Cyp11b1-bm3 fusion protein and application thereof

Abstract

The present application relates to a kind of CYP11B1-BM3 fusion protein, which is connected by connecting peptide by steroid 11 beta hydroxylase CYP11B1 and cytochrome P450 BM3 reduction domain.The encoding gene of the fusion protein is transformed into escherichia coli for transformation expression, can efficiently synthesize CYP11B1-BM3 fusion protein, and the prepared fusion protein has higher cytochrome P450 reductase activity and in vitro hydroxylation enzyme activity, can efficiently catalyze hydroxylation reaction, can be applied to the synthesis of cortisol drug.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and enzyme engineering technology, specifically relating to a CYP11B1-BM3 fusion protein and its applications. Background Technology

[0002] Hydrocortisone is an important corticosteroid drug in humans, obtained by oxidizing 11-deoxycortisol to a β-hydroxyl group at the 11-position via steroid 11β-hydroxylase CYP11B1. CYP11B1 is a member of the cytochrome P450 11B subfamily and exhibits regio- and stereoselectivity in the 11β-hydroxylation of steroid hormones. The hydroxylation reaction catalyzed by CYP11B1 requires the additional electron transfer from cytochrome P450 reductase. When CYP11B1 and cytochrome P450 reductase are expressed alone, their activity is poor due to low electron transfer efficiency.

[0003] In the industrial synthesis of corticosteroid drugs, the step of introducing 11-hydroxyl groups into steroid scaffolds is relatively expensive, and the formation of a large number of byproducts also leads to a waste of resources. Furthermore, the functional expression of human steroid 11β-hydroxylase CYP11B1 in bacteria has not yet been successfully carried out. Summary of the Invention

[0004] One of the objectives of this invention is to provide a CYP11B1-BM3 fusion protein that addresses the problems existing in the prior art. This fusion protein has high cytochrome P450 reductase activity and in vitro hydroxylation enzyme activity, enabling it to efficiently catalyze hydroxylation reactions and can be used in the synthesis of corticosteroid drugs.

[0005] The second objective of this invention is to provide a genetically engineered bacterium that produces the CYP11B1-BM3 fusion protein, which uses Escherichia coli as the host cell and can efficiently synthesize the CYP11B1-BM3 fusion protein.

[0006] Therefore, the first aspect of the present invention provides a CYP11B1-BM3 fusion protein, which is formed by linking a steroid 11β hydroxylase CYP11B1 and a cytochrome P450 BM3 reducing domain via a linker peptide; wherein the amino acid sequence of the steroid 11β hydroxylase CYP11B1 is shown in SEQ ID NO.1, and the amino acid sequence of the cytochrome P450 BM3 reducing domain is shown in SEQ ID NO.2.

[0007] In some embodiments of the present invention, the linker peptide is a rigid linker peptide or a flexible linker peptide; the rigid linker peptide includes linker peptide 2 and linker peptide 3; the flexible linker peptide includes linker peptide 1, linker peptide 4 and linker peptide 5; wherein the amino acid sequences of linker peptides 1 to 5 are as shown in SEQ ID NO. 5 to 9 respectively.

[0008] According to the present invention, the CYP11B1-BM3 fusion protein further comprises the following mutations in the amino acid sequence shown in SEQ ID NO.1: Ser at position 169 is mutated to Val, Phe at position 381 is mutated to Ala, and Leu at position 382 is mutated to Ser.

[0009] A second aspect of the present invention provides a gene encoding the CYP11B1-BM3 fusion protein as described in the first aspect of the present invention, which is composed of a codon-optimized steroid 11β-hydroxylase CYP11B1 gene, a linker peptide gene, and a codon-optimized P450 BM3 reduction domain gene linked in sequence; wherein the nucleotide sequence of the codon-optimized steroid 11β-hydroxylase CYP11B1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the codon-optimized P450 BM3 reduction domain gene is shown in SEQ ID NO.4.

