The Natural Products Discovery Center: Release of the First 8490 Sequenced Strains for Exploring Actinobacteria Biosynthetic Diversity.

Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.

Neogrisemycin, a Trisulfide-Bridged Angucycline, Produced upon Expressing the Thioangucycline Biosynthetic Gene Cluster in Streptomyces albus J1074.

Neogrisemycin (1) was isolated from recombinant Streptomyces albus J1074 strain SB4061 expressing an engineered thioangucycline (TAC) biosynthetic gene cluster (BGC). The structure and absolute configuration of 1 were established by a combination of mass spectrometry, nuclear magnetic resonance, and single-crystal X-ray diffraction analyses. Like the TACs, 1 was also proposed to derive non-enzymatically from the common epoxide (8), the nascent product encoded by the tac BGC, mediated by endogenous hydrogen trisulfide.

Application of a Biocatalytic Strategy for the Preparation of Tiancimycin-Based Antibody-Drug Conjugates Revealing Key Insights into Structure-Activity Relationships.

Antibody-drug conjugates (ADCs) are cancer chemotherapeutics that utilize a monoclonal antibody (mAb)-based delivery system, a cytotoxic payload, and a chemical linker. ADC payloads must be strategically functionalized to allow linker attachment without perturbing the potency required for ADC efficacy. We previously developed a biocatalytic system for the precise functionalization of tiancimycin (TNM)-based payloads. The TNMs are anthraquinone-fused enediynes (AFEs) and have yet to be translated into the clinic. Herein, we report the translation of biocatalytically functionalized TNMs into ADCs in combination with the dual-variable domain (DVD)-mAb platform. The DVD enables both site-specific conjugation and a plug-and-play modularity for antigen-targeting specificity. We evaluated three linker chemistries in terms of TNM-based ADC potency and antigen selectivity, demonstrating a trade-off between potency and selectivity. This represents the first application of AFE-based payloads to DVDs for ADC development, a workflow that is generalizable to further advance AFE-based ADCs for multiple cancer types.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/ß-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/ß-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

Functional Characterization of Cytochrome P450 Hydroxylase YpmL in Yangpumicin A Biosynthesis and Its Application for Anthraquinone-Fused Enediyne Structural Diversification.

Comparative analyses of four anthraquinone-fused enediyne biosynthetic gene clusters (BGCs) identified YpmL as a cytochrome P450 enzyme unique to the yangpumicin (YPM) BGC. In vitro characterization of YpmL established it as a hydroxylase, catalyzing C-6 hydroxylation in YPM A biosynthesis. In vivo application of YpmL enabled engineered production of four new tiancimycin analogues (14-17). Evaluation of their cytotoxicity against selected human cancer cell lines shed new insights into the enediyne structure-activity relationship.

Cytochrome P450 Hydroxylase TnmL Catalyzing Sequential Hydroxylation with an Additional Proofreading Activity in Tiancimycin Biosynthesis.

Tiancimycin (TNM) A belongs to the anthraquinone-fused subfamily of enediyne natural products, and selected enediynes have been translated into clinical drugs. Previously, inactivation of tnmL in Streptomyces sp. CB03234 resulted in the accumulation of TNM B and TNM E, supporting the functional assignment of TnmL as a cytochrome P450 hydroxylase that catalyzes A-ring modification in TNM A biosynthesis. Herein, we report in vitro characterization of TnmL, revealing that (i) TnmL catalyzes two successive hydroxylations of TNM E, resulting in sequential production of TNM F and TNM C, (ii) TnmL shows a strict substrate preference, with the C-26 side chain playing a critical role in substrate binding, and (iii) TnmL demethylates the C-7 OCH3 group of TNM G, affording TNM F, thereby channeling the shunt product TNM G back into TNM A biosynthesis and representing a rare proofreading logic for natural product biosynthesis. These findings shed new insights into anthraquinone-fused enediyne biosynthesis.

Discovery of ammosesters by mining the Streptomyces uncialis DCA2648 genome revealing new insight into ammosamide biosynthesis.

