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8-Deoxy-Rifamycin Derivatives from Amycolatopsis Mediterranei S699 ∆Rift Strain

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8-Deoxy-Rifamycin Derivatives from Amycolatopsis Mediterranei S699 ∆Rift Strain biomolecules Article 8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 DrifT Strain 1, 1, 1 1 2 1 Feng Ye y, Yanrong Shi y , Shengliang Zhao , Zhiying Li , Haoxin Wang , Chunhua Lu and Yuemao Shen 1,2,* 1 Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine, Shandong University, No. 44 West Wenhua Road, Jinan 250012, China; [email protected] (F.Y.); [email protected] (Y.S.); [email protected] (S.Z.); [email protected] (Z.L.); [email protected] (C.L.) 2 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China; [email protected] * Correspondence: [email protected]; Tel.: +86-531-8838-2108 Contributed equally to this work. y Received: 31 July 2020; Accepted: 31 August 2020; Published: 2 September 2020 Abstract: Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 DrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1–11) and seven known analogues (12–18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4–11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 µg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 µM, respectively. Keywords: rifamycin; Amycolatopsis mediterranei S699; proansamycin X; dehydrogenation; N-glycoside rifamycin 1. Introduction Ansamycins are a type of macrocyclic antibiotics formed by an aromatic moiety bridged at nonadjacent positions by an aliphatic chain [1,2], and exemplified by the antituberculosis rifamycin [3], antitumor maytansine [4,5] and the Hsp90 inhibitor geldanamycin [6]. These macrolactams are constructed by the multidomain modular type I PKSs (polyketide synthases) using 3-amino-5-hydroxybenzoic acid (AHBA) as the starter unit [7], followed by various post-PKS modifications. Rifamycins were first reported in 1957 from Amycolatopsis mediterranei S699 [8–10]. They have + good antibacterial activity against G bacteria and some G− bacteria [11]. The semi-synthetic rifamycin derivatives such as rifampicin are clinically used for the treatment of tuberculosis, adhesion and leprosy infection caused by Staphylococcus and other G+ bacteria [12,13]. In recent years, due to its widespread use, pathogens, especially Mycobacterium tuberculosis, have gradually developed resistance to rifampicin [14,15]. In order to increase the structural diversity of rifamycins, the mechanism of rifamycin biosynthesis has been continuously studied [16–18]. Biomolecules 2020, 10, 1265; doi:10.3390/biom10091265 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1265 2 of 14 Biomolecules 2020, 10, x 2 of 15 The rifT gene, located upstream of the PKS genes in the rifamycin biosynthetic gene cluster of The rifT gene, located upstream of the PKS genes in the rifamycin biosynthetic gene cluster of A. A. mediterranei S699, was proposed to encode a dehydrogenase [19] on the basis of bioinformatics mediterranei S699, was proposed to encode a dehydrogenase [19] on the basis of bioinformatics analysis. In this study, the mutant strain A. mediterranei S699 DrifT was constructed by deleting the rifT analysis. In this study, the mutant strain A. mediterranei S699 ΔrifT was constructed by deleting the gene (Supplementary Figures S1–S7). Systematical isolation of the fermentation products of the mutant rifT gene (Supplementary Figures S1–S7). Systematical isolation of the fermentation products of the strain afforded eleven new 8-deoxy-rifamycin derivatives (1–11) (Figure1) and seven known analogues mutant strain afforded eleven new 8-deoxy-rifamycin derivatives (1–11) (Figure 1) and seven known (12analogues–18) (Supplementary (12–18) (Supplementary Figure S8). Herein, Figure the S8). isolation, Herein, structure the isolation, elucidation structure and bioactivity elucidation of theseand eighteenbioactivity compounds of these eighteen are reported. compounds are reported. FigureFigure 1.1. Structures of new compounds 11––11.11. 