Ferroreductin mutants and their application in mouse deoxycholic acid synthesis

Abstract

This invention discloses a ferrugin mutant and its application in the synthesis of mouse deoxycholic acid, belonging to the field of biocatalytic enzyme technology. This invention uses *E. coli* as the chassis strain to express the P450 enzyme OleP and establishes a three-component redox chaperone library, screening for the most suitable redox chaperones PetH / PetF for OleP. A high-throughput method based on characteristic absorbance values ​​is developed, enabling rapid and efficient detection of lithocholic acid and mouse deoxycholic acid. Furthermore, through semi-rational design of ferrugin proteins, a ferrugin mutant, PetF, is screened. F64D This method can significantly improve the conversion rate of catalytic preparation of mouse deoxycholic acid. It provides a new strategy for improving the biosynthesis of hydroxylated steroidal substances.

Description

Technical Field

[0001] This invention relates to ferricoxin mutants and their application in the synthesis of deoxycholic acid in mice, belonging to the field of biocatalytic enzyme technology. Background Technology

[0002] Steroid compounds play important biological functions in cells and have been widely used in medicine. Steroid drugs are the second largest pharmaceutical product after antibiotics, with approximately 300 steroid drugs currently approved for clinical use. Bile acid steroids are essential substances for lipid digestion and absorption. Lithocholic acid is a widely available and inexpensive secondary bile acid. Currently, lithocholic acid is used to study drug metabolism pathways and as a starting material for the synthesis of clinical drugs. Mouse deoxycholic acid is a 6β-hydroxylated derivative of lithocholic acid. Mouse deoxycholic acid can dissolve gallstones and is used to treat cholesterol and liver diseases. Currently, due to the complex production process and low preparation efficiency, the price of mouse deoxycholic acid is as high as 1200 yuan / mg.

[0003] Compared to chemical methods of hydroxylation, enzymatic catalysis of hydroxylation is a more efficient and environmentally friendly method, offering advantages such as mild reaction conditions, high catalytic efficiency, and strong selectivity, thus overcoming the shortcomings of chemical synthesis. Cytochrome P450 enzymes are heme-based monooxygenases, considered highly promising biocatalysts due to their selective oxidation of inert carbon-hydrogen bonds. In recent years, the use of P450 enzymes to catalyze the hydroxylation of steroid drugs has attracted increasing attention. Efficient heterologous expression of P450 enzymes (Baodong, Hu., ADVANCED SCIENCE, 10, 2205580. 2022), increasing the supply of heme prosthetic group (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) can improve the efficiency of P450 enzymes in preparing hydroxylated steroid drugs. However, the problem of mismatch between P450 enzymes and redox chaperones remains; the low electron transfer efficiency of redox chaperones leads to low catalytic efficiency of P450 enzymes. Therefore, modifying redox chaperones to improve electron transfer efficiency, thereby enhancing the method of P450 enzyme-catalyzed synthesis of mouse deoxycholic acid, is of great significance. Summary of the Invention

[0004] This invention provides a ferricoxin mutant and its application in the synthesis of mouse deoxycholic acid, in order to solve the technical problem of low lithocholic acid hydroxylation efficiency caused by the mismatch between the redox chaperone and the P450 enzyme.

[0005] This invention provides a ferricoxine mutant, which is based on the amino acid sequence shown in SEQ ID NO.11, by mutating glutamine at position 62 to glutamic acid; or by mutating phenylalanine at position 64 to aspartic acid or proline.

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

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

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

[0009] The present invention also provides encoding the ferricopentaprotein mutant PetF. F64D The genes.

[0010] The present invention also provides a recombinant plasmid carrying the said 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 ferroredoxin reductase gene PetH from Synechocystis sp. PCC 6803.

[0013] The present invention also provides expression of the ferricoxin mutant PetF. F64D Recombinant microorganisms.

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

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

[0016] The present invention also provides a method for improving the catalytic efficiency of P450 enzymes, which is to co-express the P450 enzyme OleP with redox chaperone proteins; the redox chaperone proteins include ferric reductase PetH and ferric reductase PetF.

