Abstract

The increasing demand for mineral resources and concern about the contamination caused by acid
mine drainage near the mine site necessitate efforts to extract metals from tailings and prevent
contamination. In Canada, the Sudbury basin has 50-100 million metric tons of pyrrhotite tailings
with ~1% nickel, a critical metal required for batteries and other applications.
The conventional mining technologies are environmentally and economically unsustainable. Biomining is a potential sustainable alternative for bioremediation and recovery of remnant metals from tailings. However, there are several limitations including low rates and neutralization costs. Microbes thriving in the tailings with potential for acid mine drainage are excellent candidates for bioleaching. Recently, our lab isolated a dominant acidophile Acidithiobacillus ferridurans JAGS (JAGS) and the genomic and phenotypic characterization indicated this strain as a
potential host for metal recovery from metal-contaminated sites. However, the low extraction rates
by the microbes have restricted their application in an industrial setup. Commercialization of the
biomining process will require the development of genetically engineered biological systems with
improved biomining efficiency. To obtain this goal, the development of a synthetic biology
toolbox is prerequisite. Synthetic biology toolbox is an array of various genetic parts that could be
systematically used for metabolic engineering. Promoters are biological parts that control the
expression of genes of interest. Altering the sequence of promoters would result in different levels
of gene expression. Promoter engineering has been identified as a successful strategy for metabolic
engineering in various microbes, however, sparse knowledge is available for Acidithiobacillus
species. In this study, we have characterized and developed a synthetic promoter library in JAGS.
A set of randomly generated sequences of length 40 bp were cloned with a reporter gene (green
fluorescent protein) and the promoter strength was assessed as a function of the fluorescent protein
activity. The library consisted of ~50 constituent promoters showing a wide range (20-fold) of
promoter strength. A library with wider range of promoter activity will provide probable promoter
options to use for optimizing gene expression during pathway modification. The strongest
promoter in the library exhibited strength similar to one of the well-known strongest promoters,
Ptac. Developing a synthetic biology toolbox will have significant implications in the manipulation
of biosynthetic pathways for enhancing the biomining efficiency of Acidithiobacillus species.