Two-step enzymatic resolution process for large-scale production of (R)- and (S)-linalool

Optically pure linalool with (R)- and (S)-configurations is a pivotal chiral building block in the flavors and fragrances industry as well as in sustainable crop protection. However, current production of (R)- and (S)-linalool relies on plant extraction, which is severely restricted by the spatiotemporal differences in enantiomer distribution, leading to low yields and complex downstream separation processes. Herein, we developed a practical two-step batch resolution process based on the stereoselective linalool dehydratase-isomerase (LDI), isolated from Castellaniella defragrans strain 65Phen. Using the racemic linalool as substrate, (R)-linalool was obtained in the first batch, accompanied by the dehydration of (S)-linalool to β-myrcene. After fractional distillation, β-myrcene was stereoselectively hydrated back to (S)-linalool by LDI in the second batch. To overcome the thermodynamic constraints of enzymatic reactions and enhance the productivity, we further developed an in situ product removal strategy via selective adsorption using XAD-4 resin, which shifted the reaction equilibrium toward the formation of (S)-linalool and enhanced the hydration efficiency by 28.1-fold. Moreover, employing immobilized cells LDI@de-GA, we obtained 9.9 g of (R)-linalool with 98.0% enantiomeric excess (ee) value and 9.2 g of (S)-linalool with 97.3% ee from 20 g of racemic linalool, corresponding to a combined isolated yield of 95.5%. This work represents the first enzymatic resolution of linalool and establishes an environmentally friendly and sustainable approach for the synthesis of chiral linalool.