6. The main culture was ABC294640 nmr inoculated with 10 vol% of a starting culture, induced immediately with 0.3 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and grown

for 16 h at 30 °C. The cells were harvested, treated with DNase I, protease inhibitors (Roche) and MgCl2, and passed twice through a French pressure cell at 1000 psi. The cell lysate was centrifuged for 1 h at 4 °C and 30 000 g. The proteins were purified using a Ni-NTA gravity flow column (IBA) in 50 mM Tris/HCl buffer (pH 7.6) containing 1 M NaCl and eluted with 300 mM imidazole in the same buffer. The purified proteins were concentrated using Amicon Ultra Centrifugal Filter Devices (Millipore) and dissolved in 50 mM Tris/HCl (pH 8) containing 25 mM NaCl, 10% glycerol and 20 mM dithiothreitol. Protein concentrations were determined using a BCA™ Protein Assay Kit (Thermo Scientific). Expression plasmids for the carrier proteins KirAIIACP4, KirAIIACP5, KirAIIIPCP and KirBPCP were constructed as described in the Supporting Information. The carrier proteins were expressed in E. coli Rosetta2(DE3)pLysS at 23 °C by induction with 0.3 mM IPTG for 16 h.

The purification of the proteins was performed as described above. To analyze the function of KirP, in vitro phosphopantetheinylation assays were performed. The reactions analyzed by MS contained 20 μM KirP, 100–150 μM acyl or peptidyl carrier LGK974 protein (KirAIIACP4, KirAIIACP5, KirAIIIPCP or KirBPCP), Astemizole 300 μM CoA (or malonyl-/methylmalonyl-CoA), 50 mM MgCl2 and 50 mM Tris/HCl (pH 7.5) in a total reaction volume of 50 μL. The assays were incubated for 1.5 h

at 30 °C and then analyzed via HPLC-ESI-MS on a Reprosil Gold 300 C18 column 200 × 3 mm ID, 5 μm) in an Agilent HPLC-MS system. The analytes were separated by gradient elution as follows: (t0=40% B, t20=t35=100% B, Post-time 15 min 40% B; flow rate 500 μL min−1; injection volume 5 μL) using buffer A (0.1% formic acid) and buffer B (0.06% formic acid in methanol) as mobile phase. Mass spectra deconvolution was performed using the Zscore algorithm (Zhang & Marshall, 1998) implemented in magtran 1.03 (kindly provided by Dr Z. Zhang). For autoradiographic analysis, the reaction mixtures contained 5 μM KirP, 30 μM acyl or peptidyl carrier protein, 12.5 mM MgCl2, 50 mM Tris/HCl (pH 7.5) and 7 μM [1,3-14C](methyl)malonyl-CoA (50 mCi mmol−1/0.1 mM Ci mL−1). As a control, assays were performed without KirP. The reactions were incubated for 30 min at 32 °C and then quenched with 800 μL of cold acetone. The proteins were centrifuged and redissolved in sample buffer. The samples were loaded onto and separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. The proteins were then blotted onto a nitrocellulose membrane. The membrane was air-dried, and the signals were visualized by phosphorimaging with a GE phosphor screen. The kirP gene is encoded directly upstream of the kirromycin PKS gene kirAI.