Highly active dihydrodaidzein racemase mutants and their use in s-equol whole cell transformation systems

Abstract

The application discloses a mutant and a whole-cell transformation system for efficiently synthesizing S-equol. Specifically, a DDRC (dihydrodaidzein racemase) mutant for catalyzing production of S-equol is provided, and the catalytic activity of the DDRC mutant is significantly improved compared with a wild-type DDRC. The yield of S-equol can be significantly improved by using an engineering bacterium containing the mutant. Meanwhile, an EcN whole-cell transformation system is constructed, and the system can be used for safely and efficiently generating S-equol.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a highly active dihydrogen daidzein racemic enzyme mutant and its application in an S-estrol whole-cell transformation system. Background Technology

[0002] S-Estradiol is a naturally occurring chiral molecule formed from daidzein after it enters the human body through a series of complex metabolic transformations by specific microorganisms in the gut. Due to its strong affinity for estrogen receptors, its ability to bidirectionally regulate human estrogen levels, and its antioxidant capacity, S-Estradiol has enormous application potential in the pharmaceutical, cosmetic, and food industries.

[0003] Although estrol can be synthesized through multi-step chemical reactions, the chemical synthesis method generates a racemic mixture of R- and S-type structures. Separating the high-purity S-configuration from the mixture requires a complex chiral resolution process, resulting in high cost and low yield.

[0004] Microbial transformation is currently the mainstream technology for producing equol. Its core principle involves isolating anaerobic bacteria capable of producing equol from gut microbiota, and then converting daidzein in soybeans into S-equol through anaerobic fermentation. A significant advantage of this method is the high stereoselectivity of the equol product, producing only the S-configuration without requiring additional chiral resolution. However, the characteristics of anaerobic fermentation also present limitations: low conversion efficiency and a long fermentation cycle lead to high production costs.

[0005] The enzymatic conversion of equol involves a four-step enzyme cascade: Step 1: Dysgenin reductase (DZNR) converts daidzein to (R)-dihydrodaidzein; Step 2: Dysgenin racemic enzyme (DDRC) catalyzes the efficient conversion of (R)-dihydrodaidzein to (S)-dihydrodaidzein (spontaneous conversion efficiency is extremely low, enzyme catalysis is key); Step 3: Dysgenin reductase (DHDR) reduces (S)-dihydrodaidzein to tetrahydrodaidzein; Step 4: Tetrahydrodaidzein reductase (THDR) catalyzes the formation of (S)-Equol from tetrahydrodaidzein. Although the enzymatic synthesis of equol is theoretically feasible, the high cost due to the need for the expensive cofactor NADPH prevents its large-scale production.

[0006] Recombinant expression of the four enzymes required for the transformation of (S)-Equol in aerobic microorganisms (such as Escherichia coli) can construct an efficient whole-cell transformation system, and researchers have verified the feasibility of whole-cell catalysis using heterologous expression systems. However, a safe and efficient whole-cell transformation system for generating (S)-Equol is still needed.

[0007] In summary, there is an urgent need in this field to develop tool enzymes with high catalytic activity for the synthesis of (S)-Equol, as well as whole-cell transformation systems that can safely and efficiently generate (S)-Equol. Summary of the Invention

[0008] The purpose of this invention is to provide a highly catalytically active dihydrogen daidzein racemic enzyme (DDRC) and a safe and efficient probiotic EcN whole-cell transformation system, which can be used to efficiently synthesize S-estrol.

[0009] In a first aspect, the present invention provides a dihydrogen daidzein racemic enzyme (DDRC) mutant, wherein the DDRC mutant has amino acid mutations at the following mutation sites: position 60 and position 130; wherein the mutation sites are numbered based on the amino acid sequence of wild-type DDRC shown in SEQ ID NO:1, and the DDRC mutant has significantly higher catalytic activity than wild-type DDRC.

[0010] In another preferred embodiment, the sequence of the wild-type DDRC is as shown in SEQ ID NO: 1.

[0011] In another preferred embodiment, the DDRC mutant comprises the following amino acid mutations: D60E and E130Q, wherein the mutation sites are numbered based on the amino acid sequence of wild-type DDRC shown in SEQ ID NO:1.

[0012] In another preferred embodiment, the DDRC mutant is selected from the group consisting of: (1) A DDRC mutant having the amino acid sequence shown in SEQ ID NO:3; (2) A DDRC mutant having an amino acid sequence as shown in SEQ ID NO:3 formed by substitution, deletion or addition of one or more amino acid residues, preferably 1-20, more preferably 1-10, more preferably 1-3, and most preferably 1 amino acid residue, or formed by adding a signal peptide sequence, and having DDRC activity, and the DDRC mutant having significantly higher catalytic activity than wild-type DDRC. (3) A derivative polypeptide whose sequence contains the polypeptide sequence described in (1) or (2); and (4) A DDRC mutant with ≥85% homology (preferably ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98% or ≥99%) to the amino acid sequence shown in SEQ ID NO:3 and having DDRC activity, and the DDRC mutant having significantly higher catalytic activity than wild-type DDRC.

[0013] In another preferred embodiment, (3) is a fusion protein formed by adding a tag sequence, a signal sequence or a secretion signal sequence to (1) or (2).

[0014] In another preferred embodiment, the DDRC mutant is chemically modified and / or immobilized.

[0015] In another preferred embodiment, the chemical modification is selected from the group consisting of acetylation, amidation, cyclization, glycosylation, phosphorylation, alkylation, biotinylation, fluorescent group modification, polyethylene glycol (PEG) modification, or combinations thereof.

[0016] In another preferred embodiment, the DDRC mutant is attached with a tag for detection and / or purification.

[0017] In another preferred embodiment, the tag is selected from the group consisting of His6, GST, EGFP, MBP, Nus, HA, IgG, FLAG, c-Myc, ProfinityeXact, or combinations thereof.