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

[0018] In one embodiment, the ferroredoxin PetF has any of the amino acid sequences shown in SEQ ID NO. 11 to 14.

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

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

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

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

[0023] This invention also provides a high-throughput method for detecting lithocholic acid and deoxycholic acid, wherein the sample to be tested is mixed with 75% sulfuric acid-ethanol, reacted at 20-30°C for 30-60 min, and then the absorbance value is detected at 370-380 nm; wherein the 75% sulfuric acid-ethanol is a mixture of sulfuric acid and ethanol in a volume ratio of 3:1, which can react with bile acids containing hydroxyl groups to produce a colorimetric reaction, and the product after the reaction has a characteristic absorption peak at 370-380 nm.

[0024] In one implementation, the mouse deoxycholic acid content is calculated based on y = 0.6475*x + 0.2545.

[0025] In one embodiment, the high-throughput detection method is as follows: Lithocholic acid is added to the whole-cell catalyst for reaction. After the reaction, the mixture is centrifuged, the reaction solution is collected, and extracted three times with an equal volume of ethyl acetate. The solution is dried using a nitrogen blower and then dissolved in 200 μL of methanol to obtain the enzymatic reaction solution. 50 μL of the enzymatic reaction solution is added to 150 μL of 75% sulfuric acid ethanol for color development for 5–30 min, and the absorbance is measured at the characteristic absorption wavelength. A sample of the whole-cell catalyst without added P450 enzyme is used as a negative control; the greater the difference in absorbance between the sample and the negative control, the higher the content of mouse deoxycholic acid.

[0026] In one embodiment, the whole-cell catalyst is prepared by culturing genetically engineered bacteria at 35-37°C to OD0.05. 600 The value was 0.6-0.8. After adding 0.1-1mM isopropyl-β-D-thiogalactoside, the cells were cultured at 16-30℃ for 16-24h. The cells were then centrifuged at low temperature, collected, and washed with potassium phosphate buffer at pH 8.0. After washing, the cells were resuspended in potassium phosphate buffer (5%-10% v / v glycerol) to obtain whole-cell catalyst.

[0027] In one embodiment, the whole-cell catalytic reaction catalyzing the 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 [not specified]. 600 The concentration of lithocholic acid was 0.1-1.0 mg / mL, the reaction temperature was 20-37℃, and the reaction time was 6-24 h.

[0028] Beneficial effects:

[0029] (1) This invention screened 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 protein complexes, and whole-cell catalysis. The conversion rate of catalytic preparation of mouse deoxycholic acid was increased from 6.4% to 32.3%.

[0030] (2) This invention designs a high-throughput method for detecting lithocholic acid and deoxycholic acid. The content of lithocholic acid and deoxycholic acid can be characterized by reacting 50 μL of enzyme-catalyzed reaction solution with 150 μL of 75% sulfuric acid in ethanol for 30 min. Compared to the 30 min required for liquid chromatography to detect one sample, this method can simultaneously detect 96 samples in a total of only 30 min.

[0031] (3) A mutant library was constructed by analyzing the structure of ferricoxin PetF, and mutants that could improve electron transport efficiency were screened. Ultimately, an optimal mutant, PetF, was selected. F64D This improved electron transfer efficiency, increasing the conversion rate of catalytic preparation of mouse deoxycholic acid from 32.3% to 80.9%.

[0032] (4) This invention constructs the mutant OleP by mutating the P450 enzyme. S240A and the P450 enzyme mutant OleP S240A and the aforementioned ferricyanide mutant PetF F64D Co-expression, used for the catalytic preparation of mouse deoxycholic acid, can increase the conversion rate to 93.5%. Attached Figure Description

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

[0034] Figure 2 To screen for the optimal redox chaperone for the P450 enzyme OleP. A shows the distance between ferricoxin and the P450 enzyme calculated using Pymol; B shows the docking of the protein-protein complex using AlphaFold Multimer software to obtain the complex structure of the P450 enzyme and ferricoxin; C shows the screening of the optimal ferricoxin for the P450 enzyme OleP, with blue representing the fluorescence intensity of the strain and red representing the conversion rate catalyzed by the whole cell; D shows the observation of the fluorescence intensity of the strain.

