Dihydrodaidzein reductase mutant and application thereof in synthesis of (3S, 4R)-tetrahydrodaidzein

Abstract

The invention discloses a dihydrodaidzein reductase mutant and application of the dihydrodaidzein reductase mutant in synthesis of (3S, 4R)-tetrahydrodaidzein, and belongs to the technical field of biology. According to the mutant, wild-type dihydrodaidzein reductase DHDR is taken as a parent, single-point mutation or combined mutation is carried out, and a DHDR mutant is obtained; the parent site of the single point mutation is one of the 120th site, the 122nd site, the 170th site, the 178th site, the 214th site, the 219th site, the 220th site or the 220th site; the combined mutation adopts two modes: one mode is that a single-point mutation DHDR mutant is taken as a parent, mutation is carried out, and a combined mutation mutant I is obtained; and 2, taking the DHDR combined mutation mutant I as a parent, and performing mutation to obtain a combined mutation mutant II. The DHDR mutant disclosed by the invention is used for synthesizing (3S, 4R)-tetrahydrodaidzein by reducing dihydrodaidzein, and asymmetric reduction with high activity and high stereoselectivity can be realized.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a dihydrodaidzein reductase mutant and application thereof in synthesis of (3S, 4R)-tetrahydrodaidzein. BACKGROUND

[0002] (S)-equol is a product of isoflavones metabolism by human intestinal microorganisms, and has important biological activity. This compound was first isolated from horse urine in 1932, and was found in human urine 50 years later. Equol exists in the form of diastereoisomers, and (S)-equol is a configuration with selective affinity to estrogen receptor β. (S)-equol has a variety of important biological functions, including reducing the incidence of estrogen-dependent and aging-related diseases, such as female menopausal symptoms, osteoporosis, cardiovascular diseases, neurodegenerative diseases, and cancers of prostate, colon and breast. In addition, (S)-equol also has high antioxidant activity. It is worth noting that in Japan, companies such as Otsuka Pharmaceutical and Kobayashi Pharmaceutical have sold (S)-equol as a health product, which also reflects the recognition and emphasis on its biological activity.

[0003] Isoflavans, especially (S)-equol, have important biological activity, so how to optically synthesize such compounds is of great significance. Current methods for synthesizing (S)-equol include chemical synthesis, microbial transformation, etc. Among them, Anton Keßberg et al. published an article entitled “Enantioselective Total Synthesis of Natural Isoflavans: Asymmetric Transfer Hydrogenation / Deoxygenation of Isoflavanones with Dynamic Kinetic Resolution” in Organic Letters (2018, 20(10): 3006-3009), which adopted chemical synthesis, but its main defects are complicated steps, low yield and poor selectivity; and the main disadvantage of using intestinal microorganisms capable of metabolizing daidzein to obtain (S)-equol is low substrate concentration, for example, the optimal Slackia sp. YIT 11861 among the related strains found in the human intestine can only convert 100 µM substrate into product within 24 hours.

[0004] Although intestinal microorganisms or recombinant engineering bacteria can be used to convert isoflavones into (S)-isoflavanes, there are still some problems in the natural path of conversion and the key enzymes therein. For example, in the process from daidzein to (S)-equol, the wild-type dihydrodaidzein reductase (DHDR, belonging to ketoreductase) is the rate-limiting enzyme, and has low activity and certain reduction activity on (3R)-dihydrodaidzein. In the reduction of (3S)-dihydrodaidzein to (3S, 4R)-tetrahydrodaidzein, (3S, 4R)-tetrahydrodaidzein and (3R, 4R)-tetrahydrodaidzein are obtained, and only (3S, 4R)-tetrahydrodaidzein can be accepted as a substrate by the subsequent tetrahydrodaidzein reductase (THDR) to synthesize (S)-equol, resulting in poor atom economy of the wild-type dihydrodaidzein reductase DHDR.

[0005] Therefore, improving the activity and selectivity of dihydrodaidzein reductase and then improving the synthesis efficiency of (3S, 4R)-tetrahydrodaidzein are of great significance for the biological manufacturing of (S)-equol. SUMMARY

[0006] In view of the problems in the prior art, the present application aims to use the wild-type dihydrodaidzein reductase DHDR derived from Lactococcus garvieae to perform site-directed mutagenesis to obtain dihydrodaidzein reductase DHDR mutant enzymes, analyze the activity and stereoselectivity thereof, and use the dihydrodaidzein reductase DHDR mutant with high activity and high stereoselectivity to asymmetrically reduce dihydrodaidzein to synthesize (3S, 4R)-tetrahydrodaidzein.

