Escherichia coli genetically engineered bacteria for expressing 11beta-hsd1 enzyme mutant and application thereof

Abstract

The application discloses an escherichia coli gene engineering bacterium expressing 11beta-HSD1 enzyme mutants and application thereof. The nucleotide sequence of the 11beta-HSD1 enzyme mutant is shown in SEQ ID NO:2. The application discloses a method for preparing hydrocortisone acetate by using 11beta-HSD1 enzyme mutants. The 11beta-HSD1 enzyme mutant is obtained by directional evolution, and when the 11beta-HSD1 enzyme mutant is used for preparing hydrocortisone acetate, the activity is good, and the yield of the product is higher, reaching 88.2%.

Description

Technical Field

[0001] This invention relates to the fields of chemical substances and biotransformation, specifically to a genetically engineered Escherichia coli expressing an 11β-HSD1 enzyme mutant, a method for preparing hydrocortisone acetate using the same, and the application of the genetically engineered bacterium. Background Technology

[0002] 11β-HSD1, or 11β-hydroxysteroid dehydrogenase type 1, also known as cortisone reductase, is a key enzyme in the conversion of cortisone into the functional glucocorticoid cortisol. This activation is associated with several human diseases, particularly metabolic syndrome, in which 11β-HSD1 has been identified as a potential new target for therapeutic drugs.

[0003] According to literature reports, current methods for synthesizing cortisone-based steroidal drugs (hydrocortisone, hydrocortisone acetate, prednisolone, etc.) include: total chemical synthesis, semi-synthetic synthesis, and total biosynthesis. However, total chemical synthesis is complex, has a low overall yield, and lacks industrial production value; total biosynthesis, due to its complex synthetic pathway and low product yield, has also not achieved industrial production. Therefore, semi-synthetic synthesis has become the mainstream method for synthesizing cortisone-based steroidal drugs both domestically and internationally.

[0004] This invention discovers an 11β-HSD1 enzyme mutant that can be used for the synthesis of hydrocortisone acetate. Summary of the Invention

[0005] The technical problem this invention aims to solve is the complexity or low overall yield of the industrial-scale production of hydrocortisone acetate. To address this, this invention provides a genetically engineered *E. coli* strain expressing an 11β-HSD1 enzyme mutant, a method for preparing hydrocortisone acetate using this strain, and the applications of the genetically engineered strain. This invention obtains the 11β-HSD1 enzyme mutant through directed evolution, which exhibits good activity and a high yield (88.2%) when used to prepare hydrocortisone acetate.

[0006] The first aspect of the present invention provides a genetically engineered bacterium based on Escherichia coli, wherein the genetically engineered bacterium contains a gene expressing a mutant of the 11β-HSD1 enzyme, the nucleotide sequence of which is shown in SEQ ID NO:2.

[0007] In one embodiment, the gene is located in a recombinant expression vector; preferably, the backbone plasmid used to prepare the recombinant expression vector is pRSFDuet1.

[0008] In one embodiment, the host cell of the genetically engineered bacteria is preferably Escherichia coli BL21.

[0009] In one aspect of the invention, a method for constructing the genetically engineered bacteria is also included.

[0010] The method includes the following steps: transforming a synthesized recombinant expression vector containing the 11β-HSD1 enzyme mutant gene into a host cell, such as Escherichia coli BL21, by a transformation method such as chemical transformation (e.g., calcium chloride method).

[0011] In the method described, the preferred conditions for culturing the genetically engineered bacteria are: 37°C for at least 36 hours.

[0012] The second aspect of the present invention provides a method for producing an 11β-HSD1 enzyme mutant, wherein the 11β-HSD1 enzyme mutant is obtained by culturing genetically engineered bacteria as described in the first aspect of the present invention and fermenting them.

[0013] In one embodiment, the fermentation includes one or more of the following conditions:

[0014] (1) The culture medium was 2×YT solid selective medium;

[0015] (2) The concentration of the added seed solution is 1%;

[0016] (3) Fermentation conditions: 37℃, 200rpm shaking culture for 0.5 to 2 days;

[0017] Preferably, the 11β-HSD1 enzyme mutant is present in a suspension of genetically engineered bacteria, and the preparation of the suspension preferably includes the following steps: centrifuging to collect the genetically engineered bacteria, washing and resuspending them in PBS buffer; more preferably, the concentration of the PBS buffer is 0.1M.

[0018] The formulation of the 2×YT solid selective medium is 16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride, and 20 g / L agar; the formulation of the 2×YT liquid selective medium is 16 g / L peptone, 10 g / L yeast extract, and 5 g / L sodium chloride.

