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Summary Although in recent years there has been an increased awareness of the widespread nature of biofluorescence in the marine environment, the diversity of the molecules responsible for this luminescent phenotype has been mostly limited to green fluorescent proteins (GFPs), GFP-like proteins, and fluorescent fatty acid-binding proteins (FABPs). In the present study, we describe a previously undescribed group of brominated tryptophan-kynurenine small molecule metabolites responsible for the green biofluorescence in two species of sharks and provide their structural, antimicrobial, and spectral characterization. Multi-scale fluorescence microscopy studies guided the discovery of metabolites that were differentially produced in fluorescent versus non-fluorescent skin, as well as the species-specific structural details of their unusual light-guiding denticles. Overall, this study provides the detailed description of a family of small molecules responsible for marine biofluorescence and opens new questions related to their roles in central nervous system signaling, resilience to microbial infections, and photoprotection., Graphical Abstract, Highlights • We describe a new form of biofluorescence from the skin of catsharks • Bromo-tryptophan-kynurenines are biofluorescent and show antimicrobial activities • Specific dermal denticles in the chain catshark act as optical light-guides • This study opens questions related to biological function of shark fluorescence, Chemistry; Natural Product Chemistry; Natural Product Discovery

BackgroundGenome mining for Biosynthetic Gene Clusters (BGCs) has become an integral part of natural product discovery. The >200,000 microbial genomes now publicly available hold information on abundant novel chemistry. One way to navigate this vast genomic diversity is through comparative analysis of homologous BGCs, which allows identification of cross-species patterns that can be matched to the presence of metabolites or biological activities. However, current tools suffer from a bottleneck caused by the expensive network-based approach used to group these BGCs into Gene Cluster Families (GCFs).ResultsHere, we introduce BiG-SLiCE, a tool designed to cluster massive numbers of BGCs. By representing them in Euclidean space, BiG-SLiCE can group BGCs into GCFs in a non-pairwise, near-linear fashion. We used BiG-SLiCE to analyze 1,225,071 BGCs collected from 209,206 publicly available microbial genomes and metagenome-assembled genomes (MAGs) within ten days on a typical 36-cores CPU server. We demonstrate the utility of such analyses by reconstructing a global map of secondary metabolic diversity across taxonomy to identify uncharted biosynthetic potential. BiG-SLiCE also provides a "query mode" that can efficiently place newly sequenced BGCs into previously computed GCFs, plus a powerful output visualization engine that facilitates user-friendly data exploration.ConclusionsBiG-SLiCE opens up new possibilities to accelerate natural product discovery and offers a first step towards constructing a global, searchable interconnected network of BGCs. As more genomes get sequenced from understudied taxa, more information can be mined to highlight their potentially novel chemistry. BiG-SLiCE is available viahttps://github.com/medema-group/bigslice.

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

Decreasing discovery rates and increasing resistance have underscored the need for novel therapeutic options to treat Mycobacterium tuberculosis infection. Here, we screen extracts from previously uncultured soil microbes for specific activity against Mycobacterium tuberculosis, identifying three novel compounds. We further define the mechanism of action of one compound, amycobactin, and demonstrate that it inhibits protein secretion through the Sec translocation machinery., Mycobacterium tuberculosis, which causes tuberculosis (TB), is estimated to infect one-third of the world’s population. The overall burden and the emergence of drug-resistant strains of Mycobacterium tuberculosis underscore the need for new therapeutic options against this important human pathogen. Our recent work demonstrated the success of natural product discovery in identifying novel compounds with efficacy against Mycobacterium tuberculosis. Here, we improve on these methods by combining improved isolation and Mycobacterium tuberculosis selective screening to identify three new anti-TB compounds: streptomycobactin, kitamycobactin, and amycobactin. We were unable to obtain mutants resistant to streptomycobactin, and its target remains to be elucidated. We identify the target of kitamycobactin to be the mycobacterial ClpP1P2C1 protease and confirm that kitamycobactin is an analog of the previously identified compound lassomycin. Further, we identify the target of amycobactin to be the essential protein secretion pore SecY. We show further that amycobactin inhibits protein secretion via the SecY translocon. Importantly, this inhibition is bactericidal to nonreplicating Mycobacterium tuberculosis. This is the first compound, to our knowledge, that targets the Sec protein secretion machinery in Mycobacterium tuberculosis. This work underscores the ability of natural product discovery to deliver not only new compounds with activity against Mycobacterium tuberculosis but also compounds with novel targets.

