Engineering Streptomyces sp. CPCC 204095 for the targeted high-level production of isatropolone A by elucidating its pathway-specific regulatory mechanism.

BACKGROUND: Isatropolone A and C, produced by Streptomyces sp. CPCC 204095, belong to an unusual class of non-benzenoid aromatic compounds and contain a rare seven-membered ring structure. Isatropolone A exhibits potent activity against Leishmania donovani, comparable to the only oral drug miltefosine. However, its variably low productivity represents a limitation for this lead compound in the future development of new anti-leishmaniasis drugs to meet unmet clinical needs. RESULTS: Here we first elucidated the regulatory cascade of biosynthesis of isatropolones, which consists of two SARP family regulators, IsaF and IsaJ. Through a series of in vivo and in vitro experiments, IsaF was identified as a pathway-specific activator that orchestrates the transcription of the gene cluster essential for isatropolone biosynthesis. Interestingly, IsaJ was found to only upregulate the expression of the cytochrome P450 monooxygenase IsaS, which is crucial for the yield and proportion of isatropolone A and C. Through targeted gene deletions of isaJ or isaS, we effectively impeded the conversion of isatropolone A to C. Concurrently, the facilitation of isaF overexpression governed by selected promoters, prompted the comprehensive activation of the production of isatropolone A. Furthermore, meticulous optimization of the fermentation parameters was conducted. These strategies culminated in the attainment of an unprecedented maximum yield-980.8 mg/L of isatropolone A-achieved in small-scale solid-state fermentation utilizing the genetically modified strains, thereby establishing the highest reported titer to date. CONCLUSION: In Streptomyces sp. CPCC 204095, the production of isatropolone A and C is modulated by the SARP regulators IsaF and IsaJ. IsaF serves as a master pathway-specific regulator for the production of isatropolones. IsaJ, on the other hand, only dictates the transcription of IsaS, the enzyme responsible for the conversion of isatropolone A and C. By engineering the expression of these pivotal genes, we have devised a strategy for genetic modification aimed at the selective and high-yield biosynthesis of isatropolone A. This study not only unveils the unique regulatory mechanisms governing isatropolone biosynthesis for the first time, but also establishes an essential engineering framework for the targeted high-level production of isatropolone A.

Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications.

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

Tomato root specialized metabolites evolved through gene duplication and regulatory divergence within a biosynthetic gene cluster.

Tremendous plant metabolic diversity arises from phylogenetically restricted specialized metabolic pathways. Specialized metabolites are synthesized in dedicated cells or tissues, with pathway genes sometimes colocalizing in biosynthetic gene clusters (BGCs). However, the mechanisms by which spatial expression patterns arise and the role of BGCs in pathway evolution remain underappreciated. In this study, we investigated the mechanisms driving acylsugar evolution in the Solanaceae. Previously thought to be restricted to glandular trichomes, acylsugars were recently found in cultivated tomato roots. We demonstrated that acylsugars in cultivated tomato roots and trichomes have different sugar cores, identified root-enriched paralogs of trichome acylsugar pathway genes, and characterized a key paralog required for root acylsugar biosynthesis, SlASAT1-LIKE (SlASAT1-L), which is nested within a previously reported trichome acylsugar BGC. Last, we provided evidence that ASAT1-L arose through duplication of its paralog, ASAT1, and was trichome-expressed before acquiring root-specific expression in the Solanum genus. Our results illuminate the genomic context and molecular mechanisms underpinning metabolic diversity in plants.

Biosynthetic pathway redesign in non-conventional yeast for enhanced production of cembratriene-ol.

Cembratriene-ol (CBT-ol), a plant-derived macrocyclic diterpene with notable insecticidal activity, has attracted considerable attention with respect to the development of sustainable and green biopesticides. Currently, CBT-ol production is limited by an inefficient and costly plant extraction strategy. Herein, CBT-ol production was enhanced by redesigning the CBT-ol biosynthetic pathway in Candida tropicalis, with subsequent truncation of CBT-ol synthase further increasing CBT-ol production. Moreover, bottlenecks in the CBT-ol biosynthetic pathway were eliminated by adjusting the gene dosage of the rate-limiting enzymes. Ultimately, the resulting strain C. tropicalis CPPt-03D produced 129.17 mg/L CBT-ol in shaking flasks (a 144-fold increase relative to that of the initial strain C01-CD) with CBT-ol production reaching 1,425.76 mg/L in a 5-L bioreactor, representing the highest CBT-ol titer reported to date. These findings provide a green process and promising platform for the industrial production of CBT-ol and lays the foundation for organic farming.

