| Summary: | The majority of marine microbes remain uncultured, which hinders the identification and mining of CO<sub>2</sub>-fixing genes, pathways, and chassis from the oceans. Here, we investigated CO<sub>2</sub>-fixing microbes in seawater from the euphotic zone of the Yellow Sea of China by detecting and tracking their <sup>13</sup>C-bicarbonate (<sup>13</sup>C-HCO<sub>3</sub><sup>-</sup>) intake via single-cell Raman spectra (SCRS) analysis. The target cells were then isolated by Raman-activated Gravity-driven Encapsulation (RAGE), and their genomes were amplified and sequenced at one-cell resolution. The single-cell metabolism, phenotype and genome are consistent. We identified a not-yet-cultured <i>Pelagibacter</i> spp., which actively assimilates <sup>13</sup>C-HCO<sub>3</sub><sup>-</sup>, and also possesses most of the genes encoding enzymes of the Calvin-Benson cycle for CO<sub>2</sub> fixation, a complete gene set for a rhodopsin-based light-harvesting system, and the full genes necessary for carotenoid synthesis. The four proteorhodopsin (PR) genes identified in the <i>Pelagibacter</i> spp. were confirmed by heterologous expression in <i>E. coli</i>. These results suggest that hitherto uncultured <i>Pelagibacter</i> spp. uses light-powered metabolism to contribute to global carbon cycling.
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