. While in Maytenus, it was localized in the above-ground tissues; in Putterlickia plants this compound was accumulated in the roots (Kupchan et al., 1977; Kupchan et al., 1972). Further investigation enabled to demonstrate that maytansine was produced by an endophytic microbial community within the roots of Putterlickia verrucosa and P. retrospinosa (Kusari, S. et al., 2014). Besides that, in Maytenus serrata, maytansine is produced jointly by the endophytic bacterial community with the host plant (Kusari, P. et al., 2016). Recent studies showed detection of new maytansinoids in P. pyracantha (Eckelmann, Kusari, & Spiteller, 2016), opening up further questions addressed to reveal the ecological relevance of maytansinoids in their plant-hosts. A large number of publications about endophytic microorganisms focused on natural products sources became available and several reviews highlight the importance of endophytic fungi for the pursuit of natural products (Aly, Debbab, Kjer, & Proksch, 2010; Borges, W., Borges, K., Bonato, Said, & Pupo, 2009; Gunatilaka, 2006; Kharwar, Mishra, Gond, Stierle, & Stierle, 2011; Schulz, Boyle, Draeger, Rommert, & Krohn, 2002; Suryanarayanan et al., 2009; Verma, Kharwar, & Strobel, 2009; Wang & Dai, 2011) as well as endophytic actinobacteria (Golinska et al., 2015; Qin, Xing, Jiang, Xu, & Li, 2011; Raja & Prabakarana, 2011) or endophytes in general as sources for therapeutic uses in various fields (Gouda, Das, Sen, Shin, & Patra, 2016). Endophytes can potentially be used for peptide based drugs (Abdalla &.

(7) 3. Matasyoh,. 2014). or. even. other. anticancer compounds,. such. as the. pro-drug. deoxypodophyllotoxin (Kusari, S., Lamshoeft, & Spiteller, 2009), camptothecin (Venugopalan, Potunuru, Dixit, & Srivastava, 2016) or solamargine (El-Hawary et al., 2016) (Figure 1), amongst others. However, their use at industrial level will have to wait for further developments (Kusari, S., Pandey, & Spiteller, 2013; Kusari, S. & Spiteller, 2011). Currently, as illustrated with the paclitaxel investigations, the discussion about the real sources of several natural products have turned into what those metabolites are doing in nature (Davies, 2013; Davies & Ryan, 2012; Newman & Cragg, 2015; Talbot, 2015). An understanding of the chemical ecology behind microbial interactions of endophytes will lead to reveal their inexhaustible biosynthetic potential (Kusari, S., Hertweck, & Spitellert, 2012). The role natural products may play in the endophyte-host plant interaction is a matter of recent interest. Considering the wide range of biological activities already discovered for the reported microbial metabolites, their role in nature may be suggested. Since microorganisms use the chemical language to communicate and interact, one of the suggested roles may be the interference in quorum sensing by attenuating virulence factors of pathogens, which means quenching of pathogen quorum molecules (Kusari, P. et al., 2014). In nature, microbes and plants are in a close association, and the study of population dinamics, gene expression as well as metabolite production involving microbe-microbe and microbe-plant quorum sensing phenomena will give further directions to completely understand these ecosystems (Kusari, P., Kusari, S., Spiteller, & Kayser, 2015). One example where a tripartite interaction mediated by quorum sensing molecules illustrates the importance of chemical communication in ecological systems. Pantoea agglomerans (olive plant epiphyte) and Erwinia toletana (endophyte), nonpathogenic bacteria, are associated with olive knot disease. Both microorganisms release homoserine lactones analogues (Figure 1), signals that modulate the virulence of Pseudomonas savastanoi pv. Savastanoi, responsible pathogen for the disease (Hosni et al., 2011). The interference in quorum sensing is generally mediated mainly by two enzymes, lactonase and acylase, for quenching or degradating of homoserine lactone autoinducers (Hong, Koh, Sam, Yin, & Chan, 2012). Interestingly, homoserine lactone acylase has been reported from endophytic Streptomyces, which may be involved in suppressing the soft rot infection by Pectobacterium carotovorum spp. carotovorum on potato (Chankhamhaengdecha, Hongvijit, Srichaisupakit, Charnchai, & Panbangred, 2013). These findings show the complexity of chemical communication in ecological systems. Biotechnological applications of natural products for drug resistance can be fully exploited if factors such as autoinducer triggers, production/release signals or even degradation systems are considered (Kusari, P. et al., 2015)..

(8) 4. Figure 1. Illustrative examples of natural products from endophytes.. The role of endophytes in plant growth has been also investigated (Gaiero et al., 2013; Miliute et al., 2016). Plant promoting traits, such as production of indole-3-acetic acid (Figure 1), were observed in endophytes from apple phyllosphere (Miliute et al., 2016). Indole-3-acetic acid is a phytohormone that stimulates cell division as well as formation of plant roots (Davies, 2010). The effect of bacterial endophytes was also investigated in Trigonella foenum-graecum L. Four out of nine endophytic Bacillus sp., associated with that medicinal plant, showed a significant impact on plant growth and besides that, contributed to the increase in diosgenin biosynthesis, a steroid sapogenin (Jasim, Geethu, Mathew, & Radhakrishnan, 2015). In another medicinal plant, Euphorbia pekinensis, the effective growth promotion as a consequence of treatment with endophytic fungi was demonstrated. Interestingly, it was also proved that treatment of plants with non-endophytic fungi, was not as effective as the treatment with endophytes. These findings could be attributed to the production of phytohormones, such as indole-3 acetic acid (Dai, Yu, & Li, 2008). The mechanisms behind the plant-growth promotion by endophytic bacteria have been proposed as phytostimulation (through production of phytohormones), biofertilization (by increasing access to nutrients) and biocontrol (by protection against phytopathogens) (Bloemberg & Lugtenberg, 2001). A complete understanding of the microbial community composition of the plant system is necessary to predict the effectiveness of an endophyte to promote plant growth (Gaiero et al., 2013). Of.

(9) 5. course, more comprehensive tools, such as “omics” approaches, should be used to study endophytes and reveal their role in the plant host (Kaul, Sharma, & Dhar, 2016). Even more relevant for future research, after understanding the endophyte community dynamics, is how these populations will be affected in an environment influenced by climate change (Gaiero et al., 2013). As microorganisms are still underexplored reservoirs of natural products, endophytes from the Brazilian medicinal plant Lychnophora ericoides (falsa-arnica) were selected in this study for investigation of their natural products profile. L. ericoides has been used in traditional medicine for treatment of wounds, inflammation and pain and several studies focused on its chemistry have been carried out (Borella, Lopes, Vichnewski, Cunha, & Herz, 1998; dos Santos, Gobbo-Neto, Albarella, de Souza, & Lopes, 2005; Gobbo-Neto, L., dos Santos, et al., 2008; Gobbo-Neto, L., Gates, & Lopes, 2008; Gobbo-Neto, L. et al., 2010; Gobbo-Neto, L. & Lopes, 2008; Gobbo-Neto, L. et al., 2005; Sakamoto et al., 2003; Semir, Rezende, Monge, & Lopes, 2011). As part of the efforts of our research at the Laboratory of Chemistry of Microorganisms (LQMo) at the School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, coordinated by Prof. Dr. Mônica T. Pupo, endophytic microorganisms from L. ericoides were isolated and a first study including some of these actinobacteria strains was recently published by our group (Conti et al., 2016). In light of the described studies and the importance of endophytes for this research field, microbial interactions approaches were used in this PhD thesis in order to investigate natural products from endophytes of L. ericoides.. 1.2.. Natural products from microbial interactions approaches. As it was mentioned in the previous section, microorganisms inhabiting plant tissues are interacting with other microorganisms. One of the expected consequences of these interactions, which has been the focus of several studies, is the production of secondary metabolites. Several reviews have been published highlighting different strategies involving microbial interactions in order to increase the metabolite diversity (Bertrand et al., 2014; Bode, Bethe, Hofs, & Zeeck, 2002; Marmann, Aly, Lin, Wang, & Proksch, 2014; Pettit, 2009; Rutledge & Challis, 2015; Scherlach & Hertweck, 2009). These approaches are totally consistent with simulation of ecological contexts since microorganisms are in continuous interactions with other microorganisms and even macroorganisms (Suryanarayanan, T., 2013), as previously.

