Western Illinois University, Chemistry, Macomb, IL 61455
MOLECULAR CLONING OF FUNCTIONAL 3-HYDROXYACYL-COA DEHYDROGENASE FROM MICROCOCCUS LUTEUS WIUJH20, Jenq-Kuen Huang, Babu Ram Dhungana, Stephanie A. Robinson, and Lisa Wen, Western Illinois University, Department of Chemistry, Macomb, IL 61455, J-Huang3@wiu.edu
Soapstock is a by-product of edible oil refining process with low cash value. However, it is rich in oleic and linoleic acids which can be converted to value-added industrially useful compounds by certain microorganisms. Nocardia cholesterolicum strain NRRL 5767 (N. cholesterolicum NRRL 5767) is an organism known to convert oleic acid to 10-hydroxyoctadecanoic acid (10-OHOD) with the highest yield (85%) by the action of oleate hydratase. A side product of this process generated by secondary alcohol dehydrogenase (-ADH) is 10-oxo-octadecanoic (10-OXOD (10%), which complicates downstream separation and purification of 10-OHOD. The long-term goal is strain improvement of N. cholesterolicum NRRL 5767 by deleting or blocking 2-ADH to create an industrially useful microbe for the production of 10-OHOD from oleic acid. Micrococcus luteus WIUJH20 is used for 2-ADH purification due to its lack of oleate hydratase which hampers the purification of 2-ADH from N. cholesterolicum NRRL 5767. We have previously purified 2-ADH from M. luteus WIUJH20to homogeneity and determined its N-terminal sequence. Using BLAST search tool the 2-ADH gene has been identified as belonging to the 3-hydroxyacyl CoA dehydrogenase superfamily. The gene has been amplified by PCR using gene specific primers and genomic DNA of M. luteus template and cloned into pGEX2T vector. Moderate expression of the functional 2-ADH has been purified for substrate specificity and kinetics studies.
This research was supported in part by the grant from USDA CSREES 02-015470.