[0018] In another preferred embodiment, the DDRC mutant catalytically converts (R)-dihydrodaidzein to (S)-dihydrodaidzein.

[0019] In another preferred embodiment, the DDRC mutant exhibits significantly higher catalytic activity than wild-type DDRC.

[0020] In another preferred embodiment, "significantly higher" means that the catalytic activity of the DDRC mutant is increased by 10%, more preferably by 15%, and even more preferably by 20% compared with wild-type DDRC.

[0021] In another preferred embodiment, the DDRC mutant is used to catalyze the production of S-estrol.

[0022] In another preferred embodiment, in the artificially constructed strain, the DDRC mutant can increase the yield of S-estrol, preferably by 10-30%; more preferably by 15-30%; and most preferably by 15-25%.

[0023] In another preferred embodiment, the artificially constructed strain is selected from the group consisting of: Saccharomyces cerevisiae strains, Escherichia coli strains, Pichia pastoris strains, Schizosoma strains, and probiotic EcN strains.

[0024] In a second aspect, the present invention provides an isolated polynucleotide encoding a DDRC mutant as described in the first aspect of the present invention.

[0025] In another preferred embodiment, the polynucleotide is selected from: (a) Polynucleotides with sequences as shown in SEQ ID NO:4; (b) The nucleotide sequence is ≥80%, ≥81%, ≥82%, ≥83%, ≥84%, ≥85%, ≥86%, ≥87%, ≥88%, ≥89%, ≥90%, ≥91%, ≥92%, ≥93%, ≥94%, ≥95%, ≥96%, ≥97%, ≥98%, ≥99%, or ≥99.5% identical to the nucleotide sequence shown in SEQ ID NO:4, and encodes a polynucleotide as described in the first aspect of the invention; (c) A nucleotide sequence shown in SEQ ID NO:4 is truncated or augmented with 1-60 (preferably 1-30, more preferably 1-10) nucleotides at the 5' and / or 3' ends, encoding a polynucleotide of the DDRC mutant as described in the first aspect of the invention; or (d) A polynucleotide complementary to any of the polynucleotides described in (a)-(c).

[0026] In another preferred embodiment, the polynucleotide flanking the DDRC mutant further contains auxiliary elements selected from the group consisting of signal peptides, secretory peptides, tag sequences (such as 6His), or combinations thereof.

[0027] In another preferred embodiment, the polynucleotide is selected from the group consisting of DNA sequences, RNA sequences, or combinations thereof.

[0028] In a third aspect, the present invention provides a carrier containing the polynucleotides as described in the second aspect of the present invention.

[0029] In another preferred embodiment, the carrier includes an expression carrier, a shuttle carrier, and an integration carrier.

[0030] In another preferred embodiment, the expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0031] In a fourth aspect, the present invention provides a genetically engineered host cell containing a vector as described in the third aspect of the present invention, or having a genome containing polynucleotides as described in the second aspect of the present invention.

[0032] In another preferred embodiment, the host cell also contains or has integrated polynucleotides encoding daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR) into its genome.

[0033] In another preferred embodiment, the cell is a prokaryotic cell or a eukaryotic cell.

[0034] In another preferred embodiment, the host cell is a prokaryotic cell, such as Escherichia coli.

[0035] In another preferred embodiment, the host cell is a eukaryotic cell, such as a yeast cell or a plant cell.

[0036] In another preferred embodiment, the host cell is a yeast cell.

[0037] In another preferred embodiment, the host cell is a genetically engineered host cell constructed by introducing the coding sequence of the DDRC mutant as described in the first aspect of the invention into the probiotic EcN.

[0038] A fifth aspect of the present invention provides a method for preparing the DDRC mutant as described in the first aspect of the present invention, comprising the following steps: (1) Under conditions suitable for protein expression, genetically engineered host cells as described in the fourth aspect of the present invention are cultured to obtain a culture containing the DDRC mutant; and (2) Optionally, isolate and / or purify the DDRC mutant from the culture.

[0039] In a sixth aspect, the present invention provides uses of the DDRC mutant as described in the first aspect, wherein the uses are selected from the group consisting of: (a) Used in the preparation of (S)-dihydrogenainin; and / or (b) An enzyme preparation for the production of (S)-dihydrogenase.

[0040] In a seventh aspect, the present invention provides an active ingredient combination comprising: (a) a first active ingredient, wherein the first active ingredient is a genetically engineered host cell as described in the fourth aspect of the present invention; and (b) The second active ingredient is daidzein.

[0041] In another preferred embodiment, the genetically engineered host cell also contains or has integrated polynucleotides encoding daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR) into its genome.

[0042] In another preferred embodiment, the genetically engineered host cell is a genetically engineered probiotic EcN.

[0043] In another preferred embodiment, the first active ingredient and the second active ingredient may be contained in separate compositions or formulations, or together in the same composition or formulation.

[0044] In another preferred embodiment, the composition is a pharmaceutical composition.

[0045] In another preferred embodiment, the first and second active ingredients can be used for simultaneous administration, sequential administration, or intermittent administration.

[0046] In another preferred embodiment, the first and second active ingredients may each independently comprise a pharmaceutically acceptable carrier.

[0047] In another preferred embodiment, the carrier is selected from the group consisting of: sustained-release agents, excipients, fillers, diluents, binders, wetting agents, disintegrants, absorption promoters, adsorbent carriers, surfactants, or lubricants.

[0048] In another preferred embodiment, the dosage form of the preparation is selected from the group consisting of tablets, capsules, granules, pills, powders, solutions, suspensions, emulsions, liposomes, and suppositories, or combinations thereof.

[0049] In another preferred embodiment, the composition or formulation is administered orally.

[0050] An eighth aspect of the present invention provides a method for producing S-estrol, comprising the steps of: Under suitable culture conditions, genetically engineered host cells are cultured to obtain a fermentation product containing S-estrol, and S-estrol is isolated from the fermentation product. The host cell contains or has integrated into its genome polynucleotides encoding dihydrogenin racemic enzyme (DDRC) mutants, daidzein reductase (DZNR), dihydrogenin reductase (DHDR), and tetrahydrogenin reductase (THDR) as described in the first aspect of the present invention.

