Marecos, S.; Pian, B.; Medin, S. A.; Schmitz, A.; Wu, M.; Balta, J. B.; Gazel, E.; Holycross, M.; Reid, M. C.; Barstow, B.

The transition to a sustainable energy economy will require an enormous increase in the supply of rare earth elements (REE). Bioleaching offers a promising alternative to conventional hydrometallurgical methods for REE extraction from low-grade ores. However, exploiting this potential remains challenging due to large gaps in our understanding of the genetics involved, and inadequate biological tools to address them. We generated a highly non-redundant whole genome knockout collection for the bioleaching microbe Gluconobacter oxydans B58, reducing redundancy by 85% compared to the previous best collection. This new collection was directly screened for bioleaching neodymium from a synthetic monazite powder, identifying 89 genes important for bioleaching, 68 of which have not previously been associated with this mechanism. We conducted bench-scale experiments to validate the extraction efficiency of promising strains: 8 demonstrated significant increases in bioleaching by up to 111% (G. oxydans {delta}GO_1598, a disruption of the gene encoding the orotate phosphoribosyltransferase enzyme PyrE), and one strain significantly reduced it by 97% ({delta}GO_1096, a disruption of the gene encoding the GTP-binding protein TypA). Notable changes in biolixiviant pH were only observed for 3 strains, suggesting an important role for non-acid mechanisms in bioleaching. These findings provide valuable insights into further enhancing REE-bioleaching by G. oxydans\' through targeted genetic engineering.