Ferredoxin mutant and application thereof in synthesis of murine deoxycholic acid

Abstract

The invention discloses a ferredoxin mutant and application of the ferredoxin mutant in synthesis of murine deoxycholic acid, and belongs to the technical field of biological catalytic enzymes. According to the invention, Escherichia coli is used as a chassis strain to express P450 enzyme OleP, a three-component redox partner library is established, and a redox partner PetH / PetF which is most adaptive to OleP is screened. A high-throughput method based on a characteristic light absorption value is developed, and the lithocholic acid and the murine deoxycholic acid can be rapidly and efficiently detected. And the ferredoxin mutant PetFF64D is screened through semi-rational design of the ferredoxin, so that the conversion rate of catalytically preparing the murine deoxycholic acid can be greatly improved. The method provides a new strategy for improving the biosynthesis of hydroxylated steroids.

Description

Technical Field

[0001] The invention relates to a ferredoxin mutant and application thereof in the synthesis of murine deoxycholic acid, belonging to the technical field of biocatalytic enzymes. Background Art

[0002] Steroid compounds have important biological functions in cells and have been widely used in medicine. Steroid drugs are the second largest pharmaceutical product after antibiotics. Currently, about 300 steroid drugs have been approved for clinical use. Bile acid steroids are essential substances for lipid digestion and absorption. Lithocholic acid is a secondary bile acid with a wide source and low cost. At present, lithocholic acid has been used to study drug metabolic pathways and used as a starting material for the synthesis of clinical drugs. Murine deoxycholic acid is a 6β-hydroxylated derivative of lithocholic acid. Murine deoxycholic acid can dissolve gallstones and is used to treat cholesterol and liver diseases. At present, due to the complex production process and low preparation efficiency of murine deoxycholic acid, the price of murine deoxycholic acid is as high as 1,200 yuan / 1 mg.

[0003] Compared with chemical addition of hydroxyl groups, enzyme-catalyzed hydroxylation is a more efficient and environmentally friendly method with advantages such as mild reaction conditions, high catalytic efficiency and strong selectivity, which can make up for the shortcomings of chemical synthesis. Cytochrome P450 enzymes are monooxygenases with heme as a cofactor. They are considered to be highly promising biocatalysts because they can selectively oxidize inactive carbon-hydrogen bonds. In recent years, the use of P450 enzymes to catalyze the hydroxylation of steroidal drugs has attracted more and more attention. The efficiency of P450 enzyme-catalyzed preparation of hydroxylated steroidal drugs can be improved by means of efficient heterologous expression of P450 enzymes (Baodong, Hu., ADVANCED SCIENCE, 10, 2205580.2022), increasing the supply of cofactor heme (Baodong, Hu., CRITICAL REVIEWS IN BIOTECHNOLOGY, 43, 227-241.2023), directed evolution of enzymes (Grobe, Sascha., Angewandte Chemie, 60, 753-757.2021), and optimization of whole-cell catalytic conditions (Chixiang, Sun., Synthetic and Systems Biotechnology, 8, 741-748.2023). However, there is still a problem of mismatch between P450 enzymes and redox partners, because the low electron transfer efficiency of the redox partner causes the low catalytic efficiency of P450 enzymes. Therefore, it is of great significance to modify the redox partner to improve the efficiency of electron transfer and thus enhance the P450 enzyme-catalyzed synthesis of murine deoxycholic acid. Summary of the invention

[0004] The invention provides a ferredoxin mutant and application thereof in the synthesis of murine deoxycholic acid, so as to solve the technical problem of low lithocholic acid hydroxylation efficiency caused by mismatch between redox partner and P450 enzyme.

[0005] The present invention provides a ferredoxin mutant, which is based on the amino acid sequence shown in SEQ ID NO.11, wherein the glutamine at position 62 is mutated to glutamic acid; or the phenylalanine at position 64 is mutated to aspartic acid or proline.

[0006] In one embodiment, the ferredoxin mutant PetF F64D The amino acid sequence is shown in SEQ ID NO.12.

[0007] In one embodiment, the ferredoxin mutant PetF F64P The amino acid sequence is shown in SEQ ID NO.13.

[0008] In one embodiment, the ferredoxin mutant PetF Q62E The amino acid sequence is shown in SEQ ID NO.14.

[0009] The present invention also provides a protein encoding the ferredoxin mutant PetF F64D genes.

[0010] The invention also provides a recombinant plasmid carrying the gene.

[0011] In one embodiment, the plasmid is pRSFDuet.

[0012] In one embodiment, the recombinant plasmid also carries the cytochrome P450 enzyme OleP gene from Streptomyces antibioticus and the ferredoxin reductase gene PetH from Synechocystis sp. PCC 6803.

[0013] The present invention also provides a method for expressing the ferredoxin mutant PetF F64D of recombinant microorganisms.