[0035] Figure 3To establish a high-throughput method for the detection of lithocholic acid and deoxycholic acid. A) Screening chemical reagents that can specifically react with deoxycholic acid; B) Determining the characteristic detection wavelength of the product after the reaction; C) Establishing a concentration standard curve of deoxycholic acid at an absorbance of 370 nm; D) Comparing the established high-throughput detection method with a liquid chromatography method to verify the accuracy of the method.

[0036] Figure 4 A mutant library for semi-rational design of the ferroredoxin PetF was created. Pink boxes list key amino acid residues, and red boxes list the constructed mutants. Blue sites represent 17 evolutionarily conserved sites. Red sites represent 10 sites affecting the protein's catalytic activity. Blue boxes list the 7 amino acid sites for which mutations were ultimately selected.

[0037] Figure 5 To design ferroredoxin PetF based on semi-rational protein structure; A shows the structural alignment of PetF and ferroredoxin FdI, with red representing the structure of PetF and cyan representing the structure of FdI; B shows the amino acid sequence alignment of PetF and FdI, with green underlines representing regions on PetF that interact with OleP; C analyzes the evolutionary conservation of the amino acid sequence of PetF, with redder colors and higher values ​​indicating more conserved amino acid sequences; D shows the relative binding free energy calculated using FoldX for mutations at 23 sites in PetF to alanine; E shows the relative binding free energy calculated using FoldX for saturation mutations at 7 sites in PetF; F shows the fluorescence intensity and conversion rate of the constructed 23-mutant strain to mouse deoxycholic acid. Blue represents the fluorescence intensity of the strain, and red represents the conversion rate catalyzed by the whole cell. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0039] (a) 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 dipotassium 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 BioBio Biotech Co., Ltd.

[0045] (III) Whole-cell catalysis:

[0046] (1) The recombinant Escherichia coli was cultured at 35-37℃ until OD 600 The concentration was 0.6-0.8. After adding isopropyl-β-D-thiogalactoside, the mixture was cultured at 16-30℃ for 12-20 h. The concentration of isopropyl-β-D-thiogalactoside was 0.1-1 mM.

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

[0048] (3) The bacterial suspension obtained in step (2) was used to carry out the hydroxylation reaction of lithocholic acid. The whole-cell reaction system included (based on final concentration): bacterial cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. Whole-cell reaction was carried out at 20-37℃ for at least 8-16 h. After the reaction, an appropriate amount of the reaction solution was taken and extracted with an equal volume of ethyl acetate. After three extractions and separations, mouse deoxycholic acid was obtained and analyzed using a high-throughput detection method (75% sulfuric acid-ethanol solution) and high-performance liquid chromatography.

[0049] (iv) Testing methods:

[0050] High performance liquid chromatography 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: ZORBAX Eclipse XDB-C18 reverse-phase column (5μm, 4.6×250mm, Agilent, USA); column temperature: 40℃; flow rate: 0.8mL / min; elution program: 0-1min, 10% B; 1-10min, 10%-40% B; 10-20min, 40%-90% B; 20-23min, 90% B; 23-25min, 90%-10% B; 25-27min, 10% B.

[0053] (3) Use an evaporative light scattering detector, nitrogen 350 kPa, gain 6, and drift tube temperature 40℃.

[0054] Conversion rate calculation: Amount of product (mol) / Amount of initial added substrate (mol) × 100%.

[0055] Example 1: Construction of a three-component redox chaperone library and screening of redox chaperones most compatible with the P450 enzyme OleP.

