A method for synthesizing benzodiazepine derivatives by enzyme catalysis

Abstract

This invention belongs to the fields of biopharmaceutical and biochemical technologies, specifically a method for the enzyme-catalyzed synthesis of baconin. This invention uses inexpensive and readily available androstened 4-ene-3,17-dione as a substrate, via a 3-sterone-Δ... 1 The synthesis of 3-sterones via a cascade reaction catalyzed by β-dehydrogenase and 17β-hydroxydehydrogenase. This invention relates to 3-sterone-Δ 1 -Dehydrogenase mutants and their encoding genes, obtaining mutants, containing 3-sterone-Δ 1 Recombinant expression vectors and recombinant expression transformants of β-dehydrogenase gene and 17β-hydroxydehydrogenase gene, 3-sterone-Δ 1 The amino acid sequences of β-dehydrogenase and its mutants are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the amino acid sequence of 17β-hydroxydehydrogenase is shown in SEQ ID NO.5. This invention simplifies the synthesis steps of steroidal drugs, significantly improves catalytic selectivity and yield, and features high substrate concentration, mild reaction conditions, low cost, and environmental friendliness.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biopharmaceuticals and bio-chemical industry, and particularly relates to a method for synthesizing boldenone by enzyme catalysis. BACKGROUND

[0002] Boldenone (3) is an important steroid hormone drug, which can be derived from testosterone and has most of the properties of testosterone, such as promoting muscle growth, improving muscle endurance and recovery. In addition, compared with testosterone, boldenone has a lower rate of aromatization and a lower possibility of converting into estrogen. Therefore, boldenone can be used in medicine to treat muscle loss and osteoporosis, and to increase body weight, strength and appetite, retain muscle and tighten muscle for athletes out of season.

[0003] The current main preparation method of boldenone still adopts chemical synthesis, which has defects such as complicated synthesis route, need to add strong acid or toxic reagent, many by-products, low yield and high cost. In addition, a large amount of waste gas, waste water and waste material will be produced in the process of chemical synthesis of boldenone, which will seriously pollute the environment and does not meet the national environmental protection requirements. In addition to chemical method, there are also reports on the synthesis of boldenone by biological method. For example, Wang et al. produced boldenone by combined fermentation of Comamonas acidovorans and Saccharomyces strain (Chinese patent CN201910264964.X), that is, first, C1,2 dehydrogenation reaction of cheap androst-4-ene-3,17-dione (1) was carried out by Comamonas acidovorans to generate androst-1,4-diene-3,17-dione (2a), and then 2a was reduced to generate boldenone by adding Pichia pastoris genetic engineering strain capable of efficiently expressing 17β carbonyl reductase. Although this method is effective, the yield of boldenone can only reach 7.7 g / L, and two kinds of microbial liquid need to be prepared by fermentation respectively, which is complicated and low in efficiency. SUMMARY

[0004] The present application provides a method for synthesizing boldenone by enzyme catalysis with high selectivity, high yield, few by-products, mild reaction conditions, low cost, green environmental protection and high efficiency.

[0005] The present application first provides a 3-steroidone-Δ 1 dehydrogenase (Δ 1 KstD) ReKstD wild-type enzyme, and its mutant, the Δ 1The KstD mutant is selected from the group consisting of mutant ReKstD-I53L, ReKstD-I352T, ReKstD-I53L / I352T. The amino acid sequence of the wild-type enzyme ReKstD is shown as SEQ ID NO: 1, the amino acid sequence of the mutant ReKstD-I53L is shown as SEQ ID NO: 2, the amino acid sequence of the mutant ReKstD-I352T is shown as SEQ ID NO: 3, and the amino acid sequence of the mutant ReKstD-I53L / I352T is shown as SEQ ID NO: 4.

[0006] The present application provides a 17β-hydroxy dehydrogenase (17β-CR) whose amino acid sequence is shown as SEQ ID NO: 5.