[0010] In some embodiments of the present invention, the linker peptide genes include codon-optimized linker peptide 1 gene, codon-optimized linker peptide 2 gene, codon-optimized linker peptide 3 gene, codon-optimized linker peptide 4 gene, and codon-optimized linker peptide 5 gene; preferably, the nucleotide sequences of the codon-optimized linker peptide 1 to 5 genes are as shown in SEQ ID NO. 10 to 14 in sequence.

[0011] According to the present invention, the gene sequence encoding the CYP11B1-BM3 fusion protein further comprises, as shown in SEQ ID NO.3, a TCC mutation at position 504 (from 5′ to 3′) to GTA, a TTT mutation at position 1140 (from 1140 to 1143 to 1144 to 1145 to AGC mutation.

[0012] A third aspect of the present invention provides a genetically engineered bacterium that produces the CYP11B1-BM3 fusion protein as described in the first aspect of the present invention, which is a recombinant Escherichia coli expressing the gene encoding the CYP11B1-BM3 fusion protein as described in the second aspect of the present invention.

[0013] The fourth aspect of the present invention provides the use of the genetically engineered bacteria as described in the third aspect of the present invention in the preparation of CYP11B1-BM3 fusion protein.

[0014] The fifth aspect of the present invention provides the use of the CYP11B1-BM3 fusion protein as described in the first aspect of the present invention, or the gene-encoded CYP11B1-BM3 fusion protein as described in the second aspect of the present invention, or the CYP11B1-BM3 fusion protein prepared by the genetically engineered bacteria as described in the third aspect of the present invention, in the production of corticosteroid drugs.

[0015] This invention provides a CYP11B1-BM3 fusion protein, which is composed of the steroid 11β-hydroxylase CYP11B1 and the cytochrome P450 BM3 reducing domain linked by a linker peptide. This fusion protein exhibits high cytochrome P450 reductase activity and in vitro hydroxylation enzyme activity, enabling it to efficiently catalyze hydroxylation reactions and can be used in the synthesis of corticosteroid drugs. Transforming the gene encoding this fusion protein into *E. coli* for expression allows for the efficient synthesis of the CYP11B1-BM3 fusion protein, and the resulting fusion protein possesses high cytochrome P450 reductase activity and in vitro hydroxylation enzyme activity. Attached Figure Description

[0016] The present invention will now be described in further detail with reference to the accompanying drawings:

[0017] Figure 1 The construction of the CYP11B1-BM3 fusion protein expression vector is shown.

[0018] Figure 2 SDS-PAGE electrophoresis images of the mutant and wild-type CYP11B1-BM3 fusion protein; in the images, band 1 is the supernatant, band 2 is the precipitate, and band 3 is the purified protein.

[0019] Figure 3 The comparison results of the linker peptides of the CYP11B1-BM3 fusion protein are shown; among them, the amino acid sequence of linker peptide L1 is (GSG)4, and the amino acid sequences of linker peptides L2 to L5 are EAAAK, (EAAAK)3, GGGGS and (GGGGS)3, respectively.

[0020] Figure 4 This is an SDS-PAGE electrophoresis image of the CYP11B1-BM3 fusion protein; in the image, band M is the standard, band 1 is the supernatant, band 2 is the purified protein, and L2, L3, L4, and L5 are linker peptide 2, linker peptide 2, linker peptide 4, and linker peptide 5, respectively.

[0021] Figure 5 The standard curve for 7-hydroxycoumarin is shown. Detailed Implementation

[0022] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0023] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0024] I. Terminology

[0025] The term "enzyme activity" as used in this invention refers to the ability of an enzyme to catalyze a certain chemical reaction. In this invention, enzyme activity is expressed as specific activity, which refers to the enzyme activity per gram of enzyme protein, with units of mmol / mg / min.

[0026] In this invention, the terms "protein" and "protein protein" can be used interchangeably.

[0027] The term "wild-type fusion protein" as used in this invention refers to a fusion protein without the introduction of mutations.

