ACS Chemical Biology were added dichloromethane (200 μL) and triﬂuoroacetic anhydride (50 μL), and the mixture was heated for 10 min at 110 °C. The reaction was allowed to cool and was dried under a stream of nitrogen. The residue was dissolved in methanol and subjected to GC−MS. Experimental parameters for GC−MS analysis are included in Supplementary Table S2. In vitro Generation of Catenulipeptin. AciKC-modiﬁed AciA peptide (30 μM) was incubated with 2 mM cysteamine in 20 mM Tris buﬀer, pH 8.2, at RT for 12 h. The formation of peptide-cysteamine adduct was monitored by MALDI-TOF MS. Trypsin (20 μg mL−1) was then added into the mixture, and the generation of catenulipeptin was monitored by ESI-MS. The mixture was further puriﬁed by reverse-phase high-performance liquid chromatography (RP-HPLC). HPLC factions containing catenulipeptin were combined and dried by lyophilization. Bioactivity of Catenulipeptin. Wild type S. coelicolor spores were spread on a YEME agar plate, pH 7. A ﬁlter paper containing only surfactin (50 μg) or a ﬁlter paper containing both surfactin (50 μg) and catenulipeptin (25 μg) was placed in the center of the plate. The plate was incubated for 4 days at 30 °C until aerial hyphae were visible outside a zone of bald halo caused by the presence of surfactin. Detailed experimental procedures are described in the legend of Supplementary Figure S9. ■ ASSOCIATED CONTENT *S Supporting Information Description of all molecular biology procedures, protein puriﬁcations, and supporting ﬁgures. This information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: vddonk@illinois.edu. Notes The authors declare no competing ﬁnancial interest. ■ ACKNOWLEDGMENTS The authors thank W. Tang for useful suggestions regarding the generation of catenulipeptin in vitro. This work was supported by the U.S. National Institutes of Health (GM 58822 to W.A.v.d.D.). 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