Current Topics in Medicinal Chemistry, 2020, 20, 1291-1299 REVIEW ARTICLE

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ISSN: 1568-0266 eISSN: 1873-4294

Impact Factor: The Microbiome: A Reservoir to Discover New Antimicrobials Agents 3.442 The international journal for in-depth reviews on Current Topics in Medicinal Chemistry

BENTHAM SCIENCE

Sébastien Boutin1,2,* and Alexander H. Dalpke3

1Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Hei- delberg, Germany; ²German Center for Lung Research (DZL), TLRC Heidelberg, 69120 Heidelberg, Germany; 3Institute of Medical Microbiology and Hygiene, Medical Faculty, Technische Universität Dresden, 01307 Dresden, Gemany

Abstract: Nature offered mankind the first golden era of discovery of novel antimicrobials based on A R T I C L E H I S T O R Y the ability of eukaryotes or micro-organisms to produce such compounds. The microbial world proved to be a huge reservoir of such antimicrobial compounds which play important functional roles in every

Received: November 11, 2019 environment. However, most of those organisms are still uncultivable in a classical way, and therefore, Revised: February 10, 2020 the use of extended culture or DNA based methods (metagenomics) to discover novel compounds Accepted: February 17, 2020 promises usefulness. In the past decades, the advances in next-generation sequencing and bioinformat- DOI: 10.2174/1568026620666200320112731 ics revealed the enormous diversity of the microbial worlds and the functional repertoire available for studies. Thus, data-mining becomes of particular interest in the context of the increased need for new antibiotics due to antimicrobial resistance and the rush in antimicrobial discovery. In this review, an overview of principles will be presented to discover new natural compounds from the microbiome. We describe culture-based and culture-independent (metagenomic) approaches that have been developed to identify new antimicrobials and the input of those methods in the field as well as their limitations.

Keywords: Antimicrobial peptides, Metagenomics, Data mining, Bioinformatics, Microbiome, Antimicrobials agents.

1. INTRODUCTION The classical procedure to find antimicrobial compounds is to look for them in nature (Fig. 1). The first accessible From the 1930s to the 1960’s, the golden age of antimi- organisms producing antimicrobials were plants and those crobial discoveries took place. Those discoveries lead to the compounds were used for millennia. However, access to introduction in the clinics of the major classes of drugs cur- fungi and bacteria with microbial culture revolutionized the rently used. After the first discoveries, the research field fo- field [5]. The majority of antibiotics currently implemented cused more on the optimization of those drugs in the follow- in clinic originate from cultivable microorganisms [6]. In the ing 50 years [1]. The phase of optimization was always fol- environment, microbes, however, are never living in a lowed by the emergence of antimicrobial resistance leading monoculture environment and therefore, most of them have to an “arm race” to counteract those resistances [2]. Since the developed ways to communicate and/or defend themselves 2000’s, only a few drugs of entirely new classes were ap- against other bacteria/fungi. The billions of years of evolu- proved and discovered, leading to a threat in health due to tion involve in this arms race created a huge arsenal of those the steady increase of emerging pathogens and multi-drug compounds [7]. Most of them are products of “secondary resistant (MDR) bacteria [3]. The currently used drugs focus metabolism,” which are not required for survival under labo- on two major targets in 5 different modes of action. Some ratory conditions but are most likely increasing the fitness of drugs alter the structure of the cell by inhibiting the cell wall their producer in the native complex biome [5]. Those prod- synthesis or disrupting the cell membrane integrity while ucts are often encoded by a biosynthetic gene cluster (BGC), others target the molecular machinery inhibiting either the which is a physically clustered group of two or more genes DNA or RNA synthesis or the protein synthesis via the bac- as an operon within the genome. Together the BGCs will terial ribosome or metabolic pathways [4]. The emergence of encode cooperatively a complete biosynthetic pathway lead- MDR bacteria guided the field of antibiotic research into ing to the production of a specialized metabolite and possible actually two ways; understanding the molecular mechanisms