Cell-Free Total Biosynthesis of Plant Terpene Natural Products Using an Orthogonal Cofactor Regeneration System
Undramaa Bat-Erdene, John M. Billingsley, William C. Turner, Benjamin R. Lichman, Francesca M. Ippoliti, Neil K. Garg, Sarah E. O’Connor, Yi Tang
Abstract
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.