A daidzein reductase mutant and its application in preparing s-equol

Abstract

This invention discloses a daidzein reductase mutant and its application in the preparation of (S)-equorol. The amino acid sequence of the daidzein reductase mutant I53G / Q148M / Y182F is the amino acid sequence shown in SEQ ID NO: 2, with isoleucine at position 53 mutated to glycine, glutamine at position 148 mutated to methionine, and tyrosine at position 182 mutated to phenylalanine. The nucleotide sequence of the daidzein reductase mutant gene is shown in SEQ ID NO: 3. This gene was introduced into *E. coli* to obtain a recombinant genetically engineered bacterium containing this gene, enabling the preparation of a recombinant daidzein reductase mutant for the highly active and stereoselective reduction of daidzein to dihydrodaidzein, laying the foundation for the subsequent efficient bioprocessing of (S)-equorol.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and enzyme engineering, specifically relating to a soybean aglycone reductase mutant, a recombinant genetically engineered bacterium containing the mutant, and the application of the mutant in the efficient and highly stereoselective preparation of S-estrol. Background Technology

[0002] S-Equol is a naturally occurring bioactive substance produced by the metabolism of soy isoflavones (especially daidzein) under the action of specific gut microbiota. Studies have shown that S-Equol possesses significant antioxidant, anti-inflammatory, anti-cancer, menopausal syndrome-improving, osteoporosis-preventing, and cardiovascular disease-preventing physiological activities, with bioactivity far exceeding that of its precursor, daidzein. Furthermore, as a high-affinity agonist of estrogen receptor β (ER-β), S-Equol also demonstrates great potential in neuroprotection, anti-aging, and the treatment of neurodegenerative diseases such as Alzheimer's disease.

[0003] However, only about 30% to 50% of the human population can naturally produce S-equorol through their gut microbiota, severely limiting its functional expression. S-equorol does not exist directly in nature and mainly relies on chemical synthesis or microbial transformation. Chemical synthesis usually yields a racemic mixture, making it difficult to obtain the S-type product with high optical purity, and the process is complex and costly. In contrast, biotransformation using microorganisms or enzymes derived from them can specifically generate biologically active S-equorol, representing a more promising green synthetic route.

[0004] Currently, the production of S-estrol using microbial transformation of daidzein faces two major challenges: First, most naturally occurring S-estrol-producing strains are strict anaerobes, such as Proteus mirabilis LH-52 (see patent CN102925378A), Acinetobacter AUH-JLM455 (see patent CN101338294B), and Clostridium C1-6 (see patent CN104087532A), whose culture conditions are harsh and not conducive to industrial-scale production. Secondly, although key enzymes required for the complete conversion pathway from daidzein to S-estradiol (such as daidzein reductase DZNR, dihydrodaidzein racemic enzyme DDRC, dihydrodaidzein reductase DHDR, and tetrahydrodaidzein reductase THDR) have been successfully identified from strains such as Lactococcus sp. 20-92 and Eggerthella sp., and recombinant Escherichia coli engineered strains have been constructed (see patents CN104031875B and CN105861363B), the efficiency of the entire catalytic cascade reaction still needs to be improved.

[0005] Daidigin reductase is a crucial first step in the catalytic conversion of daidzein to dihydrodaidzein (DHD). However, many known daidzein reductases, such as L-DZNR derived from lactic acid bacteria (Lactococcus sp. 20-92), often exhibit reversible catalytic reactions, capable of both reducing daidzein to R-dihydrodaidzein (R-DHD) and, under specific conditions, oxidizing R-DHD back to daidzein. This bidirectional catalytic characteristic may lead to cyclic conversion between substrate and intermediates, thus affecting the efficiency of the entire cascade reaction, increasing the accumulation of byproducts, and ultimately limiting the yield and production of S-estrol. Therefore, developing an enzyme capable of unidirectional and efficient catalysis of the reduction of daidzein to R-DHD is of great significance for improving the biosynthetic efficiency of S-estrol. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a mutant obtained through rational design and modification of daidzein reductase from Firmicutes bacterium. This mutant, through specific amino acid site mutations (I53G, Q148M, Y182F), successfully transforms the original enzyme's bidirectional catalytic activity into unidirectional reductive activity, significantly reducing the occurrence of reverse reactions. Combining this highly active unidirectional mutant with downstream invertases DDRC, DHDR, and THDR enables efficient and targeted conversion from daidzein to S-estrol, providing a novel and superior biocatalyst for the industrial production of S-estrol.