chemical variants. Furthermore, genes involved in the regu- leading to the resistance to manage our use of the affected lation, such as transcription factors and transporters, are usu- drugs and developing new antibiotics targeting new path- ally also present in these clusters [8, 9]. ways or identifying new modes of action in known pathways. The field of microbiology observed in the last decades a shift in the paradigm of environmental and host-related mi- *Address correspondence to this author at the Department of Infectious crobes. The advances in culture-independent methods Diseases, Medical Microbiology and Hygiene, University Hospital Heidel- showed that microbes are part of a complex ecosystem and berg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany; the diversity of this microbiome was by far underestimated Current Topics in Medicinal Chemistry E-mail: [email protected]

Fig. (1). Summary of the pipelines used to discover microbiome derived antibiotic. The classical way consists to culture the organisms di- rectly from the environment and test them for antimicrobial production. An evolution of this way leads to the development of in situ culture where the organisms is grown in isolation in a permeable chamber put in the original environment (displayed as a iChip in the figure). The environmental DNA (eDNA) can be used to construct metagenomic library either via direct fragmentation and ligation to a vector or via a PCR targeted approach using conserved region of the biosynthetic gene clusters (BGCs) to amplify the diversity of BGCs within the environment. Those PCR products can also be integrated in a vector to construct a metagenomic library. The metagenomic data can also be explore (“data-mining”) via bio-informatic to search for new BGCs and design new primers to isolate them from the metagenome. The pre- pared library can then be transferred into a heterologous host (i.e. Escherischia coli) to perform an antimicrobial test assay. Each part of the different path is explained and discuss in details in the separate sections of the manuscript. by classical culture. It is estimated that classical culture re- capacities as well as annotation of the sequences themselves, trieves around 1% of the microbiome of environmental sam- such approaches appear to be very promising. ples, yet reaches slightly better recovery in human-associated 2. THE WORLD’S MICROBIOME AS A POTENTIAL microbiomes, due to the optimization of growth media for SOURCE FOR NEW ANTIBIOTICS detection of human pathogenic bacteria [10]. Many researchers are currently working on extending culture methods to The environmental microbiome is a source of new anti- improve culturability with impressive advances, especially in biotics, but unfortunately, culture-based approaches barely humans [11–13]. However, symbiotic microbes will still be scratch the surface of the diversity of those microbiomes difficult to culture and the production of antibiotic com- [14]. The advances in the metagenomic field pave the way pounds will be influenced by the growth conditions as well for the exploration of highly diverse microbiomes such as as other organisms growing in nature together with antimi- the soil and aquatic microbiome. The exploration of those crobial producers. Therefore, the field of drug discovery now two major environments showed that the BGCs involved in focuses on metagenomic approaches: This approach aims at the production of antimicrobial compounds by those mi- using the DNA from the environmental microbiome (eDNA) crobes are geographically heterogeneous and show a strong to discover antimicrobial functions by prediction instead of environmental specificity [15-17]. This work was the basis or prior to phenotypical identification of such compounds. of the exploration of diverse environments and geographi- Given the enormous technical developments in sequencing cally distinct biomes to mine for new antibiotics from a highly diverse environment, allowing a low re-discovery rate The Microbiome: A Reservoir to Discover New Antimicrobials Agents Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 1293

