Poster: Photosynthesis

Abs # 366: Improving CO₂ fixation by directed evolution

Presenter:	Kurek, Itzhak , itzhak.kurek@verdiainc.com
Authors	Kurek, Itzhak  (A)   Zhu, Genhai  (A)   True, Thom  (A)   Zhang, Xin  (B)   Liu, Lu  (A)   Lassner, Mike  (A)
Affiliations:	(A): Verdia (B): Maxygen Inc.

Enhancing photosynthetic CO₂ fixation is a promising approach for increasing plant productivity. Rubisco, a gateway enzyme at the entry point of CO₂ into the biosphere, catalyzes the rate-limiting step of photosynthesis. Two of Rubisco’s properties present an opportunity for improvement: 1) it has a low catalytic turnover rate, and 2) it uses O₂ as an alternative substrate in catalyzing a wasteful side reaction. An additional approach to increasing plant photosynthesis lies in increasing the activation state of Rubisco. Its activation state is regulated by Rubisco activase, a thermolabile protein. At the elevated temperatures commonly observed on hot summer days, Rubisco activase loses its activity. This can result in the reduction of Rubisco’s activation state. We hypothesize that an improvement in the thermostability of Rubisco activase may increase photosynthesic rates at high temperatures. We are using in vitro evolution to improve the catalytic efficiency of Rubisco and to improve the thermostability of Rubisco activase. To improve Rubisco, we have used DNA shuffling to construct libraries of protein variants based on the Chlamydomonas rbcL and rbcS subunits. We have identified Rubisco variants which exhibit improved carbon fixation activity and which increase the growth rate of Chlamydomonas. We are currently characterizing these proteins using a newly developed LC-MS method, which directly measures the products of the carboxylase and oxygenase reactions. We have also constructed libraries of gene variants based on the Arabidopsis Rubisco activase. After screening several thousand variants, we identified multiple enzymes exhibiting improved thermostability. Currently we are testing these thermostable proteins in transgenic Arabidopsis plants.