Method for synthesizing 11 alpha hydroxyprogesterone by using cytochrome P450 enzyme recombinant bacteria

Abstract

The invention discloses a method for synthesizing 11 alpha hydroxyprogesterone by using cytochrome P450 enzyme recombinant bacteria, and belongs to the technical field of biology. The method comprises the following steps: firstly, constructing CYP219A1 gene and reduction chaperone Pdx / Pdr gene expression vectors from neosphingosinearomaphilus, and transferring the CYP219A1 gene and reduction chaperone Pdx / Pdr gene expression vectors into escherichia coli to obtain whole-cell transformed recombinant bacteria; and after culture, feeding a substrate progesterone by using a resting cell method for biotransformation to obtain a 11 alpha-hydroxyprogesterone product. According to the CYP219A1 mutant strain provided by the invention, the conversion rate of progesterone is as high as 99%, and the 11 alpha site selectivity is 74%.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for synthesizing 11α-hydroxyprogesterone using recombinant bacteria with cytochrome P450 enzymes. Specifically, it involves using a mutant of the cytochrome P450 enzyme CYP219A1. （L298G） A method for obtaining 11α-hydroxyprogesterone compounds by biotransformation of recombinant bacteria co-expressed with the reduction partner Pdx / Pdr in a dual vector. Background Technology

[0002] Cytochrome P450 enzymes can catalyze inert C-H bonds in compounds under mild conditions, exhibiting strict regioselectivity and stereoselectivity. P450 enzymes are widely involved in drug synthesis and intermediate synthesis, such as the biosynthesis of hydrocortisone. There are numerous successful industrial applications, such as the synthesis of artemisinin, pravastatin, and other drug intermediates and drugs. This has led to an increasing number of researchers focusing their attention on the study of P450 enzymes.

[0003] As the second most used drug class globally after antibiotics, steroidal drugs are characterized by a core structural feature: a steroidal skeleton composed of three six-membered rings fused with one five-membered ring. This rigid structure provides a stable foundation for drug design, while the modification space for the C17 side chain and functional groups such as hydroxyl and carbonyl groups on the ring becomes a key target for regulating pharmacological activity. Progesterone, as the classic parent nucleus of steroidal drugs, can have its biological activity significantly altered through 11-position hydroxylation modification—for example, 11α-hydroxyprogesterone not only enhances the progesterone effect but also specifically inhibits the activity of 11β-HSD2 enzyme, which plays a gatekeeper role in cortisol metabolism. Notably, the structure-activity relationship of steroidal drugs exhibits a clear site-dependent relationship: modifications at different positions on the same parent nucleus (such as the reduction of the C3 keto group to an alcohol or the introduction of an esterification group at C17) can lead to an activity transition from anti-inflammatory drugs to sex hormones. This characteristic of fine-tuning structure to dramatic change in activity is a key reason why steroidal chemistry has become a golden field in drug development. 11α-hydroxyprogesterone binds to the active site of the 11β-HSD2 enzyme with high affinity through its unique stereoconfiguration, thereby blocking the conversion of cortisol to cortisone. Summary of the Invention

[0004] The purpose of this invention is to disclose a method for synthesizing 11α-hydroxyprogesterone compounds using cytochrome P450 enzyme mutant bacteria.

[0005] The objective of this invention is achieved through the following technical solution: This invention first provides a cytochrome P450 enzyme mutant, which is a mutant formed by mutating the amino acid sequence (as shown in SEQ ID NO:1) of the wild-type P450 enzyme CYP219A1 from *Neopsphingosine monophosphate*. Specifically, the mutation involves replacing leucine at position 298 with glycine. More preferably, the P450 enzyme mutant is CYP219A1. （L298G） That is, the leucine at position 298 in the amino acid sequence shown in SEQ ID NO:1 is mutated to glycine; the amino acid sequence of the P450 enzyme mutant is shown in SEQ ID NO.2.

[0006] The term "mutation" mentioned above includes, but is not limited to, the substitution, deletion, or addition of amino acids.

[0007] This invention also discloses a recombinant expression vector comprising the coding gene of the cytochrome P450 enzyme mutant (amino acid sequence shown in SEQ ID NO.2). Preferably, the recombinant expression vector is obtained by inserting the coding gene of the cytochrome P450 enzyme mutant into the pET28a plasmid; that is, preferably, the recombinant expression vector is pET28a- CYP219A1 （L298G） Plasmid.