[18]. Naturally, the metagenomic approach can be used with cro-organisms. Those chambers improved the culture of mi- any source material, but the main focus of the research is crobes, which are dependent on environmental/host nutrients currently soil, water and host-associated microbiomes as opening the scope of cultured microbes for antibiotic discov- supported by the major consortium performing metagenom- ery. ics and offering a huge database for data-mining [19, 20]. An updated version of the diffusion chamber is the iChip. Soil is of high interest because it is one of the most diverse The principle is the same; bacteria are serially diluted using environments and easily accessible. Many studies are cur- microfluidic diffusion chambers. However, the device pre- rently done to improve both culture and data-mining to find sents many holes containing agar where one microbial cell new pharmaceutical compounds from the soil [21]. The ma- can be isolated, then offering a high-throughput screening rine and clearwater environment is not as easy to explore, but cultivation method [36]. After isolation in the well, top and the high diversity of this environment is reflecting the bio- bottom sites of the chip are sealed with a semi-permeable synthetic potential of these biomes [22]. Finally, host- membrane and the chip is placed in the source environment associated microbiomes might be good candidates for antim- or a simulated environment allowing the nutrient and growth icrobial compounds which are harmless for the eukaryotic factors to diffuse in the agar favoring the growth of previ- hosts. This point is crucial for the translational aspect of the ously uncultivable organisms. This device allowed the isola- approach of drug discovery. The human microbiome project tion of soil Betaproteobacteria Eleftheria terrae, which pro- offers a good database for data mining and a study based on duces a new class of antibiotics called Teixobactin [37]. This the available metagenome showed that 3,118 biosynthetic antibiotic proved highly efficient against gram-positive bac- gene clusters were identified in the human microbiome and teria by binding to the lipid II and then inhibiting the produc- discovered a previously unknown antibiotic, lactocillin, pro- tion of the peptidoglycan layer. However, due to this mode duced by a vaginal commensal [23]. While human is the of action, this antimicrobial compound is inactive against target of choice, other eukaryotic hosts like sea sponges and gram-negative. coral already proved to be a good source for the identification of natural products, which may serve beneficial roles in In situ cultivation assays were modified and optimized in human health [24-26] various different ways and can be placed in solid or liquid media. Any bacteria depending only on nutrient diffusion 3. ANTIBIOTIC DISCOVERY FROM CULTURED from the native environment may grow, yet limitations have ORGANISMS to be considered [30]. Contamination from the in situ media to the chambers is a major problem as well as the moisture of The classical way to discover antimicrobial compounds solid media like soil to allow the diffusion of the nutrient used since the 1930’s consists in the screening of bacteria into the agar. The permeability of liquid or solid media to from natural sources, such as soil and water for bioactive gaseous exchange also has to be optimized to allow the or- compounds of pharmacological interest. This methodology ganisms to access the necessary gas. However, the in-situ brought a lot of promises and new drugs, however, the lack cultivation shows promises in cultivating new species and of cultivability of the microbiome leads to a low amount of therefore allowing the discovery of new compounds. As a species studied. Most of the antimicrobials currently discov- limitation, some organisms are not only dependent on the ered or under use are produced by organisms belonging to environment or host nutrients but the interactions with other myxobacteria, cyanobacteria, actinomycetes, Pseudomonas, microbes. Co-culture is currently not allowed by the in situ and Bacillus species [27]. Furthermore, secondary metabo- assays, but many studies showed that secondary metabolic lites are clades specific, and therefore limiting our focus on 5 production is influenced by the presence of other microbes clades explains the high rediscovery rates of known antibiot- and that systems must be developed to study those interac- ics [28,29]. The extension of culture conditions and media tions [38, 39]. Some researchers proposed to modify the can be laborious and has been mostly employed for the hu- iChip system to allow the diffusion within the chip of the man microbiome [11]. For environmental samples, another metabolite, allowing a full screening of the microbiome methodology is currently in use, which is in situ cultivation. while keeping the species isolated [40]. This concept basically involves the cultivation of the organisms in its natural niche [30-32]. 