Team:Slovenia/PROJECT/biosynthesis/violacein

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<p>&nbsp;</p>
 
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<p><span style="font-size: 20px; line-height: 25px;">Introduction</span></p>
 
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<p>&nbsp;</p>
 
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Biosynthesis is an enzyme catalyzed process, occurring in living cells, by which simple substrate molecules are converted into more complex products. The process often consists of several steps, in which the product of one step is used as a substrate for the following step. In synthetic biology, research and engineering of biosynthetic pathways gains more and more attention every year. One of the first great stories in the field of synthetic biology was engineering the artificial biosynthetic pathway for antimalarial drug artemisinic acid production in <em>Saccharomyces cerevisae</em>&nbsp;yeast. Production of&nbsp; artemisinic acid in genetically modified yeasts was achieved modulating regulation of specific mevalonate pathway genes and by the introduction of genes for the biosynthetic pathway from <em>Artemisia annua</em>&nbsp;plant to yeast. Another great examples of biosynthetic pathway engineering are production of fatty esters (biodiesel), fatty alcohols, and waxes by genetically modified <em>Escherichia coli</em>. Genes from different organisms were combined into completely new pathway which was introduced to <em>Escherichia coli</em>&nbsp; to produce useful fuel directly from plant biomass.
 
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<p>&nbsp;</p>
 
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For industrial applications biosynthetic pathways composed of several enzymes should be engineered in a way to achieve high yield of the desired biosynthetic products. Various strategies for optimization have been undertaken so far, such as:
 
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<p>&nbsp;</p>
 
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<ul>
 
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<li>Increasing the pool of available substrate and/or overexpression of the enzymes of the limiting biosynthetic steps,</li>
 
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<li>Introducing heterologous enzymes with preferred kinetic characteristics,</li>
 
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<li>Blocking branching of biosynthetic pathway,</li>
 
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<li>Compartmentalizing of biosynthetic pathways by directing enzymes of a particular biosynthetic pathway to a specific cell compartments or artificially made compartments (e.g. metabolosomes),</li>
 
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<li>Increasing the proximity of enzymes by assembling metabolic pathways on a protein scaffold.</li>
 
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</ul>
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<p><span style="font-size: 18px; line-height: 25px;">Scaffold-assisted biosynthetic pathway</span></p>
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__TOC__
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<p>&nbsp;</p>
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The last approach is in some way similar to solutions that nature has already evolved. In some natural biosynthetic pathways enzymes form larger complexes, which results in faster transport of intermediates from one enzyme to another. This strategy enables an organism to produce higher amounts of the final product with lower metabolic burden. Unstable and toxic intermediates can be protected from decay or can be neutralized, since they are immediately used by the next enzyme in the pathway.&nbsp;Experimental data has already shown on a case of resveratrol biosynthesis (Zhang et al., 2006) where fusing two enzymes together improves the efficiency of this particular biosynthetic pathway. However, fusing more than two enzymes may prove to be difficult on account of maintaining the functionality of the fusion protein, which is the reason that scaffolding is a better approach. A protein scaffold has already been used to improve the mevalonate pathway yield (Dueber et al. 2009). While there are many advantages of protein-based scaffold, this approach has some disadvantages. Three dimensional arrangement of polypeptides is unpredictable due to the flexibility of and between dimerization domains as represented in the scheme.&nbsp; Additionally each protein dimerization domain has different conditions under which it folds and forms the functional interaction. And perhaps the most important argument, there is a limited number of available weel-behaved protein dimerization domains available, while the biosynthetic pathways may comprise ten or more enzymes and synthetic bioengineers will want to engineer several pathways simultaneously.
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<p>&nbsp;</p>
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[[Image:Slo_dna_scafold1_1.jpg|thumb|center|700px|'''Schematic representation of advantages of DNA-guided biosynthetic scaffold. DNA imparts the linear order, while protein/polypeptide based scaffold predominantly clusters the biosynthetic enzymes without of particular order.
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<div id="okvircek" style="background-color:#CECDCD;border:1px solid #AEABAE;margin-top:9px;padding:11px">
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''']]
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<html>
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<h3>State of the art</h3>
 +
Violacein is a compound that is synthesized in five steps from tryptophan. Transfer of the biosynthetic pathway from <em>Chromobacterium violaceum</em> allows production of violacein by
 +
<em>E.Coli</em>. Parts for the complete violacein biosynthetic pathway are available in the Registry and succesful production of violacein has been demonstrated by the Cambridge 2009 iGEM team. Besides purple violacein, the same biosynthetic pathway also produces a green side product deoxychromoviridans.<br>
 +
<h3>Aims</h3>
 +
We wanted to test whether our idea can be applied to the real world application. We examined the advantages of DNA-guided protein assembly on the violacein pathway. Our goal was to construct chimeric biosynthetic enzymes and a DNA program coding for the correct order of biosynthetic enzymes.<br>
 +
<h3>Achievements</h3>
 +
We designed and constructed a modified violacein biosynthetic pathway composed of five chimeric proteins with violacein biosynthetic enzymes and different zinc fingers. Introduction of DNA program coding for the correct order of chimeric proteins into bacteria expressing violacein these chimeric enzymes resulted in 6-fold improved yield of violacein, in comparison to DNA coding for a scrambled order of biosynthetic pathway. Additionally, this arrangement resulted in a significant decrease of an unwanted side product deoxychromoviridans. These resultates demonstrate in a very convincing way the advantages of DNA scaffold platform for biosynthesis.<br>
 +
</html>
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</div>
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<br>
 +
<h2>Background</h2>
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Ever since 1882, violacein has been notable as the most obvious characteristic of a soil bacterium <em>Chromobacterium violaceum</em> 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.<br>
 +
<h2>Violacein biosynthetic pathway</h2>
 +
Genes for violacein biosynthesis (scheme below) are arranged in an operon consisting of <em>vioA, vioB, vioC, vioD</em> and <em>vioE</em>. 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 <em>E.Coli</em>. In <em>E.Coli</em> 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.
 +
 