[0007] The present application also provides a gene encoding the 3-ketosteroid-Δ 1 dehydrogenase ReKstD and its mutants, and 17β-hydroxy dehydrogenase 17β-CR; the nucleotide sequence of the wild-type ReKstD is shown as SEQ ID NO: 6, the nucleotide sequence of the mutant ReKstD-I53L is shown as SEQ ID NO: 7, the nucleotide sequence of the mutant ReKstD-I352T is shown as SEQ ID NO: 8, the nucleotide sequence of the mutant ReKstD-I53L / I352T is shown as SEQ ID NO: 9, and the nucleotide sequence of the 17β-hydroxy dehydrogenase 17β-CR is shown as SEQ ID NO: 10.

[0008] The present application also provides a vector containing the above-mentioned 3-ketosteroid-Δ 1 dehydrogenase ReKstD or its mutants, or the gene of 17β-hydroxy dehydrogenase 17β-CR.

[0009] The present application also provides an E. coli engineering bacterial cell containing the above-mentioned two vectors, one vector capable of expressing the 3-ketosteroid-Δ 1 dehydrogenase ReKstD wild-type enzyme or its mutants, and the other vector capable of expressing the gene corresponding to 17β-hydroxy dehydrogenase 17β-CR.

[0010] The present application also provides the 3-ketosteroid-Δ 1 dehydrogenase ReKstD wild-type enzyme or its mutants, or 17β-hydroxy dehydrogenase 17β-CR, or the above-mentioned vector, or the above-mentioned cell, in catalyzing the dehydrogenation of C1,2 and the reduction of 17-ketone of steroidal compounds.

[0011] Further, the steroidal compound includes but is not limited to androsta-4-ene-3,17-dione (1).

[0012] The application also provides the 3-ketosteroid-Δ 1 -Dehydrogenase ReKstD wild-type enzyme or its mutant, or 17β-hydroxy dehydrogenase 17β-CR, or the vector, or the cell, in the synthesis of baedogenone (3).

[0013] The application provides a method for synthesizing baedogenone by enzyme catalysis, which is started from 3-ketosteroid-Δ 1 -Dehydrogenase (Δ 1 -KstD) mutant enzyme - androst-4-ene-3,17-dione (1), and uses Escherichia coli engineering bacteria co-expressing Δ 1 -KstD mutant enzyme and 17β-hydroxy dehydrogenase to catalyze and convert androst-4-ene-3,17-dione (1) into baedogenone (3) in one step; the synthesis route is shown in Figure 1 . The specific steps are as follows:

[0014] Androst-4-ene-3,17-dione (1) is converted into baedogenone (3) under the action of the 3-ketosteroid-Δ 1 -Dehydrogenase ReKstD wild-type enzyme or its mutant, and 17β-hydroxy dehydrogenase 17β-CR; specifically, the wet bacteria of the genetic engineering bacteria co-expressing 3-ketosteroid-Δ 1 -Dehydrogenase ReKstD wild-type enzyme or its mutant and 17β-hydroxy dehydrogenase 17β-CR are resuspended with a buffer, and the substrate androst-4-ene-3,17-dione, isopropyl alcohol (IPA), N,N-dimethylformamide (DMF) and phenazine methosulfate (PMS) are added; the reaction is completed under the condition of 20-37°C; after the reaction solution is extracted with ethyl acetate, the ethyl acetate extract is obtained; after dehydration with anhydrous sodium sulfate, filtration and concentration under reduced pressure, the crude product is obtained; and the product baedogenone is obtained by purifying the crude product by column chromatography.

[0015] Further, in the starting reaction system, the mass percentage concentration of the substrate is 0.5%-5% (w / v), the amount of the genetic engineering bacteria (calculated according to the wet bacteria) is 300%-1000% of the mass of the substrate, the amount of IPA is 1%-10% (v / v), the amount of DMF is 5%-25% (v / v), the amount of PMS is 1-5 mM, and the pH of the reaction solution is 6-9.

[0016] Compared with the prior art, the application has the following beneficial effects:

[0017] The application first provides a 3-ketosteroid-Δ 1Genetically engineered strains of the dehydrogenase ReKstD wild-type enzyme or its mutants and 17β-hydroxy dehydrogenase (17β-CR) were used in the synthesis of boltanone, starting from the inexpensive and readily available androstened-4-ene-3,17-dione, to catalyze the synthesis of boltanone in one step.