[0014] In one embodiment, the microorganism is Escherichia coli, including but not limited to E. coli JM109, BL21 (DE3), C41 (DE3).

[0015] In one embodiment, the amino acid sequence of the cytochrome P450 enzyme OleP is shown in SEQ ID NO.15; the ferredoxin reductase PetH has the amino acid sequence shown in UniProtKB:Q55318.2; the amino acid sequence of the ferredoxin PetF is shown in any one of SEQ ID NOs.11-14.

[0016] The present invention also provides a method for improving the catalytic efficiency of P450 enzymes, which comprises co-expressing the P450 enzyme OleP and a redox chaperone protein; the redox chaperone protein comprises ferredoxin reductase PetH and ferredoxin PetF.

[0017] In one embodiment, the ferredoxin reductase PetH has the amino acid sequence shown in UniProtKB: Q55318.2.

[0018] In one embodiment, the ferredoxin PetF has an amino acid sequence shown in any one of SEQ ID NOs. 11-14.

[0019] In one embodiment, the amino acid sequence of the P450 enzyme OleP is shown in UniProtKB: Q59819, or shown in SEQ ID NO.15, or shown in SEQ ID NO.18.

[0020] In one embodiment, the P450 enzyme OleP is a P450 enzyme mutant OleP in which the serine at position 240 is mutated to alanine based on the parent, and the amino acid sequence obtained is as shown in SEQ ID NO.18. S240A .

[0021] The present invention also provides a method for whole-cell catalysis of lithocholic acid hydroxylation, using the recombinant microorganism as a whole-cell catalyst.

[0022] In one embodiment, the reaction of catalyzing 6β-hydroxylation of lithocholic acid is carried out in a 50-100 mM potassium phosphate buffer system, and the biomass (OD 600 ) is 10-35, the concentration of lithocholic acid is 0.1-0.5 mg / mL, the reaction temperature is 20-37°C, and the reaction time is 1-24h.

[0023] The present invention also provides a method for high-throughput detection of lithocholic acid and murideoxycholic acid, comprising mixing a sample to be tested with 75% sulfuric acid-ethanol, reacting at 20-30°C for 30-60 minutes, and then detecting the absorbance value at 370-380nm; the 75% sulfuric acid-ethanol is a mixed solution of sulfuric acid and ethanol in a volume ratio of 3:1, which can react with bile acid containing hydroxyl groups to develop color, and the product after the reaction has a characteristic absorption peak at 370-380nm.

[0024] In one embodiment, the murideoxycholic acid content is calculated according to y=0.6475*x+0.2545.

[0025] In one embodiment, the high-throughput detection method is as follows: adding lithocholic acid to the whole cell catalyst for reaction, centrifuging after the reaction, collecting the reaction solution, and extracting it three times with an equal volume of ethyl acetate. Drying with a nitrogen blower, and then blowing and dissolving with 200uL of methanol to obtain an enzymatic reaction solution. Take 50uL of the enzymatic reaction solution and add it to 150uL of 75% sulfuric acid ethanol for color development for 5 to 30 minutes, and measure the absorbance value at the characteristic absorption wavelength. The sample of the whole cell catalyst without the addition of P450 enzyme is used as a negative control. The greater the difference in absorbance value with the negative control group, the higher the content of murine deoxycholic acid.

[0026] In one embodiment, the whole cell catalyst is prepared by culturing the genetically engineered bacteria at 35-37°C until the OD 600 The value is 0.6-0.8, after adding isopropyl-β-D-thiogalactoside with a concentration of 0.1-1mM, continue to culture at 16-30℃ for 16-24h; centrifuge at low temperature, collect and wash the bacteria with potassium phosphate buffer at pH 8.0. After washing, resuspend the bacteria with potassium phosphate buffer (5%-10% v / v glycerol) to obtain whole-cell catalyst.

[0027] In one embodiment, the whole-cell catalytic reaction of catalyzing 6β-hydroxylation of lithocholic acid is carried out in a 50-100 mM potassium phosphate buffer system, and the biomass OD of the genetically engineered bacteria is 600 is 10-35, the concentration of lithocholic acid is 0.1-1.0 mg / mL, the reaction temperature is 20-37°C, and the reaction time is 6-24h.

[0028] Beneficial effects:

[0029] (1) The present invention screened out a pair of redox chaperones PetH / PetF that are most compatible with the P450 enzyme OleP by constructing a three-component redox chaperone library, protein-protein docking, analyzing the structure of the protein complex, and whole-cell catalysis, and the conversion rate of catalytic preparation of murine deoxycholic acid was increased from 6.4% to 32.3%.

[0030] (2) The present invention has designed a method for high-throughput detection of lithocholic acid and murodeoxycholic acid. The content of lithocholic acid and murodeoxycholic acid can be characterized by reacting 50uL of the enzymatic reaction solution with 150uL of 75% ethanolic sulfuric acid for 30 minutes. Compared with the liquid phase method which takes 30 minutes to detect one sample, this method can detect 96 samples at the same time, and only takes 30 minutes in total.

