Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach

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<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach"style="color:#000000">
<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach"style="color:#000000">
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               <p class="buttoncenter"><font color="#FFFFFF">Genetical approach</font></p>
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               <p class="buttoncenter"><font color="#FFFFFF">Genetical Approach</font></p>
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                        <br><br><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/Isobutanol"style="color:#000000">
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              <p class="buttoncenter"><font color="#FFFFFF">Results</font></p>
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   <h6>Genetical Approach</h6>
   <h6>Genetical Approach</h6>
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     <p>In the section about <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Isobutanol/Isobutanol">isobutanol</a>it is shown that isobutanol 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 <i>Escherichia coli</i>. The pathway is shown in figure 1.
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     <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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       <a href="https://static.igem.org/mediawiki/2014/8/8a/Bielefeld_CeBiTec_2014-10-16_Isobutanol_pathway.png" target="_blank">
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<font size="2" style="text-align:left;"><b>Figure 1:</b> Schematic illustration of the isobutanol pathway</font>
<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. In our project we also want to start with pyruvate which is generated from 3-phosphogylcerate in the glycolysis of the cell. For this the 3-phosphogylcerate is required which is generated by 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). <i>E. coli</i> also has a alcoholdehydrogenase (AdhE) so the only required protein for the isobutanol production is  a ketoisovalerate decarboxylase. This protein (KivD) can be received from <i>Lactococcus lactis</i>. The shown pathway in figure 1 already is an improvement of the described way. The protein IlvIH is replaced by the AlsS from <i>Bacillus subtilis</i> to increase the isobutanol production. (atsumi2008)
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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 use and improve existing BioBricks from the iGEM team NCTU Formosa 2011/2012. We use gene coding sequences of four out of five required proteins for the isobutanol production.  
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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  
These genes are  
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<li><i>kivD</i> (<a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</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 (alcoholdehydrogenase), 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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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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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>
<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 an 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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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 use the AdhE from <i>E. coli</i>, too. We pass on the temperature system and put all coding sequences in a row behind a promoter just separated by the RBS in front of each gene. The resulting part of this idea is the part <a href="http://parts.igem.org/Part:BBa_K1465306" target="_blank">BBa_K1465306</a>.
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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 alcoholdehydrogenase in literature (<a href="#Atsumi2008">Atsumi et al.</a>, 2010). For that reason we want to increase the production of isobutanol by putting the adhA gene behind our producing pathway. 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>. You can find a schematic illustration of the created BioBrick <a href="http://parts.igem.org/Part:BBa_K1465307" target="_blank">BBa_K1465307</a> in figure 2B.
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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>α-acetolactate synthase</h6>
   <h6>α-acetolactate synthase</h6>
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<p>We took the coding sequence of the gene of the α-acetolactate synthase (AlsS) from <i>B. subtilis</i> from the BioBrick <a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>.  
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<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>.  
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<br>This protein is responsible for the conversion of pyruvate into 2-acetolactate (cf. figure 1).
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<br>This protein is responsible for the conversion of pyruvate into 2-acetolactate (Figure 1).
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<tr><td>Name</td><td>α-acetolactate synthase (AlsS)</td><td><i>alsS</i></td></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>Length</td><td>554 aa</td><td>1,662 bp</td></tr>
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<tr><td>Mass</td><td>60.78 Da</td><td> -- </td></tr>
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<tr><td>Mass</td><td>62,004 Da</td><td> -- </td></tr>
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   <h6>Ketol-acid reductoisomerase</h6>
   <h6>Ketol-acid reductoisomerase</h6>
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<p>We took the coding sequence of the gene of the ketol-acid reductoisomerase (IlvC) from <i>E. coli</i> (<a href="http://parts.igem.org/Part:BBa_K539621" target="_blank">BBa_K539621</a>).  
