Team:Slovenia/PROJECT/biosynthesis/violacein

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Background

Ever since 1882, violacein has been notable as the most obvious characteristic of a soil bacterium Chromobacterium violaceum due to its deep purple color. Violacein is insoluble in water and due to its hydrophobicity retained within cells. Biosynthesis of violacein has been as well as its biological activities were investigated in detail (Duran et al., 2007). Violacein has antibacterial, antiviral, antiparasite, antitumorogenic and many other biological activities, which makes the violacein pigment a very good candidate for further research and industrial production. In addition to potential medical applications, violacein is also used as a natural purple dye.

Violacein biosynthetic pathway

Genes for violacein biosynthesis (scheme below) are arranged in an operon consisting of vioA, vioB, vioC, vioD and vioE. VioA is an FAD dependent L-tryptophan oxidase, which generates an IPA imine. The latter is a substrate for VioB, a hemoprotein oxidase, which converts the IPA imine into a dimer. VioE is a key enzyme in the violacein biosynthetic pathway that acts by converting the IPA imine dimer to intermediates, which can be taken over by VioD and VioC. VioD and VioC are FAD dependent monooxygenases that hydroxylate these compounds to form violacein and deoxyviolacein. The genes coding for the five enzymes of the violacein biosynthetic pathway have already been succesfully expressed in E.Coli. In E.Coli an additional side reaction occurs, producing a green pigment called deoxychromoviridans, which is produced before the action of VioD and VioC. We aimed to link all violacein biosynthetic enzymes into an arranged pathway assembled along the program DNA with the goal to increase the yield of the desired product.

The violacein biosynthetic pathway (Balibar and Walsh, 2006). Enzyme-catalysed biosynthetic transformation of tryptophan into violacein is shown. The purple line indicates the desired biosynthetic flux, which we aimed to achieve by introduction of a DNA program into the cells that produce chimeric biosynthetic enzymes linked to different zinc fingers. This reaction pathway should favor violacein synthesis over the side reaction leading to deoxychromoviridans.

Design of a DNA-guided biosynthetic pathway

We designed five fusion proteins by combining different zinc fingers and appropriate violacein biosynthetic enzymes. Since we wanted to create an in vivo system for violacein production, we expressed them in E. coli. Our goal was to show improvement of violacein biosynthesis by DNA program (scaffold) for which we predicted it would increase the proximity of fusion proteins and arrange their order according to the biosynthetic order of reactions. To demonstrate the significance of corect order of biosynthetic enzymes for violacein production, we compared production of violacein in the presence of correct DNA program (123456) to production in the presence of DNA programs with scrambled order for binding of chimeric proteins (341256; see the scheme below).

Correct vs. scrambled DNA program

Pictures above are an output of a 3d animation. The molecular and environmental dynamics on both scenes are the same, but there is higher amount of L-Tryptophane (marked red) on picture a) and higher amount of violacein (marked magenta) on picture b). Eventhough the system variables may not be accurate, the picture enables us to visualise the advantage of using a DNA program (more end product).

Results

E. coli containing chimeric enzymes and either correct DNA program (123456), scrambled DNA program or no program were grown for extended time. In addition, The E.coli strain carrying plasmids with violacein operon (Part:Bba_K274002), prepared by the 2009 iGEM team Cambridge, was also grown for comparison.
Bacteria containing chimeric proteins were able to produce violacein (no program), demonstrating that the addition of zinc finger domains does not interfere with their enzymatic activity (Figure 1). However, the production of violacein was delayed compared to the cultures that contained DNA programs.

Figure 1a: The bacterial culture carrying plasmids containing the DNA program roduces significantly more purple color than cultures carrying plasmids containing a scrambled DNA program or no DNA program. Cultures of bacteria carrying plasmids with BioBricks BBa_K323132, BBa_K323135 and i) plasmid containing a DNA program (123456), ii) plasmid containing a scrambled DNA program (341256) and iii) plasmid without a DNA program were incubated at 30°C in a Luria Bertani media. Purple color could be observed in the culture with the DNA program after several hours by the naked eye, while the remaining two cultures were not colored. Figure 1a: represents cultures after 24 hour incubation. As it can be determined from the picture, the color of the culture containing the plasmid with the DNA program is notably more purple than the color of other two cultures. Figure 1b: significant difference in color of violacein extracts of the incubated cultures. Violacein was extracted from the incubated cultures. A strong difference in color intensity is visible between the extracts of all three cultures, among which the extract of the culture containing a plasmid with the DNA program exhibits the most intensive purple color.

We further analyzed the extracts using more sensitive methods, e.g. thin-layer chromatography (TLC) with densitometry, mass spectrometry (for a detailed description of the protocols see the page "Methods"). Results show that in additon to violacein and deoxyviolacein, exctracts of bacterial cultures with enzymes without zinc finger domains contained many more side products (Figure 3, line 3), particularly deoxychromoviridans. The identity of all compounds was determined by mass spectrometry and absorbance spectra. However, a significant decrease of deoxychomoviridans and deoxyviolacein product formation was detected, when program DNA was present (Figure 2, compare lanes 2 and 3).

Figure 2: More violacein and less side products deoxyviolacein and deoxychromoviridans is produced when DNA program is present. Violacein in the samples +/- DNA program was extracted, analyzed by TLC and the amounts of each compound quantified by densitometry.

Figure 3: Denzitometric analysis (right) and the ratio (left) of violacein, deoxyviolacein and deoxychromoviridans in samples with DNA program and without a program. Densitometry scan of samples after TLC analysis indicate that deoxychromoviridans was only present in the sample without a program, while In the presence of DNA program, violacein was the predominant compound produced, followed by deoxyviolacein. No deoxychromoviridans was detected in samples with DNA program.

Most important, the yield of violacein when program DNA was present has improved ~6-fold and ~2 fold, in comparison to yield when no or scrambled DNA program was present, respectively (Figure 4).

Figure 4: Violacein accumulates faster in the culture with the DNA program and results in a higher yield of violacein production compared to cultures with scrambled or no DNA program. Samples were analyzed as above. Figure A shows faster production of violacein in cultures containing DNA program. Figure B shows quantification of violacein in samples taken after 24 hours of incubation. The highest yield of violacein production was determined in the culture containing a DNA program. The production was increased 6-fold in comparison to the culture without a DNA program. A significant improvement in comparison to the culture with the scrambled program is also striking, which implies that a correct arrangement of enzymes on the DNA program is important for the progress of the reaction. The experiment was repeated more than three times with similar results.

These results fully confirm the idea of DNA-guided biosynthetic assembly and its usefulness for the improvement of the biosynthetic flux. In addition to the anticipated improvement of the yield, we were very pleased to observe significant suppression of the side products (deoxychromoviridans and deoxyviolacein), which probably also contributed to the increase of the final yield of violacein.

Although the theoretical increase of the speed inthe biosynthetic pathway composed of a five reactions, the final yield depends on many different factors, including the rate limiting step, availability of the cofactors, the initial substrate - tryptophane, etc. We did not have time to perform any optimizations with respect to strains, growth media, temperature etc., therefore the maximum yield of violacein production could be significantly higher. However the experiment was repeated more than three times with similar results.

Duran N., Justo G.Z., Ferreira C.V., Melo P.S., Cordi L., Martins D. 2007. Violacein: Properties and biological activities. Biotechnology and Applied Biochemistry, 48: 127-133

Balibar CJ, Walsh CT. 2006, In vitro biosynthesis of violacein from L-tryptophan by the enzymes VioA-E from Chromobacterium violaceum, Biochemistry, 45:15444-57.