Genetic Engineering of Acidithiobacillus ferridurans Using CRISPR Systems To Mitigate Toxic Release in Biomining

Biominingprocesses utilize microorganisms, such as Acidithiobacillus, to extract valuable metals by producing sulfuric acid and ferricions that dissolve sulfidic minerals. However, excessive productionof these compounds can result in metal structure corrosion and groundwatercontamination. Synthetic biology offers a promising solution to improve Acidithiobacillus strains for sustainable, eco-friendly,and cost-effective biomining, but genetic engineering of these slow-growingmicroorganisms is challenging with current inefficient and time-consumingmethods. To address this, we established a CRISPR-dCas9 system forgene knockdown in A. ferridurans JAGS,successfully downregulating the transcriptional levels of two genesinvolved in sulfur oxidation. More importantly, we constructed anall-in-one CRISPR-Cas9 system for fast and efficient genome editingin A. ferridurans JAGS, achieving seamlessgene deletion (HdrB3), promoter substitution (Prusto Ptac), and exogenous gene insertion (GFP). Additionally,we created a HdrB-Rus double-edited strain and performed biominingexperiments to extract Ni from pyrrhotite tailings. The engineeredstrain demonstrated a similar Ni recovery rate to wild-type A. ferridurans JAGS but with significantly lowerproduction of iron ions and sulfuric acid in leachate. These high-efficientCRISPR systems provide a powerful tool for studying gene functionsand creating useful recombinants for synthetic biology-assisted biominingapplications in the future. UtilizingCRISPR-based genetic engineering, Acidithiobacillusferridurans has been modified tomitigate toxic substance release during biomining, showcasing itspotential to foster sustainable biomining practices.