Phytoene

phytoene and other carotenoids were analysed in cell extracts using a High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD), as described in previous work [12 (link)]. Commercial standards of individual carotenoids, including all-trans β-carotene, all-trans α-carotene, lutein, zeaxanthin and phytoene were obtained from Sigma-Aldrich, UK. Standards were dissolved in methanol or acetone to make a solution of 1 mg/mL and dissolved to a series of working solutions (5, 10, 25 and 50 µg/mL) to generate standard curves. To detect various pigments in the extracts, the DAD scanned wavelengths of 280 nm (colourless phytoene), 355 nm (colourless phytofluene), 450 nm (all coloured carotenoids including β-carotene, α-carotene, lutein and zeaxanthin) and 663 nm (chlorophylls). Total chlorophylls and coloured carotenoids were evaluated by extracting pigments from the harvested algal biomass of a 1 mL culture using 1 mL of 80% (v/v) acetone and absorbance was measured at 664, 647 and 480 nm, according to previous work [12 (link)].

The methanol, hexane, acetone, petroleum ether, dichloromethane, and hydrochloric acid were of analytical grade and were purchased from Labscan (Dublin, Ireland). The HPLC-grade methanol, HPLC-grade acetonitrile, HPLC–grade ethyl acetate, formic acid, sodium chloride, and potassium hydroxide were obtained from Panreac (Barcelona, Spain). The β-Carotene, all-trans-β-apo-8′-carotenal, α-carotene, phytoene, violaxanthin, lutein, β-cryptoxanthin, and lycopene were purchased from Sigma-Aldrich (Taufkirchen, Germany) and the antheraxanthin from DHI (Hørsholm, Denmark). The lutein epoxide, luteoxanthin, zeinoxanthin, 9-cis-antheraxanthin, 9-cis-violaxanthin, 13-cis-violaxanthin, and 9-cis-lutein were obtained as described elsewhere [23 (link),24 (link),25 (link),26 (link),27 (link)]. The gallic acid, p-hydroxybenzoic acid, syringic acid, caffeic acid, m-coumaric, p-coumaric, chlorogenic acid, ferulic acid, naringin, naringenin, ethyl galate, quercetin, kaempferol, crisin, vanillic acid, and myricetin were purchased from Sigma-Aldrich (Madrid, Spain). The quercitrin was obtained from Extrasynthese (Genay, France). All the aqueous solutions were prepared with purified water in a NANOpure Dlamond^TM system (Barnsted Inc., Dubuque, IO, USA).

For carotenoid extraction and HPLC analysis, transformed E. coli DH5α harboring pYS71, pYS69, or pYS76 was cultured in 250 ml flasks containing 50 ml of LB broth with 25 μg/ml gentamicin at 37°C for 24 h. After centrifugation at 10,000 g for 10 min, the cultured cells were repeatedly extracted with 3 ml of acetone for lycopene and β‐carotene or ethanol for Phytoene until the color was completely lost. The extracted solution was centrifuged and filtered through a GHP membrane (0.45 μm pore size). HPLC was performed using 20 μl of a prepared sample, with solvent A (60% acetonitrile, 38% ethyl acetate, 2% acetic acid) and solvent B (100% methanol) as the mobile phase, on a C₁₈ Shim‐pack GIS‐DOS column (4.6 × 250 mm, 5 μm; Shimadzu) as a fixed phase at a flow rate of 1.5 ml/min. β‐carotene was measured at 450 nm using a photodiode array detector. lycopene was measured at 470 nm and Phytoene at 280 nm. Phytoene, lycopene, and β‐carotene standards were purchased from Sigma‐Aldrich, Inc.