Discovery of Reactive Microbiota-Derived Metabolites that Inhibit Host Proteases.

The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis, we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals.

Ganglioside GM3 Protects Against Abdominal Aortic Aneurysm by Suppressing Ferroptosis.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially life-threatening vascular condition, but approved medical therapies to prevent AAA progression and rupture are currently lacking. Sphingolipid metabolism disorders are associated with the occurrence and development of AAA. It has been discovered that ganglioside GM3, a sialic acid-containing type of glycosphingolipid, plays a protective role in atherosclerosis, which is an important risk factor for AAA; however, the potential contribution of GM3 to AAA development has not been investigated. METHODS: We performed a metabolomics study to evaluated GM3 level in plasma of human patients with AAA. We profiled GM3 synthase (ST3GAL5) expression in the mouse model of aneurysm and human AAA tissues through Western blotting and immunofluorescence staining. RNA sequencing, affinity purification and mass spectrometry, proteomic analysis, surface plasmon resonance analysis, and functional studies were used to dissect the molecular mechanism of GM3-regulating ferroptosis. We conditionally deleted and overexpressed St3gal5 in smooth muscle cells (SMCs) in vivo to investigate its role in AAA. RESULTS: We found significantly reduced plasma levels of GM3 in human patients with AAA. GM3 content and ST3GAL5 expression were decreased in abdominal aortic vascular SMCs in patients with AAA and an AAA mouse model. RNA sequencing analysis showed that ST3GAL5 silencing in human aortic SMCs induced ferroptosis. We showed that GM3 interacted directly with the extracellular domain of TFR1 (transferrin receptor 1), a cell membrane protein critical for cellular iron uptake, and disrupted its interaction with holo-transferrin. SMC-specific St3gal5 knockout exacerbated iron accumulation at lesion sites and significantly promoted AAA development in mice, whereas GM3 supplementation suppressed lipid peroxidation, reduced iron deposition in aortic vascular SMCs, and markedly decreased AAA incidence. CONCLUSIONS: Together, these results suggest that GM3 dysregulation promotes ferroptosis of vascular SMCs in AAA. Furthermore, GM3 may constitute a new therapeutic target for AAA.

Identifying Human miRNA Target Sites via Learning the Interaction Patterns between miRNA and mRNA Segments.

miRNAs (microRNAs) target specific mRNA (messenger RNA) sites to regulate their translation expression. Although miRNA targeting can rely on seed region base pairing, animal miRNAs, including human miRNAs, typically cooperate with several cofactors, leading to various noncanonical pairing rules. Therefore, identifying the binding sites of animal miRNAs remains challenging. Because experiments for mapping miRNA targets are costly, computational methods are preferred for extracting potential miRNA-mRNA fragment binding pairs first. However, existing prediction tools can have significant false positives due to the prevalent noncanonical miRNA binding behaviors and the information-biased training negative sets that were used while constructing these tools. To overcome these obstacles, we first prepared an information-balanced miRNA binding pair ground-truth data set. A miRNA-mRNA interaction-aware model was then designed to help identify miRNA binding events. On the test set, our model (auROC = 94.4%) outperformed existing models by at least 2.8% in auROC. Furthermore, we showed that this model can suggest potential binding patterns for miRNA-mRNA sequence interacting pairs. Finally, we made the prepared data sets and the designed model available at http://cosbi2.ee.ncku.edu.tw/mirna_binding/download.

Co-designing community-level integral interventions for active ageing: a systematic review from the lens of community-based participatory research.

