Translational_Unit

Part:BBa_K1465302

Designed by: iGEM-Team Bielefeld 2014   Group: iGEM14_Bielefeld-CeBiTec   (2014-10-06)

Isobutanol pathway

Usage and Biology

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.


Figure 1: Schematic illustration of the isobutanol pathway
The shown pathway starts with pyruvate and results in isobutanol. We also start with pyruvate which is generated from 3-phosphogylcerate in the glycolysis of the cell.
The steps in the conversion of pyruvate to 2-ketoisovalerate can be executed by proteins existing in E. coli (IlvIH, IlvC and IlvD). Since E. coli also has an alcohol dehydrogenase (AdhE), the only required protein for the isobutanol production is a ketoisovalerate decarboxylase. This protein (KivD) can be received from Lactococcus lactis. The pathway shown in Figure 1 is already an improvement of the described way. The native 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 The coding sequence of the gene of Adh (alcohol dehydrogenase), 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).

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5669
    Illegal XhoI site found at 4949
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2332
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1918
    Illegal BsaI site found at 4735
    Illegal BsaI site found at 4992
    Illegal BsaI.rc site found at 320
    Illegal BsaI.rc site found at 914
    Illegal BsaI.rc site found at 2737

Characterization

For characterization we analyzed the protein expression of BBa_K1465302 under the control of T7 promoter we made a cultivation of E. coli KRX with the constructs.
Samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD were taken. Protein expression was induced with rhamnose when the culture reached a OD600 of 0.8. The first sample was taken right before the induction. Additionally we took samples two, four, 21 and 23 hours later. With these samples, we made a SDS Page. Figure 1 shows the picture of this SDS Page.


Figure 1: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD.
The mass of the overexpressed proteins is 62,004 Da (AlsS), 54,069 Da (IlvC), 65,532 Da (IlvD) and 60,947 Da (KivD)
Several bands seem to increase in their size over the time of sampling. One band at a mass of ~ 68 kD shows a significant difference between the sample of sampling right before the induction and 23 hours later. This could be an hint of the overexpression of protein IlvD (65,532 Da). As the proteins AlsS (62,004 Da) and KivD (60,947 Da) have almost the same mass one will not see a difference between the band of the proteins in the SDS Page. Right under the band at a mass of ~ 68 kD is an additional band at a mass of ~ 60 kD visible which size increases over the time of sampling, too. This could be an indication for the two proteins AlsS and KivD. Furthermore there is a band increasing over time at a mass of ~ 53 kD apparent. This could be possible the overexpressed protein IlvC (54,069 Da).
These observations fit to our expectations of possible results of this experiment, because all proteins seem to be overexpressed.