[0007] The purpose of the present application is achieved by the following technical solutions:

[0008] In a first aspect, the present application provides a dihydrodaidzein reductase mutant, which is obtained by performing single-point mutation or combined mutation on the wild-type dihydrodaidzein reductase DHDR with the amino acid sequence shown in SEQ ID NO. 1.

[0009] Further, the parent site of the single-point mutation is one of the 120th, 122nd, 170th, 178th, 214th, 219th, 220th or 220th positions.

[0010] Further, the parent proline at position 120 is mutated to alanine, phenylalanine, glycine, isoleucine, leucine, respectively, to obtain mutant DHDR-P120A, DHDR-P120F, DHDR-P120G, DHDR-P120I, DHDR-P120L;

[0011] The parent methionine at position 122 is mutated to alanine, phenylalanine, respectively, to obtain mutant DHDR-M122A, DHDR-M122F;

[0012] The parent valine at position 170 is mutated to threonine to obtain mutant DHDR-V170T;

[0013] The parent proline at position 178 is mutated to alanine, phenylalanine, respectively, to obtain mutant DHDR-P178A, DHDR-P178F;

[0014] The parent leucine at position 214 is mutated to alanine, threonine, respectively, to obtain mutant DHDR-L214A, DHDR-L214T;

[0015] The parent alanine at position 219 is mutated to phenylalanine to obtain mutant DHDR-A219F;

[0016] The parent methionine at position 220 is mutated to alanine to obtain mutant DHDR-M220A;

[0017] The parent glycine at position 230 is mutated to alanine, phenylalanine, respectively, to obtain mutant DHDR-G230A, DHDR-G230F.

[0018] Further, the combination mutation adopts the following two ways:

[0019] One is to take the single-point mutant dihydrodaidzein reductase DHDR mutant as the parent to mutate to obtain dihydrodaidzein reductase DHDR combination mutant I;

[0020] Two is to take dihydrodaidzein reductase DHDR combination mutant I as the parent to mutate to obtain dihydrodaidzein reductase DHDR combination mutant II.

[0021] Further, the dihydrodaidzein reductase DHDR single-point mutant is DHDR-P120F or DHDR-P120G;

[0022] DHDR-P120F as the parent, the parent 214th leucine is mutated to threonine, the parent 212th proline is mutated to alanine, the parent 170th valine is mutated to threonine, the obtained dihydrodaidzein reductase DHDR combined mutant I is DHDR-P120F / L214T, DHDR-P120F / P212A, DHDR-P120F / V170T respectively;

[0023] DHDR-P120G as the parent, the parent 214th leucine is mutated to threonine, the parent 212th proline is mutated to alanine, the parent 170th valine is mutated to threonine, the obtained dihydrodaidzein reductase DHDR combined mutant I is DHDR-P120G / L214T, DHDR-P120G / P212A, DHDR-P120G / V170T respectively;

[0024] Among them, the amino acid sequence of the mutant DHDR-P120F / P212A is as shown in SEQ ID NO. 3, the amino acid sequence of the mutant DHDR-P120G / L214T is as shown in SEQ ID NO. 5, and the amino acid sequence of the mutant DHDR-P120G / V170T is as shown in SEQ ID NO. 7.

[0025] Further, the dihydrodaidzein reductase DHDR combined mutant I is DHDR-P120F / P212A or DHDR-P120G / L214T;

[0026] DHDR-P120F / P212A as the parent, the parent 170th valine is mutated to threonine, the obtained dihydrodaidzein reductase DHDR combined mutant II is DHDR-P120F / P212A / V170T;

[0027] DHDR-P120G / L214T as the parent, the parent 170th valine is mutated to threonine, the obtained combined mutant II is DHDR-P120G / L214T / V170T;

[0028] Among them, the amino acid sequence of the mutant DHDR-P120G / L214T / V170T is as shown in SEQ ID NO. 9.

[0029] In a second aspect, the present invention provides a DNA molecule encoding the dihydrogen daidzein reductase mutant described in the first aspect; wherein the nucleotide sequence of the mutant DHDR-P120F / P212A is shown in SEQ ID NO.4, the nucleotide sequence of the mutant DHDR-P120G / L214T is shown in SEQ ID NO.6, the nucleotide sequence of the mutant DHDR-P120G / V170T is shown in SEQ ID NO.8, and the nucleotide sequence of the mutant DHDR-P120G / L214T / V170T is shown in SEQ ID NO.10.