[0019] A third aspect of the present invention provides a method for preparing hydrocortisone acetate, comprising the following steps: in the presence of the 11β-HSD1 enzyme mutant produced in the second aspect of the present invention, performing a reduction reaction of hydrocortisone acetate as shown below:

[0020]

[0021] Thus, the hydrocortisone acetate is obtained.

[0022] The reduction reaction also includes a solvent.

[0023] The solvent can be a conventional solvent, preferably a PBS buffer. The concentration of the PBS buffer is preferably 0.1M.

[0024] The mass-to-volume ratio of the cortisone acetate to the solvent can be 150–800 mg / L, for example 150 mg / L, 300 mg / L, 600 mg / L or 800 mg / L, preferably 150 mg / L.

[0025] The preferred temperature for the reduction reaction is 37°C.

[0026] The reduction reaction can be monitored using methods conventional in the art (e.g., TLC, HPLC), and the endpoint is generally defined as the disappearance of cortisone acetate or the cessation of reaction. The preferred duration of the reduction reaction is 1-2 days.

[0027] The reduction reaction is preferably carried out in an oscillating manner. The oscillation frequency is preferably 200 rpm.

[0028] The preferred apparatus for the reduction reaction is a 250mL Erlenmeyer flask.

[0029] After the reduction reaction is completed, the process also includes an extraction step.

[0030] The extraction process includes one or more of the following steps: adding an extractant to the reaction solution, taking the organic layer, centrifuging, and drying.

[0031] The volume ratio of the extractant to the reaction solution can be a conventional volume ratio, preferably 1:1.

[0032] The extractant is a conventional extraction solvent, which can be an alcohol solvent and / or a chlorinated organic solvent, preferably a mixture of an alcohol solvent and a chlorinated organic solvent.

[0033] The preferred alcohol solvent is methanol. The preferred chlorine-containing organic solvent is chloroform.

[0034] The preferred volume ratio of the alcohol solvent to the chlorine-containing organic solvent is 1:9.

[0035] After extraction, the product is further processed as follows: the extracted product is reconstituted with an organic solvent, centrifuged to collect the supernatant, filtered, and analyzed by HPLC.

[0036] The resolution organic solvent can be a conventional solvent in the art, preferably methanol. The volume of the resolution organic solvent is preferably 1 mL. The filtration is preferably performed using an organic filter membrane with a pore size of 0.22 μm.

[0037] Following the reduction reaction and product extraction, the preferred chromatographic column for HPLC detection is a J-01-02 Inertsil ODS-2HP 5μm, 2.1*150mm (UP). The mobile phase for HPLC detection is a conventional organic solvent, preferably 53% acetonitrile. The preferred detection wavelength for HPLC detection is 254nm.

[0038] The fourth aspect of the present invention provides the application of the genetically engineered bacteria as described in the first aspect of the present invention in the preparation of hydrocortisone acetate.

[0039] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0040] The reagents and raw materials used in this invention are all commercially available.

[0041] The positive and progressive effects of this invention are as follows: This invention obtains the 11β-HSD1 enzyme mutant through directed evolution, which has good activity and high product yield when used to prepare hydrocortisone acetate. Detailed Implementation

[0042] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0043] Example 1: Synthesis of 11β-HSD1 enzyme gene

[0044] The 11β-HSD1 enzyme gene was synthesized by gene synthesis (Nanjing Genscript Biotech Co., Ltd.), and the relevant nucleotide and amino acid sequences are shown in the sequence listing.

[0045] 11β-HSD1-HV278 amino acid sequence (SEQ ID NO:1):

[0046] MNEEFRPEMLQGKKVIVTGASKGIGREMAYHLAKMGAHVVVTARSKETLQKVVSHCLELGAASAHYIAGTMEDMTFAEQFVAQAGKLMGGLDMLILNHITNTSLNLFHDDIHHVRKSMEVNFLSYVVLTVAALPM LKQSNGSIVVVSSLAGKVAYPMVAAYSASKFALDGFFSIRKEYSVSRVNVSITLCVLGLIDTETAMKAVSGIVHMQAAPKEECALEIIKGGALRQEEVYYDSSLWTTLLIRNPCRKILEELYSTSYNMDRFINK*

[0047] 11β-HSD1-HV278 nucleotide sequence (SEQ ID NO:2):

