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Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System

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  • Undramaa Bat-Erdene
  • John M. Billingsley
  • William C. Turner
  • Benjamin R. Lichman
  • Francesca M. Ippoliti
  • Neil K. Garg
  • Sarah E. O'Connor
  • Yi Tang


Publication details

JournalACS Catalysis
DateE-pub ahead of print - 23 Jul 2021
DatePublished (current) - 6 Aug 2021
Issue number15
Number of pages6
Pages (from-to)9898-9903
Early online date23/07/21
Original languageEnglish


Here we report the one-pot, cell-free enzymatic synthesis of the plant monoterpene nepetalactol starting from the readily available geraniol. A pair of orthogonal cofactor regeneration systems permitted NAD+-dependent geraniol oxidation followed by NADPH-dependent reductive cyclization without isolation of intermediates. The orthogonal cofactor regeneration system maintained a high ratio of NAD+ to NADH and a low ratio of NADP+ to NADPH. The overall reaction contains four biosynthetic enzymes, including a soluble P450, and five accessory and cofactor regeneration enzymes. Furthermore, addition of a NAD+-dependent dehydrogenase to the one-pot mixture led to ∼1 g/L of nepetalactone, the active cat attractant in catnip.

Bibliographical note

Funding Information:
This work is support by NIH R01AT010001 to Y.T. The plasmid used for expression of NoxE was a gift from Prof. James Bowie.

© 2021 American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

    Research areas

  • biocatalysis, cofactor regeneration, enzyme cascade, natural products, synthetic biochemistry

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