Part:BBa_K2783010

Phenylalanine ammonia lyase 2

Overview

Phenylalanine ammonia lyase 2 is one of the isoforms of the PAL family from Arabidopsis thaliana. These enzymes catalyze the deamination of L-phenylalanine into ammonia and trans-cinnamic acid (Figure 1). In A. thaliana PAL2 is responsible for catalyzing the first step of the phenylpropanoid pathway by deaminating L-phenylalanine. The enzyme is used by the 2018 Groningen team in a synthetic styrene production pathway [1]. PAL2 expresses well in Escherichia coli (BL21DE3*), and functions optimally at 48°C and a pH between 8.4 - 8.9. It has an experimentally determined kcat of 3.2 s-1 and a kM of 64 µM [2].

Figure 1: schematic representation of the reaction catalyzed by PAL2. L-phenylalanine is deaminated to form ammonia and trans-cinnamic acid.

Experiments

An E.coli optimized cDNA sequence of PAL2 from A. thaliana was synthesized and cloned into pSB1C3. PAL2 from A. thaliana was chosen as it is known from literature to have a high catalytic activity, and as we attempted to make a functional improvement on existing PAL genes such as BBa_K1692004. A T7 promoter was cloned in front of PAL2 and this composite part was added separately to the registry as BBa_K2783011. The finished plasmid was introduced into E.coli BL21 with an IPTG inducible DE3 expression system. PAL2 expression was induced for 2.5 hours by adding 1 mM final concentration of Isopropyl β-D-1-thiogalactopyranoside (IPTG) during exponential growth. Cells were harvested and disrupted via french press. An SDS-PAGE (polyacrylamide gel electrophoresis) was performed to confirm protein expression (Figure 2). We chose not to tag the protein as there are indications in the literature that this abolished the majority of activity of the protein [2]. As evident from the gel image (and assays using cell lysate) AvPAL BBa_K1692004 did not express well in our host organism and therefore we were unable to directly compare the activities of PAL2 and AvPAL.

Figure 2: SDS-PAGE gel of PAL2 raw lysate soluble fraction, AvPAL BBa_K1692004 and HisEGII BBa_K2783021. PAL2 has an expected Mw of ~77 kDa and expression is clearly visible as a thick band.

To test whether PAL2 was active, we used a 20 times dilution of the raw lysate soluble fraction for an enzyme assay. This sample was incubated at 37°C with a dilution series of L-phenylalanine ranging from 3 mM to 0.3 mM concentrations. Activity was determined by measuring UV absorption on a spectrophotometer at 268 nm, the absorption wavelength of the product trans-cinnamic acid. Due to the high background signal from the cell lysate it was unfortunately impossible to estimate trans-cinnamic acid concentrations. To solve this problem, the spectrophotometer was blanked using the reaction mixture at t=0, and 268 nm absorption was measured over time. Figure 3 clearly shows that trans-cinnamic acid is produced over time by PAL2.

Figure 3: Trans-cinnamic acid production over time measured at 268 nm. Spectrophotometer was blanked using the same reaction mixture at t=0 to be able to measure the difference in absorption at 268 nm over time.

This provided the evidence that PAL2 was indeed active and produced the desired product trans-cinnamic acid. As the goal of the Groningen 2018 iGEM team was to produce styrene using yeast, the next step was to clone PAL2 into a yeast vector and see if any styrene could be produced and detected. Several culturing conditions were used: Verduyn medium, Verduyn medium with extra L-phenylalanine, Verduyn medium with L-phenylalanine and yeast with the minicellulosome with phenylalanine and a negative control without PAL2. The cultures were centrifuged and the supernatant was loaded on a HPLC for styrene detection. Figure 4 shows a plot of UV absorption intensity at 254 nm against retention time. There is a large peak around 10.4 minutes, corresponding to the absorption peak of trans-cinnamic acid. This peak was present for all samples except the negative control sample. This was expected as yeast without PAL2 is not able to produce trans-cinnamic acid. There is a second peak around 17.5 minutes retention time which corresponds to the peak of styrene.

Figure 4: UV absorption intensity at 254 nm plotted against retention time. Clear peaks are visible around 10.4 and 17.5 minutes retention time corresponding to trans-cinnamic acid and styrene, respectively.

When the styrene peak is observed in more detail (Figure 5), it becomes evident that adding extra L-phenylalanine to the growth medium of our transformed yeast improves styrene production, suggesting that L-phenylalanine production in the yeast metabolism may be a bottleneck for styrene production. A yeast containing our minicellulosome and fed with L-phenylalanine is still able to produce styrene. When no L-phenylalanine is fed to the yeast, styrene production is still observed, albeit lower than with supplementation of L-phenylalanine.

Figure 5: Close up plot of styrene peak at 254 nm. The negative control clearly does not produce any styrene. Adding extra phenylalanine to the medium leads to a higher styrene production. When the plasmids coding for the cellulosome scaffold are introduced in the yeast, styrene production decreases slightly but still remains higher than when no extra L-phenylalanine was added to the medium.

References

[1] McKenna, R., Thompson, B., Pugh, S., & Nielsen, D. R. (2014). Rational and combinatorial approaches to engineering styrene production by Saccharomyces cerevisiae. Microbial Cell Factories, 13(1), 123. https://doi.org/10.1186/s12934-014-0123-2

[2] Cochrane, F. C., Davin, L. B., & Lewis, N. G. (2004). The Arabidopsis phenylalanine ammonia lyase gene family: Kinetic characterization of the four PAL isoforms. Phytochemistry, 65(11), 1557–1564. https://doi.org/10.1016/j.phytochem.2004.05.006

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 337
  Illegal AgeI site found at 652
  Illegal AgeI site found at 676
  Illegal AgeI site found at 1651
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 184
  Illegal BsaI.rc site found at 253
  Illegal BsaI.rc site found at 439
  Illegal SapI.rc site found at 1873
  Illegal SapI.rc site found at 1999
  

Functional Parameters

family	PAL
origin	Arabidopsis thaliana