Coinciding with the increase in sequenced bacteria, mining of bacterial genomes for biosynthetic gene clusters (BGCs) has become a critical component of natural product discovery. The order Myxococcales, a reputable source of biologically active secondary metabolites, spans three suborders which all include natural product producing representatives. Utilizing the BiG-SCAPE-CORASON platform to generate a sequence similarity network that contains 994 BGCs from 36 sequenced myxobacteria deposited in the antiSMASH database, a total of 843 BGCs with lower than 75% similarity scores to characterized clusters within the MIBiG database are presented. This survey provides the biosynthetic diversity of these BGCs and an assessment of the predicted chemical space yet to be discovered. Considering the mere snapshot of myxobacteria included in this analysis, these untapped BGCs exemplify the potential for natural product discovery from myxobacteria.

Natural products, also called secondary or specialised metabolites, are compounds that are produced by cells and are intended to have an effect outside of the producer. These compounds often have potent biological activities, and humankind has been using them as medicines in their crude forms for centuries. Even today, natural products are an important source and inspiration for drug development. An especially rich source of novel natural products are marine sponges. For many compounds isolated from sponges, though, it has been shown by genome sequencing and other methods that they are produced by symbiotic microorganisms. The genes necessary for the production of secondary metabolites are often clustered together in microorganisms, a phenomenon generally called a biosynthetic gene cluster (BGC). This clustering allows for the bioinformatic identification of BGCs, so-called genome mining, and subsequent investigation of natural product biosynthesis. The focus of this thesis lies on cyanobacteria. These fascinating microorganisms are not only exceptional producers of specialised metabolites, they have also been shown to occasionally produce compounds with high similarity to natural products isolated from sponges. The goal of this thesis was to further explore the biochemical potential of cyanobacteria in natural product research. This was done by either identifying new natural products and their biosynthetic gene clusters (chapter 2, 4 and 5), by characterising novel biochemistry and new enzymes involved in natural product biosynthesis (chapter 2, 3 and 5) or by testing the potential of cyanobacteria to heterologously express biosynthetic gene clusters (chapter 5). Chapter 2 focuses on the discovery and structure elucidation of new cytotoxic cyanobacterial meroterpenes termed merosterols. Their structure was determined with a combination of nuclear magnetic resonance spectroscopy (NMR), prediction of 13C chemical shifts, and a comparison of measured and predicted circular dichroism spectra. Additionally, the biosynthetic gene cluster responsible for merosterol production was identified. The substrate of the terpene cyclase was investigated by testing different surrogates with an intrinsic tryptophan fluorescence assay. The terpene cyclase was investigated in vitro and the structure of the formed product was determined by NMR analysis. The molecule consists of a polycyclic sterol-like terpene part annelated to an aromatic benzoic acid. A notable feature is the adopted opposite stereochemistry of the one of common steroids. Known terpene cyclases of the same class consist of two or three domains, and are often membrane bound proteins. The merosterol terpene cyclase on the other hand is soluble and smaller than the known enzymes. Structural 10 modelling proposed a minimalistic monodomain architecture, which was confirmed with an X-ray crystal structure of the enzyme, as further described in chapter 3. In chapter 4 the biosynthetic potential of cyanobacteria is further explored. Analysis of cyanobacterial genomes revealed a gene cluster putatively encoding a nonribosomal peptide synthetase/polyketide synthase hybrid accompanied by seven Fe(II)/α-ketoglutarate-dependent enzymes. Bioinformatic analysis suggested that six of them are halogenases, an unprecedented number of halogenases in a single gene cluster. Mass spectrometry-guided screening of cyanobacterial extracts led to the discovery and structure elucidation of the novel, extensively halogenated nonribosomal peptide/polyketide hybrids termed aranazoles. Chapter 5 is based on previous observations that cyanobacteria can produce similar secondary metabolites as symbionts of marine macroorganisms such as sponges. A putative gene cluster encoding genes for the biosynthesis of an indolocarbazole was identified in the genome of 'Candidatus Entotheonella serta'. It contains a glutathione sulphur transferase, a class of enzymes that has not yet been described in the context of indolocarbazoles. Investigation of the presence of homologous genes in cyanobacterial genomes led to the identification of similar gene clusters in two Fischerella species. Even though no activity was detected for the glutathione sulphur transferase so far, it was confirmed that the gene cluster is indeed responsible for the biosynthesis of indolocarbazoles. Detected products included the already known indolocarbazole arcyriaflavine A and a novel compound with two additional oxygens. This product can be explained by the presence of an additional cytochrome P450 enzyme encoded in the cyanobacterial gene clusters. Oxygenations of indolocarbazoles catalysed by P450s have only been described in the context of environmental DNA libraries. Otherwise only hydroxylations catalysed by FAD-dependent enzymes have been described so far for these compounds. All in all, this thesis provides further evidence that cyanobacteria are not only capable of producing unique novel secondary metabolites, but also have a repertoire of natural products closely resembling compounds, which are produced by symbionts of marine macroorganisms.

