Molecular Cloning and Expression of a Secondary Alcohol Dehydrogenase From Nocardia Cholesterolicum NRRL 5767

Norcardia cholesterolicum NRRL 5767 (N. cholesterolicum NRRL 5767) is a well-known bacterium capable of transforming 95% of added oleic acid to a more valuable product, 10-hydroxystearic acid (10-HSA) (Appl Microbiol Biotechnol 32, 299–304, 1989). This reaction is catalyzed by oleate hydratase. However, a small amount of the 10-HSA is subsequently converted to 10-ketostearic acid (10-KSA) by secondary alcohol dehydrogenase (2o-ADH). Since 10-HSA is more valuable than 10-KSA in industry; 10-KSA is considered a side product which complicates downstream separation and purification of 10-HSA. It is attractive to hinder the production of 10-KSA through bacterium strain improvement by genetic engineering to block the production of 2o-ADH. The objective of this research is to clone, overexpress, and characterize functional recombinant 2o-ADH. An annotated 2o-ADH gene has been successfully amplified by PCR from genomic DNA of N. cholesterolicum NRRL 5767and sub-cloned into pET28a expression vector. The 2o-ADH pro...