[0056] The P450 enzyme CYP107D1 (UniProtKB: Q59819), abbreviated as OleP, derived from Streptomyces antibioticus, was synthesized by adding a histidine tag to the C-terminus through codon optimization in E. coli (nucleotide sequence shown in SEQ ID NO.1). This gene was then subcloned into the restriction enzyme sites NdeI and XhoI of plasmid pRSFDuet to obtain the recombinant plasmid pRSFDuet-OleP. Since OleP is a typical three-component P450 enzyme, requiring redox chaperone proteins to transfer electrons from the electron donor NAD(P)H to the active site of the P450 enzyme, ferredoxin reductase CamA (amino acid sequence shown in Genbank: BAA00413.1) and ferredoxin CamB (amino acid sequence shown in UniProtKB: P00259.3), derived from Pseudomonas putida, were selected. The codon-optimized ferroreductase gene CamA (SEQ ID NO.4) and ferroreductin gene CamB (SEQ ID NO.5) were synthesized and subcloned into the restriction enzyme sites between NcoI and SalI in plasmid pRSFDuet-OleP to obtain the recombinant plasmid pRSFDuet-CamA-CamB-OleP. These 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] Recombinant bacteria E. coli O and E. coli OAB were cultured in TB medium at 37°C for 2–3 hours until OD (digestion occurred). 600 The concentration of the bacterial cells was 0.6–0.8. IPTG was added to a final concentration of 0.5 mM, and the cells were incubated at 25°C for 30 h to induce expression. The cells were then washed with potassium phosphate buffer (pH 8.0) to prepare the whole-cell catalyst. The reaction system for whole-cell catalysis (based on final concentration) was: bacterial cell OD... 600 =30, lithocholic acid 0.5mg / mL, NADH 1mM, glucose dehydrogenase 1U / mL.

[0058] The whole-cell reaction was carried out at 25°C for 8 hours, and the results were as follows: Figure 1 As shown, the recombinant strain E. coli O could not catalyze the conversion of lithocholic acid to mouse deoxycholic acid, while the recombinant strain E. coli OAB could catalyze the conversion of lithocholic acid to 0.032 mg / mL mouse deoxycholic acid (conversion rate 6.4%). Therefore, redox chaperones are very important for the catalysis of OleP, and it is necessary to screen for the redox chaperones that are most compatible with OleP to improve the efficiency of catalytic preparation of mouse deoxycholic acid.

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

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

[0061]

[0062] Using AlphaFold Multimer software, 12 ferricoxins were subjected to protein-protein docking with the P450 enzyme OleP. The optimal conformation of the complex was determined based on the scoring function and orientation score. Based on the structure of the optimal conformation, the distance between the ferricoxins and OleP was calculated using Pymol software (a shorter distance indicates higher electron transfer efficiency). The results are as follows: Figure 2As shown, the distances between ferroredo proteins PetF, CamB, FldA, FldB, and Fdx and OleP are relatively short. Therefore, five suitable redox chaperone pairs were screened from the redox chaperone library. A fluorescent sensor plasmid was constructed and expressed in *E. coli* BL21(DE3). The sensor plasmid construction method involves fusing five ferrooxidase proteins to the N-terminus of sfGFP-1-10 and OleP to the C-terminus of sfGFP-11, respectively, to construct 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 (underlined genes represent fusion genes). 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.6–8, 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 five recombinant plasmids were transformed into E. coli BL21(DE3) to obtain recombinant strains. The recombinant strains were then cultured at 37℃ for 2–3 hours until OD (digestion). 600 The concentration of the culture medium was 0.6–0.8, and IPTG was added to a final concentration of 0.5 mM. The culture was then incubated at 25 °C for 24 h. 200 μL of the bacterial cells were added to a 96-well plate, and the biomass (wavelength 600 nm) and fluorescence intensity (excitation wavelength 488 nm, emission wavelength 520 nm) were measured using a microplate reader. The results showed that the ferredoxin reductase PetH and ferredoxin PetF from *Synechocystis* sp. PCC 6803 exhibited the highest fluorescence intensity, reaching 3.0 × 10⁻⁶. 6The recombinant plasmid pRSFDuet-PetH-PetF-OleP was 5.3 times more potent than that of ferroredoxin reductases CamA and CamB from *Pseudomonas putida*. The 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 exhibited the highest catalytic efficiency, increasing the conversion rate of mouse deoxycholic acid from 6.4% to 32.3%. Therefore, the optimal redox chaperones PetH / PetF for the P450 enzyme OleP were selected, resulting in the recombinant strain *E. coli* CO.

