Isopropyl β d 1 thiogalactopyranoside (iptg)

The verified plasmids were transformed into E. coli BL21 (DE3) cells, which was then cultured in LB medium supplemented with 50 μg/mL kanamycin (Sangon Biotech, Shanghai, China) at 37°C until OD₆₀₀ = 0.6–0.8, followed by inducing expressing using 0.5 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG; Sangon Biotech, Shanghai, China) for 20 h at 20°C. E. coli BL21 (DE3) cells were harvested by centrifugation and resuspended in lysis buffer (50 mM NaH₂PO₄ and 300 mM NaCl at pH 8.0). After ultrasonic fragmentation, the His-tagged proteins were purified using a Ni Sepharose 6 Fast Flow column (GE Healthcare, Uppsala, Sweden). The eluent was replaced with PC buffer (20 mM sodium phosphate and 10 mM citrate at pH 6.0) and then filtered using a 3 kDa cutoff membrane (Millipore, Billerica, MA, United States) at 4°C. The protein obtained was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein concentrations were determined using the Bradford method (Bradford, 1976 (link)).

The recombinant P protein of NoVs was expressed in a prokaryotic expression host. The activated bacterial culture was induced with an isopropyl β-D-1-thiogalactopyranoside (IPTG; Sangon Biotech, Shanghai, China) solution (final concentration 0.5 mmol/L) and grown in an LB medium (Sangon Biotech, Shanghai, China) containing 100 μg/mL of kanamycin. The bacterial culture was harvested and treated with bovine thrombin (effective cleavage ratio of 1:2000) at 37 °C for 3 h. The supernatant after enzyme digestion was purified using Ni-IDA affinity chromatography (Sangon Biotech, Shanghai, China). The purified protein was then identified using SDS-PAGE (10% separating gel and 5% concentrating gel) and confirmed to be 35 KD in size. The protein concentration was determined using Nanodrop (Thermo, Waltham, MA, USA).

Isopropyl-β-d-1-thiogalactopyranoside (IPTG) and ampicillin were purchased from Sangon (Shanghai, China). Tryptone and yeast extract were purchased from Oxoid Co. Ltd. (Basingstoke, United Kingdom). Cinnamic acid and l-phenylalanine were purchased from Sigma-Aldrich (Shanghai, China). All other chemical reagents were of analytical grade and were commercially available.

(S)-Nicotine (>99%) was obtained from Chemsky international Co., Ltd (Shanghai, China). 6HN, 6-hydroxypseudooxynicotine (6HPON), and HSP were prepared as previously described (Ma et al., 2013 (link)). TransStart® FastPfu DNA Polymerase for fragment amplification was purchased from TransGen Biotech (Beijing, China). Restriction enzymes used for plasmid construction and premixed protein marker for protein electrophoresis were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Antibiotics, isopropyl β-D-1-thiogalactopyranoside (IPTG) and other reagents were purchased from Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). The plasmid extraction, gel extraction, and DNA purification kits were obtained from Omega Bio-tek, Inc. (Norcross, GA, USA). Bacterial genomic DNA was extracted using the TIANamp Bacteria DNA Kit from Tiangen Biotech co., Ltd. (Beijing, China). All reagents and solvents were of analytical or chromatographic grade.

For the precultures of C. glutamicum, single colonies were grown in 5 mL of BHIS medium [33 ] at 30 °C and 220 rpm overnight, after which the entire resulting culture was used to inoculate 50 mL of modified CGIII medium supplemented with 10 g L⁻¹ of glucose, and grown to an OD₆₀₀ of 10.
For aerobic growth, cells were washed twice with CGXIIB medium and used to inoculate 50 ml of CGXIIA medium with 20 g L⁻¹ of xylose or glucose to an initial OD₆₀₀ = 0.8, and cultured in 500 mL shake flasks at 30 °C and 220 rpm on a rotary shaker.
A two-stage process was performed for succinate production. When the cultures reached an OD₆₀₀ of 20, the cells were harvested by centrifugation (5 000×g, 4 °C, 10 min), washed with CGXIIB medium. An appropriate amount of washed cells was suspended in 25 mL CGXIIB medium with the indicated sugars and 200 mM sodium bicarbonate, and the cell suspension was cultured in 50 mL serum bottles at 30 °C and 220 rpm on a rotary shaker. To prevent acidification, 30 g L⁻¹ of magnesium carbonate hydroxide (4MgCO₃·Mg(OH)₂·5H₂O) was added as a buffering agent. For induction, isopropyl β-d-1-thiogalactopyranoside (IPTG; Sangon Biotech, China) was added to a final concentration of 1 mM. All the fermentation experiments were performed in triplicate.

This study complies with all relevant ethical regulations and the bacterial strains and plasmids used in this study are listed in Table S2. Unless otherwise noted, S. aureus strains were grown in tryptic soy broth (TSB, Difco) at 37 °C with shaking (250 rpm), or on tryptic soy agar (TSA, Difco) at 37 °C. Escherichia coli strains were grown in Luria-Bertani (LB) broth at 37 °C with shaking (250 rpm) or on LB agar plates at 37 °C. For plasmid maintenance, antibiotics were used at the following concentrations where appropriate: for S. aureus, erythromycin (Sangon Biotech) at 10 μg/ml for RN4220 and 80 μg/ml for USA300 LAC and its derivatives, chloramphenicol (Sangon Biotech) at 15 μg/ml; for E. coli, carbenicillin (Sangon Biotech) at 150 μg/ml. For other reagents, tunicamycin and vancomycin (Dalian Meilun Biotech Co., Ltd.), targocil (Shanghai TopScience Co, Ltd.), oxacillin (Shanghai Aladdin Biochemical Technology Co.,Ltd.), imipenem, cefuroxime, ceftizoxime, cefaclor, cefoxitin, and epicatechin gallate from MedChemExpress, cefotaxime, isopropyl β-D-1-thiogalactopyranoside (IPTG), and AIP from Sangon Biotech, anhydrotetracycline (aTc) from APExBIO, Tarocin A1 from Merck, moenomycin complex from GlpBio, and nile red from Yeasen Biotechnology Co., Ltd. were used in this study.

Glutathione and 2, 2-diphenyl-1-picrylhydrazyl (DPPH) were acquired from Sigma-Aldrich (USA). Expression vector pET-30a (pET) and Rosetta (DE3) strains were preserved in our laboratory. DNA markers 2000, restriction endonucleases (Xho I and Bam HI), DNA Fragment and Plasmid Purification Kits 4.0, DNA gel extraction kits and BCA protein assay were purchased from Takara (Japan). Precast-GL gel Hepes SDS-PAGE, kanamycin, tryptone, isopropyl β-D-1-thiogalactopyranoside (IPTG) and yeast extracts were obtained from Sangon (China). Fetal bovine serum (FBS), protein markers, penicillin-streptomycin solution, Dulbecco’s modified Eagle medium (DMEM) and trypsin-EDTA were purchased from Thermo Fisher Scientific (USA). All commercially purchased chemicals were of analytical grade.