Reprogramming the enduracidin NRPS assembly line via thioesterase domain relocation
Year: 2026
Type: article
Abstract: The rational engineering of multi-megadalton nonribosomal peptide synthetase (NRPS) assembly lines remains a formidable challenge due to their immense size and complexity. Conventional module deletion often disrupts the native cyclization, limiting the generation of analogues with predictable macrocyclic cores. In this study, we identified an unusual tri-thioesterase (tri-TE) architecture in the enduracidin NRPS assembly line, comprising one type I thioesterase domain and two type II thioesterases that contribute to enduracidin biosynthesis and assembly-line function. To explore the potential of TE repositioning, we systematically relocated the terminal thioesterase domain (EndC_TE) within the 2.0 MDa enduracidin NRPS, generating a series of engineered assembly lines that produced macrocyclic peptides with programmed backbone lengths. Notably, MS/MS analyses indicate that EndC_TE retains a conserved regioselectivity across the derivatives, with macrocyclization occurring between the invariant second threonine residue and the C-terminal carboxylate across substrates of varying lengths. Overall, our work demonstrates that TE domain relocation is a feasible strategy for reprogramming the complex enduracidin NRPS assembly line, providing a useful reference for future engineering efforts. It enables the generation of macrocyclic derivatives with tailored ring sizes and highlights EndC_TE as a catalytic domain with potential for constructing cyclic peptide libraries.
Institution: Tianjin University of Science and Technology
Language: English
Subfield: Pharmacology
Field: Medicine
Domain: Health Sciences
Open Access status: gold
Estimated APC paid: $1,500
OpenAlex ID: W7165759717