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

[0065] In Example 1, it was found that when preparing deoxycholic acid from mice using the reaction of whole-cell E. coli with lithocholic acid, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are required for detection. While TLC can rapidly detect lithocholic acid and deoxycholic acid, it cannot measure their specific content, thus failing to achieve quantitative detection. HPLC, while accurately detecting their content, requires 30 minutes to analyze a single sample, making efficient screening impossible. Therefore, high-throughput detection of lithocholic acid and deoxycholic acid is crucial for the synthesis of deoxycholic acid from mice.

[0066] Lithocholic acid and deoxycholic acid were dissolved in methanol to obtain a 10 mg / mL stock solution of lithocholic acid and deoxycholic acid. 100 μL of the stock solution was added to 900 μL of methanol to obtain a 1 mg / mL solution of lithocholic acid and deoxycholic acid. According to literature reports, six chemical reagents that may specifically react with deoxycholic acid were prepared, including 3M sulfuric acid solution, 1M potassium hydroxide solution, 1.5M sodium hydroxide solution, 1% ferric chloride-ethanol solution (ferric chloride dissolved in 100 mL of ethanol at a concentration of 1 g / 100 mL), 75% sulfuric acid-ethanol solution (sulfuric acid and ethanol mixed at a volume ratio of 3:1), and cerium molybdate solution. Take 50 μL of the prepared 1 mg / mL lithocholic acid and mouse deoxycholic acid solutions, and add them separately to 150 μL of the prepared six chemical reagents to obtain six experimental reaction systems. Place each system in a microplate reader for 30 min (temperature set at 25℃, rotation speed at 800 rpm / min). Then, set the detection wavelength of the microplate reader to 230 nm to 990 nm and scan and record the full wavelength range of the reaction solution. The results are as follows: Figure 3As shown, when using 75% sulfuric acid-ethanol solution as the reaction reagent, lithocholic acid showed no characteristic absorption peak after the reaction, while deoxycholic acid showed a characteristic absorption at 370 nm. Furthermore, when a mixture of lithocholic acid and deoxycholic acid was reacted with 75% sulfuric acid-ethanol solution, the mixture still showed a characteristic absorption at 370 nm without interference in the presence of lithocholic acid. Therefore, it was found that 75% sulfuric acid-ethanol solution can react with deoxycholic acid to form a detectable substance with a characteristic absorption wavelength within a short time, which can be used to detect the content of deoxycholic acid.

[0067] Further, the prepared 10 mg / mL mouse deoxycholic acid stock solution was added to different volumes of methanol to obtain mouse deoxycholic acid standard solutions of different concentrations (0.0156, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2 mg / mL). 50 μL of each of the prepared mouse deoxycholic acid standard solutions was added to 150 μL of a prepared 75% sulfuric acid-ethanol solution to obtain eight experimental reaction systems. These systems were then placed in an ELISA reader and reacted for 30 min (temperature set to 25℃, rotation speed 800 rpm / min). The detection wavelength of the ELISA reader was then set to 370 nm, and the absorbance of the reaction solution was scanned and recorded. The results are as follows: Figure 3 As shown, a series of mouse deoxycholic acid standard solutions of known concentrations were used for detection, and the corresponding A values ​​for each concentration of standard solution were measured. 370nm The absorbance values ​​were used to obtain a standard curve equation relating the concentration of mouse deoxycholic acid to its absorbance values. The standard curve equation was obtained as follows: y = 0.6475*x + 0.2545, with a correlation R0. 2 The concentration reached 0.993. Furthermore, the reaction of mouse deoxycholic acid with 75% sulfuric acid-ethanol solution produced a yellow substance; the higher the concentration of mouse deoxycholic acid, the deeper the yellow color, indicating a positive correlation between the content and color of mouse deoxycholic acid. To further verify the accuracy of this method, 48 samples were tested simultaneously using both high-throughput methods and high-performance liquid chromatography to determine the content of mouse deoxycholic acid. The results are as follows... Figure 3 As shown, only 3 out of 48 samples contained A-type mouse deoxycholic acid. 370nm There was no correlation between absorbance and peak area; the A value of mouse deoxycholic acid in the other samples was... 370nm Absorbance and peak area are correlated; the higher the peak area, the higher the absorbance. 370nm The higher the absorbance, the higher the accuracy of the established colorimetric method based on characteristic absorption wavelengths, reaching 93%. This method can be used for high-throughput detection of lithocholic acid and deoxycholic acid.

