Itzhak Kurek and Genhai Zhu

Verdia Inc.

200 Penobscot Drive

Redwood City, CA 94063

Phone: (650)298-5864

Fax: (650)298-5812

E-mail: itzhak.kurek@verdiainc.com

Inhibition of photosynthesis due to deactivation of Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) at moderate heat stress occurs in both C₃ and C₄ plants. Rubisco is the enzyme that catalyzes the rate-limiting step in the conversion of CO₂ into carbohydrate during photosynthesis. Under non-stress conditions Rubisco activase maintains Rubisco at high activation state by loosening the binding of tightly bound RuBP. At elevated temperatures, inhibition of net photosynthesis is linked with a decrease in the activation state of Rubisco primarily due to deactivation of Rubisco activase. Since Rubisco activase is a thermo-labile enzyme, we hypothesize that an improvement in its thermo-stability may increase the photosynthetic performance of plants grown under moderate heat stress condition and the overall biomass accumulation.

To improve thermo-stability of Arabidopsis Rubisco activase we have used the MolecularBreeding^TM Directed Molecular Evolution technology, and developed a high throughput screening for activation of Rubisco by Rubisco activase at high temperatures. After two rounds of DNA shuffling and screening of several thousand variants per round, six Rubisco activase clones exhibiting improved thermo-stability have been identified. The specific activity of the shuffled clones at 40^oC was similar to or higher than the enzyme activity of Arabidopsis Rubisco activase at 25^oC. Using the Deleteagene^TM technology we have isolated Arabidopsis Rubisco activase mutant that lacks the Rubisco activase gene (ruba^-) in order to test the effect of the shuffled clones in planta. Currently we are testing the photosynthetic performance and biomass accumulation of ruba^- lines transformed with thermo-stable proteins under normal, heat stress, and limited light conditions.