Method for biologically preparing estrone by using 3-ketosteroid-delta-dehydrogenase

Abstract

The invention provides a method for biologically preparing estrone by using 3-sterone-delta-dehydrogenase, which comprises the following steps: taking 19-hydroxy-4-androstene-3, 17-diketone as a substrate, and reacting with the 3-sterone-delta-dehydrogenase KstD to obtain the estrone; wherein the amino acid sequence of the 3-ketosteroid-delta-dehydrogenase KstD is as shown in SEQ ID NO. 1. The whole synthesis process is simple, few in steps, short in period, few in side reaction, mild in reaction condition, low in equipment requirement, free of pollution to the environment and environmentally friendly.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological chemical industry, and particularly relates to a method for biologically preparing estrone by using 3-steroidone-delta1-dehydrogenase. BACKGROUND

[0002] Estrone is a natural estrogenic steroid hormone, which is one of the main estrogens secreted by the ovary, placenta and adrenal gland. It is widely used in the fields of medicine, veterinary medicine, cosmetics and food. As an important pharmaceutical raw material, estrone is mainly used for treating menopausal syndrome, osteoporosis, breast cancer, ovarian cancer and other diseases in the medical field. In addition, estrone is a key intermediate for synthesizing estrogens and 19-demethyl steroidal compounds.

[0003] Estrone: 3-hydroxyestra-1, 3, 5(10)-triene-17-one, molecular formula is C 18 H 22 O2, molecular weight 270.37, CAS number 53-16-7. Its structure is as follows:

[0004] For its synthesis, most of the current methods are chemical methods, such as patents CN118724992A and CN116640177A, which use 1, 4-androstadiene-3, 17-dione (ADD) as raw material, and convert it into estrone by aromatization reaction in organic solvent. This kind of reaction usually needs to add toxic and harmful organic reagents, the synthesis route is long, the reaction condition is harsh, and the yield is low and the cost is high, which cannot be well scaled up. There are also related patents such as CN118127114A, which uses simple arthrobacter to biotransform 19-hydroxy-4-androstene-3, 17-dione to prepare estrone, but there are problems such as long fermentation time of the strain, low feeding, long conversion time and low conversion efficiency. Patent CN117448288A uses a 3-steroidone-delta1-dehydrogenase to efficiently convert 19-nor-4-androstene dione (acid) into estrone, but it needs to be prepared into enzyme powder, and the cost of 19-nor-4-androstene dione (acid) is higher than that of 19-hydroxy-4-androstene-3, 17-dione.

[0005] Based on the above background, there is an urgent need to develop a new efficient, green and environmentally friendly preparation method for the synthesis of estrone. The present application uses a 3-steroidone-delta1-dehydrogenase KstD strain to convert 19-hydroxy-4-androstene-3, 17-dione (open ring material) into estrone, and constructs an estrone production process method with high feeding, short conversion time, high efficiency and simple operation.

[0006] The reaction is as follows: SUMMARY

[0007] Therefore, the application provides a method for preparing estrone by using 3-ketosteroid-Δ1-dehydrogenase.

