NRPS design guided by adenylation promiscuity Hajo Kries Leibniz Institute for Natural Product Research and Infection Biology (HKI), Germany Adenylation domains control the specificity of nonribosomal peptide synthetases (NRPSs), an important group of enzymes synthesizing numerous bioactive natural products. Despite great efforts invested in adenylation domain engineering in the past, progress has been restrained by the lack of suitable assays for the screening and characterization of mutants 1 . We have developed a h ydrox amate a ssay (HAMA) that detects multiple quenched products in a single reaction under substrate competition as in the cellular environment (Figure) 2 . Our assay takes advantage of hydroxylamine to quench activated carboxylates to form hydroxamic acids which are sensitively and specifically detected by UPLC-MS/MS in a multiplexed fashion. HAMA provides a fast and reliable method for simultaneously recording adenylation profiles with dozens of substrates. HAMA screening has been used to generate a promiscuous mutant of module SrfA-C from the surfactin NRPS and to profile hundreds of variants obtained by site-saturation mutagenesis (unpublished data). Dramatic specificity changes with several mutants are consistent with an important role of promiscuous enzymes for evolution. Harnessing promiscuity during directed evolution of adenylation domains will open avenues for engineering NRPS specificity towards biosynthesis of new bioactive peptides.
References 1. Stanišić, A.; Kries, H. Adenylation Domains in Nonribosomal Peptide Engineering. ChemBioChem 2019 , 20 , 1347–1356. 2. Stanišić, A.; Hüsken, A.; Kries, H. HAMA: A Multiplexed LC-MS/MS Assay for Specificity Profiling of Adenylate-Forming Enzymes. Chem.Sci. 2019 , 10 , 10395–10399.
P38F
© The Author(s), 2022
Made with FlippingBook Learn more on our blog