CENTENNIAL HONORS COLLEGE
Western Illinois University
Undergraduate Research Day 2015
Poster Presentation
Primers Design for Amplification of Putative Secondary Alcohol Dehydrogenase Gene
from Nocardia cholesterolicum NRRL5767
Joshua Diaz
Faculty Mentors: Jenq-Kuen Huang and Lisa Wen
Chemistry
Nocardia cholesterolicum NRRL5767 (NRRL 5767) is well known microorganism that converts oleic
acid to 10- hydroxystearic acid (10-HSA) with excellent yield (> 95% yield, w/w) and some minor 10ketostearic acid (10- KSA) (~5%, w/w) by oleate hydratase and secondary alcohol dehydrogenase,
respectively. This microorganism is a potential candidate for developing an industrial microbe for the
production of 10-HSA if the expression of the secondary alcohol dehydrogenase gene (2 -ADH) were
blocked. Blocking the expression of 2-ADH by genetic engineering approach should eliminate the
conversion of 10-HAS to 10-KSA therefore reducing the labor and operative costs for separation of 10HSA from 10-KSA. As a first step, the gene of 2- ADH from NRRL 5767 must be identified and
sequenced. The 2-ADH gene from Micrococcus luteus wiujh 20 (M. luteus wiujh20) (gene bank
accession number: E2D104_MICLU) and its functional recombinant enzyme have been studied in our
lab. M. luteus wiujh 20 and NRRL 5767 are different bacteria species. We used the gene sequence of 2ADH from M. luteus wiujh20 as query via BLAST (Basic Local Alignment Search Tool) to retrieve the
annotated 2-ADH genes from Nocardia species to design eight degenerate primers (four primerpairs).
These primer pairs will be used for amplification of the putative 2-ADH from genomic DNA of NRRL
5767 by Polymerase Chain Reaction (PCR). In this study, the design of degenerate primers by
bioinformatics and the preliminary data of PCR amplification will be presented.