Lycopene standard

After 120 h, a 10-mL sample was collected from culture broth and then centrifuged at 10,000×g for 20 min. The sediment was washed with distilled water and recentrifuged (triple). Dry biomass weight was determined after drying at 105 °C overnight. To measure lycopene content, the sediment was dried in a vacuum drier at 40 °C and then crushed using mortar and pestle, followed by extraction of lycopene with petroleum ether. Repeated extractions of lycopene were carried out with the above solvent until the residue became colorless. Lycopene produced was analyzed by high-performance liquid chromatography (HPLC). The HPLC was equipped with a ZORBAX Eclipse Plus C18 column (250 mm × 4.6 mm) at 30 °C. The mobile phase was acetonitrile: acetone (60:40, v/v) at a flow rate of 1 mL/min. The absorption of lycopene was measured at 470 nm. Lycopene was identified by comparing with the retention time of lycopene standard (Sigma), and quantitative analysis was performed by the single-point calibration method.

Extraction of lycopene was made according to Barba [40 (link)]; 0.3 g of tomato peel and pulp (taken from the pull described previously in Section 2.4) were added to 10 mL of a solvent solution made by hexane/acetone/ethanol (50:25:25 v/v/v) and homogenized with Ultra-Turrax (IKA^®). Subsequently, 1.5 mL of distilled water was added, and the samples were vortexed. The upper layer (1 mL) was dried under vacuum and the dry extract was resuspended in 0.4 mL of tetrahydrofuran (THF)/acetonitrile (ACN)/methanol (15:30:55 v/v/v). The mobile phase for HPLC (Perkin Elmer Nelson 3200 Series) analysis consisted of methanol/ACN (90:10 v/v) and 9 mM triethanolamine (TEA) at a flow rate of 0.9 mL/min, using a RP-C18 column (SUPELCO Kromasil 100A-5u-C18 4.6 mm × 250 mm); the absorbance was set at 475 nm and the run time was 20 min. Quantification was carried out using a standard calibration curve consisting of five points at increasing concentrations (6.25, 12.5, 25, 50, and 100 µg/mL) of lycopene standard (Sigma Chemical, St. Louis, MO, USA). The experiment was conducted in three technical replicates for each sample. Finally, the mean and standard deviation were calculated. To verify the significance of the data obtained, t-tests (* p ≤ 0.05, ** p ≤ 0.01) were carried out.

HPLC grade solvents, including methanol, 2-propanol, and tetrahydrofuran (THF) were purchased from Merck (Darmstadt, Germany). Analytical grade solvents, including acetone, n-hexane, ethanol, methanol, and triethylamine, were also purchased from Merck (Darmstadt, Germany). Potassium persulfate, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), dextran standard, pectin standard (from citrus peel), sodium chloride (NaCl), β-carotene standard, and lycopene standard (sum of isomers in ≥95% of corn oil) were purchased from Sigma Aldrich (St. Louis, MO, USA). The fresh tomatoes were purchased from the local market (Selangor, Malaysia).

The extraction of lycopene was conducted according to Barba et al., 2006 [65 (link)]; 0.3 g of tomato peel was added to 10 mL of hexane/acetone/ethanol (50:25:25 v/v/v) and the mix homogenized with Ultra-Turrax (IKA^®). Then, 1.5 mL of distilled water was added followed by vortexing. One ml of the upper layer was dried under vacuum and the dry extract was resuspended in 0.4 mL of solution tetrahydrofuran (THF)/acetonitrile (ACN)/methanol (15:30:55 v/v/v). The mobile phase for HPLC consisted of methanol/ACN (90:10 v/v) and triethanolamine (TEA) 9 mM at a flow rate of 0.9 mL/min in a RP-C18 column (SUPELCO Kromasil 100A-5u-C18 4.6 mm × 250 mm) at an absorbance of 475 nm and in a run time of 20 min. For quantification, a standard calibration curve consisting of five points at the increasing concentrations of 6.25, 12.5, 25, 50 and 100 µg/mL of lycopene standard (Sigma Chemical, St. Louis, MI, USA) was used.

The strains constructed in S. cerevisiea are listed in Table S2. The primers used for fragments, and the fragments used for strain construction are listed in Tables S3 and S4, respectively. All fragments obtained by polymerase chain reaction were gel purified using a kit (Axygen; Corning Life Science, Corning, NY, USA) before cloning. Yeast cells were transformed using lithium acetate and PEG4000^{40 (link)} for assembly cloning and gene deletion. Fragment assembly was performed using the Gibson method^{35 (link)} or yeast assembly^{41 (link)}.
Saccharomyces cerevisiae CEN.PK2-1D was used as the background strain for all constructs, and E. coli DH10B was used to propagate the recombinant plasmids. Engineered yeast strains were selected on synthetic complete medium [0.67% yeast nitrogen base with (NH₄)₂SO₄, 2% glucose, and appropriate amino acids] under auxotroph-screening conditions (uracil 20 mg L⁻¹, histidine 20 mg L⁻¹, tryptophan 20 mg L⁻¹, and leucine 100 mg L⁻¹) or yeast extract–peptone–dextrose medium (2% tryptone, 1% yeast extract, and 2% glucose) with antibiotic screening (G418 200 mg L⁻¹ and hygromycin 200 mg L⁻¹). All media were autoclaved at 115 °C for 30 min before use. Lycopene standard was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Lycopene standard, Folin–Ciocalteu reagent, sodium carbonate, gallic acid, sodium nitrite, aluminum chloride, sodium hydroxide, rutin, and L-ascorbic acid were purchased from Sigma Aldrich (Merk, Milan, Italy), as well as 2,2-diphenyl-1-picrylhydrazyl radical powder (DPPH) and all of the solvents used for the extracting and analytic procedures, such as acetone (Ac), hexane (He), dichloromethane (DCM), 2,methyl-tetrahydrofuran (Me-THF), acetonitrile (CAN), and methanol (Me-OH). All other materials and solvents used were of analytical grade. Two sauce bottles for each brand were used (750 mL/bottle) and they were chosen because they were those most used in local market. Bottles of the same brand belonged to the same production lot.