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. Author manuscript; available in PMC: 2014 Mar 13.
Published in final edited form as: J Am Chem Soc. 2013 Mar 4;135(10):3752–3755. doi: 10.1021/ja311579s

Figure 2.

Figure 2

PCR targeting of pKZ11, harboring the biosynthetic genes necessary for A-74528 production. A) The 28.7 kb san gene cluster encodes for PKS and associated genes (red), tailoring genes (blue), resistance genes (purple), regulatory genes (green), and genes of unknown function (black). B) Genes encoding oxygenases and enzymes of unknown function were individually disrupted by PCR-targeting. Only sanJ was found to be essential for A-74528 biosynthesis. C) LC-MS analysis of extracts isolated from the mutant strains revealed the presence of A-74528 in most secondary metabolite extracts but not in pKZ11/ΔsanJ. The extracted ion chromatogram (EIC) for the positive ion of A-74528 ([M+H]+ 561.1–561.15) is shown on the Y-axis. Traces for pKZ11, ΔsanJ, ΔsanU, and ΔsanQ are extracts derived from liquid cultures followed by ammonium sulfate precipitation, and are analyzed at 50 mg/mL. Traces for Δsan3, ΔsanK, ΔsanL, ΔsanP, and ΔsanX are derived from agar plate cultures, analyzed at 25 mg/mL. See SI for experimental details.