Poster: Metabolic engineering

Abs # 295: Site-directed mutagenesis influencing two separate allosteric sites of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic overproduction of isoleucine and contributes to the development of a novel selectable marker

Presenter:	Mourad, George S., mourad@ipfw.edu
Authors	Mourad, George S. (A)   Garcia, Eric L. (A)
Affiliations:	(A): Indiana-Purdue University, Department of Biology

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1, and three new site-directed mutants, omr1-5, omr1-7, and omr1-8 with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the C-terminal allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog L-O-methylthreonine (OMT) confirmed the desensitization to Ile feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7, appeared to influence one of the two Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5, omr1-7, and omr1-8 had a normal phenotype, undistinguishable from wild type. These alleles proved to be ideal as a dominant, environmentally-friendly, selectable marker for plant transformation.