[0051] In another preferred embodiment, the genetically engineered host cell is a genetically engineered probiotic EcN.

[0052] In another preferred embodiment, the culture includes test tube culture, shake flask culture, and tank fermentation culture.

[0053] In another preferred embodiment, the test tube culture uses TB medium.

[0054] In another preferred embodiment, the shake flask culture and / or tank fermentation culture uses a synthetic culture medium.

[0055] In another preferred embodiment, the inoculum size of the genetically engineered probiotic EcN is OD600 = 0.001~10, more preferably OD600 = 0.01~5, more preferably OD600 = 0.01~1, for example OD600 = 0.05.

[0056] In another preferred embodiment, the genetically engineered probiotic EcN is cultured in a synthetic culture medium for 0.1 to 10 days, more preferably 0.2 to 8 days, and even more preferably 0.5 to 5 days.

[0057] In another preferred embodiment, the genetically engineered probiotic EcN is cultured at 20–45°C, preferably 25–40°C, more preferably 25–35°C, for example, about 32°C.

[0058] In another preferred embodiment, the method further includes the step of detecting S-estrol in the fermentation product.

[0059] In another preferred embodiment, the detection is performed by liquid chromatography.

[0060] In another preferred embodiment, the S-estrol yield of the method is ≥4 g / L, more preferably ≥4.25 g / L, and even more preferably... The concentration of the substance is ≥4.8 g / L, for example, 4-8 g / L or 4-6 g / L.

[0061] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0062] Figure 1 The mass spectrometric peak areas of the catalytic product S-estradiol from wild-type DDRC and DDRC mutant are shown.

[0063] Figure 2 The liquid chromatography-mass spectra of the shake-flask test for the mutant strain Mu are shown.

[0064] Figure 3 The yields of S-estradiol, a product of shake-flask testing, are shown for wild-type strains and mutants. Detailed Implementation

[0065] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly developed a highly catalytically active dihydrodaidzein racemic enzyme (DDRC) mutant. Starting with wild-type DDRC, the inventors utilized error-prone PCR technology to modify its catalytic activity and conducted extensive screenings, obtaining a DDRC mutant with excellent high catalytic activity. Simultaneously, a whole-cell transformation system of probiotic EcN containing this DDRC mutant was constructed, which can be used for the safe and efficient generation of (S)-Equol. Based on this, the inventors completed this invention.

[0066] the term To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0067] The mutant of the present invention As used herein, the terms “mutant of the present invention”, “polypeptide of the present invention”, “DDRC mutant protein of the present invention”, “DDRC mutant of the present invention”, “mutant DDRC of the present invention”, “dihydrogenoside racemic enzyme mutant of the present invention”, and “dihydrogenoside racemic enzyme mutant protein of the present invention” are used interchangeably to refer to the DDRC (dihydrogenoside racemic enzyme) mutant described in the first aspect of the present invention.

[0068] As used herein, "isolated protein (or polypeptide)" means that the protein is substantially free of other naturally occurring or associated proteins, lipids, carbohydrates, or other substances. Those skilled in the art can purify the protein using standard protein purification techniques. A substantially pure protein will produce a single master band on a non-reducing polyacrylamide gel. The purity of the protein can be further analyzed using techniques such as amino acid sequencing.

[0069] The DDRC mutant protein of the present invention can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide. The polypeptide of the present invention can be a naturally purified product, a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (e.g., bacteria, yeast, plant) using recombinant technology. Depending on the host used in the recombinant production protocol, the polypeptide of the present invention can be glycosylated or non-glycosylated. The polypeptide of the present invention may or may not include an initial methionine residue.

[0070] The present invention also includes fragments, derivatives, and analogs of the said DDRC mutant protein. As used herein, the terms “fragment,” “derivative,” and “analyte” refer to a DDRC mutant protein that substantially retains the same biological function or activity as the said DDRC mutant protein.

[0071] The DDRC mutant protein fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, or a sequence used to purify the polypeptide, or a proteogen sequence). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art. In the present invention, the conserved substituted amino acids are preferably produced by amino acid substitutions according to Table A.

[0072] Table A The DDRC (dihydrogenase didaidzein racemic) mutant protein or its derived polypeptide of the present invention may also contain one or more polypeptide fragments at the amino or carboxyl terminus as protein tags. Any suitable tag can be used in this invention. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0073] In another embodiment, the amino acid sequence of DDRC-wild type is (SEQ ID NO:1): MIKAQLNRIALRAADADKAVEDLNKLLGVTFYGPYDDEHMGLRVALPKSGGIEVMAPMHDHDAIGAYKALQTVGEGISGIAMRVDDFDAAKEHFASLGLTPVVEFNHGKFREMIFPPQPATHNLEIAVNEFPDANGAAIQVALDMGVDWTDVCDWDAK.

[0074] In another embodiment, the amino acid sequence of the DDRC mutant of the present invention is (SEQ ID NO:3): MIKAQLNRIALRAADADKAVEDLNKLLGVTFYGPYDDEHMGLRVALPKSGGIEVMAPMHEHDAIGAYKALQTVGEGISGIAMRVDDFDAAKEHFASLGLTPVVEFNHGKFREMIFPPQPATHNLEIAVNQFPDANGAAIQVALDMGVDWTDVCDWDAK.

[0075] The polynucleotides, vectors, and host cells of the present invention The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.

[0076] The polynucleotides encoding the polypeptides described in this invention include: coding sequences that encode only the mature polypeptide; coding sequences of the mature polypeptide and various additional coding sequences; coding sequences of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0077] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0078] This invention also relates to variants of the aforementioned polynucleotides that encode polypeptides or fragments, analogs, and derivatives of polypeptides having the same amino acid sequence as those of this invention. These polynucleotide variants can be naturally occurring isoforms or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide it encodes.