(10) 6. described. However, it is well known that microbes establish different kinds of interactions such as cooperative or competitive relationships (Abrudan et al., 2015; Ghoul & Mitri, 2016; Stubbendieck & Straight, 2016), and most of in vitro microbial interactions have not been tested in more natural settings (Hibbing, Fuqua, Parsek, & Peterson, 2010). Due to the lack of knowledge of the complex regulatory factors that may influence the microbial biosynthetic machinery, a random approach named OSMAC (One Strain – MAny Compounds) was proposed in the last decade (Bode et al., 2002). Briefly, the concept establishes that by modifying culture parameters such as culture vessels, culture media, fermentor size, pH, dissolved oxygen, or even microbial interaction, the chemical diversity can be expanded. By using the OSMAC approach, more than 100 compounds from 25 different structural classes from only 6 microorganisms were isolated (Bode et al., 2002). Microbial interactions approach, or coculture strategy, has been widely used to induce microbial compounds of interest, and plenty of examples can be found in literature. For instance, increased production of paclitaxel from Paraconiothyrium SSM001 when exposed to endophytes from the same host, Taxus (yew) tree (Soliman & Raizada, 2013) was observed. The induction of istamycins (Figure 2) in Streptomyces tenjimariensis up to twice-fold as a consequence of the interaction with twelve out of 53 tested bacteria was reported (Slattery, Rajbhandari, & Wesson, 2001). Microbial interactions between Pseudomonas aeruginosa and Enterobacter sp., isolated from a marine environment, enabled the isolation of a blue compound, identified as pyocyanin (Figure 2), as well as the correction of the structural characterization of this compound by NMR due to previous misreported data in the literature (Angell, Bench, Williams, & Watanabe, 2006). Cocultivation of marine fungi have resulted in isolation of new alkaloids, marinamide and its methyl ester (Zhu & Lin, 2006), which chemical structures were later revised and attributed to pyrrolyl 4-quinolone analogues (Figure 2) (Zhu, Chen, Wu, & Pan, 2013), as well as a new xanthone, 8-hydroxy-3-methyl-9-oxo-9H-xanthene1-carboxylic acid methyl ether (Figure 2) (Li, C. et al., 2011). Microbial interaction between Aspergillus fumigatus and Streptomyces peucetius led to the induction of the new metabolites, fumiformamide. and. N,N’-((1Z,. 3Z)-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-. diyl)diformamide (Figure 2), two known N-formyl derivatives and a xanthocillin analogue BU4704 (Zuck, Shipley, & Newman, 2011). The coculturing of the endophytic fungus Fusarium tricinctum with the bacterium Bacillus subtilis led to the production of three new compounds, macrocarpon C, 2-(carboxymethylamino)benzoic acid and (-)-citreoisocumarinol (Figure 2), as well as the increased levels of known compounds of the chemical classes of pyrones, cyclic depsipeptides and lipopeptides (Ola, Thomy, Lai, Broetz-Oesterhelt, & Prolcsch, 2013). Three.

(11) 7. new decalin-type tetramic acid analogues, N-demethyl-ophiosetin and pallidorosetins A/B, were produced when Fusarium pallidoroseum was cocultured with Saccharopolyspora erythraea (Figure 2) (Whitt, Shipley, Newman, & Zuck, 2014). Three novel macrolactams with unprecedented skeletons, niizalactams A-C, were isolated when Streptomyces sp. NZ-6 was cultured in presence of mycolic acid-containing bacterium Tsukamurella pulmonis TP-B0596 (Hoshino et al., 2015). Interestingly, the same strain T. pulmonis TP-B0596 induced the production of eight new 5-alkyl-1,2,3,4-tetrahydroquinolines (Figure 2) (Sugiyama et al., 2015) as well as a novel class of lipidic metabolites named streptoaminals (Figure 2) from Streptomyces nigrescens HEK616 (Sugiyama et al., 2016)..

(12) 8. Figure 2. Illustrative examples of natural products from microbial interactions..

(13) 9. A remarkable study supported the hypothesis that not only diffusible signals but also intimate physical interactions contribute to microbial communication (Schroeckh et al., 2009). In that study, it was demonstrated that the physical interaction of Aspergillus nidulans with Streptomyces hygroscopicus led to stimulate the fungal biosynthesis of aromatic polyketides, such as orsellinic acid, lecanoric acid, and cathepsin inhibitors F-9775A and F-9775B (Figure 2) (Schroeckh et al., 2009). Another outstanding study screened 657 coculture experiments involving fungal strains by using a high-throughput UHPLC-TOF-MS-based metabolomic approach. In this study, two types of inductions were observed, de novo biosynthesis and upregulation. Interestingly, most of the detected features (peaks) from monocultures (80%) matched at least one reported compound from the DNP database ("Dictionary of Natural Products,"), while the matches were zero when the induced features, corresponding to the induced metabolites from coculture, where searched. This results suggest that the features detected at the fungal interaction zones in cocultures may correspond to novel chemical compounds, not included in natural products databases (Bertrand et al., 2013). Taking into consideration that a single microbial compound may have an impact on the metabolism of microorganisms inhabiting the same environment (Kusari, S. et al., 2013), exposing microorganisms to simulated communities where chemical exchange occurs makes the previous examples coherent with what may happen in natural contexts. At this point it is necessary to highlight that the study of microbe-microbe interactions may provide valuable information to decipher chemical communication in nature, a necessary step before validate the findings in more complex microbial communities (Lareen, Burton, & Schafer, 2016) as it may occur in natural systems. Besides that, multispecies interactions should be studied since endophytes communities are diverse and complex, and consequently, may be more involved in interactions with microbial competitors than with the plant host itself (van Overbeek & Saikkonen, 2016). This is consistent, since microbes are part of essential consortia (Newman & Cragg, 2015), no matter what the environment they inhabit. Considering the genome size of 8 Mb in actinobacteria, up to 11 Mb in myxobacteria, and more than 30 Mb in fungi, the enormous potential of microorganisms for production of small molecules is big (Bode et al., 2002). This is consistent with recent genomic approaches, such as metagenomics, showing global microbial biosynthetic diversity holds enormous potential for natural products discovery (Charlop-Powers et al., 2015). The bottleneck lays in finding the triggers for biosynthesis of the cryptic metabolites, although the regulation of antibiotic production has been extensively investigated in microbial models such as S. coelicolor, but also.

(14) 10. in nonmodel streptomycetes (Liu, Chater, Chandra, Niu, & Tan, 2013), giving directions for regulation of secondary metabolites that can be extended perhaps in other actinobacteria. Due to the relevance of small molecules as mediators of chemical responses during microbial interactions, and the fact that the levels of those chemicals are very low for detection, the need for more sensitive techniques is required. For that reason, mass spectrometry related tools were applied to the current study of endophytes from L. ericoides.. 1.3.. Mass spectrometry related tools for microbial natural products investigation. The necessity for more sensitive techniques that enable to detect small quantities of microbial compounds can be illustrated with the yet-uncharacterized secondary metabolite produced by human gut bacteria, colibactin, involved in colorectal cancer (Nougayrede et al., 2006). After more than ten years of research, the chemical structure of this cryptic metabolite remains unsolved (Bode, 2015). However, several approaches, including NMR, MS, genomics and bioinformatics tools, have enabled to tentatively propose candidates for pre-colibactins (Figure 3) (Li, Z. R. et al., 2015; Vizcaino & Crawford, 2015). A recent example given by the identification of maytansinoids in plant tissues showed clearly the need for MS/MS data that enable future studies to identify natural products without the necessity for isolating (Eckelmann et al., 2016). For that reason, techniques that enable to obtain enough chemical information from small quantities of samples are relevant for the current research, including in the field of natural products. Recent developments have occurred in the detection of microbial small molecules by using mass spectrometry (MS) approaches. A first approach recently used, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Imaging Mass Spectrometry (MALDI-TOF IMS) for detecting microbial small molecules has been developed ad widely used (Yang et al., 2012). Basically, MALDI-TOF IMS offers the possibility to detect small molecules from agar pieces containing cultured microorganisms, and also allows to visualize their distribution in two or even three dimensions (Watrous et al., 2013). Several examples and even the discovery of novel natural products and biotransformation processes have been published in the past few years (Gonzalez et al., 2012; Moree et al., 2012). This technique has been also applied for monitoring suppression of quorum sensing signals by endophytic bacteria from Cannabis sativa L. (Kusari,.

