A recombinant chassis cell with high production of equol and a construction method and application thereof

Abstract

The application discloses a recombinant chassis cell with high equol production and a construction method and application thereof, and belongs to the technical field of genetic engineering. The application carries out mutation on a key rate-limiting enzyme THDR in an (S)-equol metabolic pathway, and obtains a mutant with significantly improved catalytic efficiency and thermal stability. The mutant is introduced into an (S)-equol synthesis pathway, and through optimization of RBS and a promoter, a molecular chaperone and an NADPH regeneration module are co-expressed, so that multi-step enzymatic reactions are efficiently cooperated. After multi-dimensional modification, the recombinant strain constructed by the application has an (S)-equol production of more than 2.4 g / L, which is increased by tens of times compared with a traditional biosynthesis strain, and a substrate conversion rate is increased by about 50%, so that the existing biosynthesis yield bottleneck is broken through.

Description

Technical Field

[0001] This invention relates to a recombinant chassis cell that produces high levels of estrol, its construction method, and its application, belonging to the field of genetic engineering technology. Background Technology

[0002] Equol (7-hydroxy-3-(4'-hydroxyphenyl)-cresol) is the end product of the metabolism of soy isoflavones (mainly daidzein) by specific intestinal microorganisms, and it has significant estrogenic and antioxidant activities. As a selective estrogen receptor modulator (SERM), (S)-equol has a high affinity for estrogen receptor-β (ER-β), demonstrating unique clinical application value in relieving menopausal symptoms, improving bone density, cardiovascular health, and preventing hormone-dependent cancers.

[0003] However, not all populations are capable of converting soy isoflavones into equol. Studies have shown that only about 20%-60% of Asian populations and 25%-30% of Western populations can produce equol through gut microbiota. This individual difference severely limits the full realization of the bioefficacy of soy isoflavones. Currently, the industrial production of equol mainly relies on chemical synthesis, but chemical synthesis has the following bottlenecks: (1) the product is a racemic mixture of (R)- and (S)- equol, of which the R-type has no biological activity; (2) the reaction requires high temperature and pressure, metal catalysts and hydrogen, which requires high equipment and produces many byproducts.

[0004] In recent years, researchers have attempted to reconstruct the equol biosynthesis pathway in readily operable hosts such as *E. coli* and yeast through metabolic engineering. The biosynthesis of equol involves multiple enzymatic reactions: daidzein is first reduced to dihydrodaidzein (DHD) by daidzein reductase (DZNR), then DHD is further reduced to tetrahydrodaidzein (THD) by dihydrodaidzein reductase (DHDR), and finally (S)-equol is generated by tetrahydrodaidzein reductase (THDR). However, the existing synthetic system still faces several bottlenecks, mainly in the following four aspects: (1) The key enzymes have low catalytic efficiency and insufficient substrate affinity. Existing technologies only use methods such as direct expression of wild enzymes, conventional codon optimization, and simple overexpression to increase enzyme yield. They do not carry out rational site-directed mutagenesis and structural modification of rate-limiting enzymes such as THDR, which cannot fundamentally improve substrate binding capacity and catalytic efficiency. The yield has remained at the milligram level for a long time, and there is a clear technical ceiling.

[0005] (2) Limited supply of NADPH cofactor: Existing methods mostly involve the exogenous addition of NADPH or the single overexpression of cofactor synthesis genes. The exogenous addition is costly and difficult to industrialize; the single gene enhancement effect is limited, and the intracellular reducing power is largely diverted by the byproduct pathway. The supply of cofactors does not match the synthesis pathway, and cannot meet the needs of continuous multi-step reduction reactions.

[0006] (3) Reversible reaction leads to product degradation: Existing technologies have not made directional modifications to the catalytic direction of key enzymes, and cannot suppress reverse hydroxylation and reverse reaction. The product continues to degrade during fermentation, and it is difficult to obtain single high optical purity (S) estradiol. The purity and stability of the product cannot meet the requirements of pharmaceuticals and functional foods.

[0007] (4) Host competition for metabolic pathways to divert substrates: Existing technologies mostly use random knockout of byproduct genes, without accurately screening the core targets that directly compete with equaphrenic synthesis for carbon sources and NADPH. Improper knockout can easily lead to bacterial growth inhibition, metabolic disorders, and reduced yield, resulting in low substrate conversion rate and serious waste of resources.