[0008] The technical scheme of the application is as follows: In a first aspect, the application provides a method for preparing estrone by using 3-ketosteroid-Δ1-dehydrogenase, which comprises the following steps: taking 19-hydroxy-4-androstene-3, 17-dione as a substrate, and reacting with 3-ketosteroid-Δ1-dehydrogenase KstD to obtain estrone. The amino acid sequence of the 3-ketosteroid-Δ1-dehydrogenase KstD is shown in SEQ ID NO. 1: MTQTWDEEYDVVVIGAGGGSLTGALVAAREGLKVLVAEATDRFGGTTAYSGGGLWWPNNQALKRAGVEDTPEAAAQYYHGIVGDDSPRELQEAYLAGGPALVKYLEDNGLMEFLIYPWPDYFGKEPTAHNEGGRTMMPMYFPAEEMGDLRDQVRSGLPAERRGEPLPDMMIGGQALIGRLVLNLSKEPNVTMRRNAEGRKLIMEDGRVAGVIVSIDGQDKAIKATKGVLVAAGGFEQNQEMREKYGVPGHARDTMGAPRNFGLVQQSAIELGADTALMDQAWWSPGLTHPDGSSTFSLWFTGGIFVNNHGERFVNESWAYDKLGRAIIDLVDEGRMTLPYWMVYDNRAGERVPCNNTSVPMVETEEYREAGLWHTADTLEELADKIGVPADKLVATVERFNEFAANEKDEDFDRGGEAYDRSFSEGKSPLVPITEGPFHAAQFGLSDLGTKGGLKTDVDARVLDTGGNVIPGLYAAGNSMAPASGKVYPGGGNPIGSSMVFSYLAALDMAKN.

[0009] Further, the amino acid sequence of the 3-ketosteroid-Δ1-dehydrogenase KstD is substituted, deleted and / or increased by one or more amino acids to obtain a 3-ketosteroid-Δ1-dehydrogenase KstD with the same function.

[0010]

[0011] Based on the above technical solution, the reaction further includes the addition of Tween 80, menaquinone, and PBS at pH 7.4.

[0012] Based on the above technical solution, the following steps are further included: the engineered bacteria expressing the 3-sterone-Δ¹-dehydrogenase KstD are fermented to obtain wet cells.

[0013] Based on the above technical solutions, the engineered bacteria further include Escherichia coli.

[0014] Based on the above technical solution, the reaction is further carried out at 30°C and 200 rpm for 24 hours.

[0015] Based on the above technical solution, the amount of 19-hydroxy-4-androstene-3,17-dione used is 50g / L to 100g / L, and the mass ratio of the wet bacterial cells to 19-hydroxy-4-androstene-3,17-dione is 2:1.

[0016] Based on the above technical solution, the mass ratio of the amount of menaquinone to the amount of 19-hydroxy-4-androstene-3,17-dione is 1%:1, and the amount of PBS is 10mM.

[0017] Based on the above technical solution, the method for preparing the wet bacterial cells further includes the following steps: inoculating the engineered bacteria expressing the 3-sterone-Δ¹-dehydrogenase KstD into a test tube culture medium containing 50 μg / mL kanamycin, and culturing overnight at 37°C and 220 rpm to obtain the primary fermentation seed liquid; The primary fermentation seed culture was inoculated at a rate of 10% (v / v) into a seed shake flask culture medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm for 7 h to obtain the secondary fermentation seed culture. The secondary fermentation seed culture was added to a fermenter containing fermentation medium at an inoculum rate of 10% (v / v), and kanamycin was added to a final concentration of 50 μg / mL. The mixture was then incubated at 37°C until… The temperature was lowered to 20°C, and IPTG was added to a final concentration of 0.1 mM. The mixture was induced for 15 h, and the fermentation broth was collected. The mixture was centrifuged at 4200 rpm for 1 h, and the precipitate was collected to obtain the wet cells. The test tube culture medium comprises: 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract. The seed shake flask culture medium comprises: 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract. The fermentation medium comprises: glycerol 5 g / L, proteose peptone 18 g / L, yeast extract powder 24 g / L, potassium dihydrogen phosphate 2.3 g / L, dipotassium hydrogen phosphate 16 g / L, antifoam 0.8 g / L.

[0018] Compared with the prior art, the present application has the following beneficial effects: (1) The E. coli expressing 3-ketosteroid-Delta1-dehydrogenase KstD constructed by the present application can efficiently express high-activity KstD, and is simple to obtain. The utilization rate of 3-ketosteroid-Delta1-dehydrogenase KstD to the substrate ring-opening substance 19-hydroxy-4-androstene-3, 17-dione is as high as 98%.

