Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065

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Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065 sheng wu, Xiao-Hong Jian, Hua Yuan, Wen-Bing Jin, Yue Yin, Ling-Yun Wang, Juan Zhao, and Gong-Li Tang ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00302 • Publication Date (Web): 20 Apr 2017 Downloaded from http://pubs.acs.org on April 25, 2017

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ACS Chemical Biology

Manuscript for ACS Chemical Biology

Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065

Sheng Wu,† Xiao-Hong Jian,† Hua Yuan,† Wen-Bing Jin, Yue Yin, Ling-Yun Wang, Juan Zhao, and Gong-Li Tang*

State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China

†

These authors contributed equally.

*Correspondence: Gong-Li Tang, Email: [email protected]; Tel: 086-21-54925113, Fax: 086-21-64166128.

Data deposition: The sequence reported in this paper has been deposited into GenBank under accession no. KY379149.

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ABSTRACT

CC-1065 is the first characterized member of a family of naturally occurring antibiotics including yatakemycin and duocarmycins with exceptionally potent anti-tumor activity. CC-1065 contains three benzodipyrroles (1a-, 1b- and 1c-) of which 1a-subunit is remarkable by being composed of a cyclopropane ring, and the mechanism for the biological formation of benzodipyrrole rings remains elusive. Previously, biosynthetic studies of CC-1065 were limited to radioactively labelled precursor feeding experiments, which showed that tyrosine (Tyr) and serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits via the same mode but that this was different from the key cyclopropabenzodipyrrole (1a-) subunit with N1-C2-C3 derived from Ser. Herein, the biosynthetic gene cluster of CC-1065 has been cloned, analyzed, and characterized by a series of gene inactivation. Significantly, a key intermediate bearing a C7-OH group derived from a ∆c10C mutant exhibited improved cytotoxicity. Moreover, this data inspired us to suspect that the 1a-subunit might employ the same precursor incorporation mode as the 1band 1c-subunits. Subsequently,

13

C-labelled Tyr feeding experiments confirmed that the

N1-C2-C3 is originated from Tyr via DOPA as an intermediate. Collectively, a biosynthetic pathway of benzodipyrrole is proposed featuring a revised and unified precursor incorporation mode, which implicates an oxidative cyclization strategy for the assembly of benzodipyrrole. This work sets the stage for further study of enzymatic mechanisms and combinatorial biosynthesis for new DNA alkylating analogues.

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CC-1065, yatakemycin (YTM) and duocarmycins (Figure 1A and B) belong to a small family of cyclopropabenzodipyrrole-containing natural products that show exceptionally potent cytotoxic activity. They exhibit a characteristic AT-rich, sequence-selective DNA alkylation via a binding-induced conformational change mode.1 These agents can even directly target and alkylate the duplex DNA in nucleosome core particles.2 As the first member of this family discovered in 1978, CC-1065 has since been considered as one of the most potent antitumor agents,3-5 and subsequently spurred extraordinary enthusiasm for chemical synthesis, mechanistic studies, and drug discovery.6-11 Currently, a series of analogues have been developed as anticancer drugs via antibody-drug conjugates or other prodrugs, and have been in clinical trials exemplified by SYD985 and MDX-1203 (Figure 1C).12-14 One of the key structural features of CC-1065 is the presence of three similar benzodipyrroles (1a-, 1b- and 1c-). The structure of the 1a-subunit is different from the other two because it contains a spirocyclopropylcyclohexadienone in place of the dihydroxyindole moiety (Figure 1A).15 Biosynthetic studies of CC-1065 limited to radioactively labelled precursor feeding experiments were performed in 1982, which established the fundamental biosynthetic precursors and incorporation modes.15 According to that model, DOPA, derived from tyrosine (Tyr), together with serine (Ser) were incorporated into the two benzodipyrrole (1b- and 1c-) subunits through the same mode (Figure 1A). While only Tyr, but not DOPA, could be incorporated into the cyclopropabenzodipyrrole (1a-) subunit; moreover, this Tyr incorporation mode was different from that of other two subunits (Figure 1A). This model seemed very reasonable based upon biosynthetic considerations. With an interest in addressing how nature creates these fascinating chemicals, we have been pursuing biosynthetic studies of this family of natural products for many years. Recently, we cloned the biosynthetic gene cluster of YTM by genome scanning and proposed a primary biosynthetic pathway based on genetic and biochemical studies, which features a S-adenosylmethionine (SAM)-dependent, C-methyltransferase (MT)-like enzyme YtkT contributing to an advanced intermediate during formation of the cyclopropane ring through a radical-based mechanism.16 Additionally, genetic and biochemical characterizations revealed that a DNA glycosylase, YtkR2, specifically 3



recognizes and cleaves the YTM modified base, which further triggers the base-excision repair system to confer self-resistance.17 Here, we described the identification of the CC-1065 biosynthetic gene cluster and revision of the precursor incorporation mode guided by genetic investigation, structural elucidation of biosynthetic intermediates and Tyr-labelled precursor feeding. Furthermore, we proposed a unified pathway for the formation of the three benzodipyrrole subunits of CC-1065 via a common oxidative cyclization strategy.

