Vector Construction

In our work, we have succeeded in constructing 4 plasmids, whose enzymatic digestion results and information were shown in Tab.1 and Fig.1, individually. Specifically, piGEM23_03, designed for formaldehyde, incorporates the hps-phi pathway and was completed. piGEM23_04 has the function of degrading indole through the ycnE-FMO pathway and had been successfully assembled. piGEM23_05, crafted for butyric acid conversion, encompasses the buk-ptb-adhE2-ATF1 pathway and was finished. Lastly, piGEM23_06 for hydrogen sulfide integrates the SQR-SDO-AprBA-SAT pathway and was also completed.

We next carried out the prokaryotic expression of these target proteins using E.coli Top10 as host cell. Due to a lack of time, apart from piGEM23_04 plasmid, experiments on the other 3 plasmids didn't get satisfactory results. Therefore in this work, we focused on piGEM23_04 plasmid containing the genes encoding FMO and ycnE.

Expression of Target Proteins and Enzymatic Assay

SDS-PAGE results showed that both FMO and ycnE enzyme were successfully expressed in the partial soluble form in our engineered E.coli harboring piGEM23_04 plasmid.

Detection of Indole Degradation

In order to check the level of reactant indole, we used Kovac's reagent to react with indole to yield a product that can absorb at 571 nm. Standard curves were done at this wavelength to get information about the concentration of indole (seen in Figure 3).

Following this measurement, we detected if our engineered E.coliharboring the plasmid of piGEM23_04 can degrade endogenous and exogenous indole. As demonstrated in Figure 4A, unlike the control group, where the content of indole was continuously increased as the cell grows, E.coli expressing these two enzymes can significantly degrade indole produced from this E.coli with the presence of tryptophan in LB medium. Next, we check the degradation capability of this engineered E.coli when 1mM indole was added into LB medium. As shown in Figure 4B, there is a linear reduction of indole concentration after 6h-treatment on E.coli expressing FMO and ycnE enzymes. These phenomena suggested that the constructed engineered E.coli in our work can effectively degrade endogenous and exogenous indole.

Production of Indigo Catalyzed by FMO

Apart from the reactant indole, we also detected two products including indigo and isatin. Firstly, we can judge from the color change that indigo was produced when indole was degraded (seen from Figure 5A).

For the quantitative measurement of indigo, we used DMSO to extract and solubilize the insoluble indigo produced with the catalysis of FMO enzyme from the engineered E.coli. Because indigo molecule had a characteristic absorption peak at 620 nm, we measured the absorbance at 620 nm under different concentration of indigo and got the standard curve(shown in Figure 5B).

Based on these data, we detection the indigo level produced in the engineered E.coli expressing FMO. It was found from Figure 6 that the level of indigo was linearly increased within 28 h. Finally, our engineered E.coli can produce indigo of ~ 9.16 uM within 30h in the presence of 1mM indole. These results indicated that FMO enzyme expressed by our engineered E.coli can work.

Production of Isatin Catalyzed by ycnE

The situation became more complicated for another product isatin, where isatin has the absorption peak at 318 nm, however, LB medium also exhibited the similar absorption peak. Therefore, it is difficult to detect the production of insatin in LB (shown in Figure 7A). In order to exclude the interference from LB medium, we used M9 medium instead of LB medium. The standard curve of isatin was recorded using M9 medium as solution (seen in Figure 7B).

Next, we detected the level of isatin during the degradation of indole using our engineered E.coli. As demonstrated in Figure 8, the concentration of isatin reached 0.337 mM following 24h-cultivation. This result showed that the expressed ycnE from our engineered E.coli can perform its function.

Construction of ycnE Mutants and Enzymatic Assay

In order to enhance the enzymatic activity of ycnE, we also conducted the computer-guided modification of ycnE enzyme. Our modelling analysis revealed that E-54 site, H-65 site and F-75 site is essential for the binding of indole to ycnE. Therefore, we constructed 7 mutants at E-54 site, H-65 and F-75. Comparison analysis showed that E54S mutant exhibited the highest enzymatic activity, as seen in Figure 9. This work provided some valuable guidance for the further protein modification on this ycnE.

Apart from in vivo detection, we also made some trials on the bacteria immobilization using sodium alginate microbeads and one kind of photoreactive hydrogel. This work is still on going up to now.

All in all, our engineered E.coli harboring piGEM23_04 can not only degrade the harmful substance indole, but also can also produce the valuable products including indigo and isatin. Furthermore, some useful information has been available concerning how to modify this new ycnE enzyme to enhance the activity using protein structural prediction combined with molecular docking and MD. This strategy adopted for the degradation of indole is also suitable for the degradation of other compounds.