Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases

Ishikawa, F.; Arata, I.; Uehara, T.; Kinoshita, K.; Nakanishi, Y.; Sone, K.; Kashima, T.; Kudo, F.; Eguchi, T.; Tarada, T.; Fushinobu, S.; Tanabe, G.; Miyanaga, A.

doi:10.64898/2026.07.20.739675

bioRxiv · 10.64898/2026.07.20.739675

Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases

Ishikawa, F.·Arata, I.·Uehara, T.·Kinoshita, K.·Nakanishi, Y.·Sone, K.·Kashima, T.·Kudo, F.·Eguchi, T.·Tarada, T.·Fushinobu, S.·Tanabe, G.·Miyanaga, A.

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Abstract

Nonribosomal peptide synthetases (NRPSs) assemble structurally diverse bioactive natural products through selective communication between adenylation (A) and carrier protein (CP) domains. Although rational rewiring of these protein-protein interactions could enable customized NRPS design, this remains challenging due to the dynamic nature of protein interfaces. Here we show that structure-guided interface reprogramming enables productive non-cognate A- CP pairings across enterobactin, vibriobactin, pyochelin, and vicenistatin biosynthetic systems. Engineered interactions between the A domains EntE, VibE, or PchD and the non-cognate CPs VinL or EntB were validated by biochemical, kinetic, and structural analyses. Reconstituted pathways containing engineered VibE, EntB, and EntF restored enterobactin production and increased yield to 2.2-fold that of the native EntE-EntB-EntF system. Interface-engineered PchD also enhanced production of a non-native salicylic acid-norspermidine conjugate. An X-ray structure and molecular dynamics simulations of an engineered non-cognate A-CP complex reveal recognition principles for programmable NRPS interface design.

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Ishikawa, F., Arata, I., Uehara, T., Kinoshita, K., Nakanishi, Y., Sone, K., Kashima, T., Kudo, F., Eguchi, T., Tarada, T., Fushinobu, S., Tanabe, G., Miyanaga, A.. 2026-07-22. Reprogramming protein interfaces between adenylation and carrier protein domains in nonribosomal peptide synthetases. https://doi.org/10.64898/2026.07.20.739675

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