Engineering resilience to green biotechnology
Australia’s resource industry is currently under pressure due to declining ore grades. Finding new ways to process lower-grade ores is a necessity to maintain productivity and value in the mining industry for Australia.
This project aims to design and construct robust extremophile chassis and biobricks for the resources industry through synthetic biology and metabolic modelling. The project combines wet lab experiments (Module 1) with metabolic modelling (Module 2) to engineer biomining microorganisms for enhanced tolerance to multiple stress factors such as high metal and salt concentrations and low pH. This is expected to facilitate the mining of low grade ores and wastes using saline or brackish waters.
Literature review was conducted on the availability of molecular tools for biomining microbes and search for genetic elements responsible for stress response. The important role of ectoine biosynthesis pathway in salt tolerant biomining microbes and cusABCF and/or copAB in copper tolerant microbes has been identified. Collaborations were established to source plasmids for inserting genes of interest. The ribosome binding site of the expression plasmid was optimised and unwanted part of the plasmid was deleted. Operon encoding ectoine biosynthesis pathway (optimised for expression in Acidithiobacillus ferrooxidans) was synthesized and is currently being cloned into the expression vector of A. ferrooxidans. As a second approach, alternative sigma factors were amplified from the genome of A. ferrooxidans DSM 14882 and were successfully cloned into Escherichia coli donor strain and conjugated into two strains of A. ferrooxidans (type strain DSM 14882 and copper resistant ATCC 53933 strain). Subculturing of the constructs for verification is underway.
A comprehensive metabolic model of the pathways used in various stresses (acid, salt, metal, oxidative stress) by A. ferrooxidans has been reconstructed. This model is currently in the process of being expanded to include pathways for energy acquisition (iron and sulfur oxidation) and nutrient and biomass assimilation (carbon, nitrogen, phosphate). Furthermore, reactions and metabolites involved in cell envelope, membrane lipid, glycerophospholipid, lipopolysaccharide and purine/pyrimidine biosynthesis are being identified for inclusion into the model, as are those for exchange and transport reactions. The complete information will provide the basis for the stoichiometric matrix development.
Once all pathways have been included in the reconstruction, a metabolic network map will be drawn. Experiments to test the growth of A. ferrooxidans under different stress conditions and the effect of these environmental constraints on its biomass are in the process of being designed. The data from these experiments will assist in determining the effect of bioengineering of the microorganism.
Ultimately, this project will generate new tools for engineering extremophilic microbes and benefit mining industry by making available robust biocatalysts that enable the extraction of metals from complex, low-grade ores or wastes. This is expected to extent mine life and convert waste to value.