[0079] The polypeptides and polynucleotides in this invention are preferably provided in isolated form and more preferably purified to homogenization. The full-length nucleotide sequence or fragments of the DDRC mutant of this invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed in this invention, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared according to conventional methods known to those skilled in the art. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified from each amplification in the correct order.

[0080] Once the relevant sequence is obtained, recombinant proteins can be prepared using bioengineering methods. This typically involves cloning the polynucleotide sequence encoding the recombinant protein into an expression vector, then transforming the expression vector into cells for expression, harvesting and purifying the recombinant protein.

[0081] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0082] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or vectors) known in the art, transformed into cells, expressed, purified, and the protein obtained.

[0083] The application of PCR technology to amplify DNA / RNA is preferred for obtaining the gene of the present invention. Especially when it is difficult to obtain full-length cDNA from a library, the RACE method (RACE-cDNA end amplification method) is preferred. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods such as gel electrophoresis.

[0084] The present invention also relates to vectors containing the polynucleotides of the present invention, host cells generated by genetic engineering using the coding sequences of the vectors of the present invention or the polypeptides of the present invention or their derivatives, and methods for generating the polypeptides of the present invention by recombinant technology.

[0085] Using conventional recombinant DNA techniques, the polynucleotide sequences of this invention can be used to express or produce recombinant polypeptides of this invention or their derivatives. Generally, the following steps are involved: (1) A polynucleotide (or variant) encoding the polypeptide of the present invention or a derivative thereof, or Transform or transduce suitable host cells using a recombinant expression vector containing this polynucleotide; (2) Host cells cultured in a suitable culture medium; (3) Isolate and purify proteins from culture media or cells.

[0086] In this invention, the polynucleotide sequence encoding the polypeptide or a derivative thereof can be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well-known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable within the host. An important characteristic of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and translational control elements.

[0087] Methods well known to those skilled in the art can be used to construct expression vectors containing the coding DNA sequence of the polypeptide or a derivative thereof of the present invention and suitable transcription / translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

[0088] In addition, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for Escherichia coli.

[0089] Vectors containing the appropriate DNA sequence and appropriate promoter or control sequence can be used to transform appropriate host cells so that they can express proteins.

[0090] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; plant cells; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS, 293 cells, or Bowes melanoma cells.

[0091] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0092] Those skilled in the art are well aware of how to select appropriate vectors, promoters, enhancers, and host cells.

[0093] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0094] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0095] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0096] In another embodiment, the DDRC-wild-type polynucleotide sequence is (SEQ ID NO:2): ATGATCAAAGCGCAGCTGAACCGTATCGCGCTGCGTGCGGCGGATGCAGATAAAGCGGTTGAAGATCTGAACAAACTGCTGGGTGTTACCTTCTATGGTCCGTATGATGATGAACACATGGGTCTGCGTGTTGCGCTGCCGAAATCTGGTGGTATCGAAGTTATGGCTCCGATGCACGATCACGATGCAATCGGCGCGTATAAAGCACTGCAGACCGTTGGTGAAGGTATTTCTGG TATCGCTATGCGTGTTGATGATTTCGATGCTGCTAAAGAACACTTCGCGTCTTTAGGTCTGACCCCGGTTGTTGAATTCAACCACGGTAAATTCCGTGAAATGATCTTCCCGCCGCAGCCGGCTACCCACAACCTGGAAATCGCTGTTAACGAATTCCCGGATGCAAACGGTGCTGCAATTCAGGTTGCGCTGGATATGGGTGTTGATTGGACCGATGTTTGCGATTGGGATGCTAAAATAA.

[0097] In another preferred embodiment, the polynucleotide sequence encoding the DDRC mutant is (SEQ ID NO: 1). NO:4): ATGATCAAAGCGCAGCTGAACCGTATCGCGCTGCGTGCGGCGGATGCAGATAAAGCGGTTGAAGATCTGAACAAACTGCTGGGTGTTACCTTCTATGGTCCGTATGATGATGAAC ACATGGGTCTGCGTGTTGCGCTGCCGAAATCTGGTGGTATCGAAGTTATGGCTCCGATGCACGAGCACGATGCAATCGGCCGGTATAAAGCACTGCAGACCGTTGGTGAAGGTATTTCTGG TATCGCTATGCGTGTTGATGATTTCGATGCTGCTAAAGAACACTTCGCGTCTTTAGGTCTGACCCCGGTTGTTGAATTCAACCACGGTAAATTCCGTGAAATGATCTTCCCGCCGCAGCCGGCTACCCACAACCTGGAAATCGCTGTTAACCAATTCCCGGATGCAAACGGTGCTGCAATTCAGGTTGCGCTGGATATGGGTGTTGATTGGACCGATGTTTGCGATTGGGATGCTAAAATAA.

[0098] Probiotics EcN EcN, short for Escherichia coli Nissle 1917, is named after its inventor, Alfred Nissle. EcN is a non-pathogenic Escherichia coli strain that is sensitive to serum and easily cleared by it. Studies have confirmed that it possesses probiotic characteristics such as lacking virulence factors, producing specific adaptation factors, and strengthening the intestinal natural barrier. It is widely used in the prevention of infectious diarrhea and immune regulation. EcN strains have been used as the active pharmaceutical ingredient in a licensed medicine sold in Germany and several other countries.

[0099] The EcN 1917(T7 RNAP) strain, derived from Mutaflor's EcN probiotics, is a non-pathogenic microorganism that promotes digestive health when consumed. EcN 1917(T7 RNAP) is a T7 RNA polymerase (T7 RNAP) driven by a one-copy lacUV5 promoter introduced into the genome and integrated into the malEFG operon of EcN. It can be used in various commercial protein expression plasmids.