(15) 11. P. et al., 2014). Besides that, it enabled to visualize the spatial distribution of metabolites in situ, as illustrated by maytansine in the roots of Putterlickia verrucosa and P. retrospinosa, enabling to prove the hypothesis of microbial endophytes as actual producers of the compound (Kusari, S. et al., 2014). Another MS-based approach is molecular networking. It consists in the comparison of fragmentation patterns amongst different MS/MS spectra in order to find similarities amongst them. This approach was initially applied for peptide identification (Guthals & Bandeira, 2012) and it was then developed for microbial small molecules analysis, since every single molecule can lead to a unique MS/MS spectra, such as a fingerprint and then similarities can be found in structurally related compounds (Watrous et al., 2012; Yang et al., 2013). Molecular networking has been important for detection and partial characterization of natural products, such as an antifungal lipopeptide from the syringomycin family, thanamycin (Figure 3) (Watrous et al., 2012). As manual analysis of large MS/MS datasets results inefficient, a free access platform for the automated analysis and cross correlation of natural products MS/MS data, named Global Natural Products Social Molecular Networking (Wang, M. et al., 2016) (GNPS, gnps.ucsd.edu) was recently developed. What can be done by using the GNPS workflow include storage and organization of MS/MS data, generation and visualization of molecular networking, dereplication of natural products, contribution to MS/MS natural products collection, sharing and curate data. For dereplication purposes, which means finding known or already identified natural products, based on comparison with MS/MS data the GNPS platform includes libraries such as MassBank, ReSpect and NIST, as well as collection of natural products and pharmacologically active compounds related to the National Institutes of Health (NIH) in the United States (Wang, M. et al., 2016). Currently, the GNPS libraries contains more than 200.000 MS/MS spectra representing more than 22.000 compounds (Wang, M. et al., 2016). However, the number of published natural products can be about 300.000 to 600.000 (Berdy, 2012), which means the total chemical space still remains to be covered. To demonstrate how GNPS platform can support natural products discovery, a set of five analogues of a broadspectrum antibiotic, stenothricin, were identified and named stenothricin-GNPS 1-5. The most abundant of these analogues was isolated successfully confirming its structural similarity (Figure 3) (Wang, M. et al., 2016). Computational methods for detection and investigation of small molecules using mass spectrometry data have been developed (Hufsky, Scheubert, & Bocker, 2014). Recently, in silico approaches that enable to search experimental MS/MS.

(16) 12. spectra have been proposed and successfully tested to compensate the lack of experimental data (Allard et al., 2016).. Figure 3. Illustrative examples of natural products discovered by MS approaches and molecular networking.. Structural elucidation of new compounds based only by mass spectrometry or even elucidate the fragmentation pathway for knowns compounds are challenging tasks even for experienced mass spectrometrists (Chai, Wang, & Wang, 2016). However, as previously mentioned, fragmentation patterns are fingerprints that help in finding structural relationship amongst chemical families, as it has been widely reported by using molecular networking (Covington, McLeanab, & Bachmann, 2016; Floros, Jensen, Dorrestein, & Koyama, 2016; Garg et al., 2015; Mascuch et al., 2015; Nguyen et al., 2013; Okada, Wu, Mao, Bushin, & Seyedsayamdost, 2016; Wang, M. et al., 2016; Watrous et al., 2012). Due to the extensive application of MS/MS, it is still necessary to investigate the mechanisms for specific fragmentation reactions (Chai et al., 2016) to fully support structural elucidation. At this point, it is important to keep in mind the relevance of analyzing mass differences instead of defining the specific reactions involved in each fragment formation, in order to use mass fragmentation as a useful tool for structural elucidation (Demarque, Crotti, Vessecchi, Lopes, & Lopes, 2016)..

(17) 120. 5.. CONCLUSIONS. Demonstrating the biological role for every molecule in nature is on the way, and the investigation of natural products in microbial communities is more rational since microorganisms are not alone in nature. Here we have demonstrated the impact of microbial interactions, occurring amongst endophytes from Lychnophora ericoides, on their secondary metabolites profile. The presence of antifungal (amphotericin-analogues), anticholinesterase (physostigmine-analogues),. protease. inhibitors. (leupeptin-analogues). and. cytotoxic. (angucycline-analogues) compounds was revealed by using mass spectrometry related tools during investigation of microbial interactions. In addition, the elicitation of microbial metabolites was fully demonstrated by the action of amphotericin B, produced by S. albospinus RLe7, on two endophytic fungi, Coniochaeta sp. FLe4 and the fungus FLe9. Interestingly, elicitation was also demonstrated when Coniochaeta sp. FLe4 interacted with S. albospinus RLe3 inducing the production of angucycline derivatives. Besides identifying natural products during microbial interactions, how metabolic exchange may influence the establishment of those communities was visualized. With this study, partial annotation for the ionizable metabolome from single and interacting endophytic actinobacteria was provided. The fragmentation spectra from all microbial natural products detected here are available (“living data”) for future identification at the GNPS database repository. This is the first study of endophytic actinobacteria from L. ericoides in simulated communities, and the correlation between microbial exchange and colony growth may reflect a similar effect within their host in nature. Finally, the field of natural products has been benefited due to several advances for the support of structural elucidation and more advances will support identification of natural products in the years to come. Further work may reveal the specific biological role of small molecules from microorganisms in different environments. In conclusion, this study demonstrated elicitation of natural products from microbial interactions as well as new compounds from endophytes from L. ericoides, contributed through identification of microbial metabolites to the collaborative efforts of the scientific community in the field of natural products, and opened the possibilities for future studies involving endophytes from L. ericoides..

(18) 121. 6.. REFERENCES. Abdalla, M. A., & Matasyoh, J. C. (2014). Endophytes as producers of peptides: an overview about the recently discovered peptides from endophytic microbes. Natural Products and Bioprospecting, 4(5), 257-270.. Abdel-Mawgoud, A. M., Lepine, F., & Deziel, E. (2010). Rhamnolipids: diversity of structures, microbial origins and roles. Applied Microbiology and Biotechnology, 86(5), 13231336.. Abrudan, M. I., Smakman, F., Grimbergen, A. J., Westhoff, S., Miller, E. L., van Wezel, G. P., et al. (2015). Socially mediated induction and suppression of antibiosis during bacterial coexistence. Proceedings of the National Academy of Sciences of the United States of America, 112(35), 11054-11059.. Allard, P. M., Peresse, T., Bisson, J., Gindro, K., Marcourt, L., Pham, V. C., Roussi, F., Litaudon, M., & Wolfender, J. L. (2016). Integration of Molecular Networking and InSilico MS/MS Fragmentation for Natural Products Dereplication. Analytical Chemistry, 88(6), 3317-3323.. Alvi, K. A., Baker, D. D., Stienecker, V., Hosken, M., & Nair, B. G. (2000). Identification of inhibitors of inducible nitric oxide synthase from microbial extracts. Journal of Antibiotics, 53(5), 496-501.. Aly, A. H., Debbab, A., Kjer, J., & Proksch, P. (2010). Fungal endophytes from higher plants: a prolific source of phytochemicals and other bioactive natural products. Fungal Diversity, 41(1), 1-16. Anderson, T. M., Clay, M. C., Cioffi, A. G., Diaz, K. A., Hisao, G. S., Tuttle, M. D., Nieuwkoop, A. J., Comellas, G., Maryum, N., Wang, S., Uno, B. E., Wildeman, E. L., Gonen, T., Rienstra, C. M., & Burke, M. D. (2014). Amphotericin forms an extramembranous and fungicidal sterol sponge. Nature Chemical Biology, 10(5), 400U121. Angell, S., Bench, B. J., Williams, H., & Watanabe, C. M. H. (2006). Pyocyanin isolated from a marine microbial population: Synergistic production between two distinct bacterial species and mode of action. Chemistry & Biology, 13(12), 1349-1359.. Aoyagi, T., Takeuchi, T., Matsuzak.A, Kawamura, K., Kondo, S., Hamada, M., et al. (1969). Leupeptins, new protease inhibitors from actinomycetes. Journal of Antibiotics, 22(6), 283-286..