[0028] II. Implementation Plan

[0029] As mentioned earlier, steroid 11β-hydroxylase CYP11B1 catalyzes the conversion of 11-deoxycortisol to hydrocortisone. The hydroxylation reaction catalyzed by steroid 11β-hydroxylase CYP11B1 requires an additional reducing domain to transfer electrons. However, when steroid 11β-hydroxylase CYP11B1 and cytochrome P450 reductase are expressed alone, their activity is poor due to low electron transfer efficiency. In the industrial synthesis of corticosteroid drugs, the step of introducing 11-hydroxyl groups into the steroid scaffold is expensive, and the formation of numerous byproducts wastes resources. Furthermore, the functional expression of human-derived steroid 11β-hydroxylase CYP11B1 in bacteria has not yet been successfully achieved. Therefore, the inventors have conducted extensive research on enzymatic catalysis techniques for the synthesis of corticosteroid drugs.

[0030] The inventors have noted that cytochrome P450 BM3 (CYP102A1) is formed by the fusion of a soluble fatty acid hydroxylase and a cytochrome P450 reductase (CPR). Its reducing domain contains FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), and under the mediation of the reducing domain, electrons are transferred from NADPH to the heme group, subsequently catalyzing the oxidation of the substrate.

[0031] Through research, the inventors discovered that constructing a fusion protein by linking the cytochrome P450 BM3 reducing domain with steroid 11β hydroxylase CYP11B1 can reduce the waste of redox equivalents during the hydroxylation reaction and improve the catalytic efficiency of steroid 11β hydroxylase CYP11B1.

[0032] The inventors noted that linker peptides are present in most fusion proteins, which not only maintain the necessary distances between fusion protein components to reduce steric hindrance but also generate spatial structures favorable for catalytic reactions. Adjusting the size and structure of linker peptides in fusion proteins can effectively influence their catalytic activity; therefore, the inventors further screened linker peptides that can improve the catalytic activity of fusion proteins for the construction of fusion proteins, thereby obtaining this invention.

[0033] Therefore, the CYP11B1-BM3 fusion protein involved in the first aspect of the present invention is formed by linking the steroid 11β hydroxylase CYP11B1 and the cytochrome P450 BM3 reducing domain through a linker peptide; wherein, the amino acid sequence of the steroid 11β hydroxylase CYP11B1 is shown in SEQ ID NO.1 (Genebank No. AAA52149.1), and the amino acid sequence of the cytochrome P450 BM3 reducing domain is shown in SEQ ID NO.2 (Genebank No. WIF20057.1).

[0034] In this invention, the linker peptide is a rigid linker peptide or a flexible linker peptide; the rigid linker peptide includes linker peptide 2 and linker peptide 3; the flexible linker peptide includes linker peptide 1, linker peptide 4 and linker peptide 5; preferably, the amino acid sequences of linker peptides 1 to 5 are as shown in SEQ ID NO. 5 to 9.

[0035] The inventors discovered that by mutating the fusion protein to Val at position 169 (Ser), Ala at position 381 (Phe), and Ser at position 382 (Leu), as shown in SEQ ID NO.1, a mutant CYP11B1-BM3 fusion protein containing a set of mutant forms S169V / F381A / L382S was obtained, exhibiting improved solubility. The specific method involved PCR amplification, using the original CYP11B1-BM3 fusion protein as the template, and the primers used are shown in Table 1.

[0036] Table 1. Site-directed mutagenesis primers

[0037]

[0038]

[0039] The second aspect of the present invention relates to a gene encoding the CYP11B1-BM3 fusion protein as described in the first aspect of the present invention, which is composed of a codon-optimized steroid 11β-hydroxylase CYP11B1 gene (Nanjing Genscript Biotech Co., Ltd.), a linker peptide gene, and a codon-optimized P450 BM3 reducing domain gene (Nanjing Genscript Biotech Co., Ltd.) linked in sequence; wherein the codon-optimized steroid 11β-hydroxylase CYP11B1 gene sequence is shown in SEQ ID NO.3, and the codon-optimized P450 BM3 reducing domain gene sequence is shown in SEQ ID NO.4.

