Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach

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<h1>Module III - Isobutanol production</h1>


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   <h6>Genetical Approach</h6>

     <p><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Isobutanol/Isobutanol">Isobutanol</a> is an important substance for industry. No known organism can produce isobutanol or other branched-chain alcohols. <a href="#Atsumi2008">Atsumi et al.</a> presented a metabolic pathway to produce isobutanol in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>Escherichia coli</i></a>. The pathway is shown in Figure 1.
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<font size="2" style="text-align:left;"><b>Figure 1:</b> Schematic illustration of the isobutanol pathway</font>
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The shown pathway starts with pyruvate and results in isobutanol. We also start with pyruvate which is generated from 3-phosphogylcerate in the glycolysis of the cell. For this 3-phosphogylcerate is required which is generated in the Calvin cycle of the CO<sub>2</sub> fixation in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/CO2-fixation/CarbonDioxide">module II</a>. The steps in the conversion of pyruvate to 2-ketoisovalerate can be executed by proteins existing in <i>E. coli</i> (IlvIH, IlvC and IlvD). Since <i>E. coli</i> also has an alcohol dehydrogenase (AdhE), the only required protein for the isobutanol production is  a ketoisovalerate decarboxylase. This protein (KivD) can be received from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"><i>Lactococcus lactis</i></a>. The pathway shown in Figure 1 is already an improvement of the described way. The native protein IlvIH is replaced by the AlsS from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.subtilis" target="_blank"><i>Bacillus subtilis</i></a> to increase the isobutanol production. (<a href="#Atsumi2008">Atsumi et al.</a>)
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As we want to integrate this pathway in <i>E.coli</i> we used and improved existing BioBricks from the iGEM team NCTU Formosa 2011/2012. We used gene coding sequences of four out of five required proteins for the isobutanol production. 
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These genes are 
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<li><i>alsS</i> (<a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>)</li>
<li><i>ilvC</i> (<a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">BBa_K539621</a>)</li>
<li><i>ilvD</i> (<a href="http://parts.igem.org/Part:BBa_K539626" target="_blank">BBa_K539626</a>)</li>
<li><i>kivD</i> (<a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</a>)</li>
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The coding sequence of the gene of Adh (alcohol dehydrogenase), the fifth required protein, was not available as a BioBrick but because of <i>E.coli</i>'s own AdhE the pathway works (Atsumi et al., 2008).
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As you can see in Figure 2 we have two approaches for our producing system. 
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<font size="2" style="text-align:left;"><b>Figure 2</b>: Schematic illustration of our isobutanol constructs. <br><b>A</b> <a href="http://parts.igem.org/Part:BBa_K1465306" target="_blank">BBa_K1465306</a> <b>B</b> <a href="http://parts.igem.org/Part:BBa_K1465307" target="_blank">BBa_K1465307</a></font>
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We want to reproduce the pathway from iGEM team NCTU Formosa without their temperature system (<a href="http://parts.igem.org/Part:BBa_K887002">BBa_K887002</a>). In their system the first three proteins (AlsS, IlvC and IlvD) were generated while <i>E.coli</i> is incubated in a 37°C environment. During this the non-toxic intermediate 2-ketoisovalerate is accumulated. By shifting the temperature to a 30°C environment the missing KivD can be generated because of the non-active repressor. Together with the AdhE from <i>E. coli</i> KivD converts 2-ketoisovalerate into isobutanol. 
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In Figure 2A you can find our first approach where we also used the AdhE from <i>E. coli</i>. We disclaim the temperature system and put all coding sequences in a row behind a promoter separated by the RBS in front of each gene. We used <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson" target="_blank">Gibson</a> as the cloning method for the part <a href="http://parts.igem.org/Part:BBa_K1465302" target="_blank">BBa_K1465302</a>. For cloning the <i>ptac</i> upstream the part <a href="http://parts.igem.org/Part:BBa_K1465302" target="_blank">BBa_K1465302</a>, the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick"target="_blank">prefix insertion BioBrick assembly</a> was used. This idea resulted in the part <a href="http://parts.igem.org/Part:BBa_K1465306" target="_blank">BBa_K1465306</a>.
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We found out, that the AdhA from <i>L. Lactis</i> is the best alcohol dehydrogenase in literature (<a href="#Atsumi2010">Atsumi et al., 2010</a>). We wanted to increase the production of isobutanol by cloning the adhA gene downstream of our producing pathway. By <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Gibson">Gibson</a> we designed a new part (<a href="http://parts.igem.org/Part:BBa_K1465301" target="_blank">BBa_K1465301</a>) which contains the coding sequence of the adhA gene from <i>L. Lactis</i> and is combined with the RBS <a href="http://parts.igem.org/Part:BBa_B0034" target="_blank">BBa_B0034</a>. Afterwards we did several <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#BioBrick" target="_blank">BioBrick assemblies</a> for combining the parts <a href="http://parts.igem.org/Part:BBa_K731500" target="_blank">BBa_K731500</a>, <a href="http://parts.igem.org/Part:BBa_K1465302" target="_blank">BBa_K1465302</a> and <a href="http://parts.igem.org/Part:BBa_K1465302" target="_blank">BBa_K1465302</a>. You can find a schematic illustration of our created BioBrick <a href="http://parts.igem.org/Part:BBa_K1465307" target="_blank">BBa_K1465307</a> in Figure 2B.