(19) 122. Apsel, B., Bender, J. A., Escobar, M., Kaelin, D. E., Lopez, O. D., & Martin, S. F. (2003). General entries to C-aryl glycosides. Formal synthesis of galtamycinone. Tetrahedron Letters, 44(5), 1075-1077.. Arima, K., Kakinuma, A., & Tamura, G. (1968). Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: isolation, characterization and its inhibition of fibrin clot formation. Biochemical and Biophysical Research Communications, 31(3), 488-494.. Ayukawa, S., Takeuchi, T., Sezaki, M., Hara, T., Umezawa, H., & Nagatsu, T. (1968). Inhibition of tyrosine hydroxylase by Aquayamycin. Journal of Antibiotics, 21(5), 350353.. Barona-Gomez, F., Lautru, S., Francou, F.-X., Leblond, P., Pernodet, J.-L., & Challis, G. L. (2006). Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2) and Streptomyces ambofaciens ATCC 23877. Microbiology-Sgm, 152.. Baumgart, F., Kluge, B., Ullrich, C., Vater, J., & Ziessow, D. (1991). Identification of aminoacid substitutions in the lipopeptide surfactin using 2D NMR-spectroscopy. Biochemical and Biophysical Research Communications, 177(3), 998-1005.. Behrens, B., Engelen, J., Tiso, T., Blank, L. M., & Hayen, H. (2016). Characterization of rhamnolipids by liquid chromatography/mass spectrometry after solid-phase extraction. Analytical and Bioanalytical Chemistry, 408(10), 2505-2514.. Berdy, J. (2012). Thoughts and facts about antibiotics: Where we are now and where we are heading. Journal of Antibiotics, 65(8), 385-395.. Bergkessel, M., Basta, D. W., & Newman, D. K. (2016). The physiology of growth arrest: uniting molecular and environmental microbiology. Nature Reviews Microbiology, 14(9), 549-562.. Bertrand, S., Bohni, N., Schnee, S., Schumpp, O., Gindro, K., & Wolfender, J. L. (2014). Metabolite induction via microorganism co-culture: A potential way to enhance chemical diversity for drug discovery. Biotechnology Advances, 32(6), 1180-1204.. Bertrand, S., Schumpp, O., Bohni, N., Bujard, A., Azzollini, A., Monod, M., Gindro, K., & Wolfender, J. L. (2013). Detection of metabolite induction in fungal co-cultures on solid media by high-throughput differential ultra-high pressure liquid chromatography-timeof-flight mass spectrometry fingerprinting. Journal of Chromatography A, 1292, 219228..

(20) 123. Bindseil, K. U., Hug, P., Peter, H. H., Petersen, F., & Roggo, B. E. (1995). Balmoralmycin, a new angucyclinone and two related biosynthetic shunt products containing a novel ring system. Journal of Antibiotics, 48(6), 457-461.. Birch, A. J., Massywestropp, R. A., Rickards, R. W., & Smith, H. (1958). Studies in relation to biosynthesis. 13. Griseofulvin. Journal of the Chemical Society, 360-365.. Blank, H. (1965). Antifungal and other effects of Griseofulvin. American Journal of Medicine, 39(5), 831-838.. Bloemberg, G. V., & Lugtenberg, B. J. J. (2001). Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Current Opinion in Plant Biology, 4(4), 343-350.. Bode, H. B. (2015). The Microbes inside Us and the Race for Colibactin. Angewandte ChemieInternational Edition, 54(36), 10408-10411.. Bode, H. B., Bethe, B., Hofs, R., & Zeeck, A. (2002). Big effects from small changes: Possible ways to explore nature's chemical diversity. Chembiochem, 3(7), 619-627.. Borah, S. N., Goswami, D., Lahkar, J., Sarma, H. K., Khan, M. R., & Deka, S. (2015). Rhamnolipid produced by Pseudomonas aeruginosa SS14 causes complete suppression of wilt by Fusarium oxysporum f. sp. pisi in Pisum sativum. Biocontrol, 60(3), 375-385.. Borella, J. C., Lopes, J. L. C., Vichnewski, W., Cunha, W. R., & Herz, W. (1998). Sesquiterpene lactones, triterpenes and flavones from Lychnophora ericoides and Lychnophora pseudovillosissima. Biochemical Systematics and Ecology, 26(6), 671-676.. Borges, W. D., Borges, K. B., Bonato, P. S., Said, S., & Pupo, M. T. (2009). Endophytic fungi: Natural products, enzymes and biotransformation reactions. Current Organic Chemistry, 13(12), 1137-1163.. Brian, P. W., Curtis, P. J., & Hemming, H. G. (1949). A substance causing abnormal development of fungal hyphae produced by Penicillium janczewskii zal. III. Identity of "Curling Factor" with griseofulvin. Transactions of the British Mycological Society, 32(1), 30-33.. Brossi, A. (1990). Bioactive Alkaloids. 4. Results of recent investigations with colchicine and physostigmine. Journal of Medicinal Chemistry, 33(9), 2311-2319..

(21) 124. Brown, M. J. B., Cortes, J., Cutter, A. L., Leadlay, P. F., & Staunton, J. (1995). A mutant generated by expression of an engineered DEBS1 protein from the erythromycinproducing polyketide synthase (PKS) in Streptomyces coelicolor produces the triketide as a lactone, but the major product is the nor-analog derived from acetate as starter acid. Journal of the Chemical Society-Chemical Communications(15), 1517-1518.. Budhiraja, A., Nepali, K., Sapra, S., Gupta, S., Kumar, S., & Dhar, K. L. (2013). Bioactive metabolites from an endophytic fungus of Aspergillus species isolated from seeds of Gloriosa superba Linn. Medicinal Chemistry Research, 22(1), 323-329.. Bush, M. J., Tschowri, N., Schlimpert, S., Flardh, K., & Buttner, M. J. (2015). c-di-GMP signalling and the regulation of developmental transitions in streptomycetes. Nature Reviews Microbiology, 13(12), 749-760.. Byrne, B., Carmody, M., Gibson, E., Rawlings, B., & Caffrey, P. (2003). Biosynthesis of deoxyamphotericins and deoxyamphoteronolides by engineered strains of Streptomyces nodosus. Chemistry & Biology, 10(12), 1215-1224.. Cacho, R. A., Chooi, Y. H., Zhou, H., & Tang, Y. (2013). Complexity generation in fungal polyketide biosynthesis: A spirocycle-forming P450 in the concise pathway to the antifungal drug griseofulvin. ACS Chemical Biology, 8(10), 2322-2330.. Caffrey, P., Lynch, S., Flood, E., Finnan, S., & Oliynyk, M. (2001). Amphotericin biosynthesis in Streptomyces nodosus: deductions from analysis of polyketide synthase and late genes. Chemistry & Biology, 8(7), 713-723.. Caruso, M., Colombo, A. L., Crespi-Perellino, N., Fedeli, L., Malyszko, J., Pavesi, A., Quaroni, S., Saracchi, M., & Ventrella, G. (2000). Studies on a strain of Kitasatospora sp. paclitaxel producer Annals of Microbiology, 50(2), 89-102.. Chagas, F. O., Dias, L. G., & Pupo, M. T. (2013). A mixed culture of endophytic fungi increases production of antifungal polyketides. Journal of Chemical Ecology, 39(10), 1335-1342.. Chagas, F. O., Caraballo-Rodríguez, A. M., Dorrestein, P. C., & Pupo, M. T. (2016). Expanding the chemical repertoire of the endophyte Streptomyces albospinus RLe7 reveals amphotericin B as inducer of a fungal phenotype. Submitted for publication. Chai, Y., Wang, L., & Wang, L. (2016). How does a C=C double bond cleave in the gas phase? Fragmentation of protonated ketotifen in mass spectrometry. Journal of Mass Spectrometry, 51(12), 1105-1110..

(22) 125. Chankhamhaengdecha, S., Hongvijit, S., Srichaisupakit, A., Charnchai, P., & Panbangred, W. (2013). Endophytic actinomycetes: A novel source of potential acyl homoserine lactone degrading enzymes. Biomed Research International, 8.. Charlop-Powers, Z., Owen, J. G., Reddy, B. V. B., Ternei, M., Guimaraes, D. O., de Frias, U. A., Pupo, M. T., Seepe, P., Feng, Z. Y., & Brady, S. F. (2015). Global biogeographic sampling of bacterial secondary metabolism. Elife, 4, 18.. Chen, M., Zhou, J. R., Li, C. Y., Song, Y. Y., Xie, L. J., Chen, S., & Zeng, R. S. (2009). Isolation, identification and bioactivity of allelochemicals of Streptomyces sp. strain 6803. Allelopathy Journal, 23(2), 411-424.. Chen, Y., Peng, Z. H., Song, W., Zhu, M. M., & Han, F. M. (2007). Chromatographic tandem mass spectrometric detection of physostigmine and its major metabolites in rat urine. Analytical Letters, 40(16-18), 3256-3266.. Chooi, Y. H., Cacho, R., & Tang, Y. (2010). Identification of the viridicatumtoxin and griseofulvin gene clusters from Penicillium aethiopicum. Chemistry & Biology, 17(5), 483-494.. Clejan, S., Krulwich, T. A., Mondrus, K. R., & Setoyoung, D. (1986). Membrane lipidcomposition of obligately and facultatively alkalophilic strains of Bacillus spp. Journal of Bacteriology, 168(1), 334-340.. CLSI. (2008). Reference Method for broth dilution antifungal susceptibility testing of filamentous fungi, Approved Standard. Second Edition. CLSI Document MA38-A2. 950 West Valley Road, Suite 2500, Wayne, Pennsylvania, 19087, USA: Clinical and Laboratory Standards Institute.. Colotelo, N. (1978). Fungal Exudates. Canadian Journal of Microbiology, 24(10), 1173-1181.. Conrad, R. S., & Gilleland, H. E. (1981). Lipid alterations in cell envelopes of polymyxinresistant Pseudomonas aeruginosa Isolates. Journal of Bacteriology, 148(2), 487-497.. Conti, R. (2012). Micro-organismos de interesse farmacêutico e agrícola: estudo químico e biosintético. Universidade de São Paulo, Campus de Ribeirão Preto, Ribeirão Preto..