The ammosamides (AMMs) are a family of pyrroloquinoline alkaloids that exhibits a wide variety of bioactivities. A biosynthetic gene cluster (BGC) that is highly homologous in both gene content and genetic organization to the amm BGC was identified by mining the Streptomyces uncialis DCA2648 genome, leading to the discovery of a sub-family of new AMM congeners, named ammosesters (AMEs). The AMEs feature a C-4a methyl ester, differing from the C-4a amide functional group characteristic to AMMs, and exhibit modest cytotoxicity against a broad spectrum of human cancer cell lines, expanding the structure-activity relationship for the pyrroloquinoline family of natural products. Comparative analysis of the ame and amm BGCs supports the use of a scaffold peptide as an emerging paradigm for the biosynthesis of the pyrroloquinoline family of natural products. AME and AMM biosynthesis diverges from a common intermediate by evolving the pathway-specific Ame24 O-methyltransferase and Amm20 amide synthetase, respectively. These findings will surely inspire future efforts to mimic Nature's combinatorial biosynthetic strategies for natural product structural diversity.

Submerged fermentation of Streptomyces uncialis providing a biotechnology platform for uncialamycin biosynthesis, engineering, and production.

Uncialamycin (UCM) belongs to the anthraquinone-fused subfamily of 10-membered enediyne natural products that exhibits an extraordinary cytotoxicity against a wide spectrum of human cancer cell lines. Antibody-drug conjugates, utilizing synthetic analogues of UCM as payloads, are in preclinical development. UCM is exclusively produced by Streptomyces uncialis DCA2648 on solid agar medium with low titers (∼0.019 mg/l), limiting its supply by microbial fermentation and hampering its biosynthetic and engineering studies by in vivo pathway manipulation. Here, we report cultivation conditions that enable genetic manipulation of UCM biosynthesis in vivo and allow UCM production, with improved titers, by submerged fermentation of the engineered S. uncialis strains. Specifically, the titer of UCM was improved nearly 58-fold to ∼1.1 mg/l through the combination of deletion of biosynthetic gene clusters encoding unrelated metabolites from the S. uncialis wild-type, chemical mutagenesis and manipulation of pathway-specific regulators to generate the engineered S. uncialis strains, and finally medium optimization of the latter for UCM production. Genetic manipulation of UCM biosynthesis was demonstrated by inactivating selected genes in the engineered S. uncialis strains, one of which afforded a mutant strain accumulating tiancimycin B, a common biosynthetic intermediate known for the anthraquinone-fused subfamily of enediyne natural products. These findings highlight a biotechnology platform for UCM biosynthesis, engineering, and production that should facilitate both its fundamental studies and translational applications.

Challenges and Opportunities to Develop Enediyne Natural Products as Payloads for Antibody-Drug Conjugates.

Calicheamicin, the payload of the antibody-drug-conjugates (ADCs) gemtuzumab ozogamicin (Mylotarg®) and inotuzumab ozogamicin (Besponsa®), belongs to the class of enediyne natural products. Since the isolation and structural determination of the neocarzinostatin chromophore in 1985, the enediynes have attracted considerable attention for their value as DNA damaging agents in cancer chemotherapy. Due to their non-discriminatory cytotoxicity towards both cancer and healthy cells, the clinical utilization of enediyne natural products relies on conjugation to an appropriate delivery system, such as an antibody. Here we review the current landscape of enediynes as payloads of first-generation and next-generation ADCs.

Cryptic Sulfur Incorporation in Thioangucycline Biosynthesis.

Sulfur incorporation into natural products is a critical area of biosynthetic studies. Recently, a subset of sulfur-containing angucyclines has been discovered, and yet, the sulfur incorporation step is poorly understood. In this work, a series of thioether-bridged angucyclines were discovered, and a cryptic epoxide Michael acceptor intermediate was revealed en route to thioangucyclines (TACs) A and B. However, systematic gene deletion of the biosynthetic gene cluster (BGC) by CRISPR/Cas9 could not identify any gene responsible for the conversion of the epoxide intermediate to TACs. Instead, a series of in vitro and in vivo experiments conclusively showed that the conversion is the result of two non-enzymatic steps, possibly mediated by endogenous hydrogen sulfide. Therefore, the TACs are proposed to derive from a detoxification process. These results are expected to contribute to the study of both angucyclines and the utilization of inorganic sulfur in natural product biosynthesis.

