Discovery and Biosynthesis of Atrovimycin, an Antitubercular and

Feb 18, 2019 - Sciences, Guangzhou 510650, China. ⊥. University of Chinese Academy of Sciences, Beijing 110039, China ..... 2018, 20, 1999−2002. (...
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Discovery and Biosynthesis of Atrovimycin, an Antitubercular and Antifungal Cyclodepsipeptide Featuring Vicinal-dihydroxylated Cinnamic Acyl Chain Qing Liu,†,‡ Zhiyong Liu,§ Changli Sun,‡ Mingwei Shao,‡ Junying Ma,‡ Xiaoyi Wei,# Tianyu Zhang,*,§,⊥ Wenjun Li,*,† and Jianhua Ju*,‡,⊥

Org. Lett. Downloaded from pubs.acs.org by UNIV PARIS-SUD on 04/08/19. For personal use only.

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State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275, China ‡ CAS Key Laboratory of Tropical Marine Bio-resources and Ecology, Guangdong Key Laboratory of Marine Materia Medica, RNAM Center for Marine Microbiology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China § State Key Laboratory of Respiratory Disease, Guangzhou Regenerative Medicine and Health Guangdong Laboratory (GRMH-GDL), Guangzhou Institutes of Biomedicine and Health (GIBH), Chinese Academy of Sciences (CAS), Guangzhou 510530, China # Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China ⊥ University of Chinese Academy of Sciences, Beijing 110039, China S Supporting Information *

ABSTRACT: Atrovimycin (1), a cyclodepsipeptide containing a unique vicinal-hydroxylated cinnamic acyl chain, was isolated and elucidated from Streptomyces atrovirens LQ13. The biosynthetic pathway of 1 was achieved, revealing cytochrome P450 (Avm43) and epoxide hydrolase (Avm29) enzymes constructing the vicinal-dihydroxy substitution, as well as a tailoring P450 (Avm28) enzyme catalyzing β-hydroxylation of the L-Phe moiety. Atrovimycin shows in vitro antifungal activity and antitubercular activity against Mycobacterium tuberculosis H37Rv both in vitro (with MIC of 2.5 μg/mL) and in vivo.

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LQ13. Although cyclodepsipeptides containing cinnamic acyl chains are known (Figure S2),15,16 those containing vicinaldihydroxy substitution have not yet been reported nor has any corresponding biosynthetic logic for their assembly been disclosed. Herein, we report the isolation, structure elucidation, and biosynthetic gene cluster for atrovimycin. Also identified are genes that encode two pre-NRPS assembly enzymes associated with vicinal-dihydroxy cinnamic acyl chain construction, as well as a post-NRPS assembly cytochrome P450 enzyme responsible for the hydroxylation of atrovimycin B (2). Streptomyces atrovirens LQ13 was isolated from a saline-alkali desert soil sample collected in the Xinjiang Uygur Autonomous Region of China. The crude extract of S. atrovirens LQ13 showed antifungal activity against the agricultural pathogenic fungus Fusarium. oxysporum f. sp. cucumerinum. A 40 L scale fermentation, extraction, and bioassay-guided purification led to the isolation of atrovimycin (1, Scheme 1 and Figure S1) (titer = 17.7 mg/L). Atrovimycin (1) was isolated as a white powder, and its molecular formula was determined to be C65H90O20N10 on the

atural products play a vital role in the discovery and development of new pesticides and drugs for human applications;1,2 actinomycetes, in particular from the extreme environments, represent a large source of bioactive natural products.3 Among them, cyclopeptides have played a prominent role in the development of therapeutics, vaccines, and diagnostic agents. Indeed, over 40 cyclopeptide drugs are currently in clinical use.4 Cyclopeptides excel as pharmaceutical agents by virtue of their favorable physicochemical properties and broad spectrum of biological activities.5−7 Many bacterially derived cyclopeptides are synthesized by nonribosomal peptide synthetases (NRPSs). NRPSs assemble peptides with large structural and functional diversity based largely upon their substrate recognition, as well as pre- and postassembly modification capacities.8−10 This feature of NRPS machineries provides tremendous opportunities for structure reconstruction, optimization, and drug discovery campaigns. With our efforts to expand the diversity and utility of natural products from the extreme environment-derived actinomycetes, a number of cyclopeptides were isolated from deep-sea derived actinomycetes.11−15 Recently, we discovered a new antitubercular and antifungal drug lead, atrovimycin (1) (Scheme 1 and Figure S1), from a desert-derived strain Streptomyces atrovirens © XXXX American Chemical Society

