DNA Interaction and Cleavage Modes of Anthraquinone-Fused Enediynes: A Study on Tiancimycins, Yangpumicins, and Their Semisynthetic Analogues.

Dynemicin A has been the sole prototypical anthraquinone-fused enediyne (AFE) explored since its discovery in 1989. This study investigates the distinct DNA binding and cleavage mechanisms of emerging AFEs, represented by tiancimycins and yangpumicins, along with semisynthetic analogues. Our findings reveal their potent cytotoxicity against various tumor cell lines, while 18-methoxy tiancimycin A treatment could significantly suppress breast tumor growth with minimal toxicity. One of the most potent AFEs, i.e., tiancimycin A, preferentially targets DNA sequences 5'-ATT, 5'-CTT, 5'-GAA, 5'-GAT, and 5'-TTA. Molecular dynamics simulations suggest that emerging AFEs intercalate deeper into AT-rich DNA base pairs compared to dynemicin A. Importantly, tiancimycin A may equilibrate between insertional and intercalative modes without deintercalation, enabling selective cleavage of T and A bases. This study underscores how subtle structural variations among AFEs significantly influence their DNA recognition and cleavage, facilitating future design of novel AFEs as potent and selective payloads for antibody-drug conjugates.

Anti-HER2 Immunoliposomes: Antitumor Efficacy Attributable to Targeted Delivery of Anthraquinone-Fused Enediyne.

Although natural products are essential sources of small-molecule antitumor drugs, some can exert substantial toxicities, limiting their clinical utility. Anthraquinone-fused enediyne natural products are remarkably potent antitumor drug candidates, and uncialamycin and tiancimycin (TNM) A are under development as antibody-drug conjugates. Herein, a novel drug delivery system is introduced for TNM A using anti-human epidermal growth factor receptor 2 (HER2) immunoliposomes (ILs). Trastuzumab-coated TNM A-loaded ILs (HER2-TNM A-ILs) is engineered with an average particle size of 182.8 ± 2.1 nm and a zeta potential of 1.75 ± 0.12 mV. Compared with liposomes lacking trastuzumab, HER2-TNM A-ILs exhibited selective toxicity against HER2-positive KPL-4 and SKBR3 cells. Coumarin-6, a fluorescent TNM A surrogate, is encapsulated within anti-HER2 ILs; the resultant ILs have enhanced cellular uptake in KPL-4 and SKBR3 cells when compared with control liposomes. Furthermore, ILs loaded with more Cy5.5 accumulated in KPL-4 mouse tumors. A single HER2-TNM A-IL dose (0.02 mg kg-1) suppressed the growth of HER2-positive KPL-4 mouse tumors without apparent toxicity. This study not only provides a straightforward method for the effective delivery of TNM A against HER2-positive breast tumors but also underscores the potential of IL-based drug delivery systems when employing highly potent cytotoxins as payloads.

Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

Jejucarbosides B-E, Chlorinated Cycloaromatized Enediynes, from a Marine Streptomyces sp.

Four new chlorinated cycloaromatized enediyne compounds, jejucarbosides B-E (1-4), were discovered together with previously-identified jejucarboside A from a marine actinomycete strain. Compounds 1-4 were identified as new chlorinated cyclopenta[a]indene glycosides based on 1D and 2D nuclear magnetic resonance, high-resolution mass spectrometry, and circular dichroism (CD) spectra. Jejucarbosides B and E bear a carbonate functional group whereas jejucarbosides C and D are variants possessing 1,2-diol by losing the carbonate functionality. It is proposed that the production of 1-4 occurs via Bergman cycloaromatization capturing Cl- and H+ in the alternative positions of a p-benzyne intermediate derived from a 9-membered enediyne core. Jejucarboside E (4) displayed significant cytotoxicity against human cancer cell lines including SNU-638, SK-HEP-1, A549, HCT116, and MDA-MB-231, with IC50 values of 0.31, 0.40, 0.25, 0.29, and 0.48 µM, respectively, while jejucarbosides B-D (1-3) showed moderate or no cytotoxic effects.

Normal and Aberrant Methyltransferase Activities Give Insights into the Final Steps of Dynemicin A Biosynthesis.