[0007] The technical solution adopted in this invention is: A daidzein reductase mutant I53G / Q148M / Y182F, wherein the daidzein reductase mutant is a mutant in which isoleucine at position 53 is mutated to glycine, glutamine at position 148 is mutated to methionine, and tyrosine at position 182 is mutated to phenylalanine; its amino acid sequence is shown in SEQ ID NO.4.

[0008] The gene encoding the soybean aglycone reductase mutant I53G / Q148M / Y182F has the nucleotide sequence shown in SEQ ID NO.3.

[0009] The present invention also relates to an expression vector containing the gene of the daidzein reductase mutant I53G / Q148M / Y182F.

[0010] The present invention also relates to recombinant genetically engineered bacteria containing the expression vector.

[0011] This invention also relates to the application of the aforementioned soybean aglycone reductase mutant and recombinant genetically engineered bacteria in the preparation of S-estrol.

[0012] The application is as follows: using daidzein as a substrate, and utilizing the daidzein reductase mutant or recombinant genetically engineered bacteria containing it, in conjunction with dihydrodaidzein racemic enzyme DDRC, dihydrodaidzein reductase DHDR, and tetrahydrodaidzein reductase THDR, to produce S-estradiol.

[0013] Preferably, the engineered bacteria are obtained by cloning the L-DDRC, L-DHDR, and L-THDR genes derived from Lactococcus sp. 20-92 into Escherichia coli BL21 (DE3) to obtain a second recombinant genetically engineered bacteria.

[0014] This invention also relates to a method for preparing S-estrol, comprising the following steps: (1) The recombinant genetically engineered bacteria are fermented to obtain a first bacterial cell or its lysate containing a daidzein reductase mutant. (2) The second recombinant genetically engineered bacteria containing dihydrogen daidzein racemic enzyme DDRC, dihydrogen daidzein reductase DHDR and tetrahydrogen daidzein reductase THDR were fermented to obtain a second bacterial cell or its lysate containing DDRC, DHDR and THDR. (3) Using daidzein as a substrate, the bacterial cells or their lysate obtained in steps (1) and (2) are added to the reaction system, and the reaction is carried out in the presence of coenzyme at a suitable temperature and pH to generate S-estrol. (4) Separate and purify the reaction solution to obtain S-estrol.

[0015] Preferably, in step (3) of the method, the ratio of the amount of the first bacterial cell or its lysate to the amount of the second bacterial cell or its lysate (based on the weight of the wet bacterial cell) is 1:1 to 1:3.

[0016] Preferably, the conditions for the catalytic reaction are: temperature 37°C, pH 6.5-7.5, and reaction time 12-24 hours.

[0017] Beneficial effects:

[0018] This invention successfully transforms the bidirectional catalytic activity of the original enzyme into a unidirectional reducing activity through specific amino acid site mutations, significantly reducing the occurrence of reverse reactions. It can unidirectionally and efficiently catalyze the reduction of daidzein to R-DHD, thereby improving the biosynthetic efficiency of S-estradiol. Attached Figure Description

[0019] Figure 1SDS-PAGE electrophoresis images of crude enzymes and precipitates from the cell wall disruption supernatants of wild-type E. coli BL21(DE3)-pET-22b-FbDZNR and mutant E. coli BL21 (DE3)-pET-22b-I53G / Q148M / Y182F fermentation cells; M: standard molecular weight protein; 1: wild-type cell wall disruption precipitate; 2: wild-type supernatant crude enzymes; 3: mutant cell wall disruption precipitate; 4: mutant supernatant crude enzymes. Detailed Implementation

