Heterologous Reconstitution of 4-Amidopentadienoate Biosynthesis Identifies the PKS Machinery Responsible for Warhead Formation
- 1Aarhus University
Abstract
The 4-amidopentadienoate (APD) motif is a rare and bioactive pharmacophore found in the cyclic lipodepsipeptide natural products BE-43547, rakicidins, microtermolides, and related compounds. Although several biosynthetic gene clusters associated with this family have been identified, the enzymatic origin of the APD warhead has remained unknown. Here, we investigated APD formation using NRPS engineering to install the unusual APD-associated polyketide synthase (PKS) module from the BE-43547 biosynthetic pathway into heterologous nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly lines expressed in Escherichia coli. The engineered hybrids produced the designed lipopeptide containing the APD moiety, demonstrating that the core biosynthetic machinery is sufficient for APD installation and that no trans-acting enzymes are required. Total synthesis of the target compound enabled structural validation by co-elution, MS/MS analysis, and NMR spectroscopy. Production titers of 59 ug/L were achieved in the heterologous host. Systematic mutagenesis further revealed that the conserved dehydratase His-Asp catalytic dyad is essential for APD formation, whereas an additional highly conserved histidine is dispensable. These findings establish the biosynthetic origin of the APD warhead and provide a foundation for incorporating this privileged pharmacophore into engineered peptide scaffolds.
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BibTeX
@misc{tvilum2026heterologous,
title = {{Heterologous Reconstitution of 4-Amidopentadienoate Biosynthesis Identifies the PKS Machinery Responsible for Warhead Formation}},
author = {M. Tvilum and J. L. Veiler and T. T. Paulsen and C. Kegler and C. B. Nielsen and M. Johannsen and T. B. Poulsen and H. B. Bode and T. Toerring},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.17.752105},
url = {https://www.biorxiv.org/content/10.64898/2026.09.17.752105v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Heterologous Reconstitution of 4-Amidopentadienoate Biosynthesis Identifies the PKS Machinery Responsible for Warhead Formation
AU - M. Tvilum
AU - J. L. Veiler
AU - T. T. Paulsen
AU - C. Kegler
AU - C. B. Nielsen
AU - M. Johannsen
AU - T. B. Poulsen
AU - H. B. Bode
AU - T. Toerring
PY - 2026
DA - 2026/09/17
PB - bioRxiv
DO - 10.64898/2026.09.17.752105
UR - https://www.biorxiv.org/content/10.64898/2026.09.17.752105v1
AB - The 4-amidopentadienoate (APD) motif is a rare and bioactive pharmacophore found in the cyclic lipodepsipeptide natural products BE-43547, rakicidins, microtermolides, and related compounds. Although several biosynthetic gene clusters associated with this family have been identified, the enzymatic origin of the APD warhead has remained unknown. Here, we investigated APD formation using NRPS engineering to install the unusual APD-associated polyketide synthase (PKS) module from the BE-43547 biosynthetic pathway into heterologous nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) assembly lines expressed in Escherichia coli. The engineered hybrids produced the designed lipopeptide containing the APD moiety, demonstrating that the core biosynthetic machinery is sufficient for APD installation and that no trans-acting enzymes are required. Total synthesis of the target compound enabled structural validation by co-elution, MS/MS analysis, and NMR spectroscopy. Production titers of 59 ug/L were achieved in the heterologous host. Systematic mutagenesis further revealed that the conserved dehydratase His-Asp catalytic dyad is essential for APD formation, whereas an additional highly conserved histidine is dispensable. These findings establish the biosynthetic origin of the APD warhead and provide a foundation for incorporating this privileged pharmacophore into engineered peptide scaffolds.
ER -