The naturally occurring enediynes are notable for their complex structures, potent DNA cleaving ability, and emerging usefulness in cancer chemotherapy. They can be classified into three distinct structural families, but all are thought to originate from a common linear C15-heptaene. Dynemicin A (DYN) is the paradigm member of anthraquinone-fused enediynes, one of the three main classes and exceptional among them for derivation of both its enediyne and anthraquinone portions from this same early biosynthetic building block. Evidence is growing about how two structurally dissimilar, but biosynthetically related, intermediates combine in two heterodimerization reactions to create a nitrogen-containing C30-coupled product. We report here deletions of two genes that encode biosynthetic proteins that are annotated as S-adenosylmethionine (SAM)-dependent methyltransferases. While one, DynO6, is indeed the required O-methyltransferase implicated long ago in the first studies of DYN biosynthesis, the other, DynA5, functions in an unanticipated manner in the post-heterodimerization events that complete the biosynthesis of DYN. Despite its removal from the genome of Micromonospora chersina, the ΔdynA5 strain retains the ability to synthesize DYN, albeit in reduced titers, accompanied by two unusual co-metabolites. We link the appearance of these unexpected structures to a substantial and contradictory body of other recent experimental data to advance a biogenetic rationale for the downstream steps that lead to the final formation of DYN. A sequence of product-forming transformations that is in line with new and existing experimental results is proposed and supported by a model reaction that also encompasses the formation of the crucial epoxide essential for the activation of DYN for DNA cleavage.

A discrete intermediate for the biosynthesis of both the enediyne core and the anthraquinone moiety of enediyne natural products.

The enediynes are structurally characterized by a 1,5-diyne-3-ene motif within a 9- or 10-membered enediyne core. The anthraquinone-fused enediynes (AFEs) are a subclass of 10-membered enediynes that contain an anthraquinone moiety fused to the enediyne core as exemplified by dynemicins and tiancimycins. A conserved iterative type I polyketide synthase (PKSE) is known to initiate the biosynthesis of all enediyne cores, and evidence has recently been reported to suggest that the anthraquinone moiety also originates from the PKSE product. However, the identity of the PKSE product that is converted to the enediyne core or anthraquinone moiety has not been established. Here, we report the utilization of recombinant E. coli coexpressing various combinations of genes that encode a PKSE and a thioesterase (TE) from either 9- or 10-membered enediyne biosynthetic gene clusters to chemically complement ΔPKSE mutant strains of the producers of dynemicins and tiancimycins. Additionally, 13C-labeling experiments were performed to track the fate of the PKSE/TE product in the ΔPKSE mutants. These studies reveal that 1,3,5,7,9,11,13-pentadecaheptaene is the nascent, discrete product of the PKSE/TE that is converted to the enediyne core. Furthermore, a second molecule of 1,3,5,7,9,11,13-pentadecaheptaene is demonstrated to serve as the precursor of the anthraquinone moiety. The results establish a unified biosynthetic paradigm for AFEs, solidify an unprecedented biosynthetic logic for aromatic polyketides, and have implications for the biosynthesis of not only AFEs but all enediynes.

Examining Heterodimerization by Aryl C-N Coupling in Dynemicin Biosynthesis.

Distinct among the enediyne antitumor antibiotics, the dynemicin subgroup is comprised of two discrete halves, an enediyne and an anthraquinone, but each is ultimately derived from the same linear ß-hydroxyhexaene precursor. The linkage of these two halves by an aryl C-N bond is examined here using a variety of experimental approaches. We demonstrate that this heterodimerization is specific for anthracenyl iodide as the corresponding bromo- and amino-substituted anthracenes do not support dynemicin biosynthesis. Furthermore, biochemical experiments and chemical model reactions support an SRN1 mechanism for the aryl C-N coupling in which electron transfer occurs to the iodoanthracene, followed by loss of an anthracenyl iodide and partition of the resulting aryl radical between C-N coupling and reduction by hydrogen abstraction. An enzyme pull-down experiment aiming to capture the protein(s) involved in the coupling reaction is described in which two proteins, Orf14 and Orf16, encoded by the dynemicin biosynthetic gene cluster, are specifically isolated. Deletion of orf14 from the genome abolished dynemicin production accompanied by a 3-fold increased accumulation of the iodoanthracene coupling partner, indicating the plausible involvement of this protein in the heterodimerization process. On the other hand, the deletion of orf16 only reduced dynemicin production to 55%, implying a noncatalytic, auxiliary role of the protein. Structural comparisons using AlphaFold imply key similarities between Orf14 and X-ray crystal structures of several proteins from enediyne BGCs believed to bind hydrophobic polyene or enediyne motifs suggest Orf14 templates aryl C-N bond formation during the central heterodimerization in dynemicin biosynthesis.

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.

Targeted Discovery of an Enediyne-Derived Cycloaromatized Compound, Jejucarboside A, from a Marine Actinomycete.