[0008] Given the aforementioned deficiencies in existing technologies, there is an urgent need to develop a recombinant biomanufacturing system capable of efficiently, stably, and on a large scale producing high-purity (S)-estrol. Summary of the Invention

[0009] This patent addresses key shortcomings of existing (S)-equol production technologies, such as the chemical synthesis method producing an inactive racemic mixture, low yield (only mg level) of the biosynthetic method, metabolic flux being diverted by byproducts, and instability of the plasmid expression system. It constructs a recombinant cell based on *E. coli* BL21(DE3): by integrating the equol synthesis gene cluster into the genome and introducing the THDR enzyme A142T / Q256R double mutant (increasing catalytic efficiency by approximately 2 times), combined with CRISPRi precisely inhibiting the competitive pathway and the NADPH regeneration module enhancing reducing power supply, achieving highly efficient synthesis of (S)-equol with a yield ≥2.4 g / L, optical purity ≥99%, and substrate conversion rate ≥50%. This solves the core bottleneck of existing technologies from laboratory to industrial scale-up, providing a high-purity, low-cost, stable, and controllable biomanufacturing solution for functional foods and pharmaceuticals.

[0010] This invention provides a tetrahydrodaidzein reductase mutant with A142T and / or Q256R mutations based on the parent.

[0011] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.14, with alanine at position 142 mutated to threonine.

[0012] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.14, with glutamine at position 256 mutated to arginine.

[0013] In one embodiment, the mutant is based on the amino acid sequence shown in SEQ ID NO.14, with alanine at position 142 mutated to threonine and glutamine at position 256 mutated to arginine.

[0014] The present invention also provides a gene encoding the mutant.

[0015] The present invention also provides recombinant microorganisms expressing the mutants.

[0016] In one embodiment, the recombinant microorganism also expresses the dznr, ddr, and dhdR genes.

[0017] In one embodiment, the nucleotide sequences of the dznr, ddr, and dhdR genes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

[0018] In one embodiment, the expression of an estrol synthesis gene cluster is regulated by the pTrc promoter; the estrol synthesis gene cluster includes... dznr , ddr , dhdR , thdr The mutant gene; the nucleotide sequence of the pTrc promoter is shown in SEQ ID NO. 5.

[0019] In one embodiment, the recombinant microorganism further comprises one or more of the following modifications: (a) The thdr mutant gene is regulated by the T7 promoter (SEQ ID NO.6); (b) Replace the RBS sequence between the promoter and the thdr gene with TAAGGAG; (c) Introducing an NADPH regeneration module; the NADPH regeneration module includes the zwf gene and the gnd gene; (d) Introducing a molecular chaperone co-expression module; the molecular chaperone co-expression module includes the groEL gene and the groES gene.

[0020] In one embodiment, the nucleotide sequences of the zwf gene and the gnd gene are shown in SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0021] In one embodiment, the nucleotide sequences of the groEL gene and the groES gene are shown in SEQ ID NO.9 and SEQ ID NO.10, respectively.

[0022] In one embodiment, the competitive pathway gene in the recombinant microorganism that competes with equadol synthesis for reducing power and / or carbon source is inhibited or inactivated; the competitive pathway gene is selected from one or more of ydjG, yqhD, and dhaT.

[0023] In one embodiment, the CRISPRi system is used to suppress the expression of the competing pathway genes, and the sgRNA sequences for suppressing ydjG, yqhD, and dhaT are shown in SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13, respectively.

[0024] In one embodiment, the equadol synthesis gene cluster is integrated into the genome of the recombinant microorganism.

[0025] In one implementation, the integration site is the rrnB operon site.

[0026] In one embodiment, the recombinant microorganism uses Escherichia coli BL21(DE3) as a host.

[0027] The present invention also provides a method for preparing (S)-estrol, comprising: culturing the recombinant microorganism, and collecting, extracting and / or purifying (S)-estrol from the fermentation system.

[0028] In one embodiment, the fermentation system contains the substrate daidzein.

[0029] In one embodiment, the fermentation system uses glucose as a carbon source.

[0030] In one embodiment, the fermentation is carried out in a fed-batch fermentation tank of 5 L or larger.

[0031] In one implementation, glucose is added as a feed when the bacterial cell OD reaches 15-18.

[0032] In one embodiment, the fermentation time is not less than 60 hours, preferably 60-72 hours.

[0033] The present invention also provides the use of the tetrahydrodaidzein reductase mutant, the recombinant microorganism, or the method in the preparation of (S)-estrol or products containing (S)-estrol.

[0034] Beneficial effects: (1) Achieving precise guidance of metabolic flux and efficient utilization of substrates: This invention utilizes the CRISPRi system to precisely inhibit competing metabolic pathways such as ydjG, yqhD, and dhaT, using adhE and ackA as negative controls. The system demonstrates that not all inhibition of competing metabolic genes promotes equol synthesis. Results showed that inhibition of ydjG / yqhD / dhaT increased equol production to 721.4 mg / L, a 4.6-fold increase, while adhE inhibition led to impaired cell growth and a 43% reduction in production, and ackA inhibition caused metabolic imbalance and a 34% reduction in production. Precise identification of nodes directly competing with target pathways for NADPH and carbon sources significantly reduced the flow of carbon sources and reducing power to byproducts, resulting in a significant improvement in substrate conversion. Intracellular reducing power allocation significantly shifted towards (S)-equol synthesis, byproduct accumulation decreased, and metabolic resource utilization efficiency was significantly improved.