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<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>).  
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<br>This protein converts 2-acetolactate into 2,3-dihydroxyisovalerate (cf. figure 1).
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<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 1:</b> General information about the ketol-acid reductoisomerase (IlvC) (<a href="#UniProt">UniProt</a>)</font>
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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;">
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<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>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>Length</td><td>491 aa</td><td>1,473 bp</td></tr>
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<tr><td>Mass</td><td>54.07 Da</td><td> -- </td></tr>
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<tr><td>Mass</td><td>54,069 Da</td><td> -- </td></tr>
</table>
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   <h6>Dihydroxyacid dehydratase</h6>
   <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>.  
<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>.  
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<br>This protein is responsible for the conversion of  of 2,3-dihydroxyisovalerate into 2-ketoisovalerate (cf. figure 1).
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<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 1:</b> General information about the dihydroxyacid dehydratase (IlvD) (<a href="#UniProt">UniProt</a>)</font>
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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>
<table style="background-color:transparent; cellspacing=3;">
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<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>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>Length</td><td>616 aa</td><td>1,848 bp</td></tr>
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<tr><td>Mass</td><td>65.53 Da</td><td> -- </td></tr>
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<tr><td>Mass</td><td>65,532 Da</td><td> -- </td></tr>
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   <h6>α-ketoisovalerate decarboxylase</h6>
   <h6>α-ketoisovalerate decarboxylase</h6>
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<p>We took the coding sequence of the gene of the α-ketoisovalerate decarboxylase (KivD) from <i>L. lactis</i> from <a href="http://parts.igem.org/Part:BBa_K539742" target="_blank">BBa_K539742</a>.  
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<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>.  
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<br>This protein catalyzes the reaction from 2-ketoisovalerate into isobutyraldehyde (cf. figure 1).
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<br>This protein catalyzes the reaction from 2-ketoisovalerate into isobutyraldehyde (Figure 1).
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<center>
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<font size="2" style="text-align:left;"><b>Table 1:</b> General information about the α-ketoisovalerate decarboxylase (KivD) (<a href="#UniProt">UniProt</a>)</font>
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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;">
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<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>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>Length</td><td>548 aa</td><td>1,644 bp</td></tr>
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<tr><td>Mass</td><td>60.95 Da</td><td> -- </td></tr>
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<tr><td>Mass</td><td>60,947 Da</td><td> -- </td></tr>
</table>
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   <h6>alcoholdehydrogenase</h6>
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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>).  
<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>).  
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<br>This protein is responsible for the conversion of isobutyraldehyde into isobutanol (cf. figure 1).
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<br>This protein is responsible for the conversion of isobutyraldehyde into isobutanol (Figure 1).
<center>
<center>
<div class="element" style="margin:10px; padding:10px; width:550px;">
<div class="element" style="margin:10px; padding:10px; width:550px;">
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<font size="2" style="text-align:left;"><b>Table 1:</b> General information about the alcoholdehydrogenase (AdhA) (<a href="#UniProt">UniProt</a>)</font>
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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;">
<table style="background-color:transparent; cellspacing=3;">
<tr><th></th><th>Protein</th><th>Gene</th></tr>
<tr><th></th><th>Protein</th><th>Gene</th></tr>
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<tr><td>Name</td><td>alcoholdehydrogenase 1 (AdhA)</td><td><i>adhA</i></td></tr>
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<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>Length</td><td>340 aa</td><td>1,020 bp</td></tr>
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<tr><td>Mass</td><td>35.78 Da</td><td> -- </td></tr>
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<tr><td>Mass</td><td>35,776 Da</td><td> -- </td></tr>
</table>
</table>
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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">Nature 451</a>, 86–89.  
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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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   <a href="http://www.uniprot.org/">UniProt</a>, version 10/2014
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   <a href="http://www.uniprot.org/" target="_blank">UniProt</a>, version 10/2014
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Latest revision as of 10:03, 2 December 2014


