Light-driven Enzymatic Decarboxylation

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Several of the chemicals used in these syntheses are acutely toxic and carcinogenic. All steps involving harmful organic solvents, particularly the extraction of the reaction products and the derivatization of fatty acids must be carried out under a fume hood using personal protective equipment (safety glasses, gloves, lab coat, full-length pants, closed-toe shoes). All operations involving genetically modified organisms in this work require installations that are approved for handling of genetically modified organisms of the safety level S1.

1. Expression of the Recombinant Decarboxylase OleT_JE で E. coli

1. Preparation of the production strain
   1. Prepare a synthetic gene of OleT_JE from Jeotgalicoccus and cloned into the expression vector pASK-IBA37plus as described.⁸
      NOTE: To avoid degradation of fatty acids by enzymes from the bacterial metabolism, E. coli strain JW5020 from the Keio collection with a dysfunctional pathway for fatty acid degradation was used.
2. Cultivation and induction of enzyme expression
   1. Prepare a pre-culture by inoculating the E. coli JW5020 OleT mutant from an LB-plate (containing 100 µg ml^-1 ampicillin) in two 3 ml LB-medium in test tubes. Pipette ampicillin (100 µg ml^-1) from a stock solution into the medium for selection and incubate at 37 °C for 15 hr in a shaking incubator at 180 rpm.
   2. Add 2 ml of the pre-cultures to two 200 ml LB-medium, containing ampicillin (100 µg ml^-1), in 1 L shake flasks. Incubation proceeds at 37 °C and 180 rpm.
   3. Monitor the change in OD_600nm after the inoculation. When the OD₆₀₀ reaches a value between 0.6 and 0.8 induce the expression by adding tetracycline (0.2 µg ml^-1). Incubate the cultures for 15 hr at 20 °C and 180 rpm.
      NOTE: Since OleT_JE contains a heme cofactor, addition of δ-Amino levulinic acid (0.5 mM) prior to induction is needed to provide the culture with a precursor for cofactor synthesis.
3. Cell lysis
   1. Carry out the following steps on ice. Transfer the cultures by decanting or pipetting into centrifuge tubes and use a scale to ensure equal distribution. Centrifuge the cultures for 20 min at 12,000 x g at 4 °C.
   2. Carefully discard the supernatant and resuspend the pellets in 50 ml buffer (Tris 50 mM, NaCl 200 mM, pH 7.5) by pipetting and transfer the suspension in a conical centrifuge tube.
   3. After centrifugation for 15 min at 4,000 x g at 4 °C discard the supernatant, resuspend each pellet in 3 ml buffer by pipetting.
   4. Perform cell lysis by sonicating the solutions on ice (three cycles x 30 sec) leaving a 1 min pause in between the cycles. Centrifuge the solutions for 20 min at 15,000 x g at 4 °C to remove cell debris and transfer the supernatant into a conical centrifugation tube without disturbing the pellet by pipetting. Discard the pellet.
      NOTE: One solvent fraction will be used directly for biocatalysis after small molecules are removed. The second fraction will be used for purification of the His-tagged OleT_JE.
4. Removal of small molecules of cell-extracts
   1. Before using the crude extract in biocatalysis remove small molecules which might interfere with hydrogen peroxide formation or electron transfer by pipetting 3 ml of cell-free extract into a centrifuge filter unit with a 10 kDa membrane and centrifuge at 4,000 x g at 4 °C. Resuspend the remaining protein in 3 ml Tris-HCl-buffer.
5. Purification of OleT
   1. Apply 3 ml of the second cell extract to His Pur Ni-NTA spin columns, which have been equilibrated before with equilibration buffer (Tris 20 mM, NaCl 300 mM, imidazole 10 mM).
   2. Seal the columns with the bottom plugs and upper screw-caps and shake them lightly until the resin is distributed equally with the cell-free extract. Incubate the loaded columns for 30 min in an overhead shaker at 4 °C.
   3. Wash the columns with 1 ml of washing buffer (Tris 20 mM, NaCl 300 mM, imidazole 20 mM, pH 7.4) by centrifuging at 700 x g for 2 min. Repeat this step twice.
   4. Place the columns into a fresh conical centrifugation tube and add 1 ml of elution buffer (Tris 20 mM, NaCl 300 mM, imidazole 250 mM, pH 7.4) OleT_JE . Centrifuge at 700 x g for 2 min and repeat this step two times.
      NOTE: Imidazole will bind to nickel and thus remove OleT_JE from the column.
   5. Transfer the elution to a centrifuge filter unit (10 kDa membrane) and centrifuge at 4,000 x g at 4 °C to remove imidazole.
   6. Check the purity of the enzyme by an SDS-Page (15%).⁹ Mix 3 µl of the elution with a SDS-buffer (final concentration 1x) and incubate the solution at 95 °C for 5 min for denaturation. Subsequently apply the total amount on a SDS-Page. Run the gel at 35 mA using a commercial protein standard. Detect the protein at 50 kDa.
   7. To determine the protein concentration use a commercial available BSA-kit. Pipette 50 µl of diluted protein samples (1:100, 1:200, 1:500) and BSA standard (0, 20, 30, 40,50, 60, 80, 100 mg ml^-1) in a 96-well plate. Add 200 µl of Bradford reagent, measure the absorbance at 595 nm after 15 min with a fluorimeter and calculate the concentrations using the standard curve.