[0035] (2) Enhance the catalytic performance of key enzymes and make the product configuration more uniform: This invention utilizes rational site-directed mutagenesis of the rate-limiting enzyme THDR to obtain the A142T / Q256R double mutant, achieving an approximately 2.0-fold increase in catalytic efficiency (kcat / Km) and a simultaneous improvement in thermal stability. This modification enables the directed synthesis of (S)-equorol with an optical purity ≥99%, avoiding the problems associated with the chemical synthesis of racemic mixtures, and significantly improving product purity and bioactivity. In contrast, the W189A mutation leads to a 42% decrease in catalytic efficiency and a 43% reduction in yield; while the E50D mutation maintains positive catalytic efficiency, it enhances reverse hydroxylation activity by 3.5 times, resulting in continuous product degradation and a 74% reduction in yield.

[0036] (3) Significantly improves the efficiency and yield of (S)-estrol synthesis: This invention achieves highly efficient synergistic multi-step enzymatic reactions by enhancing the expression of key gene clusters in (S)-estrol synthesis, optimizing RBS and promoters, and co-expressing molecular chaperones and NADPH regeneration modules. After multi-dimensional modification, the strain yield increased from the initial mg level to over 2.4 g / L, which is tens of times higher than that of traditional biosynthetic strains, and the substrate conversion rate increased by about 50%. This breakthrough overcomes the current bottleneck in biosynthetic yield. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the recombinant plasmid. Detailed Implementation

[0038] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments are merely illustrative of the invention and should not be considered as specific limitations thereof. Information on the raw materials and equipment involved in the specific embodiments is shown in Table 1.

[0039] Table 1. Materials and Instrument Information

[0040] Chassis strains Escherichia coli BL21(DE3) is a commonly used host strain in this field.

[0041] Culture medium: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH 7.0.

[0042] (S)-Estrol content determination: Detection was performed by HPLC using a Daicel Chiralcel OJ-H column (250 × 4.6 mm, 5 μm), with a mobile phase of n-hexane / isopropanol (85:15), a flow rate of 0.8 mL / min, a detection wavelength of 280 nm, and a column temperature of 25℃. The retention time of (S)-Estrol was approximately 8.2 min, and that of (R)-Estrol was approximately 9.5 min. The optical purity of the (S)-configuration was confirmed to be 99.2%.

[0043] Substrate conversion rate: The conversion rate of daidzein to (S)-estradiol. It is calculated using the following formula: Substrate conversion rate (%) = (Initial total mass of daidzein - Residual total mass of daidzein at the end of fermentation) / Initial total mass of daidzein × 100%.

[0044] Example 1: Construction of (S)-Equisetol Chassis Strains (1) Strains and culture conditions in the chassis: Select Escherichia coli BL21(DE3) was used as the engineered chassis strain. It was cultured in LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and grown at 37 ℃ with shaking at 220 rpm.

[0045] (2) Gene source and cloning of the synthetic pathway: (S)-Equivalerate synthesis gene cluster ( dznr , ddr , dhdR , thdr (Source) Slackia isoflavoniconvertens DSM 22006. The optimized codons were synthesized by Genewiz (the optimized GC content was 52.8%, and the rare codons AGG, AGA, and CUA were deleted). The nucleotide sequence of the gene is shown in Table 2.

[0046] Table 2 Gene and nucleotide sequences

[0047] Based on the pET-28a(+) vector as the backbone, the original T7 promoter was replaced by the pTrc promoter (shown in SEQ ID NO.5), and integration was performed downstream of the pTrc promoter. dznr - ddr - dhdR - thdr The operon was used to construct the pET28a-pTrc-EQU plasmid. PCR reaction conditions: amplification program of 95 ℃ preheating for 3 min, followed by 30 cycles of 95 ℃ for 30 s, 55 ℃ for 30 s, and 72 ℃ for 90 s. Primers used are shown in Table 3. Vector diagram is shown below. Figure 1 .

[0048] Table 3 Primer sequences

[0049] (3) Construction of recombinant strains: The recombinant plasmid pET28a-pTrc-EQU constructed in step (2) was introduced into Escherichia coli BL21(DE3) by electroporation, and strains containing the plasmid were screened. E. coli -EQU. Validated by qPCR. dznr - thdr Transcription was successfully achieved in all cases, and sequencing confirmed that the insertion was correct.

