Autoflex 3

For expression of the NTR fragment, we used the N^pro(EDDIE) expression system, a mutant of the N‐terminal auto‐protease from classical swine fever virus [12 (link)]. The gene encoding N^pro(EDDIE)‐NTR was cloned into the pET‐11a plasmid vector (Novagen) using an In‐Fusion HD cloning kit (Takara Bio). E. coli BL21 (DE3) pLysS cells (Novagen) harboring the expression plasmid were cultured in LB medium, and gene expression was induced by IPTG. Subsequent purification using the N^pro(EDDIE) expression system followed the procedures described by Goda et al. [13 (link)]. The final purification step was AEX, using a HiTrap Q column. Protein purity was confirmed by SDS/PAGE. MALDI‐TOF MS measurements, using a Bruker Autoflex III mass spectrometer, showed that the molecular weight of the NTR fragment was 13 039, which matched well with the molecular weight calculated from the amino acid sequence (13 076). Protein concentrations were determined by measuring absorbance at 280 nm using a NanoDrop2000 UV–Vis spectrometer [molecular weight 13 076; molar extinction coefficient (ε) = 6400 m⁻¹·cm⁻¹].

To test whether FabW condenses acyl-ACPs with malonyl-ACP, the acyl-ACPs (C₆-ACP to C₁₀-ACP) were synthesized from the fatty acids, ATP, and E. coli holo-ACP by V. harveyi acyl-ACP synthetase (AasS), as described previously [29 (link),30 (link)]. The function of RsFabH or RsFabW in the initiation of fatty acid synthesis was tested in reaction mixtures containing 0.1 M sodium phosphate (pH 7.0), 0.1 μg each of EcFabD, EcFabG, EcFabZ, and EcFabI, 50 μM NADH, 50 μM NADPH, 1 mM β-mercaptoethanol, 100 μM acyl-CoA (or acetyl-CoA, or acyl-ACP), 100 μM malonyl-CoA, and 50 μM holo-ACP in a final volume of 40 μL. The reactions were initiated by addition of 0.1μg 3-ketoacyl-ACP synthase III (EcFabH, RsFabH, or RsFabW). After incubation for 2 h at 37°C, the reaction products were resolved by conformationally sensitive gel electrophoresis as described previously [29 (link),30 (link)]. To verify the products of RsFabW catalyzed reaction, 500 μL of the above reaction mixture was used, and acyl-ACPs derivatives were purified according to the method of Zhao et al. [31 (link)]. Their molecular masses were determined by matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) (Bruker Autoflex III; Freemont, CA, USA) according to the methods described previously [32 (link)].

Proton nuclear magnetic resonance (¹H-NMR) spectra were recorded on Bruker Avance 300 and 400 MHz (Zurich, Switzerland) spectrometers using DSMO-d₆ or CDCl₃ as the solvent. The ¹H-NMR were referenced to residual solvent signals at 7.26 ppm (CHCl₃) or 2.5 ppm (DMSO). Solid-state ¹³C-NMR spectra were collected on a JNM-ECZ600R (Akishima, Tokyo, Japan). The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum was collected on a Bruker Autoflex III (Karlsruhe, Germany) at Tsinghua University. Gas adsorption isotherm was measured on a Quadrasorb SI-MP (Florida, FI, USA) surface area analyzer. FTIR spectra were collected using a Thermo Nicolet Nexus470 FTIR instrument. The crystalline phase structure of all samples were observed on a Bruker-D8-Focus powder diffractometer with (Cu Kα irradiation λ = 0.15406 nm) in the range of 2 to 40° (2θ). Inductively coupled plasma mass spectrometry (ICP-MS) was collected on an Aglient 7800. GC-MS was collected on a SHIMADZU GCMS-QP2010 SE (Kyoto, Japan). The morphology and composition of samples were characterized on a field emission scanning electron microscope (FE-SEM, JEOL JSM-7500F, Akishima, Japan) coupled with an energy-dispersive X-ray spectroscope (EDS).