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Item Gluconobacter oxydans Knockout Collection Sequencing DataSchmitz, Alexa M.; Pian, Brooke; Medin, Sean; Reid, Matthew C.; Wu, Mingming; Gazel, Esteban; Barstow, Buz (2021-08-17)Data in support of research: Rare earth elements (REEs) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REEs from mineral ores or recycled materials are costly and environmentally harmful1, and thus, more sustainable extraction methods require exploration. Bioleaching offers a promising alternative to conventional REE extraction2-4 and is already used to extract 5% of the world’s gold and ≈ 15% of the world’s copper supply5,6. However, the performance of REE bioleaching lags far behind thermochemical processes2,7-9. Despite this, to the best of our knowledge, no genetic engineering strategies have yet been used to enhance REE bioleaching, and little is known about the genetics that confer this capability. Here, we build a whole genome knockout collection for Gluconobacter oxydans B58, one of the most promising organisms for REE bioleaching10, and use it to comprehensively characterize the genomics of REE bioleaching. In total, we found 304 genes that notably altered the production of G. oxydans’ acidic biolixiviant, including 165 that held up under statistical comparison with the wild type. The two most impactful groups of genes involved in REE bioleaching have opposing influences on acid production and REE bioleaching. Disruption of genes underlying the synthesis of the cofactor pyrroloquinoline quinone (PQQ) and PQQ-dependent membrane-bound glucose dehydrogenase all but eliminates bioleaching. In contrast, disruption of the phosphate-specific transport system accelerates acid production and enhances bioleaching. We identified 6 disruption mutants that increase bioleaching by at least 11%. Most significantly, disruption of pstC, encoding part of the phosphate-specific transporter pstSCAB, enhanced bioleaching by 18%. Taken together, these results provide a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.