L phenylalanine

Ultrapure water was generated from a Milli-Q Plus purification system (18.2 MΩ cm, Merck Millipore, Bayswater, VIC, Australia). Methanol (HPLC grade, ≥99.9%), acetone (HPLC grade, 99.8%), formic acid (American Chemical Society reagent, ≥98%), trifluoroacetic acid (ReagentPlus^®, 99%), phloroglucinol (HPLC grade, ≥99%), l-ascorbic acid (reagent grade, ≥98%), ammonium formate (reagent grade, ≥99.9%), (−)-epicatechin (≥90%), quercetin-3-O-glucoside (analytical standard, 98%), corticosterone (≥98.5%), ampicillin trihydrate (analytical standard, ≥98%), l-tyrosine (≥99%), l-phenylalanine (≥99%), glutathione reduced (BioReagent, ≥98.0%), caffeic acid (≥98.0%) and gallic acid (≥97.5%), methyl cellulose (1500 cP), and ammonium sulfate (ReagentPlus^®, ≥99%) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Hydrochloric acid (Trace SELECT, 34%–37%) was purchased from chem-supply (Port Adelaide, SA, Australia). (+)-Catechin, (−)-epicatechin gallate, (−)-epigallocatechin, malvidin-3-O-glucoside, dimer B2, and taxifolin (all ≥99.9%) were obtained from Extrasynthese (Genay, France).

L-Tyr (≥98%), potassium chloride (≥99.0%) (KCl), FeCN (≥99.5%), SDS (≥99.0%), NP (≥99%), pyrrole (98%), L-tryptophan, L-cysteine, and L-phenylalanine were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The preparation of the solutions of these compounds was performed with ultrapure water (18.3 MΩ × cm, Milli–Q Simplicity^® Water Purification System from Millipore Corporation, Burlington, MA, USA).

Sulfuric acid, sodium hydroxide, acetonitrile (Ultra high performance liquid chromatography (UHPLC) grade), ethylenediaminetetraacetic acid (EDTA), sodium borate, and hydrochloric acid were obtained from Panreac (Castelar del Vallés, Barcelona, Spain). Sodium acetate (anhydrous), trimethylamine (TEA), phosphoric acid, ammonium molybdate, and methyl cellulose were purchased from Sigma-Aldrich (Darmstadt, Germany). Standard of amino acids (L-arginine (Arg), L-alanine (Ala), L-asparagine (Asn), L-aspartic acid (Asp), glycine (Gly), L-glutamic acid (Glu), L-glutamine (Gln), L-histidine (His), L-isoLeucine (Ileu), Leucine (Leu), L-Lysine (Lys), L-norvaline (Nor), L-phenylalanine (Phe), L-proline (Pro), L-serine (Ser), L-threonine (Thr), L-tryptophan (Trp), L-tyrosine (Tyr), and L-valine (Val)) were purchased from Sigma-Aldrich (Steinheim, Germany). All the other chemicals and reagents used were of analytical grade.

The plasmid AroHhis, which contains the entire AroH cDNA along with a C-terminal 6xHis tag cloned in the BamHI/NotI sites of a pET22b prokaryotic expression vector, was purchased from GenScript (New Jersey, USA). PLP, L-tryptophan, L-tyrosine, L-phenylalanine, α-aminoadipate, α-ketoglutarate, α-ketoadipate, L-glutamate dehydrogenase from bovine liver (GDH), 3-acetylpyridine adenine dinucleotide (APAD⁺), L-lactic dehydrogenase (LDH), and isopropyl-β-D-thiogalactopyranoside, were purchased from Sigma. All other chemicals were of the highest purity available.

