Progesterone C21 hydroxylase, coding nucleic acid, expression vector, bioengineering strain, composition, method and application

Abstract

The invention provides progesterone C21 hydroxylase, coding nucleic acid, an expression vector, a bioengineering strain, a composition, a method and application. The amino acid sequence of the hydroxylase at the C21 site of the progesterone is as shown in SEQ ID NO. 1. The hydroxylase CYP46A35 at the progesterone C21 site can be used for specifically and efficiently catalyzing the progesterone C21 to synthesize the 11-deoxycorticosterone under mild and environment-friendly conditions. The invention also establishes a bioengineering strain based on CYP46A35 and a biocatalytic synthesis method, and realizes a novel biocatalytic approach for synthesizing 11-deoxycorticosterone, which is simple, efficient, low in cost and environment-friendly.

Description

Technical Field

[0001] The present invention relates to the field of biosynthesis, and in particular to a progesterone C21 hydroxylase, encoding nucleic acid, expression vector, bioengineering strain, composition, method and application. Background Art

[0002] Steroidal compounds are widely found in animals, plants, and microorganisms and possess important biological activities. Global sales of steroidal products currently exceed $150 billion annually, and market demand in the pharmaceutical sector is second only to antibiotics. Over 400 licensed steroid drugs are in circulation, and demand is expected to increase in the future. Steroid hormones are a very important class of compounds, essential for maintaining normal human physiological functions. Steroid hormones primarily include sex hormones and adrenal cortex hormones. Sex hormones can be divided into three categories: androgens, estrogens, and progestins. Androgens and estrogens promote the development of male and female sexual organs and are crucial for maintaining secondary sexual characteristics. Progestins primarily regulate the cyclical changes in the endometrium and support pregnancy. Adrenal cortex hormones are divided into glucocorticoids and mineralocorticoids. Glucocorticoids are involved in carbohydrate, protein, and lipid metabolism and can exert anti-allergic, anti-shock, and anti-inflammatory effects, while mineralocorticoids maintain electrolyte balance and regulate blood pressure. 11-Deoxycorticosterone exhibits mineralocorticoid-like chemical activity, promoting potassium ion efflux while also regulating the synthesis and secretion of the mineralocorticoid aldosterone to maintain electrolyte balance. 11-Deoxycorticosterone also exhibits partial progestogenic activity. This compound is an important intermediate linking progesterone and adrenal cortical hormones and a key synthetic precursor for numerous corticosteroid compounds, such as the mineralocorticoid aldosterone.

[0003] Structurally, steroids are a class of compounds consisting of a cyclopentahydrophenanthrene nucleus consisting of three adjacent six-membered rings and one five-membered ring, with various functional groups substituted at different positions on the nucleus. The type, configuration, and location of the substituents on the nucleus determine the properties and functions of different steroid compounds. The hydroxyl group is often a characteristic functional group of a particular class of steroid compounds; for example, the C21 hydroxyl group is a common feature of all adrenocortical hormones.

[0004] How to achieve specific and efficient hydroxylation at specific sites in steroidal compounds has always been a core issue in the field of steroidal drug synthesis. Currently, there are two main methods for hydroxylation modification on the steroidal backbone: chemical synthesis and biocatalysis. Currently, for hydroxylation modification at the C21 position, the chemical method mainly involves halogenation of the C21 methyl group, followed by alkaline hydrolysis to form the 21-hydroxyl group. However, halogenating agents are highly environmentally polluting and are not environmentally friendly. Compared with chemical synthesis methods, biocatalysis has the advantages of mild reaction conditions, environmental friendliness, and higher regioselectivity and stereoselectivity. However, hydroxylation modification at the C21 position has only been reported for the animal-derived enzyme CYP21A2, and the characterization of its use in the biocatalytic production of 11-deoxycorticosterone from progesterone in Saccharomyces cerevisiae is unclear. Summary of the Invention

[0005] To solve the problems existing in the above-mentioned prior art, the present invention provides a progesterone C21 hydroxylase, encoding nucleic acid, expression vector, bioengineering strain, composition, method and application.

[0006] Specifically, the present invention provides:

[0007] (1) A progesterone C21 hydroxylase, characterized in that the amino acid sequence of the progesterone C21 hydroxylase is shown in SEQ ID NO.1.

[0008] (2) A nucleic acid encoding the progesterone C21-hydroxylase according to (1).

[0009] (3) The nucleic acid according to (2), wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.2.

[0010] (4) An expression vector, characterized in that the expression vector comprises the nucleic acid according to (2) or (3).

[0011] (5) The expression vector according to (4), wherein the expression vector comprises a constitutive promoter HXT7p upstream of the nucleic acid encoding the progesterone C21-hydroxylase according to (1).

[0012] (6) The expression vector according to (4), wherein the backbone of the expression vector is pRS426 or pRS425.

[0013] (7) An expression vector composition comprising a first expression vector and a second expression vector, wherein the first expression vector is the expression vector according to any one of (4) to (6), and the second expression vector comprises a coding sequence for a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0014] (8) The expression vector composition according to (7), wherein the second expression vector comprises a constitutive promoter TEF1p upstream of the coding sequence of the cytochrome P450 enzyme reductase; preferably, the backbone of the second expression vector is pRS426 or pRS425.

