Minisymposium 24: Metabolic Engineering

Abs # 38002: Metabolic shunt of myo-inositol reduces seed phytic acid accumulation in Brassica napus – Over-expression of the ice plant-derived gene for myo-inositol methyltransferase (IMT).

Presenter:	Georges, Fawzy , Fawzy.Georges@nrc-cnrc.gc.ca
Authors	Georges, Fawzy  (A)   Dong, Jinzhuo  (C)   Hussain, Atta  (A) (B)  Patterson, Thomas  (B)   Webb, Steve  (B)   Keller, Wilf A (A)
Affiliations:	(A): Plant Biotechnology Institute, National Research Council Canada (B): Daw AgroSciences, Inc., Indianapolis, IN (C): Monsanto Company
Web Site:	http://www.nrc-cnrc.gc.ca

The sequential phosphorylation of myo-inositol (MI) is believed to be the main biosynthetic pathway of phytic acid (myo-inositol 1,2,3,4,5,6-hexakisphosphate, IP6) in both animals and plants. Due to its high affinity towards various cationic species, such as proteins and mineral ions, the phytate molecule can form strong chelation complexes with these, rendering them inaccessible for utilization by monogastric animals. Minimizing the formation of plant phytate can either be accomplished through the action of phytic acid degrading enzymes (phytases) or by blocking the phosphorylation of myo-inositol at an early point of the synthesis. We have designed a molecular approach, in which the gene for myo-inositol methyltransferase (IMT), isolated from Mesembryanthemum crystallinum (ice plant), was transferred to Brassica napus (canola) with the aim of diverting MI to produce the corresponding methyl ether, ononitol. We presumed the latter to be inert towards phosphorylation by MI kinases since, to our knowledge no naturally occurring phosphorylated cyclitol methyl ethers have been reported. Thus, by over-expressing the IMT gene in canola under a variety of different promoters (e.g., CamV35S, napin and phaseolin) we were able to produce homozygous transgenic lines with 25% - 45% reduction in phytic acid contents. There were no obvious phenotypic abnormalities observed and all seeds were viable and germinated normally. In addition, there were no measurable yield penalties in either seed count or seed composition as a result. The presence of active IMT in these lines was confirmed by enzymatic assays performed in the presence of S-adenosyl-L-methionine (SAM) with crude protein extracts, which were able to convert MI to ononitol, as analyzed by HPLC. This activity appeared to be lacking in crude proteins extracted from wild type plants.