Plant Engineering to Enable Platforms for Sustainable Bioproduction of Terpenoids.

Terpenoids represent the most diverse class of natural products, with a broad spectrum of industrial relevance including applications in green solvents, flavors and fragrances, nutraceuticals, colorants, and therapeutics. They are typically challenging to extract from their natural sources, where they occur in small amounts and mixtures of related but unwanted byproducts. Formal chemical synthesis, where established, is reliant on petrochemistry. Hence, there is great interest in developing sustainable solutions to assemble biosynthetic pathways in engineered host organisms. Metabolic engineering for chemical production has largely focused on microbial hosts, yet plants offer a sustainable production platform. In addition to containing the precursor pathways that generate the terpenoid building blocks as well as the cell structures and compartments required, or tractable localization for the enzymes involved, plants may provide a low input system to produce these chemicals using carbon dioxide and sunlight only. There have been significant recent advancements in the discovery of pathways to terpenoids of interest as well as strategies to boost yields in host plants. While part of the phytochemical field is focusing on the discovery of biosynthetic pathways, this review will focus on advancements using the pathway toolbox and toward engineering plants for the production of terpenoids. We will highlight strategies currently used to produce target products, optimization of known pathways to improve yields, compartmentalization of pathways within cells, and genetic tools developed to facilitate complex engineering of biosynthetic pathways. These advancements in Synthetic Biology are bringing engineered plant systems closer to commercially relevant hosts for the bioproduction of terpenoids.

Non-ribosomal peptide synthetase (NRPS)-encoding products and their biosynthetic logics in Fusarium.

Fungal non-ribosomal peptide synthetase (NRPS)-encoding products play a paramount role in new drug discovery. Fusarium, one of the most common filamentous fungi, is well-known for its biosynthetic potential of NRPS-type compounds with diverse structural motifs and various biological properties. With the continuous improvement and extensive application of bioinformatic tools (e.g., anti-SMASH, NCBI, UniProt), more and more biosynthetic gene clusters (BGCs) of secondary metabolites (SMs) have been identified in Fusarium strains. However, the biosynthetic logics of these SMs have not yet been well investigated till now. With the aim to increase our knowledge of the biosynthetic logics of NPRS-encoding products in Fusarium, this review firstly provides an overview of research advances in elucidating their biosynthetic pathways.

Employing synthetic biology to expand antibiotic discovery.

Antimicrobial-resistant (AMR) bacterial pathogens are a continually growing threat as our methods for combating these infections continue to be overcome by the evolution of resistance mechanisms. Recent therapeutic methods have not staved off the concern of AMR infections, so continued research focuses on new ways of identifying small molecules to treat AMR pathogens. While chemical modification of existing antibiotics is possible, there has been rapid development of resistance by pathogens that were initially susceptible to these compounds. Synthetic biology is becoming a key strategy in trying to predict and induce novel, natural antibiotics. Advances in cloning and mutagenesis techniques applied through a synthetic biology lens can help characterize the native regulation of antibiotic biosynthetic gene clusters (BGCs) to identify potential modifications leading to more potent antibiotic activity. Additionally, many cryptic antibiotic BGCs are derived from non-ribosomal peptide synthase (NRPS) and polyketide synthase (PKS) biosynthetic pathways; complex, clustered genetic sequences that give rise to amino acid-derived natural products. Synthetic biology can be applied to modify and metabolically engineer these enzyme-based systems to promote rapid and sustainable production of natural products and their variants. This review will focus on recent advances related to synthetic biology as applied to genetic pathway characterization and identification of antibiotics from naturally occurring BGCs. Specifically, we will summarize recent efforts to characterize BGCs via general genomic mutagenesis, endogenous gene expression, and heterologous gene expression.

A self-regulated network for dynamically balancing multiple precursors in complex biosynthetic pathways.