[0040] Preferably, the linker peptide genes include codon-optimized linker peptide 1 gene, codon-optimized linker peptide 2 gene, codon-optimized linker peptide 3 gene, codon-optimized linker peptide 4 gene, and codon-optimized linker peptide 5 gene; preferably, the nucleotide sequences of the codon-optimized linker peptide 1 to 5 genes are as shown in SEQ ID NO. 10 to 14.

[0041] According to the present invention, the gene sequence encoding the CYP11B1-BM3 fusion protein further comprises, as shown in SEQ ID NO.3, a TCC mutation at position 504 (from 5′ to 3′) to GTA, a TTT mutation at position 1140 (from 1140 to 1143 to 1144 to 1145 to AGC mutation.

[0042] The genetically engineered bacterium producing the CYP11B1-BM3 fusion protein according to the third aspect of the present invention is a recombinant Escherichia coli expressing the gene encoding the CYP11B1-BM3 fusion protein as described in the third aspect of the present invention.

[0043] The gene encoding the CYP11B1-BM3 fusion protein is composed of a codon-optimized steroid 11β-hydroxylase CYP11B1 gene, a linker peptide gene, and a codon-optimized P450 BM3 reducing domain gene linked in sequence; wherein, the nucleotide sequence of the codon-optimized steroid 11β-hydroxylase CYP11B1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the codon-optimized P450 BM3 reducing domain gene is shown in SEQ ID NO.4.

[0044] Preferably, the linker peptide genes include codon-optimized linker peptide 1 gene, linker peptide 2 gene, codon-optimized linker peptide 3 gene, codon-optimized linker peptide 4 gene, and codon-optimized linker peptide 5 gene; preferably, the nucleotide sequences of the codon-optimized linker peptide 1 to 5 genes are as shown in SEQ ID NO. 10 to 14.

[0045] According to the present invention, the gene sequence encoding the CYP11B1-BM3 fusion protein further comprises, as shown in SEQ ID NO.3, a TCC mutation at position 504 (from 5′ to 3′) to GTA, a TTT mutation at position 1140 (from 1140 to 1143 to 1144 to 1145 to AGC mutation.

[0046] In this invention, the gene encoding the CYP11B1-BM3 fusion protein is placed in a tandem combination between the T7 promoter and the T7 terminator, so that it is fully expressed in Escherichia coli.

[0047] Specifically, the codon-optimized linker peptide 1 gene was first used to link the codon-optimized steroid 11β-hydroxylase CYP11B1 gene and the codon-optimized P450 BM3 reducing domain gene to obtain the gene encoding the CYP11B1-BM3 fusion protein. This gene was then expressed in *E. coli* to obtain the CYP11B1-BM3 fusion protein. Next, the codon-optimized linker peptide 1 gene in the fusion protein encoding gene was replaced by codon-optimized linker peptides 2-5, respectively, through PCR amplification and Gibson homologous recombination. Expression in *E. coli* yielded fusion proteins containing linker peptides of varying lengths and rigidity / flexibility.

[0048] The application of the genetically engineered bacteria as described in the third aspect of this invention in the preparation of CYP11B1-BM3 fusion protein can also be understood as a method for preparing or expressing CYP11B1-BM3 fusion protein using the genetically engineered bacteria as described in the third aspect of this invention.

[0049] This invention provides the induction expression conditions for producing the CYP11B1-BM3 fusion protein, specifically as follows:

[0050] The engineered bacteria were inoculated into seed culture medium and activated at 37℃ and 250 rpm for 14-16 h. Fermentation was carried out at 37℃ and 250 rpm until OD600 = 0.6-0.8. 1 mM IPTG and 1 mM hemoglobin precursor deta-aminolevulinic acid were added, and expression was induced at 30℃ and 200 rpm for 21 h. Cells were then collected and washed. After cell resuspension, cell lysis and centrifugation, crude enzyme solution was obtained. After purification by nickel column chromatography, pure enzyme (CYP11B1-BM3 fusion protein) solution was obtained.

