Figure 1  Schematic representation of the overall principle of PAL gene expression.

Phenylalanine ammonia-lyase (PAL) is an important enzyme that catalyzes the deamination of phenylalanine (Phe), removing the amino group that cannot be processed by PKU patients.The principle is illustrated in Figure 2G.Initially, we used a constitutive promoter to express the pal gene and measured Phe levels using a phenylalanine ELISA kit, while TCA concentration was determined by measuring OD290 with an enzyme immunoassay analyzer.   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.   Considering the role of PAL in anaerobic environments, we decided to test its expression using an anaerobic promoter, and the results, as shown in Figure 2F, indicated normal expression under anaerobic conditions. Similarly, Phe and TCA levels were measured, as shown in Figure 2C and 2D.  When oxygen concentration was lower, TCA production was higher, 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 2E.  Within the range of pH 5 to 8, PAL activity increased with increasing pH, with pH 8 being the optimal pH.

Figure 2  Hypoxia-induced promoter pPep drives PAL expression.

We designed a trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system to efficiently express PAL in an in vitro transformation system. The schematic diagram of the trans-cinnamic acid (TCA) promoter is shown in Figure 3A, and the schematic diagram of the trans-cinnamic acid (TCA) promoter and TP901 amplification signal switch system is shown in Figure 3B. Firstly, we constructed mRFP downstream of the pTCA promoter and detected its expression, as shown in Figure 3C. After adding 1mM 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 3D and E. After adding 1mM TCA, the Phe content decreased from around 1mM to around 0mM, while the TCA content increased from 0mM to around 2mM. 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 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 3F, 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 40A.U, which was significantly higher compared to the control group. This indicates that TP901 has a significant effect in promoting downstream gene expression.

Figure3   Utilizing the trans-cinnamic acid biosensor pSenCA and signal amplification switch for efficient gene expression.

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