Received: February 18, 2019

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DOI: 10.1021/acs.orglett.9b00618 Org. Lett. XXXX, XXX, XXX−XXX

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a (A) Composition of the avm cluster in S. atrovirens LQ13; (B) predicted pathway for backbone construction in 1; (C) predicted HPG biosynthesis; (D) proposed “pre-NRPS” biosynthesis for the cinnamic acyl chain.

basis of the HRESIMS ion peak at m/z = 1329.6265 ([M − H]−, calcd 1329.6260, Figure S3). This data, combined with 1H and 13 C NMR data analyses, suggested that 1 contains 26 degrees of unsaturation. Detailed analyses of the complicated 1D and 2D 1 H and 13C NMR spectra implied that 1 contains one valine (Val), one 4-hydroxyphenylglycine (HPG), one phenylserine (β-OH-Phe), two threonines (Thr), two leucines (Leu), and three serines (Ser) (Table S1 and Figure S4). The redundant 14 13 C signals were ascribed to a 3-[2-(1,2-diol-3-(E)-pentenyl)phenyl]-2(E)-propenoic acid group due to clear HMBC correlations of H-2 with C-1/C-3, H-3 with C-4/C-5/C-9, H10 with C-4/C-8/C-9/C-11, and H-13 with C-11/C-12/C-14, together with 1H−1H COSY correlations of H-2/H-3, H-5/H6/H-7/H-8, and H-10/H-11 (Figure S4). The configurations of 2E, 12E were verified by the coupling constants17 of H-2 (J2,3 =

15.4 Hz) and NOESY correlations of H-11/H-13, H-12/H-14, respectively. The sequence of the amino acids and the planar (2D) structure of atrovimycin were elucidated by methanolysis and subsequent MS analysis. The compound was subjected to NaOMe/MeOH to yield the linearized methyl ester derivative (Figure S5C). (+)-HRESIMS/MS experiment from the parent ion at m/z 1363.7 ([M + H]+, Figure S5A) provided a variety of fragmentation ions for the derivative (Figure S5B). The linkage sequence for all residues was elucidated by scission of amido bonds in the MS/MS spectra (Figure S5C). LC−MS analyses of the derivatives generated from partial acid hydrolysis and subsequent reaction with L-FDAA further verified the amino acid sequence (Figure S6). The one additional degree of unsaturation and chemical shifts (Table S1) for H-3 and C-3 at B

DOI: 10.1021/acs.orglett.9b00618 Org. Lett. XXXX, XXX, XXX−XXX

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targeting mutagenesis method. Fermentation and HPLC-based extract analysis for this Δavm36 mutant strain revealed that Avm36 inactivation abolished the production of atrovimycin and related derivatives further demonstrating its indispensability to atrovimycin backbone biosynthesis (Figure 1A).

