When, where, and why specialised metabolites are produced: inferring function from expression control.

Chapter 19 - Regulation and Role of Metal Ions in Secondary Metabolite Production by Microorganisms

Genome sequencing has accelerated our understanding about organization of biosynthetic clusters in bacterial genomes which are responsible for the production of secondary metabolites. These metabolites are sources of important drugs and act like anticancer, antibacterial and anti-infective agents. In this context actinomycetes are notable as nearly 70% of the economically important metabolites are produced by them. Genome sequences of many actinomycetes have suggested the presence of many cryptic biosynthetic gene clusters that are not expressed or poorly expressed [1]. These cryptic gene clusters may encode for novel products that can prove to be beneficial for mankind. In a recent study published in PNAS [2] Laureti et al. have identified one such cryptic gene cluster in genome of Streptomyces ambofaciens (producer of antibiotics-spiramycine and congocidine). This cluster is not associated with any metabolite production and is found to be poorly expressed. These workers expressed this cryptic gene cluster using large ATP-binding regulator of the LuxR family (LAL) protein, the members of which are known to be activator of pikromycin [3] and rapamycin [4]. NMR analysis of the expressed product suggested that the product is a novel macrolide which was named stambomycin. These workers identified four such macrolidesstambomycin A to D. Bioactivity of the novel macrolide suggested that the product can provide new insights into anticancer therapy owing to its antiproliferative activities against adenocarcinoma, human breast, lung and prostate cancer cell lines. Researchers have been looking for better drugs for treatment of antibiotic resistant strains and different types of cancers. This study has opened up new realms in the discovery of several novel natural products that find application in medicine.

Bioinformatics guided synthesis of natural product cyclic peptides.

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature.IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression.

Recent advancements in genome sequencing and bioinformatic analysis of fungal genomes have revealed that fungi possess cryptic or silent biosynthetic gene clusters (BGCs). This discovery suggests that our understanding of the universe of fungal secondary metabolomes is limited. In this review, we summarize recent strategies for activating cryptic BGCs in fungi, identify fungal secondary metabolites, and highlight their biological activities. We also briefly discuss the isolation and purification methods for these compounds. Our focus is on genetics-dependent and genetics-independent approaches for activating cryptic BGCs in fungi. Using the strategies discussed here, cryptic fungal natural products hold significant potential as a source for the discovery of new drug candidates.

Emerging from millions of years of evolutionary optimization, Natural products (NPs) remain unique, unparalleled sources of bioactive scaffolds. Unlike synthetic molecules engineered around single therapeutic targets, NPs often exhibit multi-target, system-level bioactivity, aligned with the principles of network pharmacology, which modulates pathways in a coordinated, non-disruptive manner. This approach reduces resistance, buffers compensatory feedback loops, and enhances therapeutic resilience. Fungal endophytes represent one of the most chemically diverse and biologically sophisticated NP reservoirs known, producing polyketides, alkaloids, terpenoids, and peptides with intricate three-dimensional architectures and emergent bioactivity patterns that remain exceptionally difficult to design de novo. Advances in artificial intelligence (AI), machine learning, deep learning, and multi-omics integration have redefined the discovery landscape, transforming previously intractable fungal metabolomes and cryptic biosynthetic gene clusters (BGCs) into tractable, predictable, and engineerable systems. AI accelerates genome mining, metabolomic annotation, BGC-metabolite linking, structure prediction, and activation of silent pathways. Generative AI and diffusion models now enable de novo design of NP-inspired scaffolds while preserving biosynthetic feasibility, opening new opportunities for direct evolution, pathway refactoring, and precision biomanufacturing. This review synthesizes the chemical and biosynthetic diversity of major NP classes from fungal endophytes and maps them onto the rapidly expanding ecosystem of AI-driven tools. We outline how AI transforms NP discovery from empirical screening into a predictive, hypothesis-driven discipline with direct industrial implications for drug discovery and synthetic biology. By coupling evolutionarily refined chemistry with modern computational intelligence, the field is poised for a new era in which natural-product leads are not only rediscovered but systematically expanded, engineered, and industrialized to address urgent biomedical and sustainability challenges.

Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.

Lichen natural products are a tremendous source of new bioactive chemical entities for drug discovery. The ability to survive in harsh conditions can be directly correlated with the production of some unique lichen metabolites. Despite the potential applications, these unique metabolites have been underutilized by pharmaceutical and agrochemical industries due to their slow growth, low biomass availability, and technical challenges involved in their artificial cultivation. At the same time, DNA sequence data have revealed that the number of encoded biosynthetic gene clusters in a lichen is much higher than in natural products, and the majority of them are silent or poorly expressed. To meet these challenges, the one strain many compounds (OSMAC) strategy, as a comprehensive and powerful tool, has been developed to stimulate the activation of silent or cryptic biosynthetic gene clusters and exploit interesting lichen compounds for industrial applications. Furthermore, the development of molecular network techniques, modern bioinformatics, and genetic tools is opening up a new opportunity for the mining, modification, and production of lichen metabolites, rather than merely using traditional separation and purification techniques to obtain small amounts of chemical compounds. Heterologous expressed lichen-derived biosynthetic gene clusters in a cultivatable host offer a promising means for a sustainable supply of specialized metabolites. In this review, we summarized the known lichen bioactive metabolites and highlighted the application of OSMAC, molecular network, and genome mining-based strategies in lichen-forming fungi for the discovery of new cryptic lichen compounds.

