Application of Rhodococcus ethereans in the conversion of cholesterol to 4-androstenedione

Abstract

This invention discloses the application of *Rhodococcus etherealis* in the conversion of cholesterol to 4-androstenedione. Experiments have demonstrated that *Rhodococcus etherealis* has a highly efficient function in converting cholesterol to 4-androstenedione, and the product is easy to extract and separate, with high purity in the final product. The yield of 4-androstenedione was found to be above 0.62 mg / L. The method for obtaining 4-androstenedione is simple, environmentally friendly, and inexpensive.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the application of Rhodococcus aetherivorans in the conversion of cholesterol to produce 4-androstenedione. Background Technology

[0002] Steroid drugs are drugs whose molecular structure contains a cyclopentane-polyhydrophenanthrene core. Steroid drugs occupy an important position in the chemical drug system, ranking second only to antibiotics. They play extremely important roles in maintaining life, body development, and immune regulation, hence the term "key to life." Based on pharmacological action, they can be divided into two main types: corticosteroids and sex hormones. Corticosteroids are mainly used to treat various inflammations, autoimmune diseases, and allergic diseases. They also help maintain the body's water and electrolyte balance, such as the anti-inflammatory drug dexamethasone and the anti-allergic dermatitis drug fluocinolone acetonide. In recent years, steroid drugs have also been found to have anti-cancer, osteoporosis treatment, and HIV infection-fighting effects, such as the novel anticancer drug abiraterone. With the expanding range of diseases treated by steroid drugs, they have a broad prospect in the pharmaceutical market. Currently, more than 400 types of steroid drugs are produced globally, among which steroid hormones are the most important and possess great potential.

[0003] 4-Androstenedione (4-AD) is an irreplaceable intermediate in the synthesis of important steroid hormone drugs, and almost all steroid hormone drugs can be produced using 4-AD as a starting material. The microbial transformation of cholesterol, sterols, and other inexpensive and readily available steroidal compounds to synthesize steroid hormone drugs or the key intermediate 4-AD is of great significance for the production of steroid hormone drugs. Furthermore, microbial transformation has advantages that chemical synthesis cannot match, including high efficiency, high specificity, mild reaction conditions, no dependence on organic solvents, and environmental friendliness. Microorganisms commonly used to degrade steroidal compounds include *Mycobacterium* spp., *Rhodococcus* spp., *Streptomyces* spp., and *Nocardia* spp., with *Mycobacterium* strains being the most studied. Currently, there are no reports of using *Rhodococcus* to degrade cholesterol to produce 4-androstenedione. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide an application of Rhodococcus ethereans in the conversion of cholesterol to produce 4-androstenedione.

[0005] The technical solution of the present invention is as follows:

[0006] Application of Rhodococcus ethereans in the conversion of cholesterol to 4-androstenedione.

[0007] The above application includes the following steps:

[0008] 1) Culture of the strain: Inoculate Rhodococcus aetherivorans into the seed culture medium and culture to obtain seed culture solution;

[0009] 2) Inoculate the seed culture medium obtained in step 1) into the fermentation medium, add cholesterol solution, and convert to obtain a conversion solution containing the product 4-androstenedione.

[0010] Step 1) The preferred method for culturing the strain is to inoculate Rhodococcus aetherivorans into a seed culture medium, culture it, and obtain OD. 600 Seed culture medium with a value of 1.2-2.0.

[0011] Step 2) is preferably: the seed culture obtained in step 1) is inoculated into the fermentation medium at an inoculation amount of (6-12) v / v%, and then a cholesterol solution is added to make the final cholesterol concentration 0.2-0.8 g / L. The mixture is then converted at 28-32℃ and 180-220 rpm for 72-96 h to obtain a conversion solution containing the product 4-androstenedione. The cholesterol solution is in the form of an organic solvent.

[0012] The preferred organic solvents are ethanol, methanol, or dimethyl sulfoxide.

[0013] The preferred composition of the seed culture medium is: 20 g / L glucose, 1.0 g / L yeast extract, 7 g / L tryptone, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, 1.0 g / L sodium glutamate; the balance is water, adjusted to pH 7.2, and sterilized.

[0014] The preferred composition of the fermentation medium is: 20 g / L glucose, 8.0 g / L yeast extract, 7.0 g / L urea, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, 1.0 g / L sodium glutamate; the balance is water, adjusted to pH 7.2, and sterilized.