2.2. Materials Materials andand MethodsMethods 2.1.2.1. StrainsStrains andand PlasmidsPlasmids AmycolatopsisAmycolatopsis mediterraneimediterranei S699S699 strainstrain was a gift from Prof. Linquan Linquan Bai Bai at at Shanghai Shanghai Jiaotong Jiaotong University.University. A.A. mediterraneimediterranei S699 DΔrifT strain was constructed by by deleting deleting the the rifTrifT genegene which which was was predictedpredicted to to be be responsible responsible forfor thethe dehydrogenationdehydrogenation of putative proansamycin X X [20–22]. [20–22]. The The strain strain waswas initially initially propagated propagated onon ISP2ISP2 agaragar mediummedium (4 gg/L/L yeast extract, 10 10 g/L g/L malt malt extract, extract, 4 4 g/L g/L glucose glucose andand 20 20 g g/L/L agar). agar). Then, Then, a a single single colony colony of of each each strain strain was was inoculated inoculated in in 50 50 mL mL of of ISP2 ISP2 medium medium with with 8 g of8 glassg of glass beads beads (Ø 3 (Ø0.3 3 mm) ± 0.3 in mm) a 250 in mL a ba250ffl mLed flask baffled and flask cultivated and cultivated at 28 ◦C in at a shaking28 °C in incubator a shaking at ± 200incubator rpm. Escherichia at 200 rpm. coli EscherichiaDH5α strain coli wasDH5 usedα strain as the was general used as cloning the general host andcloning grown host in and Luria-Bertani grown in (LB) medium. Cell stocks were prepared with 20% glycerol and stored at 80 C. Apramycin was Luria-Bertani (LB) medium. Cell stocks were prepared with 20% glycerol− and◦ stored at −80 °C. addedApramycin into media was added as an antibioticinto media at as a an final antibiotic concentration at a final of 50concentrationµg/mL for allof strains50 μg/mL in thisfor all study. strains PUC in thisSuicide study. vector pOJ260 [23] (containing aac (3) IV, oriT, rep , lacZ) was used throughout the study for in-frameSuicide genevector inactivation. pOJ260 [23] The (containing integrating aac vector (3) IV, pSET152 oriT, rep (containingPUC, lacZ) wasaac used (3) IV, throughout oriT (RK2), the ori (pUC18),study for int in-frame ('C31), gene attP inactivation. ('C31), lacZ αThe) was integrating used for vector gene expression pSET152 (containing in the DrifT::rifT aac (3) strain.IV, oriT (RK2), ori (pUC18), int (φC31), attP (φC31), lacZα) was used for gene expression in the ΔrifT::rifT strain. Biomolecules 2020, 10, 1265 3 of 14 2.2. DNA Manipulation 2.2.1. Gene Knock-Out The knock-out plasmid for the rifT gene was generated as the following steps. The ~2 kb upstream and downstream homologous arms of the target genes were amplified by polymerase chain reaction (PCR) using A. mediterranei S699 genomic DNA as a template, respectively. Purified PCR fragments were inserted into the linearized pOJ260 by Gibson assembly [24]. The assembled product was then transformed into 100 µL DH5α-competent cells. Positive clones were identified by restriction enzyme digestion (Supplementary Figure S1) and DNA sequencing. The knock-out plasmid was propagated in DH5α and transformed into A. mediterranei S699 competent cells by electroporation, as described by Ding et al. [25]. The apramycin-resistant recombinants resulting from the homologous recombination between the knock-out plasmid and genomic DNA of A. mediterranei S699 were selected (Supplementary Figure S2) and transferred to ISP2 agar for several rounds of nonselective growth. Apramycin-sensitive recombinants derived from double-crossover recombination were screened, from which the targeted gene knockout mutant was verified by PCR (Supplementary Figure S3). The specific process is shown in the Supplementary Information (Supplementary Figure S4). 2.2.2. Gene Complementation The integrating vector pSET152 [23] was used for gene complementation in the A. mediterranei S699 DrifT strain. Synthesized rifK promoter fragment was digested with NdeI and XbaI, and inserted into XbaI-pretreated pSET152 vector to yield pSET152-rifKp. The rifT gene was amplified by PCR using the genomic DNA of A. mediterranei S699 as a template. The NdeI/EcoRI rifT PCR fragment was inserted into the downstream of the rifKp promoter in pSET152. Positive clones were identified by restriction enzyme digestion and DNA sequencing. The resultant plasmid pSET152-rifKp-rifT was transformed into A. mediterranei S699 DrifT-competent cells by electroporation to obtain the rifT gene complementation mutant. 2.3. Detection and Analysis of the Metabolites in Mutants For rifamycins production, A. mediterranei S699 mutants were inoculated on ISP2 agar media (100 mL) and cultivated for 7 days at 28 ◦C. The culture was diced and extracted overnight with EtOAc/MeOH (4:1, v/v) at room temperature. The concentrated crude extract was dissolved in 1 mL MeOH, and analyzed by high-pressure liquid chromatography (HPLC; Agilent 1200). Chromatographic conditions were as follows: solvents: (A) water, and (B) CH3CN, samples were eluted with a linear gradient from 20% to 35% B in the first 5 min, increased to 55% B at 19 min, to 65% B at 20 min, to 100% B at 23 min, followed by 4 min with 100% B, flow rate was 1 mL/min, and UV detection at 254 nm. In order to specify compound peaks, the concentrated crude extract was analyzed by liquid chromatography–electrospray ionization–high-resolution mass spectrometry (LC-ESI-HRMS; Finnigan).
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