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   <h6>Involved Proteins</h6>
<p>In the following section you will find some information about the five proteins involved in the isobutanol production.</p>
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   <h6>α-acetolactate synthase</h6>
<p>We took the coding sequence of the gene of the α-acetolactate synthase (AlsS) from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.subtilis" target="_blank"><i>B. subtilis</i></a> from the BioBrick <a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>. 
<br>This protein is responsible for the conversion of pyruvate into 2-acetolactate (Figure 1).
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<font size="2" style="text-align:left;"><b>Table 1:</b> General information about the α-acetolactate synthase (AlsS) (<a href="#UniProt">UniProt</a>)</font>
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<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><td>Name</td><td>α-acetolactate synthase (AlsS)</td><td><i>alsS</i></td></tr>
<tr><td>Length</td><td>554 aa</td><td>1,662 bp</td></tr>
<tr><td>Mass</td><td>62,004 Da</td><td> -- </td></tr>
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   <h6>Ketol-acid reductoisomerase</h6>
<p>We took the coding sequence of the gene of the ketol-acid reductoisomerase (IlvC) from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> (<a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">BBa_K539621</a>). 
<br>This protein converts 2-acetolactate into 2,3-dihydroxyisovalerate (Figure 1).
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<font size="2" style="text-align:left;"><b>Table 2:</b> General information about the ketol-acid reductoisomerase (IlvC) (<a href="#UniProt">UniProt</a>)</font>
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><td>Name</td><td>ketol-acid reductoisomerase (IlvC)</td><td><i>ilvC</i></td></tr>
<tr><td>Length</td><td>491 aa</td><td>1,473 bp</td></tr>
<tr><td>Mass</td><td>54,069 Da</td><td> -- </td></tr>
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   <h6>Dihydroxyacid dehydratase</h6>
<p>We took the coding sequence of the gene of the dihydroxyacid dehydratase (IlvD) from <i>E. coli</i> from <a href="http://parts.igem.org/Part:BBa_K539626" target="_blank">BBa_K539626</a>. 
<br>This protein is responsible for the conversion of  of 2,3-dihydroxyisovalerate into 2-ketoisovalerate (Figure 1).
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<font size="2" style="text-align:left;"><b>Table 3:</b> General information about the dihydroxyacid dehydratase (IlvD) (<a href="#UniProt">UniProt</a>)</font>
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<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><td>Name</td><td>dihydroxyacid dehydratase (IlvD)</td><td><i>ilvD</i></td></tr>
<tr><td>Length</td><td>616 aa</td><td>1,848 bp</td></tr>
<tr><td>Mass</td><td>65,532 Da</td><td> -- </td></tr>
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   <h6>α-ketoisovalerate decarboxylase</h6>
<p>We took the coding sequence of the gene of the α-ketoisovalerate decarboxylase (KivD) from <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#L.lactis" target="_blank"><i>L. lactis</i></a> from <a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</a>. 
<br>This protein catalyzes the reaction from 2-ketoisovalerate into isobutyraldehyde (Figure 1).
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<font size="2" style="text-align:left;"><b>Table 4:</b> General information about the α-ketoisovalerate decarboxylase (KivD) (<a href="#UniProt">UniProt</a>)</font>
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><td>Name</td><td>α-ketoisovalerate decarboxylase (KivD)</td><td><i>kivD</i></td></tr>
<tr><td>Length</td><td>548 aa</td><td>1,644 bp</td></tr>
<tr><td>Mass</td><td>60,947 Da</td><td> -- </td></tr>
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   <h6>alcohol dehydrogenase</h6>
<p>We designed a new part which contains the coding sequence of the adhA gene from <i>L. Lactis</i> (<a href="http://parts.igem.org/Part:BBa_K1465301" target="_blank">BBa_K1465301</a>). 
<br>This protein is responsible for the conversion of isobutyraldehyde into isobutanol (Figure 1).
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<font size="2" style="text-align:left;"><b>Table 5:</b> General information about the alcohol dehydrogenase (AdhA) (<a href="#UniProt">UniProt</a>)</font>
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><td>Name</td><td>alcohol dehydrogenase 1 (AdhA)</td><td><i>adhA</i></td></tr>
<tr><td>Length</td><td>340 aa</td><td>1,020 bp</td></tr>
<tr><td>Mass</td><td>35,776 Da</td><td> -- </td></tr>
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   <h6>References</h6>
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<li id="Atsumi2008">
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  Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: <a href="http://www.nature.com/nature/journal/v451/n7174/full/nature06450.html" target="_blank">Nature 451</a>, 86–89. 
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<li id="Atsumi2010">
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Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC. 2010. Engineering the isobutanol biosynthetic pathway in <i>Escherichia coli</i> by comparison three aldehyde reductase/alcohol dehydrogenase genes. In: <a href="http://link.springer.com/article/10.1007%2Fs00253-009-2085-6" target="_blank">Appl. Microbiol. Biotechnol 85</a>, 651–657
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  <a href="http://www.uniprot.org/" target="_blank">UniProt</a>, version 10/2014
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