4. SEQUENCE-BASED METAGENOMICS TO DIS- COVER NEW BIOSYNTHETIC GENE CLUSTER One approach consists of capturing bacterial cells in a diffusion chamber which has access to a flow of nutrients Extended culture will still face issues of unreliable identi- simulating the natural environment of their origin. The sam- fication due to the absence of known close relative organ- ples are serially diluted to minimize the amount of cells per isms helping to identify the cultured one. Therefore, identifi- chambers and inoculated in agar. The agar is then covered by cation will rely on genome sequencing or phenotypic charac- permeable membranes allowing the passage of nutrients terization by an expert. Furthermore, culture in a simulated from the simulated environment. This technique allows to environment or medium remains an artificial model that culture more microorganisms than the classical culture-based might lead to irreproducibility or loss of function in a non- techniques [33, 34]. The development of diffusion chambers selective medium [41]. Therefore, a phenotype observed in was particularly helpful in growing species from the phylum the natural environment cannot be recapitulated in the lab for Verrucomicrobia which grow after subsequent incubation in production. To be able to avoid such non-transferability to the diffusion chambers [34]. Diffusion chambers were also the lab, working with the DNA seems to be a good option. improving the culture of symbiotic organisms from hosts like The advances in genomics and bioinformatics associated sponges [35]. The growth chambers can be implanted in the with the development of cheaper and faster sequencing tech- tissue of the host allowing the growth of host-dependent mi- nologies allow now research to explore the full gene reper- 1294 Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 Boutin and Dalpke toire from any microbiome without culture [42]. Metage- the extract of condensation and ketosynthase domains pre- nomics can be applied in two different approaches to study sent in respectively NRPS and PKS genes from the sequence the diversity of the microbiome: targeted (16S, ITS,…) or data. The sequence is then queried to a set of manually cu- not targeted (Whole genome sequencing)[43]. In the field of rated reference genes from well-characterized chemical antibiotic discovery, the term metagenomic is slightly differ- pathways to identify potential secondary metabolite do- ent than the one used in microbial ecology. The term de- mains. Then a phylogeny is inferred to predict if the product scribed the exploration of the full content of the genome is similar to or different from previously known biosynthetic within the microbial community in the ecology field. In con- pathways [51]. eSNaPD is designed in a same way but works trast, in the antibiotic discovery field, it means the functional mostly with PCR-generated sequence tags using degenerate screening of antibiotic production via bioinformatic predic- primers targeting conserved biosynthetic genes. This relies tion or the creation of clones containing a fragment of the on the high similarities seen in natural product biosynthetic metagenome. However, both fields are using either the full genes which allow the design of primers targeting those genomic repertoire or the targeted region of the metagenome. genes to explore the structure of the biosynthetic domains. Advances in metagenomic sequencing, library preparation, Those genes clusters are then identified as potential candi- screening and engineering, which allow the study of more dates with a curated database as well as a phylogeny to infer organisms, will be the focus of the following section. potential new functionality [18]. Targeted methods, often named sequence-based methods The use of degenerate primers to amplify a conserved re- are relying on the premise that homologous sequences will gion of the biosynthetic genes of interest allows to create produce a similar protein and, therefore, phenotype. There- libraries for amplicon sequencing to identify new variants of fore, it becomes easy to screen metagenomic data for homol- the genes which might lead to a new antimicrobial product. ogy with known biosynthetic pathways in a high-throughput The PCR-based sequence has the advantage of being much manner. The challenging step is to identify the homologous more sensitive in a high diverse microbiome; estimates are sequences and predict structural proteins during the data- by 10 to 100 fold increase in sensitivity compared to shotgun mining of the metagenome. However, advances in computa- sequencing [52]. In most of the cases, the