Natural products (NPs) are a major source of compounds for medical, agricultural, and biotechnological industries. Many of these compounds are of microbial origin, and, in particular, from Actinobacteria or filamentous fungi. To successfully identify novel compounds that correlate to a bioactivity of interest, or discover new enzymes with desired functions, systematic multiomics approaches have been developed over the years. Bioinformatics tools harness the rapidly expanding wealth of genome sequence information, revealing previously unsuspected biosynthetic diversity. Varying growth conditions or application of elicitors are applied to activate cryptic biosynthetic gene clusters, and metabolomics provide detailed insights into the NPs they specify. Combining these technologies with proteomics‐based approaches to profile the biosynthetic enzymes provides scientists with insights into the full biosynthetic potential of microorganisms. The proteomics approaches include enrichment strategies such as employing activity‐based probes designed by chemical biology, as well as unbiased (quantitative) proteomics methods. In this review, the opportunities and challenges in microbial NP research are discussed, and, in particular, the application of proteomics to link biosynthetic enzymes to the molecules they produce, and vice versa.

Microbial natural products have been a source for new drugs for many decades and are unrivaled in their capacity to generate not only future therapeutic agents, but also providing key agents for agricultural and industrial use. LC-MS/MS based metabolomic tools and technologies have been developed that can rapidly dereplicate nonribosomal peptides and statistically identify related congeners in an automated nontargeted process from complex natural product extracts with nanogram sensitivity. This data-base search approach is designed to handle linear, cyclic and cyclic-branched nonribosomal peptides from proteinogenic and nonproteinogenic amino acids without genomic data or traditional bioactivity directed fractionation. Chemometric work-flows combined with a comprehensive metabolomic guided discovery strategy were used to profile the chemical space of a diverse collection of understudied fungal endophytes from fruiting plants. This approach allowed for the prioritization of unique isolates and for the focused discovery, isolation and characterization of distinct outlier metabolites by LC-SPE, 1D and 2D NMR, HRMS and single crystal X-ray analysis. These metabolomic tools and strategies have led to the discovery and characterization of 35 new and over 40 known natural products, many of which are biologically active. This thesis with enabling metabolomic tools and novel discoveries has demonstrated the utility of these analytical methodologies as an effective strategy for the untargeted discovery of new natural products from bacteria and endophytic fungi. Thesis Doctor of Philosophy (PhD)

Since the onset of microbiology in the late 19th century, scientists have been growing microorganisms almost exclusively as pure cultures, resulting in a limited and biased view of the microbial world. Only a paradigm shift in cultivation techniques – from axenic to mixed cultures – can allow a full comprehension of the (chemical) communication of microorganisms, with profound consequences for natural product discovery, microbial ecology, symbiosis, and pathogenesis, to name a few areas. Three main technical advances during the last decade are fueling the realization of this revolution in microbiology: microfluidics, next-generation 3D-bioprinting, and single-cell metabolomics. These technological advances can be implemented for large-scale, systematic cocultivation studies involving three or more microorganisms. In this review, we present recent trends in microbiology tools and discuss how these can be employed to decode the chemical language that microorganisms use to communicate.

