Genetical Approach

In the section about isobutanolit is shown that 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.

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 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). E. coli 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 Lactococcus lactis. The shown pathway in figure 1 already is an improvement of the described way. The 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 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.
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 AdhE 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 (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 each gene. 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_K1465307 in figure 2B.

Involved Proteins

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

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