[0018] This invention provides a method for the bio-enzymatic synthesis of bolanone. This method not only simplifies the synthesis steps of steroid drugs and significantly improves catalytic selectivity and yield, but also features high substrate concentration, mild reaction conditions, low cost, and environmental friendliness. It plays an important role in promoting the industrialization of bolanone-type steroid drugs in my country. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthetic route for the enzyme-catalyzed co-synthetic synthesis of baconinol in this invention.

[0020] Figure 2 The engineered bacteria in Example 4 of this invention E. coli Schematic diagram of the reaction process of whole-cell catalytic conversion of androst-4-ene-3,17-dione to boldanone using BL21(DE3) / ReKstD-I53L / I352T / pRSFDuet-1-17β-CR. Detailed Implementation

[0021] The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments.

[0022] Example 1, 3-Sterone-Δ 1 Whole-gene synthesis of - dehydrogenase ReKstD and 17β-hydroxy dehydrogenase 17β-CR

[0023] Obtaining a gene from whole-genome synthesis Rhodococcus erythropolis 3-Sterone-Δ of WY 1406 1 - Dehydrogenase gene (ReKstD, GenBank: KX645867) and a gene derived from Empedobacter stercoris The 17β-hydroxydehydrogenase gene (17β-CR, GenBank: WP171621727) was codon-optimized based on its amino acid sequences (SEQ ID NO:1 and SEQ ID NO:5) to obtain 3-sterone-Δ 1 The nucleotide sequences of the β-dehydrogenase and 17β-hydroxydehydrogenase genes are shown in SEQ ID NO: 6 and SEQ ID NO: 10, respectively. These two synthetic genes were ligated into plasmids pET21a and pRSFDuet-1, respectively, to construct recombinant plasmids pET21a-ReKstD and pRSFDuet-1-17β-CR.

[0024] Example 2, 3-ketosteroid-Δ 1 - Combination active center saturation mutation and iterative saturation mutation of dehydrogenase ReKstD

[0025] The combination active center saturation mutation was completed by using TransStart FastPfu Fly DNA polymerase. The primers containing mutation site information were designed and used, and wild type pET21a-ReKstD was used as a template.

[0026] The PCR reaction system (50 μL) was as follows: 50 ng of template, 10 μL of 5×TransStart FastPfu Fly Buffer, 4 μL of dNTP (2.5 mM each), 1 μL of each of the mutant primers (10 μM), 10 μL of 5×PCR Stimulant, 1 μL of MgSO4 (50 mM), 2.5 units of TransStart FastPfu Fly DNA polymerase, and sterile distilled water to 50 μL. ® FastPfu Fly Buffer, 4 μLdNTP (each 2.5 mM), 1 μL of each of the mutant primers (10 μM), 10 μL of 5×PCR Stimulant, 1 μL of MgSO4 (50 mM), 2.5 units of TransStart FastPfu Fly DNA polymerase, and sterile distilled water to 50 μL.

[0027] The PCR amplification program was as follows: (1) denaturation at 98 ℃ for 3 min; (2) denaturation at 98 ℃ for 20 s, (3) annealing at 65 ℃ for 30 s, (4) extension at 72 ℃ for 8 min, steps (2)-(4) for a total of 20 cycles, and finally extension at 72 ℃ for 10 min, and preservation at 4 ℃.

[0028] The PCR product obtained by amplification was digested with endonuclease DpnI at 37 ℃ for 1 hour, and then transformed into E. coli BL21 (DE3) and coated on LB solid medium containing ampicillin (100 μg / mL) and incubated at 37 ℃ overnight to construct a saturation mutant library and perform screening. Mutants with significantly improved catalytic activity were screened, and DNA sequencing was performed to determine that they were single-point mutants ReKstD-I53L and ReKstD-I352T, and then iterative saturation mutation was performed to obtain a better double-point mutant ReKstD-I53L / I352T.

