A daidzein reductase, enzyme combination thereof and application thereof

Abstract

The application discloses a soybean isoflavone reductase, an enzyme combination thereof and application thereof. The soybean isoflavone reductase has higher dihydrodaidzein yield than wild-type enzymes. On this basis, the enzyme combination of the soybean isoflavone reductase, dihydrodaidzein racemase, dihydrodaidzein reductase and tetrahydrodaidzein reductase realizes efficient biosynthesis of S-equol.

Description

Technical Field

[0001] This invention belongs to the field of bio-enzyme catalysis, specifically relating to a soybean aglycone reductase, its enzyme combination, and its application. Background Technology

[0002] S-Equol (CAS: 531-95-3) is a nonsteroidal estrogen with significant physiological activity, far exceeding that of R-Equol. It exhibits unique advantages in antioxidant, anti-inflammatory, anti-tumor, and menopausal syndrome relief, and has been widely regarded as a high-value functional ingredient. However, its natural sources are extremely limited, relying mainly on micro-metabolism by gut microbiota, making it difficult to meet the demands of large-scale applications.

[0003] Currently, the preparation of S-estrol mainly relies on chemical synthesis and biosynthesis. Chemical synthesis routes typically use daidzein (CAS: 486-66-8) as a substrate, involving multiple reduction, protection, and deprotection reactions. Representative methods include Tetrahedron 74 (2018) 2020–2029, CN110590727A, CN102633763A, and CN105777693A. These methods generally suffer from harsh reaction conditions (high temperature and pressure, precious metal catalysts), cumbersome process steps, and the production of racemic products. This not only makes it difficult to remove ineffective byproducts of R-estrol but also significantly increases separation and purification costs and environmental burden, limiting its application in the food and pharmaceutical fields.

[0004] In recent years, biosynthetic routes based on synthetic biology have gradually become a research hotspot. While natural strains, such as Lactococcus sp. 20-92, possess a complete equadol synthesis pathway, their growth requires strict anaerobic conditions, has a long fermentation cycle, and low yield, making it difficult to meet the needs of industrial scale-up (CN1251692C, CN101338294A). Therefore, researchers have constructed artificial metabolic pathways by heterologously expressing key enzymes. A typical pathway is as follows: Dydaidzein → (DZNR) → Dihydrodaidzein → (DDRC) → Chiral dihydrodaidzein → (DHDR) → Tetrahydrodaidzein → (THDR) → S-Estrol In this cascade system, DHDR (dihydrogenase) and THDR (tetrahydrogenase) are key enzymes determining the stereoselectivity of the final product. Extensive research has focused on modifying the downstream reductase: Lee et al. obtained DHDR-P212A through site-directed mutagenesis, efficiently converting 1 mM genistein to S-estradiol with a yield of 85%; Qin et al. constructed the DHDR-S118G / T169A combined mutant, achieving a conversion rate of 84.5% under 2 mM substrate conditions; CN121160648A performed multiple site mutations on THDR (such as M461A), and when used in combination with upstream DHDR-P120F / P212A, achieved a conversion rate of 93.4%, with only 1.2% intermediate residue.

[0005] In addition, to improve substrate solubility and cofactor supply, some studies have introduced PVP-40k (yield 1.22 g / L, Appl Microbiol Biotechnol, 2018, 102: 6915-6921) or enhanced NADPH regeneration through metabolic engineering (CN115747240A, 66h yield 1.9 g / L). Hangzhou Hetanchuang (CN121628799A) achieved a S-estrol yield of 2.2 g / L in a 5L fermenter through multi-enzyme co-expression and zwf overexpression.

[0006] However, most of the aforementioned studies focused on optimizing the downstream DHDR / THDR process, while relatively little attention was paid to the two key upstream enzymes—DZNR (daidzein reductase) and DDRC (dihydrodaidzein racemic enzyme). In fact, DZNR is responsible for reducing the highly hydrophobic daidzein to dihydrodaidzein, and is the first rate-limiting reaction in the entire pathway; DDRC determines the chiral flow of the intermediate, having a decisive impact on subsequent stereoselective reduction. Insufficient upstream conversion efficiency will lead to substrate accumulation or intermediate imbalance, severely limiting the overall yield.

[0007] Recent studies have begun to focus on the role of upstream enzymes: CN120966777A and Tianjin University of Commerce CN120966778A reported mutants of DZNR and DHDR, respectively, which can improve tolerance to high concentrations of substrates to some extent; CN121064140A further suggests that by synergistically regulating the expression levels of DDRC and DZNR, the coupling efficiency of the first two steps of the reaction can be significantly improved. However, this patent did not perform deep mutation modification on the enzyme protein itself, and there is still considerable room for improvement in its catalytic kinetic parameters and stereoselectivity.

[0008] In summary, although significant progress has been made in the biosynthesis of S-estrol, the lack of highly efficient mutants of upstream DZNR and DDRC limits the overall throughput of the entire cascade system. Therefore, there is an urgent need in this field to develop DZNR mutants with higher catalytic efficiency, better stereoselectivity, and stronger resistance to feedback inhibition to fill the upstream bottleneck and provide key enzyme elements for constructing efficient and stable S-estrol cell factories. Summary of the Invention

[0009] To address the lack of efficient upstream DZNR mutants in existing technologies, this invention provides a soybean aglycone reductase, its enzyme combination, and its applications. This invention utilizes rational design and directed evolutionary modification of DZNR to construct a high-performance mutant and its enzyme combination, overcoming the rate-limiting upstream step and achieving efficient biosynthesis of S-equorol.

[0010] The present invention solves the above-mentioned technical problems by adopting the following technical solution.