Characterization of TnmH as an O-Methyltransferase Revealing Insights into Tiancimycin Biosynthesis and Enabling a Biocatalytic Strategy To Prepare Antibody-Tiancimycin Conjugates.

The enediynes are among the most cytotoxic molecules known, and their use as anticancer drugs has been successfully demonstrated by targeted delivery. Clinical advancement of the anthraquinone-fused enediynes has been hindered by their low titers and lack of functional groups to enable the preparation of antibody-drug conjugates (ADCs). Here we report biochemical and structural characterization of TnmH from the tiancimycin (TNM) biosynthetic pathway, revealing that (i) TnmH catalyzes regiospecific methylation at the C-7 hydroxyl group, (ii) TnmH exhibits broad substrate promiscuity toward hydroxyanthraquinones and S-alkylated SAM analogues and catalyzes efficient installation of reactive alkyl handles, (iii) the X-ray crystal structure of TnmH provides the molecular basis to account for its broad substrate promiscuity, and (iv) TnmH as a biocatalyst enables the development of novel conjugation strategies to prepare antibody-TNM conjugates. These findings should greatly facilitate the construction and evaluation of antibody-TNM conjugates as next-generation ADCs for targeted chemotherapy.

Challenges and opportunities for natural product discovery, production, and engineering in native producers versus heterologous hosts.

Recent advances and emerging technologies for metabolic pathway engineering and synthetic biology have transformed the field of natural product discovery, production, and engineering. Despite these advancements, there remain many challenges in understanding how biosynthetic gene clusters are silenced or activated, including changes in the transcription of key biosynthetic and regulatory genes. This knowledge gap is highlighted by the success and failed attempts of manipulating regulatory genes within biosynthetic gene clusters in both native producers and heterologous hosts. These complexities make the choice of native producers versus heterologous hosts, fermentation medium, and supply of precursors crucial factors in achieving the production of the target natural products and engineering designer analogs. Nature continues to serve as inspiration for filling the knowledge gaps and developing new research strategies. By exploiting the evolutionary power of nature, alternative producers, with the desired genetic amenability and higher titers of the target natural products, and new strains, harboring gene clusters that encode evolutionary optimized congeners of the targeted natural product scaffolds, can be discovered. These newly identified strains can serve as an outstanding biotechnology platform for the engineered production of sufficient quantities of the target natural products and their analogs, enabling biosynthetic studies and potential therapeutic applications. These challenges and opportunities are showcased herein using fredericamycin, iso-migrastatin, platencin and platensimycin, the enediynes of C-1027, tiancimycin, and yangpumicin, and the leinamycin family of natural products.

Comparative Studies of the Biosynthetic Gene Clusters for Anthraquinone-Fused Enediynes Shedding Light into the Tailoring Steps of Tiancimycin Biosynthesis.

Comparative analyses of the four known anthraquinone-fused enediynes biosynthetic gene clusters identified four genes, tnmE6, tnmH, tnmL, and tnmQ, unique to the tnm gene cluster. Larger scale fermentation of both the S. sp. CB03234 wild-type and the Δ tnmH and Δ tnmL mutant strains resulted in the characterization of 20 new tiancimycin (TNM) congeners, including five enediynes. These findings enabled a proposal for the late stage of TNM biosynthesis featuring an intermediate possibly common for all anthraquinone-fused enediynes.

Genome Mining of Micromonospora yangpuensis DSM 45577 as a Producer of an Anthraquinone-Fused Enediyne.

A new anthraquinone-fused enediyne, yangpumicin A (YPM A, 1), along with four Bergman cyclization congeners (YPM B-E, 2-5), was isolated from Micromonospora yangpuensis DSM 45577 after mining enediyne biosynthetic gene clusters from public actinobacterial genome databases and prioritizing the hits by an enediyne genome neighborhood network analysis for discovery. YPM A is potent against a broad spectrum of human cancer cell lines. The discovery of 1 provides new opportunities for the functionalization of enediynes to develop new conjugation chemistries for antibody-drug conjugates.