(643d) Development of Genetic Tools for the Biomining Bacterium, Acidithiobacillus Ferrooxidans

The chemolithoautotroph, Acidithiobacillus ferrooxidans, is a key species involved in the biomining of metals from ores. As mines worldwide face decreasing ore grades and the depletion of copper oxide ores, there is interest in finding methods for leaching copper from sulfide minerals which account for a majority of the world’s copper reserves. There is significant effort aimed at engineering A. ferrooxidans and other related species for applications in bioenergy and biomining (1). The development of this organism has been hampered by the limited genetic tools compatible with acidic, oxidative conditions. There are few resistance markers usable for plasmid-driven expression and the expression of genes has been constrained to the use of a single constitutive promoter, tac. To better control gene expression within A. ferrooxidans, we have characterized two endogenous promoters, cycA1 and tusA, to differentially express heterologous genes in response to the presence of ferrous iron or sulfur in growth medium. Both promoters in the repressed state are indistinguishable from the negative control indicating tight regulation. While these promoters are weak relative to tac, they demonstrate a range of expression comparable to the lac promoter (2). Although plasmid-driven expression is useful for developing genetic circuits, methods to stably maintain heterologous genes on the chromosome are necessary for industrial applications of A. ferrooxidans. We have developed a chromosomal integration technique using a suicide plasmid harboring a hyperactive transposase. With this method, we have created mutants with integrated genes using either a fluorescent marker, super-folder Green Fluorescent Protein, or a metabolic gene, 2-keto decarboxylase (KDC). Inverse PCR was used to identify the integration sites of the KDC gene, identifying a potential site in the genome for future insertions that appeared not to affect the viability of the strain (3). While the pJRD215 plasmid belonging to the family of IncQ-type plasmids has been commonly used as a vector in this species, room for additional genes on this vector is limited by the inherent maximum size of IncQ plasmids. We have developed an alternative vector derived from A. ferrooxidans in a different subfamily of IncQ plasmids and have demonstrated that it can co-reside with the pJRD215 vector expanding the genetic space available to express additional genes (4). These genetic tools will facilitate the development of even more advanced tools and future engineering of this important industrial bacterium.

1. Banerjee I, Burrell B, Reed C, West AC, Banta S. 2017. Metals and minerals as a biotechnology feedstock: engineering biomining microbiology for bioenergy applications. Current Opinion in Biotechnology 45:144-155.
2. Kernan T, West AC, Banta S. 2017. Characterization of endogenous promoters for control of recombinant gene expression in Acidithiobacillus ferrooxidans. Biotechnology and Applied Biochemistry 64:793-802.
3. Inaba Y, Banerjee I, Kernan T, Banta S. Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans. Applied and Environmental Microbiology (Submitted).
4. Inaba Y, Kernan T, Banta S. Alternative Vector for Improved Genetic Engineering in Chemolithoautotrophs. (In preparation).