Chemicals and enzymes
All sulfides, sulfoxides, and sulfones were kindly provided by Aosaikang Pharmaceutical Co., (Nanjing, China). Primer STAR HS and restriction enzymes (EcoR I, and Not I), and T4 DNA ligase were purchased from Takara Bio-technology Co., (Dalian, China). Primers were synthesized by Generay Biotech Co., (Shanghai, China). Unless otherwise stated, all other chemicals and reagents used in this work were obtained commercially and were of reagent grade.
Strains, plasmids and media
Strains of E. coli BL21 (DE3) and E. coli DH5α were purchased from TransGen Biotech Co., Ltd (Beijing, China). E. coli DH5α was used for the construction of recombinant plasmids. The E. coli strain BL21 (DE3) and the plasmid pET-28a(+) were used for protein expression. Pichia pastoris X33 and pPICZαA/pGAPZαA were used for the secretory expression of CHMOAcineto-Mut.
Luria broth medium (LB, 1% tryptone, 0.5% yeast extract, 1% NaCl), LBK (supplied with kanamycin), Yeast extract−peptone dextrose medium (YPD, 1% yeast extract, 2% peptone and glucose), YPDZ (supplied with zeocin), buﬀered glycerol−complex medium (BMGY, 1% yeast extract, 2% peptone, 100 mM potassium phosphate (pH 6.0), 0.4 ppm of biotin, 1.34% yeast nitrogen base without amino acids, 1% glycerol), buﬀered methanol−complex medium (BMMY, 1% yeast extract, 2% peptone, 200 mM potassium phosphate (pH 6.0), 0.4 ppm of biotin, 1.34% yeast nitrogen base, 1% methanol), fermentation basal salts medium (BSM: phosphoric acid (85%, 21 mL/L), CaSO4 (0.9 g/L), K2SO4 (14.3 g/L), MgSO4 (6.0 g/L), potassium hydroxide (3.3 g/L) and glycerol (40 g/L)), after autoclaved, 8 mL/L of PTM1 solution was supplied and the pH of the medium was adjusted to 6.0 by ammonia, PTM1 trace salts solution: biotin (0.2 g/L), CuSO4·5H2O (6.0 g/L), KI (0.09 g/L), MnSO4·H2O (3 g/L), Na2MoO4·2H2O (0.2 g/L), H3BO3 (0.02 g/L), CoCl2·6H2O (0.9 g/L), ZnSO4·7H2O (42.2 g/L), concentrated H2SO4 (5 mL/L), FeSO4·7H2O (65 g/L). Glycerol feeding medium: 50% glycerol supplied with 12 mL/L of PTM1 trace salts solution. Methanol feeding medium: neat methanol supplied with 12 mL/L PTM1 trace salts solution. Bacterial fermentation medium: 0.5% peptone, 0.5 yeast extract, 0.5% glycerol, 0.9% Na2HPO4·12H2O, 0.07% Na2SO4, 0.34% KH2PO4, 0.025% MgSO4, 0.27% NH4Cl. Complex feeding medium: 6% peptone, 6% yeast extract, 25% glycerol.
Expression and purification of CHMOAcineto-Mut expressed by E. coli BL21 (DE3)
The sequence of the engineered CHMOAcineto-Mut gene was designed with a histidine tag at the N-terminal, synthesized and subsequently cloned into pET-28a(+) by Genscript Biotech (Nanjing) Co., Ltd (Nanjing, China). Transformants were cultured for 12 h in test tubes containing 4 mL of LB medium with 50 μg/mL kanamycin at 37°C and 180 rpm, and then 1 mL of the culture was inoculated into 100 mL of fresh LB medium containing 50 μg/mL kanamycin. After cultivation at 37°C, 180 rpm for 2.5 h, isopropyl-β-D-thiogalactoside (IPTG) and vitamin B solutions were added to a ﬁnal concentration of 0.2 mM, and 50 mg /L, respectively. Induction was started when the optical density (OD600) of the E. coli cells arrived at 0.6 to 0.8, and further proceeded for 20 h at 16°C, 180 rpm. The induced cells were harvested by centrifugation and lysed by ultra-sonication, and the cell lysate was centrifuged at 10,000×g and 4°C for 30 min. The supernatant was then loaded onto a HisTrap HP (GE, USA) column which was pre-equilibrated with Ni-NTA buffer A (potassium phosphate, 20 mM, pH 7.4; sodium chloride, 500 mM; 2-mercaptoethanol, 5 mM). Samples were eluted by gradient imidazole by employing Ni-NTA buffer B (Ni-NTA buffer A containing 500 mM of imidazole). Elution fractions which contained highly pure CHMOAcineto-Mut were pooled, desalted with Ni-NTA buffer C (potassium phosphate, 20 mM, pH 7.4; sodium chloride, 150 mM; dithiothreitol, 1 mM) and flash frozen in liquid nitrogen, stored at ‒80°C for further analysis.