A genomic and spectroscopic signature-based search revealed a cycloaromatized enediyne, jejucarboside A (1), from a marine actinomycete strain. The structure of 1 was determined as a new cyclopenta[a]indene glycoside bearing carbonate functionality by nuclear magnetic resonance, high-resolution mass spectrometry (MS), MS/MS, infrared spectroscopy, and a modified Mosher's method. An iterative enediyne synthase pathway has been proposed for the putative biosynthesis of 1 by genomic analysis. Jejucarboside A exhibited cytotoxicity against the HCT116 colon carcinoma cells.

Deciphering the pathway-specific regulatory network for production of ten-membered enediyne Tiancimycins in Streptomyces sp. CB03234-S.

BACKGROUND: The anthraquinone-fused 10-membered enediynes (AFEs), represented by tiancimycins (TNMs), possess a unique structural feature and promising potentials as payloads of antitumor antibody-drug conjugates. Despite many efforts, the insufficient yields remain a practical challenge for development of AFEs. Recent studies have suggested a unified basic biosynthetic route for AFEs, those core genes involved in the formation of essential common AFE intermediates, together with multiple regulatory genes, are highly conserved among the reported biosynthetic gene clusters (BGCs) of AFEs. The extreme cytotoxicities of AFEs have compelled hosts to evolve strict regulations to control their productions, but the exact roles of related regulatory genes are still uncertain. RESULTS: In this study, the genetic validations of five putative regulatory genes present in the BGC of TNMs revealed that only three (tnmR1, tnmR3 and tnmR7) of them were involved in the regulation of TNMs biosynthesis. The bioinformatic analysis also revealed that they represented three major but distinct groups of regulatory genes conserved in all BGCs of AFEs. Further transcriptional analyses suggested that TnmR7 could promote the expressions of core enzymes TnmD/G and TnmN/O/P, while TnmR3 may act as a sensor kinase to work with TnmR1 and form a higher class unconventional orphan two-component regulatory system, which dynamically represses the expressions of TnmR7, core enzymes TnmD/G/J/K1/K2 and auxiliary proteins TnmT2/S2/T1/S1. Therefore, the biosynthesis of TNMs was stringently restricted by this cascade regulatory network at early stage to ensure the normal cell growth, and then partially released at the stationary phase for product accumulation. CONCLUSION: The pathway-specific cascade regulatory network consisting with TnmR3/R1 and TnmR7 was deciphered to orchestrate the production of TNMs. And it could be speculated as a common regulatory mechanism for productions of AFEs, which shall provide us new insights in future titer improvement of AFEs and potential dynamic regulatory applications in synthetic biology.

Synthesis and biological properties of maleimide-based macrocyclic lactone enediynes.

Natural enediyne antibiotics are powerful DNA-cleavage agents due to the presence of the highly reactive hex-3-ene-1,5-diyne units. However, the complicated chemical structure and thermal instability make their synthesis, derivatization, and storage challenging. Heterocycle-fused enediynes, which exhibit strong antineoplastic activity, are promising analogues of natural enediynes for medicinal applications. To this end, a series of maleimide-based enediynes with macrocyclic lactone moieties were synthesized through the Sonagashira coupling reaction. Differential scanning calorimetry and electron paramagnetic resonance results showed that these macrocyclic enediynes exhibited a rather low onset temperature and the ability to generate radicals at physiological temperature. In addition, the structure-activity relationship of enediynes was analyzed by changing the ring size and the substituents on the propargyl group. Cellular experiments indicated that the diradicals produced by these enediynes efficiently cleaved DNA and disrupted the cell cycle distribution, and consequently induced tumor cell death via an apoptosis pathway at low half inhibitory concentrations. Computational studies suggested that the maleimide moiety promoted the propargyl-allenyl rearrangement of the cyclic enediyne, enabling the generation of diradical species through the Myers-Saito cyclization, and then abstracted hydrogen atoms from the H-donors.

Synthesis of 14-membered enediyne-embedded macrocycles.

A concise and practical strategy towards a novel class of 14-membered macrocycles containing an enediyne (Z-3-ene-1,5-diyne) structural unit is described. A highly modular assembly of various precursors via sequential Ugi/Sonogashira reactions allowed the preparation of hybrid enediyne-peptide macrocycles in most cases as single diastereoisomers. Selected macrocyclic compounds showed moderate antiproliferative activity, and can be considered as templates suitable for further diversification in terms of ring size, shape, and stereochemistry.

A recombinant scFv antibody-based fusion protein that targets EGFR associated with IMPDH2 downregulation and its drug conjugate show therapeutic efficacy against esophageal cancer.