[0031] (3) By analyzing the structure of ferredoxin PetF, a mutant library was constructed to screen mutants that can improve electron transfer efficiency. Finally, an optimal mutant PetF was screened. F64D , enhanced the efficiency of electron transfer, and the conversion rate of catalytic preparation of murine deoxycholic acid increased from 32.3% to 80.9%.

[0032] (4) The present invention constructs a mutant OleP by mutating the P450 enzyme S240A and the P450 enzyme mutant OleP S240A and the ferredoxin mutant PetF F64D Co-expression and use in catalytic preparation of murine deoxycholic acid can increase the conversion rate to 93.5%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 To detect the product after whole cell catalysis by thin layer chromatography. Spot S is a mixed standard of lithocholic acid and murine deoxycholic acid; Spot R is a sample catalyzed by whole cells of E. coli C41 (DE3) expressing empty plasmid pRSFDuet; Spot O is a sample catalyzed by whole cells of E. coli C41 (DE3) expressing P450 enzyme OleP plasmid pRSFDuet-OleP; Spot AB is a sample catalyzed by whole cells of E. coli C41 (DE3) expressing P450 enzyme OleP and redox chaperone plasmid pRSFDuet-CamA-CamB-OleP;

[0034] Figure 2 To screen the most compatible redox partner for the P450 enzyme OleP. A is the distance between ferredoxin and P450 enzyme calculated using Pymol, B is the docking of protein-protein complex using AlphaFold Multimer software to obtain the complex structure of P450 enzyme and ferredoxin; C is the screening of ferredoxin that is most compatible with the P450 enzyme OleP, blue represents the fluorescence intensity of the strain, and red represents the conversion rate of whole cell catalysis; D is the fluorescence intensity of the observed strain.

[0035] Figure 3In order to establish a high-throughput detection method for lithocholic acid and murodeoxycholic acid. A is to screen chemical reagents that can react specifically with murodeoxycholic acid; B is to determine the characteristic detection wavelength of the product after the reaction; C is to establish a concentration standard curve of murodeoxycholic acid at an absorbance of 370nm; D is to compare the established high-throughput detection method with the liquid phase method to verify the accuracy of the method.

[0036] Figure 4 Semi-rational design of mutant library of ferredoxin PetF. Pink boxes list key amino acid residues, and red boxes list constructed mutants. Blue sites represent 17 sites that are conserved in sequence evolution. Red sites represent 10 sites that affect protein catalytic activity. Blue boxes list the 7 amino acid sites that were finally selected for mutation.

[0037] Figure 5 To semi-rationally design ferredoxin PetF based on protein structure; A is the structural comparison of ferredoxin PetF and ferredoxin Fd I, the red area represents the structure of PetF, and the cyan area represents the structure of Fd I; B is the comparison of the amino acid sequences of PetF and Fd I, the green underline represents the area on PetF that interacts with OleP; C is the analysis of the evolutionary conservation of the amino acid sequence of PetF, the redder the color and the higher the value, the more conservative the amino acid sequence; D is the relative binding free energy of the mutation of 23 sites of PetF to alanine calculated by FoldX; E is the relative binding free energy of the saturation mutation of 7 sites of PetF calculated by FoldX; F is the fluorescence intensity of the constructed 23 mutants and the conversion rate of catalyzing the generation of murine deoxycholic acid. Blue represents the fluorescence intensity of the strain, and red represents the conversion rate of whole cell catalysis. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0039] (I) Culture medium:

[0040] LB medium: 10 g / L peptone, 10 g / L sodium chloride, 5 g / L yeast extract, sterilized at 121°C for 15 min.

[0041] TB medium: 12 g / L peptone, 5 g / L glycerol, 24 g / L yeast extract, 17 mM potassium dihydrogen phosphate, 72 mM potassium hydrogen phosphate, sterilized at 121°C for 15 min.

[0042] (II) Plasmids and cells:

[0043] The strains E. coli JM109, E. coli BL21 (DE3) and E. coli C41 (DE3) and the plasmid pRSFDuet are commercial strains and plasmids.

[0044] Primer sequence synthesis, reagent purchase and gene sequencing verification were all purchased and completed at Shanghai Biotech Co., Ltd.

[0045] (III) Whole-cell catalysis:

[0046] (1) Cultivate the recombinant E. coli at 35-37°C until OD 600 The value is 0.6-0.8, and after adding isopropyl-β-D-thiogalactoside, the culture is continued at 16-30°C for 12-20 hours. Among them, the concentration of isopropyl-β-D-thiogalactoside is 0.1-1mM.