RESULTS AND DISCUSSION Identification and Analysis of the CC-1065 Biosynthetic Gene Cluster. Previously, YtkT, a radical SAM-dependent enzyme was identified as essential for the cyclopropane ring formation in YTM biosynthesis (Figure 2A).16 Considering the similar structures of CC-1065 and YTM, we reasoned that the cyclopropane moiety should be formed by a similar pathway and a homologous protein may be involved in CC-1065 biosynthesis. Therefore, we adopted a PCR-based approach using degenerate primers designed according to the conserved motifs among YtkT and its homologues (see Methods) to amplify the gene encoding a YtkT-like enzyme. By screening of the Streptomyces zelensis (NRRL-11183) genomic library, a cosmid pTG1401 containing a ~40 kb DNA region was identified as a candidate to harbor the biosynthetic gene cluster, which was verified by successful heterologous expression of the cosmid pTG1401 in S. lividans 1326 to produce CC-1065, although the yield was very low compared with that of wild-type (WT, Figure S1). Sequencing of the cosmid pTG1401 yielded a 38.6 kb contiguous DNA sequence (GenBank KY379149), and further bioinformatic analysis revealed the presence of 33 ORFs (Table 1). The CC-1065 gene cluster was assigned from c10R1 to c10R6, encompassing 28 ORFs, based on the analysis and comparison with the YTM gene cluster (Figure 2B). It includes one regulatory gene, five resistance genes, and several genes encoding proteins with unknown function. Obviously, a carbamoyltransferase C10W, two MTs (C10D and C10Q), and another radical SAM-dependent MT C10T could be assigned for carbamoylation, O-methylation, and C-methylation of presumed benzodipyrrole 4


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building blocks 15, 14, 13 (Figure 2C); a 4-hydroxyphenylacetate 3-monooxygenase homolog C10J was likely involved in the conversion of Tyr into DOPA (Figure 2C). Surprisingly, only 12 ORFs, including two resistance proteins (C10R5 and C10R6) and a ferredoxin (C10H), exhibited sequence homology to the corresponding proteins involved in YTM biosynthesis. Among them, C10A (tryptophan synthase β-subunit, homologous to YtkJ) is presumably responsible for condensation of benzopyrrole 10 and Ser, and C10N (decarboxylase, YtkL-like) is assigned for subsequent decarboxylation to yield 12 following the YTM biosynthetic pathway (Figure 2C).16 Additionally, two CoA-ligases, C10G and C10K (homologs of YtkQ and YtkN), might work with nitrilase-like enzymes C10L/M or C10O/S (homolog of YtkV) together to link the three building blocks (17, 18, 19) via amide bond formation (Figure 2C). Similar enzymatic reactions have been biochemically verified in thiomarinol biosynthesis,18 while the amide ligase TmlU in this case shows no homology to the nitrilase-like enzymes in CC-1065 or YTM biosynthesis.

Genetic Characterization of a Radical SAM Protein C10P. Analogous to YTM biosynthesis, the YtkT-homolog radical SAM-dependent protein C10P was suggested for the cyclopropanation via a radical mechanism. To validate this, the c10P gene was inactivated by in-frame deletion resulting in a mutant strain S. zelensis TG1402 (Figure S2). As expected, the ∆c10P mutant did not produce CC-1065 anymore. However, HPLC analysis of the metabolite profile revealed a new peak at 16 min and two obviously increased peaks at 6.3 and 10 min (Figure 3AII), which showed that a new compound 8 and two possible intermediates 6 and 7 were produced by mutant S. zelensis TG1402. After large scale fermentation (40 L) of this mutant, we could isolate sufficient quantities of comounds 6, 7, and 8, whose structures were further determined by evaluation of the HRMS and NMR spectra and comparison with those of CC-1065 (Figure 3B, Table S4, S6-S8, and Figure S3-S5). Compound 7 (PDE-I dimer) is a known compound, which has ever been chemically synthesized during the total synthesis of CC-1065.19,20 Compound 6 may be derived from the central building block 14 by oxidation during purification; and 7 5



could be generated from 20, both of which are intermediates during the biosynthetic pathway (Figure 2C) and also produced by the WT strain (Figure 3AI). Thus, the discovery of compound 8 confirmed that C10P participates in the formation of the cyclopropane moiety in CC-1065 biosynthesis. Such a radical SAM-dependent enzyme catalyzed cyclopropanation reaction has ever been proposed in the biosynthesis of YTM and jawsamycin.16,21