[0030] Thirdly, the present invention provides an expression vector containing the DNA molecule described in the second aspect.

[0031] Fourthly, the present invention provides a host cell containing the expression vector described in the third aspect, wherein the host cell is Escherichia coli.

[0032] Fifthly, this invention provides the application of a dihydrogenistein reductase mutant in the synthesis of (3S,4R)-tetrahydrogenistein. The aforementioned DHDR-P120F / P212A, DHDR-P120G / L214T, DHDR-P120G / V170T, or DHDR-P120G / L214T / V170T dihydrogenistein reductase mutants are used for asymmetric reduction of dihydrogenistein to synthesize (3S,4R)-tetrahydrogenistein; the reaction formula for the reduction reaction is shown in Formula I:

[0033]

[0034] Advantages and effects of the present invention:

[0035] This invention modifies the wild-type dihydrogenistein reductase DHDR, studies its activity and selectivity, and obtains mutants with good activity and selectivity. When used to reduce dihydrogenistein to synthesize (3S,4R)-tetrahydrogenistein, it achieves highly active and stereoselective asymmetric reduction, with the (3S,4R)-tetrahydrogenistein product accounting for over 92.7%, and the (3S)-dihydrogenistein conversion rate exceeding 72.9%, demonstrating good atom economy and laying the foundation for the subsequent biomanufacturing of (S)-estradiol. Attached Figure Description

[0036] Figure 1 This is a comparative HPLC assay of the activity and selectivity of dihydrogen daidzein reductase DHDR wild-type (WT) and mutant P120F / P212A in Example 2 when reducing (±)-dihydrogen daidzein (DHD) to (3S,4R)-tetrahydrogen daidzein (THD). DETAILED DESCRIPTION

[0037] The specific embodiments of the present application are described in further detail below in conjunction with the accompanying drawings and examples.

[0038] The purpose of the present application is achieved by the following technical solutions:

[0039] A dihydrogenisoinucucinol reductase mutant, taking wild-type dihydrogenisoinucucinol reductase DHDR with an amino acid sequence as shown in SEQ ID NO. 1 as a parent, performing single-point mutation or combined mutation to obtain dihydrogenisoinucucinol reductase DHDR mutant.

[0040] The single-point mutation mutant is as follows:

[0041] The parent 120th proline is mutated into alanine, or phenylalanine, or glycine, or isoleucine, or leucine to obtain mutant DHDR-P120A, DHDR-P120F, DHDR-P120G, DHDR-P120I, DHDR-P120L; the parent 122nd methionine is mutated into alanine, or phenylalanine to obtain mutant DHDR-M122A, DHDR-M122F; the parent 170th valine is mutated into threonine to obtain mutant DHDR-V170T; the parent 178th proline is mutated into alanine, or phenylalanine to obtain mutant DHDR-P178A, DHDR-P178F; the parent 214th leucine is mutated into alanine, or threonine to obtain mutant DHDR-L214A, DHDR-L214T; the parent 219th alanine is mutated into phenylalanine to obtain mutant DHDR-A219F; the parent 220th methionine is mutated into alanine to obtain mutant DHDR-M220A; the parent 230th glycine is mutated into alanine, or phenylalanine to obtain mutant DHDR-G230A, DHDR-G230F.

[0042] The combined mutation adopts the following two ways:

[0043] One is to take DHDR-P120F or DHDR-P120G in the single-point mutation mutant as a parent to mutate to obtain dihydrogenisoinucucinol reductase DHDR combined mutation mutant I as follows:

[0044] Taking DHDR-P120F as a parent, the parent 214th leucine is mutated into threonine to obtain mutant DHDR-P120F / L214T; the parent 212th proline is mutated into alanine to obtain mutant DHDR-P120F / P212A; the parent 170th valine is mutated into threonine to obtain mutant DHDR-P120F / V170T;

[0045] DHDR-P120G / L214T, DHDR-P120G / P212A and DHDR-P120G / V170T.

[0046] DHDR-P120F / P212A and DHDR-P120G / L214T as the parent, and then mutations are made to obtain the following dihydrodaidzein reductase DHDR combined mutant II:

[0047] DHDR-P120F / P212A / V170T.

[0048] DHDR-P120G / L214T / V170T.

[0049] A DNA molecule encoding the above dihydrodaidzein reductase mutant.

[0050] An expression vector containing the above DNA molecule.