[0048] ATGAACGAGGAATTCAGACCAGAGATGCTCCAAGGAAAGAAAGTGATTGTCACAGGGGCCAGCAAAGGGATCGGAAGAGAGATGGCTTATCATCTGGCGAAGATGGGAGCCCATGTGGTGGTGACAGCGAGGTCAAAAGAAACTCTACAGAAGGTGGTATCCCACTGCCTGGAGCTTGGAGCAGCCTCAGCACACTACATTGCTGGCACCATGGAAGACATGACCTTCGCAGAGCAATTTGTTGCCCAAGCAGGAAAGCTCATGGGAGGACTAGACATGCTCATTCTCAACCACATCACCAACACTTCTTTGAATCTTTTTCATGATGATATTCACCATGTGCGCAAAAGCATGGAAGTCAACTTCCTCAGTTACGTGGTCCTGACTGTAGCTGCCTTGCCCATGCTGAAGCAGAGCAATGGAAGCATTGTTGTCGTCTCCTCTCTGGCTGGGAAAGTGGCTTATCCAATGGTTGCTGCCTATTCTGCAAGCAAGTTTGCTTTGGATGGGTTCTTCTCCTCCATCAGAAAGGAATATTCAGTGTCCAGGGTCAATGTATCAATCACTCTCTGTGTTCTTGGCCTCATAGACACAGAAACAGCCATGAAGGCAGTTTCTGGGATAGTCCATATGCAAGCAGCTCCAAAGGAGGAATGTGCCCTGGAGATCATCAAAGGGGGAGCTCTGCGCCAAGAAGAAGTGTATTATGACAGCTCACTCTGGACCACTCTTCTGATCAGAAATCCATGCAGGAAGATCCTGGAAGAACTCTACTCAACGAGCTATAATATGGACAGATTCATAAACAAGTAG

[0049] Example 2: Construction of Escherichia coli engineered strain HCA11

[0050] The synthesized expression plasmid pRSFDuet1-HCA11 containing the 11β-HSD1 enzyme gene was transformed into Escherichia coli BL21(DE3) via chemical transformation. The selection culture conditions were: 37℃, cultured for at least 36 hours. Correct positive clones were identified by PCR, and five clones were randomly selected and named strains HCA11-1, HCA11-2, HCA11-3, HCA11-4, and HCA11-5.

[0051] Example 3: Synthesis of hydrocortisone acetate catalyzed by engineered Escherichia coli strains HCA11-1 to HCA11-5

[0052] Shake-flask fermentation for cell catalysis: *E. coli* strains HCA11-1 to HCA11-5 were activated in 2×YT solid selective medium (formulation: 16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride, 20 g / L agar). Seed culture was prepared in 2×YT liquid selective medium (formulation: 16 g / L peptone, 10 g / L yeast extract, 5 g / L sodium chloride) (37℃, 200 rpm, 16 h). 1 mL of this culture was inoculated into three 500 mL Erlenmeyer flasks containing 100 mL of 2×YT liquid medium. The flasks were incubated at 37℃ with shaking at 200 rpm for 2 days. After centrifugation at 4000-5000 rpm for 5 min, *E. coli* cells were collected, washed with PBS buffer, and finally resuspended in 30 mL of PBS buffer. Add the substrate cortisone acetate (CA) to a 250 mL Erlenmeyer flask containing 0.1 M PBS to a final concentration of 150 mg / L, and carry out the catalytic reaction. Incubate at 37 °C with shaking at 200 rpm for 1–2 days.

[0053] Product extraction: Take 5 mL of the catalytic reaction solution into a separatory funnel, add an equal volume of extractant (methanol:chloroform = 1:9, volume ratio), take 4 mL of the lower organic phase, put it into a 10 mL centrifuge tube and dry it, redissolve it with 1 mL of methanol, centrifuge and take the supernatant and filter it through an organic filter membrane with a pore size of 0.22 μm, and perform HPLC detection (chromatographic column: J-01-02 Inertsil ODS-2HP 5 μm, 2.1*150 mm (UP); mobile phase: 53% acetonitrile; detection wavelength: 254 nm). The detection showed that hydrocortisone acetate was generated, and the HCA11-1 strain had the highest HCA yield of 88.2% during cell transformation.

[0054] Example 4: The ability of engineered Escherichia coli HCA11 to catalyze the synthesis of hydrocortisone acetate under different substrate concentrations

[0055] Cell catalysis by shake-flask fermentation: The HCA11-1 strain of *E. coli* was activated in 2×YT solid selective medium, and a seed culture was prepared in 2×YT liquid selective medium (37℃, 200 rpm, 16 h). 1 mL of the seed culture was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of 2×YT liquid medium. The flask was incubated at 37℃ with shaking at 200 rpm for 2 days. After centrifugation at 4000-5000 rpm for 5 min, *E. coli* cells were collected, washed with PBS buffer, and finally resuspended in a 250 mL Erlenmeyer flask containing 30 mL of 0.1 M PBS. Cortisone acetate (CA) was added at final concentrations of 150 mg / L, 300 mg / L, 600 mg / L, and 800 mg / L, respectively, to initiate the catalytic reaction. The flask was incubated at 37℃ with shaking at 200 rpm for 1–2 days.

