Split inteins for generating combinatorial non-ribosomal peptide libraries

Engineering Non-Ribosomal Peptide Synthetases (NRPS) is a promising strategy for discovering new bioactive compounds, which can serve as valuable leads for drug development, such as new antibiotics. However, their engineering is hampered by the limited availability of molecular tools for the efficient heterologous expression of their large biosynthetic gene clusters. In fact, a single NRPS gene can already exceed the size limits of standard cloning vectors. In this study, we establish split inteins as a novel tool for NRPS engineering to enable the expression of single, covalently linked NRPS proteins from multiple plasmids and to perform cloning-free module swapping. Using the xenotetrapeptide synthetase as model system, we show that an NRPS can be split into three parts and reconstituted via trans-splicing using two orthogonal inteins at four different engineering sites. Based on this tripartite platform we build a library comprising 21 plasmids and generated 324 hybrid NRPS by combinatorial transformation. More than half were catalytically active, producing over 200 novel peptides. This intein-based technology provides a modular platform for generating natural product-like peptide libraries, expanding biocatalytically accessible chemical space.