[0051] A host cell containing the above expression vector, and the host cell is Escherichia coli.

[0052] The application of the above dihydrodaidzein reductase mutant, DNA molecule, expression vector and host cell in the synthesis of (3S, 4R)-tetrahydrodaidzein, which is used for asymmetric reduction of dihydrodaidzein to synthesize (3S, 4R)-tetrahydrodaidzein, and the reaction formula of the reduction reaction is shown as formula I:

[0053]

[0054] The product of (3S, 4R)-tetrahydrodaidzein accounts for more than 50%, and asymmetric reduction of dihydrodaidzein to synthesize (3S, 4R)-tetrahydrodaidzein is realized.

[0055] The wild-type amino acid sequence of dihydrodaidzein reductase DHDR is shown as SEQ ID NO. 1, and is specifically as follows:

[0056] MAQEVKVPKMPGAPVFGKWISPEESVGQRLKGKKILLTGTTKGVGRVTQELLCAHGAFVCGSGRTPGVAASVADELKAKGYQAAGMDVDLSDYDAVKKWVEECAELMGGIDVVINNASHPGMAPFGEMTPEIWNYGIKNELDLVYNVCNCAWPYLQKADGASIIITSSTVALQGSNSPQACHAACKGACLSLARQLAAEGGPFGIRCNSVTPGLVWTEAMSNIPKEMASGLVAAQTTQQAVDPMDIAYAYLFLASDESRQITAANIPVDGGCAGAVTGGMQGEIEV.

[0057] The wild-type nucleotide sequence of dihydrodaidzein reductase DHDR is shown as SEQ ID NO. 2, and is specifically as follows:

[0058] atggcacaggaagtcaaagtccccaagatgcccggcgcacccgtgttcggcaagtggatctcccccgaggagtccgtcggccagcgcctgaagggcaagaagatcctgctcaccggcaccaccaagggcgtcggcagggtcacccaggagctgctgtgcgcacacggcgccttcgtctgcggctccggccgcacccccggcgtggcagcctccgtcgccgacgagctgaaggccaagggctaccaggccgccggcatggacgtcgacctgtctgactacgacgccgtgaagaagtgggttgaggagtgcgccgagctcatgggcggcatcgacgtcgtcatcaacaacgcgtcccaccccggcatggcccccttcggcgagatgaccccggagatctggaactacggcatcaagaacgagctcgacctcgtctacaacgtctgcaactgcgcatggccctacctgcagaaggcagacggcgcctccatcatcatcacctcctccaccgtcgccctccagggcagcaactcccctcaggcctgtcacgctgcctgcaagggcgcctgcctgtccctggcccgccagctcgccgctgagggcggccccttcggcatccgctgcaactccgtcaccccgggcctggtctggaccgaggccatgtccaacatccccaaggagatggcaagcggcctggtcgcagcccagaccacccagcaggctgtcgacccgatggacatcgcctacgcctacctgttcctggcatccgacgagtcccgccagatcaccgctgccaacatccccgtcgacggcggctgcgccggcgctgtgaccggcggcatgcagggcgagatcgaggtc.

[0059] The amino acid sequence of the dihydrodaidzein reductase DHDR-P120F / P212A mutant is shown as SEQ ID NO. 3, and is specifically as follows:

[0060] MAQEVKVPKMPGAPVFGKWISPEESVGQRLKGKKILLTGTTKGVGRVTQELLCAHGAFVCGSGRTPGVAASVADELKAKGYQAAGMDVDLSDYDAVKKWVEECAELMGGIDVVINNASHFGMAPFGEMTPEIWNYGIKNELDLVYNVCNCAWPYLQKADGASIIITSSTVALQGSNSPQACHAACKGACLSLARQLAAEGGPFGIRCNSVTAGLVWTEAMSNIPKEMASGLVAAQTTQQAVDPMDIAYAYLFLASDESRQITAANIPVDGGCAGAVTGGMQGEIEV.

