Molecular cloning and functional identification of a high-efficiency (+)-borneol dehydrogenase from Cinnamomum...

Cinnamomum camphora (L.) Presl, rich in terpenoids, is an important commercial plant. The monoterpenes borneol and camphor are highly desired compounds that have been widely and diversely used in medicine and spices since ancient times. However, the key enzymes in the biosynthetic pathway of borneol and camphor in C. camphora remains unknown, which limits access to these natural products. Here, the chirality of borneol and camphor were identified in C. camphora leaves. Besides the main (+)-borneol and (+)-camphor, C. camphora also contains small amounts of (-)-borneol and (-)-camphor. Then, CcBDH3 - an efficient (+)-borneol dehydrogenase (BDH) - was identified that catalyzed (+)-borneol into (+)-camphor in the presence of NAD⁺. The K_m value was 25.1 μM with a k_cat value of 5.4 × 10^-3 s^-1 at pH 8.5 and 30 °C. CcBDH3, which also yields (-)-camphor from (-)-borneol as a substrate, had a K_m value of 36.9 μM with a k_cat of 2.1 × 10^-3 s^-1, and pH of 8.0 and temperature of 32 °C. We further compared the conformational specificity of two other reported BDHs, ZSD1 and ADH2, and found that ZSD1 had the highest conversion rate with (-)-borneol. These findings provide a new way for the production of camphor with various optical activities by metabolic engineering, and the identified camphor biosynthesis pathway provides the foundation for using genetic engineering to improve the production and purity of (+)-borneol in planta.