Module III - Isobutanol production


Genetical Approach

Isobutanol is an important substance for industry. No known organism can produce isobutanol or other branched-chain alcohols. Atsumi et al. presented a metabolic pathway to produce isobutanol in Escherichia coli. The pathway is shown in Figure 1.


Figure 1: Schematic illustration of the isobutanol pathway
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 CO2 fixation in module II. The steps in the conversion of pyruvate to 2-ketoisovalerate can be executed by proteins existing in E. coli (IlvIH, IlvC and IlvD). Since E. coli 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 Lactococcus lactis. The pathway shown in Figure 1 is already an improvement of the described way. The native protein IlvIH is replaced by the AlsS from Bacillus subtilis to increase the isobutanol production. (Atsumi et al.)
As we want to integrate this pathway in E.coli 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.
These genes are The coding sequence of the gene of Adh (alcohol dehydrogenase), the fifth required protein, was not available as a BioBrick but because of E.coli's own AdhE the pathway works (Atsumi et al., 2008).

As you can see in Figure 2 we have two approaches for our producing system.

Figure 2: Schematic illustration of our isobutanol constructs.
A BBa_K1465306 B BBa_K1465307
We want to reproduce the pathway from iGEM team NCTU Formosa without their temperature system (BBa_K887002). In their system the first three proteins (AlsS, IlvC and IlvD) were generated while E.coli 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 E. coli KivD converts 2-ketoisovalerate into isobutanol.

In Figure 2A you can find our first approach where we also used the AdhE from E. coli. 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 Gibson as the cloning method for the part BBa_K1465302. For cloning the ptac upstream the part BBa_K1465302, the prefix insertion BioBrick assembly was used. This idea resulted in the part BBa_K1465306.

We found out, that the AdhA from L. Lactis is the best alcohol dehydrogenase in literature (Atsumi et al., 2010). We wanted to increase the production of isobutanol by cloning the adhA gene downstream of our producing pathway. By Gibson we designed a new part (BBa_K1465301) which contains the coding sequence of the adhA gene from L. Lactis and is combined with the RBS BBa_B0034. Afterwards we did several BioBrick assemblies for combining the parts BBa_K731500, BBa_K1465302 and BBa_K1465302. You can find a schematic illustration of our created BioBrick BBa_K1465307 in Figure 2B.

Involved Proteins

In the following section you will find some information about the five proteins involved in the isobutanol production.

α-acetolactate synthase

We took the coding sequence of the gene of the α-acetolactate synthase (AlsS) from B. subtilis from the BioBrick BBa_K539627.
This protein is responsible for the conversion of pyruvate into 2-acetolactate (Figure 1).

Table 1: General information about the α-acetolactate synthase (AlsS) (UniProt)
ProteinGene
Nameα-acetolactate synthase (AlsS)alsS
Length554 aa1,662 bp
Mass62,004 Da --

Ketol-acid reductoisomerase

We took the coding sequence of the gene of the ketol-acid reductoisomerase (IlvC) from E. coli (BBa_K539621).
This protein converts 2-acetolactate into 2,3-dihydroxyisovalerate (Figure 1).

Table 2: General information about the ketol-acid reductoisomerase (IlvC) (UniProt)
ProteinGene
Nameketol-acid reductoisomerase (IlvC)ilvC
Length491 aa1,473 bp
Mass54,069 Da --

Dihydroxyacid dehydratase

We took the coding sequence of the gene of the dihydroxyacid dehydratase (IlvD) from E. coli from BBa_K539626.
This protein is responsible for the conversion of of 2,3-dihydroxyisovalerate into 2-ketoisovalerate (Figure 1).

Table 3: General information about the dihydroxyacid dehydratase (IlvD) (UniProt)
ProteinGene
Namedihydroxyacid dehydratase (IlvD)ilvD
Length616 aa1,848 bp
Mass65,532 Da --

α-ketoisovalerate decarboxylase

We took the coding sequence of the gene of the α-ketoisovalerate decarboxylase (KivD) from L. lactis from BBa_K539742.
This protein catalyzes the reaction from 2-ketoisovalerate into isobutyraldehyde (Figure 1).

Table 4: General information about the α-ketoisovalerate decarboxylase (KivD) (UniProt)
ProteinGene
Nameα-ketoisovalerate decarboxylase (KivD)kivD
Length548 aa1,644 bp
Mass60,947 Da --

alcohol dehydrogenase

We designed a new part which contains the coding sequence of the adhA gene from L. Lactis (BBa_K1465301).
This protein is responsible for the conversion of isobutyraldehyde into isobutanol (Figure 1).

Table 5: General information about the alcohol dehydrogenase (AdhA) (UniProt)
ProteinGene
Namealcohol dehydrogenase 1 (AdhA)adhA
Length340 aa1,020 bp
Mass35,776 Da --


References
  • Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.
  • Atsumi S, Wu TY, Eckl EM, Hawkins SD, Buelter T, Liao JC. 2010. Engineering the isobutanol biosynthetic pathway in Escherichia coli by comparison three aldehyde reductase/alcohol dehydrogenase genes. In: Appl. Microbiol. Biotechnol 85, 651–657
  • UniProt, version 10/2014