BACKGROUND: While community-level interventions for promoting active ageing have received increasing attention and there is a trend to leverage technology to support traditional physical or social interventions, little hands-on guidance exists for designing these integral interventions. This study aimed to examine the interventions reported in the literature guided by Community-Based Participatory Research (CBPR) principles. The goal is to extract insights that inform future practices in co-designing integral interventions for active ageing. METHODS: The systematic review focused on community-level interventions promoting active ageing that integrated physical, social, and digital elements, i.e., integral interventions. Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed. The included interventions were analysed abductively based on the CBPR principles. RESULTS: A total of 13 studies were included, and 24 design considerations were generated under eight categories. Further reflection identified the interrelated nature of these design considerations and pinpointed the gaps in current research. This study highlights the urgency and importance of sharing recruitment methods and resource allocation details, recording and reporting collaboration specifics, and disseminating findings to stakeholders beyond academia. CONCLUSIONS: This study offers valuable insights and practical guidance to researchers and practitioners developing community-level integral interventions for active ageing. The findings also serve as a starting point for accumulating knowledge and practice in co-designing integral interventions for active ageing at the community level. The next crucial phase involves evaluating these design considerations within real-world cases to assess their applicability and identify potential areas for improvement.

Domesticating Social Alarm Systems in Nursing Homes: Qualitative Study of Differences in the Perspectives of Assistant Nurses.

BACKGROUND: New social alarm solutions are viewed as a promising approach to alleviate the global challenge of an aging population and a shortage of care staff. However, the uptake of social alarm systems in nursing homes has proven both complex and difficult. Current studies have recognized the benefits of involving actors such as assistant nurses in advancing these implementations, but the dynamics by which implementations are created and shaped in their daily practices and relations have received less attention. OBJECTIVE: Based on domestication theory, this paper aims to identify the differences in the perspectives of assistant nurses when integrating a social alarm system into daily practices. METHODS: We interviewed assistant nurses (n=23) working in nursing homes to understand their perceptions and practices during the uptake of social alarm systems. RESULTS: During the four domestication phases, assistant nurses were facing different challenges including (1) system conceptualization; (2) spatial employment of social alarm devices; (3) treatment of unexpected issues; and (4) evaluation of inconsistent competence in technology use. Our findings elaborate on how assistant nurses have distinct goals, focus on different facets, and developed diverse coping strategies to facilitate the system domestication in different phases. CONCLUSIONS: Our findings reveal a divide among assistant nurses in terms of domesticating social alarm systems and stress the potential of learning from each other to facilitate the whole process. Further studies could focus on the role of collective practices during different domestication phases to enhance the understanding of technology implementation in the contexts of complex interactions within a group.

The atypical binding mechanism of second calcium on phospholipase A2 group IIE.

Secreted phospholipase A2s (sPLA2s) are calcium dependent enzymes involved in various biological events such as lipid metabolism and inflammation. We previously identified the second calcium (Ca2) binding site of human sPLA2 Group IIE (hGIIE) by structural study and suggested that Asn21 act as the switch of Ca2 binding to modulate the enzymatic activity, but the detailed Ca2 binding mechanism is still unclear. Combined with enzymatic assay, model analysis and calcium binding affinity data for mutated hGIIE proteins, we herein further demonstrate that the flexibly bound Ca2 is essential for the catalysis of hGIIE, unlike the stable binding of Ca2 in hGIIA that replenishes the calcium in the typical loop during the reaction. The atypical Ca2 binding feature of hGIIE will provide a better understanding on the catalytic mechanism of hGIIE.

Hydrogen production driven by formate oxidation in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a potent hydrogen producer in the deficiency of exogenous electron acceptors. The electron transfer pathway for hydrogen production remains unclear, although enzymes for hydrogen production have been identified in S. oneidensis MR-1. In this study, we investigated the electron transfer pathway from formate to hydrogen, given that formate is commonly a key chemical for bacterial hydrogen production. We revealed that two formate dehydrogenases FdhA1B1C1 and FdhA2B2C2, rather than FdnGHI, played a dominant role in formate-driven hydrogen production. Menaquinone was indispensable for the electron transfer from formate to hydrogen, which excluded the presence of formate hydrogen-lyase in S. oneidensis MR-1. A previously proposed formate dehydrogenase subunit HydC was identified as a menaquinone-binding subunit of [FeFe] hydrogenase HydAB, and the hydABC operon is conserved in bacteria living in diverse environments. A formate exporter FocA and transcriptional regulator FhlA were identified for their effect on formate metabolism and hydrogen production. FhlA positively affected the metabolism of formate and hydrogen by regulating the expression of fdhA2B2C2, fdnGHI, focA, and dld-II. Overall, the electron transfer pathway deciphered in this work will facilitate the improvement of biohydrogen production by S. oneidensis MR-1.Key Points• The electron transfer pathway from formate to hydrogen in MR-1 is deciphered.• Menaquinone is indispensable for hydrogen production.• A cytochrome b subunit transfers electrons from menaquinone to [FeFe] hydrogenase.