[0047] (2) The fermentation broth obtained in step (1) was centrifuged at 4°C and 8000 rpm for 10-20 min to collect the bacterial cells, and the bacterial cells were washed with a pH 8.0 potassium phosphate buffer. After washing, the washed bacterial cells were resuspended with a pH 8.0 potassium phosphate buffer (containing 5%-10% v / v glycerol).

[0048] (3) Using the bacterial suspension obtained in step (2) to carry out the hydroxylation reaction of lithocholic acid, the whole cell reaction system includes (calculated by final concentration): strain cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. The whole cell reaction was carried out at 20-37°C for at least 8-16 hours. After the reaction, an appropriate amount of the reaction solution was taken out and added with an equal volume of ethyl acetate for extraction. After three extractions and separations, murine deoxycholic acid was obtained and analyzed and detected by a high-throughput detection method (75% sulfuric acid-ethanol solution) and high-performance liquid chromatography.

[0049] (IV) Detection method:

[0050] HPLC analysis:

[0051] (1) Mobile phase: Phase A is ultrapure water containing 0.1% trifluoroacetic acid, and phase B is methanol containing 0.1% trifluoroacetic acid.

[0052] (2) Chromatographic column: Reverse phase column ZORBAX Eclipse XDB-C18 (5 μm, 4.6×250 mm, Agilent, USA); column temperature was 40°C; flow rate was 0.8 mL / min; elution program was: 0-1 min, 10% B; 1-10 min, 10%-40% B; 10-20 min, 40%-90% B; 20-23 min, 90% B; 23-25 ​​min, 90%-10% B; 25-27 min, 10% B.

[0053] (3) An evaporative light scattering detector was used, with nitrogen at 350 kPa, gain 6, and drift tube temperature of 40 °C.

[0054] Calculation of conversion rate: amount of product (mol) / amount of initial added substrate (mol) × 100%.

[0055] Example 1: Construction of a three-component redox chaperone library and screening for the redox chaperone that best matches the P450 enzyme OleP

[0056] The P450 enzyme CYP107D1 (UniProtKB: Q59819) from Streptomyces antibioticus was selected, abbreviated as OleP, and the gene was synthesized by adding a histidine tag at the C-terminus through codon optimization in Escherichia coli (nucleotide sequence as shown in SEQ ID NO.1), and the gene was subcloned into the restriction sites NdeI and XhoI of the plasmid pRSFDuet to obtain the recombinant plasmid pRSFDuet-OleP. Since OleP is a typical three-component P450 enzyme, it requires a redox partner protein to transfer electrons from the electron donor NAD(P)H to the active center of the P450 enzyme. Therefore, the ferredoxin reductase CamA (amino acid sequence as shown in Genbank: BAA00413.1) and ferredoxin CamB (amino acid sequence as shown in UniProtKB: P00259.3) from Pseudomonas putida were selected. The codon-optimized ferredoxin reductase CamA gene (SEQ ID NO.4) and ferredoxin gene CamB gene (SEQ ID NO.5) were synthesized, and the CamA and CamB genes were subcloned into the restriction site between NcoI and SalI of the plasmid pRSFDuet-OleP to obtain the recombinant plasmid pRSFDuet-CamA-CamB-OleP. The above recombinant plasmids pRSFDuet-OleP and pRSFDuet-CamA-CamB-OleP were then transformed into the E. coli expression host E. coli C41 (DE3) to obtain two recombinant strains, named E. coli O and E. coli OAB.

[0057] The recombinant E. coli O and E. coli OAB were cultured in TB medium at 37°C for 2-3 h until OD 600 The final concentration of IPTG was 0.6-0.8, and the expression was induced by adding IPTG at a final concentration of 0.5 mM. The cells were cultured at 25°C for 30 h, and the whole cell catalyst was prepared by washing the cells with potassium phosphate buffer at pH 8.0. The reaction system of whole cell catalysis was (calculated by final concentration): strain cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL.

[0058] The whole cell reaction was carried out at 25°C for 8 h. Figure 1 As shown, the recombinant strain E. coli O cannot catalyze lithocholic acid to produce murodeoxycholic acid, while the recombinant strain E. coli OAB can catalyze lithocholic acid to produce 0.032 mg / mL murodeoxycholic acid (conversion rate 6.4%). Therefore, the redox partner is very important for the catalysis of OleP, and it is necessary to screen and obtain the redox partner that is most compatible with OleP, so as to improve the efficiency of catalytic preparation of murodeoxycholic acid.

