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

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.

Keywords: biocatalysis; cofactor regeneration; enzyme cascade; natural products; synthetic biochemistry.

Figure 1.

Cis-trans nepetalactol serves as the ten-carbon terpene core of strictosidine, the biosynthetic precursor to vinblastine, ibogaine, and ~3,000 additional monoterpene indole alkaloids.

Figure 2.

Biosynthesis of nepetalactol and nepetalactone along with possible shunt products. On pathway intermediates are boxed. Cofactor regeneration enzymes are only shown for main pathway reactions.

Figure 3.

One pot enzymatic synthesis of nepetalactol and nepetalactone using an orthogonal cofactor regeneration system. (A) GC-MS chromatograms for 10 mL-scale one-pot conversion of 6 mM geraniol to nepetalactol and nepetalactone. Final reaction contained 925.5 mg/L geraniol 1, 5 μM TfG8H, 10 μM FpR, 10 μM YkuN, 10 μM GOR, 0.5 μM ISY, 5 μM MLPL, 1 μM FumC, 10 μM MaeB, 100 μM NADPH, 100 μM NAD⁺ and 18 mM fumarate in BTP buffer (pH 9.0) unless otherwise specified. (i) starting material, 2 mM 1, (ii) 1.5-hour reaction with TfG8H, (iii) additional 1.5-hour reaction with TfG8H and an additional aliquot of 2 mM 1 added, (iv) additional 1.5-hour reaction with TfG8H and an additional aliquot of 2 mM 1 added, (v) 2-hour reaction after GOR was added to (iv), (vi) 2-hour reaction after ISY and MLPL were added to (v), (vii) 2-hour reaction after ISY/MLPL and NEPS1 were added to (v). Peak identities were deduced from GC-MS and by comparison to authentic standards (see Supporting Information). (B) Substrate (1, blue circle) and products' (2, orange star; 5, green diamond; 6, purple cross) concentrations measured over time, with timing of added enzymes indicated with arrows atop. The reaction condition is as specified above with starting 1 concentration of 957 mg/L (6.2 mM).