[0019] (2) The whole synthesis process of the present application is simple, has few steps, short cycle, few side reactions, and mild reaction conditions, and has low requirements on equipment.

[0020] (3) The present application is pollution-free and green. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0022] Figure 1 SDS-PAGE detection results of 3-ketosteroid-Delta1-dehydrogenase KstD; Figure 2 HPLC detection results of estriol in Example 3; Figure 3 And Figure 4 NMR measurement results of the enzyme conversion reaction solution in Example 3. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0024] In the following specific embodiments, the experimental materials used, such as no special instructions, are purchased through conventional channels, and the specific experiments are carried out according to conventional methods and conditions in the art, or according to the instructions of the reagent kit.

[0025] In the present application, the moisture content of the wet bacterial cells is 75% to 80%.

[0026] In the following specific embodiments, the HPLC detection method of the estrone includes: Chromatographic column: InfinityLab Poroshell 120 EC-C18 (4.6x250 mm, 5 Micron), Agilent. or equivalent chromatographic column; Mobile phase: water: acetonitrile = 50:50; Preparation of test solution: take an appropriate amount of sample, accurately weigh and determine, dissolve and dilute to prepare a solution containing about 1 mg per 1 mL as a test sample; Flow rate: 1.0 mL / min; Detector: UV detector (VWD), wavelength: 280 nm; Column oven temperature: 35°C; injection volume: 20 μL; collection time: 25 min; The area normalization method is used for detection of substrate and product, and the conversion rate is calculated: conversion rate = [C1 / (C1+C2)]x100%, wherein C1 is the peak area content% of the product, and C2 is the peak area content% of the substrate.

[0027] In the following specific embodiments, the post-treatment method of the enzyme conversion reaction solution includes: (1) Take the estrone enzyme conversion reaction solution and heat it to 60°C for one hour; (2) After quenching, pad diatomite and filter, and the water phase filtrate is detected by plate detection without product and discarded; (3) Add 10 times of methanol to the filter cake and heat it to 65°C for one hour; (4) After refluxing, heat the filter cake and add 10 times of methanol to it, heat it to 65°C for one hour, and collect the filtrate; (5) After the second refluxing, heat the filter cake and add 10 times of methanol to it, heat it to 65°C for one hour, and collect the filtrate; (6) After the third refluxing, heat the filter cake and add 20 times of methanol to it, heat it to 65°C for one hour, and collect the filtrate; (7) After the fourth refluxing, heat the filter cake and add 30 times of methanol to it, heat it to 65°C for one hour, and collect the filtrate; (8) After the fifth refluxing, heat the filter cake and add 30 times of methanol to it, heat it to 65°C for one hour, and collect the filtrate; (9) Combine all methanol extracts, add 50% activated carbon, heat to 65°C and reflux for one hour to decolorize; (10) After decolorization, filter out the filtrate, concentrate it to 3 times its volume, cool it down and crystallize for half an hour; (11) After crystallization, the white solid was filtered out and dried in a 60°C drying oven for 5 hours to obtain estradiol solid.

[0028] Example 1: Obtaining the recombinant plasmid KstD of 3-sterone-Δ¹-dehydrogenase and its expression strain 3-Sterone-Δ¹-dehydrogenase KstD, with the amino acid sequence shown in SEQ ID NO.1 and the corresponding nucleotide sequence shown in SEQ ID NO.2, was sequenced by Shanghai Sangon Biotech Co., Ltd. and inserted into the pET28a(+) vector to obtain the recombinant plasmid pET28a(+)-KstD.

[0029] The obtained recombinant plasmid pET28a(+)-KstD was transformed into BL21(DE3) competent cells, plated onto LB solid medium containing 50 μg / mL kanamycin (Kan), and incubated at 37°C for 18 h.