(23) 126. Conti, R., Chagas, F. O., Caraballo-Rodriguez, A. M., Melo, W. G. D., do Nascimento, A. M., Cavalcanti, B. C., de Moraes, M. O., Pessoa, C., Costa-Lotufo, L. V., Krogh, R., Andricopulo, A. D., Lopes, N. P., & Pupo, M. T. (2016). Endophytic actinobacteria from the brazilian medicinal plant Lychnophora ericoides Mart. and the biological potential of their secondary metabolites. Chemistry & Biodiversity, 13(6), 727-736.. Covington, B. C., McLeanab, J. A., & Bachmann, B. O. (2016). Comparative mass spectrometry-based metabolomics strategies for the investigation of microbial secondary metabolites. Natural Product Reports, Advance Article.. Cragg, G. M., Grothaus, P. G., & Newman, D. J. (2014). New horizons for old drugs and drug leads. Journal of Natural Products, 77(3), 703-723.. D'Onofrio, A., Crawford, J. M., Stewart, E. J., Witt, K., Gavrish, E., Epstein, S., Clardy, J., & Lewis, K. (2010). Siderophores from neighboring organisms promote the growth of uncultured bacteria. Chemistry & Biology, 17(3), 254-264.. Dai, C. C., Yu, B. Y., & Li, X. (2008). Screening of endophytic fungi that promote the growth of Euphorbia pekinensis. African Journal of Biotechnology, 7(19), 3505-3510.. Davies, J. (2013). Specialized microbial metabolites: functions and origins. Journal of Antibiotics, 66(7), 361-364.. Davies, J., & Ryan, K. S. (2012). Introducing the parvome: bioactive compounds in the microbial world. ACS Chemical Biology, 7(2), 252-259.. Davies, P. J. (2010). Plant hormones: their nature, occurrence, and functions. In P. J. Davies (Ed.), Plant hormones (3 ed., pp. 1-15).. Demarque, D. P., Crotti, A. E. M., Vessecchi, R., Lopes, J. L. C., & Lopes, N. P. (2016). Fragmentation reactions using electrospray ionization mass spectrometry: an important tool for the structural elucidation and characterization of synthetic and natural products. Natural Product Reports, 33(3), 432-455.. Dhanya, K. I., Swati, V. I., Swaroop, V. K., & Osborne, W. J. (2016). Antimicrobial activity of Ulva reticulata and its endophytes. Journal of Ocean University of China, 15(2), 363369.. Dictionary of Natural Products, http://dnp.chemnetbase.com/ (2013)..

(24) 127. Diggle, S. P., Matthijs, S., Wright, V. J., Fletcher, M. P., Chhabra, S. R., Lamont, I. L., Kong, X. L., Hider, R. C., Cornelis, P., Camara, M., & Williams, P. (2007). The Pseudomonas aeruginosa 4-quinolone signal molecules HHQ and PQS play multifunctional roles in quorum sensing and iron entrapment. Chemistry & Biology, 14(1), 87-96.. Domenech, C. E., Garrido, M. N., & Lisa, T. A. (1991). Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase - 2 enzymes contributing to corneal infection. FEMS Microbiology Letters, 82(2), 131-135.. dos Santos, M. D., Gobbo-Neto, L., Albarella, L., de Souza, G. E. P., & Lopes, N. P. (2005). Analgesic activity of di-caffeoylquinic acids from roots of Lychnophora ericoides (Arnica da serra). Journal of Ethnopharmacology, 96(3), 545-549.. Eckelmann, D., Kusari, S., & Spiteller, M. (2016). Occurrence and spatial distribution of maytansinoids in Putterlickia pyracantha, an unexplored resource of anticancer compounds Fitoterapia, 113, 175-181.. El-Hawary, S. S., Mohammed, R., AbouZid, S. F., Bakeer, W., Ebel, R., Sayed, A. M., Rateb, M. E. (2016). Solamargine production by a fungal endophyte of Solanum nigrum. Journal of Applied Microbiology, 120(4), 900-911.. Epand, R. F., Savage, P. B., & Epand, R. M. (2007). Bacterial lipid composition and the antimicrobial efficacy of cationic steroid compounds (Ceragenins). Biochimica Et Biophysica Acta-Biomembranes, 1768(10), 2500-2509.. Epand, R. F., Schmitt, M. A., Gellman, S. H., & Epand, R. M. (2006). Role. of membrane lipids in the mechanism of bacterial species selective toxicity by two alpha/beta-antimicrobial peptides. Biochimica Et Biophysica Acta-Biomembranes, 1758(9), 1343-1350.. Epand, R. F.; Schmitt, M. A.; Gellman, S. H.; Sen, A.; Auger, M.; Hughes, D. W.; Epand, R. M. (2005). Bacterial species selective toxicity of two isomeric alpha/beta-peptides: Role of membrane lipids. Molecular Membrane Biology, 22(6), 457-469.. Fang, J. S., & Barcelona, M. J. (1998). Structural determination and quantitative analysis of bacterial phospholipids using liquid chromatography electrospray ionization mass spectrometry. Journal of Microbiological Methods, 33(1), 23-35.. Floros, D. J., Jensen, P. R., Dorrestein, P. C., & Koyama, N. (2016). A metabolomics guided exploration of marine natural product chemical space. Metabolomics, 12(145), 1-11..

(25) 128. Freeman, E. M. (1904). The seed-fungus of Lolium temulentum, L., the Darnel. Philosophical Transactions of the Royal Society of London, Series B(196), 1-27.. Gaiero, J. R., McCall, C. A., Thompson, K. A., Day, N. J., Best, A. S., & Dunfield, K. E. (2013). Inside the root microbiome: Bacterial root endophytes and plant growth promotion. American Journal of Botany, 100(9), 1738-1750.. Garg, N., Kapono, C. A., Lim, Y. W., Koyama, N., Vermeij, M. J. A., Conrad, D., Rohwer, F., Dorrestein, P. C. (2015). Mass spectral similarity for untargeted metabolomics data analysis of complex mixtures. International Journal of Mass Spectrometry, 377, 719727.. Gentles, J. C. (1958). Experimental ringworm in guinea pigs - Oral treatment with griseofulvin. Nature, 182(4633), 476-477.. Ghoul, M., & Mitri, S. (2016). The ecology and evolution of microbial competition. Trends in Microbiology, 24(10), 833-845.. Gidden, J., Denson, J., Liyanage, R., Ivey, D. M., & Lay, J. O. (2009). Lipid compositions in Escherichia coli and Bacillus subtilis during growth as determined by MALDI-TOF and TOF/TOF mass spectrometry. International Journal of Mass Spectrometry, 283(1-3), 178-184.. Gobbo-Neto, L., dos Santos, M. D., Albarella, L., Zollo, F., Pizza, C., & Lopes, N. P. (2008). Glycosides, caffeoylquinic acids and flavonoids from the polar extract of leaves of Lychnophora ericoides Mart. (Asteraceae). Biochemical Systematics and Ecology, 36(56), 473-475.. Gobbo-Neto, L., Gates, P. J., & Lopes, N. P. (2008). Negative ion 'chip-based' nanospray tandem mass spectrometry for the analysis of flavonoids in glandular trichomes of Lychnophora ericoides Mart. (Asteraceae). Rapid Communications in Mass Spectrometry, 22(23), 3802-3808.. Gobbo-Neto, L., Guaratini, T., Pessoa, C., de Moraes, M. O., Costa-Lotufo, L. V., Vieira, R. F., Colepicolo, P., & Lopes, N. P. (2010). Differential metabolic and biological profiles of Lychnophora ericoides Mart. (Asteraceae) from different localities in the Brazilian "campos rupestres". Journal of the Brazilian Chemical Society, 21(4), 750-759.. Gobbo-Neto, L., & Lopes, N. P. (2008). Online identification of chlorogenic acids, sesquiterpene lactones, and flavonoids in the Brazilian arnica Lychnophora ericoides.