 +
[[Image:SLOviolacein8.jpg|thumb|center|600px|'''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.]]<br>
 +
 
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<h2>Design of a DNA-guided biosynthetic pathway</h2>
 +
We designed five fusion proteins by combining different zinc fingers and appropriate violacein biosynthetic enzymes. Since we wanted to create an <em>in vivo</em> system for violacein production, we expressed them&nbsp;in&nbsp;<em>E. coli</em>. 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).
 +
 
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[[Image:SLOviolaceinprogram2.png|thumb|center|600px|'''Correct vs. scrambled DNA program''']]
 +
<br>
 +
[[Image:Animacija biosinteza.jpg|thumb|center|600px|'''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).]]
 +
<br><br>
 +
<h2>Results</h2>
 +
<em>E. coli</em> 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 ([http://partsregistry.org/Part:BBa_K274002 Part:Bba_K274002]), prepared by the 2009 iGEM team Cambridge, was also grown for comparison. <br>
 +
Bacteria containing chimeric proteins were able to produce violacein (no program), demonstrating that the <strong>addition of zinc finger domains does not interfere with their enzymatic activity</strong> (Figure 1). However, the production of violacein was delayed compared to the cultures that contained DNA programs.
 +
 
 +
[[Image:SLO_violacein01.png|thumb|center|700px|'''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.]]<br>
 +
 
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We further analyzed the extracts using more sensitive methods, e.g. thin-layer chromatography (TLC) with densitometry, [https://2010.igem.org/Image:Slo_hptlc20.JPG 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).
 +
 
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[[Image:SLO_violacein03a.jpg|thumb|center|700px|'''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.]]
 +
 
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[[Image:SLO_violacein02.jpg|thumb|center|700px|'''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.]]<br>
 +
 
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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).
 +
 
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[[Image:SLOrastnaviolacein.JPG|thumb|center|700px|'''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.]]<br>
 +
 
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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.<br>
 +
 
 +
<hr>
 +
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
 +
 
 +
 
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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.
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<p>&nbsp;</p>
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Keeping in mind all potential weaknesses of protein-based scaffolding, we came to an idea that DNA molecule could also be used as a scaffold for bringing biosynthetic pathway enzymes in the correct order. We further discussed the idea and fond out that DNA molecule in fact has several special advantages (see Table).
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<p>&nbsp;</p>
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<ul>
 