[0015] (9) An enzyme composition comprising the progesterone C21-hydroxylase according to (1) and a cytochrome P450 enzyme reductase.

[0016] (10) The enzyme composition according to (9), wherein the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0017] (11) A bioengineering strain, characterized in that the bioengineering strain expresses the progesterone C21 hydroxylase according to (1); preferably, the bioengineering strain also expresses a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0018] (12) The bioengineered strain according to (11), wherein the bioengineered strain is selected from Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

[0019] (13) A method for preparing 11-deoxycorticosterone, comprising contacting the progesterone C21-hydroxylase according to (1) with progesterone in the presence of an electron donor, thereby performing a catalytic reaction to produce 11-deoxycorticosterone.

[0020] (14) The method according to (13), wherein the electron donor is a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0021] (15) The method according to (13), wherein the catalytic reaction is carried out at 28-30°C for 2-3 days, preferably for 2 days.

[0022] (16) The method according to (13), wherein the method comprises the following steps:

[0023] 1) Providing the bioengineered strain according to (11) or (12);

[0024] 2) Providing a mixed culture of the bioengineered strain and progesterone, and incubating the mixed culture; preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

[0025] (17) The method according to (16), wherein in step 2), the OD of the bioengineered strain is 600 The final concentration of progesterone in the mixed culture is 200 μM to 1 mM; the final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is 0.5-1 mM.

[0026] (18) The method according to (16), wherein the incubation is carried out at 28-30°C and 220-250 rpm for 2-3 days, preferably, the incubation is carried out at 30°C and 220 rpm for 2 days.

[0027] (19) The method according to (13), wherein the method further comprises purifying 11-deoxycorticosterone from the system obtained by the catalytic reaction.

[0028] (20) Use of the progesterone C21 hydroxylase described in (1), the nucleic acid described in (2) or (3), the expression vector described in any one of (4) to (6), the expression vector composition described in (7) or (8), the enzyme composition described in (9) or (10), or the bioengineered strain described in (11) or (12) in the preparation of 11-deoxycorticosterone.

[0029] Compared with the prior art, the present invention has the following advantages and positive effects:

[0030] 1. Through enzyme exploration and screening, the present invention discovered a new progesterone C21-hydroxylase CYP46A35, which can catalyze the reaction under mild and environmentally friendly conditions; and the enzyme has strong regioselectivity and can specifically and efficiently catalyze the hydroxylation of progesterone C21, thereby synthesizing 11-deoxycorticosterone.

[0031] 2. The present invention also developed a bioengineered strain expressing the progesterone C21-hydroxylase of the present invention and a biocatalytic synthesis method, which can biocatalyze the synthesis of 11-deoxycorticosterone from progesterone with a yield of up to 87%. Furthermore, strain growth and biocatalytic synthesis require only two days, without the need for medium or buffer changes, to achieve a progesterone conversion rate of 95.1±0.1% and a product selectivity of 91.6±0.6%. The reaction system of the present invention is simple, efficient, easy to operate, low-cost, and environmentally friendly, with broad prospects for industrial application.

[0032] 3. The progesterone C21 hydroxylase of the present invention provides a new candidate enzyme for synthesizing 11-deoxycorticosterone; the bioengineered strain and biocatalytic synthesis method of the present invention provide a new biocatalytic pathway for synthesizing 11-deoxycorticosterone.

[0033] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The reaction equation for the synthesis of 11-deoxycorticosterone from progesterone catalyzed by the progesterone C21 hydroxylase CYP46A35 of the present invention is shown.

[0035] Figure 2 The HPLC chromatograms of the products obtained by the catalytic reaction using the bioengineered strain of the present invention in Example 4 of this application are shown. i) is the HPLC curve of a progesterone standard; ii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain F (WAT11U (RnCPR)); and iii) is the HPLC curve of the product obtained by the catalytic reaction using bioengineered strain A (WAT11U (RnCPR+CYP46A35)).

[0036] Figure 3 The diagram shows an amino acid sequence alignment of the progesterone C21 hydroxylase CYP46A35 and the enzyme CYP21A2 of the present invention.

[0037] Figure 4 Shown is the high-resolution mass spectrum of 11-deoxycorticosterone obtained in Example 4 of the present application.

[0038] Figure 5 The nuclear magnetic resonance spectrum of the product 11-deoxycorticosterone obtained in Example 4 of the present application (deuterated methanol solution) is shown. 1 H NMR spectrum (600MHz); Figure B is 13 C NMR spectrum (150MHz); Figure C is 1 H- 1 H COSY NMR spectrum; Figure D is the HSQC NMR spectrum; Figure E is the HMBC NMR spectrum. The structural formula of the product 11-deoxycorticosterone is shown below:

[0039] DETAILED DESCRIPTION

[0040] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0041] It should be understood that the above-mentioned technical features of the present invention and the technical features specifically described below (including but not limited to implementation plans and examples) can be combined with each other in any appropriate manner to form a new or preferred technical solution, as long as there is no contradiction and the combined technical solution can be successfully implemented and can solve the technical problem of the present invention. The any appropriate manner shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the corresponding technical effect.

[0042] In the present invention, words such as “including”, “further”, “having”, and “further” merely indicate better or more specific implementation methods or examples, and should be understood that they do not constitute a limitation on the scope of protection of the present invention.