(26) 129. Mart. (Asteraceae) leaves by HPLC-DAD-MS and HPLC-DAD-MS/MS and a validated HPLC-DAD method for their simultaneous analysis. Journal of Agricultural and Food Chemistry, 56(4), 1193-1204.. Gobbo-Neto, L., Santos, M. D., Kanashiro, A., Almeida, M. C., Lucisano-Valim, Y. M., Lopes, J. L. C., Souza, G. E. P., & Lopes, N. P. (2005). Evaluation of the anti-inflammatory and antioxidant activities of di-C-glucosylflavones from Lychnophora ericoides (Asteraceae). Planta Medica, 71(1), 3-6.. Golinska, P., Wypij, M., Agarkar, G., Rathod, D., Dahm, H., & Rai, M. (2015). Endophytic actinobacteria of medicinal plants: diversity and bioactivity. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 108(2), 267-289.. Gonzalez, D. J., Xu, Y. Q., Yang, Y. L., Esquenazi, E., Liu, W. T., Edlund, A., et al. (2012). Observing the invisible through imaging mass spectrometry, a window into the metabolic exchange patterns of microbes. Journal of Proteomics, 75(16), 5069-5076.. Gouda, A., Fateen, E., & Nazim, W. (2012). Porphyrins profile by high performance liquid chromatography/electrospray ionization tandem mass spectrometry for the diagnosis of porphyria. Journal of Inherited Metabolic Disease, 35, S161-S161.. Gouda, S., Das, G., Sen, S. K., Shin, H. S., & Patra, J. K. (2016). Endophytes: A treasure house of bioactive compounds of medicinal importance. Frontiers in Microbiology, 7, 8.. Gray, K. C., Palacios, D. S., Dailey, I., Endo, M. M., Uno, B. E., Wilcock, B. C., Burke, M. D. (2012). Amphotericin primarily kills yeast by simply binding ergosterol. Proceedings of the National Academy of Sciences of the United States of America, 109(7), 22342239.. Grove, J. F., & McGowan, J. C. (1947). Identity of griseofulvin and "Curling Factor". Nature, 160(4069), 574-574.. Gunatilaka, A. A. L. (2006). Natural products from plant-associated microorganisms: Distribution, structural diversity, bioactivity, and implications of their occurrence. Journal of Natural Products, 69(3), 509-526.. Guthals, A., & Bandeira, N. (2012). Peptide identification by tandem mass spectrometry with alternate fragmentation modes. Molecular & Cellular Proteomics, 11(9), 550-557..

(27) 130. Hamato, N., Takano, R., Kameihayashi, K., Imada, C., & Hara, S. (1992). Leupeptins produced by the marine Alteromonas sp. B-10-31. [Note]. Bioscience Biotechnology and Biochemistry, 56(8), 1316-1318.. Heeb, S., Fletcher, M. P., Chhabra, S. R., Diggle, S. P., Williams, P., & Camara, M. (2011). Quinolones: from antibiotics to autoinducers. FEMS Microbiology Reviews, 35(2), 247274.. Henkel, T., Ciesiolka, T., Rohr, J., & Zeeck, A. (1989). Urdamycins, new angucycline antibiotics from Streptomyces fradiae. 5. Derivatives of Urdamycin A. Journal of Antibiotics, 42(2), 299-311.. Hibbing, M. E., Fuqua, C., Parsek, M. R., & Peterson, S. B. (2010). Bacterial competition: surviving and thriving in the microbial jungle. Nature Reviews Microbiology, 8(1), 1525.. Ho, Y. S., Duh, J. S., Jeng, J. H., Wang, Y. J., Liang, Y. C., Lin, C. H., Tseng, C. J., Yu, C. F., Chen, R. J., Lin, J. K. (2001). Griseofulvin potentiates antitumorigenesis effects of nocodazole through induction of apoptosis and G2/M cell cycle arrest in human colorectal cancer cells. International Journal of Cancer, 91(3), 393-401.. Hong, K. W., Koh, C. L., Sam, C. K., Yin, W. F., & Chan, K. G. (2012). Quorum quenching revisited-from signal decays to signalling confusion. Sensors, 12(4), 4661-4696.. Hori, M., Hemmi, H., Suzukake, K., Hayashi, H., Uehara, Y., Takeuchi, T., Umezawa, H. (1978). Biosynthesis of leupeptin. Journal of Antibiotics, 31(1), 95-98.. Hoshino, S., Okada, M., Wakirnoto, T., Zhang, H. P., Hayashi, F., Onaka, H., Abe, I. (2015). Niizalactams A-C, Multicyclic macrolactams isolated from combined culture of streptomyces with mycolic acid-containing bacterium. Journal of Natural Products, 78(12), 3011-3017.. Hosni, T., Moretti, C., Devescovi, G., Suarez-Moreno, Z. R., Fatmi, M. B., Guarnaccia, C., Pongor, S., Onofri, A., Buonaurio, R., & Venturi, V. (2011). Sharing of quorum-sensing signals and role of interspecies communities in a bacterial plant disease. ISME Journal, 5(12), 1857-1870.. Hue, N., Serani, L., & Laprevote, O. (2001). Structural investigation of cyclic peptidolipids from Bacillus subtilis by high-energy tandem mass spectrometry Rapid Communications in Mass Spectrometry, 15(3), 203-209..

(28) 131. Hufsky, F., Scheubert, K., & Bocker, S. (2014). New kids on the block: novel informatics methods for natural product discovery. Natural Product Reports, 31(6), 807-817.. Hutchinson, C. R. (1982). The use of Isotopic Hydrogen and Nuclear Magnetic-Resonance spectroscopic techniques for the analysis of biosynthetic pathways. Journal of Natural Products, 45(1), 27-37.. Jasim, B., Geethu, P. R., Mathew, J., & Radhakrishnan, E. K. (2015). Effect of endophytic Bacillus sp from selected medicinal plants on growth promotion and diosgenin production in Trigonella foenum-graecum. Plant Cell Tissue and Organ Culture, 122(3), 565-572.. Kalyon, B., Tan, G. Y. A., Pinto, J. M., Foo, C. Y., Wiese, J., Imhoff, J. F., Sussmuth, R. D., Sabaratnam, V., Fiedler, H. P. (2013). Langkocyclines: novel angucycline antibiotics from Streptomyces sp Acta 3034. Journal of Antibiotics, 66(10), 609-616.. Kaul, S., Sharma, T., & Dhar, M. K. (2016). "Omics" Tools for better understanding the plantendophyte interactions. Frontiers in Plant Science, 7, 9.. Kawashima, A., Hamaguchi, T., Akama, T., & Hanada, K. (1992). Antibiotic A-7884. Japan Patent Application No. 02-306537.. Kazutoshi, S. (1995). New antibiotic substance DQ112-A, production thereof and antitumor agent. Japan Patent No. 07-324082.. Kelsic, E. D., Zhao, J., Vetsigian, K., & Kishony, R. (2015). Counteraction of antibiotic production and degradation stabilizes microbial communities. Nature, 521(7553), 516U208.. Kenichi, K., Masaji, N., Junji, N., & Makoto, Y. (1994). Antibiotic SNA 6850-7. Japan Patent No. 05-044585.. Kharazmi, A., Bibi, Z., Nielsen, H., Hoiby, N., & Doring, G. (1989). Effect of Pseudomonas aeruginosa rhamnolipid on human neutrophil and monocyte function. APMIS, 97(12), 1068-1072.. Kharel, M. K.; Pahari, P., Shepherd, M. D., Tibrewal, N., Nybo, S. E., Shaaban, K. A., & Rohr, J. (2012). Angucyclines: Biosynthesis, mode-of-action, new natural products, and synthesis. Natural Product Reports, 29(2), 264-325..

