Fluorescent enzyme-coupled activity assay for phenylalanine ammonia-lyases

Phenylalanine ammonia-lyases (PALs) catalyse the non-oxidative deamination of l-phenylalanine to trans-cinnamic acid, while in the presence of high ammonia concentration the reverse reaction occurs. PALs have been intensively studied, however, their industrial applications for amino acids synthesis remained limited, mainly due to their decreased operational stability or limited substrate specificity. The application of extensive directed evolution procedures to improve their stability, activity or selectivity, is hindered by the lack of reliable activity assays allowing facile screening of PAL-activity within large-sized mutant libraries. Herein, we describe the development of an enzyme-coupled fluorescent assay applicable for PAL-activity screens at whole cell level, involving decarboxylation of trans-cinnamic acid (the product of the PAL reaction) by ferulic acid decarboxylase (FDC1) and a photochemical reaction of the produced styrene with a diaryltetrazole, that generates a detectable, fluorescent pyrazoline product. The general applicability of the fluorescent assay for PALs of different origin, as well as its versatility for the detection of tyrosine ammonia-lyase (TAL) activity have been also demonstrated. Accordingly, the developed procedure provides a facile tool for the efficient activity screens of large mutant libraries of PALs in presence of non-natural substrates of interest, being essential for the substrate-specificity modifications/tailoring of PALs through directed evolution-based protein engineering.

. Testing/set-up of the fluorescence assay starting from cinnamic acid 2, the product of the natural PAL-reaction.

PBS versus PBS/Acetonitrile system Assay conditions:
The FDC1-mediated whole cell-biotransformations were performed by incubating the reaction mixture of 0.5 mM cinnamic acid 2 and whole cell-FDC1-biocatalyst of cell density of OD 600 ~1 in 1 mL PBS buffer (20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.5) for 12 h at 200 rpm, 30 0 C. After 12h reaction time, 1 mL MeCN (PBS/MeCN system) or PBS (PBS system) was added to the reaction mixture. To 100 µL sample from the obtained reaction mixture addition of different equivalents (0.01 eq., 0.1 eq. or 1 eq. relatively to the concentration of cinnamic acid 2) of the fluorogenic probe 4 (from its DMSO stock solution) was performed, followed by irradiation at 302 nm for 1 min and fluorescence measurements.

Testing the PBS, PBS/hexane and PBS/methanol systems Assay conditions:
The FDC1-mediated whole cell-biotransformations were performed by incubating the reaction mixture of 0.5 mM cinnamic acid 2 and whole cell-FDC1-biocatalyst of OD 600~1 in 1 mL PBS buffer (20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.5) for 12 h at 200 rpm and 30 0 C. After 12h reaction time, a) 1 mL methanol was added to the reaction mixture (PBS/methanol) or b) extraction with 2 x 1 mL n-hexane (PBS/hexane) or c) dilution with additional 1 mL PBS was performed (PBS). In other variants of the procedure, in order to test the effect of whole cell-biocatalyst removal, after 12h reaction time a centrifugation step (13000 rpm, 12000 x g) was included and the obtained supernatant (SN_PBS) was processed further with addition of a) methanol, b) n-hexane or c) PBS, similarly as described above. Into 100 µL of the obtained mixtures/extracts of the different a, b and c procedure variants, the addition of 50 µL of the fluorogenic probe 4 was added from its stock solutions in n-octane (Fig. S3a) or DMSO (Fig. S3b) for a final concentration of 0.25 mM (corresponding to 1 eq. of 4 relatively to 2), followed by irradiation at 302 nm for 1 min and fluorescence measurements.

PBS/n-hexane procedure and negative controls Assay conditions:
The FDC1-mediated whole cell biotransformations were performed by incubating the reaction mixture of 1 mM cinnamic acid 2 and whole cell-FDC1-biocatalyst of OD 600~1 in 1.5 mL PBS buffer (20 mM NaH 2 PO 4 , 100 mM NaCl, pH 7.5) for 12 h at 200 rpm and 30 0 C. After 12h reaction time the reaction mixture was extracted with 3 x 0.5 mL n-hexane. Into the 100 µL of n-hexane extract 50 µL of the fluorogenic probe 4 was added from its stock solution in n-octane in final concentration of 1 mM, corresponding to 1 eq. of 4 relatively to 2, followed by irradiation at 302 nm for 1 min and fluorescence measurements. Negative controls consisted of: 1) assay performed under similar conditions, adding instead of the fluorogenic probe (FP) 4 solution only n-octane (NC1); 2) assay performed with initial solution without substrate, cinnamic acid 2 (NC2); 3) assay performed without the decarboxylation step, using 100 µL 1 mM styrene 3 solution in n-hexane and in the absence of fluorogenic probe (FP) 4 (NC3).  Figure S4. Fluorescent signals of the optimal assay condition of the PBS/n-hexane procedure in comparison with the corresponding negative controls.