[0043] In the present invention, "and / or" means any one or any combination of the listed items.

[0044] Unless otherwise specified, the numerical ranges in the present invention include both endpoints.

[0045] According to one aspect of the present invention, a new progesterone C21 hydroxylase is provided, which is a newly discovered progesterone C21 hydroxylase CYP46A35 from the Chinese giant toad (Bufo bufo gargarizans). It belongs to a different family from the enzyme CYP21A2, and its sequence identity is only 20.29% (e.g. Figure 3 The progesterone C21 hydroxylase of the present invention can specifically and efficiently catalyze the synthesis of 11-deoxycorticosterone from progesterone, and the reaction equation is as follows: Figure 1 shown.

[0046] The amino acid sequence of the progesterone C21 hydroxylase CYP46A35 according to the present invention is shown in SEQ ID NO.1.

[0047] SEQ ID NO.1:

[0048] MELLGLVSWLLLLLLLTLVVTCFLLYCGYIHYQHMKYDHIPGPPRESFLFGHTSAIWKVMRKNQLVYDLFLNWVETYGPVIRINALHKVTILSVSPESVKDVLMSPKYRKDWFYDNLHTLFGVRLMG NGLVTDRDNDHWYKQRRIMDPAFSRTYLIGLLGPFNEKAEELMERLAEEADGRSHVVMHTMMSRVTLDVISKVAFGMETNSLKDDGTPLPRAISLVMRAFVEMRDPFIRYSREKQAFIRDVQESIRL LRKTGRECIERRQKAIQDGEEIPVDILTQILKGAALEGDCDMEDLLDNFVTFFIAGQETTANQLAFTIMELARNPKILEKTQAEVDEVIGVKRDIEYNDLGKLQYLSQVLKESLRLYPTAPGTSRE IEEETIIEGFRIPPKVNLMFNSYIMGRMQQLYPDPLTFNPERFHPDAPKPYYSYFPFSLGPRSCIGQVFAQMEAKVIMAKLLQRFQFELVEGQSFGIIDTASLRPKSGVICRLTIRTNPGEGKKVD*

[0049] The present invention also provides a nucleic acid encoding the progesterone C21 hydroxylase of the present invention. The nucleic acid may be DNA or RNA.

[0050] In some embodiments of the present invention, the nucleic acid comprises a nucleotide sequence encoding the progesterone C21 hydroxylase CYP46A35 of the present invention. Preferably, the nucleotide sequence is as shown in SEQ ID NO. 2.

[0051] SEQ ID NO.2:

[0052]

[0053] According to another aspect of the present invention, an expression vector is provided, which comprises the nucleic acid of the present invention to encode the progesterone C21 hydroxylase of the present invention.

[0054] Preferably, in the expression vector, the present invention utilizes the constitutive strong promoter HXT7p to enhance the expression of C21 hydroxylase CYP46A35. In some preferred embodiments, the constitutive strong promoter HXT7p is located upstream of the nucleotide sequence encoding progesterone C21 hydroxylase CYP46A35.

[0055] The backbone of the expression vector can be pRS426 or pRS425. According to some specific embodiments of the present invention, the backbone of the vector can be modified to be more suitable for expressing the enzyme CYP46A35 of the present invention by inserting a specific promoter and terminator. For example, pRS426 is constructed as pRS426-HXT7p-FBA1t by inserting the strong constitutive promoter HXT7p and the terminator FBA1t (the vector comes with Ura3 for screening positive clones and retaining plasmids).

[0056] In some preferred embodiments, the present invention also constructs an expression vector comprising a coding sequence for a cytochrome P450 reductase. Preferably, the cytochrome P450 reductase is selected from Ncp1 and RnCPR. These cytochrome P450 reductases can provide efficient electron donors for the biocatalysis of progesterone C21 hydroxylase CYP46A35.

[0057] Preferably, the cytochrome P450 enzyme reductase Ncp1 is derived from Saccharomyces cerevisiae, and the RnCPR is derived from Rattus norvegicus.

[0058] Preferably, the amino acid sequences of cytochrome P450 enzyme reductase Ncp1 and RnCPR are shown as SEQ ID NOs. 3-4, respectively.

[0059] The nucleotide sequences encoding the above cytochrome P450 enzyme reductase are shown in SEQ ID NOs. 5-6 respectively.

[0060] Also preferably, the expression vector further comprises a constitutive strong promoter TEF1p upstream of the nucleic acid encoding the cytochrome P450 enzyme reductase to overexpress the cytochrome P450 enzyme reductase.

[0061] The backbone of the expression vector containing the cytochrome P450 enzyme reductase can be pRS426 or pRS425. According to some specific embodiments of the present invention, the backbone of the vector can be modified to be more suitable for expressing the cytochrome P450 enzyme reductase by inserting a specific promoter and terminator. For example, pRS425 is constructed as pRS425-TEF1p-PGK1t by inserting the constitutive promoter TEF1p and the terminator PGK1t (the vector comes with Leu2 for screening positive clones and retaining plasmids).

[0062] Therefore, according to another aspect of the present invention, an expression vector composition is provided, comprising a first expression vector and a second expression vector, wherein the first expression vector is the expression vector comprising the nucleic acid encoding the progesterone C21 hydroxylase as described in the present invention, and the second expression vector is the above-mentioned expression vector comprising the coding sequence of the cytochrome P450 enzyme reductase.