[0008] This invention also discloses a recombinant bacterium containing a cytochrome P450 enzyme mutant, wherein the recombinant bacterium comprises the aforementioned recombinant expression vector. Preferably, the recombinant bacterium further comprises pACYCDuet- Pdx / PdR plasmid, pACYCDuet- Pdx / PdR The gene sequence is shown in SEQ ID NO.3.

[0009] Furthermore, the recombinant bacteria were constructed using the following method: Using the synthesized pET28a-CYP219A1 plasmid as a template, pET28a- containing the mutation site was obtained. CYP219A1 (L298G) PCR products. Products containing pET28a- CYP219A1 (L298G) plasmid transformation into a plasmid containing PACYCDuet- Pdx / PdR Obtaining pET28a- in BL21(DE3) competent cells containing plasmid CYP219A1 （L298G） +pACYCDuet- Pdx / PdR Whole-cell transformation of recombinant bacteria.

[0010] The present invention also provides the application of the cytochrome P450 enzyme mutant in the synthesis of 11α-hydroxyprogesterone, specifically, in the presence of the cytochrome P450 enzyme mutant, 11α-hydroxyprogesterone is obtained by biotransformation using progesterone as a substrate.

[0011] This invention also provides a method for synthesizing 11α-hydroxyprogesterone compounds using the recombinant bacteria, specifically: Utilizing CYP219A1 (L298G) The fermentation broth of whole-cell transformed recombinant bacteria was used to biotransform the substrate progesterone to obtain 11α-hydroxyprogesterone.

[0012] Furthermore, the CYP219A1 (L298G) The fermentation broth of the whole-cell transformed recombinant bacteria was prepared by the following method: The recombinant bacteria were added to LB medium and cultured at 37°C for 12 h to obtain a seed culture of recombinant Escherichia coli. This seed culture was then inoculated into Terrific Broth medium at a volume ratio of 1:100 and cultured at 37°C until the bacterial culture reached its OD value. 600 When the pH reached 0.8, IPTG was added to induce protein expression in the bacteria. Then, 5-aminolevulinate and trace elements were added to aid protein expression. Simultaneously, the temperature was lowered to 25°C, and the culture was continued for 20 h. The bacteria were then collected by centrifugation. The final concentration of IPTG in Terrific Broth medium was 0.1 mM, the concentration of 5-aminolevulinate in Terrific Broth medium was 0.5 mM, and the concentration of trace elements in Terrific Broth medium was 0.3%.

[0013] Preferably, the trace element formula is: CaCl2·H2O 0.74 g, ZnSO4·7H2O 0.18 g, MnSO4·4H2O 0.132 g, Na2EDTA 20.1 g, CoCl2·6H2O 0.25 g, FeCl3·6H2O 16.7 g, CuSO4·5H2O 0.1 g, with purified water added to a final volume of 1 L.

[0014] Furthermore, using CYP219A1 (L298G) The conditions for obtaining 11α-hydroxyprogesterone by biotransformation of the substrate progesterone by whole-cell transforming recombinant bacteria are as follows: The collected bacterial cells were suspended in a buffer solution at pH 7.5, and progesterone was added for biotransformation. After shaking at 30°C for 24 h, an equal volume of ethyl acetate was added to the reaction solution for extraction. The organic phase was dried under reduced pressure (repeated three times and the organic phases were combined) to obtain the 11α-hydroxyprogesterone product. The concentration of the substrate in the buffer solution was 100 µM.

[0015] Preferably, the buffer solution is formulated with 40 mM dipotassium hydrogen phosphate, 10 mM potassium dihydrogen phosphate and 10% (v / v) glycerol.

[0016] The beneficial effects of this invention are: The present invention relates to CYP219A1 (L298G) The enzyme mutant (cytochrome P450 enzyme mutant) exhibits high regioselectivity and stereoselectivity for progesterone, producing the product 11α-hydroxyprogesterone with high conversion rate and selectivity. The recombinant bacteria constructed in this invention can selectively carry out oxidation reactions under mild conditions, which is less environmentally polluting and less costly compared to traditional chemical synthesis. The CYP219A1 constructed in this invention (L298G) The fermentation broth of whole-cell transformed recombinant bacteria, used as a catalytic system, achieved a conversion rate of approximately 74% for the catalytic production of 11α-hydroxyprogesterone from progesterone, improving the efficiency of raw material and energy utilization and demonstrating market potential. Attached Figure Description

[0017] Figure 1 It is a cytochrome P450 enzyme mutant CYP219A1 (L298G) Response diagram of the biotransformation of the substrate progesterone by the reduction partners Pdx / PdR; Figure 2 This is an HPLC chromatogram using progesterone as a substrate. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to embodiments. These embodiments are provided to facilitate a better understanding of the present invention, but are not intended to limit the scope of the invention.

