L rhamnose monohydrate

The ∆argF∆argI double knockout E. coli strain was constructed by the Coli Genetic Stock Center at Yale. ATUM Bio supplied the backbone vector (pD884) for cloning. Terrific Broth (TB), M9 minimal medium, carbenicillin (100 mg/mL), kanamycin (50 mg/mL), and Luria broth (LB)/agar plates containing carbenicillin/kanamycin were obtained from Teknova. Ampicillin, L-rhamnose monohydrate, triethanolamine hydrochloride, sulfuric acid, acetic acid, lithium carbamoyl phosphate dibasic hydrate, L-ornithine monohydrochloride, diacetyl monoxime and L-citrulline were purchased from Sigma Aldrich. Thermo Fisher Scientific provided B-PER Complete Bacterial Protein Extraction Reagent, HisPur Ni–NTA spin plates, antipyrine, SYPRO Orange (5000X) dye, Lipofectamine 2000 and the bicinchoninic acid (BCA) assay kit. Novus Biologicals provided the rabbit polyclonal OTC antibody (NBP1-87408) for western blot analysis. All oligonucleotides in this work were purchased from IDT and Q5 DNA polymerase (NEB) was used for all PCR reactions.

All reagents were analytical grade unless otherwise stated. Standards of different monosaccharides were used to perform the calibration curves. D-(+)-Fucose > 98%, L-rhamnose monohydrate > 99%, 2-O-methyl D-xylose > 99%, L-(+)-arabinose > 99%, D-(+)-xylose > 99%, D-(+)-galactose > 99%, D-(+)-glucose 99.5%, D-(+)-mannose ≥ 99% and Kdo (2-keto-3-deoxy-D-manno-octulosonic acid) ≥ 97% were supplied by Sigma (Beerse, Belgium); and D-(+)-galacturonic acid > 93%, D-glucuronic acid ≥ 97% and myo-Inositol ≥ 98% (internal standard) were obtained from Fluka (Buch, Switzerland).
Ethanol 96% (v/v), hexane-(n) 99+%, HPLC grade and acetyl chloride ≥ 98.0% were supplied by Merck (Darmstadt, Germany); and mEthanol anhydrous 99.8%, pyridine 99.5+%, hexamethyldisilazane ≥ 99.0% and trimethylchlorosilane ≥ 98.0% were supplied by Merck (Darmstadt, Germany).

Fresh commercial state watermelon (Citrullus lanatus) fruits were kindly supplied by Anecoop S. Coop. during the summer season of 2019 from Almeria, Spain. The fruits were processed, removing the red part of the fruit and keeping the white rinds, which were chopped into pieces of 0.5–2.5 cm and immersed in 4 volumes of distilled water for 10 min with gentle agitation. After that, the water was drained, and the rinds were freeze-dried. The dried material was milled in liquid nitrogen using an A11 Basic IKA mill (IKA, Staufen, Germany) and stored in desiccators at 0% humidity until used. Citrus and apple pectin (CP and AP, respectively) were used for comparative purposes (Sigma-Aldrich, Stenheim, Germany). Phenol red (ACS grade) and sodium hydroxide (pharma grade) were supplied by Sigma-Aldrich (Stenheim, Germany). Acetone (99%) was from WVR chemicals. N-hexane (>95% purity) and ethanol (96% v/v, USP grade) were purchased from Panreac Applichem (Darmstadt, Germany). Analytical standards: D-(+)-Galactose CAS:59-23-4, D-(+)-Galacturonic Acid monohydrate CAS:91510-62-2, L-Rhamnose monohydrate CAS: 10030-85-0, D-Glucoronic Acid CAS:6556-12-3, D-(+)-Mannose CAS: 3458-28-4, D-(+)-Glucose CAS: 50-99-7, D-(-)-Fructose CAS: 57-48-7, L-(-)-Fucose CAS: 2438-80-4, D-(+)-Xylose CAS:58-86-6, L-(+)-Arabinose CAS: 5328-37-0 were purchased from (Sigma-Aldrich).

The brown algae Sargassum thunbergii was collected in Qingdao, China, on 28 May 2014. The l-fucose, d-galactose, d-mannose, d-glucuronic acid, l-rhamnose monohydrate, d-xylose, d-glucose, and 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, Missouri, MO, USA).

Trifluoroacetic acid (TFA, Sigma-Aldrich) was used to hydrolyze LJ fucoidan as described previously [35 (link)]. The LJ fucoidan and 2 M TFA were incubated in screw-cap vials, kept at 121 °C for 2 h and dried under a nitrogen stream. The dried samples were diluted with distilled water and passed through a 0.20-µm filter, which was used for high-performance liquid chromatography (HPLC) analysis.
Dionex ICS-5000 ion chromatography (Thermo Scientific Dionex; Waltham, MA, USA) with a CarboPac SA 10 column (4 × 250 mm, Dionex; Sunnyvale, CA, USA) was used to quantify the monosaccharide of the LJ fucoidan. 200 mM NaOH was used as the mobile phase and the flow rate was 0.5 mL/min. Standard monosaccharides such as L-fucose, D-galactose, D-glucose, D-mannose, L-rhamnose monohydrate and D-xylose were obtained from Sigma-Aldrich.

Oligonucleotide primers were synthesized by Eurofins Genomics (Ebersberg, Germany) and are listed in Table S1 in the supplemental material. l-(+)-Arabinose (99% purity; Acros Organics, Thermo Fisher Scientific, USA; catalogue no. 365181000), l-rhamnose monohydrate (≥99% purity; Sigma-Aldrich, USA; catalogue no. R3875), magnesium acrylate (95% purity; Alfa Aesar, Thermo Fisher Scientific, USA; catalogue no. 42002; lot no. L14619), 4-isopropylbenzoic acid (cumic acid; ≥98% purity; Acros Organics, Thermo Fisher Scientific, USA; catalogue no. 412800050), and d-(−)-fructose (≥99% purity; Sigma-Aldrich, USA; catalogue no. F0127) were used as inducers for assaying inducible systems or substrates in metabolite consumption assays. Isoprene (99% purity; Alfa Aesar, Thermo Fisher Scientific, USA; catalogue no. L14619) was used as a standard for Isoprene yield quantification. Restriction enzymes were purchased from New England BioLabs (USA).