Gluconobacter oxydans Knockout Collection Finds Improved Rare Earth Element Extraction

Abstract

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.

Harnessing synthetic biology for sustainable biomining with Fe/S-oxidizing microbes

“…A deeper understanding of Fe/S-oxidizing microbes' genes is nonetheless still needed for more detailed metabolic engineering towards desirable biomining properties, compared to the model chassis Escherichia coli and Saccharomyces cerevisiae. Schmitz et al (2021) harnessed high-throughput genome editing and sequencing approaches in the study of heterotrophic bacterium Gluconobacter oxydans to produce organic acids for rare earth elements biomining. They created a library of single-gene transposon mutants in G. oxydans and found the bioleaching rates of rare earth elements increased up to 18% when phosphatespecific transport systems genes were disrupted.…”

“…(2) Development of high-throughput technologies to culture and screen of Fe/S-oxidizing microbes will accelerate the engineering of strains with more desirable industrial properties such as heavy metal tolerance, robustness under large bioreactor conditions. Example studies could include designing synthetic histidine kinases that can sense heavy-metals (Sarnaik et al, 2020), creating a whole-genome single-gene mutant library to enable screens for desired properties (Schmitz et al, 2021), or using CRISPRi system as an efficient platform for rapid identification of target genes (Wu et al, 2020).…”