[0061] The nucleotide sequence of the dihydrogenisoin DHR-P120F / P212A mutant is shown as SEQ ID NO. 4, and is specifically as follows:

[0062] atggcacaggaagtcaaagtccccaagatgcccggcgcacccgtgttcggcaagtggatctcccccgaggagtccgtcggccagcgcctgaagggcaagaagatcctgctcaccggcaccaccaagggcgtcggcagggtcacccaggagctgctgtgcgcacacggcgccttcgtctgcggctccggccgcacccccggcgtggcagcctccgtcgccgacgagctgaaggccaagggctaccaggccgccggcatggacgtcgacctgtctgactacgacgccgtgaagaagtgggttgaggagtgcgccgagctcatgggcggcatcgacgtcgtcatcaacaacgcgtcccactttggcatggcccccttcggcgagatgaccccggagatctggaactacggcatcaagaacgagctcgacctcgtctacaacgtctgcaactgcgcatggccctacctgcagaaggcagacggcgcctccatcatcatcacctcctccaccgtcgccctccagggcagcaactcccctcaggcctgtcacgctgcctgcaagggcgcctgcctgtccctggcccgccagctcgccgctgagggcggccccttcggcatccgctgcaactccgtcaccgcgggcctggtctggaccgaggccatgtccaacatccccaaggagatggcaagcggcctggtcgcagcccagaccacccagcaggctgtcgacccgatggacatcgcctacgcctacctgttcctggcatccgacgagtcccgccagatcaccgctgccaacatccccgtcgacggcggctgcgccggcgctgtgaccggcggcatgcagggcgagatcgaggtc.

[0063] The amino acid sequence of the dihydrodaidzein reductase DHDR-P120G / L214T mutant is shown as SEQ ID NO. 5, and is specifically as follows:

[0064] MAQEVKVPKMPGAPVFGKWISPEESVGQRLKGKKILLTGTTKGVGRVTQELLCAHGAFVCGSGRTPGVAASVADELKAKGYQAAGMDVDLSDYDAVKKWVEECAELMGGIDVVINNASHGGMAPFGEMTPEIWNYGIKNELDLVYNVCNCAWPYLQKADGASIIITSSTVALQGSNSPQACHAACKGACLSLARQLAAEGGPFGIRCNSVTPGTVWTEAMSNIPKEMASGLVAAQTTQQAVDPMDIAYAYLFLASDESRQITAANIPVDGGCAGAVTGGMQGEIEV.

[0065] The nucleic acid sequence of the dihydrogenisoin DHR-P120G / L214T mutant is shown as SEQ ID NO. 6, and is specifically as follows:

[0066] atggcacaggaagtcaaagtccccaagatgcccggcgcacccgtgttcggcaagtggatctcccccgaggagtccgtcggccagcgcctgaagggcaagaagatcctgctcaccggcaccaccaagggcgtcggcagggtcacccaggagctgctgtgcgcacacggcgccttcgtctgcggctccggccgcacccccggcgtggcagcctccgtcgccgacgagctgaaggccaagggctaccaggccgccggcatggacgtcgacctgtctgactacgacgccgtgaagaagtgggttgaggagtgcgccgagctcatgggcggcatcgacgtcgtcatcaacaacgcgtcccacggcggcatggcccccttcggcgagatgaccccggagatctggaactacggcatcaagaacgagctcgacctcgtctacaacgtctgcaactgcgcatggccctacctgcagaaggcagacggcgcctccatcatcatcacctcctccaccgtcgccctccagggcagcaactcccctcaggcctgtcacgctgcctgcaagggcgcctgcctgtccctggcccgccagctcgccgctgagggcggccccttcggcatccgctgcaactccgtcaccccgggcaccgtctggaccgaggccatgtccaacatccccaaggagatggcaagcggcctggtcgcagcccagaccacccagcaggctgtcgacccgatggacatcgcctacgcctacctgttcctggcatccgacgagtcccgccagatcaccgctgccaacatccccgtcgacggcggctgcgccggcgctgtgaccggcggcatgcagggcgagatcgaggtc.

[0067] The amino acid sequence of the dihydrodaidzein reductase DHDR-P120G / V170T mutant is shown as SEQ ID NO. 7, and is specifically as follows:

[0068] MAQEVKVPKMPGAPVFGKWISPEESVGQRLKGKKILLTGTTKGVGRVTQELLCAHGAFVCGSGRTPGVAASVADELKAKGYQAAGMDVDLSDYDAVKKWVEECAELMGGIDVVINNASHGGMAPFGEMTPEIWNYGIKNELDLVYNVCNCAWPYLQKADGASIIITSSTTALQGSNSPQACHAACKGACLSLARQLAAEGGPFGIRCNSVTPGLVWTEAMSNIPKEMASGLVAAQTTQQAVDPMDIAYAYLFLASDESRQITAANIPVDGGCAGAVTGGMQGEIEV.

