Part:BBa_K5477047
Detoxification device against dioxin and PCBs
In this system, we have integrated two detoxification modules in yeast to create a device for the removal of environmental pollutants, including dioxins and polychlorinated biphenyls (PCBs). These modules, CYP1A1-pGAL1/10-POR and UDPD-pGAL1/10-UGT1A1, work in tandem under the control of the pGAL1/10 bidirectional promoter, which allows for the expression of the enzymes in opposite directions.
The CYP1A1-pGAL1/10-POR module addresses phase I metabolism. CYP1A1 catalyzes the oxidation of compounds like dioxin and PCBs, converting these toxic hydrophobic molecules into more reactive and soluble intermediates (2) (3). This reaction requires electrons supplied by cytochrome P450 oxidoreductase (POR), which transfers electrons from NADPH to CYP1A1. This step transforms hydrophobic environmental pollutants into more hydrophilic compounds. The UDPD-pGAL1/10-UGT1A1 module complements this system by facilitating phase II metabolism. UGT1A1 (UDP-glucuronosyltransferase 1A1) conjugates glucuronic acid to the oxidized intermediates produced by CYP1A1. However, for this reaction to proceed, a constant supply of UDP-glucuronic acid is needed, which is generated by UDP-glucose dehydrogenase (UDPD). UDPD converts UDP-glucose into UDP-glucuronic acid, providing UGT1A1 with the necessary substrate for the glucuronidation process (1).
Together, these two modules, regulated by the bidirectional pGAL1/10 promoter, form an integrated detoxification system that mimics both phase I (oxidation) and phase II (conjugation) of human metabolism. By expressing these systems in yeast, we can efficiently detoxify environmental pollutants such as dioxins, and PCBs, transforming them into less toxic, more water-soluble metabolites that can be readily excreted. The following composites were used to build this device: BBa_K5477037 and BBa_K5477036
Results
Objective: To evaluate the detoxification system by incubating it with specific contaminants and performing LC-MS analysis to determine whether new compounds are produced.
Methodology: The detoxification module CYP1A1-pGAL1/10-POR with UDPD-pGAL1/10-UGT1A1 was induced with galactose and incubated with PCB. Following overnight incubation, each detoxification system was centrifuged in Eppendorf tubes to separate the cells from the supernatant. The supernatant, henceforth referred to as the “media” samples, was collected. Absolute ethanol was added to the cell pellets, which were vortexed with micro glass beads to lyse the cells. The mixtures were centrifuged to separate cellular debris from the lysate, and the resulting supernatant was collected as the “pellet” samples.
All samples were filtered prior to being loaded into LC-MS glass vials.
Result: We did not have optimized LC-MS methods available for detecting highly hydrophobic compounds, even with the use of a Reverse Phase Column. Additionally, it took some time to identify ethanol as a suitable solvent for the system available in the department. Therefore, the results presented here are based on data generated from a single run of the samples on the LC-MS system. A previous run, where DMSO was used as a solvent, was excluded from the experiment due to the presence of multiple nonspecific peaks.
| Sample Number | Sample Name | Purpose | Colour in Chromatogram (also mentioned in chromatogram legend) |
|---|---|---|---|
| B2 | Empty yeast strain | Control for yeast strain | Dark Green |
| B3 | PCB standard in Absolute Ethanol | Standard | Black |
| B4 | C1A1-U1A1 yeast + PCB pellet | Sample | Brown |
| B5 | C1A1-U1A1 yeast + PCB media | Sample | Light pink |
| B6 | Empty yeast + PCB pellet | Control for PCB incubation | Gray-Blue |
| B7 | Empty yeast + PCB media | Control for PCB incubation | Dark Gray |
| B15 | C1A1-U1A1 yeast strain | Control for detox system expression | Light Green |
Figure 1 Base Peak Chromatogram for C1A1-U1A1 detox system. Pellet, media, control samples are included.
The base peak chromatogram for C1A1-U1A1 detox system shows several peaks for C1A1-U1A1 yeast + PCB pellet sample ( iGEM B4, brown in chromatogram) has several unique peaks between retention time 20 and 30 minutes. The same peaks are seen for PCB pellet with empty strain (B6, blue Gray). The trend from previous detox system is repeated here and so we believe that the peaks are due to pellet contents.
Figure 2 Base Peak Chromatogram for C1A1-U1A1 detox system zoomed in to 20.0 minutes to 21.0 minutes retention time.
Here we see some unique peaks in C1A1-U1A1 yeast + PCB pellet sample ( iGEM B4, brown in chromatogram). The peak at retention time 20.8 has the highest intensity. On further investigating the Mass spectrum of that particular peak we see intesne signal due to an ion of molecular mass 212.1626. As this mass was not detected in other mass spectrums, we believe we can call it unique to the system. The peak could signify a by product or transformed product of PCB#3. However, it could also be a peak due to enzyme expression. We would thus further want to investigate this ion and the mass before assuming that this detoxification module indeed works.
Figure 3 Mass spectrum for C1A1-U1A1 detox system zoomed at specific peak between 20.8 retention time.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 7148
Illegal PstI site found at 232
Illegal PstI site found at 1434
Illegal PstI site found at 2548
Illegal PstI site found at 2745
Illegal PstI site found at 3129
Illegal PstI site found at 3569
Illegal PstI site found at 3629 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 7148
Illegal PstI site found at 232
Illegal PstI site found at 1434
Illegal PstI site found at 2548
Illegal PstI site found at 2745
Illegal PstI site found at 3129
Illegal PstI site found at 3569
Illegal PstI site found at 3629 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 7148
Illegal BglII site found at 5440 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 7148
Illegal PstI site found at 232
Illegal PstI site found at 1434
Illegal PstI site found at 2548
Illegal PstI site found at 2745
Illegal PstI site found at 3129
Illegal PstI site found at 3569
Illegal PstI site found at 3629 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 7148
Illegal PstI site found at 232
Illegal PstI site found at 1434
Illegal PstI site found at 2548
Illegal PstI site found at 2745
Illegal PstI site found at 3129
Illegal PstI site found at 3569
Illegal PstI site found at 3629
Illegal NgoMIV site found at 326
Illegal NgoMIV site found at 413
Illegal NgoMIV site found at 2951
Illegal NgoMIV site found at 3070
Illegal AgeI site found at 1956
Illegal AgeI site found at 6146 - 1000COMPATIBLE WITH RFC[1000]
References
1. Inui H, Itoh T, Yamamoto K, Ikushiro S, Sakaki T. Mammalian cytochrome P450-dependent metabolism of polychlorinated dibenzo-p-dioxins and coplanar polychlorinated biphenyls. Int J Mol Sci. 2014 Aug 13;15(8):14044-57. doi: 10.3390/ijms150814044. PMID: 25123135; PMCID: PMC4159838.
2. Grimm FA, Hu D, Kania-Korwel I, Lehmler HJ, Ludewig G, Hornbuckle KC, et al. Metabolism and metabolites of polychlorinated biphenyls. Crit Rev Toxicol. 2015 Mar;45(3):245–72.
3. Liu J, Tan Y, Song E, Song Y. A Critical Review of Polychlorinated Biphenyls Metabolism, Metabolites, and Their Correlation with Oxidative Stress. Chem Res Toxicol. 2020 Aug 17;33(8):2022–42.
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