[0050] (4) Fermentation and product detection: The recombinant strain constructed in step (3) E. coli -EQU was induced to express at 37 °C for 48 h in LB medium containing 0.5 mM IPTG and 2 g / L daidzein (based on final concentration). The cells were then collected by centrifugation and intracellular metabolites were extracted with ethyl acetate. The characteristic ion peak of (S)-equorol was detected by LC-MS / MS (MRM mode) using an Agilent 6495C triple quadrupole mass spectrometer with a ZORBAX SB-C18 column (2.1 × 100 mm, 1.8 μm). Mobile phase A was 0.1% formic acid, and mobile phase B was a 0.1% formic acid in acetonitrile solution. Gradient elution was used: 0–3 min 10% B, 3–8 min 10–90% B, 8–10 min 90% B, flow rate 0.3 mL / min, column temperature 35 ℃. Ion source temperature was 200 ℃, drying gas flow rate was 14 L / min, nebulizer pressure was 24 psi, sheath gas temperature was 300 ℃, flow rate was 11 L / min, electrospray nozzle voltage was 500 V, and capillary voltage was 2500 V. The quantitative ion pair of (S)-equorol was m / z The qualitative ion pair m / z 243.1→121.0 (243.1→135.0) indicates that the synthesized product is (S)-estrol, with a yield of 18.5 mg / L.

[0051] Example 2: Enhancing key enzyme expression and NADPH regeneration to improve metabolic flux (1) Screening of key enzymes: Based on pathway metabolic flux analysis (FBA) and literature comparison, determine thdr (Tetrahydrogenase) is the flux-limiting step.

[0052] (2) Enhanced expression regulation and cofactor regeneration: Based on the recombinant plasmid pET28a-pTrc-EQU from Example 1, thdr The promoter was replaced with the strong promoter P_T7 (nucleotide sequence shown in SEQ ID NO. 6), and the original downstream RBS sequence (AAGGAG, ΔG = -5.3 kcal / mol) was optimized to the current RBS sequence (TAAGGAGG, ΔG = -9.2 kcal / mol). The lower RBS free energy ΔG results in stronger ribosome-mRNA binding stability, effectively improving translation initiation efficiency. Furthermore, it was integrated upstream of the gene. zwf / gnd NADPH regeneration module ( zwf Nucleotide sequence as shown in SEQ ID NO.7; gnd Nucleotide sequences as shown in SEQ ID NO.8) and groEL / groES Folded companion co-expression module ( groEL Nucleotide sequence as shown in SEQ ID NO.9; groES The nucleotide sequence is as shown in SEQ ID NO.10, which enhances reducing power supply and protein folding efficiency. The specific steps are as follows: Using the pET28a-pTrc-EQU plasmid constructed in Example 1 as a template, the original promoter of the thdr gene was replaced with the strong promoter P_T7 (nucleotide sequence as shown in SEQ ID NO.6) by PCR site-directed mutagenesis, and an optimized RBS sequence (ΔG=-9.2 kcal / mol) was introduced to obtain the intermediate plasmid pET28a-pTrc-EQU-PT7.

[0053] Using Escherichia coli BL21(DE3) genomic DNA as a template, PCR amplification was performed. zwf Gene (SEQ ID NO.7) gnd Gene (SEQ ID NO.8), obtained by overlap PCR zwf / gndThe tandem fragment was digested with two enzymes and then ligated to the multiple cloning site of pET28a-pTrc-EQU-PT7 to obtain the recombinant plasmid pET28a-pTrc-EQU-PT7-NADPH containing the NADPH regeneration module.

[0054] Using Escherichia coli BL21(DE3) genomic DNA as a template, PCR amplification was performed. groEL Gene (SEQ ID NO.9) and groES Gene (SEQ ID NO.10), constructed via overlap PCR groEL / groES Fold the partner co-expression fragment and insert it into the specified site of the plasmid obtained in step 2 to obtain the final recombinant expression plasmid pET28a-pTrc-EQU-opt.

[0055] The constructed pET28a-pTrc-EQU-opt plasmid was introduced into Escherichia coli BL21(DE3) competent cells via electroporation. The cells were plated on LB agar containing 50 μg / mL kanamycin and incubated at 37 °C inverted for 12–16 h. Single colonies were picked and verified by PCR and sequencing to confirm their correctness, thus obtaining the recombinant strain with enhanced expression and cofactor regeneration. E. coli EQU-opt.

[0056] Table 4 Gene and nucleotide sequences

[0057] (3) Verification of expression: The constructed recombinant bacteria E. coli EQU-opt was fermented according to the method in Example 1. The fermentation broth was collected, and SDS-PAGE showed that the recombinant bacteria showed a significantly enhanced specific protein band at the theoretical molecular weight position of THDR, proving that THDR was significantly enhanced in the soluble component.

[0058] THDR enzyme activity assay: Using E. coli-EQU strain from Example 1 as a control, induced bacterial cells were collected and ultrasonically disrupted to prepare a soluble crude enzyme solution. Protein concentration was quantified using BCA. The 1 mL enzyme activity reaction system consisted of: 700 μL of 50 mM pH 7.0 PBS buffer, 100 μL of 10 mM NADPH solution, 100 μL of 10 mM tetrahydrogenase (THD) solution, and 100 μL of crude enzyme solution. After preheating at 30°C, the enzyme solution was added to initiate the reaction. The absorbance at 340 nm was continuously monitored using a spectrophotometer, and the THDR catalytic activity was characterized by the NADPH oxidation consumption rate. The test results showed that… E. coliThe THDR enzyme activity of the EQU-opt strain was 3.5 times that of the control strain.