Microbial synthesis has emerged as a promising and sustainable alternative to traditional chemical synthesis and plant extraction. However, the competition between synthetic pathways and central metabolic pathways for cellular resources may impair final production efficiency. Moreover, when the synthesis of target product requires multiple precursors from the same node, the conflicts of carbon flux have further negative impacts on yields. In this study, a self-regulated network was developed to relieve the competition of precursors in complex synthetic pathways. Using 4-hydroxycoumarin (4-HC) synthetic pathway as a proof of concept, we employed an intermediate as a trigger to dynamically rewire the metabolic flux of pyruvate and control the expression levels of genes in 4-HC synthetic pathway, achieving self-regulation of multiple precursors and enhanced titer. Transcriptomic analysis results additionally demonstrated that the gene transcriptional levels of both pyruvate kinase PykF and synthetic pathway enzyme SdgA dynamically changed according to the intermediate concentrations. Overall, our work established a self-regulated network to dynamically balance the metabolic flux of two precursors in 4-HC biosynthesis, providing insight into balancing biosynthetic pathways where multiple precursors compete and interfere with each other.

Combinatorial optimization of gene expression through recombinase-mediated promoter and terminator shuffling in yeast.

Microbes are increasingly employed as cell factories to produce biomolecules. This often involves the expression of complex heterologous biosynthesis pathways in host strains. Achieving maximal product yields and avoiding build-up of (toxic) intermediates requires balanced expression of every pathway gene. However, despite progress in metabolic modeling, the optimization of gene expression still heavily relies on trial-and-error. Here, we report an approach for in vivo, multiplexed Gene Expression Modification by LoxPsym-Cre Recombination (GEMbLeR). GEMbLeR exploits orthogonal LoxPsym sites to independently shuffle promoter and terminator modules at distinct genomic loci. This approach facilitates creation of large strain libraries, in which expression of every pathway gene ranges over 120-fold and each strain harbors a unique expression profile. When applied to the biosynthetic pathway of astaxanthin, an industrially relevant antioxidant, a single round of GEMbLeR improved pathway flux and doubled production titers. Together, this shows that GEMbLeR allows rapid and efficient gene expression optimization in heterologous biosynthetic pathways, offering possibilities for enhancing the performance of microbial cell factories.

Promoter engineering of natural product biosynthetic gene clusters in actinomycetes: concepts and applications.

Covering 2011 to 2022Low titers of natural products in laboratory culture or fermentation conditions have been one of the challenging issues in natural products research. Many natural product biosynthetic gene clusters (BGCs) are also transcriptionally silent in laboratory culture conditions, making it challenging to characterize the structures and activities of their metabolites. Promoter engineering offers a potential solution to this problem by providing tools for transcriptional activation or optimization of biosynthetic genes. In this review, we summarize the 10 years of progress in promoter engineering approaches in natural products research focusing on the most metabolically talented group of bacteria actinomycetes.

Global characterization of biosynthetic gene clusters in non-model eukaryotes using domain architectures.

The majority of pharmaceuticals are derived from natural products, bioactive compounds naturally synthesized by organisms to provide evolutionary advantages. Although the rich evolutionary history of eukaryotic algal species implicates a high potential for natural product-based drug discovery, it remains largely untouched. This study investigates 2762 putative biosynthetic gene clusters (BGCs) from 212 eukaryotic algal genomes. To analyze a vast set of structurally diverse BGCs, we employed comparative analysis based on the vectorization of biosynthetic domains, referred to as biosynthetic domain architecture (BDA). By characterizing core biosynthetic machineries through BDA, we identified key BDAs of modular BGCs in diverse eukaryotes and introduced 16 candidate modular BGCs with similar BDAs to previously validated BGCs. This study provides a global characterization of eukaryotic algal BGCs, offering an alternative to laborious manual curation for BGC prioritization.

Predicting fungal secondary metabolite activity from biosynthetic gene cluster data using machine learning.