[0068] The whole-cell catalyst prepared in Example 1 was reacted in a whole-cell catalytic reaction system (the reaction system was the same as in Example 1) at 25°C for at least 8 hours. An appropriate amount of the reaction solution was then extracted with an equal volume of ethyl acetate. After three extractions and separations, the solution was redissolved in 200 μL of methanol to obtain the enzyme-catalyzed reaction solution to be tested. Next, 50 μL of the reaction solution was analyzed using the established high-throughput method. The measured absorbance was substituted into the standard curve equation to calculate the content of mouse deoxycholic acid in the sample. Samples with high mouse deoxycholic acid content were quickly screened. Then, the remaining 150 μL of the reaction solution was filtered through a 0.22 μm microporous membrane to obtain the filtrate. This filtrate was used as the sample to be tested, and the specific contents of lithocholic acid and mouse deoxycholic acid were further determined using high-performance liquid chromatography (HPLC).

[0069] The high-throughput method established in this invention achieves an accuracy of 93%. Using a 96-well plate, the content of lithocholic acid and deoxycholic acid in 96 samples can be detected simultaneously in just 30 minutes, significantly improving the sample detection efficiency.

[0070] Example 3: Semi-rational design of ferricoxin PetF improved the catalytic ability to prepare mouse deoxycholic acid.

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

[0072] like Figure 5As shown, the ferroredoxin PetF and the P450 enzyme OleP were first docked using AlphaFold Multimer software to obtain the PetF-OleP protein complex, and then the surface interaction forces of the complex were analyzed. The PetF sequence was BLAST-aligned in the NCBI database to find the most similar ferroredoxin, FdI. The crystal structure and functional information of FdI reported in the PDB database were analyzed, dividing the PetF sequence into five protein-protein interaction regions containing a total of 48 amino acid sites. Among these, 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 preserved. Analysis of the PetF sequence using ConSurf software revealed that 17 amino acid sites are evolutionarily highly conserved and also need to be preserved. 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. The seven amino acid sites with the highest scores were selected as key amino acids with significant mutation effects. Next, these seven amino acid sites were subjected to virtual saturation mutations using FoldX software, resulting in 133 mutants. The relative binding free energy ΔΔG of the protein complex was calculated again, and 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 key mutants with enhanced interaction forces after mutation. Using the nucleotide sequence encoding PetF as the starting sequence (as shown in SEQ ID NO.2), 23 recombinant plasmids containing mutations, pRSFDuet-PetH-PetF, were constructed by designing 23 pairs of primers (primer sequences are shown in Table 2). mut -OleP was then transformed into E. coli C41(DE3) and cultured at 37°C for 2–3 hours until OD600 was achieved. 600 The concentration of IPTG was 0.6–0.8, and a final concentration of 0.5 mM was added. The mixture was incubated at 25°C for 30 h to induce expression. Fermentation conditions were the same as in Example 1, and the whole-cell catalyst was prepared according to the method in Example 1, followed by whole-cell catalysis. The whole-cell catalytic reaction system was as follows (based on final concentration): [Dose of strain cells OD] 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. React at 25℃ for 8 h.

