Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach
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<img src="https://static.igem.org/mediawiki/2014/9/96/Bielefeld_CeBiTec_2014-10-16_Isobutanol_constructs.png" width="400px" align="center"> | <img src="https://static.igem.org/mediawiki/2014/9/96/Bielefeld_CeBiTec_2014-10-16_Isobutanol_constructs.png" width="400px" align="center"> | ||
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- | <font size="2" style="text-align:left;"><b>Figure 2</b>: Schematic illustration of our isobutanol constructs. <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. | 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. | ||
<br> | <br> | ||
- | 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 the genes. . | + | 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 the genes. The resulting part of this idea is the part <a href="http://parts.igem.org/Part:BBa_K1465306" target="_blank">BBa_K1465306</a>. |
<br> | <br> | ||
- | 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 | + | 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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<h6>α-acetolactate synthase</h6> | <h6>α-acetolactate synthase</h6> | ||
- | <p>We took the coding sequence of the gene of the α-acetolactate synthase (AlsS) from <i>B. subtilis</i> from <a href="http://parts.igem.org/Part:BBa_K539627" target="_blank">BBa_K539627</a>. | + | <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>. |
- | <br>This protein is responsible for the | + | <br>This protein is responsible for the conversion of pyruvate into 2-acetolactate (cf. figure 1). |
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<h6>Ketol-acid reductoisomerase</h6> | <h6>Ketol-acid reductoisomerase</h6> | ||
- | <p>We took the coding sequence of the gene of the ketol-acid reductoisomerase (IlvC) from <i>E. coli</i> | + | <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>). |
- | <br>This protein | + | <br>This protein converts 2-acetolactate into 2,3-dihydroxyisovalerate (cf. figure 1). |
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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>. | ||
- | <br>This protein is responsible for the | + | <br>This protein is responsible for the conversion of of 2,3-dihydroxyisovalerate into 2-ketoisovalerate (cf. figure 1). |
<center> | <center> | ||
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<h6>α-ketoisovalerate decarboxylase</h6> | <h6>α-ketoisovalerate decarboxylase</h6> | ||
<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>. | <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>. | ||
- | <br>This protein | + | <br>This protein catalyzes the reaction from 2-ketoisovalerate into isobutyraldehyde (cf. figure 1). |
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<div class="element" style="margin:10px; padding:10px; width:600px;"> | <div class="element" style="margin:10px; padding:10px; width:600px;"> | ||
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<h6>alcoholdehydrogenase</h6> | <h6>alcoholdehydrogenase</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>). | ||
- | <br>This protein is responsible for the | + | <br>This protein is responsible for the conversion of isobutyraldehyde into isobutanol (cf. figure 1). |
<center> | <center> | ||
<div class="element" style="margin:10px; padding:10px; width:550px;"> | <div class="element" style="margin:10px; padding:10px; width:550px;"> |
Revision as of 15:39, 17 October 2014
Module III - Isobutanol production
Genetical Approach
As shown in the section about isobutanol 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 E.coli. The pathway is shown in figure 1.
In this pathway 2-ketoisovalerate is first decarboxylated into isobutyraldehyde by the ketoacid decarboxylase and then reduced to alcohols. The 2-keto-acid intermediates are produced by the host's amino acid biosynthetic pathway. The starting point of the whole isobutanol producing pathway is pyruvate which is generated by glycolysis of the cell. For this the 3-phosphogylcerate is required which is generated by the Calvin cycle of the CO2 fixation of module II.As we want to integrate this pathway in E.coli we use and improve existing BioBricks from the iGEM team NCTU Formosa 2011/2012. We use the coding sequences of the genes of four out of five required proteins for the isobutanol production.
These genes are
- alsS (BBa_K539627)
- ilvC (BBa_K539621)
- ilvD (BBa_K539626)
- kivD (BBa_K539742)
As you can see in figure 2 we have two approaches for our producing system. 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 an 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 use the AdhE from E. coli, 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 the genes. The resulting part of this idea is the part BBa_K1465306.
We found out, that the AdhA from L. Lactis is the best alcoholdehydrogenase in literature (Atsumi et al., 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 (BBa_K1465301) which contains the coding sequence of the adhA gene from L. Lactis and is combined with the RBS BBa_B0034. You can find a schematic illustration of the created BioBrick BBa_K1465307in 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 (cf. figure 1).
Protein | Gene | |
---|---|---|
Name | α-acetolactate synthase (AlsS) | alsS |
Length | 554 aa | 1,662 bp |
Mass | 60.78 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 (cf. figure 1).
Protein | Gene | |
---|---|---|
Name | ketol-acid reductoisomerase (IlvC) | ilvC |
Length | 491 aa | 1,473 bp |
Mass | 54.07 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 (cf. figure 1).
Protein | Gene | |
---|---|---|
Name | dihydroxyacid dehydratase (IlvD) | ilvD |
Length | 616 aa | 1,848 bp |
Mass | 65.53 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 (cf. figure 1).
Protein | Gene | |
---|---|---|
Name | α-ketoisovalerate decarboxylase (KivD) | kivD |
Length | 548 aa | 1,644 bp |
Mass | 60.95 Da | -- |
alcoholdehydrogenase
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 (cf. figure 1).
Protein | Gene | |
---|---|---|
Name | alcoholdehydrogenase 1 (AdhA) | adhA |
Length | 340 aa | 1,020 bp |
Mass | 35.78 Da | -- |
References
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Atsumi S, Hanai T, Liao JC., 2008. Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels. In: Nature 451, 86–89.
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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
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UniProt, version 10/2014