Combinatorial metabolic engineering of Streptomyces sp. CB03234-S for the enhanced production of anthraquinone-fused enediyne tiancimycins.

BACKGROUND: Anthraquinone-fused enediynes (AFEs) are excellent payloads for antibody-drug conjugates (ADCs). The yields of AFEs in the original bacterial hosts are extremely low. Multiple traditional methods had been adopted to enhance the production of the AFEs. Despite these efforts, the production titers of these compounds are still low, presenting a practical challenge for their development. Tiancimycins (TNMs) are a class of AFEs produced by Streptomyces sp. CB03234. One of their salient features is that they exhibit rapid and complete cell killing ability against various cancer cell lines. RESULTS: In this study, a combinatorial metabolic engineering strategy guided by the CB03234-S genome and transcriptome was employed to improve the titers of TNMs. First, re-sequencing of CB03234-S (Ribosome engineered mutant strains) genome revealed the deletion of a 583-kb DNA fragment, accounting for about 7.5% of its genome. Second, by individual or combined inactivation of seven potential precursor competitive biosynthetic gene clusters (BGCs) in CB03234-S, a double-BGC inactivation mutant, S1009, was identified with an improved TNMs titer of 28.2 ± 0.8 mg/L. Third, overexpression of five essential biosynthetic genes, including two post-modification genes, and three self-resistance auxiliary genes, was also conducted, through which we discovered that mutants carrying the core genes, tnmE or tnmE10, exhibited enhanced TNMs production. The average TNMs yield reached 43.5 ± 2.4 mg/L in a 30-L fermenter, representing an approximately 360% increase over CB03234-S and the highest titer among all AFEs to date. Moreover, the resulting mutant produced TNM-W, a unique TNM derivative with a double bond instead of a common ethylene oxide moiety. Preliminary studies suggested that TNM-W was probably converted from TNM-A by both TnmE and TnmE10. CONCLUSIONS: Based on the genome and transcriptome analyses, we adopted a combined metabolic engineering strategy for precursor enrichment and biosynthetic pathway reorganization to construct a high-yield strain of TNMs based on CB03234-S. Our study establishes a solid basis for the clinical development of AFE-based ADCs.

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.

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.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded ß-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

Structural characterization of DynU16, a START/Bet v1-like protein involved in dynemicin biosynthesis.

The 1.5 Šresolution crystal structure of DynU16, a protein identified in the dynemicin-biosynthetic gene cluster, is reported. The structure adopts a di-domain helix-grip fold with a uniquely positioned open cavity connecting the domains. The elongated dimensions of the cavity appear to be compatible with the geometry of a linear polyene, suggesting the involvement of DynU16 in the upstream steps of dynemicin biosynthesis.

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.

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.

Genome shuffling based on different types of ribosome engineering mutants for enhanced production of 10-membered enediyne tiancimycin-A.

Tiancimycin-A (TNM-A) is an anthraquinone-fused ten-membered enediyne produced by Streptomyces sp. CB03234, which is very promising for the development of anticancer antibody-drug conjugates (ADCs). To improve the titer of TNM-A, we have generated high-producing mutants CB03234-S and CB03234-R through ribosome engineering, but still not sufficient for pilot production of TNM-A. As the follow-up work, gentamycin-induced ribosome engineering was further adopted here to generate the mutant CB03234-G, which produced similar level of TNM-A as in CB03234-S and CB03234-R. Benefiting from the distinct antibiotic resistances of three ribosome engineering mutants, genome shuffling between any two of them was respectively carried out, and finally obtained the recombinant CB03234-GS26. Under optimal conditions, CB03234-GS26 produced 40.6 ± 1.0 mg/L TNM-A in shaking flasks and 20.8 ± 0.4 mg/L in a scaled-up 30-L fermentor. Comparing with the parental high-producing mutants, the over 1.6-fold titer improvement of CB03234-GS26 in fermentor was more promising for pilot production of TNM-A. Besides the distinctive morphological features, genetic characterization revealed that CB03234-GS26 possessed 1.8 kb rsmG related deletion just the same as CB03234-S, but no mutation was found in rpsL. Subsequent knockouts proved that rsmG was unrelated to titer improvement of TNM-A, which implied other genomic variations and mechanisms rather than ribosome engineering to enhance the biosynthesis of TNM-A. Therefore, CB03234-GS26 provided a basis to locate potential novel genetic targets, and explore the interactions between complex metabolic network and TNM biosynthetic pathway, which shall promote future construction of high-yielding systems for TNM-A and other anthraquinone-fused enediynes.Key Points •United genome shuffling and ribosome engineering help further strain improvement. •CB03234-GS26 with improved titer is practical for the pilot production of TNM-A. •Enhanced TNM-A production should attribute to novel genetic features/mechanisms.

