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 cyanide³ This enzyme belongs to the oxidoreductase class as it oxidizes the amine (\(CH-NH_2\)) functional group into imine (\(C=NH\)) and consecutively cleaves the molecule into HCN and carbon dioxide (\(CO_2\)) (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 In addition, this system also involved 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

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

1. Cyanide-producing system

   This system consists of an operon hcnABC as HCN synthase-encoding genes, pBAD promoter, and araC regulator.

2. Cyanide-degrading system

   This rhodanese expression system involved not only araC regulator and pBAD promoter but also tetR regulator and pTet inducible promoter. The system was designed so that araC regulates the tetR expression as well as tetR regulates rhodanese expression.

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 J23106, the L-AIs were slowly expressed to convert arabinose into the inactive form.

The Practical Utilization of The Auviola System

Figure 4 summarizes the on-off mechanism of the Auviola system. The use of our Auviola system was conducted in a closed compartment and depended on the level of glucose and arabinose.

In the condition with low level of glucose and high level of arabinose, the HCN production will be turned on and the HCN degradation will not. The added arabinose regulates the expression through binding of AraC protein. This complex activates pBAD to express HCN synthase. pBAD in the HCN degradation system is also activated by this complex to express TetR that represses rhodanese expression. The L-AI expression which involves the constitutive promoter is not altered by this condition.

This condition by adding the arabinose into the bioreactor is suitable for the gold bioleaching process, knowing that the HCN synthases will be expressed while the rhodanese enzymes will not (Figure 5).

In the condition with high level of glucose and low level of arabinose, the HCN synthase will be switched off meanwhile the HCN degradation will be turned on. The glucose used by the cells for metabolism will also control the on-off system through suppressing pBAD activation via cAMP-CAP pathway, so the HCN synthase will not be expressed. On the other hand, this pBAD repression will activate the pTET that subsequently activates the rhodanese expression. The L-AI expression is also not altered by this condition.

This condition is achieved by the L-AI activity that converts the arabinose in a considerable amount. This condition is suitable for the cyanide waste treatment as the rhodanese enzymes start being produced while the HCN synthase expression will be stopped (Figure 6).