[0059] As shown in Table 1, based on database mining and literature research, a three-component redox chaperone library containing 12 pairs of redox chaperones was constructed, including ferredoxin reductase PetH (amino acid sequence as shown in UniProtKB: Q55318.2) and ferredoxin PetF (amino acid sequence as shown in UniProtKB: Q55318.2) from Synechocystis sp. PCC 6803, ferredoxin reductase CamA (amino acid sequence as shown in Genbank: BAA00413.1) and ferredoxin CamB (amino acid sequence as shown in UniProtKB: P00259.3) from Pseudomonas putida, ferredoxin reductase FNR (amino acid sequence as shown in Genbank: BAA00413.1) and ferredoxin Fd I (amino acid sequence as shown in PDB: 1A70_A) from spinach, and ferredoxin reductase FdI (amino acid sequence as shown in Genbank: BAA00413.1) and ferredoxin FdI (amino acid sequence as shown in PDB: 1A70_A) from Escherichia coli. coli), flavodoxin reductase Fpr (amino acid sequence as shown in Genbank: QJZ14319) and flavodoxin Fld (amino acid sequence as shown in Genbank: QJZ13227.1), flavodoxin FldA (amino acid sequence as shown in Genbank: QJZ11404.1) and FldB (amino acid sequence as shown in Genbank: QJZ13309.1) from Escherichia coli, flavodoxin YkuN (amino acid sequence as shown in Genbank: WP_003232386.1) and YkuP (amino acid sequence as shown in Genbank: NP_389300) from Bacillus subtilis, Synethococcus elongatus PCC 7942) derived from ferredoxin reductase Fdr (amino acid sequence as shown in Genbank: WP_011242878.1) and ferredoxin Fdx (amino acid sequence as shown in UniProtKB: P0A3D2.2), ferredoxin reductase Adr (amino acid sequence as shown in PDB:1CJC_A) and ferredoxin Adx (amino acid sequence as shown in UniProtKB:P00257) from bovine adrenocortical mitochondria, ferredoxin reductase Arh1 (amino acid sequence as shown in UniProtKB:O59710) and ferredoxin Etp1 (amino acid sequence as shown in UniProtKB:Q10361) from Schizosaccharomyces pombe, ferredoxin reductase FoR (amino acid sequence as shown in Genbank:CDF80891.1) and ferredoxin FoX (amino acid sequence as shown in Genbank:WP_038530300.1) from Formosa agariphila.

[0060] Table 1 Constructed three-component redox chaperone library

[0061]

[0062] AlphaFold Multimer software was used to perform protein-protein docking of 12 ferredoxins with the P450 enzyme OleP, and the optimal conformation of the complex was found based on the scoring function and the affinity score. Based on the structure of the optimal conformation, the distance between ferredoxin and OleP was calculated using Pymol software (the shorter the distance, the higher the electron transfer efficiency). The results are shown in Figure 2. Figure 2As shown, the distances between ferredoxin PetF, CamB, FldA, FldB, and Fdx and OleP are relatively short. Therefore, 5 pairs of more suitable redox chaperones were screened from the redox chaperone library. The fluorescent sensor plasmid was constructed and expressed in E. coli BL21 (DE3). The sensor plasmid construction method is to fuse 5 kinds of ferric redox proteins to the N-terminus of sfGFP-1-10 and OleP to the C-terminus of sfGFP-11, and construct the recombinant plasmids pRSFDuet-PetH-PetF-GFP-1-10-GFP-11-OleP, pRSFDuet-CamA-CamB-GFP-1-10-GFP-11-OleP, pRSFDuet-FpR-FldA-GFP-1-10-GFP-11-OleP, pRSFDuet-FpR-FldB-GFP-1-10-GFP-11-OleP, and pRSFDuet-FdR-Fdx-GFP-1-10-GFP-11-olep (the underline represents the fusion gene). Among them, the nucleotide sequence encoding PetH is shown in SEQ ID NO.3, the nucleotide sequence encoding PetF is shown in SEQ ID NO.2, and the nucleotide sequence encoding OleP is shown in SEQ ID NO.1; the nucleotide sequences encoding CamA and CamB are shown in SEQ ID NO.4 and SEQ ID NO.5, respectively; the nucleotide sequences encoding FpR, FldA, and FldB are shown in SEQ ID NO.6 to 8, respectively; the nucleotide sequences encoding FdR and Fdx are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively; the nucleotide sequence of sfGFP-1-10 is shown in SEQ ID NO.16; and the nucleotide sequence of sfGFP-11 is shown in SEQ ID NO.17.

[0063] The above five recombinant plasmids were transformed into E. coli BL21 (DE3) to obtain recombinant strains. The recombinant strains were cultured at 37°C for 2-3 hours until OD 600 The final concentration of IPTG was 0.6-0.8, and the final concentration of IPTG was 0.5 mM. The cells were cultured at 25 °C for 24 h. 200 ul of the cells were added to a 96-well plate and the biomass (wavelength 600 nm) and fluorescence value (excitation wavelength 488 nm, emission wavelength 520 nm) were measured using a microplate reader. The results showed that the fluorescence intensity of ferredoxin reductase PetH and ferredoxin PetF from Synechocystis sp. PCC 6803 was the highest, reaching 3.0×10 6, which is 5.3 times that of the ferredoxin reductase CamA and ferredoxin CamB from Pseudomonas putida. The above recombinant plasmid pRSFDuet-PetH-PetF-OleP was then transformed into the E. coli expression host E. coli C41 (DE3) to obtain a recombinant strain named E. coli CO. This strain has the highest catalytic efficiency, and the conversion rate of murine deoxycholic acid increased from 6.4% to 32.3%. Therefore, the most suitable redox partner PetH / PetF for the P450 enzyme OleP was screened, and a recombinant strain E. coli CO was obtained.

