Aviii 500 nmr instrument

Optical rotations were measured on a Jasco P-1020 polarimeter (Jasco, Tokyo, Japan). A UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan) was applied for the measurement of UV spectra. ECD and IR data were collected on Jasco J-810 spectrometer and Bruker Tensor 27 spectrometer (Bruker, Karlsruhe, Germany), respectively. Preparative HPLC was performed on a Shimadzu LC-8A system equipped with Shim-pack RP-C₁₈ column (10 μm, 200 mm × 20 mm i.d., Shimadzu, Tokyo, Japan), using a binary channel UV detector at the wavelengths of 210 and 330 nm, respectively. With TMS as internal standard, NMR data were recorded on a Bruker AVIII-500 NMR instrument (¹H NMR, 500 MHz; ¹³C NMR, 125 MHz). HRESIMS data were obtained using Agilent 6520B Q-TOF mass instrument and Agilent 1100 series LC/MSD-Trap-SL mass analyzer (Agilent Technologies, Santa Clara, USA). The extracts were fractionated through CCs with multiple packing materials such as silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia, Stockholm, Sweden) and ODS (40−63 μm, Fuji, Tokyo, Japan). After fluorescence observation under UV light (254 and 356 nm), silica gel GF₂₅₄ plates were sprayed with vanillin−sulfuric acid to visualize the spots. Optical density (OD) values were determined by a microplate reader (Tecan, Mannedorf, Switzerland).

The optical rotations were measured on a JASCO P-1020 polarimeter at room temperature. The melting points were measured using an X-4 digital display micromelting apparatus and are uncorrected. The IR spectra were recorded on a Bruker Tensor 27 spectrometer using KBr pellets. The 1D- and 2D-NMR spectra were measured on a Bruker AVIII-500 NMR instrument (¹H: 500 MHz, ¹³C: 125 MHz) using TMS as the internal standards. HRESIMS was performed on an Agilent 6529B Q-TOF mass instrument using electrospray ionization. All of the solvents used were of analytical grade (Jiangsu Hanbang Science and Technology. Co., Ltd.). Silica gel (200–300 mesh, Qingdao Haiyang Chemical Co., Ltd, China), Sephadex LH-20 (Pharmacia, Sweden), MCI (Mitsubishi, Japan) and RP-C18 silica (40–63 μm, Fuji, Japan) were used for the column chromatography. Preparative HPLC was conducted using an Agilent 1260 Series instrument with a Shim-Pak RP-C18 column (20 × 200 mm), at a flow rate of 10.0 mL/min, and detection by a binary channel UV detector. The fractions obtained from CC were monitored by TLC with precoated Silica gel GF₂₅₄ (Qingdao Haiyang Chemical Co., Ltd, China) plates.

All bacterial strains and plasmids used in the study are listed in Table 1. The E. coli strains were routinely cultured in lysogeny broth (LB) supplemented with 100 mg/L ampicillin. The engineered strains were cultured in terrific broth (TB) to produce alkaloid products. All the chemicals were purchased from TEDIA (Tedia Co., Fairfield, OH, USA). The structure of the synthesized 2,3,9,10-tetrahydroxyberbine hydrochloride was confirmed using ¹H- and ¹³C-NMR. The primers were synthesized by Tsingke (Nanjing, China). The PCR polymerases and DNA ligases were purchased from Vazyme (Nanjing, China). Optical rotations were recorded with a JASCO P-1020 polarimeter (JASCO, Tokyo, Japan). The NMR experiments (¹H: 500 MHz, ¹³C: 125 MHz) were measured using a Bruker AVIII-500 NMR instrument (Bruker, Bremen, Germany) equipped with a broadband observe probe. TMS was used as an internal standard, and either MeOD or CDC_l3 was used as solvent for dissolving compounds.