Pentaketide Ansamycin Microansamycins A–I from Micromonospora

Letter Cite This: Org. Lett. 2018, 20, 1058−1061

pubs.acs.org/OrgLett

Pentaketide Ansamycin Microansamycins A−I from Micromonospora sp. Reveal Diverse Post-PKS Modifications Jianxiong Wang,†,§ Wen Li,‡,§ Haoxin Wang,‡ and Chunhua Lu*,† †

Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, China ‡ State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China S Supporting Information *

ABSTRACT: Overexpression of the pathway-specific positive regulator gene mas13 activated the cryptic gene cluster mas, resulting in the isolation of nine novel pentaketide ansamycins, namely, microansamycins A−I (1−9). These results not only revealed a biosynthetic gene cluster of pentaketide ansamycins for the first time but also presented an unprecedented scenario of diverse post-PKS modifications in ansamycin biosynthesis.

A

nsamycins are a family of macrolactams with remarkable bioactivities exemplified by the anti-tuberculous agents rifamycins,1−3 antitumor agents maytansinoids,4−6 and geldanamycins.7−10 Ansamycins are biosynthesized by type I polyketide synthase (PKS) using 3-amino-5-hydroxybenzoic acid (AHBA) as the starter unit and tailored by various postPKS modifications.11 Through the AHBA synthase gene-based screenings, several cryptic ansamycin biosynthetic gene clusters were identified, one of which is the mas gene cluster of Micromonospora sp. HK160111 (Figure 1a).12 Bioinformatics analysis revealed that the mas gene cluster encoded the putative PKSs and amide synthase responsible for the biosynthesis of pentaketide ansamycins.12 Pentaketide ansamycins are few. The known ones are the antioxidant Q-1047,13,14 the lipoxygenase inhibitor tetrapetalones,15 the radical scavenger ansaetherones,16 cebulactams,17 and the macrodilactams juanlimycins (Figure S1).18 Detailed biosynthetic mechanisms of pentaketides were not reported for the biosynthesis of pentaketide ansamycins. Herein, we report the activation of the mas gene cluster and identification of nine novel pentaketide ansamycins, namely, microansamycins A−I (1−9) (Figure 2). The mas gene cluster was annotated to contain 27 open reading frames (Figure 1a and Table S2, GenBank no. MG018799). This cluster consists of a subset of AHBA biosynthetic genes (masH, masJ−N, and masG) and the genes (mas4, mas11, and mas12) encoding three type I modular PKSs associated with the mas10 gene encoding amide synthase; these © 2018 American Chemical Society

Figure 1. (a) Organization of the biosynthetic gene cluster of microansamycins; (b) HPLC profiles of the metabolites of Micromonospora spp. HK160111WT and HK160111mas13OE.

are sufficient for the biosynthesis of an ansamycin backbone. This ansamycin backbone was predicted to be a novel Received: December 26, 2017 Published: February 7, 2018 1058

DOI: 10.1021/acs.orglett.7b04018 Org. Lett. 2018, 20, 1058−1061

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of a single crystal obtained from MeOH (CCDC no. 1548031) (Figure 3).

Figure 3. Single-crystal X-ray structures of 1−3 and 5.

Figure 2. Structures of microansamycins A−I (1−9).

Microansamycin B (2) was obtained as a colorless crystal with [α]20 D = +125.36 (c 0.11, MeOH), UV λmax 271 nm, and the same molecular formula C17H21NO5 ([M + H]+, m/z 320.1492) and similar NMR spectra as that of 1. The difference between 2 and 1 was that the C9/C10 epoxy in 2 was opened and formed the C1/C9 ether bond in 1. The presence of C9/ C10 epoxy in 2 was confirmed by the upfield chemical shifts of C9 (δC 57.4) and C10 (δC 56.4) (Table S4). Finally, the structure of 2 with absolute configurations was fully determined by X-ray diffraction analysis of a single crystal obtained from ethyl acetate (CCDC no. 1555914) (Figure 3). Microansamycin C (3) was obtained as a colorless crystal. Its molecular formula was determined to be C17H19NO5 on the basis of the HR ESIMS m/z 318.1338 [M + H]+ (calcd for C17H20NO5+, 318.1336). NMR comparison with that of 1 and 2 indicated the presence of a quinone instead of a dihydroquinone moiety in 3. The C1/C10 ether bond was revealed by the HMBC correlation from H10 to C1 (Figure S5, Table S5). The structure and absolute configuration of 3 was fully determined by X-ray diffraction analysis of a single crystal obtained from MeOH (CCDC no. 1548032) (Figure 3). Microansamycin D (4) was obtained as a colorless oil with [α]20 D = +169.6 (c 0.10, MeOH), UV λmax 218 and 278 nm. The molecular formula of 4 was determined to be C17H21NO4 by the HR ESIMS at m/z 304.1543 [M + H]+ (calcd for C17H22NO4+, 304.1543) (Figure S33). A nine-carbon fragment from C3 to C11 was revealed by the 1H−1H COSY correlations (Figure S5, Table S6). The presence of a dihydroquinone moiety was determined on the basis of the HMBC correlations from H3, H5, and H7 to the corresponding carbons. C9/C10 and C11/C12 double bonds were assigned to be E-form by the NOE correlations between H9 and H11 and H10 and H12a and the large coupling constant (J = 15.2 Hz) between H9 and H10 (Table S6). The relative configuration of C6 was assumed to be the same as that of 1 and 2 on the basis of biosynthetic logic. The relative configuration of C4 was suggested on the basis of the NOE correlations of H4/H6 (Figure S5). However,