[0051] The CYP11B1-BM3 fusion protein expression medium of the present invention is TB medium, specifically containing 12g peptone, 24g yeast extract, 4mL glycerol, 4.62g KH2PO4 and 25g K2HPO4 per liter of distilled water.

[0052] The fifth aspect of the present invention provides the use of the CYP11B1-BM3 fusion protein as described in the first aspect of the present invention, or the gene-encoded CYP11B1-BM3 fusion protein as described in the second aspect of the present invention, or the CYP11B1-BM3 fusion protein prepared by the genetically engineered bacteria as described in the third aspect of the present invention, in the production of corticosteroid drugs.

[0053] In this invention, the corticosteroid drugs include, but are not limited to, dexamethasone, prednisone, triamcinolone, betamethasone, etc.

[0054] The relevant detection methods of this invention are as follows:

[0055] (1) Method for determining the enzyme activity of CYP11B1-BM3 fusion protein reductase

[0056] When oxidized cytochrome c accepts electrons, its product, reduced cytochrome c, has a maximum absorption peak at 550 nm. The increase in light absorption at 550 nm per unit time was detected using an enzyme-linked immunosorbent assay (ELISA) reader (Multiskan FC, Thermo Fisher Scientific (China) Co., Ltd.) at room temperature. The production rate of the product, reduced cytochrome c, was calculated by substituting it into equation (1) to calculate the kinetic parameters.

[0057]

[0058] In the above formula:

[0059] ΔA 550 It is the difference in light absorption at 550nm;

[0060] 21.1 is the millimolecular extinction coefficient of reduced cytochrome c, in mM. -1 cm -1

[0061] Protein content refers to the amount of fusion protease added in the reaction, expressed in mg.

[0062] In this invention, the solvent for the oxidized cytochrome c solution is 10 mM KH2PO4 (pH 7.7), the reaction buffer is 0.3 mM KH2PO4 (pH 7.7), the substrate concentration gradient is 0–800 μmol, the enzyme addition amount is 80 μg, the final NADPH solution concentration is 1 mM, and the reaction time is 3 min.

[0063] (2) In vitro enzyme activity assay of CYP11B1-BM3 fusion protein

[0064] The in vitro activity of the fusion protein was determined by reacting the CYP11B1-BM3 fusion protein with 7-ethoxycoumarin and using the product 7-hydroxycoumarin. The enzyme activity was calculated by formula (2) to obtain the fusion protein with the best in vitro enzyme activity.

[0065]

[0066] In the above formula:

[0067] "7-Hydroxycoumarin" refers to the amount of "7-hydroxycoumarin" product produced, in mmol.

[0068] Protein content refers to the amount of fusion protease added in the reaction, expressed in mg.

[0069] In this invention, the in vitro enzyme activity reaction system was 200 μL, the substrate addition was 0.5 mM, the enzyme addition was 80 μg, the final concentration of NADPH solution was 10 mM, the reaction was carried out at 37 °C for 5 min, and the fluorescence value was measured (excitation wavelength was 380 nm, absorption wavelength was 460 nm).

[0070] Example

[0071] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0072] Example 1: Construction and expression of fusion protein particles

[0073] The gene sequences of CYP11B1, linker peptide 1, and BM3 domains were codon-optimized. The optimized gene sequences were then fully synthesized and ligated into the pET-28a(+) plasmid vector (Cellray (Beijing) Life Science Technology Co., Ltd.). NdeII / HindIII was selected as the cloning site. Gene synthesis and plasmid construction were completed by Nanjing GenScript Biotech Co., Ltd. The fused protein plasmid map is shown below. Figure 1As shown. The recombinant plasmid was diluted to 100 ng / μL, and 1 μL was transferred into competent Escherichia coli BL21(DE3) (Shanghai Weidi Biotechnology Co., Ltd.). After heat shock, it was incubated for 1-2 h and then concentrated and plated.