[0063] According to another aspect of the present invention, an enzyme composition is provided, comprising the progesterone C21 hydroxylase according to the present invention and a cytochrome P450 enzyme reductase. Preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0064] The progesterone C21 hydroxylase of the present invention can efficiently synthesize 11-deoxycorticosterone with cytochrome P450 enzyme reductase as an electron donor.

[0065] According to another aspect of the present invention, a bioengineering strain is provided, which expresses the progesterone C21 hydroxylase according to the present invention.

[0066] Preferably, the bioengineered strain further expresses a cytochrome P450 enzyme reductase. Preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

[0067] The bioengineering strain can be made to express the progesterone C21 hydroxylase and / or cytochrome P450 reductase by comprising the expression vector of the present invention, for example, by transforming the expression vector of the present invention into the bioengineering strain.

[0068] In some specific embodiments, the bioengineered strains of the present invention can be constructed by the following methods:

[0069] Step (1): obtaining the progesterone C21 hydroxylase CYP46A35 gene from Bufo bufo gargarizans; obtaining the cytochrome P450 enzyme reductase CPR gene;

[0070] Step (2): cloning the progesterone C21-hydroxylase CYP46A35 gene and the CPR gene obtained in step (1) into expression vectors respectively, and screening positive plasmids;

[0071] Step (3): The positive plasmids obtained in step (2) are respectively transferred into host bacteria to construct the bioengineered strain according to the present invention.

[0072] In some embodiments of the present invention, the cytochrome P450 enzyme reductase (CPR) gene in step (1) above can be selected from Ncp1 obtained by genome amplification of Saccharomyces cerevisiae strain BY4742 and RnCPR from Rattus norvegicus.

[0073] In some embodiments of the present invention, the host bacteria in step (3) above can be selected from Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

[0074] According to a preferred embodiment of the present invention, the bioengineered strain of the present invention can be constructed by the following method:

[0075] Step (1): using the cDNA of the postauricular gland tissue of Bufo bufo gargarizans as a template, obtaining the progesterone C21-hydroxylase CYP46A35 gene cyp46a35 by PCR amplification; using the genome of the yeast Saccharomyces cerevisiae strain BY4742 as a template, obtaining the cytochrome P450 enzyme reductase CPR gene ncp1 by PCR amplification; or obtaining the cytochrome P450 enzyme reductase CPR gene RnCPR of Rattus norvegicus by gene synthesis;

[0076] Step (2): The cyp46a35 gene and CPR gene obtained in step (1) were cloned into the yeast expression vectors pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2, respectively, and positive plasmids were screened;

[0077] Step (3): The positive plasmids obtained in step (2) are respectively transferred into host bacteria, thereby constructing the bioengineered strain of the present invention.

[0078] In some embodiments, the bioengineered strains of the present invention can be cultured by the following method, comprising: culturing the bioengineered strain in YPD medium at 28°C to 30°C, 220 rpm to 250 rpm, for 2 to 3 days. Preferably, the culture conditions are 30°C, 220 rpm, and 2 days. SD-Ura-Leu medium can also be used for culture.

[0079] According to another aspect of the present invention, a method for preparing 11-deoxycorticosterone is provided, wherein the progesterone C21 hydroxylase of the present invention is contacted with progesterone in the presence of an electron donor, thereby performing a catalytic reaction to produce 11-deoxycorticosterone.

[0080] The structural formulas of the substrate progesterone and the reaction product 11-deoxycorticosterone are shown below:

[0081]

[0082] Preferably, the electron donor is the cytochrome P450 enzyme reductase.

[0083] Preferably, the catalytic reaction is carried out at 28-30°C for 2-3 days, more preferably at 30°C for 2 days.

[0084] In the method for preparing 11-deoxycorticosterone, the progesterone C21 hydroxylase can be extracted and purified from the bioengineered bacteria of the present invention, or can be present in the bioengineered bacteria. The extraction and purification can be performed using methods known in the art.

[0085] In a preferred embodiment of the present invention, the progesterone C21 hydroxylase is present in the bioengineered bacteria of the present invention, and the method comprises the following steps:

[0086] 1) Providing the bioengineered strain of the present invention;

[0087] 2) providing the bioengineered strain, progesterone, and a mixed culture, and incubating the mixed culture. Preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

[0088] Preferably, in step 2), the OD of the bioengineered strain is 600The final concentration of progesterone in the mixed culture is 0.1 to 0.2; when the final concentration of progesterone is higher than 1 mM, the reaction conversion rate may decrease. 5-aminolevulinic acid hydrochloride is a precursor for heme synthesis, and heme is a coenzyme for the P450 enzyme (progesterone C21 hydroxylase). When the P450 enzyme is overexpressed, the heme synthesized in the body may be insufficient. Therefore, the additional addition of 5-aminolevulinic acid hydrochloride can supplement heme. The final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is preferably 0.5-1 mM.

[0089] Also preferably, the incubation is performed at 28-30° C. and 220-250 rpm for 2-3 days; more preferably, the incubation is performed at 30° C. and 220 rpm for 2 days.

[0090] The incubation can be performed on a thermostatic shaker.

