Overview of Chromobacterium violaceum

Chromobacterium violaceum is a bacterium able to regulate cyanide according to the presence of cyanide-producing and degrading enzymes in the wild-type ones. These enzymes are encoded by some genes, such as hcnABC for cyanide production and rhodanese for cyanide degradation.^1,2

The hcnABC is an operon that consists of a cluster of three genes; i.e., hcnA, hcnB, and hcnC. This operon encodes HCN synthase that facilitates the conversion of an amino acid glycine into cyanide.³ This enzyme belongs to the oxidoreductase class as it oxidizes the amine (CH-NH2) functional group into imine (C=NH) and consecutively cleaves the molecule into HCN and carbon dioxide (CO2) (Figure 1).⁴

Rhodanese is one of the cyanide degrading enzymes found in C. violaceum. This enzyme is also known as sulfurtransferase, as it catalyzes sulfur transfer from thiosulfate to cyanide and leads to the formation of the less toxic thiocyanate (Figure 2).^5,6 Compared to the other enzymes, the regulation of rhodanese expression is not affected by the presence of glycine and methionine, so that seems to be easily controlled.⁷

Auviola: The Engineered Chromobacterium violaceum

With the concepts of synthetic biology, we developed an engineered C. violaceum to create an on-off system for cyanide regulation in the gold bioleaching process. Compared to the wild-type, the new C. violaceum was engineered to have more cyanide-regulating genes, resulting in a better gold dissolution and cyanide waste treatment.

Our Auviola on-off system involved a regulator gene of araC since it exists on the plasmid. This regulator works dependably to arabinose level which acts as both an activator in the presence of arabinose and a repressor in the absence of arabinose.⁸ An inducible promoter of PBAD was utilized for the on-off mechanism regulated by the araC.⁹

Our circuitry design consisted of three expression systems explained below (Figure 3):

1. Cyanide-producing system

   The HCN synthase enzymes were expressed by this system in the presence of arabinose.

2. Cyanide-degrading system

   This rhodanese expression system involved not only araC regulator and PBAD promoter but also tetR regulator and PTET inducible promoter. These genes are presented in the wild-type of C. violaceum that acts as a repressor and normally play a role in tetracycline resistance.^10,11 The system was designed so that araC regulates the tetR expression as well as tetR regulates rhodanese expression. In the presence of arabinose, the TetR proteins are expressed and subsequently repress the production of rhodanese, and vice versa.

3. L-arabinose isomerase (L-AI) expression system

   The L-AI is an enzyme that catalyzes the conversion of L-arabinose into L-ribulose.¹² This system was utilized for our on-off system since C. violaceum is not able to ferment arabinose. Through utilizing a constitutive weak promoter, the L-AIs were slowly expressed to convert arabinose into the inactive form and subsequently control the on-off system.

The Practical Utilization of The Auviola System

The use of our Auviola system was conducted in a closed compartment and depended on the level of arabinose. The gold bioleaching process was conducted by adding the arabinose as the HCN synthases are expressed while the rhodanese enzymes are not. After the L-AI converted the arabinose in a considerable amount, the system continued to degrade the cyanide as the rhodanese enzymes started being produced while the HCN synthases were stopped (Figure 4).

Demonstration of Auviola System

The demonstration was conducted by analytical procedure to determine the cyanide concentration in several defined points. This procedure gave results that cyanide presents in a high concentration after adding the arabinose that represents the gold bioleaching process. Afterward, the cyanide concentration started to decrease as the L-AIs were slowly produced to convert the arabinose and subsequently the rhodanese enzymes were expressed to degrade the cyanide.

In addition, fluorimetry was also conducted to demonstrate the Auviola system. Firstly, the genes of interest were replaced by some fluorescent protein-encoded. After that, some samples were taken in several defined points then the intensities were read by the spectrofluorometer. The results showed that the protein presents in a high concentration at the point that represents the gold bioleaching process. Afterward, the protein level started to decrease as long as the rhodanese enzymes were expressed to degrade the cyanide.