The present study aimed to evaluate the anti-tumor efficacy of the epidermal growth factor receptor (EGFR)-targeting recombinant fusion protein Fv-LDP-D3 and its antibody-drug conjugate Fv-LDP-D3-AE against esophageal cancer. Fv-LDP-D3, consisting of the fragment variable (Fv) of an anti-EGFR antibody, the apoprotein of lidamycin (LDP), and the third domain of human serum albumin (D3), exhibited a high binding affinity for EGFR-overexpressing esophageal cancer cells, inhibited EGFR phosphorylation and down-regulated inosine monophosphate dehydrogenase type II (IMPDH2) expression. Fv-LDP-D3 was taken up by cancer cells through intensive macropinocytosis; it inhibited the proliferation and induced the apoptosis of esophageal cancer cells. In vivo imaging revealed that Fv-LDP-D3 displayed specific tumor-site accumulation and a long-lasting retention over a 26-day period. Furthermore, Fv-LDP-D3-AE, a pertinent antibody-drug conjugate prepared by integrating the enediyne chromophore of lidamycin into the Fv-LDP-D3 molecule, displayed highly potent cytotoxicity, inhibited migration and invasion, induced apoptosis and DNA damage, arrested cells at G2/M phase, and caused mitochondrial damage in esophageal cancer cells. More importantly, both of Fv-LDP-D3 and Fv-LDP-D3-AE markedly inhibited the growth of esophageal cancer xenografts in athymic mice at well tolerated doses. The present results indicate that Fv-LDP-D3, and Fv-LDP-D3-AE exert prominent antitumor efficacy associated with targeting EGFR, suggesting their potential as promising candidates for targeted therapy against esophageal cancer.

Characterization and initial demonstration of in vivo efficacy of a novel heat-activated metalloenediyne anti-cancer agent.

BACKGROUND: Enediynes are anti-cancer agents that are highly cytotoxic due to their propensity for low thermal activation of radical generation. The diradical intermediate produced from Bergman cyclization of the enediyne moiety may induce DNA damage and cell lethality. The cytotoxicity of enediynes and difficulties in controlling their thermal cyclization has limited their clinical use. We recently showed that enediyne toxicity at 37 °C can be mitigated by metallation, but cytotoxic effects of 'metalloenediynes' on cultured tumor cells are potentiated by hyperthermia. Reduction of cytotoxicity at normothermia suggests metalloenediynes will have a large therapeutic margin, with cell death occurring primarily in the heated tumor. Based on our previous in vitro findings, FeSO4-PyED, an Fe co-factor complex of (Z)-N,N'-bis[1-pyridin-2-yl-meth-(E)-ylidene]oct-4-ene-2,6-diyne-1,8-diamine, was prioritized for further in vitro and in vivo testing in normal human melanocytes and melanoma cells. METHODS: Clonogenic survival, apopotosis and DNA binding assays were used to determine mechanisms of enhancement of FeSO4-PyED cytotoxicity by hyperthermia. A murine human melanoma xenograft model was used to assess in vivo efficacy of FeSO4-PyED at 37 or 42.5 °C. RESULTS: FeSO4-PyED is a DNA-binding compound. Enhancement of FeSO4-PyED cytotoxicity by hyperthermia in melanoma cells was due to Bergman cyclization, diradical formation, and increased apoptosis. Thermal enhancement, however, was not observed in melanocytes. FeSO4-PyED inhibited tumor growth when melanomas were heated during drug treatment, without inducing normal tissue damage. CONCLUSION: By leveraging the unique thermal activation properties of metalloenediynes, we propose that localized moderate hyperthermia can be used to confine the cytotoxicity of these compounds to tumors, while sparing normal tissue.

A carrier-free nanoparticle with dual NIR/acid responsiveness by co-assembly of enediyne and IR820 for combined PTT/chemotherapy.

Combined photothermal therapy/chemotherapy by co-delivery of a photosensitizer (PS) and a chemotherapeutic drug has demonstrated great potential for cancer treatment. The intrinsic drawbacks of traditional drug delivery systems (DDSs), such as tedious synthetic procedures, side effects originated from the carrier materials, low loading efficiency, and uncontrolled drug release, however, have impaired their further advancement. On the other hand, enediyne antibiotics are highly cytotoxic toward cancer cells through the generation of lethal carbon radicals via thermal-induced cyclization, endowing them with great potential to achieve enhanced synergistic anticancer performance by incorporation with the photothermal effect of PS. To this end, a carrier-free and NIR/acid dual-responsive DDS was constructed for combined photothermal therapy/chemotherapy. The facile co-assembly of maleimide-based enediyne and PS IR820 was achieved in aqueous solution to give nanoparticles (EICN) with a hydrodynamic diameter of 90 nm and high stability. In vitro study confirmed the acid/NIR dual-responsive degradation and drug release, free radical generation and DNA-cleaving ability of EICN, which was accomplished by the corporation of enediyne and IR820 moieties. Further tests on HeLa cells verified the excellent synergistic anticancer performance of EICN including the improved cellular uptake, NIR-enhanced drug release, DNA damage and histone deacetylase inhibitor capacity. Overall, this carrier-free DDS with dual acid/NIR-responsivity would potentially provide new insights for the development of combined photothermal/chemotherapy.

