Engineering of an NADP+-dependent glycolate dehydrogenase through directed evolution and in vivo assessment of its applicability for decreasing plant photorespiration

• Applicant: Professor Dr. Martin Engqvist
• Subject Area: Plant Biochemistry and Biophysics
• Term: from 2010 to 2014
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 191228634

Photorespiration is a highly wasteful process that reduces yield in major crops such as potato, wheat and rice. Recent approaches to decrease photorespiration by introducing novel metabolic pathways in plant chloroplasts show great promise - increasing biomass accumulation as well as photosynthetic rates in Arabidopsis thaliana plants - but suffer from the lack of a suitable glycolate dehydrogenase (GDH) to catalyze the committing step. To fill this gap, the overall goal in this project is to engineer an NADP+-dependent GDH with high in vivo activity in plants and to test its efficiency for improving these pathways. The project comprises four specific aims. The first aim is to clone putative target genes in Escherichia coli and to evaluate their enzyme products. NADP+-dependent dehydrogenases from plants, yeast and bacteria will be screened for activity with glycolate. The best enzymes will be chosen as targets together with the A. thaliana glyoxylate reductase, an enzyme known to oxidize glycolate using NADP+ as co-factor. The second aim is to improve the GDH activity of target the enzymes. Directed evolution - a powerful method comprising cycles of mutation and selection - will be employed to improve target enzyme affinity and activity towards glycolate. In this process, beneficial mutations will be identified by sequencing, resulting in a deepened knowledge about the enzymes structure-function relationship. As a final step, each possible combination of beneficial mutations will be tested through PCR shuffling with subsequent screening. The third aim is to biochemically characterize the novel GDHs. Key properties such as pH- and temperature optimum, Km and Vmax with glycolate and related substrates will be determined for the best enzymes resulting from the directed evolution process. The fourth aim is to determine the in vivo efficiency of the engineered GDHs for catalyzing the committing step in pathways designed to reduce photorespiration in A. thaliana.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Frances H. Arnold, Ph.D.