Team:Bielefeld-CeBiTec/Results/Pathway

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Module III - Isobutanol production

Cloning

Coding sequences

We started with the pSB1C3_alsS_ilvC_ilvD_kivD construct which can be found in the parts registry as BBa_K1465302. Therefore we used the CDS of the NCTU team Formosa 2011/2012 BioBricks alsS (BBa_K539627), ilvC (BBa_K539621), ilvD (BBa_K539626) and kivD (BBa_K539742). We used the parts of the parts distribution to combine them by Gibson Assembly. For this purpose we amplified the various CDS and combined them with the RBS (BBa_B0034) by PCR.
In the beginning it did not work with pSB1C3 so we used pSB1K3.
When we had pSB1K3_alsS_ilvC_ilvD_kivD, we wanted to reclone it in pSB1C3 and combine it with a promoter. We wanted to do this by BioBrick Assembly. While doing this we identified an illegal restriction site in the end of alsS because of the combination with the following RBS. The restriction site was removed by PCR amplification and Gibson assembly with new primer (rv_ilvC_alsS-new, fw_alsS_ilvC-new). In this approach we were able to amplify pSB1C3 as backbone, so no recloning was necessary.

Additionally we wanted to combine our part BBa_K1465302 with the adhA from Lactococcus lactis. This alcoholdehydrogenase was identified as the best described enzyme for the last step in the 2-keto-acid pathway (Atsumi2008, Atsumi2010). This pathway is responsible for the isobutanol production.
We wanted to add the adhA to pSB1C3_alsS_ilvC_ilvD_kivD by BioBrick Assembly, but adhA was not available as a BioBrick. So we decided to design it as a new BioBrick (BBa_K1465301). The approach can be found in the Results section of adhA. We did a BioBrick Sufffix Insertion and the result was the pSB1C3_alsS_ilvC_ilvD_kivD_adhA construct which is the part BBa_K1465303.

Constructs with promoter

To characterize our BioBricks, we wanted to add different promoter to them. We chose the Ptac promoter (BBa_K731500) for characterizations during cultivations and the stronger T7 promoter (BBa_I719005) for SDS pages.
We performed different BioBrick Assemblies, because in the beginning it was not working.
We tried BioBrick Suffix Assembly where the Ptac was cut out of the gel and BioBrick Prefix Assembly where we cut the alsS_ilvC_ilvD_kivD part out of the gel. In the end, the suffix version worked out and we created the BioBrick device pSB1C3_Ptac_alsS_ilvC_ilvD_kivD (BBa_ K1465306)
The same BioBrick Assemblies we performed with the Ptac promoter and the alsS_ilvC_ilvD_kivD_adhA part, and the BioBrick Suffix Assembly worked out again. We created the BioBrick device pSB1C3_Ptac_alsS_ilvC_ilvD_kivD_adhA (BBa_ K1465307)
For the combination with the T7 promoter we were only performing successful BioBrick Suffix Assembly for both constructs.

Expression

For the protein expression analysis of or two created constructs we made a cultivation of E. coli KRX with respectively one of the constructs.

pSB1A2_T7_alsS_ilvC_ilvD_kivD

Samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD were taken like explained in the cell lysis for a SDS-PAGE Protocol. 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 possibly 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.

pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA

We took samples of E. coli KRX with our construct pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA like explained in the cell lysis for a SDS-PAGE Protocol. The induction of the protein expression with rhamnose happened when a OD600 of 0.8 was reached. Right before the induction the first sample was taken and additional samples Protein expression was induced with rhamnose when the culture reached a OD600 of 0.8. The samples were taken two, four, 21 and 23 hours later. These samples were used for a SDS Page. In Figure 2 you can find the picture of this SDS Page.


Figure 2: SDS page from pSB1A2_T7_alsS_ilvC_ilvD_kivD_adhA.
The mass of the overexpressed proteins is 62,004 Da (AlsS), 54,069 Da (IlvC), 65,532 Da (IlvD), 60,947 Da (KivD) and 35,776 Da (AdhA)
In the picture of the SDS Page several bands look increasing in their size over the time of sampling. Again bands of approximately the mass of the four proteins AlsS (62,004 Da), IlvC (54,069 Da), IlvD (65,532 Da)and KivD (60,947 Da) seem to be present. The band at a mass of ~ 68 kD could be an evidence of the overexpression of protein IlvD. The next band right under ~ 60 kD could be an indication for the two proteins AlsS (62,004 Da) and KivD (60,947 Da). In addition the over time increasing band at a mass of ~ 53 kD is apparent and could be possible the overexpressed protein IlvC (54,069 Da). Furthermore the size of the band at a mass of ~ 38 kD increases over time and is comparable to the possible band of the AdhA in the SDS Page of pSB1A2_T7_adhA. This band could be the overexpressed protein AdhA (35,776 Da).
As in the SDS Page of pSB1A2_T7_alsS_ilvC_ilvD_kivD all proteins seem to be overexpressed and present. This experiment measured up to our expectations.

Cultivation

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 and treated them as described in the protocol. 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.


Figure 3: Calibration curve for the calculation of the isobutanol amount in the GC-MS data.
The first cultivation for producing isobutanol was made at a temperature of 37°C. We induced our construct carrying cultures after 1.5 h at an OD600 of 0.8. As controls, we had the KRX wildtype and not induced cultures carrying BBa_K1465306 and BBa_K1465307. We also had controls, which just were analyzed in the point of induction and in the end. These were the induced BBa_K1465306 and BBa_K1465307 cultures, the not induced ones and the KRX wildtype.
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.

Figure 4: Comparison of the growth of the analyzed cultures at 37°C.
BBa_K1465306 is the construct without the adhA and
BBa_K1465307 the construct with the adhA
Using GC-MS analysis we could prove the isobutanol production. The amount of production is shown in Figure 5. It is displayed that the isobutanol production increases over the cultivation time. In the stationary phase, the isobutanol amount decreases. Evaporation might be an explanation for this.
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 5: Comparison of the growth and the isobutanol production for cultures at 37°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
The second cultivation was performed at a temperature of 30°C. At this temperature we expected a better protein expression, thus a better isobutanol production. In Figure 6 a comparison of the OD600 of the induced constructs at the different temperatures is shown. The growth at the different temperatures is nearly the same. During this cultivation, we took samples at more time points to get an indication for a product formation rate. We took samples in the point of induction and then after two, four, six, eight, ten and 20 hours. In Figure 7 the isobutanol production is shown in comparison to the OD600. Here you can see the expected trend, that the culture carrying BBa_K1465307, the one with adhA, produces more isobutanol than BBa_K1465306. Nevertheless, the amount is much lower than the amount of BBa_K1465306 at 37°C.

Figure 6:Comparison of the growth of the induced cultures carying BBa_K1465306 and BBa_K1465307 (with adhA) at the temperatures 30°C and 37°C.

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.

Figure 8: Product formation rate of the cultures with BBa_K1465306 (without AdhA) and BBa_K1465307 (with AdhA) during the 30°C cultivation.

Conclusion

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.


References
  • 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

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