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  • angiotensin ii receptor blockers br Acknowledgments This dat

    2018-10-29


    Acknowledgments This data-set was generated with the financial support of the Partnerships for Enhanced Engagement in Science (PEER) initiative from the United States Agency for International Development [Grant no. PGA-2000003446] to TP.
    Data, experimental design, materials and methods The data shown here substantiate the exploration of the mutated polyketide synthase which directs the biosynthesis of erythryomcin in Saccharopolyspora erythraea. In the sixth module of this polyketide synthase, the acyltransferase domain was recently mutated to accept propargylmalonyl-SNAC (2, Fig. 1) as substrate next to the native substrate methylmalonyl-CoA [1]. The critical mutation in the acyltransferase domain was V295A, located in the heart of the active center [2]. The mutation reduced the sterical hindrance on the substrate, allowing for the accommodation of 2. Now, we explored the substrate flexibility of the DEBS AT6 V295A variant using a number of thioester-activated and differently substituted malonic angiotensin ii receptor blockers derivatives. Biomolecular modeling was able to further the design and implementation of additional mutations in the active site of DEBS AT6, which decrease the steric constraints and improve the incorporation of the synthetic substrate 2 into the resulting polyketide. In this article, the synthesis of artificial extender unit analogs for polyketide biosynthesis and the mutagenesis of an acyltransferase domain for acceptance of these building blocks are described. Furthermore, the data on feeding experiments in S. erythraea are shown.
    General information Unless otherwise stated, materials for chemical synthesis were obtained from commercial suppliers (Sigma Aldrich, Alfa Aesar, Fluka, Acros) in the highest purity available and used without further purification. Solvents were dried following standard procedures [3]. Solvents used for extraction and chromatography were purchased from Thermo Fisher Scientific. Flash chromatography was carried out using Acros silica gel 60 (35–70μm mesh). Thin-layer chromatography (TLC) was performed on aluminum-backed, precoated silica gel (60 F245) from Merck with cyclohexane/EtOAc or DCM/MeOH mixtures as mobile phases. Spots were detected by staining with KMnO4 solution (5.0g KMnO4, 33g K2CO3, 10mL 5% aqueous NaOH in 500mL H2O) and subsequent heat treatment. NMR spectra were recorded by using a Varian Mercury 400 (400MHz, 1H; 100MHz, 13C) spectrometer and calibrated using residual undeuterated solvent as an internal reference. Data are shown in Supplementary File 1. High-resolution mass spectra were recorded on a LTQ Orbitrap with Accela HPLC-System (column Hypersil Gold, length 50mm, inside diameter 1mm, particle size 1.9μm, ionization method: Electrospray Ionization). Products were characterized by NMR (1H, 13C) and HRMS. For mass spectrometric detection the electrospray ionization was carried out in positive ionization mode by using a source voltage of 4kV. The capillary voltage was set to 18V, the capillary temperature to 275°C, and the tube lens voltage to 115V. Spectra were acquired in full scan centroid mode with a mass-to-charge range from 200 to 2000.
    S. erythraea NRRL-B-24071, S. erythraeaΔAT6hygR[1] and S. erythraea AT6* were used for fermentation. The alterations of the selected residues in the YASH motif [1] were accomplished by oligonucleotide-mediated mutagenesis and overlap-extension PCR using the Phusion Flash Master Mix (Thermo Fisher). Briefly, mutagenesis was achieved by performing PCR with designed oligonucleotide primers (Table 1) that include the desired mutation in their sequence (oligonucleotides 3 and 4) and flanking oligonucleotides (1 and 2) in a Piko™ Thermocycler with the following program: 3min denaturation at 99°C, 5 cycles of 15s at 99°C, annealing for 15s at 65°C and 40s extension at 72°C, 25 cycles of 15s 99°C, 40s at 72°C, and a final extension of 60s at 72°C. The EcoRV digested plasmid pKSSU89 was used as template [1].The PCR products were DpnI digested, purified and precipitated using SureClean (Bioline, German) and redissolved in water. The two overlapping fragments were fused together in a subsequent extension reaction. The inclusion of flanking primers 1 and 2 in the extension reaction allowed the amplification of the fused product by PCR: 3min at 99°C, 25 cycles of 15s at 99°C and 40s of 72°C, 60s of 72°C. The final PCR products were gel-purified and cloned into ScaI linerarized pKSSU96 via SLIC-MIX [7]. Insert-containing clones were identified by colony PCR and analysis of isolated plasmids. Identity of the plasmids was confirmed by DNA sequencing. The DEBS3-encoding plasmids carrying the desired mutations were transformed into E. coli ET12567/pUZ8002 and then conjugated into S. erythraea ΔAT6hygR. Conjugation and propagation of resulting clones was performed as in reference [8].