(29) 132. Kharwar, R. N., Mishra, A., Gond, S. K., Stierle, A., & Stierle, D. (2011). Anticancer compounds derived from fungal endophytes: their importance and future challenges. Natural Product Reports, 28(7), 1208-1228.. Kim, D. W., Kang, S. G., Kim, I. S., Lee, B. K., Rho, Y. T., & Lee, K. J. (2006). Proteases and protease inhibitors produced in streptomycetes and their roles in morphological differentiation. Journal of Microbiology and Biotechnology, 16(1), 5-14.. Kim, I. S., & Lee, K. J. (1995a). Physiological roles of leupeptin and extracellular proteases in mycelium development of Streptomyces-exfoliatus SMF13. Microbiology-Uk, 141, 1017-1025.. Kim, I. S., & Lee, K. J. (1995b). Regulation of production of leupeptin, leupeptin-inactivating enzime, and trypsin-like protease in Streptomyces-exfoliatus SMF13. Journal of Fermentation and Bioengineering, 80(5), 434-439.. Kondo, S., Kawamura, K., Iwanaga, J., Hamada, M., Aoyagi, T., Maeda, K., Takeuchi, T., & Umezawa, H. (1969). Isolation and characterization of leupeptins produced by actinomycetes. Chemical & Pharmaceutical Bulletin, 17(9), 1896-1901.. Kowall, M., Vater, J., Kluge, B., Stein, T., Franke, P., & Ziessow, D. (1998). Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105. Journal of Colloid and Interface Science, 204(1), 1-8.. Kupchan, S. M., Komoda, Y., Branfman, A. R., Sneden, A. T., Court, W. A., Thomas, G. J., Hintz, H. P. J., Smith, R. M., Karim, A., Howie, G. A., Verma, A. K., Nagao, Y., Dailey, R. G., Zimmerly, V. A., & Sumner, W. C. (1977). Maytansinoids-Isolation, structural elucidation and chemical interrelation of novel ansa macrolides. Journal of Organic Chemistry, 42(14), 2349-2357.. Kupchan, S. M., Komoda, Y., Thomas, G. J., Smith, R. M., Bryan, R. F., Haltiwan., R. C., Karim, A., Court, W. A., & Gilmore, C. J. (1972). Maytansine, a novel antileukemic ansa macrolide from Maytenus ovatus. Journal of the American Chemical Society, 94(4), 1354-1356.. Kusari, P., Kusari, S., Eckelmann, D., Zuhlke, S., Kayser, O., & Spiteller, M. (2016). Crossspecies biosynthesis of maytansine in Maytenus serrata. RSC Advances, 6(12), 1001110016..

(30) 133. Kusari, P., Kusari, S., Lamshoft, M., Sezgin, S., Spiteller, M., & Kayser, O. (2014). Quorum quenching is an antivirulence strategy employed by endophytic bacteria. Applied Microbiology and Biotechnology, 98(16), 7173-7183.. Kusari, P., Kusari, S., Spiteller, M., & Kayser, O. (2015). Implications of endophyte-plant crosstalk in light of quorum responses for plant biotechnology. Applied Microbiology and Biotechnology, 99(13), 5383-5390.. Kusari, S., Hertweck, C., & Spitellert, M. (2012). Chemical ecology of endophytic fungi: origins of secondary metabolites. Chemistry & Biology, 19(7), 792-798.. Kusari, S., Lamshoeft, M., Kusari, P., Gottfried, S., Zuehlke, S., Louven, K., Hentschel, U., Kayser, O., & Spiteller, M. (2014). Endophytes are hidden producers of maytansine in Putterlickia Roots. Journal of Natural Products, 77(12), 2577-2584.. Kusari, S., Lamshoeft, M., & Spiteller, M. (2009). Aspergillus fumigatus Fresenius, an endophytic fungus from Juniperus communis L. Horstmann as a novel source of the anticancer pro-drug deoxypodophyllotoxin. Journal of Applied Microbiology, 107(3), 1019-1030.. Kusari, S., Pandey, S. P., & Spiteller, M. (2013). Untapped mutualistic paradigms linking host plant and endophytic fungal production of similar bioactive secondary metabolites. Phytochemistry, 91, 81-87.. Kusari, S., & Spiteller, M. (2011). Are we ready for industrial production of bioactive plant secondary metabolites utilizing endophytes? Natural Product Reports, 28(7), 12031207.. Laatsch, H. (2014). AntiBase 2014: The Natural Compound Identifiers (Wiley-VCH, 2014).. Lareen, A., Burton, F., & Schafer, P. (2016). Plant root-microbe communication in shaping root microbiomes. Plant Molecular Biology, 90(6), 575-587.. Larsen, T. O., Smedsgaard, J., Nielsen, K. F., Hansen, M. E., & Frisvad, J. C. (2005). Phenotypic taxonomy and metabolite profiling in microbial drug discovery. Natural Product Reports, 22(6), 672-695.. Lepine, F., Milot, S., Deziel, E., He, J. X., & Rahme, L. G. (2004). Electrospray/mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines (HAQs).

(31) 134. produced by Pseudomonas aeruginosa. Journal of the American Society for Mass Spectrometry, 15(6), 862-869.. Li, C., Zhang, J., Shao, C., Ding, W., She, Z., & Lin, Y. (2011). A new xanthone derivative from the co-culture broth of two marine fungi (Strain No. E33 and K38). Chemistry of Natural Compounds, 47(3), 382-384.. Li, J., Zhu, H. Q., Li, J. Y., Jin, S. H., & Hu, C. Q. (2007). Isolation, structure elucidation and activity of an unknown impurity of amphotericin B. Journal of Antibiotics, 60(4), 272276.. Li, Z. R., Li, Y. X., Lai, J. Y. H., Tang, J. Q., Wang, B., Lu, L., Zhu, G. Q., Wu, X. Y., Xu, Y., & Qian, P. Y. (2015). Critical intermediates reveal new biosynthetic events in the enigmatic colibactin pathway. Chembiochem, 16(12), 1715-1719.. Liu, G., Chater, K. F., Chandra, G., Niu, G. Q., & Tan, H. R. (2013). Molecular regulation of antibiotic biosynthesis in Streptomyces. Microbiology and Molecular Biology Reviews, 77(1), 112-143.. Liu, H.-z., Chen, W., & Liu, Q.-Y. (1990). Fermentation, extract and the activity of thrombin inhibitor in Streptomyces S-254. Zhongguo Kangshengsu Zazhi, 15(6), 407-411.. Liu, J., Ng, T., Rui, Z., Ad, O., & Zhang, W. J. (2014). Unusual acetylation-dependent reaction cascade in the biosynthesis of the pyrroloindole drug physostigmine. Angewandte Chemie-International Edition, 53(1), 136-139.. Liu, J. F., Mbadinga, S. M., Yang, S. Z., Gu, J. D., & Mu, B. Z. (2015). Chemical structure, property and potential applications of biosurfactants produced by Bacillus subtilis in petroleum recovery and spill mitigation. International Journal of Molecular Sciences, 16(3), 4814-4837.. Liu, Q.-y., Liu, H.-z., Chen, W., & Chi, Z.-w. (1990). The Component separation and structure of thrombin inhibitor produced by Streptomyces S-254. Zhongguo Kangshengsu Zazhi, 15(5), 342-346.. Liu, W. C., Gong, T., & Zhu, P. (2016). Advances in exploring alternative Taxol sources. RSC Advances, 6(54), 48800-48809.. Macmillan, J. (1953). Griseofulvin. 7. Dechlorogriseofulvin. Journal of the Chemical Society, 1697-1702..

(32) 135. Mangrich, A. S., Lermen, A. W., Santos, E. J., Gomes, R. C., Coelho, R. R. R., Linhares, L. F., & Senesi, N. (1998). Electron paramagnetic resonance and ultraviolet-visible spectroscopic evidence for copper porphyrin in actinomycete melanins. Biology and Fertility of Soils, 26(4), 341-345.. Marmann, A., Aly, A. H., Lin, W. H., Wang, B. G., & Proksch, P. (2014). Co-Cultivation-A powerful emerging tool for enhancing the chemical diversity of microorganisms. Marine Drugs, 12(2), 1043-1065.. Marsden, A. F. A., Wilkinson, B., Cortes, J., Dunster, N. J., Staunton, J., & Leadlay, P. F. (1998). Engineering broader specificity into an antibiotic-producing polyketide synthase. Science, 279(5348), 199-202.. Mascuch, S. J., Moree, W. J., Hsu, C. C., Turner, G. G., Cheng, T. L., Blehert, D. S., Kilpatrick, A. M., Frick, W. F., Meehan, M. J., Dorrestein, P. C., Gerwick, L. (2015). Direct detection of fungal siderophores on bats with White-Nose Syndrome via fluorescence microscopy-guided ambient ionization mass spectrometry. Plos One, 10(3), 12.. McConnell, R. M., York, J. L., Frizzell, D., & Ezell, C. (1993). Inhibition studies of some serine and thiol proteinases by new leupeptin analogues. Journal of Medicinal Chemistry, 36(8), 1084-1089.. McNamara, C. M., Box, S., Crawforth, J. M., Hickman, B. S., Norwood, T. J., & Rawlings, B. J. (1998). Biosynthesis of amphotericin B. Journal of the Chemical Society-Perkin Transactions 1(1), 83-87.. McPhee, W. J., & Colotelo, N. (1977). Fungal Exudates. 1. Characteristics of hyphal exudates in Fusarium culmorum. Canadian Journal of Botany-Revue Canadienne De Botanique, 55(3), 358-365.. Miliute, I., Buzaite, O., Gelvonauskiene, D., Sasnauskas, A., Stanys, V., & Baniulis, D. (2016). Plant growth promoting and antagonistic properties of endophytic bacteria isolated from domestic apple. Zemdirbyste-Agriculture, 103(1), 77-82.. Moree, W. J., Phelan, V. V., Wu, C.-H., Bandeira, N., Cornett, D. S., Duggan, B. M., & Dorrestein, P. C. (2012). Interkingdom metabolic transformations captured by microbial imaging mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 109(34), 13811-13816..