Discovery of 7-Hydroxy-CC-1065 (9) from a ∆c10C Mutant Strain. The pair of proteins C10B/C10C display homology to YtkS/YtkK, and belong to a superfamily of molybdopterin oxidoredoreductases, which includes three subfamilies exemplified by sulphite oxidase, xanthine oxidase and dimethylsulphoxide reductase.22,23 However, it is difficult to assign their functions in the biosynthetic pathway based on sequence analysis alone. To further explore its role in CC-1065 biosynthesis, we inactivated c10C and obtained the mutant strain S. zelensis TG1403 (Figure S2). This ∆c10C mutant abolished the production of CC-1065, but led to the production of another new compound 9 combined with an increase in the levels of intermediate 6 (Figure 3AIV). Further introduction of plasmid pTG1404 expressing a functional copy of c10C under the control of ermE* promoter in trans could restore the normal production of CC-1065 (Figure 3AIII). We finally obtained 8 mg of pure compound 9 from 40 L fermentation broth of S. zelensis TG1403 (∆c10C) strain, then analyzed the HRMS and NMR spectra and compared with CC-1065 (Table S3, S5, S8, and Figure S6), which led assignment of the chemical structure as 7-hydroxy-CC-1065 (Figure 3B). These results demonstrated that C10C is necessary for CC-1065 biosynthesis; and this molybdopterin oxidoredoreductase pair most likely catalyzes a dehydroxylation of benzodipyrrole at a late stage of the pathway (Figure 2C). While enzymatic dehydroxylation of the aromatic ring is a chemically challenging reaction, similar processes catalyzed by Mo-flavo-Fe/S dependent proteins have only been observed in anaerobic metabolism of hydroxybenzenes.24-26 Therefore, it will be intriguing to further study this enzymatic reaction in the near future.

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Considering that 9 harbors the cyclopropane warhead, we reasoned that this compound should exhibit cytotoxic activity. Subsequent cytotoxicity assays against different tumor cell lines were performed using 1 as positive control. Surprisingly, the newly discovered analogue showed even higher biological activity than that of CC-1065 (Figure 3C), providing another candidate for the further derivation and evaluation. Previous chemical synthesis of CC-1065 and duocarmycin derivatives revealed that the electron-withdrawing C7-substituents decrease the DNA alkylation reactivity of the cyclopropane ring.27-28 Herein, the biosynthesized 7-hydroxy-CC-1065 by the engineered mutant suggested that the electron-donating C7-substituent likely increases its biological activity.

Revision of the Precursor and Mode of Incorporation. Inactivation of c10C leading to the production of 7-hydroxy-CC-1065 raised a further hypothesis that the 1a-subunit seems to be constructed by the same mode as 1b- and 1c-subunits, all of which might be incorporated from Tyr via a DOPA intermediate. This notion disagrees with the radioactively labelled precursor feeding results which suggested that only 1b- and 1c-subunits are constructed by DOPA and Ser; whereas the 1a-subunit originates from Tyr but not DOPA, and the N1-C2-C3 unit is from Ser (Figure 1A).15 Additionally, two pathways (“a” and “d”) were originally proposed for the incorporation of Tyr into the 1a-subunit: the “d”-pathway was ruled out based on the DOPA feeding results, and the “a”-pathway was suggested to account for the location of the quinone oxygen in the correct position relative to the phenolic hydroxyl of Tyr (Figure 4A).15 To address this confusion, we

performed

a

labelled

precursor

feeding

experiment

using

L-[1-13C]-

and

[RING-13C6]-DOPA. However, no incorporation could be observed even in 1b- and 1c-subunits despite several attempts. Given the fact that DOPA is easily oxidized then subsequently polymerized, and could be bio-transformed from Tyr, we then had to take an alternative approach using L-[2-13C]-Tyr to conduct the feeding experiment.

13

C-NMR

spectroscopy of the labelled CC-1065 clearly revealed strong signal enrichment at positions C-2 (5.0%), C-16 (5.8%), and C-29 (4.5%) (Figures 4B, 4C and S7, Table S9). Obviously, the labelling of C-16 and C-29 agreed well with the radioactively labelled 7



precursor feeding results; however, the significant signal enrichment of C-2 not C-12 unambiguously demonstrated that Tyr is incorporated into the 1a-subunit via the “d”-pathway, which suggested that the previously proposed “a”-pathway is not correct (Figure 4A). Reasonably, incorporation via pathway “d” would require a DOPA intermediate followed by loss of the tyrosine phenolic hydroxyl group. Additionally, these results also indicated that the N1-C2-C3 unit is from Tyr, which supports the idea that all three subunits are constructed by Ser and DOPA using the same incorporation mode during the biosynthetic process (Figure 4C). Thus, the previously confusing results from L-[3- or 5-3H]-Tyr can be explained properly. On the basis of the above genetic and chemical data, we proposed a unified biosynthetic pathway for benzodipyrrole. First, Tyr is oxidized to DOPA by C10J; then the ortho-diphenol is oxidized into the highly electrophilic ortho-quinone, which is necessary for conjugate addition via the amino group to generate 10 after tautomerization (Figure 2C). Similar oxidation-addition-tautomerization reactions are repeated to form the second C-N bond to yield benzodipyrrole 13, which may serve as a common intermediate for all of the three subunits (Figure 2C). For the 1a-subunit, additional dehydroxylation by C10B/C10C is required, then cyclopropanation by C10P after the linkage of three subunits (Figure 2C).