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<li>DNA molecule has highly predictable structure. Therefore scaffold based on it provides not only higher local concentration of biosynthetic pathway enzymes, but can also arrange them into a predefined order,</li>
 
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<li>Because double helix of DNA molecule makes a turn every 10 nucleotides or so, the proper spatial orientation of bounded proteins can be achieved by varying the number of spacer nucleotides between the two binding sites for DNA-binding proteins,</li>
 
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<li>A large number of different DNA binding proteins exists in nature. Some of them, such as zinc fingers or TAL effectors, have modular structure and can be engineered to bind any given nucleotide sequence. There are already more than 700 experimentally tested zinc fingers available to choose from. Therefore DNA scaffolding offers virually unlimited number of distinct combinations.</li>
 
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</ul>
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<p>Comparison od advantages and disadvantages between DNA and protein-based biosynthetic scaffold:</p>
 
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<table style="border: 0px;text-align:left;background-color:transparent" border="0" width="720" height="270">
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">&nbsp;</td>
 
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<td style="border: 1px solid #bbbbbb;"><strong>DNA scaffold</strong>&nbsp;</td>
 
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<td style="border: 1px solid #bbbbbb;"><strong>Protein scaffold&nbsp;</strong></td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p><strong>Spatial orientation</strong></p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Linear</p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Bundled</p>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p><strong>Order</strong></p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Highly predictable order</p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Unpredictable</p>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p><strong>Scaffold : enzyme ratio</strong></p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Difficult to achieve substantial amount of scaffold, ratio in favour of enzymes</p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Easy to achieve favorable ratio with gene expression regulation.</p>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p><strong>Scaffold: enzyme interactions</strong></p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Similar well characterized, predictable interactions&nbsp;</p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Variations in strength, limited number of well-characterized interactions</p>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p><strong>Variability, number of available elements</strong></p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Large number of zinc finger and other DNA binding domains is readily available, engineered zinc finger domains</p>
 
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</td>
 
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<td style="border: 1px solid #bbbbbb;">
 
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<p>Limited number of protein dimerization domains</p>
 
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</td>
 
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</tr>
 
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<tr>
 
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<td style="border: 1px solid #bbbbbb;"><strong>Interference with cellular metabolism</strong></td>
 
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<td style="border: 1px solid #bbbbbb;">May bind to chromatin, selecti rom sequences that do not affect growh</td>
 
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<td style="border: 1px solid #bbbbbb;">Signal transduction domains usually do not interfere with bacteria</td>
 
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</tr>
 
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</table>
 
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<p>&nbsp;</p>
 
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<p><span style="font-size: 20px; line-height: 25px;">Selection of the biosynthetic pathways</span></p>
 
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Registry of standard  biological parts comprises several biosynthetic pathways. Two of the more characterised pathways are violacein&nbsp; and carotenoid biosynthetic pathway. Violacein pathway was chosen because the product  has many potential medical applications . Additionaly, violacein pathway is very convenient  choice due to the colored products. The carotenoid pathway  was selected because of a increasing significance of these compounds in food  and health industr and similar to violacein its products are colored, facilitating assays. By selecting different carotenoid pathways we wanted to point  out the possible application of our system at an industrial level. We  were surprised how easy it was find appropriate genes in a  Registry of standard biological parts and it was a the testimony of the usefulness of the  Registry as a practical and valuable tool in the field of  synthetic biology.
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<p><span style="font-size: 20px; line-height: 25px;">Design of DNA-guided biosynthetic pathway</span></p>
 
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<p>&nbsp;</p>
 
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After the biosynthetic pathways were chosen we fused all enzymes to DNA binding proteins. We decided to use zinc fingers as the DNA binding proteins, since there is a large number of experimentally available zinc fingers available.We introduced the obtained chimeric biosynthetic pathway enzymes into <em>E. coli</em> and tested if enzymes fused with zinc fingers are still functional, which was the case in all tested enzymes. We designed scaffold DNA molecule we called DNA program.Zinc finger binding sequences were arranged on a program DNA in a way that they enabled arrangement of chimeric biosynthetic pathway enzymes in the correct order and proper spatial orientation. We also designed the scrambled variant of DNA program, where all zinc finger binding sequences were still present but were not arranged in a correct order. We introduced program DNA, scrambled DNA and random DNA molecules into cells containing all chimeric biosynthetic pathway enzymes and determined the kinetics, productivity and yield of biosynthetic pathway products. To our great pleasure strategy proved to be successful. Yield of the product was significantly higher in cells that contained program DNA than in cells that contained scrambled one or no program DNA at all.
 
