Engineering artificial photosynthesis based on rhodopsin for CO2 fixation

Abstract Microbial rhodopsin, a significant contributor to sustaining life through light harvesting, holds untapped potential for carbon fixation. Here, we construct an artificial photosynthesis system which combines the proton-pumping ability of rhodopsin with an extracellular electron uptake mechanism, establishing a pathway to drive photoelectrosynthetic CO 2 fixation by Ralstonia eutropha (also known as Cupriavidus necator ) H16, a facultatively chemolithoautotrophic soil bacterium. R. eutropha is engineered to heterologously express an extracellular electron transfer pathway of Shewanella oneidensis MR-1 and Gloeobacter rhodopsin (GR). Employing GR and the outer-membrane conduit MtrCAB from S. oneidensis , extracellular electrons and GR-driven proton motive force are integrated into R. eutropha ’s native electron transport chain (ETC). Inspired by natural photosynthesis, the photoelectrochemical system splits water to supply electrons to R. eutropha via the Mtr outer-membrane route. The light-activated proton pump - GR, supported by canthaxanthin as an antenna, powers ATP synthesis and reverses the ETC to regenerate NADH/NADPH, facilitating R. eutropha ’s biomass synthesis from CO 2 . Overexpression of a carbonic anhydrase further enhances CO 2 fixation. This artificial photosynthesis system has the potential to advance the development of efficient photosynthesis, redefining our understanding of the ecological role of microbial rhodopsins in nature.