Fungal secondary metabolites (SMs) contribute to the diversity of fungal ecological communities, niches, and lifestyles. Many fungal SMs have one or more medically and industrially important activities (e.g., antifungal, antibacterial, and antitumor). The genes necessary for fungal SM biosynthesis are typically located right next to each other in the genome and are known as biosynthetic gene clusters (BGCs). However, whether fungal SM bioactivity can be predicted from specific attributes of genes in BGCs remains an open question. We adapted machine learning models that predicted SM bioactivity from bacterial BGC data with accuracies as high as 80% to fungal BGC data. We trained our models to predict the antibacterial, antifungal, and cytotoxic/antitumor bioactivity of fungal SMs on two data sets: (i) fungal BGCs (data set comprised of 314 BGCs) and (ii) fungal (314 BGCs) and bacterial BGCs (1,003 BGCs). We found that models trained on fungal BGCs had balanced accuracies between 51% and 68%, whereas training on bacterial and fungal BGCs had balanced accuracies between 56% and 68%. The low prediction accuracy of fungal SM bioactivities likely stems from the small size of the data set; this lack of data, coupled with our finding that including bacterial BGC data in the training data did not substantially change accuracies currently limits the application of machine learning approaches to fungal SM studies. With >15,000 characterized fungal SMs, millions of putative BGCs in fungal genomes, and increased demand for novel drugs, efforts that systematically link fungal SM bioactivity to BGCs are urgently needed.IMPORTANCEFungi are key sources of natural products and iconic drugs, including penicillin and statins. DNA sequencing has revealed that there are likely millions of biosynthetic pathways in fungal genomes, but the chemical structures and bioactivities of >99% of natural products produced by these pathways remain unknown. We used artificial intelligence to predict the bioactivities of diverse fungal biosynthetic pathways. We found that the accuracies of our predictions were generally low, between 51% and 68%, likely because the natural products and bioactivities of only very few fungal pathways are known. With >15,000 characterized fungal natural products, millions of putative biosynthetic pathways present in fungal genomes, and increased demand for novel drugs, our study suggests that there is an urgent need for efforts that systematically identify fungal biosynthetic pathways, their natural products, and their bioactivities.

Engineered and total biosynthesis of fungal specialized metabolites.

Filamentous fungi produce a very wide range of complex and often bioactive metabolites, demonstrating their inherent ability as hosts of complex biosynthetic pathways. Recent advances in molecular sciences related to fungi have afforded the development of new tools that allow the rational total biosynthesis of highly complex specialized metabolites in a single process. Increasingly, these pathways can also be engineered to produce new metabolites. Engineering can be at the level of gene deletion, gene addition, formation of mixed pathways, engineering of scaffold synthases and engineering of tailoring enzymes. Combination of these approaches with hosts that can metabolize low-value waste streams opens the prospect of one-step syntheses from garbage.

Manipulation and epigenetic control of silent biosynthetic pathways in actinobacteria.

Most biosynthetic gene clusters (BGCs) of Actinobacteria are either silent or expressed less than the detectable level. The non-genetic approaches including biological interactions, chemical agents, and physical stresses that can be used to awaken silenced pathways are compared in this paper. These non-genetic induction strategies often need screening approaches, including one strain many compounds (OSMAC), reporter-guided mutant selection, and high throughput elicitor screening (HiTES) have been developed. Different types of genetic manipulations applied in the induction of cryptic BGCs of Actinobacteria can be categorized as genome-wide pleiotropic and targeted approaches like manipulation of global regulatory systems, modulation of regulatory genes, ribosome and engineering of RNA polymerase or phosphopantheteine transferases. Targeted approaches including genome editing by CRISPR, mutation in transcription factors and modification of BGCs promoters, inactivation of the highly expressed biosynthetic pathways, deleting the suppressors or awakening the activators, heterologous expression, or refactoring of gene clusters can be applied for activation of pathways which are predicted to synthesize new bioactive structures in genome mining studies of Acinobacteria. In this review, the challenges and advantages of employing these approaches in induction of Actinobacteria BGCs are discussed. Further, novel natural products needed as drug for pharmaceutical industry or as biofertilizers in agricultural industry can be discovered even from known species of Actinobactera by the innovative approaches of metabolite biosynthesis elicitation.

Genome Mining of Cinnamoyl-Containing Nonribosomal Peptide Gene Clusters Directs the Production of Malacinnamycin.

Cinnamoyl-containing nonribosomal peptides (CCNPs) constitute a unique family of actinobacterial secondary metabolites that display a broad spectrum of biological activities. Here, we present a genome mining approach targeting cyclase and is isomerase to discover new CCNPs, which led to the identification of 207 putative CCNP gene clusters from public bacterial genome databases. After strain prioritization, a novel class of CCNP-type glycopeptides named malacinnamycin was identified. A plausible biosynthetic pathway for malacinnamycin was deduced by bioinformatics analysis.

Genome-wide identification of the alkaloid synthesis gene family CYP450, gives new insights into alkaloid resource utilization in medicinal Dendrobium.