[0059] Intracellular NADPH content detection: Fermentation cells were collected by low-temperature centrifugation, and the precipitate was rapidly quenched to remove extracellular impurities; intracellular coenzymes were extracted by lysing the cells, and the reaction solution was prepared according to the WST-8 kit instructions. The cells were incubated in the dark, and the absorbance at 450 nm was measured using a microplate reader. The strain from Example 1 was used as an example. E. coli-EQU As a parallel control, the intracellular NADPH content was calculated using a standard curve. The results showed that the intracellular NADPH content of the modified strain was 2.8 times higher than that of the original strain.

[0060] (4) Production analysis: After 48 hours of fermentation, E. coli The yield of EQU-opt (S)-equadol reached 156.3 mg / L, which is E. coli The 8.4-fold increase in EQU validates the effectiveness of key enzyme fortification and cofactor regeneration.

[0061] Example 3: Inhibiting competitive metabolic pathways to enhance the selective synthesis of (S)-estrol (1) Identification of competing pathways: (S)-Estrol synthesis competes with byproduct pathways such as ethanol, acetic acid, and lactic acid for carbon sources and reducing power. Competing genes include: ydjG (Alcohol dehydrogenase, nucleotide sequence as shown in Gene ID: 946359) yqhD (Aldehyde reductase, nucleotide sequence as shown in Gene ID: 947235) dhaT (1,3-Propanediol oxidoreductase, nucleotide sequence as shown in Gene ID: 946677).

[0062] (2) Competitive pathway inhibition strategy: The strain constructed in Example 2 E. coli EQU-opt was the starting strain, and the CRISPRi system was used to inhibit it. ydjG , yqhD and dhaT Expression was performed, and a positive inhibitory bacterium containing the dCas9-sgRNA expression vector pCas-SC was constructed and named [name missing]. E. coli EQU-opt-Cri. To verify the specificity of CRISPRi in inhibiting the competitive pathway, the alcohol dehydrogenase gene, which does not directly compete with (S)-estrol synthesis for reducing power, was selected. adhE and acetate kinase gene ackA As a negative control, sgRNA expression vectors pCas-adhE and pCas-ackA were constructed, and recombinant bacteria with the corresponding genes suppressed were also tested. E. coli EQU-opt-adhE, E. coliEQU-opt-ackA. The sgRNA sequence is detailed in Table 3. The sgRNA expression vector (pCas-) will be used. ydjG pCas- yqhD pCas- dhaT Transformed into the recombinant strain constructed in Example 2 E. coli In EQU-opt, the culture was incubated at 37℃ and 220 rpm with shaking for 1 h for recovery. The bacterial culture was then spread onto LB solid medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and incubated upside down at 37℃ for 12–16 h. Single colonies were picked and verified by PCR and sequencing to obtain positive recombinant strains. E. coli EQU-opt-CRi.

[0063] Table 5 sgRNA and its function

[0064] (3) Detection of target gene activity and metabolic flux changes: positive inhibitory strains E. coli EQU-opt-CRi, negative control strain E. coli EQU-opt- adhE , E. coli EQU-opt- ackA and the originating strain E. coli EQU-opt was inoculated into LB liquid medium and cultured at 37°C and 220 rpm until OD. 600 ≈0.6–0.8, add 0.5 mM IPTG + 0.2 μg / mL anhydrous tetracycline for induction, continue induction culture at 37℃ for 12 h, and achieve CRISPRi initiation by tetracycline. ydjG Gene transcriptional repression.

[0065] Crude enzyme solution was prepared by ultrasonically disrupting bacterial cells, using ethanol as a substrate and NAD+. + As a coenzyme, the NADH production rate was monitored at 340 nm. ydjG Activity. Due to ydjG Catalytic dehydrogenation of ethanol to acetaldehyde, simultaneously with NAD+ + Reduced to NADH, NADH exhibits characteristic absorption at 340 nm, and the rate of increase in absorbance at 340 nm characterizes enzyme activity. Results showed that after induction... ydjG Activity decreased by 75%, and acetic acid accumulation decreased by 62%.

[0066] The fermentation supernatant was filtered through a 0.22 μm filter membrane and the intracellular ethanol and acetic acid contents were measured by GC-MS, which decreased by 68% and 55%, respectively. Combined with the total intracellular NADPH, the proportion of NADPH consumed by byproducts and the (S)-estrol synthesis flux measured in Example 2, the metabolic flux allocation ratio was calculated: the proportion of NADPH consumed by byproducts decreased from 35% to 8%, and more reducing power was directed to (S)-estrol synthesis.