[0011] The present invention provides a daidzein reductase, wherein the amino acid sequence of the daidzein reductase, compared with the amino acid sequence shown in SEQ ID NO: 1, includes one or more of the following amino acid residue differences: K12P, T20L, M529K, M280V and K14R.

[0012] In some embodiments, the daidzein reductase has enhanced enzymatic activity in catalyzing the conversion of daidzein to dihydrodaidzein compared to the wild-type daidzein reductase with the amino acid sequence shown in SEQ ID NO: 1.

[0013] In some embodiments, the increased enzyme activity is at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0014] In some embodiments, the amino acid sequence of the daidzein reductase, compared to the amino acid sequence shown in SEQ ID NO: 1, includes one of the following amino acid residue differences or combinations of amino acid residue differences: (1) K12P / M529K / T20L; (2) K12P / M280V; (3) K12P / M529K; (4) K12P / T20L; (5) M529K / M280V; (6) M529K / T20L; (7) M280V / T20L; (8) K12P; (9) K14R; (10) T20L; (11) M280V; and, (12) M529K; The " / " indicates that the differences in amino acid residues before and after the symbol coexist.

[0015] The amino acid residue difference “K12P” means that, based on the amino acid sequence shown in SEQ ID NO: 1, the 12th lysine (K) residue is replaced with a proline (P) residue; the remaining amino acid residue differences are defined according to the same rules.

[0016] In some embodiments, the amino acid sequence of the daidzein reductase contains one or more of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 1: K12P, T20L, and M529K.

[0017] In some embodiments, the amino acid sequence of the daidzein reductase, compared to the amino acid sequence shown in SEQ ID NO: 1, includes one of the following amino acid residue differences or combinations of amino acid residue differences: (1) K12P / M529K / T20L; (2) K12P / M529K; (3) K12P / T20L; (4) M529K / T20L; (5) K12P; (6) T20L; and, (7) M529K; The " / " indicates that the differences in amino acid residues before and after the symbol coexist.

[0018] In some embodiments, the daidzein reductase contains an amino acid residue difference K12P compared to the amino acid sequence shown in SEQ ID NO: 1, and optionally also contains an amino acid residue difference M529K and / or T20L.

[0019] As used in this invention, "optionally" means that the event or condition described thereafter may or may not occur, and the description includes both the occurrence and non-occurrence of the event or condition. For example, "optionally also contains amino acid residue differences M529K and / or T20L" means that the daidzein reductase contains amino acid residue differences K12P compared to the amino acid sequence shown in SEQ ID NO: 1; or, the daidzein reductase contains amino acid residue differences K12P / M529K, K12P / T20L, or K12P / M529K / T20L compared to the amino acid sequence shown in SEQ ID NO: 1.

[0020] In some embodiments, the daidzein reductase, compared to the amino acid sequence shown in SEQ ID NO: 1, includes one of the following amino acid residue differences or combinations of amino acid residue differences: (1) K12P / M529K / T20L; (2) K12P / M280V; (3) K12P / M529K; (4) K12P / T20L; and, (5) K12P; The " / " indicates that the differences in amino acid residues before and after the symbol coexist.

[0021] In another aspect, the present invention provides an isolated nucleic acid that encodes the daidzein reductase described above.

[0022] Another aspect of the present invention provides an enzyme combination comprising wild-type daidzein reductase with the amino acid sequence as shown in SEQ ID NO: 1 and / or daidzein reductase as described above, as well as dihydrodaidzein reductase (DHDR) and tetrahydrodaidzein reductase (THDR); optionally, the enzyme combination further comprises dihydrodaidzein racemic enzyme (DDRC).

[0023] In some embodiments, the dihydrogenin racemase is selected from one or more of the dihydrogenin racems with GenBank accession numbers HJI80516.1, WRQ72387.1, WP_294440774.1, WP_201738867.1, WP_173363555.1, and WP_427882528.1; preferably the dihydrogenin racemase with GenBank accession number WP_173363555.1; and / or, The dihydrogenoside reductase is selected from one or more of the dihydrogenoside reductases with GenBank accession numbers WP_294375638.1, WP_279056040.1, and WP_481432641.1; preferably, it is the dihydrogenoside reductase with GenBank accession number WP_294375638.1; and / or, The tetrahydrodaidzein reductase is one or more of the tetrahydrodaidzein reductases with GenBank accession numbers WP_281623898.1, WP_481432642.1, and WP_294375640.1; preferably, it is the tetrahydrodaidzein reductase with GenBank accession number WP_281623898.1; and / or, The amino acid sequence of the soybean aglycone reductase differs from that shown in SEQ ID NO: 1 by K12P / M529K / T20L.

[0024] In some embodiments, the enzyme combination includes the daidzein reductase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase; and the amino acid sequence of the daidzein reductase differs from the amino acid sequence shown in SEQ ID NO: 1 by K12P / M529K / T20L, the dihydrodaidzein reductase is the dihydrodaidzein reductase with Genbank accession number WP_294375638.1, and the tetrahydrodaidzein reductase is the tetrahydrodaidzein reductase with Genbank accession number WP_281623898.1.

[0025] In some embodiments, the enzyme combination further includes dihydrogenin racemic enzyme (DDRC), which is selected from one or more of the dihydrogenin racemic enzymes with GenBank accession numbers HJI80516.1, WRQ72387.1, WP_294440774.1, WP_201738867.1, WP_173363555.1 and WP_427882528.1; preferably the dihydrogenin racemic enzyme with GenBank accession number WP_173363555.1.

[0026] Another aspect of the present invention provides a recombinant expression vector comprising the nucleic acid as described above or a nucleic acid encoding the enzyme combination as described above; The nucleic acids encoding the enzyme combination are located in the same or different recombinant expression vectors.

