E coli bl21 de3 plyss

The pCDFDuet-AtaPT-CstDXS transformed into E. coli BL21(DE3)pLysS (Promega, Madison, WI, USA) by heat shock method. The engineered strains carrying pCDFDuet-AtaPT-CstDXS were cultured in the LB medium containing streptomycin (50 μg/mL) and chloramphenicol (34 μg/mL) at 37 °C, 200 rpm for 18 h. The 1.5 mL of culture was then inoculated into the 500 mL Erlenmeyer flask containing 150 mL of the same medium and was further cultivated at 37 °C (200 rpm) until the OD₆₀₀ reached 1.0. The gene expression was induced by adding 1 mM IPTG (final concentration); after that, cells were grown at 18 °C, 250 rpm for 5 h. To start the bioconversion process, 3 mM 4HC (1) (TCI, Tokyo, Japan) and 3 mM MgCl₂ were supplied to the induced culture and the cells were further cultivated at the same condition for 18 h. After that, the culture medium was harvested by centrifugation at 4 °C, 8000 rpm for 10 min. E. coli BL21(DE3)pLysS carried pCDFDuet-AtaPT-CstDXS that was grown in parallel at the same condition, except without the supplement of 4HC (1) used as the control in this study. Since ferulenol (2) is light-sensitive, the bioconversion experiment throughout this study took place in the darkness to minimize the product degradation.

His-tagged fibronectin (domains 7–10) was expressed from pET15bFN-III_7-10RGE [19 (link)] in E.coli BL21(DE3)pLysS (Promega, cat. no. L1195) and purified using Ni-NTA chromatography. Aliquots of the purified protein were frozen in liquid nitrogen and stored in PBS (pH 7.4) at a concentration of 1 mg ml⁻¹ at −80 °C.

Construction of expression plasmids of peach UGT genes were carried out by cloning the full-length ORFs with the N-terminal His-tag into the pET6xHN expression vector (Clontech, Palo, CA, USA). Primers for cloning were listed in Supplementary Table S1. The identity of the cloned gene was confirmed by sequencing of the complete insert. Recombinant proteins were heterologous expressed in E. coli BL21 (DE3) pLysS (Promega, Madison, WI, USA). The transformed cells were precultured at 37°C overnight in 20 mL Luria-Bertani (LB) medium containing 100 μg mL^-1 ampicillin, and then inoculated into 1 L LB medium containing the same antibiotics. The culture was grown at 37°C at 150 rpm to an OD (600 nm) = 0.5 to 0.6, and the protein expression was induced with 1 mM isopropylthio-β-galactoside (IPTG). The culture was incubated at 16°C at 150 rpm for 20 h. The cells were harvested by centrifugation (4,000 g, 4°C, and 15 min) and resuspended in 1× PBS (1.37 M NaCl, 26.8 mM KCl, 20.3 mM Na₂HPO4, and 17.6 mM KH₂PO4, pH 7.2–7.4). After storing at -80°C overnight, the cells were disrupted by sonication. The supernatant was obtained by centrifugation (10,000 g, 4°C, and 30 min), and then purified using TALON Spin column (Clontech, Palo, CA, USA) following the manufacturer’s instructions.