[0030] Pick a fresh single colony and inoculate it into an Erlenmeyer flask containing 10 mL of LB liquid medium containing 50 μg / mL Kan. Incubate overnight at 37°C and 220 rpm. Transfer to fresh LB medium containing Kan resistance and incubate at 37°C. Observe the OD of the bacterial culture. 600nm The concentration was 0.6-0.8. IPTG was added (final concentration 0.1 mM), and the cells were incubated at 20°C for approximately 15 hours. The cells were collected by centrifugation, resuspended in Tris-HCl (50 mM pH=8.0), and the cells were sonicated to disrupt the cell structure. SDS-PAGE was then performed to observe protein expression. Figure 1 As shown, the 3-sterone-Δ¹-dehydrogenase KstD is approximately 55 kDa in size and is mainly expressed in the supernatant.

[0031] Example 2: Fermentation of pET28a(+)-KstD BL21(DE3) strain to prepare wet cells expressing KstD. Select freshly streaked single colonies of pET28a(+)-KstD BL21(DE3) strain, inoculate them into 10 mL test tube culture medium (add kanamycin to a final concentration of 50 μg / mL), and incubate overnight at 37℃ and 220 rpm to obtain the primary fermentation seed liquid; Inoculate the primary fermentation seed culture at an inoculum rate of 10% (v / v) into seed shake flask culture medium (with kanamycin added to a final concentration of 50 μg / mL), and incubate at 37°C and 220 rpm for 7 h to obtain the secondary fermentation seed culture.

[0032] The secondary seed culture was inoculated into a fermenter containing fermentation medium at an inoculation rate of 10% (v / v), and kanamycin was added to a final concentration of 50 μg / mL.

[0033] Adjust the air volume and speed in a timely manner, and maintain the dissolved oxygen (DO) in the tank within the range of 30%-50% by controlling the stirring speed of 200-600 rpm, the air flow rate of 15-20 L / min and the feeding rate.

[0034] As the biomass of the strain increased, the pH in the fermenter first dropped to its lowest point, then rebounded before feeding was initiated. The feeding rate was slowly adjusted according to the pH trend (increasing feeding as pH rises, decreasing feeding as pH falls). Samples were taken and measured every 30 minutes after feeding. The value and glycerol content determine whether to cool down to the induction temperature. After cooling, samples are taken every 1 hour for measurement. .

[0035] Incubate at 37℃ until The temperature was lowered to 20°C, and IPTG was added to a final concentration of 0.1 mM. Induction was performed for 15 hours. The fermentation broth was collected, centrifuged at 4200 rpm for 1 hour, and the precipitate was collected to obtain the wet cells. The cells were then frozen at -20°C for later use.

[0036] The test tube culture medium comprises: 10 g / L peptone, 10 g / L sodium chloride, and 5 g / L yeast extract. Feed culture medium preparation: 600 g / L glycerol, 50 g / L diammonium hydrogen phosphate; Seed culture medium preparation: peptone 10 g / L, sodium chloride 10 g / L, yeast extract 5 g / L; Fermentation medium preparation: glycerol 5g / L, peptone 18g / L, yeast extract 24g / L, potassium dihydrogen phosphate 2.3g / L, dipotassium hydrogen phosphate 16g / L, defoamer (silicone oil) 0.8g / L; All the above culture media were sterilized at 121℃ for 30 minutes after preparation.

[0037] Example 3: Method for biopreparing estrone using 3-sterone-Δ¹-dehydrogenase In a 250 mL shake flask, add 2 g of the substrate open-ring compound 19-hydroxy-4-androstene-3,17-dione, 0.1 g of Tween 80, 0.1 g of 2-hydroxypropyl-β-cyclodextrin, and 30 mL of PBS buffer (10 mM pH 7.4). Sonicate the substrate with microwave to make it homogeneous, then add 4 g of KstD wet cells and 0.02 g of the electron transporter menadione. Incubate the mixture at 30 °C and 200 rpm on a shaker for 24 h.