Traditionally, natural products have been isolated by bioactivity-guided fractionation. This strategy, however, led to the frequent re-isolation of known metabolites. An alternative strategy for the discovery of novel specialized metabolites is genome mining. Genome mining is an in-silico natural product discovery strategy which uses genetic information to identify natural product biosynthetic gene clusters (BGCs). The identified BGCs are annotated and the information about the BGC constitution is used to predict the structure of the corresponding secondary metabolite. Based on these predictions the corresponding natural product can be isolated in a targeted fashion. Trans-acyltransferase polyketide synthases (trans-AT PKSs) are giant modular multi-domain enzymes that are responsible for the biosynthesis of complex polyketides. These bacterial assembly-lines, ubiquitously distributed across the bacterial branch of life, were shown to produce bioactive polyketides of pharmaceutical and agricultural value. Phylogenetic analysis of ketosynthase (KS) domains from characterized PKSs, responsible for polyketide chain elongation, revealed a correlation between KS domains that phylogenetically cluster together and the substrates they recognize. Based on the correlation between phylogenetic origin and substrate specificity, referred to as the trans-AT PKS correlation rule, we developed the trans-AT PKS-derived polyketide predictor (TransATor) web application for the precise predictions of trans-AT PKS-derived polyketide structures. The TransATor web-application can be used to conduct in-silico dereplication studies. The TransATor-based in-silico annotation of a BGC to a known metabolite prevents the re-isolation of known metabolites, as exemplified by the isolation of tartrolon D from Gynuella sunshinyii. Moreover, TransATor predictions were employed to prioritize isolation efforts and, most importantly, to guide the purification and structure elucidation workflow. This has led to the isolation of a novel polyketide scaffold from Brevibacillus sp. Leaf182 and a new phormidolide-like polyketide, leptolyngbialide, from Leptolyngbia sp. PCC 7375. The prediction of KS substrate specificities furthermore resulted in the revision of the biosynthetic model for oocydin from Serratia plymuthica, the proposal of a model for sesbanimide biosynthesis in Azorhizobium doebereinerae and the reinvestigation and revision of the absolute stereochemistry of phormidolide isolated from a Leptolyngbia sp. ISBN3-Nov-94-8. A previous study on misakinolide-like polyketides suggested that the corresponding PKS family has evolved through the exchange, deletion or insertion of conserved sequential arrangements of PKS modules (PKS motifs). Driven by the hypothesis that trans-AT PKSs evolved in a mosaic-like manner, through combinatorial recombination of conserved motifs from different pathways, we analyzed all 1724 trans-AT PKSs deposited in GenBank for the presence of motifs shared between different PKSs. In total, 327 conserved sequential arrangements of PKS modules were identified. Based on these conserved motifs, also reflected in the polyketide structure, we were able to isolate new polyketides with conserved substructures. These included the novel glutarimide-containing polyketide secimide from the plant pathogen Pseudomonas syringae pv. syringae and the new phormidolide, gynuellalide, from G. sunshinyii. Moreover, the identification of a conserved PKS gene fragment encoding part of the PKS responsible for the biosynthesis of the cytotoxic spliceostatin in the plant pathogen Xanthomonas campestris pv. cannabis resulted in the identification of the pathogenicity factor of the bacterium. In addition, PKSs that share conserved motifs with other orphan PKSs but do not show any conserved motifs with known polyketides are good candidates for the discovery of structural novelty in the corresponding polyketides. The analysis of conserved PKS motifs shared between different PKSs furthermore paves the way towards evolutionary inspired engineering of non-natural hybrid PKSs to produce novel polyketides. 8 A complementary strategy to genome mining is the activation of silent BGCs. Here, we studied the potential of the actinobacterial signaling metabolite hormaomycin to activate silent BGCs in the producing organism and the model strain Streptomyces coelicolor. The effect of hormaomycin on the metabolome of the producing organism, Streptomyces griseoflavus W-384 was analyzed, and compared to the metabolome of hormaomycin production deficient- and hormaomycin overproducing mutants. Two novel metabolites, xanthoductin and xanthotryptin were only produced in the hormaomycin overproducing mutant. Moreover, the production of pharmaceutically important natural products was either strongly up- or downregulated in the presence of hormaomycin. In order to study its effect on other Streptomyces, we subjected S. coelicolor, either challenged with the hormaomcyin overproducer or purified hormaomycin to matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry and nanospray desorption ionization mass spectrometry (nanoDESI) coupled to molecular network analysis. These analyses revealed that hormaomycin induces the production of siderophores, antibiotics, morphogens and pigments belonging to different classes of natural products. In addition, hormaomycin was able to trigger the production of unknown metabolites. These studies show that hormaomycin can be used as a tool to activate silent BGCs and to increase the production of pharmaceutically relevant specialized metabolites. Intrigued by the chemotype triggered by hormaomycin, we were interested in identifying its molecular target. Using a mutasynthesis strategy, hormaomycin analogs with either alkyne or azide handles were generated. These analogs were coupled to different tags and the obtained probes used for pull-down assays to identify the target of hormaomcyin. These assays revealed the the F-ATP synthase as the putative target molecule. Quantification of intracellular ATP levels in the presence or absence of hormaomycin and the phenotypic comparison of the effect of hormaomycin with the decoupling agent carbonylcyanide m-chlorophenylhydrazone supported our initial hypothesis that hormaomycin exerts its effect by interacting with the F-ATP synthase. Moreover, we conducted a high-throughput bipartite interaction screen of all strains of the At-LSPHERE strain collection (50,000 interactions) of bacterial leaf isolates to identify talented antibiotic producers. Linking these data with genome mining studies (AntiSMASH and BigScape analysis), resulted in the identification of antibiotic producers harboring BGCs that encode for putative biosynthetic and structural novelty. The most promising inhibiting strain, Brevibacillus sp. Leaf182 that contains a dozen BGCs was selected for bioactivity-guided fractionation to identify the antibiotic(s) responsible for the observed inhibition of one third of all strains of the strain collection. Isolation of the metabolites responsible for the majority of the observed antibiotic activity yielded tyrocidin analogs and marthiapeptide A. To our surprise, marthiapeptide A is putatively produced via the non-ribosomal route of peptide biosynthesis, which is remarkable given that two structurally close homologs were previously reported to be ribosomally-synthesized and post-translationally modified peptides. The biosynthesis of the same class of polythiazole/polyoxazole-containing 7-8 membered cyclopeptides from two biorthogonal pathways displays an extreme case of convergent evolution. Moreover, the isolation of a novel trans-AT PKS scaffold from the same organism highlights the huge untapped biosynthetic potential of bacterial isolates from the plant phyllosphere.