2. Light-catalyzed Biotransformation

1. Biocatalytic reactions without hydrogen peroxide addition
   1. Prepare a 10 ml stock solution of 10 mM stearic acid (M_r 284.5 g mol^-1) by adding 10%(v/v) tergitol and 0.0284 g stearic acid to distilled water. Heat the solution in a heating chamber to 60 °C until the fatty acid is completely dissolved.
   2. Biocatalysis and sampling
      1. Prepare two 2 ml reaction mixtures containing 50 mM EDTA, 0.01 mM FMN, 0.5 mM stearic acid in Tris-HCl-buffer. Add a magnetic stirring bar for sufficient oxygen supply and place the glass tubes in a water bath, heated to 25 °C.
      2. Add 200 µg ml^-1 of purified enzyme or 10% (v/v) cell-free extract to each of the reaction mixtures and illuminate them with a clear glass light bulb (120 W) in 15 cm distance of the reaction tubes.
      3. Take 200 µl samples at specific time points (0, 10, 30, 60 and 120 min and over night) stopping the reaction with 20 µl 37% HCl. Add 5 µl myristic acid (10 mM stock solution) as internal standard.
2. Analysis of reaction products
   1. For extraction add 500 µl ethyl acetate to the samples twice, invert the tubes and centrifuge for 1 min at 13,000 x g.
   2. Take off 400 µl of the supernatant and let the solvent completely evaporate.
   3. Derivatize the carboxylic acids into trimethylsilylcarboxylic acid by adding 200 µl N-methyl-N-(trimethylsilyl)trifluoro acetamide (MSTFA). Incubate the solution at 60 °C for 30 min in order to convert hydroxyl groups to trimethylsilyl ethers.
   4. Determination of conversion by GC/FID
      NOTE: Determine the formation of 1-heptadecene (RT: 8.34 min), α- and β-hydroxy stearic acid (RT: 12.05 and 12.1 min) and decrease of the substrate (RT: 11.15 min) using GC/FID.
      1. Set the temperature profile described below Set the injection temperature to 340 °C. Hold the initial oven temperature is at 90 °C for 3.5 min and subsequently increase with 45 °C min^-1. At 220 °C, hold the temperature for 2 min. After a repeated increase of 45 °C min^-1 , hold the temperature at 280 °C for 2 min and then increase with 60 °C min^-1.
         NOTE: Maximum temperature of 330 °C is held for 1.44 min.
      2. Inject 4 µl of the sample, with a split ratio of 5 to get rapid volatilization and homogenous mixing with the carrier gas helium.
         NOTE: In dependence on the increasing temperature the substrate and products will enter the gas phase. The substances are detected by the GC/FID detector after passing the column at specific retention times and are displayed as peaks on the screen. Observe the peak formation on the monitor.
      3. Calculate the concentration of formed product by the following formula:
         
         NOTE: The calibration factor has been determined specifically for this column for each substrate and product by calculating the standard curve from known amounts of the derivatized substances and their corresponding peak area.
   5. Identify olefin and β-hydroxy acid peaks by GC/MS.
      1. Identify olefin and β-hydroxy acid peaks by GC/MS. Set the temperature profile. Set the injection temperature to 250 °C. Hold the oven temperature at 100 °C for 5 min and subsequently increase with 20 °C min^-1.
         NOTE: Maximum temperature is held for 7.5 min.
      2. Set the mass spectrometer detector temperature to 250 °C and scan at 50 to 500 m/z in electron impact mode.
         NOTE: Electron impact ionization removes a single electron resulting in the formation of a radical cation which will be passed forward for mass analysis. Due to the high intramolar energy covalent bonds within the molecule break creating fragments of lower m/z. These fragments form a fingerprint specific for a substance.
      3. Inject 1 µl of the sample. Detect 1-Heptadecene after 11.4 min retention time by the corresponding fingerprint (Figure 2).