[0029] Example 3, 3-ketosteroid-Δ 1 - Construction and expression of genetic engineering bacteria of vectors of wild type enzyme or mutant genes of dehydrogenase ReKstD and vectors of 17β-hydroxy dehydrogenase genes

[0030] As an example of the mutant ReKstD-I53L / I352T, the plasmids pET21a-ReKstD-I53L / I352T and pRSFDuet-1-17β-CR were mixed with E. coli competent cells BL21 (DE3), heat shocked at 42°C for 45 seconds, then ice-bathed for 2 min, followed by recovery at 37°C for 1 h, and then spread on LB agar medium containing ampicillin and kanamycin, and cultured to obtain the target co-expression transformant. A single colony was picked from the plate and inoculated into 25 mL of LB liquid medium (containing ampicillin and kanamycin), and cultured at 37°C, 200 rpm overnight to obtain a seed solution. 5 mL of the seed solution was transferred into 500 mL of LB liquid medium (containing ampicillin and kanamycin), and cultured at 37°C, 200 rpm. When the OD600 of the culture solution of the recombinant strain reached 0.6-0.8, 0.1 mM IPTG (Isopropyl β-D-Thiogalactoside) was added, and induction was performed at 25°C for 12-14 h. The cells (wet bacteria) were collected by centrifugation at 4°C, 5000 rpm for 10 min, and the whole-cell biocatalyst of the engineered bacteria was obtained.

[0031] β-D-Thiogalactoside), inducted at 25°C for 12-14 h. The cells (wet bacteria) were collected by centrifugation at 4°C, 5000 rpm for 10 min, and the whole-cell biocatalyst of the engineered bacteria was obtained.

[0032] Example 4, engineered bacteria E. coli BL21 (DE3) / ReKstD-I53L / I352T / pRSFDuet-1-17β-CR whole-cell catalysis for asymmetric synthesis of baodanone

[0033] Freshly prepared wet bacteria of the engineered bacteria 40 g were weighed into a 1 L conical flask, 164 mL of phosphate buffer (50 mM, pH 7.5) was added, and the whole-cell suspension was prepared by stirring, 10 g of androst-4-ene-3, 17-dione, 6 mL of isopropyl alcohol, 30 mL of DMF, and 122 mg of phenazine methosulfate were added. The reaction mixture was shaken at 37°C, 200 rpm, and a small amount of sample was taken at intervals for HPLC analysis to track the reaction progress, and the reaction was terminated after 6 hours. The reaction solution was extracted with ethyl acetate (3 x 200 mL), the organic layers were combined, dried with Na2SO4, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain 9.56 g of the product baodanone, with a separation yield of 95.6%. 1H NMR (500 MHz, CDC13) δ / ppm 7.03 (d, J = 10.2 Hz, 1H), 6.19 (dd, J = 10.2, 2.0 Hz, 1H), 6.03 (d, J = 1.8 Hz, 1H), 3.60 (t, J = 8.5 Hz, 1H), 2.43 (tdd, J = 13.5, 5.2, 1.6 Hz, 1H), 2.32 (ddd, J = 13.3, 4.5, 2.6 Hz, 1H), 2.12-1.97 (m, 2H), 1.96-1.88 (m, 1H), 1.88-1.80 (m, 1H), 1.77-1.69 (m, 1H), 1.69-1.52 (m, 3H), 1.44 (dddd, J = 13.5, 11.7, 8.1, 3.4 Hz, 1H), 1.29 (qd, J = 12.1, 5.7 Hz, 1H), 1.20 (s, 3H), 1.09-0.95 (m, 3H), 0.91 (ddd, J = 12.2, 10.7, 7.1 Hz, 1H), 0.78 (s, 3H). 13 C NMR (126 MHz, CDC13) δ / ppm 186.52, 169.48, 156.14, 127.46, 123.84, 81.38, 52.57, 50.14, 43.70, 43.15, 36.37, 35.60, 33.20, 32.83, 30.31, 23.57, 22.56, 18.76, 11.24.