Synthesis of maleimide-based enediynes with cyclopropane moieties for enhanced cytotoxicity under normoxic and hypoxic conditions.

Myers-Saito cycloaromatization (MSC) is the working mechanism of many natural enediyne antibiotics with high antitumor potency. However, the presence of the equilibrium between diradical and zwitterionic intermediates in MSC severely hinders further improvement in cytotoxicity toward tumor cells. To this end, a series of maleimide-based enediynes with cyclopropane moieties were synthesized for enhanced cytotoxicity toward tumor cells. By taking advantage of radical clock reactions, the diradical intermediates generated from MSC would rearrange to new diradicals with much longer separation and weaker interactions between two radical centers. The computational study suggested a low energy barrier (4.4 kcal mol-1) for the radical rearrangement through the cyclopropane ring-opening process. Thermolysis experiments confirmed that this radical rearrangement results in the formation of a new diradical intermediate, followed by abstracting hydrogen atoms from 1,4-cyclohexadiene. Interestingly, the DNA cleavage ability and cytotoxicity of enediynes were significantly enhanced after the introduction of cyclopropane moieties. In addition, these maleimide-based enediynes exhibited a similar cytotoxicity under hypoxic conditions to that under normoxic conditions, which is beneficial for treating solid tumors where hypoxic environments frequently lead to deteriorated efficiency of many antitumor drugs. Docking studies indicated that the diradical intermediate was located between the minor groove of DNA with a binding energy of -7.40 kcal mol-1, which is in favor of intracellular DNA damage, and thereby inducing cell death via an apoptosis pathway as suggested by immunofluorescence analysis.

Synthesis and Biological Evaluation of Shishijimicin A-Type Linker-Drugs and Antibody-Drug Conjugates.

Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

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.

Construction of Inducible Genetic Switch for the Global Regulator WblA To Sustain Both Overproduction of Tiancimycins and On-Demand Sporulation in Streptomyces sp. CB03234.

The complex life cycle of streptomycetes is closely related to their secondary metabolisms, all controlled by cascade regulations. Tiancimycins (TNMs) are ten-membered enediynes possessing great potential for antitumor drug development. However, their low yields in Streptomyces sp. CB03234 have greatly limited subsequent studies. Through transcriptome analysis and genetic characterization, we proved that WblA is one pivotal global regulator to repress the biosynthesis of TNMs. The deletion of wblA could significantly enhance the production of TNMs, but also abolish the sporulation in CB03234. By constructing the NitR-ε-caprolactam inducible genetic switch, the expression of wblA was governed in CB03234-NRW, thereby sustaining the overproduction of TNMs and recovering the normal sporulation upon induction, which were practical for the scaled-up production of TNMs. Considering the prevalence and conserved regulatory roles of WblA in streptomycetes, our developed strategy shall provide an effective and practical approach to facilitate titer improvement and discovery of natural products.

C-N-Coupled Metabolites Yield Insights into Dynemicin A Biosynthesis.

The biosynthesis of the three structural subclasses of enediyne antitumor antibiotics remains largely unknown beyond a common C16 -hexaene precursor. For the anthraquinone-fused subtype, however, an unexpected iodoanthracene γ-thiolactone was established to be a mid-pathway intermediate to dynemicin A. Having deleted a putative flavin-dependent oxidoreductase from the dynemicin biosynthetic gene cluster, we can now report four metabolites that incorporate the iodoanthracene and reveal the formation of the C-N bond linking the anthraquinone and enediyne halves emblematic of this structural subclass. The coupling of an aryl iodide and an amine is familiar from organometallic chemistry, but has little or no precedent in natural product biosynthesis. These metabolites suggest further that enediyne formation occurs early in the overall biosynthesis, and that even earlier events might convert the C16 -hexaene to a common C15 intermediate that partitions to enediyne and anthraquinone building blocks for the heterodimerization.