[0019] Example 1 Figure 1 For CYP219A1 (L298G) The flowchart of biotransformation with substrate includes the following steps: 1. Preparation of recombinant Escherichia coli for co-expression: (1) The nucleotide sequence of CYP219A1 was downloaded from JGI Genome Portal - Home and ligated into the pET28a vector via gene synthesis to obtain pET28a- CYP219A1The amino acid sequence of CYP219A1 is shown in SEQ ID NO.1.

[0020] (2) CYP219A1 (L298G) The amino acid sequence is shown in SEQ ID NO.2, which is obtained by PCR through a mutation of leucine at position 298 of the amino acid sequence shown in SEQ ID NO.1 to glycine. The template is pET28a- CYP219A1 The forward primer sequence used is 5′- gctcgggaattgccacctaccgcaccg-3′ (SEQ ID NO.4), and the reverse primer sequence used is 5′- aattcccgagctttgatggcgcagcgctt-3′ (SEQ ID NO.5).

[0021] (3) The contents of pET28a- CYP219A1 (L298G) plasmid transformation into a plasmid containing PACYCDuet- Pdx / PdR Recombinant Escherichia coli co-expressed in BL21(DE3) competent cells containing plasmids were obtained.

[0022] 2. The multi-enzyme expression of recombinant Escherichia coli includes the following steps: The co-expressed recombinant *E. coli* was inoculated into LB broth (pH 7.2) and cultured at 37 °C and 220 rpm for 12 hours to obtain a seed culture of recombinant *E. coli*. The seed culture was then inoculated into Terrific Broth medium for scale-up culture and fermentation; the volume ratio of seed culture to Terrific Broth medium was 1:100. After inoculation, the recombinant *E. coli* was further cultured at 37 °C until the bacterial culture reached its OD value. 600 When the pH reached 0.8, IPTG was added to induce protein overexpression in the cells. Then, 5-aminolevulinate and trace elements were added to aid protein expression. Simultaneously, the temperature was lowered to 25 °C, the rotation speed was reduced to 180 rpm, and the cells were cultured for another 20 h. The cells were then collected by centrifugation. The concentration of IPTG in Terrific Broth medium was 0.1 mM, the concentration of 5-aminolevulinate in Terrific Broth medium was 0.5 mM, and the concentration of trace elements in Terrific Broth medium was 0.3%.

[0023] 3. Biotransformation to synthesize 11α-hydroxyprogesterone: The collected bacterial cells were suspended in a buffer solution with a pre-centrifugation broth-to-buffer solution volume ratio of 10:1, maintaining a pH of 7.5. Progesterone was added for biotransformation, and the reaction was carried out at 30°C with shaking for 24 h. An equal volume of ethyl acetate was added to the reaction solution for extraction with thorough shaking. The organic phase was dried under reduced pressure (repeated three times, with the organic phases combined) to obtain the 11α-hydroxyprogesterone product. The substrate concentration in the buffer solution was 100 µM, and the buffer solution consisted of 40 mM dipotassium hydrogen phosphate, 10 mM potassium dihydrogen phosphate, and 10% (v / v) glycerol.

[0024] Example 2 The 11α-hydroxyprogesterone obtained in Example 1 was dissolved in 100 μl of methanol, filtered through a 0.22 μM organic filter membrane, and detected by high-performance liquid chromatography (HPLC). In this invention, the HPLC analysis used a Waters E2695 HPLC system and a COSMOSIL packed column. The mobile phase was a 25%-100% methanol-water gradient elution for 28 min, the detection wavelength was 240 nm, and the flow rate was 1 mL / min.

