Part:BBa_K2932000

CYP2E1, rabbit cytochrome P450 2E1 ( optimized for Bacillus preferred codon)

Biological function

CYP2E1 is a member of cytochrome P450(CYP), which plays a role in metabolizing the toxin and drug including alcohol, benzene, chloroform, 4-nitrophenol, acetone, etc. in the animal body

Benzene oxidation

Phenol is a product from benzene oxidation which is catalyzed by CYP2E1.

Gene cloning and protein expression

We optimized the gene codon of rabbit cytochrome P450 2E1 (CYP2E1) based on Bacillus subtilis codon usage preference and synthesized the DNA fragment by Twist Bioscience. We made a Basic BioBrick Part of CYP2E1. Then, to make a Composite BioBrick Part for testing the function, DNA fragments were amplified by PCR and assembled with a terminator, followed by inserting to PliaI-RBS/pSB1C3.

Construct of PliaI-RBS-CYP2E1-Tr/pSB1C3

DNA has been checked by PCR, restriction enzymes and sequencing with primers of VF2 and VR.

To express CYP2E1 gene in Bacillus subtilis, we transfer the DNA fragments of PliaI-RBS-CYP2E1-Tr to pBS0E vector and transformed Bacillus subtilis 168 with the resulting plasmids.

Protein induction procedure

↓ culture Bacillus subtilis 168 carrying the plasmid of PliaI + RBS + CYP2E1 + terminator/pBS0E in LB + Amp (100 μg/ml) O/N at 37°C, shaking at 170 rpm, supplemented with 1mM of thiamine and 75mg/L of 5-aminolevulinic acid
↓ transfer 3 ml to 50 ml LB+Antibiotics with all the supplements in 250 ml flask
↓ measure OD650
↓ shake at 200rpm,37°C until OD650 between 0.5~0.7
↓ add 50 μl of 30ug/ml Bacitracin for induction at 25°C, shaking at 100 rpm for 18.5 hr

After protein induction by bacitracin, the total lysates of Bacillus expressing CYP2E1 were subjected to SDS-PAGE and stained with Coomassie blue. CYP2E1 protein has a molecular weight of 55kDa and shown as a band overlapped with a lot of proteins located between 48-63 kDa. Further confirmation is needed to make sure the expression of CYP2E1 and optimal induction procedure. (The work of SDS-PAGE was collaborated with team NCTU-Formosa)

Emerson reaction assay (4-aminoantipyrine colorimetric reaction)

Emerson reaction is describing 4-aminoantipyrine (4-AAP) oxidation with phenol in alkaline condition catalyzed by oxidative potassium ferricyanide K3[Fe(CN)6] to produce p-quinoneimide adduct in red color.

To test Emerson reaction, we prepare phenol solution (83g/L, equals to 0.88M, i.e., phenol solubility in water) with 10X serial dilution. The red color product of the reaction can be measured at OD580. The color changed from yellow, orange to red color depends on the concentration of phenol.

The calibration curve presented in the figure below is made in the scatter plot. The concentration of phenol (mg/L) (Y) can be calculated from the values of OD580 by the equation of Y = 0.00003*e^(16.202*X)

CYP2E1 functional assay

Benzene is one of the substrates of CYP2E1. Because of the toxicity of benzene, a collegiate iGEM team, Tunghai-TAPG helped us conduct the analysis in a specialized laboratory in the Department of Chemistry in the university.

Experiment procedure

↓ prepare CYP2E1 and WT lysates
↓ prepare 1.79 g/L of benzene (i.e., benzene solubility in water)
↓ add 90μl of benzene solution with 10X serial dilution to each well
↓ incubate with 40μl of CYP2E1 or WT Bacillus lysates at room temperature for 30 min
↓ transfer 90μl of the mixture to a new well
↓ add 90μl of solution I (1% of 4-aminoantipyrine in KOH solution, pH=9~10
↓ then add 45μl of solution II (4% of K3[Fe(CN)6])
↓ measure at OD580

The data showed that the values measured at OD580 are higher in the group of benzene plus CYP2E1 compared to controls of benzene without CYP2E1, indicating that the phenol is converted from benzene by CYP2E1. The benzene has a basal effect in Emerson reaction. The values of OD580 of Benzene are regarded as background and are subtracted for analyzing the phenol generation.

