Guidelines for High-Throughput Engineering of Nonribosomal Peptide Synthetase Libraries

Nonribosomal peptide synthetases (NRPS) are highly versatile enzymatic machineries responsible for the synthesis of complex peptides. These enzymes' characteristic modular architecture and synthesis pathway have prompted considerable research into their manipulation to generate new-to-nature peptides. Recent advances in the engineering of NRPS offer new avenues for application as a sustainable approach for the production of defined complex peptides, providing a viable alternative to the conventional chemical synthesis methods currently employed. In this protocol, we provide detailed guidelines for the modular assembly of different NRPS by using the eXchange Unit Thiolation domain (XUT) concept to generate hybrid NRPS. The engineering concept was adapted to modern Golden Gate assembly cloning that provides a flexible and sustainable way to assemble NRPS in high-throughput that produces predictable peptides.