Part:BBa_K5013003
This is a phenylalanine degrading enzyme controlled by a hypoxic promoter
Considering the functionality of phenylalanine lyase (PAL) in an anaerobic environment, we decided to position PAL downstream of the hypoxia promoter (pPepT), as this part allows for the regulation of PAL expression in such conditions. Ultimately, this composite part facilitates the transformation of phenylalanine into trans-cinnamic acid.
Phenylketonuria (PKU) is a common amino acid metabolism disorder caused by an enzyme deficiency in the phenylalanine (PA) metabolic pathway, preventing the conversion of phenylalanine into tyrosine. This leads to the accumulation of phenylalanine and its ketones, which are excreted in large quantities in the urine. The phenylalanine degradation enzyme (PAL) encoded by StlA can metabolize Phe into trans-cinnamic acid. Trans-cinnamic acid in the intestines is easily absorbed by the body, and the blood transports it to the liver. In the liver, trans-cinnamic acid is converted into hippuric acid (HA), which is eventually excreted in the urine. The anaerobic inducible promoter pPepT originates from Escherichia coli. Under anaerobic conditions, FNR aggregates to form an active regulatory factor, thereby activating transcription of downstream genes under the control of pPepT.
Usage and Biology
Firtly, we express the pal gene by utilizing the pLac and the ribosome binding site B0034. The pET23b vector will be employed, and the engineered plasmid will be introduced into E.coli Rosetta for efficient expression.
Figure 1 The design of gene circuit for PAL overexpression.
Figure 2 Gel electrophoresis of the pal gene.
By measuring the Phe content and TCA content after expressing the engineered strains, the degradation ability of PAL was evaluated. The strains were resuspended in 1 mL of Phe experimental buffer (M9 0.5% glucose with 1 mM Phe) to an OD600 of 0.1, and the Phe content was determined using a phenylalanine ELISA kit.
Based on the Phe content measurement experiment, we used an microplate reader reader to measure OD290 and calculate the concentration of TCA. The results are shown in Figure 3. After expressing PAL, the Phe content decreased while the TCA content significantly increased, indicating that PAL can effectively degrade Phe into TCA.
Figure 3 PAL degradation capability.
Additionally, we also validated the effect of environmental pH on the metabolic capacity of PAL. As shown in Figure 4, when the environmental pH value ranged from 5 to 8, the content of TCA increased with the increase in pH value. Therefore, this experiment can demonstrate that within the pH range of 5 to 8, the activity of PAL increases with the increase in pH value, with pH 8 being the optimal pH.
Figure 4 Testing the impact of pH environment on the degradation ability of PAL. .
After them, we used a hypoxia-sensing promoter (pPepT), constructed phenylalanine lyase (PAL) gene downstream of it, and used B0015 as gene circuit terminator. The constructed plasmid based on pSB1A3 was then introduced into E. coli BL21.
Figure 5 Design of pPepT and PAL.
Characterization
We tested the expression of the anaerobic promoter, as shown in Figure 6A, and found that it was expressed normally under anaerobic conditions. By measuring the Phe content and TCA content after expressing the engineered strains, the degradation ability of PAL was evaluated. The strains were resuspended in 1 mL of Phe experimental buffer (M9 0.5% glucose with 1 mM Phe) to an OD600 of 0.1, and the Phe content was determined using a phenylalanine ELISA kit. Based on the Phe content measurement experiment, we used an microplate reader reader to measure OD290 and calculate the concentration of TCA. We measured the levels of Phe and TCA, as shown in Figures 6B and 6C. We observed higher TCA production under lower oxygen concentrations, indicating that PAL can play a significant role under anaerobic conditions. Furthermore, we validated the effect of environmental pH on PAL metabolic capacity, as shown in Figure 6D. PAL activity increased with increasing pH within the range of pH 5 to 8, with pH 8 being the optimal pH.
Figure 6 Expression data graph of the hypoxic promoter.
Potential application directions
This experiment demonstrated the efficacy of pPepT-PAL in regulating PAL expression in an anaerobic environment. This finding has potential applications in future probiotic production, as it addresses the issue of nutritional depletion caused by protein expression during bacterial cultivation and storage. By extending the growth cycle and reducing shelf-life, probiotic bacteria can now effectively express relevant proteins in the anaerobic intestinal environment. This not only lowers production costs but also enhances patient efficacy. Therefore, this research holds promising development prospects.
References
Chien, Tiffany, et al. "Enhancing the tropism of bacteria via genetically programmed biosensors." Nature biomedical engineering 6.1 (2022): 94-104. Isabella, Vincent M., et al. "Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria." Nature biotechnology 36.9 (2018): 857-864.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2346
Illegal BamHI site found at 608
Illegal XhoI site found at 659
Illegal XhoI site found at 722
Illegal XhoI site found at 740
Illegal XhoI site found at 818
Illegal XhoI site found at 1019
Illegal XhoI site found at 1262
Illegal XhoI site found at 2009 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1178
Illegal NgoMIV site found at 1424
Illegal NgoMIV site found at 1592
Illegal NgoMIV site found at 1735
Illegal NgoMIV site found at 2069 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 719
Illegal BsaI site found at 1049
Illegal BsaI site found at 1055
Illegal BsaI.rc site found at 2180
Illegal SapI site found at 1009
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