[0091] In some specific embodiments of the present invention, step 2) comprises the following steps:

[0092] a) inoculating the bioengineered strain into SD-Ura-Leu medium and culturing until OD 600 is about 2.0;

[0093] b) Inoculate into YPD medium at 5% (volume) to make OD 600 is about 0.1;

[0094] c) adding progesterone and 5-aminolevulinic acid hydrochloride, and incubating the mixed culture.

[0095] In some specific embodiments of the present invention, the method of catalyzing the synthesis of 11-deoxycorticosterone from progesterone of the present invention comprises the following steps:

[0096] Step (1): dissolving progesterone in a reaction solvent to obtain a progesterone solution;

[0097] Step (2): mixing the progesterone solution obtained in step (1) at a concentration of 1% (V / V) with the bioengineered strain culture of the present invention and incubating the mixture;

[0098] Step (3): The mixture of step (2) is shaken on a shaker for microbial catalysis to obtain the product 11-deoxycorticosterone.

[0099] The reaction solvent in step (1) can be methanol or N,N-dimethylformamide. Those skilled in the art will appreciate that other solvents can also be used in the method of the present invention, as long as the biocatalytic reaction can be achieved to obtain 11-deoxycorticosterone.

[0100] The method for preparing 11-deoxycorticosterone of the present invention may further comprise the step of purifying 11-deoxycorticosterone from the obtained reaction product. The purification method may be performed by ethyl acetate extraction, followed by separation and purification of 11-deoxycorticosterone from the catalytic reaction product by, for example, preparative reverse phase column chromatography.

[0101] Preferably, the ethyl acetate extraction method is carried out by the following steps:

[0102] 1) The reaction product was centrifuged and the supernatant was extracted twice with twice the volume of ethyl acetate;

[0103] 2) Add acetone to the cells and then perform ultrasonic disruption;

[0104] 3) removing ethyl acetate and acetone by rotary evaporation to obtain a crude product;

[0105] 4) The crude product was redissolved in methanol and subjected to coarse separation by normal phase column chromatography, and then subjected to secondary separation by preparative liquid chromatography.

[0106] In step 4), separation can also be performed directly using preparative liquid chromatography.

[0107] The progesterone C21-hydroxylase CYP46A35 discovered in the present invention can efficiently catalyze progesterone to produce the product 11-deoxycorticosterone. This enzyme has strong regioselectivity and can specifically catalyze the C21-hydroxylation of progesterone, producing only a small amount of byproducts (95.1±0.1% conversion rate, 91.6±0.6% target product selectivity). In addition, the method for preparing 11-deoxycorticosterone of the present invention improves the yield of biocatalytic progesterone C21-hydroxylation to 11-deoxycorticosterone through chassis strain optimization and CPR screening and comparison strategies, achieving a yield as high as 87%, and achieving a progesterone conversion rate of 95.1±0.1% and a product selectivity of 91.6±0.6%.

[0108] According to another aspect of the present invention, further provided are uses of the progesterone C21 hydroxylase, the nucleic acid, the expression vector, the expression vector composition, the enzyme composition, and the bioengineered strain of the present invention in the preparation of 11-deoxycorticosterone.

[0109] The present invention will be further described in detail below in conjunction with the examples of the present invention. The following examples are illustrative, not restrictive, and the scope of protection of the present invention should not be limited by the following examples. The examples of the present invention are implemented under the premise of the technical solution of the present invention, and detailed implementation schemes and specific operating processes are given, but the scope of protection of the present invention is not limited to the following examples.

[0110] Example

[0111] In the following examples, the experimental methods without specific steps and conditions were carried out according to conventional methods and conditions known in the art, or according to the methods and conditions recommended by the manufacturers.

[0112] Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples can be obtained from conventional commercial sources.

[0113] The sources or formulas of the following reagents are as follows:

[0114] The pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2 plasmids can be constructed according to the reference "Wang, WF; Xiao, H.; Zhong, JJ, Biosynthesis of a ganoderic acid in Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganodermalucidum. Biotechnol Bioeng 2018, 115(7), 1842-1854"; they can also be constructed by inserting the yeast-derived promoter HXT7p and terminator FBA1t into the pRS426 plasmid, and by inserting the yeast-derived promoter TEF1p and terminator PGK1t into the pRS425 plasmid. Plasmids pRS425 and pRS426 can be purchased from Addgene.

[0115] Progesterone: purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0116] Methanol: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0117] YPD medium: 10 g yeast extract, 20 g peptone, 20 g glucose, dilute to 1 L.

[0118] SD-Ura-Leu medium: Purchased from Shanghai Shaoxin Biotechnology Co., Ltd. Add 8 g of powder and dilute to 950 mL. For solid medium, add 20 g of agar powder per liter. Adjust the pH to 6-6.5. Add glucose to a final concentration of 2% before use.

[0119] Ethyl acetate was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0120] 5-Aminolevulinic acid hydrochloride was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.

[0121] Saccharomyces cerevisiae BY4742 strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0122] Saccharomyces cerevisiae BJ5464-NpgA strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0123] Saccharomyces cerevisiae YRC01 strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0124] Saccharomyces cerevisiae WAT11U strain: purchased from Shanghai Baosai Biotechnology Co., Ltd.