Cloning and expression of the CHMOAcineto-Mut gene in P. pastoris X33
The CHMOAcineto-Mut gene was ampliﬁed using pET-28a(+)-CHMOAcineto-Mut as the template by PCR under the following conditions: pre-denaturation at 95°C for 3 min; 29 cycles at 98°C for 10 s, 55°C for 15 s, and 72°C for 105 s; followed by a ﬁnal extension at 72°C for 10 min. The primers used in this study were shown in Table S1. The PCR product was ligated into pPICZαA/pGAPZαA and then cloned into E. coli DH5α. About 5 μg plasmids were linearized by Sac I for 6 h and recovered with PCR purification Kit (Aidlab Biotechnologies Co., Ltd, Beijing, China), and then electrotransformed into P. pastoris X33 with a Micropulser (BioRad, Hercules, CA, USA). Transformants were selected on YPD (supplemented with zeocin) plates after incubation for 48 h at 30°C.
Positive yeast transformants were cultured for 24 h in test tubes containing 4 mL of YPD medium with 100 μg/mL zeocin at 28°C and 200 rpm, and then 1 mL of the culture was inoculated into 100 mL of BMGY medium containing 100 μg/mL ampicillin. After cultivation at 28°C, 200 rpm until optical density of the yeast cells arrived at 1–2, cells were resuspended in BMMY medium and further induced at 28°C, 200 rpm for 96 h. Neat methanol (1%, v/v) was added into the medium at further 24, 48, 72 and 96 h.
Purification of CHMOAcineto-Mut expressed in yeast
After cultivation, cells were removed from the induced yeast fermentation broth by centrifugation at 6,000×g, 4°C for 40 min. About 500 mL fermentation clear broth was subjected to microfiltration (0.45 μm), concentrated by ultrafiltration on a LabscaleTM Tangential Flow Filtration System (Merck Millipore, German) equipped with a 30 kDa cut off module (Pellicon® XL, 50 cm2, Millipore, Germany). The concentrated broth was diluted and concentrated twice with Ni-NTA buffer A. Samples were centrifugated at 10,000×g, 4°C for 30 min to remove deactivated proteins, then further purified by Ni2+-affinity chromatography (Ren et al. 2020) and flash frozen in liquid nitrogen, stored at ‒80°C for further analysis.
High level production of CHMOAcineto-Mut by high cell density fermentation
CHMOAcineto-Mut-P: single recombinant yeast colony was isolated on YPDZ agar plates and pre-cultured (200 rpm, 30°C) in 200 mL liquid YPD medium which contained ampicillin (100 μg/mL) until the optical density of the cells arrived at 2.0, then inoculated into a 5-L bioreactor (BXBIO, shanghai, China) which contained 1.8 L BSM medium. The fermentation was carried out at 28°C, DO>20%, and pH 6.0. After about 18 h of the fermentation, the DO value was suddenly rebounded to 100%, indicating the consumption of glycerol in the initial medium, and then glycerol feeding was started until an OD600 of about 200. A starvation was kept for 1 h to make sure the metabolism intermediates of glycerol were consumed. Then methanol induction was started with an initial rate of 4 mL/h during the first 4 h to make the yeast adapt to methanol before being stepped to a higher rate of about 18 mL/h in 12 h. After the fermentation, cells were removed and the yeast secretion supernatant was kept on ice for further analysis.