Conclusions. In summary, we have identified a common biosynthetic origin of the three benzodipyrrole subunits in CC-1065. We have genetically characterized the CC-1065 biosynthetic gene cluster and revised the biosynthetic pathway based on the elucidation of 7-hydroxy-CC-1065 and labelled precursor feeding. These studies described herein indicate that the different benzodipyrroles in CC-1065 share the same mechanism for their formation in the early stage of the pathway. We have therefore outlined a proposal for the formation of benzodipyrrole by an oxidative cyclization strategy. On the other hand, given the fact that the same family of natural products usually governed by evolutionarily-related biosynthetic gene clusters with high homology,

it is indeed unexpected that more than half of the 8


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genes in the CC-1065 and YTM biosynthetic clusters exhibit un-relationship with each other. Therefore, it is intriguing to ascertain how nature creates similar chemical structures by different mechanisms through comparative biosynthetic studies of CC-1065 and YTM. The similarities and differences between CC-1065 and YTM will allow us to discern their evolutionary relationships. Additionally, the available gene clusters of YTM and CC-1065 not only set the stage to create new analogues for drug development, but also provide a great opportunity to access the enzymatic chemistry of radical-based cyclopropanation.

METHODS Materials, Bacteria Strains and Plasmids. Isotopically labeled precursors were purchased from Cambridge Isotope Laboratories, Andover (USA). A CC-1065 producer, Streptomyces zelensis NRRL 11183 was purchased from American Agricultural Research Service (ARS). Escherichia coli DH5α competent cells were used for routine subcloning and plasmid preparations and it were grown in LB medium with appropriate antibiotics. PCR amplification was carried out using either Taq DNA polymerase or PfuUltraTM DNA polymerase with genomic DNA or fosmid pTG1401 as a template, degenerate or specific primers were listed in Table S2. Primer synthesis was performed at GENEWIZ, Inc. DNA sequencing was performed at the Shanghai Majorbio Pharm Technology Co., Ltd. Bacterial strains and plasmids used in this study are summarized in Table S1. Primers used in this study are listed in Table S2. Sequence Analysis. The open reading frames (ORFs) were deduced from the sequence by performing Frame Plot 4.0 beta program (http://nocardia.nih.go.jp/fp4) and BLAST methods (http://www.ncbi.nlm.nih.gov/blast/). Metabolites Analysis and Purify. HPLC analysis was carried out on Agilent 1200series. Semipreparative was carried out on SHIMADZU LC-20-AT. NMR spectra was measured on a Bruker AV-500 with the residual C2H6OS (δH 2.50 ppm; δC 39.51 ppm) as an internal standard. ESI-MS was recorded on a ThermoScientific LCQ Fleet ion trap spectrometer with an Agilent LC system. High-resolution mass spectral analysis was acquired on Agilent 6230 TOF-MS. A commercial silica gel (siliaFlash®, 230−400 mesh) and reverse phase silica gel (chromatorex®, 230−400 mesh) was used for column chromatography. 9



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Genomic Library Construction and Screening. For the generation of cosmid libraries, total DNA samples were partially digested with Sau3AI. The DNA fragments between 30 and 40 kb were recovered using Pulse-field gel electrophoresis (PFGE). The recover fragments were dephosphorylated with Heat-Labile alkaline phosphatase (EPICENTRE Biotechnologies). Then the fragments were mixed and ligated with a cosmid vector pJTU2463 (A derivative of pOJ446 with the SCP2 replicon was replaced by int and attp from pSET152, which is kindly given by Prof. Zixin Deng) digested by HpaI and CIAP (NEB) and BamHI sequentially which is pSET152-derived. Packaging was done according to a standard protocol. Degenerate primers were designed

based

on

the

radical-SAM

dependent

5′-GGCTTCTACGTGCACATHCCNTTYTG-3′

enzyme and

YtkT16

(ytkT-1, ytkT-2,

5′-ACCTGGTCGTGGAAGSWYTGNACNCC-3′). A 0.37-kb fragment was obtained by using the primers ytkT-1 and ytkT-2, which was further used as probe for library screening. Six cosmids was obtained, and pTG1401 (8G5) was selected for sequence through analysis.

Construction of Gene-inactivation and Complementary Mutant Strains In-frame Deletion of orf c10C. A 2.0 Kb HindIII/NdeI fragment (primers C10C-L-for and C10C-L-rev) and a 1.6kb NdeI/EcoRI fragment (primers C10C-R-for and C10C-L-rev) were cloned into the HindIII/EcoRI sites of the thermosensitive plasmid pKC1139, giving the recombinant plasmid pTG1403. Then, it was introduced into S. zelensis NRRL 11183 by conjugation from E. coli S17-1, and apramycin-resistant exconjugants were selected at 30°C. These exconjugants were first grown in TSB media with apramycin at 37 °C to obtain single-crossover mutants, which were further inoculated in TSB media without apramycin for generations to screen for apramycin-sensitive clones. The double-crossover mutants S. zelensis TG1403 (∆c10C) were verified by PCR analysis, as judged by a 616 bp desired product using the primers C10C-gt-For and C10C-gt-Rev (Figure S2.) In-frame Deletion of orf c10P. For c10P inactivation, a 2.0 kb HindIII/PstI fragment (primers C10P-L-for and C10P-L-rev) and a 1.7 kb PstI/EcoRI fragment (primers C10P-R-for and C10P-L-rev) were cloned into the HindIII/EcoRI sites of pKC1139, giving the recombinant 10