[0125] Example 1. Construction of progesterone C21 hydroxylase CYP46A35 expression plasmid

[0126] 1. Extract RNA from the post-auricular gland tissue of Bufo bufo gargarizans and obtain cDNA through reverse transcription. According to the DNA sequence of CYP46A35 (SEQ ID NO.2), primers CYP46A35-F and CYP46A35-R were designed to amplify the CYP46A35 gene, and the CYP46A35 gene fragment was obtained by PCR amplification. Using the genomic DNA of Saccharomyces cerevisiae BY4742 strain as a template, the cytochrome P450 enzyme reductase gene ncp1 fragment was amplified using primers Ncp1-F and Ncp1-R. Jiutian Company synthesized the cytochrome P450 enzyme reductase gene RnCPR fragment of Rattus norvegicus, and amplified the gene fragment using primers RnCPR-F and RnCPR-R. The nucleotide sequences of the above CPR are shown in SEQ ID NO.5 and 6, respectively, and the primers used are as follows:

[0127] CYP46A35-F:tttttaattttaatcaaaaaATGGAGCTGCTGGGCCTGG (SEQ ID NO.7)

[0128] CYP46A35-R:atcaattaatttgaattaacgtttTTAGTCAACCTTCTTCCTTC (SEQ ID NO.8)

[0129] Ncp1-F:atctaagttttaattacaaaatgccgtttggaatagac (SEQ ID NO.9)

[0130] Ncp1-R: cgatttcaattcaattcaatgtttttaccagacatcttcttg (SEQ ID NO.10)

[0131] RnCPR-F:atctaagttttaattacaaaATGGGTGATTCTCATGAAG(SEQ ID NO.11)

[0132] RnCPR-R:cgatttcaattcaattcaatgtttTTAAGACCAAACATCTAAAG (SEQ ID NO.12)

[0133] 2. Construction of expression vector containing CYP46A35 enzyme gene and CPR gene

[0134] The linearized plasmid fragments pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2 were obtained by single-restriction digestion with the restriction endonuclease MssI. Using a one-step cloning method, the CPR gene amplified fragment was homologously recombined with the linearized plasmid vector pRS425-TEF1p-PGK1t-Leu2; the CYP46A35 gene was homologously recombined with the linearized plasmid vector pRS426-HXT7p-FBA1t-Ura3. The products were transformed into competent Escherichia coli DH5α cells. Transformants were screened on LB plates containing 100 μg / mL ampicillin, and positive clones were identified by sequencing to obtain the recombinant expression vector.

[0135] Example 2. Construction of a bioengineered strain expressing CYP46A35

[0136] The recombinant expression vector obtained in Example 1 was extracted. The pRS426-HXT7p-FBA1t-Ura3 and pRS425-TEF1p-PGK1t-Leu2 empty vectors were used as controls to transform competent cells of four Saccharomyces cerevisiae strains, BY4742, BJ5464-NpgA, YRC01, and WAT11U, respectively. These Saccharomyces cerevisiae strains do not express the CYP46A35 enzyme and the cytochrome P450 enzymes ncp1 and RnCPR. Transformants were screened using uracil and leucine double-deficient plates. Table 1 shows the resulting bioengineered strains.

[0137] Table 1. Bioengineered strains AH

[0138] Bioengineered strains Transformed strains Progesterone C21-hydroxylase Cytochrome P450 enzyme reductase A WAT11U CYP46A35 RnCPR B BY4742 CYP46A35 Ncp1 C BJ5464-NpgA CYP46A35 Ncp1 D YRC01 CYP46A35 Ncp1 E WAT11U CYP46A35 Ncp1 F (control group) WAT11U none RnCPR G (pRS426 blank group) WAT11U none none H (pRS425 blank group) WAT11U none none

[0139] Bioengineered strains AE were transformed with recombinant expression vectors containing the CYP46A35 gene and the corresponding CPR gene. As a control, bioengineered strain F was transformed only with the recombinant expression vector containing the cytochrome P450 reductase RnCPR, thus failing to express the progesterone C21-hydroxylase CYP46A35. Bioengineered strains G and H were transformed only with the empty vectors pRS426-HXT7p-FBA1t-Ura3 or pRS425-TEF1p-PGK1t-Leu2, thus failing to express either the progesterone C21-hydroxylase CYP46A35 or the cytochrome P450 reductase.

[0140] Example 3. Homology Analysis of Progesterone C21 Hydroxylase CYP46A35

[0141] Progesterone C21 hydroxylase CYP46A35 and CYP21A2 belong to different P450 enzyme families. Figure 3 As shown, the amino acid sequence alignment of progesterone C21 hydroxylase CYP46A35 and enzyme CYP21A2 showed that the sequence identity between the two was only 20.29%, with significant differences.

[0142] Example 4. Using the bioengineered strain of the present invention to catalyze the synthesis of 11-deoxycorticosterone from progesterone

[0143] Progesterone was dissolved in methanol at a progesterone concentration of 20 mM.

[0144] The bioengineered strain A constructed in Example 2 was inoculated into SD-Ura-Leu medium and cultured at 30°C and 220 rpm until the OD 600 The OD value is about 2.0; 5% (volume) inoculation was inoculated into YPD medium (1% yeast extract, 2% peptone, 2% glucose). 600 The final concentration of 5-aminolevulinic acid hydrochloride and progesterone in methanol were about 0.1, and the final concentration of 5-aminolevulinic acid hydrochloride and progesterone in methanol were added at the same time, so that the final concentration of progesterone was 200 μM; the culture was shaken at 30°C and 220 rpm for 2 days and cultured until the OD 600 The HgCl2O3 concentration was about 6.67, and a biocatalytic system solution was obtained.