[0100] The probiotic EcN whole-cell transformation system of the present invention contains or integrates in its genome a polynucleotide encoding the dihydrogenin racemic enzyme (DDRC) mutant described in the first aspect of the present invention, as well as other related proteins encoding wild-type or mutant proteins, including: daidzein reductase (DZNR), dihydrogenin reductase (DHDR) and tetrahydrogenin reductase (THDR) or combinations thereof, and can be used for the safe and efficient production of S-estrol.

[0101] Combinations of active ingredients and their applications The present invention also provides an active ingredient combination comprising: a genetically engineered host cell as described in the fourth aspect of the invention as a first active ingredient; and daidzein as a second active ingredient. Preferably, the active ingredient combination is a composition or formulation; more preferably, the composition is a pharmaceutical composition.

[0102] In another preferred embodiment, the genetically engineered host cell is a whole-cell transformation system of probiotic EcN.

[0103] In another preferred embodiment, the composition further includes probiotics selected from the group consisting of lactic acid bacteria, bifidobacteria, or combinations thereof; and / or prebiotics selected from the group consisting of fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), lactulose oligosaccharides (LACT), soybean oligosaccharides (SOS), inulin, or combinations thereof.

[0104] In a preferred embodiment, the composition is a liquid formulation, a solid formulation, or a semi-solid formulation.

[0105] In a preferred embodiment, the liquid formulation is selected from the group consisting of solution products or suspension products.

[0106] In a preferred embodiment, the dosage form of the composition is selected from the group consisting of powders, granules, tablets, sugar-coated tablets, capsules, granules, suspensions, solutions, syrups, drops, and sublingual tablets.

[0107] The pharmaceutical compositions of the present invention can be administered in any form, such as tablets, injections, or capsules. The pharmaceutical formulation includes excipients, drug-permitted media, and carriers, which may be selected according to the route of administration. The pharmaceutical formulations of the present invention may further comprise auxiliary active ingredients.

[0108] Lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone (PVP), cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, or mineral oil can all be used as carriers, excipients, or diluents for the pharmaceutical composition in this invention.

[0109] Furthermore, the pharmaceutical compositions of the present invention may further include lubricants, wetting agents, emulsifiers, suspension stabilizers, preservatives, sweeteners, and flavorings. The pharmaceutical compositions of the present invention can be produced as enteric-coated formulations using various known methods, so that the active ingredient of the pharmaceutical composition, i.e., the microorganism, can pass smoothly through the stomach without being destroyed by gastric acid.

[0110] Furthermore, the microorganisms of the present invention can be used in capsule form prepared by conventional methods. For example, standard excipients and the cryogenically dried microorganisms of the present invention are mixed to form small pellets, which are then filled into gelatin capsules. Additionally, the microorganisms of the present invention and permitted excipients for pharmaceuticals, such as liquid gums, cellulose, silicates, or mineral oils, can be mixed to form suspensions or dispersions, which can be filled into soft gelatin capsules.

[0111] The pharmaceutical compositions of the present invention can be formulated into enteric-coated tablets for oral administration. The term "enteric-coated" as used in this application includes all coatings permitted for use in conventional pharmaceuticals that are not degraded by gastric acid but are readily decomposed in the small intestine, rapidly releasing the microorganisms of the present invention. The enteric-coated tablets of the present invention can be maintained at 36-38°C for more than 2 hours in synthetic gastric acid such as an HCl solution with pH=1, and preferably decompose within 1 hour in synthetic intestinal fluid such as a buffer solution with pH=7.0.

[0112] The casings of this invention are coated with approximately 16-30 mg per tablet, preferably 16-25 mg, and more preferably 16-20 mg. The casing thickness in this invention is 5-100 μm, ideally 20-80 μm. The casing components are selected from conventionally known polymers.

[0113] The preferred casings of the present invention are prepared from a copolymer of cellulose acetate phthalate polymer or trimellitate polymer and isobutylene acid (e.g., a copolymer of isobutylene acid containing more than 40% isobutylene acid and isobutylene acid containing methylcellulose phthalate hydroxypropyl ester or its ester derivative).

[0114] The cellulose phthalate acetate used in the casings of this invention has a viscosity of approximately 45-90 cp, an acetyl content of 17-26%, and a phthalic acid content of 30-40%. The cellulose trimellitate acetate used in the casings has a viscosity of approximately 5-21 cs and an acetyl content of 17-26%. The cellulose trimellitate acetate is produced by Eastman Kodak and can be used in the casing materials of this invention.

[0115] The hydroxypropyl methylcellulose phthalate used in the casings of this invention has a molecular weight of generally 20,000-130,000 Daltons, with an ideal molecular weight of 80,000-100,000 Daltons. The hydroxypropyl content is 5-10%, the methoxy content is 18-24%, and the phthaloyl content is 21-35%.

[0116] The hydroxypropyl methylcellulose phthalate used in the casings of this invention is HP50, manufactured by Shin Etsu-Chemidnl Co., Ltd. of Japan. HP50 contains 6-10% hydroxypropyl, 20-24% methoxy, and 21-27% propyl, with a molecular weight of 84,000 Daltons. Another casing material is HP55, which contains 5-9% hydroxypropyl methylcellulose phthalate, 18-22% methoxy, and 27-35% phthalic acid, with a molecular weight of 78,000 Daltons.

[0117] The sausage casings of this invention are prepared as follows: a casing solution is sprayed onto the core using conventional methods. All solvents used in this casing coating method are alcohols (such as ethanol), ketones (such as acetone), halogenated hydrocarbon compounds (such as dichloromethane), or combinations thereof. A softener, such as di-n-butyl phthalate and triacetin, is added to the casing solution in a ratio of 1 part casing to approximately 0.05 parts or approximately 0.3 parts softener. The spraying method is preferably performed continuously, and the amount of material sprayed can be controlled according to the conditions used for coating. The spraying pressure can be adjusted arbitrarily; generally, ideal results can be obtained at an average pressure of 1-1.5 bar.