Natural products are considered to be the lifeline treatment for several diseases where their structural complexity makes them a source of potential lead molecules. As a producer of antibiotics, food colorants, enzymes, and nutritious food, fungi are beneficial to humans. Fungi, as a source of novel natural products, draw attention of scientists. However, redundant isolation of metabolite retards the rate of discovery. So, apart from the standard conditions for the production of secondary metabolites, certain induction strategies are used to trigger biosynthetic genes in fungi. Advancement in the computational tools helps in connecting gene clusters and their metabolite production. Therefore, modern analytical tools and the genomic era in hand leads to the identification of manifold of cryptic metabolites. The cryptic biosynthetic gene cluster (BGC) has become a treasure hunt for new metabolites representing biosynthetic pathways, regulatory mechanisms, and other factors. This review includes the use of chemical inducers/epigenetic modifiers and co-culture (species interaction) techniques to induce these BGCs. Furthermore, it cites a detailed representation of molecules isolated using these strategies. Since the induction occurs on the genomic molecular DNA and histones, this together brings a significant exploration of the biosynthetic pathways.Graphical Abstract.

BackgroundHeterologous expression in Streptomyces provides a platform for mining natural products (NPs) encoded by cryptic biosynthetic gene clusters (BGCs) of bacteria. The BGCs are first engineered in hosts with robust recombineering systems, such as Escherichia coli, followed by expression in optimized heterologous hosts, such as Streptomyces, with defined metabolic backgrounds.ResultsWe developed a highly efficient heterologous expression platform, named Micro-HEP (microbial heterologous expression platform), that uses versatile E. coli strains capable of both modification and conjugation transfer of foreign BGCs and optimized chassis Streptomyces strain for expression. The stability of repeat sequences in these E. coli strains was superior to that of the commonly used conjugative transfer system E. coli ET12567 (pUZ8002). For optimizing expression of foreign BGCs, the chassis strain S. coelicolor A3(2)-2023 was generated by deleting four endogenous BGCs followed by introducing multiple recombinase-mediated cassette exchange (RMCE) sites in the S. coelicolor A3(2) chromosome. Additionally, modular RMCE cassettes (Cre-lox, Vika-vox, Dre-rox, and phiBT1-attP) were constructed for integrating BGCs into the chassis strain. Micro-HEP was tested using BGCs for the anti-fibrotic compound xiamenmycin and griseorhodins. Two to four copies of the xim BGC were integrated by RMCE, with increasing copy number associated with increasing yield of xiamenmycin. The grh BGC was also efficiently expressed, and the new compound griseorhodin H was identified.ConclusionWe demonstrated that our Micro-HEP system enables the efficient expression of foreign BGCs, facilitating the discovery of new NPs and increasing yields.Graphical

BackgroundMarine microorganisms are an important source of new drug leads. However, the discovery and sustainable production of these compounds are often hampered due to the unavailable expression of cryptic biosynthetic gene clusters or limited titer. Ribosome engineering and response surface methodology (RSM) integrated strategy was developed in this study to activate cryptic gene cluster in the deepsea-derived Streptomyces somaliensis SCSIO ZH66, and subsequently isolation, structural analysis, and the yield enhancement of the activated compound, anticancer drug lead Fredericamycin A (FDM A), were performed.ResultsIn order to discover novel natural products from marine Streptomyces strains by genome mining strategy, the deepsea-derived S. somaliensis SCSIO ZH66 was subject to ribosome engineering to activate the expression of cryptic gene clusters. A resistant strain ZH66-RIF1 was thereby obtained with 300 μg/mL rifampicin, which accumulated a brown pigment with cytotoxicity on MS plate while absent in the wild type strain. After screening of fermentation conditions, the compound with pigment was purified and identified to be FDM A, indicating that the activation of a cryptic FDM A biosynthetic gene cluster was taken place in strain ZH66-RIF1, and then it was identified to be ascribed to the mutation of R444H in the β subunit of RNA polymerase. To further improve the yield efficiently, nine fermentation medium components were examined for their significance on FDM A production by Plackett–Burman design and Box-Behnken design. The optimum medium composition was achieved by RSM strategy, under which the titer of FDM A reached 679.5 ± 15.8 mg/L after 7 days of fermentation, representing a 3-fold increase compared to the original medium. In terms of short fermentation time and low-cost fermentation medium, strain ZH66-RIF1 would be an ideal alternative source for FDM A production.ConclusionsOur results would hasten the efforts for further development of FDM A as a drug candidate. Moreover, this ribosome engineering and RSM integrated methodology is effective, fast and efficient; it would be applicable to genome mining for novel natural products from other strains.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0244-2) contains supplementary material, which is available to authorized users.