Thr implied the existence and linkage position of the cyclodepsipeptide. Absolute amino acid configurations of the amino acid residues in atrovimycin were determined using Marfey’s method and chiral-phase HPLC analysis; the results are summarized in Table S2. The two Leu and one HPG corresponded to D-amino acids; Val possessed the L-configuration; and phenylserine was found to have the (2S,3R) configuration (Table S3, Figures S7 and S8). Both L- and D-Ser were found in atrovimycin, and the peak area ratio was about 2.0, implying the existence of two L-Ser and one D-Ser in the structure of atrovimycin. The two threonines in 1 were confirmed as L-alloThr and D-alloThr following construction of their L-FDAA and L-FDLA derivatives. In order to elucidate the exact positions of these five amino acids, the fragments (Leu-Val-HPG-Ser)-L-FDAA and (Ser-Thr)-L-FDAA were prepared from the acid hydrolysates noted above. Further hydrolysis of the two fragments and Marfey’s analysis confirmed the D-Ser and D-Ser, D-alloThr in the two fragments (Figures S9 and S10), respectively. Thus, the structure of atrovimycin was assigned as 3-[2-(1,2-diol-3-(E)-pentenyl)phenyl]-2(E)-propenoic acid-[L-alloThr-L-Ser-D-Leu-(2S,3R)-β-OH-Phe-L-Ser-DalloThr-D-Ser-D-HPG-L-Val-D-Leu-O]. The configurations of C-10 and C-11 in the cinnamic acyl chain were determined by chemical derivatization and electrical circular dichroism (ECD) calculations (Table S4).18 Reaction of atrovimycin (1) with 2,2-dimethoxypropane afforded acetonide derivative atrovimycin A-1 (1a) (Figure S12A). The NOESY correlations of H-10/H-11/H-17 and the coupling constant of J10,11 (br. s) implied the relative configuration as 10R*,11S*. The negative Cotton effect at 280 nm in the CD spectrum supported the absolute configurations of 10R,11S by calculation; the experimental ECD data for 1a at 280 nm was in good accordance with the values calculated for (10R,11S)-N-methyl-3-[2-(1,2diol-3-(E)-pentenyl)phenyl]-2(E)-acrylamide (1b) (Figures S11−S13). Whole genome sequencing of S. atrovirens LQ13 was carried out using a combination of second generation 454 and third generation PacBio sequencing technologies to facilitate identification of the putative atrovimycin biosynthetic gene cluster. Bioinformatic analyses of the S. atrovirens LQ13 genome data using online antiSMASH software revealed a putative avm cluster with 51% similarity to skyllamycin (sky) cluster.16b Open reading frame (ORF) identifications and gene annotation using FramePlot 4.0beta software and NCBI searches made clear that the putative avm cluster encodes four discrete NRPSs for peptide core construction (Avm30, Avm31, Avm35, and Avm36), four β-ketosynthases [type II PKSs] responsible for cinnamic acyl chain precursor synthesis (Avm17, Avm20, Avm21, and Avm22), two cytochrome P450 enzymes (Avm28 and Avm43), six regulatory proteins (Avm4, Avm5, Avm7, Avm12, Avm13, and Avm25), three transport proteins (Avm26, Avm32, and Avm33), and other proteins having unclear biosynthetic functions (Scheme 1 and Table S5). At the same time, a cosmid library of S. atrovirens LQ13 genomic DNA was constructed, using the SuperCos1 vector system; we picked up 2496 clones in total. The four discrete NRPS genes within the avm cluster were analyzed using online PKS/NRPS analysis software. The results showed that the four genes encode a total of 11 modules (Scheme 1B), and the substrate-specific sequence of binding pockets of the NRPS A domains are listed in Table S8. Avm36 containing two domains composed of A-ACP is the starting module responsible for the Thr activation step (Table S8). A Δavm36 mutant strain was constructed using PCR-

Figure 1. (A) HPLC analysis of the wild-type and mutant strains. (B) HPLC-HRESIMS extracted ion chromatogram (EICs) for [M − H]− ions corresponding to the compounds of the mutant strains Δavm29 and Δavm43. (C) Proposed structures of polyene 19 (Δavm43 adduct) and epoxide 20 (Δavm29 adduct).

The atrovimycin gene cluster contains four genes, avm37, avm39, avm40, and avm41, homologous to genes encoding NAD(P)-dependent oxidoreductase, 4-hydroxyphenylpyruvate dioxygenase, aminotransferase, and prephenate dehydrogenase, respectively (Table S5); these four enzymes are organized into the biosynthetic pathway of HPG moiety (Scheme 1C).19 Gene inactivation trials revealed that mutant strains Δavm37, Δavm39, and Δavm40 failed to produce atrovimycin, whereas the Δavm41 strain still generated atrovimycin, but in severely limited titers (Figure S14). These data indicate that avm41 is not essential for 1 biosynthesis, providing that 4-hydroxyphenylpyruvate 15 from primary metabolism is available. In avm gene cluster are located four ketosynthases, avm17, avm20, avm21, and avm22 with sequence homologies to sky22 (69% similarity), sky19 (59% similarity), sky18 (74% similarity), and sky17 (84% similarity), respectively (Scheme 1A and Table S5). The ketosynthases encoded by four sky genes along with reductase and dehydratase are proposed to catalyze formation of the polyene precursor.16b In vitro experiments20 revealed that the polyene-producing type II PKSs, Iga11 (79% similarity to Avm22), and Iga12 (60% similarity to Avm17), possessed the ability to generate highly reduced polyketides. As predicted, inactivation of avm20 and avm21 afforded strains unable to produce atrovimycin and related derivatives (Figure S14). The cinnamic acyl precursor of atrovimycin was envisioned to employ malonyl-ACP 5 as a building block, generated by the carboxylation of acetyl-CoA 3 to afford malonyl-CoA 4 by carboxyltransferase Avm10 or acyl-CoA carboxylase Avm11 (or both). Transfer of 4 to the ACP, catalyzed by acyltransferase Avm6 or Avm34 (or both) (Scheme 1D), is in keeping with standard biosynthetic logic. Continued inactivation efforts entailed disruption of avm6, avm10, avm11, and avm34 respectively. Notably, none of these inactivations abolished atrovimycin production by the respecC