[0015] Experiments have shown that *Rhodococcus etherealis* has a highly efficient function in converting cholesterol into 4-androstenedione, and the product is easy to extract and separate, with high purity in the final product. The yield of 4-androstenedione was measured to be above 0.62 mg / L. The method for obtaining 4-androstenedione is simple, environmentally friendly, and inexpensive. Attached Figure Description

[0016] Figure 1 The high-performance liquid chromatogram (254 nm) of the product 4-AD is shown.

[0017] Figure 2 This is the reaction formula for the degradation of cholesterol to produce product 4-AD.

[0018] Figure 3 The LCMS-IT-TOF spectrum of product 4-AD is shown.

[0019] Figure 4 For product 4-AD 1 H-NMR nuclear magnetic resonance spectrum.

[0020] Figure 5 For product 4-AD 13 C-NMR nuclear magnetic resonance spectrum.

[0021] Figure 6 The Dept-135 NMR spectrum of product 4-AD. Detailed Implementation

[0022] The present invention will be further described below through specific embodiments.

[0023] The Rhodococcus aetherivorans strain used in the embodiments of this invention, with accession number CGMCC1.12425, was purchased in July 2020 from the China General Microbiological Culture Collection Center (website: https: / / cgmcc.net / ).

[0024] Alternatively, strains of Rhodococcus aetherivorans CGMCC 1.10521 and Rhodococcus aetherivorans CGMCC 1.6146, purchased in July 2020 from the China General Microbiological Culture Collection Center (website: https: / / cgmcc.net / ), can be used in this invention.

[0025] Example 1

[0026] The seed culture medium consisted of: 20 g / L glucose, 1.0 g / L yeast extract, 7 g / L tryptone, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 1.0 g / L sodium glutamate; the remainder was water. The pH was adjusted to 7.2 and then sterilized.

[0027] Example 2

[0028] The fermentation medium consisted of: 20 g / L glucose, 8.0 g / L yeast extract, 7.0 g / L urea, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, and 1.0 g / L sodium glutamate; the remainder was water. The pH was adjusted to 7.2 and then sterilized.

[0029] Example 3

[0030] The application of Rhodococcus ethereans in the conversion of cholesterol to 4-androstenedione includes the following steps:

[0031] 1) Culture of the strain: Rhodococcus aetherivorans CGMCC 1.12425 was inoculated into seed culture medium (Example 1) and cultured to obtain OD. 600 Seed culture medium with a value of 1.68;

[0032] 2) The seed culture obtained in step 1) was inoculated into the fermentation medium (Example 2) at an inoculation rate of 10 v / v%, and then cholesterol solution was added to make the final cholesterol concentration 0.5 g / L. The mixture was converted at 30°C and 180 rpm for 84 h to obtain a conversion solution containing the product 4-androstenedione. The solvent of the cholesterol solution was ethanol.

[0033] 3) Product separation and purification: Centrifuge the conversion solution from step 2) at 4000 rpm for 15 min. Extract the bacterial culture three times with an equal volume of ethyl acetate, 15 min each time, and collect the ethyl acetate layer extract as extract 1.

[0034] After ultrasonic disruption of the bacterial cells by adding a certain amount of methanol for 30 min, centrifugation at 4000 rpm for 10 min was performed. The supernatant was evaporated to dryness using a rotary evaporator, and then extracted twice with an equal volume of a mixture of water and ethyl acetate (water to ethyl acetate volume ratio of 1:1). The ethyl acetate layer was collected as extract 2. Extracts 1 and 2 were combined, and anhydrous sodium sulfate was added to remove water. The extract was then concentrated under reduced pressure using a rotary evaporator until dry to obtain a paste. The paste was mixed with chromatographic methanol to obtain a mixture.

[0035] The mixture was separated using preparative liquid chromatography to obtain a product with high purity. Separation conditions: Column: XBridge BEH C 18 OBD Prep Column (10×150mm, 5μm); Mobile phase: 35% acetonitrile-water solution isocratic elution; Flow rate: 2mL / min; Injection volume: 200μL; Column temperature: 35℃; PDA detector.

[0036] 4) Product analysis and structural identification: High-purity products are analyzed by mass spectrometry, etc. 1 H NMR,13 ¹³C NMR and Dept-135 analyses confirmed that the obtained product was AD (see...). Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 ). Figure 1 Control group: No cholesterol was added in step 2) of this embodiment.

[0037] 5) By constructing a standard curve for AD, the yield of AD converted from cholesterol by Rhodococcus etherealis was further analyzed. High-performance liquid chromatography (HPLC) analysis conditions: Column: Waters CORTECS C 18 (4.6×50mm, 2.7μm); Mobile phase: 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution gradient elution; Flow rate: 0.5mL / min; Injection volume: 5μL; Column temperature: 35℃; PDA detector.