[0118] The term "effective amount of drug" in the instructions refers to an amount that can produce function or activity in humans and / or animals and is acceptable to humans and / or animals. For example, in this invention, a drug containing 1×10⁻¹⁰ to 1×10⁻¹⁰⁻¹ can be prepared. 20 CFU / ml or CFU / g (specifically, it may contain 1×10⁻⁶ CFU / ml). 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 The formulation contains genetically engineered host cells as described in this invention (cfu / ml or cfu / g) and 1-2000 mg (particularly, may contain 10-1000 mg, more particularly, may contain 20-500 mg) of daidzein.

[0119] When used to prepare pharmaceutical compositions, the effective doses of the genetically engineered host cells and daidzein as described in this invention can vary depending on the administration regimen and the severity of the disease to be treated. A suitable dosage form for oral administration comprises approximately 1 × 10⁻¹ to 1 × 10⁻¹ of [amount missing] in close mixture with a pharmaceutically acceptable solid or liquid carrier.20 CFU / ml or CFU / g (specifically, it may contain 1×10⁻⁶ CFU / ml). 4 -1×10 15 cfu / ml or cfu / g; more specifically, it may contain 1×10 6 -1×10 11 Genetically engineered host cells as described in this invention (cfu / ml or cfu / g) and about 1-2000 mg (particularly, may contain 10-1000 mg, more particularly, may contain 20-500 mg) of daidzein. This dosage regimen can be adjusted to provide an optimal therapeutic response. For example, due to the urgent requirements of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0120] The compositions described herein can be administered orally or otherwise. Solid carriers include starch, lactose, dicalcium phosphate, microcrystalline cellulose, sucrose, and kaolin, while liquid carriers include culture media, polyethylene glycol, nonionic surfactants, and edible oils (such as corn oil, peanut oil, and sesame oil), provided they are suitable for the genetically engineered host cells and daidzein characteristics as described in this invention and the desired specific route of administration. Adjuvants commonly used in the preparation of pharmaceutical compositions may also be advantageously included, such as flavoring agents, colorings, preservatives, and antioxidants such as vitamin E, vitamin C, BHT, and BHA.

[0121] From the perspective of ease of preparation and administration, preferred pharmaceutical compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. Oral administration is preferred.

[0122] The composition of the invention is administered to the individual once or more daily. Dosage units represent doses that are formally divisible and applicable to human or all other mammalian individuals. Each unit contains a pharmaceutically permissible carrier and an effective therapeutic amount of the microorganisms of the invention. Dosage varies depending on the patient's weight and severity of obesity, the included supplemental active ingredients, and the microorganisms used. Furthermore, administration can be split if possible, and continuous if necessary. Therefore, the dosage does not limit the invention. In a preferred embodiment, the composition comprises: pharmaceuticals, animal feed, etc.

[0123] In a preferred embodiment, the composition is formulated as follows: 1×10⁻¹×10⁻¹ 20 Genetically engineered host cells as described in this invention at cfu / mL and 1-2000 mg of daidzein; and pharmaceutically acceptable carriers and / or excipients.

[0124] In another preferred embodiment, the composition is formulated as follows: 1×10 6 -1×10 11Genetically engineered host cells as described in this invention at cfu / mL and 10-1000 mg of daidzein; and pharmaceutically acceptable carriers and / or excipients.

[0125] The main advantages of this invention include: (1) A DDRC (dihydrogen daidzein racemic enzyme) mutant with significantly improved catalytic activity compared to the wild type was obtained, with catalytic efficiency increased by at least 20% or more.

[0126] (2) A recombinant strain containing the DDRC mutant was constructed, and its catalytic production of (S)-Equol increased by 20.3% compared with the wild type.

[0127] (3) A safe and efficient whole-cell transformation system for probiotic EcN was constructed to generate (S)-Equol.

[0128] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0129] Example 1: Construction of a random mutant library of dihydrogen daidzein racemic enzyme (DDRC) In this embodiment, a random mutant library of dihydrogen daidzein racemic enzyme (DDRC) was constructed using a random mutant kit, with approximately 2000 transformants.

[0130] 1.1 Construction of a random mutant library for DDRC Using pETDuet-1-DDRC-DZNR-THDR-DHDR (a plasmid containing the DDRC gene fragment) as a template, random mutations were performed using the Takara Diversify® PCR Random Mutagenesis Kit. The PCR system and PCR program are shown in Tables 1 and 2, respectively. The PCR products were recovered after agarose gel electrophoresis to obtain the error-prone PCR product of DDRC.

[0131] Table 1. PCR system Table 2. PCR Procedure 1.2 Amplification of homologous arm fragments of DDRC for plasmid construction The PCR system used was the standard system of Novizan's high-fidelity PCR enzyme 2×Phanta Max Master Mix (Dye Plus) to amplify the homologous arm fragment of DDRC for plasmid construction. The primer sequences used are shown in SEQ ID NO:8-9. The specific PCR system and PCR procedure are shown in Tables 3 and 4, respectively. The desired PCR products were obtained after recovery by agarose gel electrophoresis.

[0132] Table 3. PCR system Table 4. PCR Procedure 1.3 Construction of recombinant strain W4 containing DDRC gene mutation The pCDFDuet-1 vector was digested with the fast digesters SacI and Hind III, and then the linearized fragment of the pCDFDuet-1 vector containing the SacI and Hind III recombination sites was obtained by agarose gel extraction.

[0133] The mutant library gene fragment obtained in 1.2 was recombined into the pCDFDuet-1 vector using homologous recombinase. The recombinant product was then transferred into strain W2 (pETDuet-1-DDRC-DZNR-THDR-DHDR / BL21(DE3)) which already contains genes for the four pathways of S-estradiol synthesis, to obtain recombinant strain W4 containing the DDRC gene mutation.

[0134] Example 2: 96-well plate fermentation and automated high-throughput screening of DDRC mutant library Transformants were screened using automated and high-throughput screening equipment, and a mutant Mu with increased WT product compared to the wild-type strain was identified.