[0034] The reaction progress of this example is shown in Figure 2

[0035] Example 5, engineered bacteria E. coli BL21(DE3) / ReKstD-I53L / pRSFDuet-1-17β-CR whole cell catalyzed asymmetric synthesis of baodanone

[0036] ​Take 80 g of the freshly prepared wet bacteria of the engineered bacteria in a 1 L conical flask, add 172 mL of phosphate buffer (50 mM, pH 7.0), stir evenly to prepare a whole cell suspension, add 10 g of androst-4-ene-3, 17-dione, 8 mL of isopropyl alcohol, 20 mL of DMF, and 122 mg of phenazine methosulfate. The above reaction mixture is shaken at 37°C and 200 rpm, and a small amount of sample is taken every hour to track the reaction process by HPLC analysis. The reaction is terminated after 6 hours. The reaction solution is extracted with ethyl acetate (3 x 200 mL), the organic layers are combined, dried with Na2SO4, and concentrated to obtain the crude product. The crude product is purified by column chromatography to obtain 9.45 g of the product, bardanone, with a separation yield of 94.5%.

[0037] Example 6, engineered bacteria E. coli BL21 (DE3) / ReKstD-I352T / pRSFDuet-1-17β-CR whole cell catalyzed asymmetric synthesis of bardanone

[0038] Take 60 g of the freshly prepared wet bacteria of the engineered bacteria in a 1 L conical flask, add 170 mL of phosphate buffer (50 mM, pH 7.5), stir evenly to prepare a whole cell suspension, add 8 g of androst-4-ene-3, 17-dione, 5 mL of isopropyl alcohol, 25 mL of DMF, and 98 mg of phenazine methosulfate. The above reaction mixture is shaken at 37°C and 200 rpm, and a small amount of sample is taken every hour to track the reaction process by HPLC analysis. The reaction is terminated after 6 hours. The reaction solution is extracted with ethyl acetate (3 x 200 mL), the organic layers are combined, dried with Na2SO4, and concentrated to obtain the crude product. The crude product is purified by column chromatography to obtain 7.54 g of the product, bardanone, with a separation yield of 94.2%.

[0039] The above description is only the preferred embodiment of the present application, which is used to illustrate the technical concept and characteristics of the present application, and its purpose is to enable those skilled in the art to understand the content of the present application and implement it, and it cannot limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made according to the spirit and essence of the present application should be covered within the protection scope of the present application.

Claims

1. A process for the enzymatic catalytic synthesis of Bao Dan ketone, characterized in that, The specific steps are as follows: Androst-4-ene-3,17-dione is placed in a 3-sterone-Δ... 1 A mutant of the wild-type dehydrogenase ReKstD, along with 17β-hydroxy dehydrogenase, generates boltanone; specifically, the co-expressed 3-sterone-Δ 1 Mutants of the wild-type ReKstD dehydrogenase and genetically engineered cells of 17β-hydroxy dehydrogenase were resuspended in buffer, and the substrate androster-4-ene-3,17-dione, isopropanol (IPA), N,N-dimethylformamide (DMF), and methyl phenazine sulfate (PMS) were added. The reaction was carried out at 20–37°C until complete. The reaction mixture was extracted with ethyl acetate to obtain an ethyl acetate extract, which was then dehydrated with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography to obtain the product, baconin. wherein; the 3-ketosteroid-Δ 1 - the mutant of the wild-type enzyme ReKstD is ReKstD-I53L, ReKstD-I352T or ReKstD-I53L / I352T; the amino acid sequence of the wild-type enzyme ReKstD is shown in SEQ ID NO: 1, the amino acid sequence of the mutant ReKstD-I53L is shown in SEQ ID NO: 2, the amino acid sequence of the mutant ReKstD-I352T is shown in SEQ ID NO: 3, and the amino acid sequence of the mutant ReKstD-I53L / I352T is shown in SEQ ID NO: 4; the amino acid sequence of the 17beta-hydroxy dehydrogenase is shown as SEQ ID NO: 5; In the initial reaction system, the mass percentage concentration of the substrate is 0.5%-5% (w / v), the amount of the genetically engineered bacteria is 300%-1000% of the mass of the substrate, the amount of IPA is 1%-10% (v / v), the amount of DMF is 5%-25% (v / v), the amount of PMS is 1-5 mM, and the pH of the reaction solution is 6-9.

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