The strain S. haloaromaticamans used in the current study was isolated from soil of a wastewater disposal site and it was able to degrade the fungicide OPP only when supplemented with CA (Perruchon et al., 2016 (link)). The bacterium was routinely cultivated in minimal salts media supplemented with nitrogen (MSMN), OPP (30–50 mg L^–1) and CA in a shaking incubator at 27°C in the dark. MSMN preparation and OPP chromatographic analysis was as described by Perruchon et al. (2016) (link), while bacterial growth was determined by measurement of the optical density at 600 nm (OD₆₀₀). Inoculation of flasks was performed by fresh bacterial cells grown at the mid-log phase, pelleted by centrifugation, washed three times with sterile ddH₂O and resuspended with MSMN to an OD₆₀₀ of 0.1.
L-methionine, L-isoleucine, L-tyrosine, L-phenylalanine, L-homoserine, O-succinyl-L-homoserine, L-cystathionine, L-homocysteine and cyanocobalamin (synonym to vitamin B12 in the manuscript), were purchased by Sigma-Aldrich (Taufkirchen, Germany) and they were used for the preparation of aqueous solutions (0.05 mM). These were filter sterilized and used for the preparation of MSMN containing amino acids, B12 and intermediates of methionine biosynthesis at the desired concentrations.

The parental wild-type yeast strain used in this study was BY4741. All gene deletions, genomic integrations, and plasmid transformations were done in this background using standard methods. For a complete list of strains used, see S1 Table.
Yeast cultures were grown at 30°C in YPD or synthetic defined (SD) media with appropriate nutrient dropouts. SD media used for growth of yeast cultures contained: 2% w/v dextrose (Thermo Fisher Scientific, Waltham, MA), 13.4 g/L Yeast Nitrogen Base without Amino Acids (BD Biosciences, San Jose, CA), 0.03 g/L L-isoleucine (Sigma-Aldrich, St. Louis, MO), 0.15 g/L L-valine (Sigma-Aldrich), 0.04 g/L adenine hemisulfate (Sigma-Aldrich), 0.02 g/L L-arginine (Sigma-Aldrich), 0.03 g/L L-lysine (Sigma-Aldrich), 0.05 g/L L-phenylalanine (Sigma-Aldrich), 0.2 g/L L-Threonine (Sigma-Aldrich), 0.03 g/L L-tyrosine (Sigma-Aldrich), 0.018 g/L L-histidine (Sigma-Aldrich), 0.09 g/L L-leucine (Sigma-Aldrich), 0.018 g/L L-methionine (Sigma-Aldrich), 0.036 g/L L-tryptophan (Sigma-Aldrich), and 0.018 g/L uracil (Sigma-Aldrich). Media additionally contained 5 mM or 10 mM guanidinium hydrochloride (Sigma-Aldrich) where indicated and bortezomib (LC Laboratories, Woburn, MA) treatment lasted 4 hours.

The PAL enzyme was extracted and partially purified by the method of Suzuki et al.³⁹ . Fresh leaves (1 g) were ground at 4 °C in 5 mL of 0.1 M sodium borate buffer (pH 8.8). Homogenates were centrifuged at 12,000 × g for 15 min at 4 °C and the supernatant was used as the enzyme extract. Reaction mixtures consisting of 500 μL sodium borate buffer (pH 8.7) and 250 μL enzyme extracts were pre-incubated for 5 min at 40 °C. The reaction was started by the addition of 300 μL of 50 mM l-phenylalanine (SIGMA-ALDRICH) and, after incubation for 1 h at 40 °C, stopped by adding 50 μL of 5 N HCl. The reaction mixture was centrifuged again (12,000 × g for 15 min) prior to injection into an HPLC (SHIMADZU, C-R4A Chromatopac; SCL-6B system controller) featuring a ZORBAX SB-C18 analytical column (4.6 × 150 mm, 5 μm particle size, AGILENT, Germany) and a U.V. detector at room temperature. The mobile phase consisted of 57% acetonitrile in water with a flow rate of 0.5 mL min⁻¹. Detection of trans-cinnamic acid (t-CA) was based on retention time and performed at 275 nm. The activity of PAL was expressed as nmol t-CA min⁻¹ g⁻¹ of fresh mass in relation to the peak area of a t-CA standard solution (1 mg/100 mL sodium borate buffer, pH 8.7).