[0064] Example 2: Establishment of a high-throughput detection method for lithocholic acid and murodeoxycholic acid

[0065] In Example 1, it is found that when Escherichia coli whole cell catalyst and lithocholic acid are reacted to prepare murodeoxycholic acid, thin layer chromatography and high performance liquid chromatography are needed to detect. Although thin layer chromatography can quickly detect lithocholic acid and murodeoxycholic acid, the specific content of lithocholic acid and murodeoxycholic acid cannot be measured, and the purpose of quantitative detection cannot be reached. Although high performance liquid chromatography can accurately detect the content of lithocholic acid and murodeoxycholic acid, 30min is needed to detect a sample, and the purpose of efficient screening cannot be reached. Therefore, the high throughput detection method of lithocholic acid and murodeoxycholic acid is a key factor in the synthesis of murodeoxycholic acid.

[0066] Dissolve lithocholic acid and murodeoxycholic acid in methanol to obtain 10 mg / mL lithocholic acid and murodeoxycholic acid mother solutions. Take 100uL of lithocholic acid and murodeoxycholic acid mother solutions and add them to 900uL of methanol to obtain 1mg / mL lithocholic acid and murodeoxycholic acid solutions. According to literature reports, prepare 6 chemical reagents that may react specifically with murodeoxycholic acid, including 3M sulfuric acid solution, 1M potassium hydroxide solution, 1.5M sodium hydroxide solution, 1% ferric chloride-ethanol solution (ferric chloride is dissolved in 100mL ethanol at a concentration of 1g / 100mL), 75% sulfuric acid-ethanol solution (sulfuric acid and ethanol are mixed in a volume ratio of 3:1), and cerium molybdate solution. Take 50uL of the prepared 1mg / mL lithocholic acid and murine deoxycholic acid solutions, add them to 150uL of the prepared 6 chemical reagents, and get 6 experimental groups of reaction systems, and place them in the microplate reader for reaction for 30min (temperature set to 25℃, speed set to 800rpm / min). Then set the detection wavelength of the microplate reader to 230nm to 990nm, and scan and record the full wavelength of the reaction solution. The results are as follows Figure 3As shown, when 75% sulfuric acid-ethanol solution is used as a reaction reagent, there is no characteristic absorption peak after the reaction of lithocholic acid, while there is characteristic absorption at 370nm after the reaction of murodeoxycholic acid. And a mixture of lithocholic acid and murodeoxycholic acid is reacted with 75% sulfuric acid-ethanol solution. In the presence of lithocholic acid, the mixture is not disturbed and still has characteristic absorption at 370nm. Therefore, it is screened that 75% sulfuric acid-ethanol solution can form a detectable substance with a characteristic absorption wavelength with murodeoxycholic acid in a short time, which can be used to detect the content of murodeoxycholic acid.

[0067] Further, the above-prepared 10 mg / mL murine deoxycholic acid mother solution was added to different volumes of methanol to obtain standard solutions of murine deoxycholic acid of different concentrations (0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 mg / mL). Take 50uL of the prepared standard solutions of murine deoxycholic acid of different concentrations and add them to 150uL of the prepared 75% sulfuric acid-ethanol solution to obtain 8 experimental groups of reaction systems, and place them in an ELISA reader for reaction for 30 minutes (the temperature was set to 25°C and the speed was 800rpm / min). Then set the detection wavelength of the ELISA reader to 370nm, and scan and record the absorbance value of the reaction solution. The results are as follows: Figure 3 As shown, a series of known concentrations of murine deoxycholic acid standard solutions were used for detection, and the A corresponding to each concentration of the standard solution was measured. 370nm The absorbance value of murodeoxycholic acid is obtained to obtain the standard curve equation between the concentration and absorbance value of murodeoxycholic acid. The standard curve equation is obtained as follows: y = 0.6475*x + 0.2545, correlation R 2 Reached 0.993. Moreover, after the reaction of murodeoxycholic acid with 75% sulfuric acid-ethanol solution, a yellow substance was generated. The higher the concentration of murodeoxycholic acid, the darker the yellow color, indicating that the content of murodeoxycholic acid is positively correlated with the color. In order to further verify the accuracy of this method, 48 samples to be tested were taken and the content of murodeoxycholic acid was detected by high-throughput method and high-performance liquid chromatography. The results are as follows Figure 3 As shown, only 3 of the 48 samples had A of murine deoxycholic acid. 370nm There is no correlation between absorbance and peak area. 370nm There is a correlation between the absorbance value and the peak area. The higher the peak area, the higher the A 370nm The higher the absorbance value, the higher the colorimetric value. Therefore, the accuracy of the colorimetric method based on the characteristic absorption wavelength was 93%, which can be used as a method for high-throughput detection of lithocholic acid and murodeoxycholic acid.