[0067] The results are shown in Table 6. Not all inhibition of competing metabolic genes promotes (S)-estrol synthesis; only precise identification of nodes that directly compete with the target pathway for NADPH and carbon sources is effective. ydjG / yqhD / dhaT Only in this way can the effective redirection of metabolic flux be achieved.

[0068] Table 6 Effects of different groups

[0069] Example 4: Targeted Mutation of Key Enzyme Catalytic Sites (1) Target enzyme selection and structural modeling: Using the THDR enzyme encoded by the gene shown in SEQ ID NO.4 (amino acid sequence shown in SEQ ID NO.14) as the target for modification, a three-dimensional structure was constructed using AlphaFold2. The key residues in the substrate tetrahydrodaidzein binding region were located by molecular docking (AutoDock Vina): Ala142, Gln256, and Glu50, which are key residues for the reverse hydroxylation reaction.

[0070] (2) Mutation design and mutant construction: By combining protein structure simulation and conservation analysis, the functions of the predicted sites were determined: A142 and Q256 were candidate positive mutation sites; W189A was predicted as a null mutation and E50D was predicted as a harmful mutation. The latter two were used as comparative experiments for verification.

[0071] The strain constructed in Example 3 E. coli Using EQU-opt-CRi as the host, overlap extension PCR was employed to construct double mutants A142T / Q256R by replacing Ala142 with Thr (A142T) and Gln256 with Arg (Q256R). Simultaneously, to verify the specificity of the mutation sites, Trp189 was replaced with Ala (W189A) and Glu50 with Asp (E50D). The strains were inoculated into LB medium and cultured at 37°C and 220 rpm until OD200. 600=0.6~0.8, add 0.5 mM IPTG and induce at 37℃ for 12 h. Collect bacterial cells by centrifugation, sonicate and purify by Ni-NTA affinity chromatography to obtain pure mutant protein.

[0072] Using the unmutated original THDR protein as a control, the specific enzyme activity of each mutant was determined by the NADPH-dependent enzyme activity assay (refer to Example 1). The results showed that the specific enzyme activity of the original THDR protein was 126.4 U / mg; A142T was 215.8 U / mg, Q256R was 202.1 U / mg, and the double mutant A142T / Q256R was 289.6 U / mg. Positive mutation can significantly improve catalytic activity.

[0073] (3) Enzymatic verification: After purifying the mutant protein, the catalytic efficiency (kcat / Km) and thermal stability (Tm) were measured in vitro. kcat / Km is the enzyme specificity constant, which comprehensively reflects the enzyme's catalytic ability and substrate affinity. The higher the value, the better the overall catalytic performance of the enzyme. Tm is the protein melting temperature, which refers to the temperature at which half of the protein molecules undergo conformational unwinding and denaturation. The higher the value, the stronger the protein's thermal stability.

[0074] Enzymatic reactions were carried out with different concentrations of substrate. The absorbance of NADPH was monitored at 340 nm. The Michaelis-Menten equation was fitted to calculate kcat and Km, and the kcat / Km ratio was obtained. Differential scanning fluorescence (DSF) was used to detect fluorescence signals with a gradient temperature increase from 25℃ to 85℃, and Tm was determined based on the melting curve.

[0075] The results showed that the double mutant A142T / Q256R had a 3.5-fold increase in catalytic efficiency and a 6.3-fold increase in thermal stability (Tm) compared to the wild type. The control mutant W189A had a 42% decrease in catalytic efficiency and a 3.1-fold decrease in thermal stability (Tm) compared to the wild type; the control mutant E50D had a similar catalytic efficiency to the wild type, but its reverse hydroxylation activity was approximately 3.5 times stronger.

[0076] (4) Fermentation results of recombinant strains: Will thdr The genes of mutants A142T / Q256R, W189A, and E50D were inserted into the original wild-type vector pET28a-pTrc-EQU-opt. thdr The gene location was determined, and the site-specific replacement was completed. The recombinant plasmid containing the above mutant was then sequentially introduced into the engineered strain constructed in Example 3. E. coli EQU-opt-CRi, named E. coliEQU-Mut was used for 48 h of fermentation. The results showed that the recombinant strain expressing the double mutant A142T / Q256R produced 1,441.6 mg / L (1.44 g / L) of (S)-equorol, which was higher than... E. coli The EQU-opt-Cri yield was increased by 2.0-fold, with a purity >97% and an S-configuration optical purity >99.0%. The reduction metabolic byproducts mediated by ydjG, yqhD, and dhaT were significantly reduced, with ethanol content at 18.6 mg / L, acetic acid at 22.3 mg / L, and total polyols at 11.5 mg / L, representing a 79.2% decrease in total byproducts compared to the starting strain. In contrast, the control mutant W189A (S)-equol yielded 412.5 mg / L, a 43% decrease compared to the starting strain; ethanol content was 76.4 mg / L, acetic acid at 85.1 mg / L, and total polyols at 49.8 mg / L, with significantly higher byproduct accumulation. The control mutant E50D (S)-equol yielded 189.4 mg / L, which was 74% lower than the original strain. Ethanol, acetic acid, and polyols accumulated in large quantities, and the total by-product content was 2.1 times that of the original strain. At the same time, due to the enhanced reverse hydroxylation activity of this mutant, the target product (S)-equol was continuously degraded.