[0145] The biocatalytic system solution was extracted twice with twice the volume of ethyl acetate and centrifuged at 15,000 rpm for 15 minutes. The ethyl acetate layer was evaporated in a rotary concentrator (1,000 rpm, 30°C). Methanol was added for reconstitution and centrifuged at 15,000 rpm for 15 minutes. The supernatant was filtered through a 0.22 μm filter to obtain the product solution.

[0146] The product solution was detected by high performance liquid chromatography (HPLC). HPLC detection conditions were as follows: Agilent 1260 InfinityII liquid chromatography system, C18 reverse phase column (model: InfinityLab Poroshell 120); mobile phase A: 0.1% formic acid in water, mobile phase B: acetonitrile, gradient elution; column temperature: 30°C, elution rate: 0.5 mL / min, diode array detector (DAD) UV detection wavelength: 240 nm.

[0147] As a control, the bioengineered strains F, G, and H constructed in Example 2 were used to react with progesterone as a substrate using the same method as above and detected by HPLC.

[0148] The HPLC results were as follows Figure 2 As shown, i) is the HPLC curve of the progesterone standard; iii) is the HPLC curve of the biocatalytic reaction product of the bioengineered strain A (WAT11U (RnCPR+CYP46A35)), which has been transformed with the progesterone C21 hydroxylase CYP46A35 gene and the cytochrome P450 enzyme reductase RnCPR gene. The reaction product curve shows a new peak different from progesterone. The components of this peak were verified by high-resolution mass spectrometry and nuclear magnetic resonance (see the data in Example 5 and Figure 4-5 ), confirming its structure as 11-deoxycorticosterone. Curve iii) shows that only a very small amount of progesterone remains in the product, indicating that bioengineered strain A can efficiently and specifically biocatalyze the conversion of progesterone to 11-deoxycorticosterone. ii) is the HPLC curve of the biocatalytic reaction product of control bioengineered strain F (WAT11U(RnCPR)), which was transformed with only the cytochrome P450 reductase RnCPR gene but not the progesterone C21-hydroxylase CYP46A35 gene. The reaction product curve shows only a progesterone peak, but no 11-deoxycorticosterone peak, indicating that bioengineered strain F, which does not have the progesterone C21-hydroxylase CYP46A35 gene, cannot catalyze the conversion of progesterone to 11-deoxycorticosterone.

[0149] In addition, HPLC detection found that yeast engineered strains G and H were also unable to catalyze the biotransformation of progesterone.

[0150] The above results also prove that the bioengineered strain constructed by the present invention successfully transcribed and expressed the transferred progesterone C21-hydroxylase CYP46A35 gene and cytochrome P450 enzyme reductase gene, and that both expressed enzymes can exert the desired biological activity and effectively play the role of substrate catalysis and electron transfer.

[0151] The results of catalytic synthesis of 11-deoxycorticosterone using the bioengineered strain AE constructed in Example 2 by the same method as in this example are listed in Table 2.

[0152] Table 2. Results of 11-deoxycorticosterone synthesis catalyzed by bioengineered strain AE

[0153] Bioengineered strains Progesterone conversion rate (%) Product selectivity (%) 11-Deoxycorticosterone yield (%) A 95.1±0.1 91.6±0.6 87.1 B 22.7±0.1 92±0.5 20.9 C 10.9±0.7 94.1±0.2 10.3 D 53±0.5 97.1±0.1 51.5 E 76.1±7.6 91.7±0.3 69.8

[0154] The above results indicate that the bioengineered strains of the present invention can catalyze the synthesis of 11-deoxycorticosterone with product selectivity exceeding 91%. Strains A and E, whose chassis is WAT11U, exhibit higher progesterone conversion rates and 11-deoxycorticosterone yields than other chassis strains. In particular, strain A, which expresses RnCPR, exhibits higher progesterone conversion rates and 11-deoxycorticosterone yields than strain E, which expresses Ncp1.

[0155] Therefore, the present invention utilizes the newly discovered progesterone C21 hydroxylase CYP46A35 to optimize the fermentation chassis strain and conduct CPR comparative screening. The catalytic reaction can be carried out under mild and environmentally friendly conditions. In addition, the enzyme has strong regioselectivity and can specifically and efficiently catalyze the hydroxylation of progesterone C21, thereby synthesizing 11-deoxycorticosterone.

[0156] Example 5. High-resolution mass spectrometry and nuclear magnetic resonance verification of the product

[0157] The product solution obtained in Example 4 was separated on a C18 X-bridge (5 μm, 10 × 250 mm) chromatographic column using an Agilent 1260 Infinity II preparative HPLC system (Agilent Technologies, USA). The purity and precise molecular weight of the compound were determined using an Agilent 6120 Quadruple HPLC-HRMS system (Agilent Technologies, USA) under essentially the same conditions as for the HPLC detection of the compound (except that the flow rate of the HPLC-HRMS was 0.4 mL / min). Finally, the isolated high-purity compound was evaporated to dryness, dissolved in deuterated methanol, and subjected to nuclear magnetic resonance (NMR) spectroscopy. Its structure was analyzed based on its molecular weight and nuclear magnetic spectrum.