A Pressure Test to Make 10 Molecules in 90 Days: External Evaluation of Methods to Engineer Biology.

Centralized facilities for genetic engineering, or "biofoundries", offer the potential to design organisms to address emerging needs in medicine, agriculture, industry, and defense. The field has seen rapid advances in technology, but it is difficult to gauge current capabilities or identify gaps across projects. To this end, our foundry was assessed via a timed "pressure test", in which 3 months were given to build organisms to produce 10 molecules unknown to us in advance. By applying a diversity of new approaches, we produced the desired molecule or a closely related one for six out of 10 targets during the performance period and made advances toward production of the others as well. Specifically, we increased the titers of 1-hexadecanol, pyrrolnitrin, and pacidamycin D, found novel routes to the enediyne warhead underlying powerful antimicrobials, established a cell-free system for monoterpene production, produced an intermediate toward vincristine biosynthesis, and encoded 7802 individually retrievable pathways to 540 bisindoles in a DNA pool. Pathways to tetrahydrofuran and barbamide were designed and constructed, but toxicity or analytical tools inhibited further progress. In sum, we constructed 1.2 Mb DNA, built 215 strains spanning five species ( Saccharomyces cerevisiae, Escherichia coli, Streptomyces albidoflavus, Streptomyces coelicolor, and Streptomyces albovinaceus), established two cell-free systems, and performed 690 assays developed in-house for the molecules.

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters.

Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

Discovery of indolotryptoline antiproliferative agents by homology-guided metagenomic screening.

Natural product discovery by random screening of broth extracts derived from cultured bacteria often suffers from high rates of redundant isolation, making it ever more challenging to identify novel biologically interesting natural products. Here we show that homology-based screening of soil metagenomes can be used to specifically target the discovery of new members of traditionally rare, biomedically relevant natural product families. Phylogenetic analysis of oxy-tryptophan dimerization gene homologs found within a large soil DNA library enabled the identification and recovery of a unique tryptophan dimerization biosynthetic gene cluster, which we have termed the bor cluster. When heterologously expressed in Streptomyces albus, this cluster produced an indolotryptoline antiproliferative agent with CaMKIIδ kinase inhibitory activity (borregomycin A), along with several dihydroxyindolocarbazole anticancer/antibiotics (borregomycins B-D). Similar homology-based screening of large environmental DNA libraries is likely to permit the directed discovery of new members within other previously rare families of bioactive natural products.

Targeted metagenomics: finding rare tryptophan dimer natural products in the environment.

Natural product discovery from environmental genomes (metagenomics) has largely been limited to the screening of existing environmental DNA (eDNA) libraries. Here, we have coupled a chemical-biogeographic survey of chromopyrrolic acid synthase (CPAS) gene diversity with targeted eDNA library production to more efficiently access rare tryptophan dimer (TD) biosynthetic gene clusters. A combination of traditional and synthetic biology-based heterologous expression efforts using eDNA-derived gene clusters led to the production of hydroxysporine (1) and reductasporine (2), two bioactive TDs. As suggested by our phylogenetic analysis of CPAS genes, identified in our survey of crude eDNA extracts, reductasporine (2) contains an unprecedented TD core structure: a pyrrolinium indolocarbazole core that is likely key to its unusual bioactivity profile. This work demonstrates the potential for the discovery of structurally rare and biologically interesting natural products using targeted metagenomics, where environmental samples are prescreened to identify the most phylogenetically unique gene sequences and molecules associated with these genes are accessed through targeted metagenomic library construction and heterologous expression.

