Results

Find the five target enzymes sequence to degrade pyrethroids

Pyrethroid insecticides are reported to be neurotoxic to both insects and mammals, to find more enzymes have functions in pyrethroids degradation, we searched in previous iGEM projects and published papers, the following are some of the targets we found:
1) iGEM Warwick 2022 team presented a project, namely Pyre-Pyrethroid Remediation, involved the design of an integrated system to test and degrade build-up of λ-cyhalothrin (a type of pyrethroid) in the environment. The enzyme they used is our first target: Pyre1. [Pyre1]
2) A paper published on scientific reports mentioned the EstPS1 gene, which encodes a novel carboxylesterase of Pseudomonas synxantha PS1 isolated from oil well-produced water, showed high biodegradability towards pyrethroid pesticides, so we chose EstPS1 as our second target. [EstPS1]
3) Pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112, which is our third target, could not only degrade pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyze rho-nitrophenyl esters of various fatty acids, indicating EstP as an esterase with broad substrates. [EstP]
4) EstA-associated biodegradation of pyrethroids was confirmed in 2019, which used the key degrading gene estA and related carboxylesterase EstA from Bacillus cereus BCC01. We selected EstA as the forth target. [EstA]
5) A fifth novel esterase gene, pytH, isolated from Sphingobiumsp. strain JZ-1, encoding a pyrethroid-hydrolyzing carboxylesterase, which was able to transform p-nitrophenyl esters of short-chain fatty acids and a wide range of pyrethroid pesticides. [PytH]
the above five enzymes have been reported to degrade pyrethroids, their research is still insufficient. We do not know whether these enzymes can be expressed in more basic chassis organisms, whether there are more suitable expression conditions, and whether their expression amounts meet the needs of water purification. Therefore, we launched this year’s project. We chose to use E.coli, the most common strain in synthetic biology research, as our carrier. E.coli can meet our needs in terms of safety as a common strain living in the gut of humans and animals, so can E.coli express these five enzymes?

Expression of the five target enzymes

The first step in our experiments is to do a preliminary test of enzyme expression, to do this, we connected with the company to construct those enzymes into pET28a(+) plasmids. After which, the plasmids were transfected into E.coli BL21(DE3) strains and used IPTG as induction. The preliminary expression conditions were set as 1) induction for 20 h under 16℃; 2) induction for 4 h under 37℃.
The following are the results:

SDS-Page Results of EstA

SDS-Page Results of PytH

SDS-Page Results of EstPS1

SDS-Page Results of EstP

As we observed through SDS-PAGE, protein EstA and PytH showed clear bands around expected molecular weight, protein EstPS1 and EstP can be expressed but precipitated in the inclusion body, however, protein Pyre1 was not expressed.

In order to confirm those bands we pointed out indicated our target proteins, we did an interview with Prof. Geng from Minzu University of China, who suggested western blot assays to provide evidence, so we asked for collaboration and received her kind support in providing the Western Blot runs to confirm our findings. The WB in the above plots clearly provided us positive results.

Find the best expression temperature of EstA, PytH, EstPS1 and EstP

After we confirmed the expression of protein EstA, PytH, EstPS1 and EstP, we wanted to find the optimized expression conditions for these proteins.
We have communicated with the person in charge of the sewage treatment company and learned that the current temperature of the sewage treatment tank is generally 16℃ in winter and 37℃ in summer. Therefore, we hope to find the most suitable temperature in the range of 16-37℃.

So we firstly fixed the induction time and IPTG concentration, then set a series of temperature gradient. The SDS-PAGE result suggested:
1) 20℃ lead to the highest EstA expression level.
2) 16℃ lead to the highest PytH expression level. As the temperature arises, PytH comes to the inclusion body, so it prefers lower temperature.
3) Although EstP also precipitated in the inclusion body, but 22℃ expression expression produced more soluble protein.
4) No matter what the temperature is, EstPS1 is left in the inclusion body.

