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).