Poster: Metabolic engineering

Abs # 283: Metabolic Engineering via the Chloroplast Genome to Produce p-Hydroxybenzoic Acid, a Principal Monomer of Liquid Crystal Polymers

Presenter:	Devine, Andrew L, adevine@mail.ucf.edu
Authors	Devine, Andrew L (A)   Khan, Muhammad S (A)   Deuel, Deborah L (B)   Van Dyk, Drew E (B)   Viitanen, Paul V (B)   Daniell, Henry  (A)
Affiliations:	(A): University of Central Florida (B): DuPont Experimental Station

The p-hydroxybenzoic acid (pHBA) is the major monomer in liquid crystal polymers (LCPs). E. coli chorismate pyruvate lyase (CPL) is an enzyme encoded by the ubiC gene that converts 1 mol of chorismate to 1 mol each of pyruvate and pHBA. In the present study the ubiC gene has been stably integrated into the chloroplast genome using plastid transformation. Transgenic tobacco plants were obtained by particle bombardment of chloroplast vectors, and site-specific integration and homoplasmy were confirmed by PCR and Southern blot analyses. The pHBA accumulated in the form of glucose conjugates, similar to the situation reported for nuclear-transgenic plants. The maximal pHBA level reached in individual leaf samples was 26.5% dry weight (DW). Maximal levels of monomer accumulation in the entire plant was 18.3% DW in the total leaf material and 3.1% DW in the total stalk, when fully mature plants were analyzed. The chloroplast transgenic lines were morphologically indistinguishable from control plants. The highest CPL enzyme activity observed in total leaf material was 50,783 pkat/mg of protein, which corresponds to ~35% of the total soluble protein. Leaf levels of pHBA and CPL enzyme activity were 50-fold and 240-fold higher, respectively, than values previously reported for nuclear-transgenic plants. These observations suggest that substrate availability becomes rate-limiting for pHBA production in plants at very high levels of CPL gene expression, at least in the leaf tissue. LCPs have a number of superior properties over conventional resins, but are currently too expensive to exploit in all of their potential applications. This is largely due to ingredient costs. The present study provides an alternate route to produce pHBA via metabolic engineering of the chloroplast.