[0038] 6) The yield of AD was 0.62 mg / L, and the purity of AD was 96.67%.

[0039] Example 4

[0040] The application of Rhodococcus ethereans in the conversion of cholesterol to 4-androstenedione includes the following steps:

[0041] 1) Culture of the strain: Rhodococcus aetherivorans CGMCC 1.12425 was inoculated into seed culture medium (Example 1) and cultured to obtain OD. 600 Seed culture medium with a pH of 1.2;

[0042] 2) The seed culture obtained in step 1) was inoculated into the fermentation medium (Example 2) at an inoculation rate of 6 v / v%, and then cholesterol solution was added to make the final cholesterol concentration 0.2 g / L. The mixture was converted at 32°C and 220 rpm for 96 h to obtain a conversion solution containing the product 4-androstenedione. The solvent of the cholesterol solution was methanol.

[0043] 3) Same as step 3) of Example 3;

[0044] Step 4 omitted

[0045] 5. Same as step 5 in Example 3;

[0046] 6. The yield of AD was found to be 0.65 mg / L, and the purity of AD was 96.32%.

[0047] Example 5

[0048] The application of Rhodococcus ethereans in the conversion of cholesterol to 4-androstenedione includes the following steps:

[0049] 1) Culture of the strain: Rhodococcus aetherivorans CGMCC 1.12425 was inoculated into seed culture medium (Example 1) and cultured to obtain OD. 600 Seed culture medium with a pH of 2.0;

[0050] 2) The seed culture obtained in step 1) was inoculated into the fermentation medium (Example 2) at an inoculation rate of 12 v / v%, and then cholesterol solution was added to make the final cholesterol concentration 0.8 g / L. The mixture was converted at 28°C and 200 rpm for 72 h to obtain a conversion solution containing the product 4-androstenedione. The solvent of the cholesterol solution was dimethyl sulfoxide.

[0051] 3) Same as step 3) of Example 3;

[0052] Step 4 omitted

[0053] 5. Same as step 5 in Example 3;

[0054] 6. The yield of AD was 0.63 mg / L, and the purity of AD was 93.78%.

[0055] Separation and purification methods include, but are not limited to, commonly used methods such as filtration, extraction, crystallization, chromatography, and possible combinations thereof.

[0056] Experiments have shown that replacing Rhodococcus aetherivorans CGMCC 1.12425 in Example 5 with Rhodococcus aetherivorans CGMCC 1.10521 or Rhodococcus aetherivorans CGMCC 1.6146, while maintaining the same other conditions as in Example 5, yields and purity of AD are similar to those in Example 5.

Claims

1. Use of Rhodococcus aetherolicus for converting cholesterol to 4-androstenedione, characterized in that Includes the following steps: 1) Cultivation of the strain: inoculate the seed culture medium with Rhodococcus aetherolicus (CGMCC 1.12425), cultivate, and obtain the seed culture solution; Rhodococcus aetherivorans ) 2) The seed culture obtained in step 1) is inoculated into the fermentation medium, and then cholesterol solution is added for conversion to obtain a conversion solution containing the product 4-androstenedione. The cholesterol solution is in the form of an organic solvent, such as ethanol, methanol or dimethyl sulfoxide.

2. The application according to claim 1, characterized in that: Step 1) The strain is cultured by inoculating the seed culture medium with *Rhodococcus etherealis* (…). Rhodococcus aetherivorans CGMCC 1.12425, cultured, yielding OD. 600 Seed culture medium with a value of 1.2-2.

0.

3. The application according to claim 1, characterized in that: Step 2) involves inoculating the seed culture obtained in step 1) into the fermentation medium at an inoculation rate of (6-12) v / v%, then adding cholesterol solution to make the final cholesterol concentration 0.2-0.8 g / L, and converting at 28-32℃ and 180-220 rpm for 72-96 h to obtain a conversion solution containing the product 4-androstenedione.

4. The application according to claim 1 or 2, characterized in that: The seed culture medium consisted of: 20 g / L glucose, 1.0 g / L yeast extract, 7 g / L tryptone, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, and 1.0 g / L sodium glutamate; the remainder was water. The pH was adjusted to 7.2 and then sterilized.

5. The application according to claim 1 or 3, characterized in that: The fermentation medium consisted of: 20 g / L glucose, 8.0 g / L yeast extract, 7.0 g / L urea, 0.5 g / L K2HPO4, 0.5 g / L KH2PO4, 1.0 g / L MgSO4·7H2O, and 1.0 g / L sodium glutamate; the remainder was water. The pH was adjusted to 7.2, and the medium was sterilized.

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