[0135] 2.1 Methods 2.1.1 Selection and culture of bacterial strains Using Multidrop, LB medium (100 μg / ml Amp + 50 μg / ml Spe) was dispensed into 96-well plates at 500 μL per well. Mutant strains growing on the plates were selected using a fully automated high-throughput cloning selection system, Qpix400, and single colonies were inoculated into the corresponding wells. Each 96-well plate contained a blank control (well A1) and three wild-type controls (wells A2-A4). The 96-well plates were placed in a high-speed shaking incubator and incubated overnight at 37°C and 1000 rpm.

[0136] 2.1.2 Preservation, subculturing, and catalytic reaction of seed culture The seed culture from Example 2.1.1 was transferred at 4% to a new 96-well plate containing 500 μL of TB medium (100 μg / ml Amp + 50 μg / l Spe) per well and cultured at 37°C and 1000 rpm for 2 h until the OD was approximately 0.8. 0.2 mM IMPTG (10 mM stock solution, 10 μL per well) was added for induction, and the plate was cultured overnight at 25°C and 800 rpm. The bacteria were collected by centrifugation (3500 rpm, 5 min), and 95 μL of Mix solution (400 g / L glucose-stock solution (10 μL), 85 μL pH 8.0, 200 mM PBS buffer to make up to 100 μL of reaction system) was added. The plate was shaken at 1500 rpm for 30 s, and 5 μL of 100 g / L daidzein (dissolved in DMSO) was added. The plate was then reacted at 32°C and 800 rpm for 7 h.

[0137] 2.1.3 Product Extraction After the reaction was complete, 400 μL of ethanol was added for extraction (shaking at 32℃ and 800 rpm for 1 h), and the supernatant was collected by centrifugation. The supernatant was then transferred from every four 96-well plates to one 384-well plate for mass spectrometry analysis.

[0138] 2.1.4 Mass Spectrometry Analysis of Compounds Echo-MS was used to quantitatively analyze the products, thereby determining the catalytic efficiency of the mutants and completing a large-scale preliminary screening.

[0139] High-throughput mass spectrometry (HMS) was used to detect the relevant compounds. The HMS settings were as follows: mobile phase: methanol (with 4 mM ammonium formate); carrier solvent: MeOH100; flow rate: 250 μl / min; sample fluid class: SP standard mode; injection volume: 5 nl; injection interval: 2000 ms. The mass spectrometer (SCIEX Triple Quad 6500+) settings were: ESI ion source, MRM negative ion mode; temperature: 300℃; ion source gas 1: 90 psi; ion source gas 2: 45 psi; curtain gas: 20 psi; CAD gas: 9; spray voltage: 4500 V.

[0140] MRM conditions for S-estrol: Dewll time 50 ms, DP voltage -90.0, EP voltage -10.0, CE voltage -21.0, CXP voltage -17.0, and the detected ion pair is 241.066+120.946 (241.066 is the parent ion, and 120.946 is the fragment ion).

[0141] 2.2 Results From 2000 samples, screening was conducted based on the yield of the product S-estradiol. Most mutants showed little or no change in yield, while six mutants exhibited yields exceeding 10% relative to the wild type. Specific yields are as follows: Figure 1 As shown in the figure, M1 saw the largest increase, at 36.1%.

[0142] Example 3: Fermentation test of mutant Mu Two rounds of shake-flask tests were performed on the mutants M1-M6 screened in Example 2 to achieve secondary screening. Comparison of the two rounds of tests showed that M1 produced the most significant increase in S-equorol yield compared to the wild-type strain. This producing strain was named Mu, and fermentation tests were conducted on it in this example.

[0143] 3.1 Materials LB medium: Weigh 5 g yeast extract, 10 g tryptone and 10 g NaCl, dissolve in distilled water and bring the volume to 1 L, then store in a wide-mouth bottle. If preparing a solid medium, add 2% agar.

[0144] TB medium: Weigh 11.8g tryptone and 23.6g yeast extract on a balance and dissolve in 900 mL deionized water. Add 5 mL of 80% glycerol (pre-prepared) and autoclave at 115℃ for 20 minutes. When the medium cools to below 60℃, pour in the phosphate solution (weigh 9.4g K2HPO4 and 2.2g KH2PO4, dissolve in 100 mL deionized water, and autoclave at 115℃ for 20 minutes).

[0145] 3.2 Methods 3.2.1 Small-scale shake-flask experiment with mutant Mu 1) Inoculate the bacterial culture from the strain preservation tube into a 5ml LB test tube containing Amp and Spec antibiotics, and incubate at 37℃ and 190rpm for about 8-12 hours; 2) Inoculate 2% of the bacterial culture from the test tube into a 25ml Erlenmeyer flask containing Amp and Spec antibiotics. Incubate at 37℃ and 190rpm for about 1.5-2.5h. When OD600 = 0.8-1, add 0.2mM IPTG. Continue incubation at 25℃ and 220rpm for 10-12h, then centrifuge to collect the bacteria. 3) Centrifuge, discard the supernatant, add pH=8.0, 200mM potassium phosphate buffer to concentrate the bacterial cells to 100 OD, and add 4% glucose (mother liquor concentration 400g / L) and 5 g / L daidzein (mother liquor concentration 100g / L, dissolved in DMSO) to a total system of 500ul. React at 32℃ for about 7h. 4) Add an equal volume of ethanol to terminate the reaction, vortex for 30 seconds, centrifuge, and take the supernatant for dilution and HPLC detection.