DOI: 10.1021/acs.orglett.9b00618 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters tive mutant strains (Figure S14). Interestingly, this finding suggests that genes associated with the transformation of 3 to 5 (Scheme 1D) may have compensatory genes elsewhere within the genome. Repetitive condensation by Avm17, Avm20, Avm21, and Avm22, substrate reduction by Avm16, and dehydration by Avm42 affords C14-polyene intermediate 9. Consistent with this idea, inactivation of avm16 afforded a strain unable to generate atrovimycin and its derivatives. The conversion of the linear polyene to an aromatic ring requires double bond isomerization and aromatization; Avm15 showed 88% similarity to a putative isomerase in Streptomyces sp. W007. Thus, Avm15 likely plays a role in generating the (4E,6Z,8E)product 10, as well as the subsequent aromatized species. Not surprisingly, the avm15 deletion mutant strain failed to produce atrovimycin, thus supporting the indispensability of functional Avm15 in 1 biosynthesis (Figure S14). The cinnamic acyl chain in atrovimycin possesses an vicinaldihydroxy moiety, suggesting involvement of multiple enzymecatalyzed steps. From the avm gene cluster, we identified two enzyme candidates, Avm29 and Avm43, likely involved in vicinal-dihydroxy installation. Avm29 showed 68% similarity with epoxide hydrolase from S. sp. WM4235, and Avm43 showed 71% similarity with a cytochrome P450 from S. rapamycinicus NRRL5491 (Table S5). Both Δavm29 and Δavm43 mutant strains were constructed. Notably, fermentation and HPLC-based extract analyses revealed the failure of both mutant strains to generate 1 or its derivatives. It was consequently speculated that the vicinal-dihydroxyl group of the cinnamic acyl chain is pre-NRPS assembled rather than a posttailoring modification. Further HPLC-HRESIMS analyses of crude extracts of Δavm29, Δavm43, and wild-type strains (Figures 1A and S15) revealed that both the Δavm29 and Δavm43 strains yielded trace amounts of atrovimycin analogue 19 with an [M − H]− at m/z = 1295.6 Da, consistent with a pendant (1E,3E)-1,3-dien-pentenyl group instead of the usual cinnamic acyl side chain (Figure S16). Similarly, the Δavm29 mutant produced small amounts of an analogue with [M − H]− at m/z = 1311.6 Da, consistent with epoxide 20 (Figure 1B). It is proposed that Avm43 catalyzes an epoxidation to afford intermediate 12, and Avm29 serves to hydrolyze 12 en route to diol 13 (Scheme 1D). The putative avm cluster contains two cytochrome P450 encoding genes (avm28 and avm43) initially suspected of coordinating Phe hydroxylation following NRPS assembly of the atrovimycin core. To interrogate this idea early on, two mutant strains Δavm28 and Δavm43 were constructed and fermented, and extracts from each respective fermentation were then subjected to metabolomic analyses. As noted earlier, efforts aimed at understanding avm43 revealed its importance with respect to cinnamic acid vicinal-dihydroxylation. Consequently, our interest in Phe β-hydroxylation focused more heavily on avm28. HPLC analyses of each extract revealed that avm28 inactivation abolished the production of atrovimycin (1) while affording a closely related species (Figure 1A). From the Δavm28 mutant strain extract, a new analogue, atrovimycin B (2), with a molecular formula of C 65 H 90 O 19 N 10 was unambiguously identified. Careful comparisons of 1D and 2D 1 H and 13C NMR data with those of atrovimycin (1) (Table S1) revealed the replacement of the β-OH-Phe in 1 with a simple Phe in 2. As might be predicted, Marfey’s analysis with Δavm28 mutant adduct 2 revealed the absolute configuration of the new residue to be L-Phe (Tables S2 and S3, Figures S7 and S8).

