Supor membrane

The extraction protocol used was taken from Bell et al. (2015) (link). Briefly, two experimental replicates of each biological rep (n = 6) were prepared as follows: 40 mg of ground powder was heated in a dry-block at 75°C for 5 min; 1 mL of preheated 70% (v/v) methanol (70°C) was then added to each sample and placed in a water bath for 20 min at 70°C. Samples were centrifuged for 5 min (12,000 rpm, 20°C) to collect loose material into a pellet. The supernatant was then filtered using 0.22 μm Acrodisc syringe filters with Supor membrane (hydrophilic polyethersulfone; VWR, Lutterworth, United Kingdom) into fresh Eppendorf tubes. Samples were frozen at −80°C until analysis by LC–MS.

The extraction method was adapted from [20 (link)]. Three replicates of each batch were prepared as follows: 40 mg of ground steamed-pureed turnip powder was heated in a dry-block at 75 °C for 2 min to ensure inactivation of any remaining active myrosinase enzyme. Preheated 70% (v/v) methanol (1.2 mL; 70 °C) was added and the sample placed in a water bath for 20 min at 70 °C. Samples were then centrifuged for 10 min (10,000 rpm, 18 °C) to collect loose material into a pellet. The supernatant was then filtered through 0.22 μm Acrodisc syringe filters with Supor membrane (hydrophilic polyethersulfone; VWR, Lutterworth, UK) and frozen (−80 °C) in Eppendorf tubes until analysis by LC-MS.

For each strain, at least sixteen single colonies originating from independent transformants were inoculated in 0.5 mL drop-out liquid medium without uracil, histidine, and leucine in a 96 deep-well plate with air-penetrable lid (EnzyScreen, NL). Plates were incubated at 30°C with 300 rpm agitation at 5.1 cm orbit cast overnight (Eppendorf innova 44). 20 μL of the overnight cultures were used to inoculate synthetic fed-batch medium (Feed-In-Time fed-batch medium, m2p-Labs) in a 96 deep-well plate. Fermentation was carried out for 72 hours at the same conditions as above.
At the end of the cultivation OD₆₀₀ was measured using the microtiter plate reader BioTek Synergy MX (BioTek). The culture broth was filtered (Acroprep 0.2 μm, Supor membrane, VWR) and the supernatant analyzed for 3-hydroxypropionic acid concentration on HPLC. For measuring 3HP on HPLC, 30 μL of the sample was analyzed for 30 min using an Aminex HPX-87H ion exclusion column with a 1 mM H₂SO₄ flow of 0.6 mL.min^-1. The temperature of the column was 60°C. The refractive index at 45°C and the UV absorption at 210 nm were recorded. 3HP quantification was performed based on RI chromatograms. The UV spectrum was recorded with a diode array and the identity of 3-hydroxypropionic acid was additionally verified by comparison with the spectrum of a commercial standard.