Chemicals

3-Acetyloxy-androsta-3,5-dien-17-one and 5-AD were obtained from Aurisco Pharmaceutical Co. Ltd. (Taizhou, China). DHEA, 4-AD, and other chemicals were purchased from Sigma Chemical Reagent Co. Ltd. (Shanghai, China). His60 Ni Superflow Resin and Gravity Columns (TaKaRa Bio, Dalian, China) were purchased from TaKaRa Bio (Dalian, China). Thin-layer chromatography plate HSGF 254 was purchased from Jiangyou Silica Gel Open Co. Ltd. (Yantai, China). All chemicals were of analytical grade as commercially available.

Synthesis of 3-acetyloxy-androsta-3,5-dien-17-one

Under nitrogen protection, 50 g of 4-AD, 50 g of toluene-p-sulfonic acid, and 200 mL of acetic anhydride were combined in the reactor and stirred at 40 °C until the complete reaction of the raw materials was confirmed by thin-layer chromatography (TLC). Subsequently, the reaction mixture was cooled to room temperature, and the product was precipitated by adding a substantial amount of water, filtered, and dried to yield 3-acetyloxy-androsta-3,5-dien-17-one.

Identification of 3-acetyloxy-androsta-3,5-dien-17-one by LC−MS

LC−MS was performed using a SCIEX X500R Q-TOF MS system equipped with an electrospray ionization (ESI) source interface coupled with an ExionLC 30A UHPLC. Chromatographic separation was accomplished employing an ACQUITY UPLC HSS T3 column (100 Å, 1.8 μm, 2.1 mm × 100 mm) with a flow rate of 0.3 mL/min over a 12-min period. The mobile phases consisted of 0.1% (v/v) formic acid in water (A) and methanol (B) in an isocratic elution mode with a ratio of A:B = 1:9. MS was in positive-ion electrospray mode, and operating parameters were as follows: ion spray voltage, 5500 V; temperature, 500 °C; ion source gas, 50 psi; scan range, 80–800 Da; and MS collision energy, 10 eV.

Screening of lipases to hydrolyze 3-acetyloxy-androsta-3,5-dien-17-one

The hydrolyzing 3-acetyloxy-androsta-3,5-dien-17-one capacity was assayed by using 0.1 g immobilized lipases and 20 g/L 3-acetyloxy-androsta-3,5-dien-17-one, added into 2 mL 0.2 M phosphate buffer saline (PBS) buffer (pH 6.0); the reaction mixture was incubated at 30 °C for 24 h. After the reaction, the supernatant was extracted by ethyl acetate for TLC analysis. Enzymes exhibiting hydrolytic activity were additionally screened by introducing 0.1 g of lipase into 2 mL of 0.2 M PBS at pH 6, supplemented with 50 g/L of 3-acetyloxy-androsta-3,5-dien-17-one. The reaction mixture was incubated at 30 °C for 8 h. Subsequently, the supernatant was extracted by ethyl acetate and substrate conversions were analyzed by HPLC.

Optimization of lipase hydrolysis reaction

The product 5-AD generation rate was determined by measuring in the 1 mL assay mixture in different conditions for 2 h, composed of 25 g/L 3-acetyloxy-androsta-3,5-dien-17-one and 0.1 g Lipase PS Amano SD. The effect of pH was determined by measuring over the range from pH 4.0 to 10.0, the effect of temperature was determined in different temperatures from 20 to 50 °C, and the effect of organic solvent was determined by measuring in assay mixture 0.2 M PBS at pH 7 and 20% (v/v) different organic solvents. Following the reaction, the supernatant was extracted using ethyl acetate for subsequent HPLC analysis.

Plasmids and strain construction

KR genes from five different sources, the GDH gene from Bacillus subtilis, and the alcohol dehydrogenase gene from Lactobacillus brevis (LbADH) were selected from the NCBI database (Table S1). The corresponding nucleotide sequences of these enzymes were codon-optimized, synthesized, and then inserted into the plasmid pET-28a (+), providing an N-terminal histidine-tag by Suzhou Genewiz Company. Then, the cloning vector transformed into competent Escherichia coli BL21(DE3) cells to obtain the recombinant E. coli strains.