[0077] In summary, neither the non-catalytic site W189A nor the incorrect mutation direction E50D increased (S)-estrol production; instead, they led to a significant decrease, demonstrating that THDR directional mutations have strict site and direction specificity. Only the A142T / Q256R double mutant... E. coli EQU-Mut achieves a synergistic improvement in catalytic efficiency and thermal stability, providing a key enzyme element for the efficient synthesis of (S)-estradiol.

[0078] Example 5: Genome Integration and Fermentation (1) Genome integration strategy: The pTrc-EQU-wt expression cassette was constructed, containing the pTrc promoter, dznr gene, ddr gene, dhdR gene, and the thdr mutant A142T / Q256R gene.

[0079] The pTrc-EQU-wt expression cassette was integrated into the CRISPR-Cas9-mediated homologous recombination. E. coli EQU-Mut strain rrnB Operator sites; rrnB The operon contains the 16S rRNA gene. rrsB (Gene ID: 948346), 23S rRNA gene rrlB (Gene ID: 948472), 5S rRNA gene rrfB(Gene ID: 948471) and glutamate tRNA gene gltT (Gene ID: 947808) Obtain plasmid-independent stable strain E. coli -EQU-G.

[0080] (2) Fermentation parameters: The fermentation process was carried out at the following conditions: glucose 20 g / L, pH 7.0, dissolved oxygen 30%, cultured until OD600 reached 15-18 for induction, IPTG final concentration 0.5 mM, and fermentation temperature 37℃.

[0081] (3) Magnification verification: The constructed strain E. coli -EQU-G was fed-batch fermented in a 5 L fermenter. The specific steps were as follows: The strain was... E. coli -EQU-G was inoculated at a rate of 5% (v / v) into LB medium (pH 7.0) containing 20 g / L glucose and incubated at 37°C, maintaining dissolved oxygen at 30% (controlled range 20%-40%). When the bacterial OD... 600 When the concentration reached 15-18, IPTG was added to a final concentration of 0.5 mM for induction, and a peristaltic pump was started to continuously feed 600 g / L glucose solution at a constant rate of 8 mL / h until the end of fermentation. Daidzein was added in three stages: 2 g / L at induction, 1.5 g / L 24 h after induction, and 1.5 g / L 48 h after induction, for a total addition of 5 g / L. To assess product stability, samples were continuously taken and tested until 72 h of fermentation. The results showed that the yield was 2,280 mg / L at 60 h of fermentation, and (S)-estradiol yield reached 2,402 mg / L at 72 h of fermentation, with a substrate conversion rate of 50% and no significant decrease in yield, demonstrating that the product remained stable in the later stages of fermentation.

[0082] (4) Product extraction and testing: After centrifugation to collect bacterial cells, extraction with ethyl acetate, and purification by silica gel column chromatography, the purity was determined by HPLC to be 99.0%. Chiral HPLC (using a Daicel Chiralcel OJ-H column (250×4.6 mm, 5 μm), mobile phase n-hexane / isopropanol (85:15), flow rate 0.8 mL / min, detection wavelength 280 nm, column temperature 25℃; (S)-equorol retention time approximately 8.2 min, (R)-equorol approximately 9.5 min) confirmed the S-configuration optical purity to be 99.2%. LC-MS / MS confirmed that the characteristic ion peaks were consistent with the standards.

[0083] Comparative Example 1: The specific implementation method is the same as in Example 2, except that, based on the pET28a-pTrc-EQU plasmid constructed in Example 1, only the following is changed: thdr The promoter was replaced with the strong promoter P_T7 (nucleotide sequence shown in SEQ ID NO. 6), and the RBS sequence was optimized (ΔG = -9.2 kcal / mol) without integration. zwf / gnd The NADPH regeneration module also does not co-express. groEL / groES Molecular chaperone module, construct recombinant plasmid pET28a-P_T7-thdr-EQU.

[0084] RBS before optimization (original): AAGGAG, ΔG = -5.3 kcal / mol; RBS after optimization: TAAGGAGG, ΔG = -9.2 kcal / mol.

[0085] Fermentation was carried out according to the method in Example 2. The results showed that the yield of (S)-equorol was 68.4 mg / L, which was 56.2% lower than that in Example 2. This indicates that a single promoter and RBS optimization can improve the expression level of the target enzyme, but cannot overcome the dual bottlenecks of NADPH supply and protein folding. It is necessary to synergistically integrate the NADPH regeneration module and the chaperone protein co-expression module to maximize metabolic flux.