These realizations made clear that Avm28 serves the role of a tailoring hydroxylase during atrovimicin biosynthesis. A series of bioactivity assays was carried out to assess the biological activities of atrovimycins. In determining potential antifungal activities of atrovimycin, we employed seven phytopathogenic fungi as fungal targets using a standard agar medium assay (Table S9).21 Data obtained for atrovimycin indicated a high level of activity against Fusarium. oxysporum f. sp. Cucumerinum, the causative agent driving cucumber fusarium wilt disease; we measured an IC50 value of 6.99 μM, which is more effective than the positive control, cycloheximide (8.17 μM). We also evaluated compounds 1 and 2 for antitubercular activities22 against M. tuberculosis H37Rv using rifampicin (MIC = 0.25 μg mL−1) as a positive control. The MICs of both 1 and 2 were 2.5 μg mL−1. Based on the in vitro activities, we further evaluated in vivo antitubercular activity of atrovimycin. We performed a dose escalation efficacy assay (100 mg kg−1 and 300 mg kg−1) with 1 in BALB/c mice infected with M. tuberculosis H37Rv by aerosol route for 12 d. Rifampicin (10 mg kg−1) and ethambutol (100 mg kg−1) were used as positive controls, compared to the untreated group (CMC-Na solvent); treatment with 300 mg kg−1 of 1 resulted in a 0.86 log10 reduction in colony forming units (CFUs) with respect to bacterial burden in lung (Figure 2). Especially notable is that the in vivo activity displayed

Figure 2. In vivo efficacy assay showing bacterial lung burdens (log10 CFU) in mice infected with M. tuberculosis H37Rv. (D0, the day treatment initiated; Ut, the negative control group treated with CMCNa solvent; RIF, rifampicin; EMB, ethambutol; dosage, mg kg−1; qd, daily; bid, twice a day.) Mice received treatment for 12 continuous days. Data are expressed as mean ± SD of five samples. Statistical analysis was performed using unpaired Student’s t test. ***, P < 0.0001. NS: insignificant.

by 1 is essentially identical to that observed for the first line therapeutic ethambutol; moreover, mice treated with 1 showed no signs of toxicity while showing clear therapeutic benefits. In summary, a novel cyclodepsipeptide, atrovimycin A (1), was isolated from the fermentation broth of Streptomyces atrovirens LQ13. Subsequently, from one of its mutant strains (Δavm28), an atrovimycin analogue, atrovimycin B (2), was isolated and elucidated. Because both compounds 1 and 2 showed impressive antitubercular activities and compound 1 also displayed antifungal activity against the plant pathogen Fusarium oxysporum f. sp. cucumerinum, we can highlight the potential application of atrovimycins as important antitubercular and antifungal drug leads. Moreover, this work expands the compound library of cinnamic acid-modified cyclodepsipeptides and provides significant insight into pre- and postassembly line chemistries and unique structural units with respect to depsipeptide natural products. Most importantly, the elucidation of atrovimycin structures and biosynthetic machineries D

DOI: 10.1021/acs.orglett.9b00618 Org. Lett. XXXX, XXX, XXX−XXX

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illuminates new approaches by which to engineer and produce new drug candidates for increasingly challenging diseases.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.9b00618. Experimental details, figures, tables, and 1D and 2D NMR spectra of 1 and 2 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Tianyu Zhang: 0000-0001-5647-6014 Jianhua Ju: 0000-0001-7712-8027 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (81425022, U1501223, U1706206), the Chinese Academy of Sciences (XDA11030403, YJKYYQ20170036), and the Guangdong NSF (2016A030312014) to J.J., and Guangdong Province Higher Vocational Colleges & Schools Pearl River Scholar Funded Scheme (2014) to W.L. T.Z. received Science and Technology Innovation Leader of Guangdong Province (2016TX03R095). We thank support from the Guangzhou Branch of the Supercomputing Center of the Chinese Academy of Sciences, we also thank the equipment public service center (Mr. Y. Gao) and the analytical facility center (Ms. Sun, Ms. Zhang, Ms. Ma, Dr. Xiao, and Mr. Li) of SCSIO for helping with the microbial fermentor operation and recording spectroscopic data, respectively.

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DOI: 10.1021/acs.orglett.9b00618 Org. Lett. XXXX, XXX, XXX−XXX