[0038] The enzyme transfection reaction solution was collected, post-processed, and the contents of the ring-opening compound and estradiol were determined by HPLC. (See Table 1.) Figure 2 As shown, the conversion rate is approximately 99.86%. The NMR results are as follows... Figure 3 and Figure 4 As shown, the purity is 99.5%.

[0039] Example 4: Method for biopreparing estrone using 3-sterone-Δ¹-dehydrogenase In a 250 mL shake flask, add 1.5 g of the substrate open-ring compound 19-hydroxy-4-androstene-3,17-dione, 0.1 g of Tween 80, 0.1 g of 2-hydroxypropyl-β-cyclodextrin, and 30 mL of PBS buffer (10 mM pH 7.4). Sonicate the substrate with microwave to achieve a homogeneous state, then add 3 g of KstD wet cells and 0.015 g of the electron transporter menadione. Incubate the mixture at 30 °C and 200 rpm on a shaker for 24 h.

[0040] The enzyme transfection reaction solution was collected and post-processed. The contents of the ring-opening compound and estradiol were determined by HPLC. As shown in Table 1, the conversion rate was approximately 99.82%.

[0041] Example 5: Method for biopreparing estrone using 3-sterone-Δ¹-dehydrogenase In a 250 mL shake flask, add 3 g of the substrate ring opener, 0.1 g of Tween 80, 0.1 g of 2-hydroxypropyl-β-cyclodextrin, and 30 mL of PBS buffer (10 mM pH 7.4). Sonicate the substrate with microwave to make it homogeneous, then add 6 g of KstD wet cells and 0.03 g of the electron transporter menadione. Incubate the mixture at 30 °C and 200 rpm on a shaker for 24 h.

[0042] The enzyme transfection reaction solution was collected and post-processed. The contents of the ring-opening compound and estradiol were determined by HPLC. As shown in Table 1, the conversion rate was approximately 98.74%.

[0043] Example 6: Scale-up of the process for the biosynthesis of estrone using 3-sterone-Δ¹-dehydrogenase Add 80 g of the substrate open-ring compound 19-hydroxy-4-androstene-3,17-dione, 4 g of Tween 80, 4 g of 2-hydroxypropyl-β-cyclodextrin, and 1200 mL of PBS buffer (10 mM pH 7.4). Sonicate the substrate with microwave to make it homogeneous. Then add 160 g of KstD wet cells and 0.8 g of the electron transporter menadione. Incubate the mixture at 30 °C and 200 rpm on a shaker for 24 h.

[0044] The enzyme transfer reaction solution was collected, and enzyme transfer reaction solution post-treatment was performed. The content of the ring-opening product and the estrone in the enzyme transfer reaction solution was detected by HPLC. The conversion rate was about 98.52%.

[0045] Comparative Example 1 The difference between this comparative example and Example 3 is that the amount of KstD wet mycelium is too small, which is 0.1 g.

[0046] Comparative Example 2 The difference between this comparative example and Example 3 is that the amount of KstD wet mycelium is too large, which is 10 g.

[0047] Comparative Example 3 The difference between this comparative example and Example 3 is that the 3-steroidone-Δ1-dehydrogenase with the amino acid sequence shown in SEQ ID NO. 3 is used to replace the KstD wet mycelium in Example 3, and the wet mycelium is prepared according to Examples 1-2.