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.

Sungeidines from a Non-canonical Enediyne Biosynthetic Pathway.

We report the genome-guided discovery of sungeidines, a class of microbial secondary metabolites with unique structural features. Despite evolutionary relationships with dynemicin-type enediynes, the sungeidines are produced by a biosynthetic gene cluster (BGC) that exhibits distinct differences from known enediyne BGCs. Our studies suggest that the sungeidines are assembled from two octaketide chains that are processed differently than those of the dynemicin-type enediynes. The biosynthesis also involves a unique activating sulfotransferase that promotes a dehydration reaction. The loss of genes, including a putative epoxidase gene, is likely to be the main cause of the divergence of the sungeidine pathway from other canonical enediyne pathways. The findings disclose the surprising evolvability of enediyne pathways and set the stage for characterizing the intriguing enzymatic steps in sungeidine biosynthesis.

Streptomycin-induced ribosome engineering complemented with fermentation optimization for enhanced production of 10-membered enediynes tiancimycin-A and tiancimycin-D.

Tiancimycins (TNMs) are a group of 10-membered anthraquinone-fused enediynes, newly discovered from Streptomyces sp. CB03234. Among them, TNM-A and TNM-D have exhibited excellent antitumor performances and could be exploited as very promising warheads for the development of anticancer antibody-drug conjugates (ADCs). However, their low titers, especially TNM-D, have severely limited following progress. Therefore, the streptomycin-induced ribosome engineering was adopted in this work for strain improvement of CB03234, and a TNMs high producer S. sp. CB03234-S with the K43N mutation at 30S ribosomal protein S12 was successfully screened out. Subsequent media optimization revealed the essential effects of iodide and copper ion on the production of TNMs, while the substitution of nitrogen source could evidently promote the accumulation of TNM-D, and the ratio of produced TNM-A and TNM-D was responsive to the change of carbon and nitrogen ratio in the medium. Further amelioration of the pH control in scaled up 25 L fermentation increased the average titers of TNM-A and TNM-D up to 13.7 ± 0.3 and 19.2 ± 0.4 mg/L, respectively. The achieved over 45-fold titer improvement of TNM-A, and 109-fold total titer improvement of TNM-A and TNM-D enabled the efficient purification of over 200 mg of each target molecule from 25 L fermentation. Our efforts have demonstrated a practical strategy for titer improvement of anthraquinone-fused enediynes and set up a solid base for the pilot scale production and preclinical studies of TNMs to expedite the future development of anticancer ADC drugs.

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.

EGFR-targeting, ß-defensin-tailored fusion protein exhibits high therapeutic efficacy against EGFR-expressed human carcinoma via mitochondria-mediated apoptosis.

