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)

The coding sequence of the gene of Adh (alcoholdehydrogenase), the fifth required protein, was not available as a BioBrick but because of E.coli's own Adh the pathway works (Atsumi et al., 2008).

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.

AlsS (α-acetolactate synthase)

IlvC (Ketol-acid reductoisomerase)

IlvD (Dihydroxyacid dehydratase)

KivD (α-ketoisovalerate decarboxylase)

AdhA (Alcoholdehydrogenase)

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.