gene of interest is tional biology result in the development of several good an NRPS or a PKS gene. One of the most recent examples of tools to identify and annotate potential natural products. sequence targeted discovery is the identification of malacid- Those tools are based on the idea that the natural product ins [53]. This new natural product was found in soil micro- results from biosynthetic gene clusters that follow a highly biomes and is active against multidrug-resistant pathogens conserved logic. Therefore, the algorithms are looking for including methicillin-resistant Staphylococcus aureus. The core enzyme/gene-based sequences similarities within the authors used the targeted method to screen several soil mi- databases [44]. One of the most used programs is antiS- crobiomes and selected an environment rich in a new clade MASH, which uses a Hidden Markov Models (HMM) based of malacidins to follow up with the functional metagenomic algorithm named ClusterFinder to identify the BGCs [45, approach that will be reviewed in the next section. 46]. The advantage of an HMM-based algorithm is that it The main disadvantage of sequence based approaches is will identify BGCs that lack high similarity in domain struc- that the scope of research is limited to genes we already ture to any known biosynthetic gene clusters, circumventing know due to the design of primers. Furthermore, most of the the problem that had previously been posed in the identifica- candidate identification is depending on either homology to a tion of biosynthetic gene clusters from sequence data. There- database or a ruling system and therefore, the identification fore, by focusing on the active site via an active site finder of completely novel products is merely impossible. We will module, the program is able to identify conserved amino acid always discover related products or derivate from the exist- motifs in the enzymes of interest like non-ribosomal peptide ing product. For example, the main pathways studied are synthetase (NRPS) and polyketide synthase (PKS). The pro- polyketides, or nonribosomally synthesized peptides, there- gram is also able to predict the structure of the natural prod- fore only pathways which are closely related and fall into the uct based on the sequence of the annotated BGCs. However, rules are implemented in the mining software. The im- such accuracy came with the need for a well-annotated and provement in the knowledge of the evolution of BGCs will assembled metagenome. This step might be a high burden help to extend the rules to identify new pathways. That is the and is not a trivial topic. The need for computational power basis of new software like EvoMining to use evolutionary and expertise in the assembly of high-quality genomes knowledge to relax the rules to identify new compounds makes it extremely complex [47, 48]. Many improvements [54]. Basically, it is considered that BGCs often evolved by have been made to make metagenome assembly more “user duplication and divergence of primary metabolism enzymes. friendly” and almost fully automated programs are now Therefore, the software uses the core metabolism pathways available such as MetaSort or MetaWrap [49, 50]. However, shared between many bacterial species to calculate the di- the quality of the assembly and the reliability of the contigs vergence to identify BGCs that have likely been repurposed remain a problem to be solved. Furthermore, it is still diffi- for secondary metabolite biosynthesis [55]. cult to sequence a highly diverse microbiome like the one from the soil, which contains 104-5 unique species with suffi- cient coverage to assemble the reads correctly. Therefore, a 5. FUNCTIONAL METAGENOMICS TO TEST NEW BIOSYNTHETIC GENE CLUSTER FOR ANTIBIOTIC new set of tools was developed to circumvent the need of longer contigs. Those tools like NaPDos or eSNaPD can DISCOVERY identify secondary metabolite genes based on short reads by Finding the sequence of a new potential antibiotic is not focusing on core domains [18, 51]. NaPDos is focusing on an end per se. Those sequences need to be validated in a The Microbiome: A Reservoir to Discover New Antimicrobials Agents Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 1295 functional assay to ensure the expression and antimicrobial RNA-polymerase of E. coli [67]. To overcome that problem, function of the product. Therefore, most of the studies com- some works are done to express other sigma factors in the E. bine sequence-based to functional metagenomics in the dis- coli host [68, 69]. Another option is to use a