EstA Temperature Condition Tests

PytH Temperature Conditions Tests

EstP Temperature Conditions Tests

EstPS1 Temperature Conditions Tests

Find the best induction time of EstA, PytH, EstPS1 and EstP

After solve the problem of temperature, we would like to know how long is it appropriate for sewage to circulate in the treatment tank? The worker in the sewage treatment company said that according to his experience, 16 h is more appropriate in winter and 4 h is major indicated in summer. So the next experiment we did was to fixed the induction temperature and IPTG concentration, then set a series of time gradient around 16 h or 4 h. The SDS-PAGE result suggested:

1) EstA expressed pretty well under 20℃ and the induction time seems not influence the expression level..
2) The highest PytH expression level was achieved with 18 h or 20 h induction under the temperature of 16℃. .
3) More soluble EstP protein were produced after 24 h induction under the temperature of 22℃. .
4) EstPS1 was expressed under 16℃, we observed little soluble bands after 3 h induction..

EstA Induction Time Tests

PytH Induction Time Tests

EstP Induction Time Tests

EstPS1 Induction Time Tests

The improvement of Pyre sequence

The last part of our project is to solve the problem of Pyre expression. In the preliminary test, we downloaded the sequence from Warwick 2022 team, however, it did not work. We then carefully reviewed the whole project and related papers, we finally found out the key issue lies in the promoter and RBS part. The sequence Warwick uploaded contains promoter and RBS, we then destructed the original promoter and RBS on pET28a(+), which may lead to the incompatibility for E.coli BL21(DE3). So we isolated Pyre to construct a new plasmid named pET28a(+)-Pyre, which showed the expression band of Pyre protein, indicating the successful improvement of the existing part.

Plan to Test the degradation ability of the five target enzymes

After successfully expressing five types of pyrethroid degradation enzymes, we plan to test the degradation ability by the following procedures.
Induce the expression of these proteins again under the optimal conditions obtained in the previous experiments. Then mix the 4 ml bacterial solution with 1 ml of specified concentration pyrethroid solution and subjected it to 16 hours of rotational mixing. To conveniently and quickly detect the pyrethroid content within the system, we used commercially available test strips. A control group without active pyrethroid is also needed for comparison.

Future plan of pyrethroids degradion Implementation

We still need to consider more aspects if we are to put our project into practice.
Firstly, how to check the activity of pyrethroids degrading enzymes? We found that this experiment can be performed using a solution of p-nitrophenol acetate. If there is esterase activity, the solution will change from colorless to yellow. Then we can measure the activity of five enzymes in order to obtain more accurate experimental conditions through calculation.
Secondly, how can our reactions be stopped? If pyrethroids is not currently present in the sewage, or if the sewage treatment plant is facing special circumstances such as holidays, we need to stop the engineered E. coli from working. After searching for related publication, we noticed antibiotic signal-based CRISPR-Cas9-based kill switches (CRISPRks) and temperature signal-based T-switch systems, and if we can integrate suicide systems into the E. coli genome in the future, then we will have the potential for more precise and efficient human control.

==== Reference ====

[PytH] Zuo Z, Gong T, Che Y, Liu R, Xu P, Jiang H, Qiao C, Song C, Yang C. Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil. Biodegradation. 2015 Jun;26(3):223-33.
[EstA] Hu W, Lu Q, Zhong G, Hu M, Yi X. Biodegradation of Pyrethroids by a Hydrolyzing Carboxylesterase EstA from Bacillus cereus BCC01. Applied Sciences. 2019; 9(3):477.
[EstPS1] Cai, X., Wang, W., Lin, L. et al. Autotransporter domain-dependent enzymatic analysis of a novel extremely thermostable carboxylesterase with high biodegradability towards pyrethroid pesticides. Sci Rep 7, 3461 (2017).
[EstP] Pei C. Wu, Yu H. Liu, Zhuo Y. Wang, Xiao Y. Zhang, He Li, Wei Q. Liang, Na Luo, Ji M. Hu, Jia Q. Lu, Tian G. Luan, and Li X. Cao. Journal of Agricultural and Food Chemistry 2006 54 (3), 836-842
[Pyre1] DING, J., LIU, Y., GAO, Y., ZHANG, C., WANG, Y., XU, B., YANG, Y., WU, Q. & HUANG, Z. 2022. Biodegradation of λ-cyhalothrin through cell surface display of bacterial carboxylesterase. Chemosphere, 289, 133130.