CHMOAcineto-Mut-E: single recombinant E. coli colony was isolated on LBK agar plates and precultured (180 rpm, 37°C) in 300 mL liquid bacterial fermentation medium supplied with kanamycin (50 μg/mL) in shake flask until the optical density arrived at 1.5, then inoculated into 5-L bioreactor which contained 2.7 L bacterial fermentation medium. The fermentation was carried out at 37°C, DO>20%, and pH 7.0. After 4 h, the complexed feeding medium was supplied with a constant rate of 25 mL/h. Cells were induced when the OD600 arrived about 8.0 by adding IPTG stock solution to a final concentration of 200 μM at 25°C.
Protein assays and determination of the FAD occupation rate
The concentration of CHMOAcineto-Mut was determined by Bradford method, using bovine serum albumin (BSA) as the standard. UV-vis scanning was performed on a Molecular Devices SpectraMax M2 Microplate Reader (USA). Determination of FAD occupation was modified from Fraaije’s work [Fraaije et al. 2005], purified CHMOAcineto-Mut samples were diluted to 5 mg/mL by denature buffer (potassium phosphate, 20 mM, pH 8.0; NaCl, 150 mM; DTT, 1 mM; SDS, 1%, w/v) and incubated at 95°C for 5 min, then the absorbance was analyzed in the wavelength range of 280-447 nm, respectively.
Determination of intracellular FAD content of E. coli and P. pastoris
At the scheduled time, cells (E. coli or P. pastoris, 132 OD) were resuspended in 100 mM potassium phosphate buffer (pH 9.0, 0.5 mL), the suspension was subjected to agitation at 1500 rpm, 4°C for 30 min by 200 mg glass beads (Φ 1 mm). A clear cell lysate was obtained by centrifugation of the sample at 10,000×g, 5 min. Samples were further incubated at 95°C for 10 min and centrifugated again at 10,000×g for 10 min to remove the protein. The resultant supernatant was subjected to HPLC analysis by a SHIMADAZU LC2010A system equipped with a C18 column (250 mm × 4.6 mm, 10 μm particle size, DEAIC), under 30°C, 447 nm, using methanol/water (6/4, v/v, 0.6 mL min-1) as the mobile phase (tR FAD = 4.531 min).
Glycosylation characterization of CHMOAcineto-Mut
Purified CHMOAcineto-Mut-E and CHMOAcineto-Mut-P was diluted with denature buffer to 1 mg/mL and incubated at 95°C for 10 min, then cooled to room temperature. 10 μg of the sample was mixed with 10 μg of the SpEndo H and incubated at 37°C for 1 h. Expression, purification of SpEndo H was conducted according to our previous work [Zheng et al. 2020]. Samples were further analysed by SDS-PAGE.
Bio-asymmetric oxidation of pyrmetazole
After cultivation, cells were removed by centrifugation at 6,000×g, 4°C for 30 min. The pH value of the resulting fermentation clear broth was adjusted to 8.0 by slowly dropping 1 M K2CO3 aqueous solution at 0°C. For 10-mL scale reactions, the reactions were performed in closed 50 mL shake flask which contained 100 mM potassium phosphate buffer (pH 8.0), 0.2 mM NADP+, lyophilized BstFDH preparation. The reaction was initiated by adding 1 mL pyrmetazole methanol stock solution and shaken at 25°C, 180 rpm. For 0.6 L scale bio-oxidation reaction, the reaction was performed in a jacked 1 L fermenter, and the reaction mixture was scaled up. At the schedule time, 100 μL of the reaction mixture was taken, extracted by ethyl acetate (0.5 mL) and analyzed by chiral HPLC [Xu et al., 2020].
Purification and characterization of esomeprazole sodium salt
Purification of the esomeprazole sodium was conducted according to the modified procedure by Xu et al [Xu et al., 2020]. After the reaction was finished, the pH was adjusted to 11 by NaOH and subjected to centrifugation at 6000×g for 0.5 h. The supernatant was neutralized by acetic acid to pH 7, then extracted by ethyl acetate. The combined organic layers were dried over anhydrous Na2SO4, concentrated under reduced pressure. The resultant residual was dissolved in ethanol, added with 1 equiv. of NaOH powers, and the resulting solution was crystalized in cold ethanol/methyl-tert-butyl-ester to afford esomeprazole sodium salt.