[0027] In this document, the terms "located in the same or different recombinant expression vectors," "located in the same or different transformants," and "expressed in the same or different transformants" refer to the following: the coding nucleic acid fragments constituting the enzyme assemblage can be assembled together into the same recombinant expression vector, or they can be assembled separately into multiple independent recombinant expression vectors; correspondingly, each recombinant expression vector can be introduced into the same transformant for co-expression, or it can be introduced into multiple independent transformants, with the transformants collectively completing the enzyme assemblage expression; the soybean aglycone reductase and each enzyme component in the enzyme assemblage can be expressed in the same transformant or expressed separately in different transformants. In some embodiments, the backbone of the recombinant expression vector is the pET-22B plasmid backbone.

[0028] In another aspect, the present invention provides a recombinant expression vector combination, the recombinant expression vector combination comprising a recombinant expression vector containing nucleic acid encoding the above-mentioned daidzein reductase or wild-type daidzein reductase with an amino acid sequence as shown in SEQ ID NO: 1, a recombinant expression vector containing nucleic acid encoding dihydrodaidzein reductase, and a recombinant expression vector encoding tetrahydrodaidzein reductase.

[0029] In some embodiments, the recombinant expression vector combination further includes a recombinant expression vector containing a nucleic acid encoding dihydrogen daidzein racemic enzyme.

[0030] In some embodiments, the dihydrogenin racemic enzyme, dihydrogenin reductase, and tetrahydrogenin reductase are each independently defined in the enzyme combination as described above. Specifically: In some embodiments, the dihydrogenin racemase is selected from one or more of the dihydrogenin racems with GenBank accession numbers HJI80516.1, WRQ72387.1, WP_294440774.1, WP_201738867.1, WP_173363555.1, and WP_427882528.1; preferably the dihydrogenin racemase with GenBank accession number WP_173363555.1; and / or, The dihydrogenoside reductase is selected from one or more of the dihydrogenoside reductases with GenBank accession numbers WP_294375638.1, WP_279056040.1, and WP_481432641.1; preferably, it is the dihydrogenoside reductase with GenBank accession number WP_294375638.1; and / or, The tetrahydrodaidzein reductase is one or more of the tetrahydrodaidzein reductases with Genbank accession numbers WP_281623898.1, WP_481432642.1 and WP_294375640.1; preferably the tetrahydrodaidzein reductase with Genbank accession number WP_281623898.1.

[0031] In some specific embodiments, the amino acid sequence of the daidzein reductase differs from that shown in SEQ ID NO: 1 by K12P / M529K / T20L, the dihydrodaidzein reductase is the dihydrodaidzein reductase with Genbank accession number WP_294375638.1, and the tetrahydrodaidzein reductase is the tetrahydrodaidzein reductase with Genbank accession number WP_281623898.1.

[0032] In some specific embodiments, the dihydrogen daidzein racemase is the dihydrogen daidzein racemase with Genbank accession number WP_173363555.1.

[0033] In another aspect, the present invention provides a transformant comprising the nucleic acid as described above, the recombinant expression vector as described above, or a combination of recombinant expression vectors as described above; or expressing the daidzein reductase as described above or a combination of enzymes as described above.

[0034] In some implementations, the transformant is a non-animal / plant variety.

[0035] In some embodiments, the host cell used in the construction of the transformant is *Escherichia coli* (E. coli). Escherichia coli The preferred Escherichia coli is Escherichia coli BL21(DE3).

[0036] In some implementations, the nucleic acids are located in the same or different transformants; The recombinant expression vectors are located in the same or different transformants; The daidzein reductase is expressed in the same or different transformants; The enzyme combination is expressed in the same or different transformants.

[0037] Another aspect of the present invention provides a combination of transformants, the combination of transformants comprising a transformant containing nucleic acid encoding the above-mentioned daidzein reductase or wild-type daidzein reductase with an amino acid sequence as shown in SEQ ID NO: 1, and transformants containing nucleic acids encoding dihydrodaidzein reductase and tetrahydrodaidzein reductase, respectively. Optionally, the transformant assembly may further include a transformant containing a racemic enzyme encoding dihydrogenin.

[0038] In some embodiments, the dihydrogenin racemic enzyme, dihydrogenin reductase, and tetrahydrogenin reductase are each independently defined in the enzyme combination as described above.

[0039] In some implementations, the transformant is a non-animal / plant variety.

[0040] In some embodiments, the host cell used in the construction of the transformant is *Escherichia coli* (E. coli). Escherichia coli The *Escherichia coli* is further preferably *Escherichia coli* BL21(DE3).

[0041] Another aspect of the present invention provides a method for preparing daidzein reductase or enzyme combination, the method comprising culturing the transformants and / or transformant combinations as described above, and obtaining the daidzein reductase or enzyme combination from the fermentation product.

[0042] In some embodiments, the method includes: culturing the seed culture of the transformant and / or a combination of transformants at 37±1°C and 150-250 rpm until OD reaches zero. 600 Value > 0.8; then induce culture at 26±2℃ for 4-24h, for example 16h, with IPTG being the preferred inducer.

[0043] In some embodiments, the culture medium used for the culture is a fermentation medium, and / or the final concentration of IPTG is 0.5-1.5 mM, for example 1 mM or 0.5 mM.

[0044] In some embodiments, the fermentation medium comprises 1.5-2.5% (w / v) yeast extract, 0.8-1.6% (w / v) tryptone, 0.1-0.5% (w / v) sodium chloride, 0.5-1.5% (w / v) glycerol, 0.1-0.3% (w / v) dipotassium hydrogen phosphate, 2-7% (w / v) PVP-K25, and optionally, also contains 40-60 μg / mL ampicillin.