[0146] 3.2.2 Mutant Mutant Fermenter Test Primary seed culture: 1 ml of glycerol-based bacterial culture was added to 100 ml of LB medium and cultured at 37°C and 200 rpm for 9 hours on a shaker. Secondary seed culture was inoculated in a clean bench at a rate of 1%, and cultured at 37°C and 200 rpm for 14 hours on a shaker. The secondary seed culture was then transferred to a 1 L fermenter. Initial fermentation conditions were: temperature 30°C, stirring speed 200 rpm, pH 7.0, air flow rate 50 L / min, dissolved oxygen set at 30% and linked to stirring and aeration. Initially, the reaction aid (560 g / L glucose monohydrate solution) was added to the fermentation broth and stirred until homogeneous. Then, the substrate solution was slowly added. Reaction time: 15 hours. After the catalytic reaction was complete, an equal volume of 95% ethanol was added to the fermentation broth, and extraction was performed at 250°C and 300 rpm for 1 hour.

[0147] 3.2.3 Liquid Chromatography Analysis of Compounds The relevant compounds were detected using Shimadzu LC20A XR high-performance liquid chromatography (HPLC) under the following conditions: the HPLC column was a Boltimate C18 Core-Shell column (100 mm × 2.1 mm, 2.7 μm); the mobile phase was water (A) and acetonitrile (B); the detection wavelength was 290 nm; the mobile phase gradient program was: 0–2 min (15% B), 2–16 min (15%–70% B), 16–18 min (70%–95% B), and 16–20 min (95%–15% B).

[0148] 3.3 Results Shake-flask test results of the mutant Mu-producing strain are as follows: Figure 2 and Figure 3 As shown. Compared with the wild-type strain (which contains only wild-type DDRC and has the same genes as the mutant Mu), the chromatographic peak area of ​​the catalytic product S-estradiol of mutant Mu is increased. Further calculation of the product of mutant Mu shows that its yield reached 4.51 g / L, which is 20.3% higher than that of wild type.

[0149] In addition, fermenter tests were conducted on the mutant Mu, and its fermenter yield was 4.92 g / L, with a conversion rate of 98.4%.

[0150] Example 4: Sequencing of mutant Mu 4.1 Methods Colony PCR was performed on the mutant Mu, and the PCR products were sequenced using first-generation sequencing. The primer sequences used are shown in SEQ ID NO:10-11. The colony PCR procedure was as follows: the PCR system used was the Novizan high-fidelity PCR enzyme 2 × PhantaMax Master Mix (Dye Plus) standard system. The PCR system and PCR program are shown in Tables 5 and 6, respectively.

[0151] Table 5. PCR system: Table 6. PCR Procedure 4.2 Results The base sequence of the DDRC mutant Mu is shown in SEQ IN NO:4, and the amino acid sequence is shown in SEQ IN NO:3. The changes in mutant Mu compared to the wild type are shown in Table 7.

[0152] Table 7. Mutation sites of mutant Mu Example 5: Biosynthesis of S-estrol using EcN 1917 strain 5.1 Methods The EcN 1917 (T7 RNAP) strain was selected as the chassis cell for its combination of biosafety and application flexibility. The constructed plasmids pETDuet-1-DDRC-DZNR-THDR-DHDR and pCDFDuet-1-DDRC-Mu were transformed into competent EcN1917 (T7 RNAP) cells to obtain the new strain N4. Information on the introduced exogenous genes in N4 is shown in Table 8. Fermentation tests were performed on strain N4 to determine its S-equadolphin production.

[0153] Table 8. Amino acid sequence information corresponding to exogenous genes 5.2 Results The S-estradiol yield of strain N4 (EcN whole-cell transformation system) in a 1L fermenter was 4.87 g / L, with a conversion rate of 97.4%.

[0154] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A dihydrogenin racemic enzyme (DDRC) mutant, characterized in that, The DDRC mutant uses the wild-type DDRC with the amino acid sequence shown in SEQ ID NO:1 as the starting sequence, and undergoes amino acid mutations at the following sites: D60E and E130Q; wherein, the DDRC mutant has significantly higher catalytic activity than the wild-type DDRC.

2. The DDRC mutant as described in claim 1, characterized in that, The DDRC mutant is tagged with a tag for detection and / or purification.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the DDRC mutant as described in claim 1 or 2.

4. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 3.

5. A genetically engineered host cell, characterized in that, The host cell contains the vector as described in claim 4, or its genome is integrated with the polynucleotide as described in claim 3.

6. The genetically engineered host cell as described in claim 5, characterized in that, The host cell also contains polynucleotides encoding daidzein reductase (DZNR), dihydrodaidzein reductase (DHDR), and tetrahydrodaidzein reductase (THDR).

7. The genetically engineered host cell as described in claim 5, characterized in that, The host cell is a genetically engineered host cell constructed by introducing a polynucleotide encoding the DDRC mutant as described in claim 1 into the probiotic EcN.

8. The method for preparing the DDRC mutant as described in claim 1, characterized in that, Includes the following steps: (1) Under conditions suitable for protein expression, culture the genetically engineered host cells as described in any one of claims 5-7 to obtain a culture containing the DDRC mutant; and (2) Optionally, isolate and / or purify the DDRC mutant from the culture.

9. The use of the DDRC mutant as described in claim 1, characterized in that, The intended use is selected from the following group: (a) Used in the preparation of (S)-dihydrogenainin; and / or (b) An enzyme preparation for the production of (S)-dihydrogenase.

10. A combination of active ingredients, characterized in that, The combination of active ingredients includes: (a) a first active ingredient, wherein the first active ingredient is the genetically engineered host cell as described in claim 7; and (b) The second active ingredient, wherein the second active ingredient is daidzein.

11. A method for producing S-estrol, characterized in that, Including the following steps: Under suitable culture conditions, genetically engineered host cells are cultured to obtain a fermentation product containing S-estrol, and S-estrol is isolated from the fermentation product. The host cell contains polynucleotides encoding the dihydrogen daidzein racemic enzyme (DDRC) mutant, daidzein reductase (DZNR), dihydrogen daidzein reductase (DHDR), and tetrahydrogen daidzein reductase (THDR) as described in claim 1, and is capable of synthesizing S-estradiol.

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