Part:BBa_K4759002:Experience

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Applications of BBa_K4759002

① Plasmid construction P450 enzyme CYP107D1 derived from Streptomyces, abbreviated as Olep, is selected to synthesize the above gene by adding histidine tags at the C-terminus through E. coli codon optimization and subcloning the above genes onto the plasmid pET-28a to obtain the recombinant plasmid pET-28a-oleP. Since the CYP107 family is a typical three-component P450 enzyme that requires a pair of redox partner to transfer electrons from the electron donor NAD(P)H to the P450 enzyme activity center, ferredoxin reductase CamA and ferredoxin CamB are selected as redox partner. The codon-optimized camA gene and camB gene are synthesized, and they are subcloned to the plasmid pACYCDuet to obtain the recombinant plasmid pACYCDuet-camA-camB.

② Recombinant strain construction The above two recombinant plasmids are then transformed into the E. coli expression host C43 (DE3) to obtain one recombinant strain, named E. coli O1.

③Experimental results of CO spectra To verify the activity of Olep, we scanned the solution of Olep, Olep-CO, and Olep-CO-sodium dithionite at full wavelength. The characteristic absorption peak of pure enzyme Olep at 450 nm was found by CO spectroscopy. The results showed that Olep is an active P450 enzyme.

Fig1-1: Full wavelength scan of purified Olep in three states. The blue, orange and red circles mark the characteristic absorbance of the purified Olep, Olep-CO complex and Olep-CO-sodium dithionite complex, respectively.

1.2 The expression situation of Olep The recombinant strain E. coli O1 is cultivated using TB culture and grown at 37 °C until an OD600 value of 0.6-0.8 is reached. 0.5 mM IPTG(isopropyl-β-D- thiogalactopyranoside) is added and the culture was incubated for another 20 h at 25 °C. After the fermentation is completed, the fermentation broth is centrifuged at 4°C, 8000 rpm for 10 min, the bacteria are collected, and the bacteria are washed with potassium phosphate buffer at pH 8.0. After the bacterial body is complete, resuspend the bacterial body with potassium phosphate buffer (containing 500 mM sodium chloride, and 20 mM imidazole) at pH 8.0 and perform cell disruption with an ultrasonic disruptor on ice. The procedure of the ultrasonic crusher is as follows: POWER, 38%; 2 s on /3 s off; 5 min. The cell disruption solution is centrifuged at 4°C, 8000 rpm for 10 min, and the supernatant is collected as Olep crude enzyme. Olep pure enzyme is obtained by Ni-NTA affinity chromatography with a pH 8.0 potassium phosphate buffer (containing 500 mM sodium chloride and 200 mM imidazole). The purified Olep is desalted with Amicon Ultra 30 K ultrafiltration centrifuge tubes.

SDS-PAGE showed that Olep would form a large number of inclusion bodies expressed in E. coli, with only 32.4% soluble protein.

Fig1-2: SDS-PAGE analysis of Olep. Lane 1, soluble expression of Olep; Lane 2, inclusion body of Olep; Lane 3, purified soluble Olep. M, marker

1.3 Identification of whole-cell biocatalytic products ① Whole-cell biocatalysis After the washing of the bacteria, the washed bacteria are resuspended with potassium phosphate at pH 8.0 (glycerol containing 10% v/v) to be the whole cell catalyst. The whole cell catalyzed reaction system is (in terms of final concentration): strain cell OD600=30, deoxycholic acid 1 mg/mL, NADPH 1 mM, glucose dehydrogenase 1 U/mL.

② The analysis of hydroxylated product of DCA catalyzed by TLC Thin layer chromatography (TLC) is a type of chromatography used to separate mixtures by covering a glass sheet, plastic sheet, or aluminum foil with a thin layer of adsorbent. In our project, we also explored TLC methods for detecting DCA and 6β-OH DCA. Among them, the formula of the developing agent is a ratio of dichloromethane to methanol of 10:1. The color developer is CAM stain (225 mL of water + 12.5 g ammonium molybdate tetrahydrate + 2.5 g of cerium molybdate, 25 mL of concentrated sulfuric acid (slowly added in ice bath)).

Fig1-3: TLC results for DCA and 6β-OH DCA From left to right, DCA, 6β-OH DCA, and their mixtures are separated

③ The analysis of hydroxylated product of DCA catalyzed by HPLC High performance liquid chromatograph (HPLC) is the application of high performance liquid chromatography principle, mainly used for the analysis of high boiling point non-volatile, heat instability and large molecular weight of organic compounds instrument equipment. With HPLC testing, we found that the recombinant strain E. coli O1 (containing recombinant plasmids pET28a-oleP and pACYCDuet-camA-camB) yields 0.048 mg/mL 6β-OH deoxycholic acid (conversion rate 4.8%). Therefore, it is necessary to improve the soluble expression of Olep in Escherichia coli to improve the efficiency of Olep-catalyzed preparation of 6β-OH deoxycholic acid.

Fig1-4: HPLC results of catalytic products.

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