[0020] Example 1: Construction of recombinant genetically engineered bacteria Based on the wild-type daidzein reductase FbDZNR (GenBank: MBP2639643.1) indexed in NCBI, the FbDZNR gene fragment (codon optimized) was commercially synthesized. Using this fragment as a template, the fragment was amplified and expanded by PCR (with Nde I and Xho I restriction endonuclease fragments added to both sides), and its nucleotide sequence is shown in SEQ ID NO.1. The gene was then inserted into the pET-22b plasmid using the Nde I and Xho I restriction endonuclease sites, resulting in the recombinant plasmid pET-22b-FbDZNR. This recombinant plasmid was then transformed into *E. coli* BL21(DE3) using conventional transformation methods to establish a daidzein reductase gene-producing bacterium. The recombinant strain *E. coli* BL21(DE3)-pET-22b-FbDZNR containing the wild-type FbDZNR gene was stored in an ultra-low temperature freezer at -80°C. The primers used for PCR amplification of the wild-type FbDZNR gene were: The upstream primer is: 5'-CATATGATGAGTTATGATATA-3' (SEQ ID NO.8) The downstream primer is: 5'-CTCGAGTTCAAATGCATACGC-3' (SEQ ID NO.9) Example 2: Site-directed mutagenesis The interaction mode between the genistein and its substrate was analyzed using homology modeling and molecular docking techniques. Based on the analysis results, isoleucine at position 53 (I53), glutamine at position 148 (Q148), and tyrosine at position 182 (Y182) were selected as key mutation sites. The aim was to lock the substrate binding mode by changing the hydrophobicity and spatial conformation of the substrate binding pocket, thereby improving the stability of the reduced intermediate and reducing the probability of the formation of the oxidation transition state, thus blocking the reverse reaction. The mutated primers were designed using CE Design V1.04 provided by Novizan Pharmaceuticals, and the mutated primers are shown in Table 1.

[0021] Table 1 Mutant Primers

[0022] Site-directed mutagenesis was performed using the pET-22b-FbDZNR recombinant plasmid as a template. The whole plasmid was amplified using Vazyme 2xphanta master mix, and the reaction system was set up according to Table 2.

[0023] Table 2 Site-directed mutagenesis system name Volume (μl) Vazyme 2xphanta master mix 12.5μL pET-22b-FbDZNR template plasmid 1μL Primer-F 1μL Primer-R 1μL <![CDATA[ddH2O]]> Add to 25 μL The PCR program was as follows: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 67°C annealing for 30 s, 72°C extension for 5 min, for 30 cycles, followed by a final extension at 72°C for 10 min, and finally incubation at 4°C. After the PCR reaction, 1 μL of DpnI digestive enzyme was added, and the reaction was carried out at 37°C for 2 h to digest the template. The purified PCR product was then recovered and transformed into E. coli BL21 (DE3) competent cells.

[0024] Transformation method: 10 μL of PCR product was mixed with 100 μL of Novizan commercial E. coli BL21 (DE3) competent cells, incubated on ice for 20 min, then heat-shocked at 42℃ for 90 s, immediately removed, incubated on ice for 2 min, and 400 μL of LB liquid medium was added. The mixture was incubated at 37℃ and 200 rpm for 60 min. 100 μL of the bacterial culture was then spread onto a solid LB agar plate containing 100 μg / mL ampicillin resistance and incubated overnight at 37℃. The next day, three recombinant E. coli BL21 strains containing mutant plasmids were selected from the plates. These recombinant bacteria were inoculated from the plates into 50 mL shakers containing 5 mL of the corresponding LB liquid medium (LB (g / L): peptone 10, sodium chloride 10, yeast extract 5), and the corresponding antibiotics were added. The mixture was incubated on a shaker at 37 °C for 12 h at 200 rpm. After incubation, plasmids were extracted and sent to General Electric for sequencing. Finally, the sequencing results were compared with the wild-type enzyme protein nucleic acid sequence to determine whether the mutation was successful.

[0025] Following the method described above, a single-site mutation was first performed to obtain the recombinant expression bacterium E. coli. The recombinant plasmid pET-22b-I53G, which had undergone one mutation, was used as a template for a second mutation to obtain the recombinant expression strain E. coli BL21 (DE3)-pET-22b-I53G / Q148M. Using the recombinant plasmid pET-22b-I53G / Q148M, which had undergone two mutations, as a template for a third mutation, a three-point mutation recombinant expression strain E. coli containing the FbDZNR mutant was obtained and sequenced correctly. BL21 (DE3)-pET-22b-I53G / Q148M / Y182F.