Discovery and development of natural products (NPs) have played important roles in the fields of human medicine and other biotechnology fields for the past several decades. Recent genome-mining approaches for the isolation of novel and cryptic NP biosynthetic gene clusters (BGCs) have led to the growing interest in NP research communities including Asian NP researchers from China, Japan, and Korea. Recently, a three-nation government-sponsored program named 'A3 Foresight Network on Chemical and Synthetic Biology of NPs' has been launched with a goal of establishing an Asian hub for NP research-&-personnel exchange program. This brief commentary describes introduction, main researchers, and future perspective of A3 NP network program.

Unlocking the rich harvest of marine microbial ecosystems has the potential to both safeguard the existence of our species for the future, while also presenting significant lifestyle benefits for commercial gain. However, while significant advances have been made in the field of marine biodiscovery, leading to the introduction of new classes of therapeutics for clinical medicine, cosmetics and industrial products, much of what this natural ecosystem has to offer is locked in, and essentially hidden from our screening methods. Releasing this silent potential represents a significant technological challenge, the key to which is a comprehensive understanding of what controls these systems. Heterologous expression systems have been successful in awakening a number of these cryptic marine biosynthetic gene clusters (BGCs). However, this approach is limited by the typically large size of the encoding sequences. More recently, focus has shifted to the regulatory proteins associated with each BGC, many of which are signal responsive raising the possibility of exogenous activation. Abundant among these are the LysR-type family of transcriptional regulators, which are known to control production of microbial aromatic systems. Although the environmental signals that activate these regulatory systems remain unknown, it offers the exciting possibility of evoking mimic molecules and synthetic expression systems to drive production of potentially novel natural products in microorganisms. Success in this field has the potential to provide a quantum leap forward in medical and industrial bio-product development. To achieve these new endpoints, it is clear that the integrated efforts of bioinformaticians and natural product chemists will be required as we strive to uncover new and potentially unique structures from silent or cryptic marine gene clusters.

BackgroundActinomycete genome sequencing has disclosed a large number of cryptic secondary metabolite biosynthetic gene clusters. However, their unavailable or limited expression severely hampered the discovery of bioactive compounds. The whiB-like (wbl) regulatory genes play important roles in morphological differentiation as well as secondary metabolism; and hence the wblAso gene was probed and set as the target to activate cryptic gene clusters in deepsea-derived Streptomyces somaliensis SCSIO ZH66.ResultswblAso from deepsea-derived S. somaliensis SCSIO ZH66 was inactivated, leading to significant changes of secondary metabolites production in the ΔwblAso mutant, from which α-pyrone compound violapyrone B (VLP B) was isolated. Subsequently, the VLP biosynthetic gene cluster was identified and characterized, which consists of a type III polyketide synthase (PKS) gene vioA and a regulatory gene vioB; delightedly, inactivation of vioB led to isolation of another four VLPs analogues, among which one was new and two exhibited improved anti-MRSA (methicillin-resistant Staphylococcus aureus, MRSA) activity than VLP B. Moreover, transcriptional analysis revealed that the expression levels of whi genes (whiD, whiG, whiH and whiI) and wbl genes (wblC, wblE, wblH, wblI and wblK) were repressed by different degrees, suggesting an intertwined regulation mechanism of wblAso in morphological differentiation and secondary metabolism of S. somaliensis SCSIO ZH66.ConclusionswblA orthologues would be effective targets for activation of cryptic gene clusters in marine-derived Streptomyces strains, notwithstanding the regulation mechanisms might be varied in different strains. Moreover, the availability of the vio gene cluster has enriched the diversity of type III PKSs, providing new opportunities to expand the chemical space of polyketides through biosynthetic engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0515-6) contains supplementary material, which is available to authorized users.

Characterization of cryptic biosynthetic gene clusters (BGCs) from microbial genomes has been proven to be a powerful approach to the discovery of new natural products. However, such a genome mining approach to the discovery of bioactive plant metabolites has been muted. The plant BGCs characterized to date encode pathways for antibiotics important in plant defense against microbial pathogens, providing a means to discover such phytoalexins by mining plant genomes. Here is reported the discovery and characterization of a minimal BGC from the medicinal plant Catharanthus roseus, consisting of an adjacent pair of genes encoding a terpene synthase (CrTPS18) and cytochrome P450 (CYP71D349). These two enzymes act sequentially, with CrTPS18 acting as a sesquiterpene synthase, producing 5-epi-jinkoheremol (1), which CYP71D349 further hydroxylates to debneyol (2). Infection studies with maize revealed that 1 and 2 exhibit more potent fungicidal activity than validamycin. Accordingly, this study demonstrates that characterization of such cryptic plant BGCs is a promising strategy for the discovery of potential agrochemical leads. Moreover, despite the observed absence of 1 and 2 in C. roseus, the observed transcriptional regulation is consistent with their differential fungicidal activity, suggesting that such conditional coexpression may be sufficient to drive BGC assembly in plants.