Mutations in the proteolipid subunits of the vacuolar H+-ATPase provide resistance to indolotryptoline natural products.

Indolotryptoline natural products represent a small family of structurally unique chromopyrrolic acid-derived antiproliferative agents. Like many prospective anticancer agents before them, the exploration of their potential clinical utility has been hindered by the limited information known about their mechanism of action. To study the mode of action of two closely related indolotryptolines (BE-54017, cladoniamide A), we selected for drug resistant mutants using a multidrug resistance-suppressed (MDR-sup) Schizosaccharomyces pombe strain. As fission yeast maintains many of the basic cancer-relevant cellular processes present in human cells, it represents an appealing model to use in determining the potential molecular target of antiproliferative natural products through resistant mutant screening. Full genome sequencing of resistant mutants identified mutations in the c and c' subunits of the proteolipid substructure of the vacuolar H(+)-ATPase complex (V-ATPase). This collection of resistance-conferring mutations maps to a site that is distant from the nucleotide-binding sites of V-ATPase and distinct from sites found to confer resistance to known V-ATPase inhibitors. Acid vacuole staining, cross-resistance studies, and direct c/c' subunit mutagenesis all suggest that indolotryptolines are likely a structurally novel class of V-ATPase inhibitors. This work demonstrates the general utility of resistant mutant selection using MDR-sup S. pombe as a rapid and potentially systematic approach for studying the modes of action of cytotoxic natural products.

Characterization of an environmental DNA-derived gene cluster that encodes the bisindolylmaleimide methylarcyriarubin.

Bisindolylmaleimides represent a naturally occurring class of metabolites that are of interest because of their protein kinase inhibition activity. From a metagenomic library constructed with soil DNA, we identified the four gene mar cluster, a bisindolylmaleimide gene cluster that encodes for methylarcyriarubin (1) production. Heterologous expression of the mar gene cluster in E. coli revealed that the Rieske dioxygenase MarC facilitates the oxidative decarboxylation of a chromopyrrolic acid (CPA) intermediate to yield the bisindolylmaleimide core. The characterization of the mar cluster defines a new role for CPA in the biosynthesis of structurally diverse bacterial tryptophan dimers.

Investigating the Integration and the Long-Term Use of Smart Speakers in Older Adults' Daily Practices: Qualitative Study.

BACKGROUND: As smart speakers become more popular, there have been an increasing number of studies on how they may benefit older adults or how older adults perceive them. Despite the increasing ownership rates of smart speakers among older adults, studies that examine their integration and the long-term use in older adults' daily practices are scarce. OBJECTIVE: This study aims to uncover the integration of smart speakers into the daily practices of older adults over the long term, contributing to an in-depth understanding of maintained technology use among this demographic. METHODS: To achieve these objectives, the study interviewed 20 older adults who had been using smart speakers for over 6 months. These semistructured interviews enabled participants to share their insights and experiences regarding the maintained use of smart speakers in the long term. RESULTS: We identified 4 dimensions of the long-term use of smart speakers among older adults, including functional integration, spatial integration, cognitive integration, and semantic integration. For the functional integration of smart speakers, the study reported different types of use, including entertainment, information collection, medication reminders, companionship, environment modification, and emergency calls. For the spatial integration of smart speakers, the study showed older adults' agency in defining, changing, and reshaping daily practices through the spatial organization of smart speakers. For the cognitive integration of smart speakers, the findings showed the cognitive processes involved in adapting to and incorporating smart speakers into daily habits and routines. For the semantic integration of smart speakers, the findings revealed that older adults' enjoyable user experience and strong bonds with the device contributed to their acceptance of occasional functional errors. Finally, the study proposed several suggestions for designers and developers to better design smart speakers that promote maintainable use behaviors among older adults. CONCLUSIONS: On the basis of the findings, this study highlighted the importance of understanding how older adults use smart speakers and the practices through which they integrate them into their daily routines. The findings suggest that smart speakers can provide significant benefits for older adults, including increased convenience and improved quality of life. However, to promote maintainable use behaviors, designers and developers should consider more about the technology use contexts and the specific needs and preferences of older adults when designing these devices.