Actinomycetes produce many pharmaceutically useful compounds through type I polyketide biosynthetic pathways. Soil has traditionally been an important source for these actinomycete-derived pharmaceuticals. As the rate of antibiotic discovery has decreased and the incidence of antibiotic resistance has increased, researchers have looked for alternatives to soil for bioprospecting. Street sediment, where actinomycetes make up a larger fraction of the bacterial population than in soil, is one such alternative environment. To determine if these differences in actinomycetal community structure are reflected in type I polyketide synthases (PKSI) distribution, environmental DNA from soils and street sediments was characterized by sequencing amplicons of PKSI-specific PCR primers. Amplicons covered two domains: the last 80 amino acids of the ketosynthase (KS) domain and the first 240 amino acids of the acyltransferase (AT) domain. One hundred and ninety clones from ten contrasting soils from six regions and nine street sediments from six cities were sequenced. Twenty-five clones from two earthworm-affected samples were also sequenced. UniFrac lineage-specific analysis identified two clades that clustered with actinomycetal GenBank matches that were street sediment-specific, one similar to the PKSI segment of the mycobactin siderophore involved in mycobacterial virulence. A clade of soil-specific sequences clustered with GenBank matches from the ambruticin and jerangolid pathways of Sorangium cellulosum. All three of these clades were found in sites >700 km apart. Street sediments are enriched in actinomycetal PKSIs. Non-actinomycetal PKSI pathways may be more chemically diverse than actinomycetal PKSIs. Common soil and street sediment PKIs are globally distributed.

In this perspective, we highlight recent examples and trends in metabolic engineering and synthetic biology that demonstrate the synthetic potential of enzyme and pathway engineering for natural product discovery. In doing so, we introduce natural paradigms of secondary metabolism whereby simple carbon substrates are combined into complex molecules through “scaffold diversification”, and subsequent “derivatization” of these scaffolds is used to synthesize distinct complex natural products. We provide examples in which modern pathway engineering efforts including combinatorial biosynthesis and biological retrosynthesis can be coupled to directed enzyme evolution and rational enzyme engineering to allow access to the “privileged” chemical space of natural products in industry-proven microbes. Finally, we forecast the potential to produce natural product-like discovery platforms in biological systems that are amenable to single-step discovery, validation, and synthesis for streamlined discovery and production of biologically active agents.