[0025] Figure 2 This is the HPLC chromatogram of the experimental sample, where red represents the sample containing pET28a- constructed in Example 1. CYP219A1 （L298G） +pACYCDuet- Pdx / PdR The blue spectrum represents the spectrum obtained after 24 hours of co-expression of recombinant *E. coli* with wild-type CYP219A1; the green spectrum represents progesterone standards. Based on the integral area of ​​the 11α-hydroxyprogesterone peak in the experimental sample divided by (integrated area of ​​11α-hydroxyprogesterone peak + integral area of ​​progesterone peak), the co-expression of recombinant *E. coli* of this invention increased the biocatalytic conversion rate of 11α-hydroxyprogesterone compounds from 2.8% in the wild type to 74.1%, and the selectivity from 8.9% in the wild type to 74.2%. This result demonstrates... CYP219A1 （L298G） The mutant exhibits high product selectivity and catalytic activity.

[0026] The 11α-hydroxyprogesterone compound obtained through this catalytic system is expected to have good application value in the biopharmaceutical field. Compared with the complex process and multiple by-products of traditional chemical synthesis, this catalytic system has better economic and environmental benefits.

Claims

1. A cytochrome P450 enzyme mutant, characterized in that, It is CYP219A1 through amino acid mutation The obtained mutant, CYP219A1, has the amino acid sequence SEQ ID NO.1, with the amino acid mutation site being leucine at position 298 mutated to glycine.

2. The cytochrome P450 enzyme mutant as described in claim 1, characterized in that, The mutant is CYP219A1 （L298G） Its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, The recombinant expression vector includes the encoding gene of the cytochrome P450 enzyme mutant as described in claim 2.

4. A recombinant bacterium containing a cytochrome P450 enzyme mutant, characterized in that, The recombinant bacteria comprises the recombinant expression vector according to claim 3.

5. The recombinant bacteria as described in claim 4, characterized in that, The recombinant bacteria also contain pACYCDuet- Pdx / PdR plasmid, pACYCDuet- Pdx / PdR The gene sequence is shown in SEQ ID NO.

3.

6. The use of the cytochrome P450 enzyme mutant of claim 2 in the synthesis of 11α-hydroxyprogesterone, characterized in that, Cytochrome P450 enzyme mutants use progesterone as a substrate and undergo biotransformation to produce 11α-hydroxyprogesterone.

7. A recombinant bacterium containing pET28a- CYP219A1 （L298G） plasmids and pACYCDuet- Pdx / PdR plasmid, pET28a- CYP219A1 （L298G） The plasmid is the cytochrome P450 enzyme mutant CYP219A1 described in claim 2. （L298G） The coding gene was inserted into the pET28a plasmid.

8. The use of the recombinant bacteria according to claim 7 in the synthesis of 11α-hydroxyprogesterone.

9. The application as described in claim 8, characterized in that, Includes the following steps: The recombinant bacteria were added to LB medium and cultured at 37°C to obtain a seed culture of the recombinant bacteria. The seed culture was then inoculated into Terrific Broth medium and cultured at 37°C until the bacterial culture reached its OD value. 600 When the value reaches 0.8, IPTG is added to induce bacterial protein expression, followed by the addition of 5-aminolevulinate and trace elements to assist protein expression. At the same time, the temperature is lowered to 25°C, and the culture is continued. The bacterial cells are then collected by centrifugation. The collected bacterial cells were suspended in a buffer solution at pH 7.

5. The volume ratio of the fermentation broth to the buffer solution before centrifugation was 10:

1. Progesterone was added for biotransformation, and the reaction was carried out by shaking at 30°C. After the reaction was completed, an equal volume of ethyl acetate was added to the reaction solution and the mixture was shaken thoroughly for extraction. The organic phase was then dried under reduced pressure to obtain the 11α-hydroxyprogesterone product.

10. The application according to claim 9, characterized in that, The final concentration of IPTG in Terrific Broth medium was 0.1 mM, the concentration of 5-aminolevulinate in Terrific Broth medium was 0.5 mM, and the concentration of trace elements in Terrific Broth medium was 0.3%. The trace element formula is as follows: CaCl2·H2O 0.74 g, ZnSO4·7H2O 0.18 g, MnSO4·4H2O 0.132 g, Na2EDTA 20.1 g, CoCl2·6H2O 0.25 g, FeCl3·6H2O 16.7 g, CuSO4·5H2O 0.1 g, with purified water added to a final volume of 1 L. The concentration of progesterone in the buffer solution is 100 µM; the buffer solution is formulated as follows: 40 mM dipotassium hydrogen phosphate, 10 mM potassium dihydrogen phosphate and 10% (v / v) glycerol.

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