[0045] In some embodiments, the fermentation medium comprises 2.0% (w / v) yeast extract, 1.2% (w / v) tryptone, 0.3% (w / v) sodium chloride, 1% (w / v) glycerol, 0.2% (w / v) dipotassium hydrogen phosphate, 5% (w / v) PVP-K25, and 50 μg / mL ampicillin.

[0046] In some embodiments, the method further includes the step of purifying the cultured product to obtain the daidzein reductase or enzyme combination.

[0047] Another aspect of the present invention provides a composition comprising a wild-type daidzein reductase with the amino acid sequence as shown in SEQ ID NO: 1 or a daidzein reductase as described above, and daidzein; optionally, the composition further comprises dihydrodaidzein reductase and tetrahydrodaidzein reductase; or, The composition comprises daidzein, and a transformant or a combination of the above transformants containing nucleic acid encoding the above-mentioned daidzein reductase or the wild-type daidzein reductase with an amino acid sequence as shown in SEQ ID NO: 1; optionally, the transformant simultaneously expresses the wild-type daidzein reductase or daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase.

[0048] In some embodiments, the composition further includes dihydrogen daidzein racemic enzyme.

[0049] In some embodiments, the transformant simultaneously expresses the wild-type daidzein reductase or daidzein reductase, dihydrodaidzein racemicase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase.

[0050] In some embodiments, the dihydrogenin racemic enzyme, dihydrogenin reductase, and tetrahydrogenin reductase are each independently defined in the enzyme combination as described above.

[0051] In some embodiments, the composition further includes NADPH and / or NADH, glucose, PVP-K25, and GDH enzyme.

[0052] In some embodiments, the amino acid sequence of the GDH enzyme is shown in SEQ ID NO: 2.

[0053] In some embodiments, the daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, and tetrahydrodaidzein reductase are each independently present as liquid enzymes, solid enzymes, or immobilized enzymes, or as cells or combinations thereof expressing the daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, or tetrahydrodaidzein reductase. The cells or combinations thereof expressing the daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, or tetrahydrodaidzein reductase are, for example, wet bacterial cells.

[0054] The liquid enzyme refers to an enzyme preparation that does not contain cells and exists in a liquid state, including but not limited to crude enzyme solution (such as the supernatant obtained by centrifugation after cell disruption) or purified enzyme solution obtained through further purification.

[0055] The solid enzyme refers to an enzyme preparation that does not contain cells and exists in a solid state, including but not limited to lyophilized powder, spray-dried granules, precipitates, or crystals.

[0056] The immobilized enzyme refers to an enzyme form that does not contain cells and is formed by binding daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, or tetrahydrodaidzein reductase to an insoluble carrier or forming cross-linked aggregates through physical or chemical means (including but not limited to adsorption, embedding, covalent binding, cross-linking, etc.), thereby facilitating recovery and reuse.

[0057] Another aspect of the present invention provides a method for preparing dihydrogenin and / or S-estradiol, the method comprising: contacting and reacting genistein with a wild-type genistein reductase with an amino acid sequence as shown in SEQ ID NO: 1 and / or a genistein reductase as described above, to obtain dihydrogenin.

[0058] In some embodiments, the method further includes reacting dihydrogenin with dihydrogenin reductase and tetrahydrogenin reductase to prepare S-estradiol.

[0059] In some embodiments, the method further includes reacting dihydrogenin with dihydrogenin racemic enzyme, dihydrogenin reductase and tetrahydrogenin reductase to prepare S-estradiol.

[0060] In some embodiments, the dihydrogenin racemic enzyme, dihydrogenin reductase, and tetrahydrogenin reductase are each independently defined in the enzyme combination as described above.

[0061] In some embodiments, the wild-type daidzein reductase and / or the daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase as described above (optionally also including the dihydrodaidzein racemicase) participate in the reaction in the form of transformants or combinations of transformants as described above.

[0062] In some embodiments, the method includes: after induction, adding daidzein and glucose to the fermentation broth to continue culturing the transformant or combination of transformants to obtain the dihydrodaidzein and / or S-estradiol; optionally, before induction, inoculating the seed culture of the transformant into the fermentation medium and culturing to OD. 600 Value > 0.8.

[0063] In some implementations, the method includes one or more of the following conditions: (1) The temperature for continued cultivation is 25-37℃, for example, 25℃; (2) The continued culture time is 20-30 h, for example, 24 h; (3) The fermentation medium comprises 1.5-2.5% (w / v) yeast extract, 0.8-1.6% (w / v) tryptone, 0.1-0.5% (w / v) sodium chloride, 0.5-1.5% (w / v) glycerol, 0.1-0.3% (w / v) dipotassium hydrogen phosphate, 2-7% (w / v) PVP-K25, and 40-60 μg / mL ampicillin; preferably 2.0% (w / v) yeast extract, 1.2% (w / v) tryptone, 0.3% sodium chloride, 1% (w / v) glycerol, 0.2% (w / v) dipotassium hydrogen phosphate, 5% (w / v) PVP-K25, and 50 μg / mL ampicillin; (4) The final concentration of glucose in the fermentation medium is 10-30 g / L, for example 20 g / L; (5) The final concentration of the daidzein in the fermentation medium is 0.5-5 g / L, for example 2 g / L; (6) The inducing agent used is IPTG, and its final concentration in the fermentation medium is preferably 0.5-1.5 mM, for example 0.5 mM; (7) The induction temperature is 25±1℃ and the induction time is 4-24h, for example 16h; (8) Fermentation culture is carried out in an Erlenmeyer flask (e.g., 250 mL).

