Production of optically pure (–)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved

Abstract (–)-Borneol is a bicyclic plant secondary metabolite. Optically pure (–)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (–)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (–)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (–)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s−1 mM−1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (–)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (–)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (–)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (–)-borneol in culture. (Patent pending).