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plasmid pTG1402. Following above procedure and conditions, this plasmid was transferred into S. zelensis NRRL 11183 to generate the double crossover mutants S. zelensis TG1402 (∆c10P). The primers C10P-gt-For and C10P-gt-Rev were used for genotype validation by PCR amplification, as judged by a 1869 bp desired product (Figure S2). Complementation of c10C into S. zelensis TG1403 (∆c10C). To express the c10C gene in mutant S. zelensis TG1403 (∆c10C), a 1.5 kb EcoRI/NdeI fragment (amplified with primers tC-For and tC-Rev) containing the intact c10C gene was cloned into the pSET152 vector to yield plasmid pTG1404. It was introduced into S. zelensis TG1403 by conjugation to produce complemented strain S. zelensis TG1404, and the fermentation broth of this strain was analyzed by HPLC for CC-1065 production as described below. The wild-type strain was used as a control.

Fermentation, Precursor Feeding and Analysis Production and Analysis of CC-1065. A well grown agar slant of S. zelensis NRRL 11183 was used to inoculate a 500-mL Erlenmeyer flask containing 100 mL TSB media (pH= 7.0).30 After incubated under 28°C, 220 rpm for 2 days, 10 mL of seeding culture was further transferred into a 500-mL flask containing 100 mL fermentation media (dextrin 30 g , fish meal 10 g , cornmeal 30 g, cottonseed meal 30 g, glucose 10 g, Na citrate 2.5 g, MgSO4·7H2O 1 g , CaCO3 5 g, FeSO4.7H2O 0.02 g, KCl 0.5 g, CoCl2·7H2O 0.02 g, Na2HPO4·12H2O 3 g, pH 7.0, distilled H2O to 1 liter) which is incubated for 6 days, under the same conditions as the seed flask. The culture broth was centrifugalized (4000 rpm for 10 min), the culture filtrates were removed, and the mycelia were extracted with acetone, and the crude extract was used for HPLC analysis. HPLC analysis was carried out on a Grace AlltimaTM C18 column (5µm, 100Å, 10×250 mm), eluting with a flow rate of 1mL/min over a 23 min gradient as follows: T = 0 min, 5% B; T = 3 min, 40% B; T = 10 min, 55% B; T = 12 min, 70% B; T = 21 min, 85% B; T = 22 min, 15% B and T = 23 min, 15% B (solvent A, H2O + 0.1% HCOOH; solvent B, CH3CN + 0.1% HCOOH) under 360 nm using an Agilent 1200 HPLC system. Precursor Feeding to S. zelensis NRRL 11183. Radioactive precursors were added at 66 hours after inoculation in the fermentation media, and continued the fermentation for another three days (The final concentration for the precursors is: L-[2-13C]-tyrosine 30 mg/L). 11



Compound Isolation and Structural Elucidation Isolation and Purification of CC-1065. After 6 days of fermentation, the fermentation broth (10 L) was centrifuged to give supernatant and mycelium cake. The mycelium cake was extracted with acetone. The extract was concentrated in vacuo to an aqueous solution. The solution was extracted twice with ethyl acetate. The organic layer was evaporated to give a crude oily material, the residue was applied to a silica gel column (siliaFlash®, 230−400 mesh) which was developed with chloroform-methanol (15:1). The active eluate was concentrated to dryness, solubilized in DMSO and then subjected to semi-preparative HPLC (Venusil XBP C18, 5 µm, 100 Å, 10 х 250 mm) with a flow rate of 3 mL/min over a 13 min gradient (T= 0 min, 50% B; T= 4 min, 50% B; T = 12 min, 70% B; T = 12.5 min, 90% B; T =13 min, 50% B; solvent A, H2O; solvent B, CH3CN) lead to purification of CC-1065. The active fractions were concentrated to dryness to give a yellow powder. Isolation and Purification of Isotopically Labelled CC-1065. The cultures feeding with each isotope-labeled precursor were extracted and treated as described above. A tentative assignment of the

I3

C NMR signals of CC-1065 has been published by Martin et al.30 However, our date

suggested that Martin’s tentative assignment of the signals had to be reversed. For all of the carbon atoms, our assignment could be unambiguously confirmed in the course of this study by HMQC- and HMBC-NMR spectra of the labeled CC-1065. Both reversals were later on confirmed by the enrichment observed after L-[2-13C]-tyrosine feeding. Compound 7-hydroxy-CC-1065 (9) from the S. zelensis TG1403 (∆c10C) mutant. About 40 L fermentation broths were extracted and treated as described above, the crude extracts were subjected to the reverse phase silica gel column chromatography. The compounds were eluted with H2O /CH3CN (80:20-20:80). All the fractions were monitored by HPLC. Desired fractions were collected and lyophilized to dryness. The active eluate was concentrated to dryness, solubilized in DMSO and then subjected to semi-preparative HPLC (Venusil XBP C18, 5 µm, 100Å, 10 х 250 mm) with a flow rate of 3 mL/min over a 16 min gradient (T= 0 min, 40% B; T= 3 min, 50% B; T= 6 min, 70% B; T = 10 min, 85% B; T = 12 min, 90% B; T =14 min, 95% B; T =16 min, 50% B;