[0068] After the whole-cell catalyst prepared in Example 1 is reacted at 25°C in a whole-cell catalytic reaction system (the reaction system is the same as Example 1) for at least 8 hours, an appropriate amount of the reaction solution is taken out and an equal volume of ethyl acetate is added for extraction. After 3 extractions and separations, 200uL of methanol is used to redissolve to obtain the enzymatic reaction solution to be detected. Next, 50uL of the reaction solution is taken and detected using the high-throughput method established above, and the measured absorbance value is substituted into the standard curve equation to calculate the content of murodeoxycholic acid in the sample. Samples with high murodeoxycholic acid content are quickly screened out, and then the remaining 150uL of the reaction solution is filtered through a 0.22μm microporous filter membrane to obtain a filtrate, and the filtrate is used as a sample to be tested, and the specific content of lithocholic acid and murodeoxycholic acid is further detected using high performance liquid chromatography.

[0069] The accuracy of the high-throughput method established by the present invention reaches 93%. It only takes 30 minutes to simultaneously detect the contents of lithocholic acid and murideoxycholic acid in 96 samples using a 96-well plate, which significantly improves the detection efficiency of the samples.

[0070] Example 3: Semi-rational design of ferredoxin PetF to improve the ability to catalyze the production of murine deoxycholic acid

[0071] In order to further enhance the interaction between ferredoxin and P450 enzymes, thereby improving the catalytic efficiency of P450 enzymes, ferredoxin PetF was modified to improve its ability to catalyze the preparation of murine deoxycholic acid. Figure 4 As shown, a semi-rational design was performed on ferredoxin PetF to construct a mutant library containing 48 amino acid sites. The high-throughput method constructed in Example 2 was used as a method for detecting murine deoxycholic acid and applied to high-throughput detection of samples, thereby quickly screening for advantageous mutants.

[0072] like Figure 5As shown, the AlphaFold Multimer software was first used to dock the ferredoxin PetF and the P450 enzyme OleP to obtain the PetF-OleP protein-protein complex, and then the surface interaction force of the complex was analyzed. The PetF sequence was BLAST-aligned in the NCBI database to find the most similar ferredoxin Fd I. The crystal structure of Fd I reported in the PDB database and its annotated functional information were analyzed, and the PetF sequence was divided into 5 protein-protein interaction regions, containing a total of 48 amino acid sites. Among them, L36, P37, C40, G43, C45, S46, T47, C48, T77, and C78 are located in the catalytic region of PetF, and these 10 amino acid sites need to be retained. The PetF sequence was analyzed in the ConSurf software, and it was found that 17 amino acid sites were highly conserved in evolution, and these amino acid sites also need to be retained. The remaining 21 amino acid sites were virtually mutated to alanine using FoldX software, and the relative binding free energy ΔΔG of the protein complex was calculated, and the 7 amino acid sites with the largest scores were selected as the key amino acids with the largest mutation effects. Next, the 7 amino acid sites were virtually saturated mutated using FoldX software to obtain 133 mutants, and the relative binding free energy ΔΔG of the protein complex was calculated. Finally, the 23 mutants with the highest scores (D21Y, D21F, D21W, D58E, D58W, D58Y, D58F, D61V, D61R D61I, D61T, Q62W, Q62Y, Q62F, Q62E, F64P, F64I, F64D, D67W, D67F, D67F, D67W, D68P) were selected as the key mutants with improved interaction after mutation. Taking the nucleotide sequence encoding PetF as the starting sequence (as shown in SEQ ID NO.2), 23 pairs of primers were designed (primer sequences are shown in Table 2) to construct 23 recombinant plasmids containing mutations pRSFDuet-PetH-PetF mut -OleP, and transformed into E. coli C41 (DE3), respectively, and cultured at 37°C for 2-3 h until OD 600 The final concentration of IPTG was 0.6-0.8, and the final concentration of IPTG was 0.5 mM. The cells were cultured at 25°C for 30 h to induce expression. The fermentation conditions were the same as those in Example 1, and the whole cell catalyst was prepared according to the method in Example 1, and the whole cell catalytic reaction was performed. The reaction system of the whole cell catalysis was (calculated by final concentration): strain cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. React at 25°C for 8 h.