[0064] In some embodiments, the method includes: after the transformant or combination of transformants has been induced in a fermenter, adding daidzein to the fermentation culture medium to react and obtain the S-estrol.

[0065] In some embodiments, the method includes: obtaining the corresponding wet cells after induction of the transformant or combination of transformants, and then reacting them with daidzein to obtain the S-estrol.

[0066] In some embodiments, the reaction further includes the step of regenerating the oxidized coenzyme into the reduced coenzyme.

[0067] In some embodiments, the reduced coenzyme is any one or more of NADH and NADPH, and the oxidized coenzyme is NAD. + and NADP + Any one or more of the following.

[0068] In some embodiments, the oxidized coenzyme is regenerated into a reduced coenzyme using NAD. + NADP + NAD + and NADP +Any one of the following, a hydrogen donor and a coenzyme regenerating enzyme; wherein the hydrogen donor and the coenzyme regenerating enzyme are selected from one or more of the following: (1) Glucose and glucose dehydrogenase; the preferred amino acid sequence of the glucose dehydrogenase is shown in SEQ ID NO:2. (2) Formic acid / formate salt and formic acid dehydrogenase; (3) Alcohols and alcohol dehydrogenases.

[0069] In some implementations, the method includes one or more of the following conditions: (1) The reaction system comprises: 2-7% (w / v) wet cells of each transformant, 2-7% (w / v) PVP-K30, 0.5-5 g / L NADPH and / or NADH, 10-30 g / L glucose, 5-20 g / L daidzein and 5-20 g / L glucose dehydrogenase; preferably comprising 5% (w / v) wet cells of each transformant, 5% (w / v) PVP-K30, 1 g / L NADPH and / or NADH, 20 g / L glucose, 10 g / L daidzein and 10 g / L glucose dehydrogenase; (2) The reaction temperature is 30-38℃, for example 35℃; (3) The reaction time is 30-55 hours, for example, 48 hours; (4) Before induction, the seed culture of each transformant was transferred to a shake flask containing fermentation medium and cultured at 37±1℃ and 150-250 rpm for 1-5 h. (5) The induction conditions used for the induction are 28±1℃ and the induction time is 4-24h, for example 16h; (6) The inducing agent used is IPTG, and its final concentration in the fermentation medium is preferably 0.8-1.5 mM, for example 1 mM; (7) The buffer solution is phosphate buffer, preferably with a concentration of 80-120 mM, for example 100 mM, and a pH of 6.8-7.2, for example 7.0.

[0070] Each of the transformants can be a transformant that simultaneously expresses daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase and tetrahydrodaidzein reductase, or four transformants that express daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively.

[0071] In another aspect, the present invention provides the use of wild-type daidzein reductase with an amino acid sequence as shown in SEQ ID NO: 1, daidzein reductase as described above, nucleic acid as described above, enzyme combination as described above, recombinant expression vector as described above, recombinant expression vector combination as described above, transformant as described above, or combination of transformants as described above in the preparation of dihydrodaidzein and / or S-estrol.

[0072] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0073] The reagents and raw materials used in this invention can be obtained commercially or prepared using conventional techniques in the field.

[0074] The positive and progressive effects of this invention are as follows: This invention involves directed evolutionary modification of DZNR, resulting in a mutant with a higher dihydrogen daidzein yield than the wild-type enzyme. Based on this, the enzyme combination of DZNR, DDRC, DHDR, and THDR provided by this invention achieves highly efficient biosynthesis of S-estradiol. Detailed Implementation

[0075] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0076] Experimental methods in the following examples, unless otherwise specified, were performed under standard conditions as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. All experimental materials used in this invention are commercially available unless otherwise specified.

[0077] Example 1 Screening of daidzein reductase DZNR

[0078] Screening for high-enzyme-activity daidzein reductases, among which

[0079] So_DZNR originates from Solobacterium sp Its login number in the Genbank database is MBR3203739.1; OB_DZNR originates from Oscillospiraceae bacterium Its login number in the Genbank database is MBQ5323586.1; HS_DZNR originates from Hungatella sp Its login number in the Genbank database is MCI8606935.1; Sl_DZNR originates from Slackia sp Its login number in the Genbank database is WP_481432640.1; Fi_DZNR originates from Faecalibacillus intestinalis Its login number in the Genbank database is WP_370805408.1.

[0080] After optimizing the above gene codons, the gene was constructed into the pET-22B vector and introduced into host Escherichia coli BL21(DE3) competent cells. The cells were cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB medium to obtain recombinant DZNR.

[0081] The recombinant DZNR strain was streaked onto a glycerol plate. Single colonies were picked and transferred to a test tube containing 5 mL of LB medium (containing 50 μg / mL ampicillin). The culture was incubated overnight at 37°C and 200 rpm to obtain the recombinant DZNR seed culture. 500 μL of the seed culture was transferred to a 250 mL Erlenmeyer flask containing 500 μL of fermentation medium (containing 50 μg / mL ampicillin) and incubated at 37°C and 200 rpm until OD500 was reached. 600 Value > 0.8. Isopropyl thiogalactoside (IPTG) at a final concentration of 0.5 mM was used to induce expression in the engineered bacteria at 25℃. After culturing for 16 h, glucose at a final concentration of 20 g / L and daidzein (dissolved in DMSO) were added to the fermentation broth, and the culture was continued for another 24 h. 0.2 ml of the fermentation broth was taken, 2 ml of methanol was added, and after filtration, the relative content of dihydrodaidzein was determined by HPLC. The shake-flask yields of each recombinant strain are shown in Table 1.

