Team:BJU China/Engineering


Overall Objective:

In our project, we hope to convert tryptophan to halogenated tryptophan through halogenase, and then through TnaA proteolysis and MaFMO protein oxidation to become halogenated indigo, and finally synthesize a variety of indigo derivatives (Figure 1).

Figure 1. Denovo synthesis indigo derivatives from tryptophan

Our Process:

In our engineering success section, we obtained the three proteins SttH, RedH, and PyrH through molecular cloning, and fused them with the Fre protein through L3 linker, thereby increasing the solubility of the three halogenated proteins in water and promoting the production of indigo derivatives. The figure below shows the relevant gene circuit diagram we constructed. 

Figure 2. Gene circuit diagram of Halogenase 

We constructed halogenase plasmids using Gibson Assembly and verified by colony PCR and gene sequencing . According to the results in Figure 3, we can roughly identify the three genes (Frr-L3-SttH, Fre-L3-PyrH, Fre-L3-RebH) comparing with DNA Marker. And then the PCR product fragments were sent for sequencing and the results were also consistent with our target fragments.

Figure 3. Molecular identification of Halogenase genes

Next, we started the protein level experiment to test gene expression. For better expression of enzymes, we optimize the induction condition of gene expression: three different temperatures (18℃, 30℃, 37℃) and inducer concentrations (0 mM, 0.1mM, 1mM) were set for test. Additionally, since the halogenases are fusion enzymes with higher solubility, we disrupted collected cells and tested both pellet and supernatant after induction (Figure 4-6). 

From the results of three SDS-PAGE electrophoresis gels, it can be seen that compared with the control group induced by 0mM IPTG, there are obvious changes in the lanes induced by IPTG. Obvious bands can be seen in the precipitated protein gels of SttH, RebH and PyrH, which proves these halogenase can be effectively expressed under the induction of IPTG.

Figure 4. SDS-PAGE Gel of Fre-L3-SttH (Left is precipitation, Right is supernatant)

Figure 5. SDS-PAGE Gel of Fre-L3-PyrH (Left is precipitation, Right is supernatant)

Figure 6. SDS-PAGE Gel of Fre-L3-RebH (Left is supernatant, Right is precipitation) 

Through the expression of halogenase at different temperatures under the same IPTG concentration, we can get the following conclusions:

(1) There is a significant decrease in RebH when induced at 18℃ compared with the expression levels at 30℃ and 37℃.

(2) While PyrH protein expressed better and has obvious bands at 18℃ compared to the expression at 30℃ and 37℃, so we can conclude that 18°C is the most suitable induction temperature for PyrH.

(3) SttH induced by IPTG could express well at three different temperatures, and as the temperature increases, the expression level also gradually increases. There is no significant difference in the expression level of SttH at 37°C with 0.1, 1, and 5 mM IPTG, and the amount of protein is significantly more compared to the other two temperatures, so we can speculate that the optimal temperature for the expression of SttH is 37°C.

From the SDS-PAGE results of the three proteins, it is not difficult to see that the content of halogenase expressed in the supernatant solution is low. This may be due to low solublity of halogenases, even through Fre-L3 protein connection, the solubility is still not effectively improved, most enzymes are still exsit in the precipitation. At the same time, it may also be related to our parameter settings in the cell disruption. Because of the insufficient ultrasonication strength, the protein is wrapped in cytoplasm then forms precipitate. 

Subsequently, we performed the protein expression induction experiment again with optimized induction conditions and ultrasonication strength, and better results were obtained (Figure 7).

Figure 7. SDS-PAGE Gel of three halogenases (Left is supernatant, Right is precipitation)

Finally, we did a fermentation experiment with E. coli BL21 ΔTnaA containing Fre-L3-SttH/PyrH/RebH and the results were shown in Figure 8, from which we can see different indigo derivatives as expected.

Figure 8. Fermentation results of three halogenases