Role of endothelial Raptor in abnormal arteriogenesis after lower limb ischaemia in type 2 diabetes.

AIMS: Proper arteriogenesis after tissue ischaemia is necessary to rebuild stable blood circulation; nevertheless, this process is impaired in type 2 diabetes mellitus (T2DM). Raptor is a scaffold protein and a component of mammalian target of rapamycin complex 1 (mTORC1). However, the role of the endothelial Raptor in arteriogenesis under the conditions of T2DM remains unknown. This study investigated the role of endothelial Raptor in ischaemia-induced arteriogenesis during T2DM. METHODS AND RESULTS: Although endothelial mTORC1 is hyperactive in T2DM, we observed a marked reduction in the expression of endothelial Raptor in two mouse models and in human vessels. Inducible endothelial-specific Raptor knockout severely exacerbated impaired hindlimb perfusion and arteriogenesis after hindlimb ischaemic injury in 12-week high-fat diet fed mice. Additionally, we found that Raptor deficiency dampened vascular endothelial growth factor receptor 2 (VEGFR2) signalling in endothelial cells (ECs) and inhibited VEGF-induced cell migration and tube formation in a PTP1B-dependent manner. Furthermore, mass spectrometry analysis indicated that Raptor interacts with neuropilin 1 (NRP1), the co-receptor of VEGFR2, and mediates VEGFR2 trafficking by facilitating the interaction between NRP1 and Synectin. Finally, we found that EC-specific overexpression of the Raptor mutant (loss of mTOR binding) reversed impaired hindlimb perfusion and arteriogenesis induced by endothelial Raptor knockout in high-fat diet fed mice. CONCLUSION: Collectively, our study demonstrated the crucial role of endothelial Raptor in promoting ischaemia-induced arteriogenesis in T2DM by mediating VEGFR2 signalling. Thus, endothelial Raptor is a novel therapeutic target for promoting arteriogenesis and ameliorating perfusion in T2DM.

Effectiveness of identity-building interventions on recovery identity and patient-reported health outcomes in chronic diseases: A meta-analysis.

This meta-analysis aimed to assess the impact of identity-building interventions on recovery identity and patient-reported health outcomes in chronic disease patients. We identified 15 relevant empirical studies (comprising 2261 patients) from 989 records through extensive keyword searches and manual screening conducted between March 2nd and March 13th, 2023. Utilizing the Cochrane tool, meta-regression, and the GRADE approach, we evaluated these studies for their characteristics, findings, and quality. The analysis revealed that identity-building interventions, encompassing recovery-oriented group, interest group, and linguistic approaches, positively influenced identity synthesis and had varying effects on health outcomes. Notably, multiple regression analysis demonstrated that identity synthesis significantly predicted health outcomes. However, the study identified mild heterogeneity, a high attrition bias risk, and insufficient data on selection and detection bias as limitations. Overall, identity-building interventions proved influential in enhancing recovery identity, a vital predictor of patient-reported health outcomes in chronic disease patients.

DMLS: an automated pipeline to extract the Drosophila modular transcription regulators and targets from massive literature articles.