Next, according to the calibration curve of phenol analysis in Emerson reaction. We converted the OD values of phenol formation to benzene degradation. The result suggested CYP2E1 in the bacterial lysate can convert 0.09 - 0.47 μg/L of benzene to phenol, implying the possibility of CYP2E1 application in benzene removal in our microalgae purification system.

Microalgae purification system demonstration

Finally, we’d like to know the phenol consumption by algae in our system. We incubated algae with a serial 10x dilution of phenol solution as did in the analysis of the calibration curve, followed by culturing algae with light at 37°C for 1hr. Then, the algae culture media was centrifuged to discard the algal cells, and the supernatants were subjected to Emerson reaction assay. As data shown, the OD values dropped significantly in the group of phenol with algae in a dose-dependent manner, indicating that phenol is consumed by algae.

Using the calibration curve of phenol concentration to OD values, we obtained the data that algae can take up 0.15 - 0.34 μg/L of phenol in culture media. The result is consistent with the study by M. Wurster, et al. in 2003, proving the algae is capable of removing phenol in the environment.

Summary

We successfully demonstrated the CYP2E1 can oxidize benzene to phenol, which can be absorbed by algae. These findings extend the possibility of applying the enzymes to microalgae purification system to remove various pollutants or toxic chemicals, which are naturally unable to be absorbed by algae.

Discussion

Cytochrome P450 2E1 (CYP2E1) used in benzene and chloroform removal is first studied by Sharon L. Doty, et al. in 2007 and published on the distinguished PNAS journal. The authors demonstrated that CYP2E1-transgenic tree, Populus alba, has the ability to remove benzene at 0.28 μg/h. Based on our study in microalgae purification system, it is for the first time to demonstrate algae is able to take up phenol converted from benzene in the process of CYP2E1 catalyzation. The algae can indirectly absorb benzene at 0.95 μg/h, which improve the efficiency up to 4.7 fold. Our result showed that the microalgae system has great potential to remove unwanted pollutants efficiently with extracellular enzymes, which convert the pollutants to substrates absorbed by algae.

References

1. Frank J. Gonzalez. “CYP2E1” Drug Metabolism and Disposition 2007, 35 (1) 1-8; doi:10.1124/dmd.106.012492
2. M. Wurster, S. Mundt, E. Hammer, F. Schauer, U. Lindequist “Extracellular degradation of phenol by the cyanobacterium Synechococcus PCC 7002” Journal of Applied Phycology. 2003; 15(2–3) pp 171–176 doi:10.1023/A:1023840503605
3. Philipp F. Popp, Mona Dotzler, Jara Radeck, Julia Bartels & Thorsten Mascher. "The Bacillus BioBrick Box 2.0: expanding the genetic toolbox for the standardized work with Bacillus subtilis" Scientific Reports 2017; 7(1):15058 doi: 10.1038/s41598-017-15107-z.
4. Selvakumar, Paulraj Mosae. “Phenol Sensing Studies by 4-Aminoantipyrine Method-A Review.” Organic & Medicinal Chemistry International Journal 2018:5(2) doi:10.19080/omcij.2018.05.555657.
5. Sharon L. Doty, C. Andrew James, Allison L. Moore, Azra Vajzovic, Glenda L. Singleton, Caiping Ma, Zareen Khan, Gang Xin, Jun Won Kang, Jin Young Park, Richard Meilan, Steven H. Strauss, Jasmine Wilkerson, Federico Farin and Stuart E. Strand. "Enhanced phytoremediation of volatile environmental pollutants with transgenic trees." Proc Natl Acad Sci USA. 2007;104(43):16816-21. doi: 10.1073/pnas.0703276104
6. Sander, R. “Compilation of Henrys Law Constants (Version 4.0) for Water as Solvent.” Atmospheric Chemistry and Physics, vol. 15, no. 8, 2015, pp. 4399–4981., doi:10.5194/acp-15-4399-2015.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 856
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 689
  Illegal SapI site found at 1338
  Illegal SapI.rc site found at 466