[0082] The fermentation medium formula is as follows: yeast extract (2.0%), tryptone (1.2%), sodium chloride (0.3%), glycerol (1%), dipotassium hydrogen phosphate (0.2%), and PVP-K25 (5%).

[0083] Table 1. Dihydrogen daidzein yield of recombinant DZNR strain

[0084] As shown in Table 1, Sl_DZNR enzyme activity was the highest.

[0085] Example 2: Screening of mutant strains of Sl_DZNR

[0086] By rationally designing mutation sites (mutation sites are shown in Table 2), the Sl_DZNR mutant glycerol bacteria were streaked onto agar plates. Single colonies were picked and transferred to test tubes containing 5 mL LB medium (containing 50 μg / mL ampicillin). The cultures were incubated overnight at 37°C and 200 rpm to obtain the Sl_DZNR mutant seed culture. Fermentation and testing were performed using the same steps as in Example 1. The shake-flask yields of each recombinant strain are shown in Table 2.

[0087] Table 2. Dihydrogenase yield of the Sl_DZNR mutant strain.

[0088] As shown in Table 2, DZNR-M19 has the highest yield.

[0089] Example 3: Construction of recombinant bacteria for producing equadol

[0090] A combined recombinant strain of DZNR-M19 with DHDR and THDR is needed to produce estrol, wherein: UsDHDR originates from uncultured Slackia sp Its login number in the Genbank database is WP_294375638.1; AEDHDR originates from Adlercreutzia equolifaciens Its login number in the Genbank database is WP_279056040.1; SsDHDR originates from Slackia sp Its login number in the Genbank database is WP_481432641.1; SaTHDR originates from Senegalimassilia anaerobia Its login number in the Genbank database is WP_281623898.1; SsTHDR originates from Slackia sp Its login number in the Genbank database is WP_481432642.1; UsTHDR originates from uncultured Slackia sp Its login number in the Genbank database is WP_294375640.1.

[0091] The S-equorol synthase combination is shown in Table 3. After codon optimization and synthesis of the S-equorol synthase combination (daidzein reductase + dihydrodaidzein reductase + tetrahydrodaidzein reductase), it was cloned into the pETDuet-1 vector, transformed into E. coli DH5α competent cells, and cultured on ampicillin-resistant plates. Single colonies of positive transformants were picked, cultured, and plasmids were extracted and sequenced. After confirmation, recombinant plasmids were extracted and introduced into host E. coli BL21(DE3) competent cells, cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB medium to obtain recombinant S-equorol synthase.

[0092] The recombinant strain of S-equorol synthase was streaked onto a glycerol plate. Single colonies were picked and transferred to a test tube containing 5 mL of LB medium (containing 50 μg / mL ampicillin). The culture was incubated overnight at 37°C and 200 rpm to obtain the recombinant strain seed culture of S-equorol synthase. Fermentation was carried out using the same procedures as in Example 1. 0.2 mL of fermentation broth was taken, 2 mL of methanol was added, and after filtration, the relative equorol content was determined by HPLC. The shake-flask yields of each recombinant strain are shown in Table 3.

[0093] Table 3. S-Estrol Yield of Enzyme Combinations and Recombinant Bacteria

[0094] As shown in Table 3, when preparing S-estradiol using a combination of three enzymes, the combination of DZNR-M19 can increase the final yield compared to the combination of wild-type enzyme Sl_DZNR. In particular, the recombinant strain SQ4 has the highest yield, reaching 769.3 mg / L.

[0095] Example 4: Construction and screening of recombinant bacteria by adding racemic enzyme DDRC

[0096] Recombinant bacteria were constructed by adding racemic enzyme DDRC, wherein: Ea_DDRC originates from Eggerthellaceae bacterium Its login number in the Genbank database is HJI80516.1; Nb_DDRC originates from uncultured bacterium Its login in the Genbank database is WRQ72387.1; Ss_DDRC originates from Slackia sp Its login in the Genbank database is WP_294440774.1; Sg_DDRC originates from Senegalimassilia Its login in the Genbank database is WP_201738867.1; Eg_DDRC originates from Eggerthellaceae Its login in the Genbank database is WP_173363555.1; Si_DDRC originates from Slackia isoflavoniconvertens Its login in the Genbank database is WP_427882528.1.

[0097] The S-equorol synthase combination is shown in Table 4. After codon optimization and synthesis of the S-equorol synthase combination (daidzein reductase + dihydrodaidzein racemic enzyme + dihydrodaidzein reductase + tetrahydrodaidzein reductase), it was cloned into the pETDuet-1 vector, transformed into *E. coli* DH5α competent cells, and cultured on ampicillin-resistant plates. Single colonies of positive transformants were picked, cultured, and plasmids were extracted and sequenced. After confirmation, the recombinant plasmid was extracted and introduced into host *E. coli* BL21(DE3) competent cells, cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB medium to obtain the recombinant S-equorol synthase.

[0098] The recombinant strain of S-estrol synthase was streaked onto a glycerol plate, and a single colony was picked and transferred to a test tube containing 5 mL of LB medium (containing 50 μg / mL ampicillin). The culture was incubated overnight at 37°C and 200 rpm to obtain the recombinant strain seed culture of S-estrol synthase. Fermentation and testing were performed using the same method as in Example 3, and the shake-flask yields of each recombinant strain are shown in Table 4.

[0099] Table 4. S-Estrol Yield of Recombinant Enzyme Bacteria

[0100] As shown in Table 4, when preparing S-estradiol using a combination of four enzymes, the combination of DZNR-M19 significantly increased the final yield compared to the wild-type enzyme Sl_DZNR combination, and the yield of S-estradiol was significantly higher when using a combination of four enzymes compared to a combination of three enzymes. Furthermore, with other enzymes remaining unchanged, the S-estradiol yield was highest when using the SQ16 recombinant strain containing Eg_DDRC.

