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bioRxiv · 10.64898/2026.02.09.704528

Artificial Recombination of Nonribosomal Peptide Synthetases Rapidly Evolves Natural Products

Zhong, L.·Wang, X.·Shi, X.·Bai, X.·Liu, Y.·Chen, H.·Seenivasan, B.·Ji, X.·Yang, Q.·Li, S.·Mueller, R.·Tu, Q.·Zhang, Y.-M.·Bian, X.
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Abstract

Efficient engineering of nonribosomal peptide synthetases (NRPSs) is a key strategy for expanding valuable peptide natural products. Current bioinformatics-guided approaches are often constrained by a set of predefined fusion sites, remaining experimentally challenging in most NRPS systems. Here we present the Recombineering Accelerated Evolution (RACE), which harnesses Red/ET recombineering mediated partially matched homologous recombination to recapitulate recombination-driven NRPS evolution on a highly accelerated timescale. Application of RACE to six known NRPS gene clusters generated 830 recombinants and yielded over 600 novel peptides including novel bis-lipopeptides. These recombinants reveal 112 previously unrecognized recombination fusion sites, providing an extensive landscape for NRPS evolution and more useful resources for guiding NRPS engineering. The RACE establishes a new paradigm for programmable NRPS evolution and enables rapid discovery of bioactive peptides.

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Zhong, L., Wang, X., Shi, X., Bai, X., Liu, Y., Chen, H., Seenivasan, B., Ji, X., Yang, Q., Li, S., Mueller, R., Tu, Q., Zhang, Y.-M., Bian, X.. 2026-02-11. Artificial Recombination of Nonribosomal Peptide Synthetases Rapidly Evolves Natural Products. https://doi.org/10.64898/2026.02.09.704528

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