Expression and purification of the enzymes

All proteins with 6-histidine tag were purified following the same procedure (Chen et al. 2022). Recombinant E. coli BL21(DE3) cells were cultivated overnight at 37 °C and transferred to 50 mL LB medium with 50 μg/mL kanamycin. Then, the strain was cultured in a fermentation medium with a ratio of 1% (v/v) at 37 °C and 200 rpm. The fermentation medium contained 12 g/L yeast extract, 15 g/L peptone, 10 g/L glycerol, 8.9 g/L NaH2PO4·12H2O, 3.4 g/L KH2PO4, 2.67 g/L NH4Cl, 0.71 g/L Na2SO4, and 0.3 g/L MgSO4·7H2O. Upon reaching midlog phase (OD600 = 0.6–0.8), 1 M isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into the expressed system, and the cultures were incubated at 24 °C and 200 rpm for an additional 18 h. Cells were collected by centrifugation at 8000 × g and 10 min at 4 °C. The harvested cells were then resuspended in 50 mM PBS at pH 6, followed by homogenization. Then, the target enzymes were purified with His60 Ni-Superflow Resin and Gravity Columns by nickel affinity chromatography according to the protocol instructions. The collected proteins were stored at −20 °C. Protein expression was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and protein concentration was measured by the Bradford method with the bovine serum albumin (BSA) as the standard.

Reduction of 5-AD by dual enzyme coupling reaction in crude enzyme extracts catalysis form

Biocatalysis 5-AD by crude enzyme extract was investigated under the following conditions: 50 mL each of GDH/ADH and KR bacterium solution was harvested when OD600 was 10; the precipitate was collected by centrifugation and resuspended in PBS (pH 6.3) to 20 mL and then crushed as the crude enzyme. The 20-mL reaction mixture contained crude enzyme, 25 g/L 5-AD, 25 g/L glucose or 10% isopropanol (v/v), and 250 mg/L NAD+ and NADP+; then, the mixture continued stirring at 35 °C. The pH of the system was maintained at pH 6.3 by using the 1.5 M K2CO3 solution.

Effect of catalytic substrate on the production of by-product 4-AD of the dual enzyme coupling reaction

The reaction mixture included 10 g/L 5-AD, 50 μL purified enzyme or 50 μL PBS as control, 450 μL 0.2 M PBS at pH 6, and different concentrations of catalytic substrates; the reaction mixture was incubated at 30 °C for 5h. After the reaction, the supernatant was extracted by ethyl acetate for HPLC analysis.

The characterization of NAD(P)+ and NAD(P)H influence on SwiKR isomerization products was performed with or without 500 mg/L NAD(P)+ or NAD(P)H. The characterization of NAD(P)+ and glucose influence on GDH isomerization products were performed with or without 50 g/L glucose and 500 mg/L NAD(P)+.

The effect of NAD(P)+ inhibition on GDH isomerization products was performed with 25 g/L glucose, 0–2 g/L NAD+, and NADP+. The effect of glucose inhibition on GDH isomerization products was performed with 500 mg/L NAD(P)+ and 0–400 g/L glucose.

Analytical methods

The products were analyzed by TLC on a silica gel developed in the solvent system of ethyl acetate-n-hexane (1:2, v/v). After drying, the chromatograms detected by stained with 0.8 M potassium permanganate standard solution, followed by heating by a heat gun.

The 4-AD, DHEA, 5-AD, and 3-acetyloxy-androsta-3,5-dien-17-one concentration was analyzed by HPLC (Agilent Technologies 1260 Infinity II, USA) with an ELSD detector (Agilent Technologies G4260B 1260 Infinity II ELSD, USA) and an InertSustain C18 (5 μm) column (Shinjuku-ku, Tokyo, Japan) set at 50 °C. The HPLC conditions were as follows: The mobile phase was acetonitrile-methanol-water (25:35:40, v/v) from 0 to 16 min, linearly changed to acetonitrile-methanol (35:65, v/v) from 16 to 18 min, and maintained this mobile phase from 18 to 23 min. The pressure of the nebulizer gas (N2) was set at 4 bar, the temperature of the detector drift tube was 50 °C, and the injection volume was 10 μL.

Structural analysis of catalytic products by NMR

After isolation, the structure of catalytic products was analyzed with the 1D NMR (1H and 13C) and 2D NMR (heteronuclear multiple-bond correlation (HMBC)) spectra. A Bruker Advance III 600 MHz spectrometer (Bruker Bio Spin Corp., Billerica, MA, USA) was applied, with chloroform-D1, and methanol-D4 as solvents. The 1H and 13C NMR spectra were recorded at 400 and 100 MHz, respectively.