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

A widely accepted species concept for bacteria has yet to be established. As a result, species designations are inconsistently applied and tied to what can be considered arbitrary metrics. Increasing access to DNA sequence data and clear evidence that bacterial genomes are dynamic entities that include large numbers of horizontally acquired genes have added a new level of insight to the ongoing species concept debate. Despite uncertainties over how to apply species concepts to bacteria, there is clear evidence that sequence-based approaches can be used to resolve cohesive groups that maintain the properties of species. This cohesion is clearly evidenced in the genus Salinispora, where three species have been discerned despite very close relationships based on 16S rRNA sequence analysis. The major phenotypic differences among the three species are associated with secondary metabolite production, which occurs in species-specific patterns. These patterns are maintained on a global basis and provide evidence that secondary metabolites have important ecological functions. These patterns also suggest that an effective strategy for natural product discovery is to target the cultivation of new Salinispora taxa. Alternatively, bioinformatic analyses of biosynthetic genes provide opportunities to predict secondary metabolite novelty and reduce the redundant isolation of well-known metabolites. Although much remains to be learned about the evolutionary relationships among bacteria and how fundamental units of diversity can be resolved, genus and species descriptions remain the most effective method of scientific communication.

Functional metagenomics, the study of the collective genome of a microbial community by expressing it in a foreign host, is an emerging field in biotechnology. Over the past years, the possibility of novel product discovery through metagenomics has developed rapidly. Thus, metagenomics has been heralded as a promising mining strategy of resources for the biotechnological and pharmaceutical industry. However, in spite of innovative work in the field of functional genomics in recent years, yields from function-based metagenomics studies still fall short of producing significant amounts of new products that are valuable for biotechnological processes. Thus, a new set of strategies is required with respect to fostering gene expression in comparison to the traditional work. These new strategies should address a major issue, that is, how to successfully express a set of unknown genes of unknown origin in a foreign host in high throughput. This article is an opinionating review of functional metagenomic screening of natural microbial communities, with a focus on the optimization of new product discovery. It first summarizes current major bottlenecks in functional metagenomics and then provides an overview of the general metagenomic assessment strategies, with a focus on the challenges that are met in the screening for, and selection of, target genes in metagenomic libraries. To identify possible screening limitations, strategies to achieve optimal gene expression are reviewed, examining the molecular events all the way from the transcription level through to the secretion of the target gene product.

Background With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30–80 kb in size, breakthrough techniques are needed to characterize this SM wealth. Results Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery. Conclusion The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1561-x) contains supplementary material, which is available to authorized users.

The development and spread of multidrug resistant pathogens have reinforced the urgency to find novel natural products with antibiotic activity. In bacteria, orphan biosynthetic gene clusters (BGCs) far outnumber the BGCs for which chemistry is known, possibly because they are transcriptionally silent under laboratory conditions. A strategy to trigger the production of this biosynthetic potential is to challenge the microorganism with low concentrations of antibiotics, and by using a Burkholderia genetic reporter strain (Seyedsayamdost, Proc Natl Acad Sci 111:7266–7271), we found BGC unsilencing activity for the antimicrobial andrimid, produced by the marine bacterium Vibrio coralliilyticus. Next, we challenged another marine Vibrionaceae, Photobacterium galatheae, carrier of seven orphan BGCs with sub-inhibitory concentrations of andrimid. A combined approach of transcriptional and chemical measurements of andrimid-treated P. galatheae cultures revealed a 10-fold upregulation of an orphan BGC and, amongst others, a 1.6–2.2-fold upregulation of the gene encoding the core enzyme for biosynthesis of holomycin. Also, addition of andrimid caused an increase, based on UV-Vis peak area, of 4-fold in production of the antibiotic holomycin. Transcriptional measurements of stress response related genes in P. galatheae showed a co-occurrence of increased transcript levels of rpoS (general stress response) and andrimid induced holomycin overproduction, while in trimethoprim treated cultures attenuation of holomycin production coincided with a transcriptional increase of recA (SOS stress response). This study shows that using antimicrobial compounds as activators of secondary metabolism can be a useful strategy in eliciting biosynthetic gene clusters and facilitate natural product discovery. Potentially, such interactions could also have ecological relevant implications.