[0101] Example 5: Fermentation of S-estrol by recombinant strain SQ16 in a fermenter

[0102] The seed culture was cultured overnight in LB medium (37°C, 220 rpm); then, a 4% inoculum was inoculated into a 5L fermenter containing 2.5 L of fermentation medium (composition as in Example 1, except that glycerol was replaced with an equal volume of glucose) and 50 μg / mL ampicillin for fermentation. After fermentation, the results were analyzed in the same manner as in Example 1 – HPLC showed that the yield of S-equorol was 6.3 g / L.

[0103] Example 6: Production of S-Estradiol by Recombinant Bacterium SQ16 Enzyme Conversion

[0104] 10 μl of recombinant glycerol bacteria strain SQ16 was inoculated into 5 mL LB medium (containing 100 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 12 h. Then, the seed culture was transferred at an inoculum volume of 1.5% to a shake flask containing 20 mL of fermentation medium (containing 50 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the engineered bacteria were induced to express their culture at 28°C for 16 h. The fermentation broth was centrifuged, and the wet cells were resuspended in 10 mL of 100 mM pH 7.0 phosphate buffer to a final concentration of 5% (w / v). Then, 5% (w / v) PVP-K30, 1 g / L NADPH, 1 g / L NADH, 20 g / L glucose, 10 g / L daidzein (dissolved in 5% DMSO) and 10 g / L GDH enzyme (glucose dehydrogenase derived from Bacillus megaterium, whose amino acid sequence is shown in SEQ ID NO: 2) were added. The mixture was transformed at 35℃ for 48 h, and the S-estradiol yield was detected by HPLC, reaching 7.7 g / L.

[0105] Example 7 Enzymatic Conversion for the Production of S-Estrol

[0106] Following Example 1, Eg_DDRC, UsDHDR, and SaTHDR were constructed into the pET-22B vector and introduced into competent *Escherichia coli* BL21(DE3) cells. The cells were cultured on ampicillin-resistant plates, and single colonies were picked and cultured in LB broth to obtain recombinant bacteria containing Eg_DDRC, UsDHDR, and SaTHDR. 10 μl of each of the following recombinant bacteria (Glyceryl DZNR-M19, Eg_DDRC, UsDHDR, and SaTHDR) were inoculated into four 5 mL LB broths (containing 100 μg / mL ampicillin) and cultured at 37°C and 200 rpm for 12 h. Then, the seed culture was transferred at an inoculum volume of 1.5% to four shake flasks containing 20 mL of fermentation medium (as in Example 1) and cultured at 37°C and 200 rpm for 3 h. Isopropyl thiogalactoside (IPTG) was added to a final concentration of 1 mM, and the engineered bacteria were induced to express the bacteria at 28 °C for 16 h. The fermentation broth was centrifuged, and each wet cell was resuspended in 10 mL of 100 mM pH 7.0 phosphate buffer to a final concentration of 5% (w / v). Then, 5% PVP-K30, 1 g / L NADPH, 1 g / L NADH, 20 g / L glucose, 10 g / L daidzein (dissolved in 5% DMSO), and 10 g / L GDH enzyme were added, and the mixture was transformed at 35 °C for 48 h. The yield of S-estradiol was detected by HPLC and found to be 8.3 g / L.

[0107] Sl_DZNR amino acid sequence (SEQ ID NO: 1): MQHAKYPHLFSKGRVGKVTTKNRVIRNSMGTYLNVGKLCDVSDRNIKHAAEAAEGGPGIVFLDNCLIVDGYHMGLAAYDDTYIPGLSMIAQAMHDHGAVAGMQLAHPGRDMGFAGGDNVVAPSAVLPEIMINAGATVPRPLTIDEIHEIEEQYGQAAARVKQAGFDIVEVHGACGCLPTNFLSPHDNQRNDIYGGSLFNRQRFLVEVIRSIKRYVGPDFPVSVKLDMDDCEPDGIRLEECIDTCRVLEREGVALLNLVTATHVTANFSTSFYPWSYCADMAAQVKEQVNIPVMVTGAIQSPEAAEKILADGKVDFIGTARQCLADQAWVEKARTGNEDDIRPCIRCQIGCTDRGILGHHPISCAVNPTLFHYYEELYPKAATPKNVAVVGAGPAGCEAALTLKQRGHNVTVFEKREIGGTMIEAGAAWYKADINRFIDYYRKQLEKQHIDVRMQEVTPQDIADGGYDACIVAIGGEPRKLNVPGIDKPIVTEGIDFLYGSKKVEGKSAVVVGGATTTAEIALDLAEKGMDVTIVKRGTKFLNPAGCQMDIEYTIRLHQLGVKLMTGYRLDSVTDSSAIAIDQYGEKVEIPTENVVISAGYLNRPGFAEQLEEISDMDVYMAGDCKKVAEIPDATHAGYAVARMI Amino acid sequence of GDH enzyme (SEQ ID NO: 2): MYPDLKGKVVVITGSSTGLGKSMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDIMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKYFVENDVKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTQYPSFQAGRG Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and substitutions can be made to those details based on all the teachings disclosed, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A daidzein reductase, characterized in that, The amino acid sequence of the soybean aglycone reductase contains one or more of the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 1: K12P, T20L, and M529K.

2. The daidzein reductase as described in claim 1, characterized in that, The amino acid sequence of the soybean aglycone reductase, compared with the amino acid sequence shown in SEQ ID NO: 1, contains one of the following amino acid residue differences or combinations of amino acid residue differences: (1) K12P / M529K / T20L; (2) K12P / M529K; (3) K12P / T20L; (4) M529K / T20L; (5) K12P; (6) T20L; and, (7) M529K.

