Chapter 20 Engineering Photosynthetic Enzymes Involved in CO2–Assimilation by Gene Shuffling

Enhancing photosynthesis is a promising approach for increasing plant productivity. Advances in plant transformation technology make it possible to manipulate photosynthesis by overexpressing particular genes for alleviating bottleneck steps, diverting the flux of Calvin cycle intermediates and photoassimilates, or introducing new enzymes and pathways that can positively influence photosynthesis. Furthermore, directed molecular evolution makes it possible to target selected key enzymes in photosynthetic pathways for modifying their specific catalytic or protein properties and tailoring them to best function under specified growth conditions. In this chapter, advances in directed molecular evolution technology and the use of gene shuffling methodology to modify Rubisco and Rubisco activase to enhance plant photosynthesis and growth are described. By shuffling the Chlamydomonas reinhardtii Rubisco large subunit and utilizing competitive growth selection, several mutated Rubisco enzymes with increased carboxylase activity or CO2/O2 specificity were identified. The mutations identified in the modified Chlamydomonas Rubisco variants were then introduced into the tobacco enzyme by site-directed mutagenesis. Enzyme kinetic assays indicated that the modified tobacco Rubisco enzymes displayed increased CO2/O2 specificity, carboxylase activity and reduced Km for CO2. Similarly, gene shuffling technology was used to generate several Arabidopsis thaliana Rubisco activase variants exhibiting improved thermostability in order to alleviate the inhibition of plant photosynthesis by elevated temperatures. The thermostable activase variants were then expressed in an Arabidopsis Rubisco activase deletion line created by fast-neutron mutagenesis. The positive effects of the shuffled thermostable Rubisco activase variants on Rubisco activation state, rates of photosynthesis, and growth under moderate heat stress were demonstrated.