Design and in vitro realization of carbon-conserving photorespiration

Significance Photorespiration limits plant carbon fixation by releasing CO 2 and using cellular resources to recycle the product of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) oxygenation, 2-phosphoglycolate. We systematically designed synthetic photorespiration bypasses that combine existing and new-to-nature enzymatic activities and that do not release CO 2 . Our computational model shows that these bypasses could enhance carbon fixation rate under a range of physiological conditions. To realize the designed bypasses, a glycolate reduction module, which does not exist in nature, is needed to be engineered. By reshaping the substrate and cofactor specificity of two natural enzymes, we established glycolate reduction to glycolaldehyde. With the addition of three natural enzymes, we observed recycling of glycolate to the key Calvin Cycle intermediate ribulose 1,5-bisphosphate with no carbon loss.