[0074] Select a single, clearly visible colony and add it to 4 mL of LB broth containing 50 μg / mL kanamycin. Incubate at 37°C and 220 rpm for 10 h. Transfer 1–2% of the culture to 200 mL of LB broth containing 50 μg / mL kanamycin and incubate at 37°C and 220 rpm until OD (occurrence limit) is reached. 600 When the value reached 0.8, IPTG was added to a final concentration of 1 mM, and the mixture was cultured at 30°C and 220 rpm for 21 h to induce protein expression.

[0075] Example 2: Construction of the mutant CYP11B1 gene

[0076] Mutation was performed using a point mutation kit from Vazyme. The mutation primers are as follows (underlined parts indicate mutation sites). The mutation primer sequences and mutation system are shown in Tables 1 and 2. The template is the plasmid CYP11B1-BM3 synthesized by Vazyme.

[0077] Table 2 Site-directed mutagenesis reaction system

[0078]

[0079] After the mutation results were verified by sequencing, the digestion product was digested with DpnI enzyme and homologous recombination was performed and then transferred into Escherichia coli BL21(DE3).

[0080] The mutant was expressed according to the method in Example 1, and the supernatant and precipitate were subjected to SDS-PAGE electrophoresis to analyze the expression of the fusion protein. Figure 2 As can be seen, the expression level of the fusion protein in the supernatant increased significantly after mutation, and a clear target band was observed at 132 kDa after purification, indicating that the solubility of the fusion protein was improved and its expression was better after mutation.

[0081] Example 3; Construction of different linker peptide fusion proteins

[0082] Using the fusion protein granules constructed in Example 1 as templates, recombinant fusion protein granules containing linker peptides 2 and 4 were constructed by PCR amplification and DNA ligase, respectively. Recombinant fusion protein granules containing linker peptides 3 and 5 were constructed by Gibson assembly. Figure 3 This is a schematic diagram comparing different linker peptide fusion proteins. Linker peptides 2 and 3 are rigid linker peptides, while linker peptides 4 and 5 are flexible linker peptides. The PCR amplification primers used are shown in Table 3; the Gibson assembly primers are shown in Table 4.

[0083] Table 3 PCR amplification primers

[0084]

[0085] Table 4 Gibson assembly primers

[0086]

[0087] Example 4: Induction and Analysis of Different Linker Peptide Fusion Proteins

[0088] The linker peptide obtained in Example 3 was transformed and induced to express using the method described in Example 1, with the correct plasmid replaced. The peptide was purified using a nickel column, and the purified enzyme protein and crude enzyme solution were analyzed by SDS-PAGE. The results are as follows: Figure 4 As shown, the purified protein bands were clear and could be used for subsequent enzyme activity measurements. The concentration of the fusion protein was detected using the BCA protein concentration assay kit (Wuhan Aidi Biotechnology Co., Ltd.).

[0089] Example 5: Detection of reductase activity in different linker peptide fusion proteins

[0090] Take a 96-well plate, add different volumes of cytochrome c to each well to achieve a final concentration of 0–200 μmol / L, then add 80 μL of enzyme solution and buffer containing 0.3 M KH₂PO₄ (pH 7.7), and finally add NADPH (final concentration 100 μM), bringing the total volume to 200 μL. After mixing thoroughly, immediately place the plate in a microplate reader and start the reaction at 25°C. Start timing after adding NADPH and measure the absorbance at 550 nm. Plot the reaction rate against substrate concentration with different concentrations of cytochrome c (μmol / L) on the x-axis and the reduced cytochrome c produced per unit time (μmol / min / mg) on ​​the y-axis. Calculate the enzymatic kinetic constant using the double reciprocal plot method. The results are shown in Table 5.