[0158] The structure of the reaction product of Example 4 is analyzed as follows.

[0159] High-resolution mass spectrometry calculated value C of 11-deoxycorticosterone 21 H 30 O3[M+H] + : 331.2268, high resolution mass spectrometry measured value 331.2273 (such as Figure 4 shown).

[0160] 1H NMR (600 MHz, methanol-d4): δ 5.71 (s, 1H, H4), 4.18 (s, 2H, H22), 1.23 (s, 3H, H19), 0.71 (s, 3H, H18).

[0161] 13 C NMR (150 MHz, methanol-d4): δ 210.49 (C20), 200.84 (C3), 173.47 (C4), 122.83 (C5), 68.71 (C21), 58.32 (C17), 55.88 (C11), 53.73 (C9), 47.21 (C13), 44.12 (C12), 38.57 (C10), 38.15 (C8), 35.39 (C1), 33.31 (C2), 32.48 (C6), 31.84 (C7), 24.09 (C16), 22.50 (C15), 20.71 (C11), 16.28 (C19), 12.47 (C18).

[0162] The nuclear magnetic resonance spectrum of 11-deoxycorticosterone obtained in Example 4 is as follows: Figure 5 As shown. Among them, Figure A is 1 H NMR spectrum (600MHz); Figure B is 13 C NMR spectrum (150MHz); Figure C is 1 H- 1 Figure D is the HSQC NMR spectrum; Figure E is the HMBC NMR spectrum.

[0163] By comparing with the NMR data of the reported compound, the structure of the reaction product of Example 4 was determined to be 11-deoxycorticosterone.

[0164] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A progesterone C21 hydroxylase, characterized in that The amino acid sequence of the progesterone C21 hydroxylase is shown in SEQ ID NO.

1.

2. A nucleic acid encoding the progesterone C21 hydroxylase according to claim 1.

3. The nucleic acid according to claim 2, wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO.

2.

4. An expression vector, characterized in that The expression vector comprises the nucleic acid according to claim 2 or 3. 5 . The expression vector according to claim 4 , wherein the expression vector comprises a constitutive promoter HXT7p upstream of the nucleic acid encoding the progesterone C21 hydroxylase according to claim 1 . The expression vector according to claim 4 , wherein the backbone of the expression vector is pRS426 or pRS425.

7. An expression vector composition comprising a first expression vector and a second expression vector, wherein the first expression vector is the expression vector according to any one of claims 4 to 6, and the second expression vector comprises a coding sequence for a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

8. The expression vector composition according to claim 7, wherein the second expression vector comprises a constitutive promoter TEF1p upstream of the coding sequence of the cytochrome P450 enzyme reductase; preferably, the backbone of the second expression vector is pRS426 or pRS425.

9. An enzyme composition comprising the progesterone C21 hydroxylase according to claim 1 and cytochrome P450 enzyme reductase.

10. The enzyme composition of claim 9, wherein the cytochrome P450 enzyme reductase is selected from the group consisting of Ncp1 and RnCPR.

11. A bioengineered strain, characterized in that: The bioengineered strain expresses the progesterone C21 hydroxylase according to claim 1; preferably, the bioengineered strain also expresses a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

12. The bioengineered strain according to claim 11, wherein the bioengineered strain is selected from the group consisting of Saccharomyces cerevisiae strains BY4742, BJ5464-NpgA, YRC01 and WAT11U.

13. A method for preparing 11-deoxycorticosterone, comprising contacting the progesterone C21 hydroxylase according to claim 1 with progesterone in the presence of an electron donor, thereby performing a catalytic reaction to produce 11-deoxycorticosterone.

14. The method according to claim 13, wherein the electron donor is a cytochrome P450 enzyme reductase; preferably, the cytochrome P450 enzyme reductase is selected from Ncp1 and RnCPR.

15. The method according to claim 13, wherein the catalytic reaction is carried out at 28-30°C for 2-3 days, preferably at 30°C for 2 days.

16. The method according to claim 13, wherein the method comprises the following steps: 1) Providing the bioengineered strain according to claim 11 or 12; 2) Providing a mixed culture of the bioengineered strain and progesterone, and incubating the mixed culture; preferably, the mixed culture further comprises 5-aminolevulinic acid hydrochloride.

17. The method according to claim 16, wherein in step 2), the OD of the bioengineering strain is 600 The final concentration of progesterone in the mixed culture is 200 μM to 1 mM; the final concentration of 5-aminolevulinic acid hydrochloride in the mixed culture is 0.5-1 mM.

18. The method according to claim 16, wherein the incubation is carried out at 28-30°C and 220-250 rpm for 2-3 days; preferably, the incubation is carried out at 30°C and 220 rpm for 2 days.

19. The method according to claim 13, wherein the method further comprises purifying 11-deoxycorticosterone from the system obtained by the catalytic reaction.

20. Use of the progesterone C21 hydroxylase according to claim 1, the nucleic acid according to claim 2 or 3, the expression vector according to any one of claims 4 to 6, the expression vector composition according to claim 7 or 8, the enzyme composition according to claim 9 or 10, and the bioengineered strain according to claim 11 or 12 in the preparation of 11-deoxycorticosterone.

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