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solvent A, H2O; solvent B, CH3CN) lead to purification of 7-hydroxy-CC-1065, which is yellow powder. Compounds 8, dimer PDE-I (7) and oxidative PDE (6) from the S. zelensis TG1402 (∆c10P) mutant. The fermentation broth (40 L) was centrifuged to give supernatant and mycelium cake. The mycelium cake was extracted with acetone. Then the solution was extracted twice with ethyl acetate. The organic layer was evaporated to give a crude oily material, the residue was applied to a silica gel column (silia Flash®, 230−400 mesh) which was developed with chloroform-methanol (15: 1). Then obtained precipitations were subjected to semi-preparative HPLC as described above. Oxidative PDE was obtained by semi-preparative HPLC with a flow rate of 3 mL/min over a 14 min gradient (T= 0 min, 20% B; T= 4 min, 30% B; T = 12 min, 50% B; T = 13 min, 80% B; T =13.6 min, 60% B; T = 14 min, 20% B; (solvent A, H2O; solvent B, CH3CN). Similarly, compound 8 and dimer PDE-I (7) was obtained by semi-preparative HPLC with a flow rate of 3 mL/min over a 13 min gradient (T= 0 min, 50% B; T= 4 min, 50% B; T = 12 min, 70% B; T = 12.5 min, 90% B; T =13 min, 50% B; solvent A, H2O; solvent B, CH3CN). Compound 8, Oxidative PDE is yellow powder and dimer PDE-I is brown powder.

Supporting Information. Table S1and S2, plasmids and primers used in this study; Table S3-S7, NMR spectroscopic data of compounds CC-1065, 8, 9, 6, and 7; Table S8, summarized HR-ESI-MS data of compounds 6-9; Table S9, analysis of incorporation of I3C-labelled CC-1065. Figure S1, LC-MS analysis of CC-1065 production by heterologous expression of pTG1401; Figure S2, generation and verification of the S. zelensis mutant strains; Figure S3-S6, NMR spectra of compounds 6-9; Figure S7, NMR spectra of compound L-[2-13C]-tyrosine labelled CC-1065.

Notes: The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank Prof. Z. Deng of Shanghai Jiao Tong University for kindly providing plasmid pJTU2463; we acknowledge the supporting grants from National Natural Science Foundation 13



of China (21632007, 21502217 and 21621002), Science and Technology Commission of Shanghai Municipality (15JC1400400 and 15ZR1449400), Chinese Academy of Sciences (XDB20000000 and QYZDJ-SSW-SLH037) and State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University (MMLKF15-11).

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(9) Boger, D. L., and Garbaccio, R. M. (1999) Shape-dependent catalysis: insights into the source of catalysis for the CC-1065 and duocarmycin DNA alkylation reaction. Acc. Chem. Res. 32, 1043-1052. (10) Ghosh, N., Sheldrake, H. M., and Pors, K. (2009) Chemical and biological explorations of the family of CC-1065 and the duocarmycin natural products. Top. Med. Chem. 9, 1494-1524. (11) Patil, P. C., Satam, V., and Lee, M. (2015) A short review on the synthetic strategies of duocarmycin analogs that are powerful DNA alkylating agents. Anti-Cancer Agent. Med. Chem. 15, 616-630. (12) Tietze, L. F., and Krewer, B. (2009) Novel analogues of CC-1065 and the duocarmycins for the use in targeted tumour therapies. Anti-Cancer Agent. Med. Chem. 9, 304-325. (13) Zhao, R. Y., Erickson, H. K., Leece, B. A., Reid, E. E., Goldmacher, V. S., Lambert, J. M., and Chari, R. V. J. (2012) Synthesis and biological evaluation of antibody conjugates of phosphate prodrugs of cytotoxic DNA alkylators for the targeted treatment of cancer. J. Med. Chem. 55, 766-782. (14) Gerber, H. P., Koehnb, F. E., and Abraham, R. T. (2013) The antibody-drug conjugate: an enabling modality for natural product-based cancer therapeutics. Nat. Prod. Rep. 30, 625-639. (15) Hurley, L. H., and Rokem, J. S. (1983) Biosynthesis of the antitumor antibiotic CC-1065 by Streptomyces zelensis. J. Antibiot. 36, 383-390. (16) Huang, W., Xu, H., Li, Y., Zhang, F., Chen, X. Y., He, Q. L., Igarashi, Y., and Tang, G. L. (2012) Characterization of yatakemycin gene cluster revealing a radical S-adenosylmethionine dependent methyltransferase and highlighting spirocyclopropane biosynthesis. J. Am. Chem. Soc. 134, 8831-8840. (17) Xu, H., Huang, W., He, Q. L., Zhao, Z. X., Zhang, F., Wang, R., Kang, J., and Tang, G. L. (2012) Self-resistance to antitumor antibiotic: a DNA glycosylase triggers the base excision repair system in yatakemycin biosynthesis. Angew. Chem. Int. Ed. 51, 10532-10536. (18) Dunn, Z. D., Wever, W. J., Economou, N. J., Bowers, A. A., and Li, B. (2015) Enzymatic basis of “hybridity” in thiomarinol biosynthesis. Angew. Chem. Int. Ed. 54, 5137-5141. (19) Carter, P., Fitzjohn, S., Halazy, S., and Magnus, P. (1987) Studies on the synthesis of the antitumor agent CC-1065. Synthesis of PDE I and PDE II, inhibitors of cyclic 15