[0091] Table 5. Enzyme kinetics analysis of reductases from different linked peptide fusion proteins

[0092]

[0093]

[0094] Example 6: In vitro enzyme activity detection of different linker peptide fusion proteins

[0095] 7-Ethoxycoumarin was dissolved in anhydrous ethanol to prepare a 20 mmol / L stock solution, which was then diluted to 0.5 mmol / L with preheated 0.1 mol / L pH 7.6 phosphate buffer. In a light-protected centrifuge tube, 500 μL of enzyme solution and 800 μL of 0.5 mmol / L 7-ethoxycoumarin were added sequentially. After thorough mixing, the tube was incubated at 30°C with shaking for 5 min. Then, 100 μL of freshly prepared 9.6 mmol / L NADPH coenzyme was added, and the reaction was timed for 15 min from the start. 200 μL of the final reaction solution was added to a black ELISA plate (for fluorescence assay), and the fluorescence values ​​of the reactants and products were measured using a fluorescence microplate reader (excitation wavelength 380 nm, emission wavelength 460 nm). This assay method was used to determine whether the CYP450 enzyme possesses deethylation activity.

[0096] The fluorescence intensity of the sample was measured using a fluorescence microplate reader, and the fluorescence was carried over... Figure 5 The product yield was calculated using the standard curve equation for 7-hydroxycoumarin. The specific activity of the enzyme was calculated using equation (2), and the results are shown in Table 6.

[0097] Table 6. In vitro enzyme activity of different linked peptide fusion proteins

[0098]

[0099] Comparison of reductase activity and in vitro enzyme activity of different linker peptide fusion proteins shows that fusion proteins containing flexible linkers (linkers 1, 4, and 5) exhibit higher reductase activity and in vitro enzyme activity than fusion proteins containing rigid linkers (linkers 2 and 3). Regarding length, fusion proteins with 15 amino acid linkers (linkers 3 and 5) demonstrate superior reductase activity and in vitro enzyme activity compared to fusion proteins with 5 amino acid linkers (linkers 2 and 4). The fusion protein with 12 linkers (linker 1) shows reductase activity and in vitro enzyme activity second only to the fusion protein containing linker 5. Therefore, it is inferred that for P450 fusion proteins, flexible linkers are preferable, with a preferred length of 12–15 amino acids, such as linkers 1 and 5.

[0100] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function.

Claims

1. A CYP11B1-BM3 fusion protein, comprising the steroid 11β-hydroxylase CYP11B1 and the cytochrome P450 BM3 reducing domain linked by a linker peptide; wherein, The amino acid sequence of the steroid 11β hydroxylase CYP11B1 is shown in SEQ ID NO.1, and the amino acid sequence of the cytochrome P450 BM3 reducing domain is shown in SEQ ID NO.

2. The linker peptide is a rigid linker peptide or a flexible linker peptide; the rigid linker peptide is linker peptide 3; the flexible linker peptides are linker peptide 1 and linker peptide 5; wherein the amino acid sequences of linker peptides 1, 3, and 5 are as shown in SEQ ID NO. 5, 7, and 9, respectively.

2. A gene encoding the CYP11B1-BM3 fusion protein as described in claim 1, comprising a codon-optimized steroid 11β-hydroxylase CYP11B1 gene, a linker peptide gene, and a codon-optimized P450 BM3 reducing domain gene linked sequentially; wherein, The nucleotide sequence of the codon-optimized steroid 11β hydroxylase CYP11B1 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the codon-optimized P450BM3 reducing domain gene is shown in SEQ ID NO.

4. The linker peptide genes are codon-optimized linker peptide 1, linker peptide 3, and linker peptide 5 genes; wherein the nucleotide sequences of the codon-optimized linker peptide 1, 3, and 5 genes are shown in SEQ ID NO. 10, 12, and 14, respectively.

3. A genetically engineered bacterium producing the CYP11B1-BM3 fusion protein as described in claim 1, wherein the bacterium is a recombinant Escherichia coli expressing the gene as described in claim 2.

4. The application of the genetically engineered bacteria as described in claim 3 in the preparation of CYP11B1-BM3 fusion protein.

5. The application of the CYP11B1-BM3 fusion protein as described in claim 1 in the production of corticosteroid drugs.

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