[0069] The nucleotide sequence of the dihydrogenisoin DHR-P120G / V170T mutant is shown as SEQ ID NO. 8, and is specifically as follows:

[0070] atggcacaggaagtcaaagtccccaagatgcccggcgcacccgtgttcggcaagtggatctcccccgaggagtccgtcggccagcgcctgaagggcaagaagatcctgctcaccggcaccaccaagggcgtcggcagggtcacccaggagctgctgtgcgcacacggcgccttcgtctgcggctccggccgcacccccggcgtggcagcctccgtcgccgacgagctgaaggccaagggctaccaggccgccggcatggacgtcgacctgtctgactacgacgccgtgaagaagtgggttgaggagtgcgccgagctcatgggcggcatcgacgtcgtcatcaacaacgcgtcccacggcggcatggcccccttcggcgagatgaccccggagatctggaactacggcatcaagaacgagctcgacctcgtctacaacgtctgcaactgcgcatggccctacctgcagaaggcagacggcgcctccatcatcatcacctcctccaccaccgccctccagggcagcaactcccctcaggcctgtcacgctgcctgcaagggcgcctgcctgtccctggcccgccagctcgccgctgagggcggccccttcggcatccgctgcaactccgtcaccccgggcctggtctggaccgaggccatgtccaacatccccaaggagatggcaagcggcctggtcgcagcccagaccacccagcaggctgtcgacccgatggacatcgcctacgcctacctgttcctggcatccgacgagtcccgccagatcaccgctgccaacatccccgtcgacggcggctgcgccggcgctgtgaccggcggcatgcagggcgagatcgaggtc.

[0071] The amino acid sequence of the dihydrodaidzein reductase DHDR-P120G / L214T / V170T mutant is shown as SEQ ID NO. 9, and is specifically as follows:

[0072] MAQEVKVPKMPGAPVFGKWISPEESVGQRLKGKKILLTGTTKGVGRVTQELLCAHGAFVCGSGRTPGVAASVADELKAKGYQAAGMDVDLSDYDAVKKWVEECAELMGGIDVVINNASHGGMAPFGEMTPEIWNYGIKNELDLVYNVCNCAWPYLQKADGASIIITSSTTALQGSNSPQACHAACKGACLSLARQLAAEGGPFGIRCNSVTPGTVWTEAMSNIPKEMASGLVAAQTTQQAVDPMDIAYAYLFLASDESRQITAANIPVDGGCAGAVTGGMQGEIEV.

[0073] The nucleotide sequence of the dihydrogenisoin DHR-P120G / L214T / V170T mutant is shown as SEQ ID NO. 10, and is specifically as follows:

[0074] atggcacaggaagtcaaagtccccaagatgcccggcgcacccgtgttcggcaagtggatctcccccgaggagtccgtcggccagcgcctgaagggcaagaagatcctgctcaccggcaccaccaagggcgtcggcagggtcacccaggagctgctgtgcgcacacggcgccttcgtctgcggctccggccgcacccccggcgtggcagcctccgtcgccgacgagctgaaggccaagggctaccaggccgccggcatggacgtcgacctgtctgactacgacgccgtgaagaagtgggttgaggagtgcgccgagctcatgggcggcatcgacgtcgtcatcaacaacgcgtcccacggcggcatggcccccttcggcgagatgaccccggagatctggaactacggcatcaagaacgagctcgacctcgtctacaacgtctgcaactgcgcatggccctacctgcagaaggcagacggcgcctccatcatcatcacctcctccaccaccgccctccagggcagcaactcccctcaggcctgtcacgctgcctgcaagggcgcctgcctgtccctggcccgccagctcgccgctgagggcggccccttcggcatccgctgcaactccgtcaccccgggcaccgtctggaccgaggccatgtccaacatccccaaggagatggcaagcggcctggtcgcagcccagaccacccagcaggctgtcgacccgatggacatcgcctacgcctacctgttcctggcatccgacgagtcccgccagatcaccgctgccaacatccccgtcgacggcggctgcgccggcgctgtgaccggcggcatgcagggcgagatcgaggtc.

[0075] Example 1

[0076] Rational design of mutants and construction of recombinant E. coli:

[0077] The wild type dihydrodauricine reductase DHDR with amino acid sequence of SEQ ID NO. 1 was rationally designed, and the related mutation was designed on the primer. The mutation site was introduced into the template sequence by overlap extension PCR technology, with the recombinant plasmid pET28b_NhisMBP containing the target gene as the template.

