Engineering Polyhistidine Tags on Surface Proteins of Acidithiobacillus ferrooxidans: Impact of Localization on the Binding and Recovery of Divalent Metal Cations

Abstract

Metal processing using microorganisms has many advantages including the potential for reduced environmental impacts as compared to conventional technologies. Acidithiobacillus ferrooxidansis an iron-and sulfur-oxidizing chemolithoautotroph that is known to participate in metal bioleaching, and its metabolic capabilities have been exploited for industrial-scale copper and gold biomining. In addition to bioleaching, microorganisms could also be engineered for selective metal binding, enabling new opportunities for metal bioseparation and recovery. Here, we explored the ability of polyhistidine (polyHis) tags appended to two recombinantly expressed endogenous proteins to enhance the metal binding capacity of A. ferrooxidans. The genetically engineered cells achieved enhanced cobalt and copper binding capacities, and the Langmuir isotherm captures their interaction behavior with these divalent metals. Additionally, the cellular localization of the recombinant proteins correlated with kinetic modeling of the binding interactions, where the outer membrane-associated polyHis-tagged licanantase peptide bound the metals faster than the periplasmically expressed polyHis-tagged rusticyanin protein. The selectivity of the polyHis sequences for cobalt over copper from mixed metal solutions suggests potential utility in practical applications, and further engineering could be used to create metal-selective bioleaching microorganisms.

Overexpression of the Licanantase Protein in Acidithiobacillus ferrooxidans Leads to Enhanced Bioleaching of Copper Minerals

“…The inclusion of the His-tag is useful, as it can be used for the validation of the recombinant expression via Western blot analysis (Figure S1). This strain has previously been evaluated for its ability to bind copper and cobalt at varying pH values through the display of the poly-His tag . Here we explored the effect of the genetic modification on the bioleaching of copper sulfides (chalcopyrite, chalcocite, and covellite), in comparison to wild type cell and abiotic control conditions.…”

“…A. ferrooxidans ATCC23270 and strain AF lic (transformed with the pYI55 plasmid encoding the A. ferrooxidans licanantase protein with a c-terminal polyhistidine tag) were obtained as previously described. 18 All A. ferrooxidans cells were maintained in 100 mL of F2S growth medium with an initial optical density of 0.001 measured at 600 nm (OD 600 ), corresponding to a cell density of 8.3 × 10 6 cells/mL, 20 at 30 °C and 140 rpm. The F2S medium (pH 1.8) contained 0.8 g/L (NH 4 ) 2 SO 4 , 0.1 g/L K 2 HPO 4 , 2.0 g/L MgSO 4 • 7H 2 O, 5 mL/L trace mineral solution (MD-TMS), 10 mM citric acid, 100 mM FeSO 4 •7H 2 O, and 0.1% (w/v) dispersed sulfur (#S789400; Toronto Research Chemicals, Toronto, ON).…”

“…A. ferrooxidans ATCC23270 and strain AF lic (transformed with the pYI55 plasmid encoding the A. ferrooxidans licanantase protein with a c-terminal polyhistidine tag) were obtained as previously described . All A. ferrooxidans cells were maintained in 100 mL of F2S growth medium with an initial optical density of 0.001 measured at 600 nm (OD 600 ), corresponding to a cell density of 8.3 × 10 6 cells/mL, at 30 °C and 140 rpm.…”

“…Mineral bioleaching via contact mechanisms can depend on the Fe 3+ concentration in biofilms, and thus the Fe 3+ chelation ability of the purified proteins with or without the His-tag was tested at pH 1.8. Both of purified licanantase proteins could bind Fe 3+ in acidic solutions, but surprisingly, the presence of His-tags in the licanantase resulted in a 1.8-fold decrease in the Fe 3+ concentration remaining in the solution as compared to licanantase without the His-tags, despite the histidine protonation 18 (Figure S1). The mechanism behind this observed difference is not clear; however, it suggests that the licanantase with His-tags results in increased binding of Fe 3+ , forming the protonated histidine−metal complexes at low pH.…”

Overexpression of AcoP in Acidithiobacillus ferrooxidans for Enhanced Copper Reclamation

“…ferrooxidans, for metal binding and recovery in bioremediation processes has been less explored. , We recently reported the enhanced adsorption of divalent metals (cobalt and copper) by engineered A. ferrooxidans via the display of polyhistidine peptide tags . The previous study validated the potential for developing A.…”