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<p>&nbsp;</p>
 
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<p>&nbsp;</p>
 
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<p><span style="font-size: 20px; line-height: 25px;">Estimation of the yield enhancement by DNA-guided biosynthesis</span></p>
 
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<p>&nbsp;</p>
 
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We wanted to estimate the acceleration of the biosynthetic pathway flux achieved by the ordered assembly of biosynthetic enzymes. In reality the overall reaction rate depends on many different factors. Each of them can be the rate limiting step. Diffusion is often the rate limiting step for biosynthesis of small molecules. Under this approximation we found that the reaction rate can be significantly accelerated if biosynthetic pathway enzymes are arranged on the DNA program. Acceleration of overall reaction kinetics can be under those approximations in direct correlation with the number of biosynthetic steps. The rationale is that the local concentration of the substrate is several orders of magnitude greater when enzymes are assembled on biosynthetic chains in comparison to soluble monomeric enzymes, which are typically present in the cell at submilimolar concentrations.
 
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<p>&nbsp;</p>
 
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A simple way to perform an&nbsp;<em>in silico</em><em>&nbsp;</em>&nbsp;simulation of the dynamics of quasi-random distributed enzymes in a cell, is to evaluate a few enzymatic reaction steps of the Michaelis-Menten kinetic law. To provide a more relevant view of enzyme and substrate dynamics, we designed a stochastic model that incorporates the mentioned law as the main kinetic constraint. To perform a valid representation of simulation&rsquo;s results, we also included the Poisson distributed time delay as an essential part of the product (substrate) accumulation, accounting for the diffusion delay. The time delay variable was assumed to be inversely proportional to the average distance and enzyme/substrate local concentration. The following figure shows the simulated difference in product formation and corresponding reaction velocity for a three reaction biosynthetic pathway, assuming equal binding affinity and diffusion-limited reaction rate.
 
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<p>&nbsp;</p>
 
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[[Image:SLOencimi3_a.jpg|thumb|center|700px|'''The comparison of a simulated product formation for a three reaction biosynthetic pathway. The "scaffold" graph shows a fast approaching of the steady state, where all the substrates are consumed and the accumulation of the final biosynthetic product reaches the maximal concentration. The graph corresponding to the , contrarily, reaches the steady state much later.''']]
 

Latest revision as of 03:59, 28 October 2010

Chuck Norris facts:

biosynthetic pathways: violacein


Contents


State of the art

Violacein is a compound that is synthesized in five steps from tryptophan. Transfer of the biosynthetic pathway from Chromobacterium violaceum allows production of violacein by E.Coli. Parts for the complete violacein biosynthetic pathway are available in the Registry and succesful production of violacein has been demonstrated by the Cambridge 2009 iGEM team. Besides purple violacein, the same biosynthetic pathway also produces a green side product deoxychromoviridans.

Aims

We wanted to test whether our idea can be applied to the real world application. We examined the advantages of DNA-guided protein assembly on the violacein pathway. Our goal was to construct chimeric biosynthetic enzymes and a DNA program coding for the correct order of biosynthetic enzymes.

Achievements

We designed and constructed a modified violacein biosynthetic pathway composed of five chimeric proteins with violacein biosynthetic enzymes and different zinc fingers. Introduction of DNA program coding for the correct order of chimeric proteins into bacteria expressing violacein these chimeric enzymes resulted in 6-fold improved yield of violacein, in comparison to DNA coding for a scrambled order of biosynthetic pathway. Additionally, this arrangement resulted in a significant decrease of an unwanted side product deoxychromoviridans. These resultates demonstrate in a very convincing way the advantages of DNA scaffold platform for biosynthesis.


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.