Bioleaching of rare earth elements (REEs), using microorganisms such as Gluconobacter oxydans, offers a sustainable alternative to environmentally harmful thermochemical extraction, but is currently not very efficient. Here, we generate a whole-genome knockout collection of single-gene transposon disruption mutants for G. oxydans B58, to identify genes affecting the efficacy of REE bioleaching. We find 304 genes whose disruption alters the production of acidic biolixiviant. Disruption of genes underlying synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the PQQ-dependent membrane-bound glucose dehydrogenase nearly eliminates bioleaching. Disruption of phosphate-specific transport system genes enhances bioleaching by up to 18%. Our results provide a comprehensive roadmap for engineering the genome of G. oxydans to further increase its bioleaching efficiency.

Rare earth elements (REE) are essential ingredients of sustainable energy technologies, but separation of individual REE is one of the hardest problems in chemistry today. Biosorption, where molecules adsorb to the surface of biological materials, offers a sustainable alternative to environmentally harmful solvent extractions currently used for separation of rare earth elements (REE). The REE-biosorption capability of some microorganisms allows for REE separations that, under specialized conditions, are already competitive with solvent extractions, suggesting that genetic engineering could allow it to leapfrog existing technologies. To identify targets for genomic improvement we screened 3,373 mutants from the whole genome knockout collection of the known REE-biosorbing microorganism Shewanella oneidensis MR-1. We found 130 genes that increased biosorption of the middle REE europium, and 112 that reduced it. We verified biosorption changes from the screen for a mixed solution of three REE (La, Eu, Yb) using Inductively Coupled Plasma Mass Spectrometry (ICP-MS) in solution conditions with a range of ionic strengths and REE concentrations. We found, among other things, that disruptions of a key regulatory component of the arc system (hptA), which regulates cellular response to anoxic environments and polysaccharide biosynthesis related genes (wbpQ, wbnJ, SO3183) consistently increase biosorption across all our solution conditions. Our largest total biosorption change comes from our SO4685, a capsular polysaccharide (CPS) synthesis gene, disruption which results in an up to 79% increase in biosorption and nusA, a regulatory protein, disruption which results in an up to 35% decrease in biosorption. Knockouts of glnA, pyrD, and SO3183 increase relative biosorption affinity for ytterbium over lanthanum in multiple solution conditions tested, while many other genes we explored have more complex binding affinity changes. Taken together, these results begin to elucidate how various genes affect the membrane chemistry of S. oneidensis and offer potential targets for improving biosorption and separation of REE.

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Rare earth elements (REE) are critical components of our technological society and essential for renewable energy technologies. Traditional thermochemical processes to extract REE 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 of 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 find 304 genes that notably alter production of G. oxydans’ acidic biolixiviant, including 165 that hold up under statistical comparison with 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 synthesis of the cofactor pyrroloquinoline quinone (PQQ) and the 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, enhances bioleaching by 18%. Taken together, these results give a comprehensive roadmap for engineering multiple sites in the genome of G. oxydans to further increase its bioleaching efficiency.

The global demand for critical rare earth elements (REE) is rising with the increase in demand for sustainable energy technologies like wind turbines, electric vehicles, and high efficiency lighting. Current processes for producing REE require high energy inputs and can produce disproportionate amounts of hazardous waste. Biological methods for REE production are a promising solution to this problem. In earlier work we identified the most important genetic mechanisms contributing to the REE-bioleaching capability of Gluconobacter oxydans B58. Here we have targeted two of these mechanisms to generate a high-efficiency bio-mining strain of G. oxydans. Disruption of the phosphate-specific transport system through a clean deletion of pstS constitutively turns on the phosphate starvation response, yielding a much more acidic biolixiviant, and increasing bioleaching by up to 30%. Coupling knockout of pstS with the over-expression of the mgdh membrane-bound glucose dehydrogenase gene, results in up to 73% improvement of REE-bioleaching.