non-model het- covery process [53]. This technique is at the interface be- erologous host such as Streptomyces, Sacharopolyspora or tween culture-based and sequence-based techniques. The Myxococcus [70–72]. In general, researchers need to con- basic idea of the functional metagenomic pipeline consists of sider whether the codons are optimized to help expression in extracting total DNA from a microbial community and shear the chosen heterologous hosts and therefore use a host which in fragments (1-5 kb) that can be shotgun cloned in an ex- is not too phylogenetically distant from the original source pression vector. The vector is transferred in the appropriate [73]. To obtain a cleaner expression of the BGCs, the ge- heterologous host (e.g. E. coli) and tested for antibiotic pro- nome of the host can also be minimized to the minimum core duction. The main advantage is that it does not requires prior genome to ensure no competition between the pathways. The knowledge of the DNA sequences or function and will allow new secondary metabolite pathway will then be the only user the discovery of completely new antibiotic or divergent of an efficient supply of primary metabolic precursors and pathways which are not in the database yet [56]. While the biochemical energy;, therefore, being optimized for the pro- theoretical concept of the technique seems simple, the reali- duction of the compound [74]. Alternatively, a co-culture of zation relies on major critical steps such as the extraction and two heterologous hosts expressing different complementary fragmentation of the DNA, the library preparation, the parts of the BGCs can be used to optimize the production. It choice of the heterologous host for expression and the func- was previously demonstrated that the combined use of an tional assay to test the natural product [57]. engineered E. coli and S. cerevisiae as a microbial community will produce a precursor of the anti-cancer drug pacli- The preparation of the DNA for functional metagenomics taxel. The taxadiene is produced by the E. coli and is trans- will struggle with the same problems that all microbiome formed into taxanes by the engineered S. cerevisiae. The studies are facing; the need for representative and pure DNA extraction. The lysis and extraction methods (precipitation or combination of the two strains increased the production of the compound by 1.8-fold [75]. affinity columns) will perform differently in gram-negative and gram-positive bacteria. Mechanical lysis associated with The final step of the functional metagenomics is to per- enzymatic lysis is mandatory to extract DNA from gram- form the culture assay to validate the expression of the an- positive bacteria and fungi [58]. Furthermore, the purity of timicrobial compounds. Antimicrobial production is a more the DNA is crucial to avoid contaminants, which will inhibit complicated phenotype to select for than antimicrobial resis- the shearing and integration into the vector, therefore, a puri- tance and therefore needs the use of appropriate screening fication step should be included [59, 60]. The BGCs are of- strategies to identify and isolate the proper clones [57]. The ten organized as an operon and therefore cloning pieces of most used technique is the double-agar layer [76]. This DNA can result in functional machinery. However, some method consists of growing the heterologous host expressing machinery requires hundreds of genes (~20kb to 100kb) for a the vector on agar plates and then a top agar containing the complex product; therefore, shearing and cloning such a big indicator strain is overlaid and scored for antimicrobial activ- piece of DNA is challenging. The use of cosmids and fos- ity by looking for a zone of inhibition. This is a high- mids helps to integrate large DNA fragments in the host in throughput technique allowing us to screen a large number an efficient way [61] but to integrate fragments of hundreds of clones (>100,000) from a metagenomic derived library of kilobases, the use of Bacterial Artificial Chromosome [77, 78]. A new method was developed where the top layer (BAC) is needed as they can integrate >300 kb fragment is replaced by a “bacteriospray”. The method is based on the [62]. As cosmids and fosmids are more efficient than BACs use of an air brush, which will spray an even mist of the in- for transformation, a recent development consists of reas- dicator bacteria on the bottom agar containing the colonies sembling pieces of overlapping clones into a BAC using of the heterologous host. As the layer is more uniform, the