Here we report the construction of two metabolically-engineered C. violaceum strains (pBAD and pTAC, respectively) that produced significantly more cyanide lixiviant compared to wild-type bacteria. Lixiviant production was effectively decoupled from quorum control through the use of exogenous promoters. In corroboration with the observed increase in cyanide production by the engineered strains, Au recovery from ESM was significantly increased in the engineered strains. Our efforts in modulating the lixiviant metabolism of C. violaceum demonstrate proof-of-concept that enhanced bioleaching microbes can be constructed as sustainable means of recovering precious metals such as Au from electronic waste.

Wild-type C. violaceum produces cyanide from glycine for short periods at the early stationary phase in its growth. The reliance of lixiviant production on cell population density is quorum-controlled and is probably co-evolved as a defense mechanism in its bid for niche colonization19. In an effort to obtain suitable strains of C. violaceum for bioleaching of Au from electronic waste, we examined if lixiviant production can be decoupled from quorum control and if so, whether cyanide production can be increased by modulating the lixiviant metabolic pathways of the organism. Normal cyanide metabolism is maintained by combined actions of the cyanogenic hcnABC operon and the cyanolytic cynTSX operon. We sought to engineer a tightly-regulated and tunable (responsive to varying concentrations of inducer) bioleaching strain by integrating a single copy of the hcnABC operon, under the transcriptional control of the exogenous promoters, into the bacterial genome. This site-specific genomic integration of an inducible cyanogenic operon was performed using Tn7-mediated transposition developed by Schweizer and coworkers20. We inserted a duplicate copy of the hcnABC operon (under the transcriptional control of exogenous promoters pBAD or pTAC, respectively) to decouple cyanogenesis from quorum control.

By design, genomic insertions can be made at specific Tn7 attachment (attTn7) sites located downstream of the highly conserved glmS gene (encoding for glucosamine 6-phosphate synthetase) in bacteria. The glmS gene of wild-type C. violaceum (GI: 34496132) is located upstream of a functionally unassigned gene (GI: 34496133) with a 45 bp intergenic region that is predicted to contain the attTn7 site. However, in contrast to studies with other bacterial species21, genomic integration in C. violaceum did not occur at the intergenic region downstream of the glmS gene; instead, the transposable segment was inserted within the 3′region of the glmS gene (after 1607 bp of the 1830 bp intact glmS gene). Despite disruption of the glmS gene, bacterial fitness in the engineered C. violaceum strains was not compromised (growth curves of wild-type and engineered strains are virtually identical, Fig. S2).

In attempts to increase the production of the cyanide lixiviant, we used a range of inducer concentrations to obtain dose-responsive profiles of cyanide production. Using L-arabinose as the exogenous inducer, cyanide production was increased above wild-type levels (Fig. 3), with maximal cyanide production (relative to uninduced levels) observed with 0.002% L-arabinose. An analogous dose-responsive profile was obtained with IPTG as the exogenous inducer (Fig. 4), where the addition of 1 mM IPTG resulted in maximal cyanide production, compared to uninduced or wild-type levels. Our observations indicated that the engineered pBAD and pTAC strains exhibited tunable, enhanced cyanide lixiviant production. The observed proto-typical dose-responsive profiles also suggested that lixiviant production in these strains had been decoupled from quorum-control. A comparison of the lixiviant profiles of wild-type versus engineered strains revealed that the pBAD and pTAC strains produced peak concentrations (at 30 hrs after inoculation) of 34.5 mg/L and 31 mg/L of cyanide, respectively (representing significant increases over the wild-type peak concentration of 20 mg/L of cyanide, Fig. 2).

With the expectation that an increase in cyanide lixiviant production would accompany a corresponding increase in bioleaching of Au from electronic waste, the engineered pBAD and pTAC C. violaceum strains were used to recover precious Au from ESM. Bioleaching studies revealed that there were significant increases in the recovery of Au from the ESM electronic waste for both strains of engineered C. violaceum, as compared to wild-type bacteria (Fig. 5). There have been studies on the use of cyanogenic bacteria in the recovery of Au from solid waste10,22,23,24: the reported modest recovery (up to 14.9% of total amount of Au present) corroborate with our observation (11% of total amount of Au present) on the limited utility of wild-type cyanogenic bacteria in bioleaching. In comparison, we were able to achieve Au recovery in excess of 30% of total amount of Au present using engineered C. violaceum. We view these significant increases in Au recovery from electronic waste (over wild-type cyanogenic bacteria) as a heartening possibility of using bioleaching as a sustainable means of recovering precious metals from electronic waste in the future.