[0048] The amino acid sequence shown in SEQ ID NO. 3 is: MTTPOHISVDLLVVGSGTGLAAALAANELGLSVLVVEKTSLVGGSLARSGGAFWMPGNSILTAAGSSDTODYGRTYLDAVVDGDAPIERAHAFVEHGPATIEMLCRTTPMKFQWAKGYSDYHPEAPGGSAVGRTCECRPFDTAVLGDALARLRPGVMESDFPMPVTGADYRWLHLMVRVPRKSWPRILLRAAOGIGGLAMRRRYAAGGOALAAGLFAGVIDARIPVWTDAPVVDLLTEEGRVTGAIVARDGVKTOESARRGVVIAAGGFDNLMSWRHKFOSERLGEHLSLGGIGNTGDGIRLGODLGADTALMDOAWWFPAFAPLPGGEPTVMLAERSLPGCLLVIOTGORFINEATDYMTFGOILLRREOAGDPVEAMWMVFDORYRNSYLMAAELFPRMPIPOSWYDAGIAHRSDDLDGLAARIGASPOTLLATISRFNDLARSGVDDDFGRGASAYDRYYGDPTVTPNPNLRPLEKGPYYAVOVVLSDLGTCGGLRADTRGRVLREDGPPIEGLYAIGNTAANAFGKSYPGAGATIGOGFVFGYIAARHAAGRLP.

[0049] Performance detection 1. The enzyme conversion reaction liquid after 24h of transformation in example 3-6, comparative example 1~3 was detected by liquid chromatography (HPLC), and the detection results are shown in table 1.

[0050] Table 1 Liquid phase detection results of example 3-6, comparative example 1~3 transformation 24h sampling

[0051] It can be known from the comparison of example 3 and comparative example 1 that when the amount of KstD wet bacteria is too small, the conversion rate of estrone is significantly reduced, because the enzyme amount is too small to completely convert the substrate; It can be known from the comparison of example 3 and comparative example 2 that when the amount of KstD wet bacteria is too much, the conversion rate of estrone is significantly reduced, because when the enzyme amount is too much, the conversion system is too thick, which cannot be mixed well, affecting mass transfer and oxygen transfer.

[0052] It can be known from the comparison of example 3 and comparative example 3 that the conversion rate of estrone, the product of 3-ketosteroid-Δ1-dehydrogenase catalyzing the substrate 19-hydroxy-4-androstene-3,17-dione, is higher.

[0053] In summary, the present application provides a method for biologically preparing estrone by using 3-ketosteroid-Δ1-dehydrogenase. The whole synthesis process of the present application is simple, has few steps, short cycle, less side reaction, mild reaction condition, does not require high equipment, and is pollution-free and green.

[0054] The above only describes the preferred embodiments of the present application, and does not limit the present application, any modification, equivalent replacement, improvement, etc. within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A method for the biosynthesis of estradiol using 3-sterone-Δ¹-dehydrogenase, characterized in that, This includes the reaction of 19-hydroxy-4-androstene-3,17-dione with 3-sterone-Δ¹-dehydrogenase KstD to obtain estradiol; The amino acid sequence of the 3-sterone-Δ¹-dehydrogenase KstD is shown in SEQ ID NO.

1.

2. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 1, characterized in that, The 3-sterone-Δ¹-dehydrogenase KstD also includes a 3-sterone-Δ¹-dehydrogenase KstD with the same function obtained by substituting, deleting and / or adding one or more amino acids to the amino acid sequence of the 3-sterone-Δ¹-dehydrogenase KstD.

3. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 1, characterized in that, The nucleotide sequence of the 3-sterone-Δ¹-dehydrogenase KstD is shown in SEQ ID NO.

2.

4. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 1, characterized in that, The reaction also includes the addition of Tween 80, menaquinone, and PBS at pH 7.

4.

5. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 1, characterized in that, The process includes the following steps: fermenting engineered bacteria expressing the 3-sterone-Δ¹-dehydrogenase KstD to obtain wet cells.

6. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 1, characterized in that, The reaction was carried out at 30°C and 200 rpm for 24 hours.

7. The method for biopreparing estradiol using 3-sterone-Δ¹-dehydrogenase as described in claim 5, characterized in that, The amount of 19-hydroxy-4-androstene-3,17-dione used is 50 g / L to 100 g / L, and the mass ratio of the wet bacterial cells to 19-hydroxy-4-androstene-3,17-dione is 2:1.

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