Defensins play an essential role in innate immunity. In this study, a novel recombinant ß-defensin that targets the epidermal growth factor receptor (EGFR) was designed and prepared. The EGFR-targeting ß-defensin consists of an EGF-derived oligopeptide (Ec), a ß-defensin-1 peptide (hBD1) and a lidamycin-derived apoprotein (LDP), which serves as the "scaffold" for the fusion protein (Ec-LDP-hBD1). Ec-LDP-hBD1 effectively bound to EGFR highly expressed human epidermoid carcinoma A431 cells. The cytotoxicity of Ec-LDP-hBD1 to EGFR highly expressed A431 cells was more potent than that to EGFR low-expressed human lung carcinoma A549 and H460 cells (the IC50 values in A431, A549, and H460 cells were 1.8 ± 0.55, 11.9 ± 0.51, and 5.19 ± 1.21 µmol/L, respectively); in addition, the cytotoxicity of Ec-LDP-hBD1 was much stronger than that of Ec-LDP and hBD1. Moreover, Ec-LDP-hBD1 suppressed cancer cell proliferation and induced mitochondria-mediated apoptosis. Its in vivo anticancer action was evaluated in athymic mice with A431 and H460 xenografts. The mice were administered Ec-LDP-hBD1 (5, 10 mg/kg, i.v.) two times with a weekly interval. Administration of Ec-LDP-hBD1 markedly inhibited the tumor growth without significant body weight changes. The in vivo imaging further revealed that Ec-LDP-hBD1 had a tumor-specific distribution with a clear image of localization. The results demonstrate that the novel recombinant EGFR-targeting ß-defensin Ec-LDP-hBD1 displays both selectivity and enhanced cytotoxicity against relevant cancer cells by inducing mitochondria-mediated apoptosis and exhibits high therapeutic efficacy against the EGFR-expressed carcinoma xenograft. This novel format of ß-defensin, which induces mitochondrial-mediated apoptosis, may play an active role in EGFR-targeting cancer therapy.

Resistance to Enediyne Antitumor Antibiotics by Sequestration.

The enediynes, microbial natural products with extraordinary cytotoxicities, have been translated into clinical drugs. Two self-resistance mechanisms are known in the enediyne producers-apoproteins for the nine-membered enediynes and self-sacrifice proteins for the ten-membered enediyne calicheamicin. Here we show that: (1) tnmS1, tnmS2, and tnmS3 encode tiancimycin (TNM) resistance in its producer Streptomyces sp. CB03234, (2) tnmS1, tnmS2, and tnmS3 homologs are found in all anthraquinone-fused enediyne producers, (3) TnmS1, TnmS2, and TnmS3 share a similar ß barrel-like structure, bind TNMs with nanomolar KD values, and confer resistance by sequestration, and (4) TnmS1, TnmS2, and TnmS3 homologs are widespread in nature, including in the human microbiome. These findings unveil an unprecedented resistance mechanism for the enediynes. Mechanisms of self-resistance in producers serve as models to predict and combat future drug resistance in clinical settings. Enediyne-based chemotherapies should now consider the fact that the human microbiome harbors genes encoding enediyne resistance.

Hot off the press.

A personal selection of 32 recent papers is presented covering various aspects of current developments in bioorganic chemistry and novel natural products such as tundrenone from Methylobacter tundripaludum.

Structural Insights into the Free-Standing Condensation Enzyme SgcC5 Catalyzing Ester-Bond Formation in the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027.

C-1027 is a chromoprotein enediyne antitumor antibiotic, consisting of the CagA apoprotein and the C-1027 chromophore. The C-1027 chromophore features a nine-membered enediyne core appended with three peripheral moieties, including an ( S)-3-chloro-5-hydroxy-ß-tyrosine. In a convergent biosynthesis of the C-1027 chromophore, the ( S)-3-chloro-5-hydroxy-ß-tyrosine moiety is appended to the enediyne core by the free-standing condensation enzyme SgcC5. Unlike canonical condensation domains from the modular nonribosomal peptide synthetases that catalyze amide-bond formation, SgcC5 catalyzes ester-bond formation, as demonstrated in vitro, between SgcC2-tethered ( S)-3-chloro-5-hydroxy-ß-tyrosine and ( R)-1-phenyl-1,2-ethanediol, a mimic of the enediyne core as an acceptor substrate. Here, we report that (i) genes encoding SgcC5 homologues are widespread among both experimentally confirmed and bioinformatically predicted enediyne biosynthetic gene clusters, forming a new clade of condensation enzymes, (ii) SgcC5 shares a similar overall structure with the canonical condensation domains but forms a homodimer in solution, the active site of which is located in a cavity rather than a tunnel typically seen in condensation domains, and (iii) the catalytic histidine of SgcC5 activates the 2-hydroxyl group, while a hydrogen-bond network in SgcC5 prefers the R-enantiomer of the acceptor substrate, accounting for the regio- and stereospecific ester-bond formation between SgcC2-tethered ( S)-3-chloro-5-hydroxy-ß-tyrosine and ( R)-1-phenyl-1,2-ethanediol upon acid-base catalysis. These findings expand the catalytic repertoire and reveal new insights into the structure and mechanism of condensation enzymes.

