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A plug-and-play system for polycyclic tetramate macrolactam production and functionalization.

Anna Glöckle1, Sebastian Schuler1, Manuel Einsiedler1,2

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|January 10, 2025
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Summary

We developed a genetic plug-and-play system for producing polycyclic tetramate macrolactams (PoTeMs) and their derivatives. This system enables high-level heterologous expression and facilitates the study of PoTeM tailoring enzymes.

Keywords:
Combinatorial biosynthesisHeterologous expressionNatural productsPolycyclic tetramate macrolactamPromoter engineering

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Area of Science:

  • Natural Product Biosynthesis
  • Synthetic Biology
  • Enzymology

Background:

  • Polycyclic tetramate macrolactams (PoTeMs) are synthesized via an unusual iterative polyketide synthase/non-ribosomal peptide synthetase (iPKS/NRPS) machinery.
  • This process yields a common precursor with a tetramic acid group and two unsaturated polyene chains.
  • Pathway-specific tailoring enzymes, such as cyclization-catalyzing oxidases, generate structural diversity by modifying these polyene chains.

Purpose of the Study:

  • To establish a versatile genetic system for the high-level heterologous expression of PoTeMs.
  • To demonstrate the utility of this system for studying post-PKS/NRPS enzymatic modifications.
  • To enable the investigation of novel PoTeM biosynthetic pathways and tailoring reactions.

Main Methods:

  • Development of a genetic plug-and-play system using the iPKS/NRPS IkaA.
  • Screening of eight strong promoters for optimizing ikaA expression in Streptomyces hosts.
  • Application of the system to heterologous expression and study of PoTeM modifying genes (ptmD, ikaD, cftA).

Main Results:

  • Successful high-level heterologous expression of PoTeMs in different Streptomyces hosts.
  • Demonstrated general utility of the plug-and-play system for studying diverse tailoring enzymes.
  • Reconstruction of ikarugamycin biosynthesis and generation of five novel ikarugamycin derivatives.

Conclusions:

  • The developed plug-and-play system facilitates the reconstruction of PoTeM biosynthesis.
  • This platform enables the generation of PoTeM derivatives and aids in studying tailoring enzymes.
  • The system is expected to accelerate future research into PoTeM cyclization and tailoring reactions.