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. 2020 May 8;11:2272. doi: 10.1038/s41467-020-16145-4

Fig. 2. Reconstitution of in-vitro lactazole A biosynthesis.

Fig. 2

a Primary amino acid sequence of LazA precursor peptide. bg Reconstitution of azole and Dha formation in FIT-Laz. LazA precursor peptide produced with the FIT system was treated with a combination of Laz enzymes as indicated in each panel and the reaction outcomes were analyzed by LC-MS. Displayed are brEIC LC-MS chromatograms and composite mass spectra integrated over a time period shaded in the corresponding chromatograms. See Methods and Supplementary Figs. 58 for details on reaction conditions and the explanation of brEIC chromatograms. hk Reconstitution of lactazole A biosynthesis in FIT-Laz. Displayed are LC-MS chromatograms (left to right: brEIC; nrEIC at m/z 1026.77 for LP-NH2 generated during the final macrocyclization step; overlaid nrEICs at m/z 701.20 for lactazole A shown in black, m/z 692.20 for Dha4-lactazole in green, and m/z 710.20 for Ser10-lactazole in olive) and overlaid mass spectra for lactazole A, Dha4-lactazole, and Ser10-lactazole. These results demonstrate that the order of enzyme addition is critical to the success of lactazole A biosynthesis.