Biosynthetic Functionalization of Nonribosomal Peptides
journal contributionposted on 11.02.2021, 22:03 authored by David L. Niquille, Ines B. Folger, Sophie Basler, Donald Hilvert
Nonribosomal peptides (NRPs) are a therapeutically important class of secondary metabolites that are produced by modular synthetases in assembly-line fashion. We previously showed that a single Trp-to-Ser mutation in the initial Phe-loading adenylation domain of tyrocidine synthetase completely switches the specificity toward clickable analogues. Here we report that this minimally invasive strategy enables efficient functionalization of the bioactive NRP on the pathway level. In a reconstituted tyrocidine synthetase, the W227S point mutation permitted selective incorporation of Phe analogues with alkyne, halogen, and benzoyl substituents by the initiation module. The respective W2742S mutation in module 4 similarly permits efficient incorporation of these functionalized substrate analogues at position 4, expanding this strategy to elongation modules. Efficient incorporation of an alkyne handle at position 1 or 4 of tyrocidine A allowed site-selective one-step fluorescent labeling of the corresponding tyrocidine analogues by Cu(I)-catalyzed alkyne–azide cycloaddition. By combining synthetic biology with bioorthogonal chemistry, this approach holds great potential for NRP isolation and molecular target elucidation as well as combinatorial optimization of NRP therapeutics.
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Biosynthetic Functionalizationtarget elucidationpathway levelNRP therapeuticsbioactive NRPEfficient incorporationclickable analoguestyrocidine analoguesPhe-loading adenylation domaincombinatorial optimizationalkynemodule 4Trp-to-Ser mutationPhe analoguesW 2742S mutationstrategytyrocidine synthetaseposition 1assembly-line fashionelongation modulesreconstituted tyrocidine synthetaseNonribosomal Peptides Nonribosomal ...benzoyl substituentsinitiation modulebioorthogonal chemistryfunctionalized substrate analoguesNRP isolationW 227S point mutation