Transcription regulation in multicellular species is mediated by modular transcription factor (TF) binding site combinations termed cis-regulatory modules (CRMs). Such CRM-mediated transcription regulation determines the gene expression patterns during development. Biologists frequently investigate CRM transcription regulation on gene expressions. However, the knowledge of the target genes and regulatory TFs participating in the CRMs under study is mostly fragmentary throughout the literature. Researchers need to afford tremendous human resources to fully surf through the articles deposited in biomedical literature databases in order to obtain the information. Although several novel text-mining systems are now available for literature triaging, these tools do not specifically focus on CRM-related literature prescreening, failing to correctly extract the information of the CRM target genes and regulatory TFs from the literature. For this reason, we constructed a supportive auto-literature prescreener called Drosophila Modular transcription-regulation Literature Screener (DMLS) that achieves the following: (i) prescreens articles describing experiments on modular transcription regulation, (ii) identifies the described target genes and TFs of the CRMs under study for each modular transcription-regulation-describing article and (iii) features an automated and extendable pipeline to perform the task. We demonstrated that the final performance of DMLS in extracting the described target gene and regulatory TF lists of CRMs under study for given articles achieved test macro area under the ROC curve (auROC) = 89.7% and area under the precision-recall curve (auPRC) = 77.6%, outperforming the intuitive gene name-occurrence-counting method by at least 19.9% in auROC and 30.5% in auPRC. The web service and the command line versions of DMLS are available at https://cobis.bme.ncku.edu.tw/DMLS/  and  https://github.com/cobisLab/DMLS/, respectively. Database Tool URL: https://cobis.bme.ncku.edu.tw/DMLS/.

Non-invasive detection of lymphoma with circulating tumor DNA features and protein tumor markers.

Background: According to GLOBOCAN 2020, lymphoma ranked as the 9th most common cancer and the 12th leading cause of cancer-related deaths worldwide. Traditional diagnostic methods rely on the invasive excisional lymph node biopsy, which is an invasive approach with some limitations. Most lymphoma patients are diagnosed at an advanced stage since they are asymptomatic at the beginning, which has significantly impacted treatment efficacy and prognosis of the disease. Method: This study assessed the performance and utility of a newly developed blood-based assay (SeekInCare) for lymphoma early detection. SeekInCare utilized protein tumor markers and a comprehensive set of cancer-associated genomic features, including copy number aberration (CNA), fragment size (FS), end motif, and lymphoma-related virus, which were profiled by shallow WGS of cfDNA. Results: Protein marker CA125 could be used for lymphoma detection independent of gender, and the sensitivity was 27.8% at specificity of 98.0%. After integrating these multi-dimensional features, 77.8% sensitivity was achieved at specificity of 98.0%, while its NPV and PPV were both more than 92% for lymphoma detection. The sensitivity of early-stage (I-II) lymphoma was up to 51.3% (47.4% and 55.0% for stage I and II respectively). After 2 cycles of treatment, the molecular response of SeekInCare was correlated with the clinical outcome. Conclusion: In summary, a blood-based assay can be an alternative to detect lymphoma with adequate performance. This approach becomes particularly valuable in cases where obtaining tissue biopsy is difficult to obtain or inconclusive.

Discovery and synthetic refactoring of tryptophan dimer gene clusters from the environment.

Here we investigate bacterial tryptophan dimer (TD) biosynthesis by probing environmental DNA (eDNA) libraries for chromopyrrolic acid (CPA) synthase genes. Functional and bioinformatics analyses of TD clusters indicate that CPA synthase gene sequences diverge in concert with the functional output of their respective clusters, making this gene a powerful tool for guiding the discovery of novel TDs from the environment. Twelve unprecedented TD biosynthetic gene clusters that can be arranged into five groups (A-E) based on their ability to generate distinct TD core substructures were recovered from eDNA libraries. Four of these groups contain clusters from both cultured and culture independent studies, while the remaining group consists entirely of eDNA-derived clusters. The complete synthetic refactoring of a representative gene cluster from the latter eDNA specific group led to the characterization of the erdasporines, cytotoxins with a novel carboxy-indolocarbazole TD substructure. Analysis of CPA synthase genes in crude eDNA suggests the presence of additional TD gene clusters in soil environments.