[0026] Example 3: Unidirectional catalytic activity of daidzein reductase mutant Wild-type recombinant E. coli BL21(DE3)-pET-22b-FbDZNR and the mutant recombinant E. coli BL21(DE3)-pET-22b-I53G / Q148M / Y182F constructed in Example 2 were induced to express the bacteria. Both were inoculated into 5 ml of LB medium containing ampicillin (30 μg / mL) resistance and cultured at 37°C for 8 h. Then, they were transferred to 400 mL of TB fermentation medium at a 2% inoculum size and cultured at 37°C and 200 rpm until the bacterial OD600 reached 0.6. The culture was then transferred to a shake flask at 30°C and 200 rpm for 18 h of induced fermentation. After fermentation, the bacterial cells were collected by centrifugation at 8000 rpm for 5 min. Weigh 2g of each of the fermented cells from the two enzymes, resuspend them in 20ml of 100mM pH 7.0 sodium phosphate buffer, sonicate (1s, 2s, 15min), and centrifuge at 12000rpm for 20min. The supernatant is the crude enzyme solution of wild-type and mutant daidzein reductase. Perform SDS-PAGE protein electrophoresis on the crude enzyme solutions of the two reductases and the cell precipitate. The results are as follows: Figure 1 As shown, both wild-type and mutant reductases exhibit distinct protein expression bands around 70 kDa, consistent with the predicted protein size, demonstrating that both reductases can achieve efficient soluble expression in recombinant E. coli.

[0027] Two 1 mL control reaction systems were set up, with appropriate amounts of crude enzyme solution, 0.2 mM daidzein, and 0.5 mM NADPH added to each. The systems were reacted in sodium phosphate buffer at 37℃ and pH 7.0 for 30 minutes. After the reaction, the products were extracted with an equal volume of ethyl acetate and detected by HPLC. HPLC detection conditions: C18 column (4.6 × 250 mm, 5 μm), mobile phase: methanol:water:acetic acid = 45:55:0.1 (v / v / v), flow rate 1.0 mL / min, detection wavelength 254 nm.

[0028] During the catalytic process, the wild-type enzyme not only detected the formation of R-DHD but also indicated that some daidzein substrate was not completely consumed. Furthermore, the peak area of ​​R-DHD did not continue to increase after the reaction time was extended, indicating the presence of a reverse reaction. In contrast, the mutant enzyme I53G / Q148M / Y182F exhibited a significantly different catalytic mode. The peak area of ​​R-DHD continuously increased over time, and no significant increase in substrate residue was detected, demonstrating that its catalytic reaction mainly proceeded in the direction of R-DHD formation, exhibiting unidirectional catalytic activity.

[0029] Example 4: Preparation of S-Estradiol by Co-catalysis of Two Bacteria General Biotechnology was commissioned to use a whole-genome artificial synthesis method to clone the L-DDRC (nucleotide sequence shown in SEQ ID NO:5), L-DHDR (nucleotide sequence shown in SEQ ID NO:6), and L-THDR (nucleotide sequence shown in SEQ ID NO:7) genes, which are derived from Lactococcus sp. 20-92 as described in patent application 201410243148.8, into the PET-22b vector between the Nde I and Xho I restriction sites. The N-terminus and C-terminus of the L-DDRC gene are Nde I and BamHI restriction sites, respectively; the N-terminus and C-terminus of the L-DHDR gene are BamHI and Not I restriction sites, respectively; and the N-terminus and C-terminus of the L-THDR gene are Not I and Xho I restriction sites, respectively. The SD-AS sequence GAAGGAGAATATACATAT was introduced at the 5' end of both the L-DHDR and L-THDR gene fragments. The addition of the SD-AS sequence ensured the simultaneous and efficient expression of multiple enzymes on the same plasmid. The vector pET-22b-DDRC-DHDR-THDR was then transformed into *E. coli* BL21 (DE3) to obtain the second recombinant genetically engineered strain, *E. coli* BL21-22b-DDRC-DHDR-THDR.

[0030] The *E. coli* BL21-22b-I53G / Q148M / Y182F and *E. coli* BL21-22b-DDRC-DHDR-THDR strains constructed in Example 2 were inoculated into LB liquid medium containing the corresponding antibiotics and cultured at 37°C with shaking until OD600≈0.6. IPTG was added to a final concentration of 0.5 mM, and the culture was induced at 25°C for 16 hours. The bacterial cells were collected by centrifugation, washed twice with sodium phosphate buffer (pH 7.0), and resuspended for later use, yielding the first and second bacterial suspensions.

