Team:Heidelberg/pal


AvPAL Assay





Phenylalanine ammonia lyase (PAL)

Background

For our project we decided to use phenylketonuria (PKU) as a model for a metabolic disease that could be treated with our therapeutic approach of natural transformation.
PKU is a hereditary disease in which the amino acid phenylalanine cannot be degraded. The molecular cause for this disease is a limited functionality or even the absence of the enzyme phenylalanine hydroxylase (PAH) which converts phenylalanine into tyrosine [1].

Figure 1: Enzymatic reaction of the phenylalanine hydroxylase.

The dysfunction of the enzyme leads primarily to an accumulation of phenylalanine associated with a tyrosine deficiency. This resulting accumulation of phenylalanine affects the development of the brain. The cause of this is not yet fully understood. Untreated, the disease leads to a severe impairment of mental development already in the first months of life.
In the 1960s the so-called Guthrie test was developed to test newborns for the occurrence of PKU [2]. Nowadays testing for PKU, as well as for 13 other diseases, is a standard part of newborn screening. Early diagnosis within the first few days is required in order to avoid a severe disability because a cure for PKU is not available. Currently the only treatment option so far is a diet in which the daily intake of phenylalanine is strictly controlled by a low-protein diet. Essential amino acids and proteins have to be supplemented.
Further Information that we received from Experts regarding PKU and newborn screening can be found in our Expert Interview - Integrated Human practices Dr. Kölker Dietmar Hopp Stoffwechselzentrum and in the description of our inspection of the diagnostics in newborn screening.

Finding an alternative to dietary restrictions

For patients, adjusting the diet means a strong renunciation in everyday activities such as a visit to a restaurant. Therefore, we aim to contribute to the development of other therapeutic solutions to treat this disease with our natural transformation approach, we want the gut bacteria to express phenylalanine ammonia lyase (PAL) as a therapeutic gene product.
This idea is based on the paper; “Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU)” [3]. The authors of the paper have cloned the AvPAL gene into Lactobacillus reuteri for oral administration as a probiotic (tested in vivo in a mouse model). Four months after treatment, the probiotic could still be detected in the stool, but eight months after treatment, it could no longer be detected. Our idea of optimization is that once the plasmid is taken up by the intestinal bacteria via natural transformation, the enzyme is permanently expressed. We will try to initiate a selective advantage for the plasmid to maintain the stable frequency. The advantage would be that the administration of drugs would be only necessary once, or very rarely and a selective advantage would require minimal adjustments of the patients diet. In addition, the very individual composition of the microbiome does not have to be changed.

Figure 2: Enzymatic reaction of the phenylalanine ammonia lyase.

Experimental design and results

To test the properties of PAL, we decided to use AvPAL from iGEM registry Part BBa_K1983000.
The AvPAL DNA with the sequence from the part BBa_K1983000 was cloned into a pET15b backbone using BamHI and NdeI restriction enzymes. We expressed the enzyme in E. coli BL21 with induction through IPTG.
We controlled the appearance of AvPAL in an SDS-PAGE as well as in a western blot (see Fig. 3). Additionally, we tried to clone AvPAL into the pUC19 Backbone but it did not work out and therefore can be seen as a negative control in the SDS-PAGE and the western blot.

Figure 3 AvPAL SDS-PAGE, Ponceau staining and Western Blot. (A) shows the SDS-PAGE with coomassie blue staining. (B) is the ponceau staining of the PDVF membrane after blotting. This staining is performed to control if the blotting was successful. The protein bands are visual detectable. (C) shows the final results after the western blot staining with anti-his tag antibodies. The stained protein size was at about 65 kDa, which correspond to the expected size of our target protein AvPAL.

In vitro assay of enzyme activity

The enzyme was expressed in a 50 mL overnight culture. The pellet was lysed in DPBS and bacteria were fracked in a french press machine. After centrifugation, one part of the supernatant and the pellet was used for the SDS-PAGE and the other part of the supernatant for the in vitro measurements (see Fig. 4). The supernatant was taken to measure the degradation of phenylalanine (Phe) to trans-cinnamic acid (tCa). We used three different phenylalanine concentrations, where 1 mM and 0,5 mM Phe were best detectable (see Fig. 4 D). Supernatant of the bacteria with AvPAL cloned into the pUC19 Backbone was used as a negative control. The absorbance was measured at 300 nm because at this wavelength the absorbance differed clearly between Phe and tCA (see Fig. 4 C).

Figure 4 Absorbance of Phe and tCa and measurments in vitro. (A-C) represent the absorbance spectrum of (A) phenylalanine (Phe), (B) trans-cinnamic acid (tCa) and in (C) the merged graphs of (A) and (B) the gray line is set to 300 nm. This wavelength was chosen to measure the degradation of Phe, because at this wavelength the absorbance differed the most. (D) shows the in vitro measurements with the concentration of 1 mM and 0,5 mM Phe added as well as the blank supernatant without any Phe (LB) and the negative control supernatant.

As can be seen in figure 4 D, the absorbance at 300 nm increases for both, positive control (supernatant of bacterial fractionation with phe added) and negative controls (supernatant of bacterial fractionation without phe, supernatant of bacterial fractionation AvPAL in pUC19). However, the increase in the negative curves is linear and no saturation is seen. It is therefore likely that this increase is due to other processes or side reactions in the solution. The positive controls show a much larger increase in absorbance at 300 nm with saturation occurring after 4-6 h, as well as it would be expected for the detection of tCa.

Discussion

The measured values indicate that PAL is present as a functional enzyme in our supernatant of the fracked cells, degrading phe to tCA. After about 4-6 h, no further increase in tCA concentration is seen, indicating complete turnover. As expected, the phase of complete conversion is reached earlier at lower phe concentration.

Further Optimization

We also aimed to clone AvPAL into a suitable vector for natural transformation. We wanted to use the in vitro assay we developed in order to prove to what extent AvPAL is expressed by A. baylyi transformed via natural transformation. Unfortunately, the natural transformation did not work out. For further information have a look at our Engineering Success chapter.
There is also the problem of PAL being expressed inside the cell, so phenylalanine has to be brought through the membrane to be degraded by the bacteria. This problem has already been described by previous iGEM teams e.g in Part BBa_K1983000. The expression of PheP (E. coli L-phenylalanine permease) was proposed as a solution. PheP is supposed to facilitate diffusion through the membrane and would therefore also be a relevant tool for our project. We want that phenylalanine can be taken up by the intestinal bacteria, so that Phe degradation succeeds. This means that we would also need to give the bacteria transformed via natural transformation the ability to facilitate the uptake of phenylalanine.

References

[1] Flydal, M. I., & Martinez, A. (2013). Phenylalanine hydroxylase: function, structure, and regulation. IUBMB life, 65(4), 341–349. https://doi.org/10.1002/iub.1150

[2] GUTHRIE, R., & SUSI, A. (1963). A SIMPLE PHENYLALANINE METHOD FOR DETECTING PHENYLKETONURIA IN LARGE POPULATIONS OF NEWBORN INFANTS. Pediatrics, 32, 338–343.

[3] Durrer, K. E., Allen, M. S., & Hunt von Herbing, I. (2017). Genetically engineered probiotic for the treatment of phenylketonuria (PKU); assessment of a novel treatment in vitro and in the PAHenu2 mouse model of PKU. PloS one, 12(5), e0176286. https://doi.org/10.1371/journal.pone.0176286