pentaketide scaffold (AHBA−C2−C2−C3−C3) based on retrobiosynthetic analysis of the substrate specificities of the PKS acyltransferases (AT) (Figure S1). Eight redox genes (mas1−2, mas5, mas7−9, mas14−15) and four regulatory factor genes (mas3, mas13, mas17, and mas20) were identified in the remaining part of the mas cluster. However, no ansamycins were isolated and detected by LC-MS analysis (Figure S4) from the fermentation products of the Micromonospora sp. HK160111 wild-type strain after medium optimization,12 indicating that the mas gene cluster was cryptic. The mas gene cluster was activated to afford the mutant HK160111mas13OE strain by overexpression of the transcriptional regulator gene mas13 (SARP), under the engineered kasOp* promoter (Figure S2),19 a strategy similar to that applied previously. 20,21 HPLC analysis revealed that HK160111mas13OE strain produced several extra components compared with that of the HK160111 wild-type strain (HK160111WT) (Figure 1b). Large-scale cultivation of HK160111mas13OE led to the isolation of nine novel ansamycins (1−9) (Figure 2). Microansamycin A (1) was obtained as a colorless crystal with [α]20 D = +122.2 (c 0.26, MeOH) and UV λmax 272 nm. The molecular formula of 1 was determined to be C17H21NO5 on the basis of the HR ESIMS m/z 320.1490 [M + H]+ (calcd for C17H22NO5+, 320.1492) (Figure S12). A nine-carbon fragment from C5 to C12a was established on the basis of the 1H−1H COSY correlations (Figure S5, Table S3). A dihydroquinone moiety was identified by the HMBC correlations from H3 to C1, C2, C4, and C5 and from H5 to C1 (Figure S5, Table S3). The linkages between C1 and C12−C15 and the presence of a γ-lactam ring were evidenced by the HMBC correlations from H12a and H14a to corresponding carbons. An ether bond between C1 and C9 was determined on the basis of the HMBC correlation from H9 to C1. Hence, a pentaketide ansamycin was suggested. Finally, the structure of 1 with absolute configurations was fully determined by X-ray diffraction analysis 1059

DOI: 10.1021/acs.orglett.7b04018 Org. Lett. 2018, 20, 1058−1061

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had features including C9/C10 and C11/C12 double bonds in E-form, an N-acetyl group, particularly, a meta-trisubstituted benzene ring. All these compounds were tested for the antimicrobial activities against Staphylococcus aureus ATCC 25923, Mycobacterium smegmatis MC2 155, and Candida albicans 5314. None of them showed inhibitory activities against these strains. Antioxidant activity of 1−9 was assayed by the DPPH radical scavenging method using vitamin C as the positive control,18,19 indicating that only 4 had moderate activity with IC50 of 0.85 mmol/L. Retro-biosynthetic analysis indicated that 1−9 were produced by employing AHBA as the starter unit, two malonyl-CoA and two methyl malonyl-CoA as the extenders to form the AHBA−C2−C2−C3−C3 pentaketide ansamycin backbone. This backbone was novel compared to the three known ones AHBA−C3−C3−C4−C3, AHBA−C3−C3−C3−C3, and AHBA−C2−C3−C2−C2 (Figure S1). Microansamycin PKSs are composed of four chain elongation modules and a loading module that are organized into three subunits Mas4, Mas11, and Mas12 (Scheme 1). Analysis of the domain compositions of each module revealed the structure of promicroansamycin. Structure comparison of promicroansamycin with that of 1−9 indicated diverse post-PKS modifications in the biosynthesis of microansamycins (Scheme 1). Sequence analysis revealed that Mas9 has 50.3% amino acid identity with that of Nam7, a hydroxylase involved in naphthalene ring formation of neoansamycins by catalyzing the hydroxylation of AHBA.22 Therefore, Mas9 was proposed to be responsible for the C1 hydroxylation of promicroansamycin (Scheme 1). Different absolute configurations of C14 in 1 (14S), 2 (14R), 3 (14R), and 5 (14S) were revealed on the basis of single-crystal X-ray diffraction analysis, indicating that keto−enol tautomerizations may occur in promicroansamycin and/or its hydroxylated products. Additionally, different absolute configurations were observed at C12 in 1 (12S), 2 (12S), 3 (12R), and 5 (12S), which was attributed to the