[0073] The results are as follows Figure 5As shown, 8 out of the 23 mutants exhibited improved catalytic efficiency. Compared to the wild-type recombinant strain E. coli CO, the mutant PetF... F64D PetF F64P PetF Q62E The catalytic efficiency was significantly improved, especially in the mutant PetF F64D The catalytic efficiency was significantly improved, with the conversion rate increasing from 32.3% to 80.9%. Furthermore, the electron transport rate could be calculated by combining 100 ns molecular dynamics simulations and the VMD "pathway" plugin. The electron transport rate of OleP-PetF was 2.8 × 10⁻⁶. -5 OleP-PetF F64D The electron transport rate is 8.1 × 10⁻⁶. -5 It is 2.9 times more potent than wild-type OleP-PetF. Therefore, the plasmid pRSFDuet-PetH-PetF, carrying a mutant with significantly higher catalytic efficiency than other proteins, was selected through screening. F64D -OleP, and the recombinant strain E. coli F64D containing this plasmid.

[0074] Table 2 Primer sequences for designing mutants

[0075]

[0076]

[0077] Example 4: Co-expression of the ferrugin mutant PetF F64D and the P450 enzyme mutant OleP S240A Improving the ability to catalyze the preparation of mouse deoxycholic acid

[0078] The primers are designed 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 at position 240 of the sequence of the P450 enzyme OleP shown in SEQ ID NO.15 to obtain pRSFDuet-PetH-PetF-OleP. S240A Furthermore, based on the optimal ferricyanide mutant PetF obtained in Example 3... F64DFollowing the method described in Example 1 for constructing pRSFDuet-PetH-PetF-OleP, the plasmid pRSFDuet-PetH-PetF expressing the double mutant was constructed. F64D -OleP S240A , and recombinant bacteria containing the plasmid.

[0082] The fermentation conditions for the recombinant bacteria were the same as in Example 1, and a whole-cell catalyst was prepared according to the method in Example 1 for whole-cell catalysis. The whole-cell catalytic reaction system was as follows (based on final concentration): bacterial cell OD 600 =30, lithocholic acid 0.5 mg / mL, NADH 1 mM, glucose dehydrogenase 1 U / mL. The reaction was carried out at 25℃ for 8 h. Results showed co-expression of PetF. F64D And OleP S240A This can further improve catalytic efficiency, increasing the conversion rate from 80.9% in Example 3 to 93.5%.

[0083] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An iron-oxido protein mutant characterized in that, the amino acid sequence shown as SEQ ID NO. 11 as a starting sequence, mutating the glutamine at position 62 into glutamic acid; or the phenylalanine at position 64 into aspartic acid or proline.

2. A gene encoding the ferredoxin mutant of claim 1.

3. A recombinant plasmid carrying the gene of claim 2.

4. A recombinant microorganism expressing the ferredoxin mutant of claim 1.

5. A recombinant E. coli bacterium, characterized in that, The cytochrome P450 OleP gene from Streptomyces sp. Streptomyces antibioticus and the ferredoxin reductase gene PetH from Synechocystis sp. Synechocystis PCC6803, and the ferredoxin mutant of claim 1.

6. A cell catalyst containing the recombinant microorganism of claim 4 or the recombinant Escherichia coli of claim 5.

7. A method for the hydroxylation of cholic acid catalyzed by whole cells, characterized in that, Using the recombinant Escherichia coli of claim 5 as a whole cell catalyst, catalyzing the preparation of muricholic acid from lithocholic acid.

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 [not specified]. 600 The concentration of lithocholic acid was 0.1-0.5 mg / mL, and the reaction temperature was 20-37℃.

9. A method of improving the catalytic efficiency of a P450 enzyme, characterized by, co-expressing the P450 enzyme OleP with an oxidoreductase partner; the oxidoreductase partner includes ferredoxin reductase PetH and the ferredoxin mutant of claim 1; the amino acid sequence of the ferredoxin reductase PetH is shown as UniProtKB: Q55318.2; the amino acid sequence of the ferredoxin mutant is shown as SEQ ID NO. 12 or SEQ ID NO. 13; the amino acid sequence of the P450 enzyme OleP is shown as SEQ ID NO. 15 or SEQ ID NO. 18.

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