Synthetic biology is an interdisciplinary field that combines biology and engineering, aiming to design and construct new biological systems and functions. It goes beyond studying the complexity of organisms in nature and seeks to optimize or reconstruct biological systems through artificial design, exploiting the programmability of life. This programmability of biological systems provides opportunities to address global challenges such as climate change, disease treatment, and food security.

In our project, we utilize a variety of components, including those previously commonly used or rarely employed, and test them multiple times to ensure their effectiveness. We have undergone numerous iterations to achieve microbial targeted therapy for diseases. The following provides detailed descriptions of the test results for each component, demonstrating the practical viability of our product.

1. Design

We aim to reduce the absorption of phenylalanine in the human body by using E. coli to absorb the phenylalanine ingested by patients. We have generated a transport system by introducing a gene expressing the PheP transporter. Simultaneously, we express PAL, an important enzyme that catalyzes the deamination of phenylalanine (Phe) and removes the amino group that cannot be processed by PKU patients.

Figure 1  Schematic representation of the overall principle of Phep and PAL.

2. Build

In this section, we constructed engineered strains expressing both PheP and PAL to produce transgenic E. coli capable of absorbing phenylalanine and degrading Phe. We used a constitutive promoter to express the gene of PheP and PAL.
 

3. Test

After expressing the engineered strains,measured Phe levels using a phenylalanine ELISA kit, while TCA concentration was determined by measuring OD290 with an microplate reader. The results, as shown in Figure 2A and 2B, demonstrate that after PAL expression, Phe levels decreased significantly, while TCA levels increased, indicating that PAL can effectively degrade Phe into TCA.    

Figure 2      Expression diagram of Phep and PAL.

4. Learn

Through this test, it is known that there is an excessive amount of phenylalanine in E. coli cells, approaching a saturated state, which prevents the cells from further absorbing phenylalanine from the intestine. Therefore, it is considered to add a metabolic system led by phenylalanine deaminase.

1. Design

Although our first version of the plan partially transferred phenylpyruvic acid into the bacterial cells, the capacity of bacteria is limited. When the production of Phe exceeds the tolerance of the bacteria, it still leads to intracellular Phe saturation and accumulation in the patient's body. Therefore, our second version of the plan replaced the promoter with the anaerobic promoter pPepT. When the engineered bacteria enter the patient's gut, the anaerobic environment activates the inducible anaerobic promoter, and the PAL gene starts to express. The engineered bacteria secrete PAL, which metabolizes Phe in the patient's body into trans-cinnamic acid (TCA). TCA is then transported through the intestinal capillaries into the liver via the bloodstream, where it further metabolizes into hippuric acid (HA), ultimately being excreted in the urine.

Figure3     Hypoxia-induced promoter pPepT drives PAL expression effect.

2. Build

In pSB1A3 vector, we placed PAL and Phep genes downstream of the hypoxia-induced promoter pPepT. After enzymatic cleavage, ligation, and transformation, they will be successfully transferred into Escherichia coli bacteria, allowing the target genes to be expressed and producing engineered bacteria capable of transporting phenylpyruvic acid and metabolizing phenylalanine into trans-cinnamic acid.

3. Test

We tested the expression of the hypoxia-induced promoter, as shown in Figure 4A, and found that it was expressed normally under anaerobic conditions. Similarly, we measured the levels of Phe and TCA, as shown in Figures 4B and 4C. 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 4D. PAL activity increased with increasing pH within the range of pH 5 to 8, with pH 8 being the optimal pH.

Figure 4    Expression data graph of the anaerobic promoter.

4. Learn

By comparison, we can conclude that the transformation ability of phe in bacteria containing Phep alone is weaker than that in bacteria containing both PheP and PAL. Therefore, it can be demonstrated that the inclusion of the metabolic system aligns with the expectations of the experiment.

1. Design

Considering that it is not feasible to directly administer Escherichia coli and that in vivo medication does not comply with local regulations on food and drug safety in China, we plan to design an ex vivo transformation system. This system includes the T7 promoter, RBS ribosome-binding site, PHEP transport protein, terminator, TCA promoter, TP901 integrase, and PAL phenylalanine ammonia-lyase. We retained the design of the transport protein gene and the overall structure of the PAL enzyme from the in vivo transformation system but made modifications to the promoter region of the PAL gene sequence. We modified the deoxy promoter to the T7 promoter and reversed its direction, adding the TP901 integrase sequence and TCA promoter before it. Prior to the entire translation process, a small amount of PAL enzyme had already been translated, converting some Phe to trans-cinnamic acid. Trans-cinnamic acid promotes the action of the TCA promoter, initiating the translation of the TP901 integrase. The TP901 integrase recognizes the attB/attP site，and enables the T7 promoter to reverse and face the PAL gene sequence, initiating the translation of PAL. The strong T7 promoter rapidly produces a large amount of PAL enzyme without inhibiting bacterial growth，the schematic diagram is shown in Figure 5.

Figure 5  Schematic representation of the overall principle of PAL gene expression.

2. Build

We designed a trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system to efficiently express PAL in an in vitro transformation system. 

3. Test

  The schematic diagram of the trans-cinnamic acid (TCA) promoter is shown in Figure 6A, and the schematic diagram of the trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system is shown in Figure 6B. Firstly, we constructed mRFP downstream of the pTCA promoter and detected its expression, as shown in Figure 6C. After adding 1 mM TCA, there was a significant increase in mRFP fluorescence intensity. Secondly, we measured the content of Phe and TCA in the engineered strain using the same method, as shown in Figure 6D and E. After adding 1mM TCA, the Phe content decreased from around 1 mM to around 0 mM, while the TCA content increased from 0 mM to around 2 mM. Therefore, pTCA has a good activation ability for PAL expression.
   To verify if the TP901 amplification signal switch system can promote gene expression downstream of pSenCA (TCA responsive promoter), we coupled mRFP downstream of pSenCA and added the TP901 amplification signal switch system. We tested the expression of fluorescent protein at different TCA concentrations. From Figure 6F, it can be observed that in an environment with 1mM TCA, the strain with the TP901 signal amplification switch system added downstream of pTCA had a fluorescence intensity of around 40 A.U, which was significantly higher compared to the control group. This indicates that TP901 has a significant effect in promoting downstream gene expression.

Figure 6   Utilizing the trans-cinnamic acid (TCA) biosensor pSenCA and signal amplification switch for efficient gene expression.

4. Learn

By analyzing the results, this design can basically meet the demand, but there is still the problem of slow degradation time. Therefore, we plan to use this technology in the factory production stage to produce edible products without phenylalanine.

Through three iterations of microbes, we have obtained an engineered microorganism that can rapidly degrade Phe and complies with regulations. These iterations are based on our exploration and research on the market, patients, and regional regulations.

1. Chien, Tiffany, et al. "Enhancing the tropism of bacteria via genetically programmed biosensors." Nature biomedical engineering 6.1 (2022): 94-104.
2. Courbet, Alexis, et al. "Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates." Science translational medicine 7.289 (2015): 289ra83-289ra83.
3. Flachbart, Lion Konstantin, Sascha Sokolowsky, and Jan Marienhagen. "Displaced by deceivers: prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns." ACS synthetic biology 8.8 (2019): 1847-1857.
4. Binder, Stephan, et al. "A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level." Genome biology 13.5 (2012): 1-12.
5. Pi, Jing, PETER J. Wookey, and A. J. Pittard. "Cloning and sequencing of the pheP gene, which encodes the phenylalanine-specific transport system of Escherichia coli." Journal of bacteriology 173.12 (1991): 3622-3629.
6. 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.