Characterization of an Anthracene Intermediate in Dynemicin Biosynthesis.

Despite the identification of a ß-hydroxyhexaene produced by the enediyne polyketide synthases (PKSs), the post-PKS biosynthetic steps to the individual members of this antitumor and antibiotic family remain largely unknown. The massive biosynthetic gene clusters (BGCs) that direct the formation of each product caution that many steps could be required. It was recently demonstrated that the enediyne PKS in the dynemicin A BGC from Micromonospora chersina gives rise to both the anthraquinone and enediyne halves of the molecule. We now present the first evidence for a mid-pathway intermediate in dynemicin A biosynthesis, an iodoanthracene bearing a fused thiolactone, which was shown to be incorporated selectively into the final product. This unusual precursor reflects just how little is understood about these biosynthetic pathways, yet constrains the mechanisms that can act to achieve the key heterodimerization to the anthraquinone-containing subclass of enediynes.

Ribosome engineering and fermentation optimization leads to overproduction of tiancimycin A, a new enediyne natural product from Streptomyces sp. CB03234.

Tiancimycin (TNM) A, a recently discovered enediyne natural product from Streptomyces sp. CB03234, showed rapid and complete killing of cancer cells and could be used as a payload in antibody drug conjugates. The low yield of TNM A in the wild-type strain promoted us to use ribosome engineering and fermentation optimization for its yield improvement. The Streptomyces sp. CB03234-R-16 mutant strain with a L422P mutation in RpoB, the RNA polymerase ß-subunit, was obtained from the rifamycin-resistant screening. After fermentation optimization, the titers of TNM A in Streptomyces sp. CB03234-R-16 reached to 22.5 ± 3.1 mg L-1 in shaking flasks, and 13 ± 1 mg L-1 in 15 L fermentors, which were at least 40-fold higher than that in the wild-type strain (~ 0.3 mg L-1). Quantitative real-time RT-PCR revealed markedly enhanced expression of key genes encoding TNM A biosynthetic enzymes and regulators in Streptomyces sp. CB03234-R-16. Our study should greatly facilitate the future efforts to develop TNM A into a clinical anticancer drug.

A dual role for a polyketide synthase in dynemicin enediyne and anthraquinone biosynthesis.

Dynemicin A is a member of a subfamily of enediyne antitumour antibiotics characterized by a 10-membered carbocycle fused to an anthraquinone, both of polyketide origin. Sequencing of the dynemicin biosynthetic gene cluster in Micromonospora chersina previously identified an enediyne polyketide synthase (PKS), but no anthraquinone PKS, suggesting gene(s) for biosynthesis of the latter were distant from the core dynemicin cluster. To identify these gene(s), we sequenced and analysed the genome of M. chersina. Sequencing produced a short list of putative PKS candidates, yet CRISPR-Cas9 mutants of each locus retained dynemicin production. Subsequently, deletion of two cytochromes P450 in the dynemicin cluster suggested that the dynemicin enediyne PKS, DynE8, may biosynthesize the anthraquinone. Together with 18O-labelling studies, we now present evidence that DynE8 produces the core scaffolds of both the enediyne and anthraquinone, and provide a working model to account for their formation from the programmed octaketide of the enediyne PKS.