To facilitate subsequent engineering efforts, we sought insights into the modulations of the lixiviant metabolic network of the pBAD strain through a comparative proteomics study of the cyanogenically-enhanced variant against the wild-type C. violaceum strain (Fig. 7). We examined if the observed levels of cyanide produced in the engineered strain could be further increased with future metabolic engineering. A logical corollary follows that an increase in cyanide production could be met with modulations in the proteome that could have decreased cyanogenesis and increased cyanolysis, leading to a limiting “cap” in the amount of cyanide production. Conversely, one could expect the observed increase in cyanogenesis to be within the inherent lixiviant capacity of the cyanogenic bacterium and minimal modulations in the cyanogenic and cyanolytic pathways of the engineered strain would be observed.

Figure 7 Comparative proteome analyses after increased cyanogenesis in Chromobacterium violaceum. The cyanogenic pathway is highlighted in blue. Up-regulations are represented in red and down-regulations are represented in green. The identities of proteins and enzymes detected in the study are shown in Supplementary Table S1. Full size image

Cyanide production through the actions of HCN synthase can be decreased by decreasing the availability of the substrate glycine; consequently, glycine flux away from cyanogenesis can be independently achieved through the actions of enzymes such as serine hydroxymethyltransferase17 (glyA, in the biosynthesis of serine from glycine), glycine decarboxylase25 (gcvP, in the glycine cleavage system), serine dehydratase26 (sdaA1, in serine catabolism), threonine aldolase and glycine C-acetyltransferase27 (CV_4309 and kbl, respectively, in the biosynthesis of threonine). Our comparative proteomics study revealed that upon an increase in cyanide production in the engineered strain, there were no significant changes in the protein levels of these enzymes (directly and indirectly) associated with the cyanolytic pathways (Fig. 7). Cyanolysis can also be directly achieved through the actions of cyanase28; our study did not detect significant changes in the levels of cyanase in the engineered strain, compared to wild-type C. violaceum. In addition, we did not observe any significant changes in the protein levels of enzymes associated with glycine biosynthesis and hence, cyanogenesis.

Cyanogenesis in bacteria and plants is associated with virulence and defense12; in the context of C. violaceum, our comparative proteomics study revealed significant modulations in metabolic pathways associated with metabolic dormancy and energy conservation. Taken together, an increase in nucleotide salvage (represented by a decrease in the enzyme purine nucleoside phosphorylase29/pnp), a decrease in deoxyribonucleotide biosynthesis (represented by a decrease in thioredoxin/trxA levels, the essential cofactor for ribonucleotide reductase30), a decrease in the capacity for oxidative stress response31 (represented by decreases in superoxide dismutase/sodB1 and thioredoxin/trxA levels) and an increase in fatty acid biosynthesis32 (represented by an increase in enoyl-ACP reductase/CV_3743 levels, an essential enzyme in the biosynthesis of saturated straight-chain fatty acids; and a decrease in 3-hydroxyacyl-CoA dehydrogenase/fadB levels, an enzyme associated with β-oxidation of fatty acids) suggested that the increased levels of cyanide production was within the cyanolytic capacity of the engineered strain. Our hypothesis was further corroborated by the observation that there was no significant change in protein levels of cioA33, a cyanide-insensitive terminal cytochrome oxidase in the respiratory electron transport chain of C. violaceum; if cyanide levels were to exceed the inherent cyanolytic capacity of the bacterium, the expectation would be a significant up-regulation of cioA, so that aerobic respiration (in particular, cytochrome c oxidase) would not be inhibited.

We have demonstrated that lixiviant metabolism in C. violaceum can be engineered for enhanced cyanide production; a decoupling of cyanogenesis from quorum control resulted in a significant increase in cyanide production and correspondingly, an increase in Au recovery from electronic waste. Comparative proteomics analyses suggested that further increase in cyanogenesis is possible and our results highlight the utility of lixiviant metabolic engineering in the construction of next-generation bioleaching microbes for the recovery of precious metals such as Au from electronic waste.