(33) 136. Murao, S., & Hayashi, H. (1986). Physostigmine and N-8-norphysostigmine, insecticidal compounds, from Streptomyces sp. Agricultural and Biological Chemistry, 50(2), 523524.. Murenets, N. V., Kudinova, M. K., Klyev, N. A., Chernyshev, A. I., & Shorshnev, S. V. (1986). Galtamycin - Galtamycinone Structure. Antibiotiki I Meditsinskaya Biotekhnologiya, 31(6), 431-434. Newman, D. J., & Cragg, G. M. (2015). Endophytic and epiphytic microbes as “sources” of bioactive agents. Frontiers in Chemistry, 3(34), 1-13.. Newman, D. J., & Cragg, G. M. (2016). Natural products as sources of new drugs from 1981 to 2014. Journal of Natural Products, 79(3), 629-661.. Nguyen, D. D., Wu, C. H., Moree, W. J., Lamsa, A., Medema, M. H., Zhao, X. L., Gavilan, R. G., Aparicio, M., Atencio, L., Jackson, C., Ballesteros, J., Sanchez, J., Watrous, J. D., Phelan, V. V., van de Wiel, C., Kersten, R. D., Mehnaz, S., De Mot, R., Shank, E. A., Charusanti, P., Nagarajan, H., Duggan, B. M., Moore, B. S., Bandeira, N., Palsson, B. O., Pogliano, K., Gutierrez, M., & Dorrestein, P. C. (2013). MS/MS networking guided analysis of molecule and gene cluster families. Proceedings of the National Academy of Sciences of the United States of America, 110(28), E2611-E2620.. Nougayrede, J. P., Homburg, S., Taieb, F., Boury, M., Brzuszkiewicz, E., Gottschalk, G., Buchrieser, C., Hacker, J., Dobrindt, U., & Oswald, E. (2006). Escherichia coli induces DNA double-strand breaks in eukaryotic cells. Science, 313(5788), 848-851.. Ogura, K., Maeda, M., Nagai, M., Tanaka, T., Nomoto, K., & Murachi, T. (1985). Purification and structure of a novel cysteine proteinase-inhibitor, Strepin P-1. Agricultural and Biological Chemistry, 49(3), 799-805.. Okada, B. K., Wu, Y., Mao, D., Bushin, L. B., & Seyedsayamdost, M. R. (2016). Mapping the Trimethoprim-Induced Secondary Metabolome of Burkholderia thailandensis ACS Chemical Biology, Article ASAP.. Oki, T., Kitamura, I., Matsuzawa, Y., Shibamoto, N., Ogasawara, T., Yoshimoto, A., Inui, T., Naganawa, H., Takeuchi, T., & Umezawa, H. (1979). Antitumor anthracycline antibiotics, aclacinomycin A and analogues. II. Structural determination. . The Journal of Antibiotics, 32(8), 801-819..

(34) 137. Ola, A. R. B., Thomy, D., Lai, D., Broetz-Oesterhelt, H., & Prolcsch, P. (2013). Inducing secondary metabolite production by the endophytic fungus Fusarium tricinctum through coculture with Bacillus subtilis. Journal of Natural Products, 76(11), 2094-2099.. Omura, S., Nakagawa, A., Fukamachi, N., Miura, S., Takahashi, Y., Komiyama, K., & Kobayashi, B. (1988). OM-4842, A new platelet-aggregation inhibitor from Streptomyces. Journal of Antibiotics, 41(6), 812-813.. Oxford, A. E., Raistrick, H., & Simonart, P. (1939). Studies in the biochemistry of microorganisms. LX. Griseofulvin, C17H17O6Cl, a metabolic product of Penicillium griseofulvum Dierckx. Biochemical Journal, 33, 240-248.. Palacios, D. S., Dailey, I., Siebert, D. M., Wilcock, B. C., & Burke, M. D. (2011). Synthesisenabled functional group deletions reveal key underpinnings of amphotericin B ion channel and antifungal activities. Proceedings of the National Academy of Sciences of the United States of America, 108(17), 6733-6738.. Palaniswamy, V. A., McCloskey, D. V., Ratych, O. T., Taylor, S. P., McCormyck, T., & Singh, P. D. (1990). Preparation and structural elucidation of amphotericin X. Planta Medica, 56(6).. Perez-Picaso, L., Rios, M. Y., Hernandez, A. N., & Martinez, J. (2006). H-1 and C-13 assignments of cyclo N-(Lys-Phe)-Orn-Val , a semicyclic imide tetrapeptide from Burkholderia cepacia. Magnetic Resonance in Chemistry, 44(10), 959-961.. Petersen, A. B., Ronnest, M. H., Larsen, T. O., & Clausen, M. H. (2014). The chemistry of griseofulvin. Chemical Reviews, 114(24), 12088-12107.. Pettit, R. K. (2009). Mixed fermentation for natural product drug discovery. Applied Microbiology and Biotechnology, 83(1), 19-25.. Peypoux, F., Bonmatin, J. M., Labbe, H., Grangemard, I., Das, B. C., Ptak, M., Wallach, J., & Michel, G. (1994). [Ala4] Surfactin, a Novel Isoform from Bacillus subtilis studied by Mass and NMR Spectroscopies. European Journal of Biochemistry, 224(1), 89-96.. Power, P., Dunne, T., Murphy, B., Lochlainn, L. N., Rai, D., Borissow, C., Rawlings, & B.; Caffrey, P. (2008). Engineered synthesis of 7-oxo- and 15-deoxy-15-Oxoamphotericins: Insights into structure-activity relationships in polyene antibiotics. Chemistry & Biology, 15(1), 78-86..

(35) 138. Proschak, A., Schultz, K., Herrmann, J., Dowling, A. J., Brachmann, A. O., Ffrench-Constant, R., Muller, R. & Bode, H. B. (2011). Cytotoxic fatty acid amides from Xenorhabdus. Chembiochem, 12(13), 2011-2015.. Qin, S., Xing, K., Jiang, J.-H., Xu, L.-H., & Li, W.-J. (2011). Biodiversity, bioactive natural products and biotechnological potential of plant-associated endophytic actinobacteria. Applied Microbiology and Biotechnology, 89(3), 457-473.. Raja, A., & Prabakarana, P. (2011). Actinomycetes and drug-An overview. American Journal of Drug Discovery and Development, 1(2), 75-84.. Rathinasamy, K., Jindal, B., Asthana, J., Singh, P., Balaji, P. V., & Panda, D. (2010). Griseofulvin stabilizes microtubule dynamics, activates p53 and inhibits the proliferation of MCF-7 cells synergistically with vinblastine. BMC Cancer, 10, 13.. Ratnaweera, P. B., de Silva, E. D., Wijesundera, R. L. C., & Andersen, R. J. (2016). Antimicrobial constituents of Hypocrea virens, an endophyte of the mangrove-associate plant Premna serratifolia L. Journal of the National Science Foundation of Sri Lanka, 44(1), 43-51.. Rohr, J., & Zeeck, A. (1987). Metabolic products of microorganisms. 240. Urdamycins, new angucycline antibiotics from Streptomyces fradiae. 2. Structural studies of urdamycin B to urdamycin F. Journal of Antibiotics, 40(4), 459-467.. Rutledge, P. J., & Challis, G. L. (2015). Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nature Reviews Microbiology, 13(8), 509-523.. Saha, A. K., Mukherjee, T., & Bhaduri, A. (1986). Mechanism of action of amphotericin B on Leishmania donovani promastigotes. Molecular and Biochemical Parasitology, 19(3), 195-200.. Sakamoto, H. T., Flausino, D., Castellano, E. E., Stark, C. B. W., Gates, P. J., & Lopes, N. P. (2003). Sesquiterpene lactones from Lychnophora ericoides. Journal of Natural Products, 66(5), 693-695.. Salway, A. H. (1912). Researches on the constitution of physostigmine Part I. Journal of the Chemical Society, 101, 978-989.. Sanchez, D. G., Otero, L. H., Hernandez, C. M., Serra, A. L., Encarnacion, S., Domenech, C. E., & Lisa, A. T. (2012). A Pseudomonas aeruginosa PA01 acetylcholinesterase is.