adenosine-3',5'-monophosphate phosphodiesterase using the 3,3'-bipyrrole strategy. J. Am. Chem. Soc. 109, 2711-2717. (20) Boger, D. L., and Colemad, R. S. (1988) Total synthesis of (±)-N2-(phenylsulfonyl)-CPI, (±)-CC-1065, (+)-CC-1065, ent-(-)-CC-1065, and the precise, functional agents (±)-CPI-CDPI2, (+)-CPI-CDPI2, and (-)-CPI-CDPI2 [(±)-(3bR*, 4aS*)-, (+)-(3bR, 4aS)-, and (-)-(3bS, 4aR)-deoxyCC-1065]. J. Am. Chem. Soc. 110, 4796-4807. (21) Hiratsuka, T., Suzuki, H., Kariya, R., Seo, T., Minami, A., and Oikawa, H. (2014) Biosynthesis of the structurally unique polycyclopropanated polyketide-nucleoside hybrid jawsamycin (FR-900848). Angew. Chem. Int. Ed. 53, 5423-5426. (22) Rothery, R. A., Workun, G. J., and Weiner, J. H. (2008) The prokaryotic complex iron-sulfur molybdoenzyme family. Biochim. Biophys. Acta 1778, 1897-1929. (23) Schwarz, G., Mendel, R. R., and Ribbe, M. W. (2009) Molybdenum cofactors, enzymes and pathways. Nature 460, 839-847. (24) Unciuleac, M., Warkentin, E., Page, C. C., Boll, M., and Ermler, U. (2004) Structure of a xanthine oxidase-related 4-hydroxybenzoyl-CoA reductase with an additional [4Fe-4S] cluster and an inverted electron flow. Structure 12, 2249-2256. (25) Messerschmidt, A., Niessen, H., Abt, D., Einsle, O., Schink, B., and Kroneck, P. M. H. (2004) Crystal structure of pyrogallol–phloroglucinol transhydroxylase, an Mo enzyme capable of intermolecular hydroxyl transfer between phenols. Proc. Natl. Acad. Sci. U. S. A. 101, 11571-11576. (26) Boll, M. (2005) Key enzymes in the anaerobic aromatic metabolism catalysing Birch-like reductions. Biochim. Biophys. Acta 1707, 34-50. (27) Amishiro, N., Okamoto, A., Okabe, M., and Saito, H. (2000) Synthesis and antitumor activity of duocarmycin derivatives: modification at the C-7 position of segment-A of A-ring pyrrole compounds. Bioorg. Med. Chem. 8, 1195-1201. (28) Boger, D. L., Brunette, S. R., and Garbaccio, R. M. (2001) Synthesis and evaluation of a series of C3-substituted CBI analogues of CC-1065 and the duocarmycins. J. Org. Chem. 66, 5163-5173.

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Figure legends Figure 1. Structures of famous cyclopropabenzodipyrrole-containing compounds. (A) Previously established precursors and incorporation mode in CC-1065 biosynthesis. (B) Structures of yatakemycin (YTM) and duocarmycins. (C) Two ADC-drug candidates in phase I clinical trial.

Figure 2. Biosynthetic gene clusters and proposed pathways. (A) Key YtkT-catalyzed C-methylation involved in YTM biosynthesis. (B) Gene organization and comparison between CC-1065 and YTM clusters (see Table 1); identical genes are indicated by dashed lines. (C) Proposed biosynthetic pathway of CC-1065.

Figure 3. Genetic characterization of the key genes involved in CC-1065 biosynthesis. (A) HPLC analysis of CC-1065 related metabolites (UV at 374 nm): I) wild type (WT, S. zelensis NRRL-11183; II) mutant strain S. zelensis TG1402 (∆c10P); III) mutant strain S. zelensis TG1404 (mutant TG1403 harboring the c10C expression plasmid pTG1404); IV) mutant strain S. zelensis TG1403 (∆c10C). (B) Chemical structures of compounds isolated from the mutants. (C) Comparative cytotoxic activity of CC-1065 and 7-hydroxy-CC-1065 against different cell lines. The genotypes of all the mutants were confirmed by PCR analysis, and the results were summarized in Figure S2.

Figure 4. Characterization of the precursor incorporation mode by feeding of L-[2-13C]-Tyr. (A) The feeding result supports the “d”-pathway for Tyr incorporation and rules out the previously proposed “a”-pathway. (B) 13C-NMR spectra of CC-1065 by fermentation without (I) and with (II) L-[2-13C]-Tyr. (C) Revision of the feeding result and proposed the incorporation mode.