transformation-associated recombination (TAR) in Sac- authors claimed that the technique is more sensitive, allow- charomyces cerevisiae [63]. Modification of the host also ing to test 5–10 times more clones per agar plate [79]. Of allows the enrichment of functionally active BGCs. Most of course, the methods used in the extended culture method, the known antimicrobial compounds (NRPS and PKS) are such as the diffusion chamber assay and the iChip, can be dependent on their own phosphopantetheine transferase used for screening as the membrane is impermeable to bacte- (PPTase) [64]. Therefore the use of a PPTase deficient host ria. The heterologous host can be grown in the chambers will screen vectors containing a PPTase and most likely an while the indicator bacteria grow in the media. However, as active BGCs [65]. discussed previously, this method is difficult to extend in a high-throughput system to test 100,000s clones. Another critical step after the preparation of the vector is to find a suitable host. Indeed, the transcriptional machinery of the host organism has to recognize promoters from the CONCLUSION metagenome to be able to translate metagenomic transcripts. The advances in extended culture and metagenomics The failure of this step is the main reason for the inability to change our way to explore microbial communities and their detect antimicrobial activity from metagenomic constructs potential. However, metagenomics is not yet “user-friendly,” [66]. Most of the studies are using Escherichia coli as a het- and many steps need to be optimized. The virus/phage world erologous host because its genetic content and transcriptional is under-explored for antimicrobial usage, although we can machinery are well understood. However, the expression of access part of their function via their DNA. The technology metagenomic DNA is limited in this model by the recogni- is still not low cost, so the coverage needed to cover function of the promoter by the sigma factor subunits of the tions of the biosphere is rarely reached by the metagenomic 1296 Current Topics in Medicinal Chemistry, 2020, Vol. 20, No. 14 Boutin and Dalpke project. However, big consortia like the human microbiome Tsunematsu, Y.; Wiemann, P.; Wyckoff, E.; Yan, X.; Yim, G.; Yu, project or the earth microbiome project will clearly help us F.; Xie, Y.; Aigle, B.; Apel, A.K.; Balibar, C.J.; Balskus, E.P.; Barona-Gómez, F.; Bechthold, A.; Bode, H.B.; Borriss, R.; Brady, access those biospheres [19, 20]. Those consortia will mostly S.F.; Brakhage, A.A.; Caffrey, P.; Cheng, Y-Q.; Clardy, J.; Cox, likely build strong and reliable Standard Operating Proce- R.J.; De Mot, R.; Donadio, S.; Donia, M.S.; van der Donk, W.A.; dure (SOPs) to extract and purify eDNA for classical and Dorrestein, P.C.; Doyle, S.; Driessen, A.J.M.; Ehling-Schulz, M.; functional metagenomic, which are still lacking currently. Entian, K-D.; Fischbach, M.A.; Gerwick, L.; Gerwick, W.H.; The length of the sequencing data is not long enough yet to Gross, H.; Gust, B.; Hertweck, C.; Höfte, M.; Jensen, S.E.; Ju, J.; Katz, L.; Kaysser, L.; Klassen, J.L.; Keller, N.P.; Kormanec, J.; sequence full BGCs in one reads, but the recent development Kuipers, O.P.; Kuzuyama, T.; Kyrpides, N.C.; Kwon, H-J.; Lautru, of long reads technology such as the SMRT sequencing or S.; Lavigne, R.; Lee, C.Y.; Linquan, B.; Liu, X.; Liu, W.; the nanopore technology will help reach this read length and Luzhetskyy, A.; Mahmud, T.; Mast, Y.; Méndez, C.; Metsä-Ketelä, facilitate the construction of active clones [80, 81]. Further- M.; Micklefield, J.; Mitchell, D.A.; Moore, B.S.; Moreira, L.M.; Müller, R.; Neilan, B.A.; Nett, M.; Nielsen, J.; O’Gara, F.; Oikawa, more, the acquisition of knowledge on bacterial interaction H.; Osbourn, A.; Osburne, M.S.; Ostash, B.; Payne, S.M.; will help us to understand the key player in competition and Pernodet, J-L.; Petricek, M.; Piel, J.; Ploux, O.; Raaijmakers, J.M.; synergism and developed more efficient and stable antimi- Salas, J.A.; Schmitt, E.K.; Scott, B.; Seipke, R.F.; Shen, B.; crobials. Sherman, D.H.; Sivonen, K.; Smanski, M.J.; Sosio, M.; Stegmann, E.; Süssmuth, R.D.; Tahlan, K.; Thomas, C.M.; Tang, Y.; Truman, A.W.; Viaud, M.; Walton, J.D.; Walsh, C.T.; Weber, T.; van CONSENT FOR PUBLICATION Wezel, G.P.; Wilkinson, B.; Willey, J.M.; Wohlleben, W.; Wright, G.D.; Ziemert, N.; Zhang, C.; Zotchev, S.B.; Breitling, R.; Takano, Not applicable. E.; Glöckner, F.O. Minimum Information about a Biosynthetic Gene cluster. Nat. Chem. Biol., 2015, 11(9), 625-631. 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