[0056] Product extraction: Take 5 mL of the catalytic reaction solution into a separatory funnel, add an equal volume of extractant (methanol:chloroform = 1:9, volume ratio), take 4 mL of the lower organic phase, put it into a 10 mL centrifuge tube and dry it, redissolve it with 1 mL of methanol, centrifuge and take the supernatant and filter it through an organic filter membrane with a pore size of 0.22 μm, and perform HPLC detection (chromatographic column: J-01-02 Inertsil ODS-2HP 5 μm, 2.1*150 mm (UP); mobile phase: 53% acetonitrile; detection wavelength: 254 nm). Hydrocortisone acetate product was detected. Under the four substrate concentration conditions, the HCA yield was 87.6%, 76.9%, 64.1%, and 59.7%, respectively.

Claims

1. A method based on Escherichia coli (E. coli) Escherichia coli Genetically engineered bacteria, characterized in that, The genetically engineered bacteria contain a gene that expresses a mutant of the 11β-HSD1 enzyme, the nucleotide sequence of which is shown in SEQ ID NO:

2.

2. The genetically engineered bacteria as described in claim 1, characterized in that, The gene is located in a recombinant expression vector.

3. The genetically engineered bacteria as described in claim 2, characterized in that, The backbone plasmid used to prepare the recombinant expression vector was pRSFDuet1.

4. The genetically engineered bacteria as described in claim 1, characterized in that, The host cell of the genetically engineered bacteria is Escherichia coli BL21.

5. A method for producing an 11β-HSD1 enzyme mutant, characterized in that, The 11β-HSD1 enzyme mutant is obtained by culturing the genetically engineered bacteria as described in any one of claims 1-4 and fermenting them.

6. The method for producing the 11β-HSD1 enzyme mutant as described in claim 5, characterized in that, The fermentation includes one or more of the following conditions: (1) The culture medium was 2×YT solid selective medium; (2) The concentration of the added seed solution is 1%; (3) Fermentation conditions: 37℃, 200rpm shaking culture for 0.5~2 days.

7. The method as described in claim 6, characterized in that, The 11β-HSD1 enzyme mutant was present in the suspension of genetically engineered bacteria.

8. The method as described in claim 7, characterized in that, The preparation of the genetically engineered bacterial suspension includes the following steps: centrifuging to collect the genetically engineered bacteria, washing and resuspending them in PBS buffer.

9. The method as described in claim 8, characterized in that, The concentration of the PBS buffer is 0.1M.

10. A method for preparing hydrocortisone acetate, characterized in that, It includes the following steps: in the presence of the 11β-HSD1 enzyme mutant produced by the method according to any one of claims 5-9, cortisone acetate is subjected to the following reduction reaction: ， Thus, the hydrocortisone acetate is obtained.

11. The method for preparing hydrocortisone acetate as described in claim 10, characterized in that, The reduction reaction also includes a solvent.

12. The method for preparing hydrocortisone acetate as described in claim 11, characterized in that, The solvent is PBS buffer.

13. The method for preparing hydrocortisone acetate as described in claim 12, characterized in that, The concentration of the PBS buffer is 0.1M; The mass-to-volume ratio of the cortisone acetate to the solvent is 150~800 mg / L; And / or, the reduction reaction takes 1 to 2 days; And / or, the reduction reaction is carried out in an oscillating manner; And / or, after the reduction reaction is completed, the step of continuing extraction is also included.

14. The method for preparing hydrocortisone acetate as described in claim 13, characterized in that, The mass-to-volume ratio of the cortisone acetate to the solvent is 150 mg / L, 300 mg / L, 600 mg / L, or 800 mg / L.

15. The method for preparing hydrocortisone acetate as described in claim 13, characterized in that, The frequency of the oscillation is 200 rpm.

16. The method for preparing hydrocortisone acetate according to any one of claims 13-15, characterized in that, The extraction process includes one or more of the following steps: adding an extractant to the reaction solution, taking the organic layer, centrifuging, and drying; The volume ratio of the extractant to the reaction solution is 1:1; And / or, the extractant is an alcohol solvent and / or a chlorine-containing organic solvent.

17. The method for preparing hydrocortisone acetate as described in claim 16, characterized in that, The alcohol solvent is methanol; the chlorine-containing organic solvent is chloroform. And / or, the volume ratio of the alcohol solvent to the chlorinated organic solvent is 1:

9.

18. The use of a genetically engineered bacterium as described in any one of claims 1-4 in the preparation of hydrocortisone acetate.

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