MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12639/m_MTBLS12639_LC-MS___metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12639/s_MTBLS12639.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12639/a_MTBLS12639_LC-MS___metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12639/i_Investigation.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12639Data analysis and peak area calculation were done on Freestyle 1.8 SP2 (Thermo Fisher Scientific).MetaboLightsPublicLiquid Chromatography MS -Ultra-high-performance LC coupled with high resolution MS was carried out using a Vanquish UHPLC system (Thermo Fisher Scientific) with a VF-P10-A binary pump and a VF-A10-A auto sampler which was set to 10 °C and equipped with a 25 µL injection syringe and a 100 µL sample loop. The column, Accucore® C-18 RP (100 × 2.1 mm; 2.6 µm, Thermo Fisher Scientific), was kept at 25 °C within the column compartment VH-C10-A. Eluent A was water with 2% acetonitrile and 0.1% formic acid. Eluent B was pure acetonitrile. All solvents and water were LC-MS grade. The gradient for sample separation started with 0% B to 2.2 min, followed by an increase to 11% B over 10 min, and 90% B over a further 4 min. It was then kept at 90% B for 2 min and re-equilibrated to 0% B over 1 min. Flow rate was kept constant at 0.5 mL/min. The Vanquish UHPLC system was also equipped with a variable wavelength detector (VC-D40-A-01). UV-Vis data was collected from 190 to 680 nm with a bunchwidth of 1 nm.Establishing betaxanthin pigment biosynthesis in cyanobacteria. 10.1016/j.ymben.2026.01.002.Friedrich Schiller University JenaDavid A. RussoPure compoundexometabolomeendometabolomemass spectrometryLC-MS measurement of phenylalanine, tyrosine and tryptophanFor measurement of intracellular levels, cell pellets were resuspended in 200 µL 50% aqueous MeOH (v/v) and sonicated on ice for 10 min. The suspension was then centrifuged for 2 min at 14, 000 x g and the supernatant was used for LC-MS analysis. The difference between the harvested culture volume (300 µL) and the resuspension volume (200 µL) was accounted for in the final calculations.StandardSynechocystis sp. PCC 6803Data analysis and peak area calculation were done on Freestyle 1.8 SP2 (Thermo Fisher Scientific).Strainhttps://www.ebi.ac.uk/metabolights/MTBLS12639david.russo@uni-jena.deSemi-synthesis of a phenylalanine-betaxanthin standardA semi-synthetic Phe-Bx standard was prepared in-house following a previously established method (Cabanes et al., 2014) with several modifications. Two grams of commercial Beta vulgaris (beet) powder were dissolved in 20 mL ddH2O. The mixture was then filtered with a 125 µm E-D-SCHNELLSIEB® nylon filter to remove remaining insoluble debris. Subsequently, 2 g of Lewatit VP OC 1065 (Sigma-Aldrich) were added to the filtrate and the pH titrated to pH 11.4 with aqueous ammonia. After a 10 min incubation step with constant stirring at room temperature, the pH was reduced to 5.0 with glacial acetic acid. The mixture was incubated for 30 min with constant stirring. Lewatit beads were then collected on a filter and thoroughly rinsed first with ddH2O followed by methanol. The beads were then carefully dried under a nitrogen gas stream. Beads were resuspended in aqueous ammonia and incubated for 30 min at pH 11.4. Following the addition of 500 mg Phe, the pH of the solution was adjusted to 5.0 with glacial acetic acid. The condensation product was stirred for another 30 min before storage at -20 °C or -80 °C until further use. Before use, the standard was quantified by UV-vis absorption spectrophotometry using a molar absorption coefficient at 480 nm of 4.8 x 104 L-1 mol-1 cm-1. LC-MS measurement of phenylalanine, tyrosine and tryptophanFor measurement of Phe, Tyr, and Trp levels, a culture volume of 300 µL was harvested daily and the cells were separated by centrifugation at 9,300 x g for 5 min at 4 °C. Before storage, the cell pellets were washed with 300 µL of their respective growth medium. All samples were stored at -80 °C until the analysis. For measurement of extracellular levels, the supernatant was thawed shortly before the analysis and used without further modification or, when necessary, diluted appropriately with P4-TES CPH medium. LC-MS measurement of Phe-BxFor measurement of Phe-Bx levels, the cells were separated by centrifugation at 9,300 x g for 5 min at 4 °C. All samples were stored at -80 °C until the analysis.MetabolomicsCyanobacteriaBetaxanthinstargeted metabolitesCyanobacteriaBetaxanthinstargeted metabolitesMass spectra were recorded with an Orbitrap Exploris 480 MS (Thermo Fisher Scientific) coupled to a heated electrospray (HESI) source. For monitoring two full scan modes were selected with the following parameters. Polarity: positive; scan range (m/z): 100 to 1500; resolution: 180,000; AGC target: standard; maximum injection time mode: auto. General settings: sheath gas flow rate (arb): 50; auxiliary gas flow rate (arb): 10; sweep gas flow rate (arb): 1; spray voltage (kV): 3.5; capillary temperature (°C): 325; RF lens (%): 50; vaporizer temperature (°C): 350; acquisition time (min): 0.7 - 16.2.TyrosinePhenylalanineTryptophanPhenyalanine-betaxanthinfalseEstablishing heterologous betaxanthin pigment biosynthesis in cyanobacteriaBetalains are water-soluble pigments with two major classes: red-violet betacyanins and yellow-orange betaxanthins. These pigments are increasingly being sought after as natural replacements for synthetic pigments in the food industry. Traditionally, betalains are extracted from cultivated plants. But due to low concentrations of native pigments, the process is inherently inefficient. Now, an increase in consumer demand calls for the development of scalable and sustainable betalain production routes. To address this challenge, we introduced a heterologous pathway for the production of betaxanthins into cyanobacteria. The pathway consists of an engineered variant of the cytochrome P450 CYP76AD1 (W13L, F309L) and the l‐DOPA 4,5‐dioxygenase DODA1 from Beta vulgaris (beet). Introduction of the two-enzyme betaxanthin pathway in Synechocystis sp. PCC 6803 did not result in detectable betaxanthins. Subsequent metabolic adjustments to the shikimate pathway, using a feedback resistant AroGfbr from E. coli, led to an overaccumulation of the aromatic amino acids phenylalanine, tryptophan, and tyrosine, and the production of low levels of phenylalanine-betaxanthin. Optimization of the cultivation conditions (i.e., growth in nutrient-rich medium and CO2-enriched air) increased titers approximately 165 times and led to the production of phenylalanine-betaxanthin with a final titer of 18.2 ± 5.1 mg L–1. Our work establishes a microbial system for photoautotrophic betaxanthin pigment production without the need for exogenous amino acid supplementation.2026-01-062025-06-26MTBLS12639MTBLC18186MTBLC27897MTBLC28044CID:101720918CHEBI:18186CHEBI:27897CHEBI:28044