Calibration with styrene
In order to determine the styrene concentration detectable by the fluorescent activity assay calibration with styrene solutions of (0-1 mM in n-hexane) and solution of 0.5 mM diaryltetrazole (in n-octane) was performed in 200 L n-hexane. The samples were photoirradiated at 302 nm in UV transparent Corning 96-well Clear Flat Bottom UV-transparent plates at room temperature for 1 min. 100 L from samples was placed into Corning 96-well Black Flat Bottom plates and using 360 nm excitation wavelength the fluorescence emission (410-520 nm) was measured. Using the values obtained at emission maxima of 460 nm, the calibration curves (Fig. S7) were obtained. Since at ≥ 0.2 mM styrene concentrations the fluorescent signal intensities exceeded the dynamic range of the instrument, these samples were 100 fold diluted with n-hexane, thus two calibration curves were obtained for a) low styrene concentrations (0.005-0.15 mM) - Fig. S7a and b)

The synthesis of phenylsulfonylhydrazone (II)
The stirred solution of the aldehyde (I, 18.9 mmol, 2 g) in ethanol (20 mL) was mixed with the solution of phenylsulfonylhydrazine (18.9 mmol, 3.3 g) in ethanol (20 mL). After stirring for 30 min at room temperature, the mixture was cooled and diluted with water (50 mL). The formed precipitate was filtered off, washed with hexane (3x10 mL) and dried.

Construction of pcpal-fdc1-pCDFDuet-1 expression vector
The pCDFDuet-1 vector containing two multiple cloning sites (MCS) was employed for the molecular cloning of both ScFDC1 and PcPAL. The gene of ScFDC1, obtained through digestion of pTfdc1Sc plasmid 3 with restriction enzymes SalI and HindIII, together with SalI and HindIII digested pCDFDuet-1 vector were extracted from the agarose gel and ligated (1 h incubation at 22 °C). 5 µL from the ligation reaction were transformed through heatshock into E. coli XL-1 Blue competent cells, the grown colonies were selected and the presence of the insert (fdc1 gene) was verified through colony PCR (Fig. S16b). The primers used for colony PCR are: T7_for: 5'AATACGACTCACTATAGGGGAATTG3' and Duet_DOWN1: 5'GATTATGCGGCCGTGTACAA3'. From one of the positive colonies an overnight culture was grown (5 mL sterile LB supplemented with tetracycline 12.5 µg/mL and streptomycin 30 µg/mL) at 37 °C and 180 rpm and the plasmid fdc1-pCDFDuet-1 was extracted.
First pcpal gene was amplified by PCR, using the pcpal-pET19b plasmid 4 as template, and restriction sites for NdeI and AatII were introduced. The primers used for the PCR experiments are NdeI_for: 5'ATTCATATGATGGAAAACGGAAACGGAGC3' and AatII_rev: 5'AATGACGTCTCAGGAAATGGGCAAGGG3'. The purified PCR product and fdc1-pCDFDuet-1 plasmid were digested with NdeI and AatII restriction enzymes, extracted from the agarose gel and then ligated as previously described. After transformation through heat-shock of the ligation mixture into E. coli XL-1 Blue competent cells, the presence of the insert (pcpal gene) in the grown colonies was tested through colony PCR using the primers: Duet_UP2: 5'TTGTACACGGCCGCATAATC3' and T7_rev: 5'TGCTAGTTATTGCTCAGCGG3'. One of the positive colonies was used to inoculate an overnight culture (5 mL sterile LB supplemented with tetracycline 12.5 µg/mL and streptomycin 30 µg/mL) at 37 °C and 180 rpm and the plasmid pcpal-fdc1-pCDFDuet-1 was extracted.