The medicinal Dendrobium species of Orchidaceae possess significant pharmaceutical value, and modern pharmacological research has shown that Dendrobium contains many important active ingredients. Alkaloids, the crucial components of medicinal Dendrobium, demonstrate beneficial healing properties in cardiovascular, cataract, gastrointestinal, and respiratory diseases. Members of the cytochrome P450 monooxygenase (CYP) gene family play essential roles in alkaloid synthesis, participating in alkaloid terpene skeleton construction and subsequent modifications. Although studies of the CYP family have been conducted in some species, genome-wide characterization and systematic analysis of the CYP family in medicinal Dendrobium remain underexplored. In this study, we identified CYP gene family members in the genomes of four medicinal Dendrobium species recorded in the Pharmacopoeia: D. nobile, D. chrysotoxum, D. catenatum, and D. huoshanense. Further, we analyzed the motif composition, gene replication events, and selection pressure of this family. Syntenic analysis revealed that members of the clan 710 were present on chromosome 18 in three medicinal Dendrobium species, except for D. nobile, indicating a loss of clan 710 occurring in D. nobile. We also conducted an initial screening of the CYP genes involved in alkaloid synthesis through transcriptome sequencing. Quantitative real-time reverse transcription PCR showed that the expression of DnoNew43 and DnoNew50, homologs of secologanin synthase involved in the alkaloid synthesis pathway, was significantly higher in the stems than in the leaves. This result coincided with the distribution of dendrobine content in Dendrobium stems and leaves, indicating that these two genes might be involved in the dendrobine synthesis pathway. Our results give insights into the CYP gene family evolution analysis in four medicinal Dendrobium species for the first time and identify two related genes that may be involved in alkaloid synthesis, providing a valuable resource for further investigations into alkaloid synthesis pathway in Dendrobium and other medicinal plants.

Amycolatopsis mediterranei: A Sixty-Year Journey from Strain Isolation to Unlocking Its Potential of Rifamycin Analogue Production by Combinatorial Biosynthesis.

Ever since the isolation of Amycolatopsis mediterranei in 1957, this strain has been the focus of research worldwide. In the last 60 years or more, our understanding of the taxonomy, development of cloning vectors and conjugation system, physiology, genetics, genomics, and biosynthetic pathway of rifamycin B production in A. mediterranei has substantially increased. In particular, the development of cloning vectors, transformation system, characterization of the rifamycin biosynthetic gene cluster, and the regulation of rifamycin B production by the pioneering work of Heinz Floss have made the rifamycin polyketide biosynthetic gene cluster (PKS) an attractive target for extensive genetic manipulations to produce rifamycin B analogues which could be effective against multi-drug-resistant tuberculosis. Additionally, a better understanding of the regulation of rifamycin B production and the application of newer genomics tools, including CRISPR-assisted genome editing systems, might prove useful to overcome the limitations associated with low production of rifamycin analogues.

The antiSMASH database version 4: additional genomes and BGCs, new sequence-based searches and more.

Many microorganisms produce natural products that are frequently used in the development of medicines and crop protection agents. Genome mining has evolved into a prominent method to access this potential. antiSMASH is the most popular tool for this task. Here we present version 4 of the antiSMASH database, providing biosynthetic gene clusters detected by antiSMASH 7.1 in publicly available, dereplicated, high-quality microbial genomes via an interactive graphical user interface. In version 4, the database contains 231 534 high quality BGC regions from 592 archaeal, 35 726 bacterial and 236 fungal genomes and is available at https://antismash-db.secondarymetabolites.org/.

ABC-HuMi: the Atlas of Biosynthetic Gene Clusters in the Human Microbiome.

The human microbiome has emerged as a rich source of diverse and bioactive natural products, harboring immense potential for therapeutic applications. To facilitate systematic exploration and analysis of its biosynthetic landscape, we present ABC-HuMi: the Atlas of Biosynthetic Gene Clusters (BGCs) in the Human Microbiome. ABC-HuMi integrates data from major human microbiome sequence databases and provides an expansive repository of BGCs compared to the limited coverage offered by existing resources. Employing state-of-the-art BGC prediction and analysis tools, our database ensures accurate annotation and enhanced prediction capabilities. ABC-HuMi empowers researchers with advanced browsing, filtering, and search functionality, enabling efficient exploration of the resource. At present, ABC-HuMi boasts a catalog of 19 218 representative BGCs derived from the human gut, oral, skin, respiratory and urogenital systems. By capturing the intricate biosynthetic potential across diverse human body sites, our database fosters profound insights into the molecular repertoire encoded within the human microbiome and offers a comprehensive resource for the discovery and characterization of novel bioactive compounds. The database is freely accessible at https://www.ccb.uni-saarland.de/abc_humi/.