Exploring the Oxygenase Function of Form II Rubisco for Production of Glycolate from CO2

Abstract The oxygenase activity of Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) converts ribulose-1,5-bisphosphate (RuBP) into 2-phosphoglycolate, which in turn channels into photorespiration, resulting in carbon and energy loss in higher plants. We observed that glycolate can be accumulated extracellularly when two genes encoding the glycolate dehydrogenase of cyanobacteria Synechocystis sp. PCC 6803 were inactivated. This inspired us to explore the oxygenase function of Rubisco for production of glycolate, an important industrial chemical, from CO2 by engineered cyanobacteria. Since the oxygenase activity of Rubisco is generally low in CO2-rich carboxysome of cyanobacteria, we introduced Form II Rubisco, which cannot be assembled in carboxysome, into the cytoplasm of cyanobacteria. Heterologous expression of a Form II Rubisco from endosymbiont of tubeworm Riftia pachyptila (RPE Rubisco) significantly increased glycolate production. We show that the RPE Rubisco is expressed in the cytoplasm. Glycolate production increased upon addition of NaHCO3 but decreased upon supplying CO2. The titer of glycolate reached 2.8 g/L in 18 days, a 14-fold increase compared with the initial strain with glycolate dehydrogenase inactivated. This is also the highest glycolate titer biotechnologically produced from CO2 ever reported. Photosynthetic production of glycolate demonstrated the oxygenase activity of Form II Rubisco can be explored for production of chemicals from CO2.