[0031] In a 100 mL reaction flask, add 50 mL of pH 7.0 phosphate buffer, 2 g / L daidzein (dissolved with a small amount of DMSO), 1.0 mM NADPH, 5 mg / mL glucose, and 0.2 mg / mL glucose dehydrogenase (for coenzyme regeneration). Add the first and second bacterial suspensions at a 1:1 ratio to each bacterial cell, resulting in a final concentration of 20 g / L (wet cells). The total reaction volume is 50 mL. Incubate at 37°C and 200 rpm for 24 hours on a shaker. Glucose and NADP are added every 6 hours during the reaction. + After the reaction was complete, 1 mL of the reaction solution was taken, extracted with an equal volume of ethyl acetate, and then analyzed by HPLC. Enantiomeric analysis was performed using a chiral column (Chiralcel OD-H) with a mobile phase of hexane / isopropanol (90:10), a flow rate of 1.0 mL / min, and a detection wavelength of 280 nm to determine the enantiomeric excess (ee) of S-estradiol.

[0032] Calculations showed that the conversion rate of daidzein was greater than 95%, the yield of S-estradiol reached 85.6%, and its enantiomeric excess (ee) was >99.9%. These results indicate that by using the unidirectional daidzein reductase mutant of this invention in combination with three downstream enzymes for catalysis, daidzein can be converted to S-estradiol efficiently and with high stereoselectivity, providing a new strategy for the industrial biosynthesis of S-estradiol.

[0033] Example 5: Effect of different bacterial cell ratios on S-estrol yield Following the method in Example 4, the wet cell weight ratios of the first and second bacterial suspensions were set to 1:0.5, 1:1, 1:2, and 1:3, respectively, while the total wet cell concentration remained constant at 20 g / L. All other reaction conditions were kept unchanged, and the yield of S-estradiol was measured after 24 hours of reaction. The results are shown in Table 3. When the ratio of the first to second bacteria was 1:1 to 1:3, the yield of S-estradiol remained at a high level (>80%), with the 1:2 ratio being the optimal yield of 89.3%.

[0034] Table 3. Effects of different bacterial cell ratios on S-estrol production

Claims

1. A soybean aglycone reductase mutant I53G / Q148M / Y182F, characterized in that, The soybean aglycone reductase mutant is characterized by the following mutations in the amino acid sequence shown in SEQ ID NO.2: isoleucine at position 53 is mutated to glycine, glutamine at position 148 is mutated to methionine, and tyrosine at position 182 is mutated to phenylalanine.

2. The gene encoding the soybean aglycone reductase mutant I53G / Q148M / Y182F as described in claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

3.

3. An expression vector containing the gene of the soybean aglycone reductase mutant I53G / Q148M / Y182F as described in claim 2.

4. Recombinant genetically engineered bacteria containing the expression vector of claim 3.

5. The application of the soybean aglycone reductase mutant I53G / Q148M / Y182F as described in claim 1 and the recombinant genetically engineered bacteria as described in claim 4 in the preparation of S-estrol.

6. The application according to claim 5, characterized in that, Using daidzein as a substrate, S-estradiol is produced by the combined action of the daidzein reductase mutant or recombinant genetically engineered bacteria containing it, along with dihydrodaidzein racemic enzyme DDRC, dihydrodaidzein reductase DHDR, and tetrahydrodaidzein reductase THDR.

7. A method for preparing S-estrol, characterized in that, Includes the following steps: (1) The recombinant genetically engineered bacteria of claim 4 are fermented to obtain a first bacterial cell or its lysate containing a daidzein reductase mutant. (2) The second recombinant genetically engineered bacteria containing dihydrogen daidzein racemic enzyme DDRC, dihydrogen daidzein reductase DHDR and tetrahydrogen daidzein reductase THDR were fermented to obtain a second bacterial cell or its lysate containing DDRC, DHDR and THDR. (3) Using daidzein as a substrate, the bacterial cells or their lysate obtained in steps (1) and (2) are added to the reaction system, and the reaction is carried out in the presence of coenzyme at a suitable temperature and pH to generate S-estrol. (4) Separate and purify the reaction solution to obtain S-estrol.

8. The method according to claim 7, characterized in that, In step (3), the ratio of the first bacterial cell or its lysate to the second bacterial cell or its lysate (based on the weight of the wet bacterial cell) is 1:1 to 1:

3.

9. The method according to claim 7 or 8, characterized in that, The conditions for the catalytic reaction are: temperature 37℃, pH 6.5-7.5, and reaction time 12-24 hours.

Images