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Table 1 Deduced functions of ORFs in CC-1065 biosynthetic gene cluster AAa

Gene *

orf(-3)

121

orf(-2)*

454

orf(-1)*

209

c10R1

337

c10A c10B

Protein homolog (accession no.), origin

S/Ib(%)

Proposed function

Putative exported protein (CBG74657); S. scabiei 87.22 β-Lactamase-like protein (EFE71864); S. ghanaensis ATCC 14672 Isochorismatase-like protein (ABM10690); Arthrobacter aurescens TC1

92/84

Hypothetical protein

92/89

Hydrolase

76/62

Isochorismatase

78/66

Regulator

449 355

AraC family regulator (ABM10694); Arthrobacter aurescens TC1 Tryptophan synthase β subunit (ACA86296); S.w.c Molybdopterin oxidoreductase (ACA86297); S.w.c

72/57 52/36

c10C c10D c10E c10F

723 228 219 324

Molybdopterin oxidoreductase (ACA86297); S.w.c Methyltransferase type 11 (ACA86298); S.w.c Hypothetical protein Swoo2015 (ACA86299); S.w.c Hypothetical protein Swoo2011 (ACA86295); S.w.c

67/49 65/44 63/45 61/48

c10G

456

Coenzyme F390 synthetase (ACA86300); S.w.c

63/51

c10H c10I c10J

81 501 508

72/53 60/43 68/53

c10R2 c10K

1019 394

Hypothetical protein Swoo2017 (ACA86301); S.w.c Hypothetical protein Swoo2018 (ACA86302); S.w.c 4-Hydroxyphenylacetate 3-hydroxylase (ACA86303); S.w.c ABC transporter related (ACA86310); S.w.c Coenzyme F390 synthetase (ACA86294); S.w.c

71/52 49/30

c10L c10M c10N c10O c10R3 c10R4 c10P c10Q c10S c10T

311 273 390 283 272 324 457 234 296 498

Nitrilase/cyanide hydratase (ACA86293); S.w.c Nitrilase/cyanide hydratase (ACA86292); S.w.c Decarboxylase (ACA86291); S.w.c Nitrilase/cyanide hydratase (ACA86289); S.w.c ABC-2 type transporter (ACA86288); S.w.c ABC transporter (ACA86287); S.w.c Coproporphyrinogen III oxidase (ACA86286); S.w.c Methyltransferase type 11 (ACA86285); S.w.c Nitrilase/cyanide hydratase (ACA86284); S.w.c Radical SAM domain protein (ACA86283); S.w.c

65/48 56/38 67/53 66/48 63/47 65/45 84/67 76/53 72/52 81/65

c10R5

251

54/36

c10U c10V

349 130

67/49 74/62

c10W

595

72/60

Carbamoyltransferase

c10R6

207

DNA alkylation repair enzyme (ACJ29188); Shewanella piezotolerans WP3 Hypothetical protein Swoo1998 (ACA86282); S.w.c Hypothetical protein CY0110_02219 (EAZ92503); Cyanothece sp. CCY0110 Carbamoyltransferase (ADI12179); S. bingchenggensis BCW-1 Hypothetical protein (ABW10861); Frankia sp. EAN1pec

Tryptophan synthase NIpC/p60-like transpeptidase Oxidoreductase Methyltransferase Unknown NIpC/p60-like transpeptidase AMP-dependent synthetase/ligase Ferredoxin Dioxygenase 4-Hydroxyphenylacetate 3-monooxygenase Eexcinuclease A subunit AMP-dependent synthetase/ligase Hydrolase Hydrolase Decarboxylase Hydrolase Transporter Transporter Radical SAM enzyme Methyltransferase Hydrolase Radical SAM Methyltransferase DNA alkylation repair enzyme Unknown Unknown

71/59

Resistance protein

orf(+1)*

290

49/33

Urea transporter

orf(+2)*

258

72/62

Urease

a

Urea transporter (EFA91372); Prevotella buccalis ATCC 35310 Putative urease accessory protein (CAL99984); Saccharopolyspora erythraea NRRL 2338

Amino acid, b Similarity/Identity, c Shewanella woodyi ATCC 51908, *orfs beyond CC-1065 gene cluster.

19



Figure 1

A

OH

CO2H

1x

H2N41

NH2

HO

1c 38

3

2

1

11 9

O

6

8

25

24

12

17 18 23

N

22

13 14 16

7

1a

O

15

N

19

33M

O

27 29 28

26

O

NH2

OH

OH

20

OH

Ser

NH2

20M

OH

2 x Dopa

CO2H

3x

O

CC-1065 (1)

OH

O

NH

OH

2 x Tyr 1b & 1c

33 32

30

21

N H

NH2

OH

34

31

4

5

35

36

1b

10

HN

O

40

N39

37 3M

O

O

O

B 2c

S

O

OH O

O

O

O

2b

HN

2a

HN O

N H

O

O

N

HN

OH

N H

Cl

N

N

HN

O

Antibody

NH N

O O

O O

O

O HN Cl

N H

Duocarmycin SA (3)

Yatakemycin (YTM, 2)

C

O

N

O

O

OH O

Duocarmycin A (3')

HN

NH

N

N

linker

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

O N SYD985

linker Antibody

N

O O

MDX-1203 (BMS-936561)

20


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Figure 2

21



Figure 3

22


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Figure 4

23




Page 24 of 24

Unified Biosynthetic Origin of the Benzodipyrrole Subunits in CC-1065

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