[0086] Comparative Example 2: The specific implementation method is the same as in Example 2, except that, based on the pET28a-P_T7-thdr-EQU constructed in Comparative Example 1, upstream integration is performed. zwf / gnd The NADPH regeneration module (nucleotide sequences such as SEQ ID NO.7 and SEQ ID NO.8) does not integrate the groEL / groES folding chaperone co-expression module.

[0087] The constructed recombinant bacteria were fermented according to the method in Example 2. The results showed that, compared with Comparative Example 1, the recombinant bacteria increased... zwf / gnd After the NADPH regeneration module was used, the intracellular NADPH concentration increased from 0.58 μmol / g DCW to 1.15 μmol / g DCW, an increase of 98.3%; the (S)-estrol production increased from 68.4 mg / L to 112.7 mg / L, an increase of 64.8%, indicating that the module can effectively enhance the supply of reducing power.

[0088] Compared to Example 2, the soluble expression rate of THDR was only 52%, which was 66.7% of that in Example 2, and the expression was missing. groEL / groESMolecular chaperones caused a large number of proteins to form inclusion bodies, resulting in a 27.9% decrease in yield (112.7 vs 156.3 mg / L). The intracellular NADPH concentration (1.15 μmol / g DCW) was basically the same as that in Example 2 (1.18 μmol / g DCW), but the yield difference was significant, demonstrating that when the reducing power is sufficient, protein folding efficiency becomes the main bottleneck, and a molecular chaperone module needs to be introduced to maximize enzyme catalytic efficiency.

[0089] Comparative Example 3: The specific implementation method is the same as in Example 4, except that the E. coli-EQU-opt-CRi constructed in Example 3 is used as the starting strain, and the wild-type strain is... thdr The mutants A142T, Q256R, W189A, and E50D, as shown in Table 7, were replaced respectively, and a systematic comparison was made with the A142T / Q256R double mutant in Example 4 to verify the specificity of the mutation site.

[0090] The recombinant bacteria were constructed according to the method in Example 4, and their ability to ferment and prepare (S)-estrol was determined. The results are shown in Table 7. The improvement in THDR catalytic efficiency and thermal stability exhibits strict site specificity. Only the A142T / Q256R double mutation can synergistically improve catalytic performance and yield, while mutations at other sites cannot achieve efficient synthesis, further demonstrating the uniqueness and advancement of the mutation modification in this invention.

[0091] Table 7. Effects of recombinant bacteria expressing different mutants

[0092] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A tetrahydrodaidin reductase mutant, characterized in that, Based on the amino acid sequence shown in SEQ ID NO.14, alanine at position 142 was mutated to threonine, and glutamine at position 256 was mutated to arginine.

2. The gene encoding the mutant of claim 1.

3. A recombinant microorganism expressing the tetrahydrodaidzein reductase mutant of claim 1.

4. The recombinant microorganism according to claim 3, characterized in that, The recombinant microorganism also expressed the dznr, ddr, and dhdR genes; the nucleotide sequences of the dznr, ddr, and dhdR genes are shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, respectively.

5. The recombinant microorganism according to claim 4, characterized in that, The expression of the equol synthesis gene cluster is regulated by the pTrc promoter; the equol synthesis gene cluster includes the daidzein reductase gene. dznr Dihydrodaibinagen racemic enzyme gene ddr Dihydrodaidin reductase gene dhdR The gene as described in claim 2; the nucleotide sequence of the pTrc promoter is shown in SEQ ID NO.

5.

6. The recombinant microorganism according to claim 5, characterized in that, It also includes one or more of the following modifications: (a) The gene of claim 2 is regulated by the T7 promoter; (b) Replace the RBS sequence between the promoter and the thdr gene with TAAGGAGG; (c) Introducing an NADPH regeneration module; the NADPH regeneration module includes the zwf gene and the gnd gene; (d) Introducing a molecular chaperone co-expression module; the molecular chaperone co-expression module includes the groEL gene and the groES gene.

7. The recombinant microorganism according to claim 6, characterized in that, In the recombinant microorganism, the competitive pathway genes that compete with equadol synthesis for reducing power and / or carbon source are inhibited or inactivated; the competitive pathway genes are selected from one or more of ydjG, yqhD, and dhaT.

8. The recombinant microorganism according to any one of claims 3 to 7, characterized in that, The host is Escherichia coli; the Escherichia coli includes Escherichia coli BL21(DE3).

9. A method for preparing (S)-estrol, characterized in that, include: The recombinant microorganisms described in any one of claims 3 to 8 are cultured, and (S)-estrol is collected, extracted, and / or purified from the fermentation system.

10. The use of the tetrahydrodaidzein reductase mutant of claim 1, the recombinant microorganism of any one of claims 3 to 8, or the method of claim 9 in the preparation of S-estrol or products containing (S)-estrol.

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