Biosynthesis - Violacein

Background

Violacein is a pigment produced by several bacteria through pathway of 5 related genes, vioA, vioB, vioC, vioD and vioE. It was initially discovered in Chromobacterium violaceum. This metabolite of tryptophan has such special applications as antibacterial, anti-trypanocidal, anti-ulcerogenic, and anticancer drugs.

There are two interesting branches in this pathway which could be utilized in service of verification of the membrane rudder. We can identify the different end-products by means of separation, such as thin-layer chromatography (TLC), or high-performance liquid chromatography (HPLC). The product proportion change demonstrates the efficiency of alteration in reaction direction.

Violacein Biosynthetic Pathway

Fig.1 :The violacein biosynthetic pathway. The purple line indicates the biosynthetic flux of pigment deoxyviolacein and violet. The green line indicates the the biosynthetic flux of pigment deoxychromoviridans.

The violacein biosynthetic pathway includes 5 key enzymes which work in conjunction. VioA, a flavoenzyme and VioB, a heme protein, they work together to oxidize and dimerize tryptophen into IPA imine dimer. Then vioE induces a indole rearrangement,producing prodeoxyviolacein, also known as PVA.

One key prerequisite in the metabolic flux is that: Automatically VioA, VioB and VioE can assemble and function to produce PVA. There exists one intrinsic E.coli enzyme that aids in an additional side reaction, further modifying PVA into a green pigment called deoxychromoviridans(1st main product).

The last two proteins, VioC and VioD are flavin-dependent oxygenases. VioC alone transfers PVA into a purple pigment named deoxyviolacein(2nd main product), while vio C could also act sequentially with vioD. vioD hydroxylates 5-position indole ring, then the other 2-position indole ring is processed by VioC to create the oxindole, and in this way violacein(3rd main product) is produced.

Design of Membrane Assembly

Theoretically our membrane rudder offers a method to fine-tune reaction direction, and in the meantime decrease side-products quantity.

Control over reaction flow

Since VioA,VioB and VioE normally aggregate to finish the transfer from tryptophen to PVA, the key to direction of the rest part of pathway is if PVA could be passed on to vioD timely.

Reduction of side-products

We chose violacein mainly because of the branching feature of its pathway.The vio genes are distributed to different membrane assembly. As demonstrated in figure 2,vioA was linked to membrane assembly 1, vio E was linked to membrane assembly 3, while vioC and vioD were linked to VIVID, the light-inducing-dimerizable protein. We inserted segments M1-vioA and VVD-vioC into pETduet, VVD-vioD and M3-vioE into pACYCduet. Since vioB and vioE normally function in dimer state, pRSFduet with rbs-vioB and rbs-vioE was transformed into E.Coli to ensure dimerization, then all five functional enzymes could be expressed now.

figure2//膜蛋白图片

As long as VVD is photo-induced and dimerized, they could act like magnets that pull vioC and vioD together,making it easier for intermediates to undergo catalyzation of vioC and vioD. In this case violacein should be the dominant product we expect.

Results and Discussion

By attaching the core enzymes to the VIVID, we succeeded to control the flow of branched chain reaction, such as violacein biosynthetic pathway. Results indicate that violacein was dominant from extracts of bacterial cultures induced by light. Extracts of bacterial cultures restrained from light, however, containd both deoxyviolacein and violacein.(Figure X, compare X and X) Moreover, a significant decrease of side products such as deoxychomoviridans was detected, when membrane system was present. (Figure X, compare lane X and X)

The switch of the reaction can be realized by the extracellular signals. Through the light induction, only the reaction producing violacein by vioD and vioC sequentially is initiated by means of dimerizing the VIVID proteins. On the other hand, as long as light is restrained from the bacteria, however, both the reaction pathways leading to the deoxyviolacein by vioC and violacein are available. In other word, by controlling the extracellular signal, such as light, we are able to manipulate the branched chain reactions thus producing the target products we desire. HPLC results show that under the induction of light, there was barely deoxyviolacein while most products were violacein. However, both violacein and deoxyviolacein were detected from the extraction of bacteria culture restrained from light.

Moreover, membrane complex system would help to reduce the amount of side-products to some extent. Under the case that the enzymes involved in the side reaction are situated in the cytoplasm, they are less competent than the core enzymes attached on the membrane to mediate the subsequent reactions due to the spatial obstacle. From the HPLC results, we detected a significant decrease of side-products deoxychomoviridans which confirmed our thesis and indicated the significance membrane complex system has in the reduction of side-products.

Reference

1. Balibar, C. J. and C. T. Walsh (2006). "In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum." Biochemistry 45(51): 15444-57.

2. Hoshino, T. "Violacein and related tryptophan metabolites produced by Chromobacterium violaceum: biosynthetic mechanism and pathway for construction of violacein core." Appl Microbiol Biotechnol 91(6): 1463-75.

3. Shrode, L. B., Z. A. Lewis, et al. (2001). "vvd is required for light adaptation of conidiation-specific genes of Neurospora crassa, but not circadian conidiation." Fungal Genet Biol 32(3): 169-81.