[0078] Table 1 PCR amplification system

[0079]

[0080] PCR amplification procedure: (1) 95°C pre-denaturation for 10 min; (2) 94°C denaturation for 30 s; (3) 50°C annealing for 30 s; (4) 72°C extension for 1 min; (5) 35 cycles of steps (2)-(4); (6) 72°C extension for 10 min.

[0081] The first round of PCR product was taken for 5 μL agarose gel electrophoresis verification, and after verifying the correct band size, the remaining DNA was subjected to agarose gel electrophoresis gel recovery. The recovered product was subjected to second round of PCR. The product of the second round of PCR was purified to obtain a DNA product containing a mutation site.

[0082] The above purified product and pET28b_NhisMBP were double-digested with EcoR I and Xho I, and the same sticky end product was obtained by agarose gel electrophoresis gel recovery. The target gene was ligated with the plasmid using T4 DNA ligase to construct a recombinant plasmid.

[0083] The ligated recombinant plasmid was transformed into E. coli Rosetta2 (DE3) competent cells, and the recombinant colonies were plated on LB solid medium containing kanamycin and incubated overnight. Single colony PCR verification was performed on the selected positive clone strain, and the positive clone strain was cultured for expression. The mutant bacteria of dihydrodauricine reductase DHDR were obtained by centrifugation.

[0084] Crude enzyme solution preparation:

[0085] The enriched bacteria were resuspended with an appropriate amount of 50 mM (pH = 6.5) phosphate buffer, and then placed in an ice water bath for ultrasonic disruption treatment (disruption program: 1 min, disruption 5 s and stop 5 s, power 50%). The disrupted bacterial solution became clear and transparent. The centrifuge was pre-cooled, and then the disrupted bacterial solution was centrifuged (4°C, 6 min, 12000 rpm). The supernatant was collected to obtain the crude enzyme solution required for subsequent experiments.

[0086] Example 2

[0087] Under the analytical scale, the mutant of dihydrodaidzein reductase DHDR asymmetrically reduced dihydrodaidzein:

[0088] The mutant strain obtained in Example 1 was fermented in a large-scale culture to induce expression, and the obtained bacterial cells were collected. The crude enzyme solution was used as a catalyst for the reduction reaction. The reaction system was as follows: (±)-dihydrodaidzein (dissolved in methanol, final concentration 2 mM, 50 μL), 0.2 mg of NADP + , 9 mg of glucose, pH = 6.5 phosphate buffer, 0.1 g of the mutant of dihydrodaidzein reductase DHDR (wet bacterial amount), and 0.02 g of glucose dehydrogenase (GDH) (wet bacterial amount), with a total volume of 1 mL. The reaction conditions were: 37°C, 200 rpm shaking for 3 h, and the reaction product was extracted with ethyl acetate to obtain the target product.

[0089] Table 2 DHDR wild type and mutant asymmetric reduction (±)-dihydrodaidzein data table

[0090]

[0091] As Figure 1 shown, compared with the DHDR wild type, the DHDR mutant P120F / P212A has significantly improved activity and selectivity in converting (±)-dihydrodaidzein, and it basically only converts (S)-dihydrodaidzein, and the selectivity of the produced (3S,4R)-tetrahydrodaidzein is also very high, and the product ratio can reach 98.4%. The DHDR wild type has poor activity and selectivity, and when converting (±)-dihydrodaidzein, it will produce various products including (3S,4R)-tetrahydrodaidzein and (3R,4R)-tetrahydrodaidzein, the selectivity is poor, and the conversion rate of (S)-dihydrodaidzein is also lower than that of the DHDR mutant P120F / P212A.

Claims

1. A dihydrogenin reductase mutant, characterized in that, Using wild-type dihydrogen daidzein reductase DHDR with the amino acid sequence shown in SEQ ID NO.1 as the parent, single-point mutations or combination mutations were performed to obtain the DHDR mutant of dihydrogen daidzein reductase.

2. The dihydrogenin reductase mutant as described in claim 1, characterized in that, The parental site for a single-point mutation is one of the following: position 120, position 122, position 170, position 178, position 214, position 219, position 220, or position 220.

