Team:Bielefeld-CeBiTec/Project/Isobutanol/GeneticalApproach
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<font size="2" style="text-align:left;"><b>Figure 2</b>: Schematic illustration of our isobutanol constructs</font> | <font size="2" style="text-align:left;"><b>Figure 2</b>: Schematic illustration of our isobutanol constructs</font> | ||
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- | + | We want to reproduce the pathway from iGEM team NCTU Formosa without their temperature system. In their system the first three proteins (AlsS, IlvC and IlvD) were generated while <i>E.coli</i> is incubated in 37°C environment. During this the non-toxic intermediate 2-ketoisovalerate is accumulated. By moving <i>E. Coli</i> to an 30°C environment the missing KivD can be generated because of the non-active repressor. Together with the Adh 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 Adh from <i>E. Coli</i> too. We pass on the temperature system and put all coding sequences in a row behind a promotor just separated by RBS. | In figure 2A you can find our first approach where we use the Adh from <i>E. Coli</i> too. We pass on the temperature system and put all coding sequences in a row behind a promotor just separated by RBS. |
Revision as of 16:09, 16 October 2014
Isobutanol
Genetical Approach
As shown here 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, a higher alcohol, in E.coli which 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. In their system the first three proteins (AlsS, IlvC and IlvD) were generated while E.coli is incubated in 37°C environment. During this the non-toxic intermediate 2-ketoisovalerate is accumulated. By moving E. Coli to an 30°C environment the missing KivD can be generated because of the non-active repressor. Together with the Adh from E. Coli KivD converts 2-ketoisovalerate into isobutanol.
In figure 2A you can find our first approach where we use the Adh from E. Coli too. We pass on the temperature system and put all coding sequences in a row behind a promotor just separated by RBS.
We found out, that the AdhA from L. Lactis is the best alcoholdehydrogenase in literature (REFERENCE!!!). 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 which contains the coding sequence of the adhA gene from L. Lactis (BBa_K1465301). You can find a schematic illustration of this approach 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 BBa_K539627.
This protein is responsible for the convertion 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 from BBa_K539621.
This protein is responsible for the convertion of 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 convertion 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 is responsible for the convertion of 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 convertion 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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UniProt, version 10/2014