Genetical Approach

In the section about isobutanol it 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 an 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 is already 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 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

• 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 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 use the AdhE from E. coli, too. We disclaim the temperature system and put all coding sequences in a row behind a promoter just 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. The resulting part of this 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 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 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