Reengineering for robust photorespiratory bypass to improve photosynthesis and crop production.

Paul F. South1,2 Berkley J. Walker1,2 and Donald R. Ort1,2

1USDA-ARS Photosynthesis Research Unit, 2Institute for Genomic Biology, University of Illinois

Over the past 50 years improvements in grain crop yield potential has maintained food supplies as demand has increased. As climate changes globally and human population increases traditional methods of crop improvement have become less effective in adapting and improve agricultural production on ever limiting land and water availability. Improving photosynthetic efficiency has been a long standing goal toward increasing output and crop yield that has to date only played a minor role in crop improvements. At 25°C and current CO2 levels roughly
25% of Rubisco activity is the fixation of oxygen instead of carbon dioxide resulting in the
conversion of RuBP to one molecule of phosphoglycerate and one molecule of glycolate. C3 plants recover the carbon in glycolate through the C2 photorespiratory pathway. The C2
pathway uses energy in the form of ATP and reducing equivalents and loses fixed CO2 resulting in a reduction in photosynthetic efficiency by 30%. Synthetic biology has provided new opportunities in altering photorespiratory metabolism to improve photosynthetic efficiency.
Indeed metabolic bypasses to photorespiration have been generated and have demonstrated improvements in growth. Using the Golden Gate synthetic biology approach we have
assembled a series of multigene constructs that contain alternate metabolic pathways to bypass
photorespiration. In addition, we designed a screen based approach to test a range of standardized parts (promoters, terminators) in model plants Arabidopsis thaliana and Nicotiana tabacum. We have successfully transformed in large multigene constructs and have demonstrated detectable gene expression. Using a fluorescence based screen we tested single construct transformed lines for rescue of photorespiratory deficient plants in Arabidopsis. Our results indicate that large multigene constructs containing a metabolic bypass to
photorespiration can rescue changes in fluorescence caused by low CO2 stress in deficient plants. Determining robust photorespiratory bypass constructs can provide insight into next generation crops and our utilization of standard parts and fluorescent screening provide a new tool kit for plant synthetic biology to engineer improvements in photosynthetic efficiency.

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