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From 2013.igem.org

Motivation

Delftibactin is a secondary metabolite naturally produced by Delftia acidovorans and has the ability to specifically precipitate gold from solutions. Today, electronic waste has become an immense environmental problem not only in developed nations, but also third-world countries through the increasing export of waste and disposal challenges.

Therefore, an efficient method to recycle electronic waste is urgently needed. From our work we concluded, that delftibactin could be used as an efficient substance to recycle gold. In fact, many different electronic scraps contain considerable gold amounts such as PC mainboards (566 ppm in terms of weight) and mobile phones (350 ppm). And those electronic devices are more and more demanded by society though their average lifetime seems to decrease steadily. To date, existing methods for gold recycling and safe disposal of electronic waste are still very energy-consuming and harmful to the environment.

Nowadays, harsh substances, such as strong acids, are conventionally used for leaching gold from electronic waste. Applied chemicals pose a potential thread to the environment as they could contaminate the biosphere. Biological agents could become an alternative to chemical clearance of metals from electronic waste. Nature's gold-altering microbes are non-pollutive and they are not even genetically modified. While bacteria such as Cupriavidus metallidurans convert the dissolved element to its metallic form inside the cell [11], species like Chromobacterium violaceum [4] or Delftia acidovorans [5] secrete substances to the surrounding medium for gold precipitation. These microorganisms can therefore be engineered to increase the yield of bioleaching substances.

Achievements

In our experiments, we successfully managed to dissolve electronic waste in aqua regia (i.e. nitro-hydrochloric acid) and neutralize the solution to receive gold chloride. When we added the supernatant of a D. acidovorans liquid culture to the gold chloride solution, pure gold was precipitated. Furthermore, we were able to melt the precipitated gold resulting in little gold flakes. The procedure already worked on a small scale in our project. With increased efforts, yields of Delftibactine for industrial applications are conceivable. In those dimensions, gold could easily be recycled. With Delftibactin there is no further need for chemical reducing reagents to purify gold from solution. Nevertheless, the efficiency of our approach still has to be improved. We were able to apply Delftibactin for the extraction of gold from electronic waste but still had to expose the CPUs with aqua regis to bring gold in solution. Ideally, one should get rid of the use of this highly corrosive mixture.

To further increase the efficiency of Delftibactin production, we aimed at transferring the entire synthesis machinery for Delftibactin into E. coli. As the NRPS producing delftibactin is a very large enzyme complex consisting of many modules, amplification, cloning and transformation of the constructs was very challenging. Nonetheless, we successfully managed to amplify, assemble and transform all of the genes (in sum 54 kbp) required for production of Delftibactin. In this process, we established protocols, including Gibson assembly of large fragments, and transformation of those constructs into E. coli via electroporation. Optimized procedures will ease the cloning of large customized NRPSs in future.

Challenges

A hurdle to overcome is that the DelH displayed an above-average mutation rate when present in E. coli without the rest of the Delftibactin pathway genes. DelH could potentially be toxic to the E. coli. Therefore, cells select for mutations that render the clones unable to express a funtional DelH protein [12]. In our experiments, the putative selection pressure towards nonfunctional DelH was manifested in deletions, insertions or substitutions in the respective ribsosome binding side or at the beginning of DelH. However, we managed to obtain a single clone, which only possessed a point mutation that potentially does not interfere with protein expression since no false stop codon was created and no frame shift was caused. In this clone, DelH was successfully expressed, yet with an amino acid exchange (Alanine to Threonine) at the N terminus of the protein. At the moment, we are aiming to obtain the gene without any mutation by lowering protein expression. To this end, the most-promising approach is to design a DelH construct containing a weak promoter and weak ribosome binding side. This would decrease the selection pressure caused by DelH. An alternative strategy would be the construction of DelH without any promoter or ribosome binding site. As soon as we would get a mutation-free DelH fragment,,it could be ligated into a plasmid backbone via conventional restriction enzyme-based cloning. A weak promoter and ribosome binding side would keep the detrimental impact of DelH as low as possible. Despite the point mutation, we were able to show that DelH is expressed in E. coli. Our prediction of the tertiary structure of DelH indicated that the.substituted amino acid in the mutant DelH is located at the outer side of the protein. Nevertheless it could still have an important function. Therefore it is not possible to foresee whether the mutation would have any effect on the function of DelH. However alanine is an unpolar amino acid whereas threonine is polar. If the mutation was in a functional region, this could have significant effects rendering DelH nonfunctional but keeping E. coli alive.