3. The dihydrogenin reductase mutant as described in claim 2, characterized in that, Mutating the 120th proline in the parental line to alanine, phenylalanine, glycine, isoleucine, and leucine yielded mutants DHDR-P120A, DHDR-P120F, DHDR-P120G, DHDR-P120I, and DHDR-P120L, respectively. The parental methionine at position 122 was mutated to alanine and phenylalanine, respectively, to obtain mutants DHDR-M122A and DHDR-M122F. The parental line was mutated from valine to threonine at position 170 to obtain the mutant DHDR-V170T; Mutating proline at position 178 of the parental line to alanine and phenylalanine yielded mutants DHDR-P178A and DHDR-P178F, respectively. The parental leucine at position 214 was mutated to alanine and threonine, respectively, to obtain mutants DHDR-L214A and DHDR-L214T. The parental alanine at position 219 was mutated to phenylalanine to obtain the mutant DHDR-A219F; The methionine at position 220 of the parental line was mutated to alanine to obtain the mutant DHDR-M220A; The parental glycine at position 230 was mutated to alanine and phenylalanine, respectively, to obtain mutants DHDR-G230A and DHDR-G230F.

4. The dihydrogenin reductase mutant as described in claim 1, characterized in that, Combinatorial mutations can be performed in the following two ways: One method involved using a single-point mutated dihydrogen daidzein reductase DHDR mutant as the parent, and then mutating it to obtain dihydrogen daidzein reductase DHDR combined mutant I. Secondly, using the dihydrogen daidzein reductase DHDR combined mutant I as the parent, mutations were performed to obtain dihydrogen daidzein reductase DHDR combined mutant II.

5. A dihydrogenin reductase mutant as described in claim 4, characterized in that, The dihydrogenogenin reductase DHDR single-point mutant is either DHDR-P120F or DHDR-P120G. Using DHDR-P120F as the parent, the parental leucine at position 214 was mutated to threonine, the parental proline at position 212 was mutated to alanine, and the parental valine at position 170 was mutated to threonine, resulting in the following DHDR combined mutant I dihydrogen daidzein reductases: DHDR-P120F / L214T, DHDR-P120F / P212A, and DHDR-P120F / V170T. Using DHDR-P120G as the parent, the parental leucine at position 214 was mutated to threonine, the parental proline at position 212 was mutated to alanine, and the parental valine at position 170 was mutated to threonine, resulting in the following DHDR combined mutant I dihydrogen daidzein reductases: DHDR-P120G / L214T, DHDR-P120G / P212A, and DHDR-P120G / V170T. The amino acid sequences of the mutant DHDR-P120F / P212A are shown in SEQ ID NO.3, the amino acid sequences of the mutant DHDR-P120G / L214T are shown in SEQ ID NO.5, and the amino acid sequences of the mutant DHDR-P120G / V170T are shown in SEQ ID NO.

7.

6. A dihydrogenin reductase mutant as described in claim 5, characterized in that, Dihydrogenase DHDR combined mutant mutant I is either DHDR-P120F / P212A or DHDR-P120G / L214T; Using the parent DHDR-P120F / P212A, the parental valine at position 170 was mutated to threonine, resulting in the DHDR-P120F / P212A / V170T, a combined mutant of dihydrogen daidzein reductase. Using the parent DHDR-P120G / L214T, the valine at position 170 of the parent was mutated to threonine, resulting in the combined mutant II DHDR-P120G / L214T / V170T. The amino acid sequence of the mutant DHDR-P120G / L214T / V170T is shown in SEQ ID NO.

9.

7. A DNA molecule, characterized in that, The DNA molecule encodes the dihydrogen daidzein reductase mutant according to any one of claims 1-6; wherein the nucleotide sequence of the mutant DHDR-P120F / P212A is shown in SEQ ID NO.4, the nucleotide sequence of the mutant DHDR-P120G / L214T is shown in SEQ ID NO.6, the nucleotide sequence of the mutant DHDR-P120G / V170T is shown in SEQ ID NO.8, and the nucleotide sequence of the mutant DHDR-P120G / L214T / V170T is shown in SEQ ID NO.

10.

8. An expression carrier, characterized in that, The expression vector contains the DNA molecule as described in claim 7.

9. A host cell, characterized in that, The present invention contains the expression vector of claim 8, wherein the host cell is Escherichia coli.

10. The application of a dihydrogenistein reductase mutant in the synthesis of (3S,4R)-tetrahydrogenistein, characterized in that, The DHDR-P120F / P212A, DHDR-P120G / L214T, DHDR-P120G / V170T, or DHDR-P120G / L214T / V170T from the dihydrogenidin reductase mutants of claim 5 or 6 were used for asymmetric reduction of dihydrogenidin to synthesize (3S,4R)-tetrahydrogenidin; the reaction formula for the reduction reaction is shown in Formula I: 。

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