Molecular Cloning and Characterization of Coniferyl Alcohol Acyltransferase (CFAT) from Ocimum Sp.

Abstract

The genus Ocimum serves as a large pool of metabolites including isoprenoids and phenylpropanoids. Phenylpropanoids, such as eugenol, isoeugenol, eugenol methylether, chavicol, anol etc., are products of the diverted pathway from the lignin biosynthesis. The phenylpropanoid biosynthesis use acetylated monolignols such as coniferyl and coumaryl acetate as their substrate. These acetylated monolignols in turn are synthesized by acyltransferases which are solely responsible in diverting the metabolic flux away from lignin biosynthesis towards phenylpropanoid biosynthesis. This study aims at studying a similar enzyme, coniferyl alcohol acyltransferase (CFAT) which catalyzes the synthesis of coniferyl acetate by using coniferyl alcohol as a substrate. Coniferyl acetate in turn serves as the unique substrate used by eugenol synthase (EGS) for eugenol biosynthesis. This study aims at developing an intensive understanding of the role of CFAT in regulating the levels of phenylpropanoids in Ocimum. Transcriptome data of Ocimum was analyzed to search for putative CFAT contigs. These contigs were analyzed and out of 3 selected sequences, contig_13604 was filtered out as the most promising candidate gene. Further, the contig was cloned and the full length gene was obtained using RACE PCR. Real time and semi-quantitative PCR results added to the understanding of expression levels of this gene across leaf (young and mature) and stem tissues of Ocimum. The work proceeded towards recombinant protein expression with enzyme activity assay and chemical characterization of enzyme underway.