[0073] The results are as follows Figure 5As shown in Figure 2, the catalytic efficiency of 8 mutants among the 23 mutants was improved. Compared with the wild-type recombinant strain E. coli CO, the mutant PetF F64D 、PetF F64P 、PetF Q62E The catalytic efficiency of the mutant PetF F64D The catalytic efficiency of OleP-PetF was improved the most, with the conversion rate increased from 32.3% to 80.9%. In addition, the electron transfer rate can be calculated by combining 100ns molecular dynamics simulation and VMD's "pathway" plug-in. The electron transfer rate of OleP-PetF is 2.8×10 -5 ,OleP-PetF F64D The electron transfer rate is 8.1×10 -5 , which is 2.9 times that of the wild-type OleP-PetF. Therefore, the plasmid pRSFDuet-PetH-PetF carrying mutants with significantly better catalytic efficiency than other proteins was screened. F64D -OleP, and the recombinant strain E. coli F64D containing the plasmid.

[0074] Table 2 Primer sequences for designing mutants

[0075]

[0076]

[0077] Example 4: Co-expression of the ferredoxin mutant PetF F64D and the P450 enzyme mutant OleP S240A Improve the ability of catalysis to produce murine deoxycholic acid

[0078] Design primers as follows:

[0079] S240A-F: ATGGGCGTTgcaCTGCTGATTGCGGGTCACGAAAC;

[0080] S240A-R: GCAATCAGCAGtgcAACGCCCATGTTCACGATCTCG

[0081] Using the pRSFDuet-PetH-PetF-OleP plasmid constructed in Example 1 as the starting sequence, a site-directed alanine mutation was performed on the 240th position of the sequence of the P450 enzyme OleP shown in SEQ ID NO.15 to obtain pRSFDuet-PetH-PetF-OleP S240A Based on the optimal ferredoxin mutant PetF obtained in Example 3 F64DReferring to the method of constructing pRSFDuet-PetH-PetF-OleP in Example 1, a plasmid pRSFDuet-PetH-PetF expressing a double mutant was constructed. F64D -OleP S240A , and recombinant bacteria containing the plasmid.

[0082] The fermentation conditions of the recombinant bacteria were the same as those in Example 1, and the whole cell catalyst was prepared according to the method in Example 1 to carry out the whole cell catalytic reaction. The reaction system of the whole cell catalysis was (based on the final concentration): strain cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. React at 25°C for 8 h. The results showed that co-expression of PetF F64D and OleP S240A The catalytic efficiency can be further improved, and the conversion rate is increased from 80.9% in Example 3 to 93.5%.

[0083] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A ferredoxin mutant, characterized in that Taking the amino acid sequence shown in SEQ ID NO.11 as the starting sequence, mutate the glutamine at position 62 to glutamic acid; or The phenylalanine at position 64 was mutated to aspartic acid or proline.

2. Encoding the ferredoxin mutant PetF according to claim 1 F64D genes.

3. A recombinant plasmid carrying the gene according to claim 2.

4. A recombinant microorganism expressing the ferredoxin mutant according to claim 1.

5. A recombinant Escherichia coli, characterized in that pRSFDuet-1 is used as an expression vector to express the cytochrome P450 enzyme OleP gene from Streptomyces antibioticus, the ferredoxin reductase gene PetH from Synechocystis sp. PCC 6803, and the ferredoxin PetF; the ferredoxin PetF has an amino acid sequence shown in any one of SEQ ID NOs. 11 to 14.

6. A cell catalyst comprising the recombinant microorganism according to claim 4 or the recombinant Escherichia coli according to claim 5.

7. A method for whole-cell catalytic lithocholic acid hydroxylation, characterized in that: The recombinant Escherichia coli according to claim 5 is used as a whole-cell catalyst to catalyze the preparation of murideoxycholic acid using lithocholic acid as a substrate.

8. The method according to claim 7, characterized in that The catalysis was carried out in a 50-100 mM potassium phosphate buffer system, and the biomass OD of the recombinant Escherichia coli was 600 is 10-35, the concentration of lithocholic acid is 0.1-0.5 mg / mL, and the reaction temperature is 20-37°C.

9. A method for improving the catalytic efficiency of P450 enzymes, characterized in that: The P450 enzyme OleP is co-expressed with a redox chaperone protein; the redox chaperone protein includes a ferredoxin reductase PetH and a ferredoxin PetF; the ferredoxin reductase PetH has the amino acids shown in UniProtKB: Q55318.2; the ferredoxin PetF has the amino acid sequence shown in any one of SEQID NO.11 to 14; optionally, the amino acid sequence of the P450 enzyme is shown in SEQ ID NO.15 or SEQ ID NO.

18.

10. A method for high-throughput detection of lithocholic acid and murideoxycholic acid, characterized in that: The sample to be tested is mixed with 75% sulfuric acid-ethanol, reacted at 20-30° C. for 30-60 minutes, and then the absorbance value is detected at 370-380 nm; optionally, the murideoxycholic acid content is calculated according to y=0.6475*x+0.2545.

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