3. An isolated nucleic acid, characterized in that, The nucleic acid encodes the daidzein reductase as described in claim 1 or 2.

4. An enzyme combination, characterized in that, The enzyme combination includes daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase as described in claim 1 or 2.

5. The enzyme combination as described in claim 4, characterized in that, The dihydrogenase reductase is selected from one or more of the dihydrogenase reductases with GenBank accession numbers WP_294375638.1, WP_279056040.1, and WP_481432641.1; and / or, The tetrahydrodaidzein reductase is one or more of the tetrahydrodaidzein reductases with GenBank accession numbers WP_281623898.1, WP_481432642.1, and WP_294375640.1; and / or, The amino acid sequence of the soybean aglycone reductase differs from that shown in SEQ ID NO: 1 by K12P / M529K / T20L.

6. The enzyme combination as described in claim 5, characterized in that, The amino acid sequence of the daidzein reductase differs from that shown in SEQ ID NO: 1 by K12P / M529K / T20L. The dihydrodaidzein reductase is the dihydrodaidzein reductase with Genbank accession number WP_294375638.1, and the tetrahydrodaidzein reductase is the tetrahydrodaidzein reductase with Genbank accession number WP_281623898.

1.

7. The enzyme combination according to any one of claims 4-6, characterized in that, The enzyme combination also includes dihydrogen daidzein racemic enzyme.

8. The enzyme combination as described in claim 7, characterized in that, The dihydrogenogenin racemase is selected from one or more of the dihydrogenogenin racems with Genbank accession numbers HJI80516.1, WRQ72387.1, WP_294440774.1, WP_201738867.1, WP_173363555.1 and WP_427882528.

1.

9. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 3 or the nucleic acid encoding the enzyme combination as described in any one of claims 4-8; The nucleic acids encoding the enzyme combination are located in the same or different recombinant expression vectors.

10. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 3 or the recombinant expression vector as described in claim 9, or expresses the daidzein reductase as described in claim 1 or 2 or the enzyme combination as described in any one of claims 4-8; The nucleic acids are located in the same or different transformants; The recombinant expression vectors are located in the same or different transformants; The daidzein reductase is expressed in the same or different transformants; The enzyme combination is expressed in the same or different transformants.

11. A method for preparing daidzein reductase or a combination of enzymes, characterized in that, The method includes culturing the transformant as described in claim 10 to obtain the daidzein reductase or enzyme combination from the fermentation product.

12. A composition, characterized in that, The composition comprises daidzein and daidzein reductase as described in claim 1 or 2; or, The composition comprises daidzein and the transformant as described in claim 10.

13. The composition according to claim 12, characterized in that, The composition comprises the daidzein reductase, daidzein, dihydrodaidzein reductase, and tetrahydrodaidzein reductase; or, The composition includes daidzein and the transformant, which simultaneously expresses daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase.

14. The composition according to claim 13, characterized in that, The composition comprises the daidzein reductase, daidzein, dihydrodaidzein reductase, tetrahydrodaidzein reductase, and dihydrodaidzein racemic enzyme; or, The composition comprises daidzein and the transformant, the transformant simultaneously expressing daidzein reductase, dihydrodaidzein reductase, tetrahydrodaidzein reductase and dihydrodaidzein racemic enzyme.

15. The composition according to any one of claims 13-14, characterized in that, The dihydrogenistein reductase, tetrahydrogenistein reductase, and dihydrogenistein racemic enzyme are each independently defined in the enzyme combination as described in claim 5, 6, or 8; and / or, The composition further comprises NADPH and / or NADH, glucose, PVP-K25 and GDH enzyme; and / or, The daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, and tetrahydrodaidzein reductase exist independently as liquid enzymes, solid enzymes, or immobilized enzymes, or as cells or combinations thereof expressing the daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase, or tetrahydrodaidzein reductase.

16. A method for preparing dihydrogen daidzein and / or S-estrol, characterized in that, The method includes: contacting and reacting daidzein reductase as described in claim 1 or 2 with daidzein to prepare dihydrodaidzein.

17. The method as described in claim 16, characterized in that, The method further includes: reacting dihydrogenin with dihydrogenin reductase and tetrahydrogenin reductase to prepare S-estradiol; or, reacting dihydrogenin with dihydrogenin racemic enzyme, dihydrogenin reductase and tetrahydrogenin reductase to prepare S-estradiol.

18. The method as described in claim 16 or 17, characterized in that, The method includes one or more of the following conditions: (1) The daidzein reductase, dihydrodaidzein racemic enzyme, dihydrodaidzein reductase and tetrahydrodaidzein reductase participate in the reaction in the form of the transformant as described in claim 10; (2) The dihydrogenin reductase, dihydrogenin racemic enzyme, and tetrahydrogenin reductase are each independently defined in the enzyme combination as described in claim 5, 6, or 8; and, (3) The method further includes the step of regenerating the oxidized coenzyme into the reduced coenzyme.

19. The method as described in claim 18, characterized in that, After induction, the transformant is further cultured with daidzein and glucose in the fermentation broth to obtain dihydrodaidzein and / or S-estrol; or, After the transformation is induced, the corresponding wet cells are obtained, and then they are reacted with daidzein to obtain the S-estrol.

20. The use of the daidzein reductase as described in claim 1 or 2, the nucleic acid as described in claim 3, the enzyme combination as described in any one of claims 4-8, the recombinant expression vector as described in claim 9, or the transformant as described in claim 10 in the preparation of dihydrodaidzein and / or S-estrol.

Images