Isobutanol pathway under Ptac control
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.
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
As you can see in Figure 2 we have two approaches for our production 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 also used the AdhE from E. coli. We disclaim the temperature system and put all coding sequences in a row behind a promoter separated by the RBS upstream of each gene.
During our literature research about the isobutanol production pathway (Figure 1) we found out, that the alcohol dehydrogenase from Lactococcus lactis (AdhA) is more efficient than the one from Escherichia coli (Atsumi et al., 2010). We wanted to increase the production of isobutanol by cloning the adhA gene downstream of our producing pathway. You can find a schematic illustration of our created BioBrick BBa_K1465307 in Figure 2B.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 6916
Illegal XhoI site found at 6196
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 3579
- 1000Illegal BsaI site found at 3165
Illegal BsaI site found at 5982
Illegal BsaI site found at 6239
Illegal BsaI.rc site found at 1567
Illegal BsaI.rc site found at 2161
Illegal BsaI.rc site found at 3984
For characterization of the protein expression of BBa_K1465302 under the control of T7 promoter look at the BioBrick BBa_K1465302.
To proof the isobutanol production of our cultures carrying BBa_ K1465306 and BBa_ K1465307 we performed two cultivations.
First we made a calibration curve, so we can quantify the production. For this, we prepared samples with the concentrations 0.001%, 0.01%, 0.05%, 0.1% and 0.5% of isobutanol in LB medium. Like shown in Figure 3, the regression curve has the function 1.1058x + 0.0005= y and R2 is 0.999. With this, we made the upcoming quantifications. The normalized peak area is the peak area of isobutanol divided by the peak area of 2-butanol, which is the internal standard. For the evaluation of the GC-MS data we did not want to calculate with percent but with mg/L. Therefore, we converted the percent values by multiplication with 10 because the samples were diluted 1:10 before the measurement. Multiplication of the resulting value with the density of isobutanol, which is 802kg/m3 yields in g/l, which can be multiplied again with 1000 to get mg/l.
We also made an evaporation experiment to check the recovery rate of isobutonal during our experiments. We performed this experiment at 37°C over 20 hours and found out that about 50% isobutanol disappeared. In this setup we compared flasks we opened ten times and flask we just opened in the end. The amount of the opened flasks was in the error margin of the not opened flask. Hence it can be said, that the isobutanol concentration is not influenced by opening the flasks.
For the GC-MS analysis we took samples at the point of induction and then four, seven, ten and 20 hours afterwards.
In Figure 4 you can see the comparison of the OD600 of the analyzed cultures. It is conspicuously, that the induced constructs are growing slower from the point of induction, which indicates that there might be an redirected flux towards the isobutanol synthesis.
The growth of the not induced cultures carrying BBa_K1465306 and BBa_K1465307 were showing a slightly decreased growth than the wildtype, which might be because of the large plasmids.
Differently than expected, the culture carrying BBa_K1465307, which includes the adhA produced less isobutanol than BBa_K1465306. The maximum amount of isobutanol produced by the strain carrying the BBa_K1465306, the BioBrick without the adhA is about 55mg/l and of BBa_K1465307 about 37 mg/l. We were able to show in a SDS Page that all proteins were expressed.
Figure 7: Comparison of the growth curves and the isobutanol production for cultures at 30°C.
BBa_K1465306 is the construct without the adhA and BBa_K1465307 the construct with the adhA. The growth is shown in black and the isobutanol production in blue In Figure 8 the product formation rate is shown. It is obvious, that the product formation is not increasing directly from the point of induction. In the first two to three hours after induction there is no production of isobutanol, Three Hours after induction the amount of isobutanol increases until about six hours after induction, from then the production works well for about four hours. After this the amount decreases.
We successfully created six new BioBricks of which four are available in the parts registry: BBa_K1465302, BBa_K1465303, BBa_K1465306 and BBa_K1465307. The two other BioBricks are pSB1A2_T7_alsS_ilvC_ilvD_kivD and pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA.
Further, it could be shown that isobutanol was produced. We could not show, that the production with AdhA taken from L. lactis is higher. The expression of the E. coli own adhE seems to be enough.
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