the relative configuration of C14 could not be determined yet. Interestingly, a pair of signals with a ratio of ∼5:1 was observed in the NMR spectra of 4 (Figures S27−S32, Table S6 and 6a), implying the tautomerization of C14. Microansamycin E (5) was obtained as a colorless crystal with [α]20 D = +158.6 (c 0.14, MeOH), UV λmax 288 nm. The molecular formula of 5 was determined to be C17H21NO5 by the HR ESIMS m/z 320.1491 [M + H]+ (calcd for C17H22NO5+, 320.1492) and 342.1307 [M + Na]+ (calcd for C17H21NO5Na+, 342.1312) (Figure S40). NMR comparison with that of 4 revealed that the C11/C12 and C9/C10 double bonds were hydrogenated and dihydroxylated, respectively. The C1/C10 ether bond was determined by the HMBC correlations from H10 to C1 (Table S7). Finally, the structure of 5 with absolute configurations was fully determined by X-ray diffraction analysis of a single crystal obtained from ethyl acetate (CCDC no. 1555915) (Figure 3). The structure of 6 was determined to be 11,12-bisdehydromicroansamycin E by NMR comparison with that of 5, which was consistent with the HR ESIMS data at m/z 318.1340 [M + H]+. The C11/C12 double bond was assigned to be E-form by the NOE correlation between H10 and H12a (Figure S5). Interpretation of the NMR spectroscopic data revealed that 7 and 8 had a substructure similar to that of 6 (from C1 to C13) (Tables S9 and S10). A 2-hydroxyacetyl moiety in 7 was assigned by the HMBC correlations from the oxymethylene protons at δH 4.04 (H2′) to C1′ (δC 172.8). A propionyl residue (C13, C14, and C14a) in 7 was evidenced by the HMBC correlations from H14a to C13 and C14 and the chemical shift of C13 at δC 204.9 (Figure S5, Table S9). The presence of a propionyl group in 8 was indicated by the HMBC correlations from H3′ to C1′ and C2′; however, that was an Nacyl group revealed by the chemical shift of C1′ at δC 175.0 (Table S10). The same relative configurations of 7 and 8 were confirmed by NMR comparison with that of 6 and the NOE correlation between H10 and H12a. By analogy with these NMR interpretations, the structure of 9 was determined and 1060

DOI: 10.1021/acs.orglett.7b04018 Org. Lett. 2018, 20, 1058−1061

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ACKNOWLEDGMENTS This research was supported in part by the National Natural Science Foundation of China (81673317, 81530091, 31570039), the Science Foundation of Two Sides of Strait (U1405223), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_17R68).

reduction of the C11/C12 double bond. Breakages of the ansa chains were observed between C13 and C14 in 8 and between C14 and C15 in 7 and 9, which may have occurred by retroClaisen cleavage of the β-dicarbonyl moiety of C13−C15. Alternatively, 9 could be conversed from promicroansamycin via amide hydrolysis, decarboxylation, and acetylation. Conversion of 6 to 7 may follow a similar procedure from 9, except for acylation with hydroxyacetyl instead of acetyl or acetylation followed by hydroxylation. Because 1−9 were isolated from 50 L cultures with the yields lower than 10 mg except for 5 (30 mg), 7−9 would be the products of nonspecific degradation of the corresponding ansamycins or ansamycin intermediates prematurely released from PKS, as observed in the biosynthesis of rifamycin B.23 No 1-oxy was observed in 9, implying either a relaxation of Mas9, a putative hydroxylase responsible for the hydroxylation of AHBA, or a reductive dehydration of an intermediate structurally similar to 4. Bioreductive capability of this microorganism was evidenced by the hydrogenations of phenol/quinone in 1, 2, and 4 and double bonds in 1−3 and 5, which was observed in other ansamycin producers previously.24 Overall, tailoring of the putative promicroansamycins to form 1−9 involved oxidations including hydroxylation and epoxylation, hydrogenations of phenol and/or quinone, keto−enol tautomerization, retro-Claisen cleavage, decarboxylation, and N-acylations (Scheme 1), indicating unprecedented diverse post-PKS modifications during the biosynthesis of microansamycins. This study has implications for future efforts to produce modified ansamycins by genetic engineering of these biosynthetic processes.25,26

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b04018. Methods, additional tables, and figures, including structure elucidation and NMR data and spectra for compounds 1−9 (PDF) Accession Codes

CCDC 1548031−1548032 and 1555914−1555915 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/ data_request/cif, or by emailing [email protected]. uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chunhua Lu: 0000-0002-3261-1020 Author Contributions §

J.W. and W.L. contributed equally to this work.

Notes

The authors declare no competing financial interest. 1061

DOI: 10.1021/acs.orglett.7b04018 Org. Lett. 2018, 20, 1058−1061