Daidzein reductase mutant and application thereof in efficient synthesis of (s)-equol

Abstract

This invention discloses a soybean aglycone reductase mutant and its application in the efficient synthesis of (S)-equol, belonging to the fields of enzyme engineering, genetic engineering, metabolic engineering, and fermentation engineering. This invention constructs a DZNR mutant with enhanced catalytic activity, which, together with DDRC, DHDR, and THDR, forms a four-enzyme cascade pathway for the synthesis of (S)-equol in recombinant E. coli containing pCoenzy. Further, Ec01-7, which additionally expresses ribA, and Ec01-22, which additionally expresses ribA and crr, were constructed. Using the strain Ec01-7 constructed in this invention, culture conditions were screened and fed-batch conversion was performed in a 5 L fermenter, yielding 8.0 g / L of (S)-equol in 70 h, providing a feasible technical route for the large-scale biomanufacturing of (S)-equol.

Description

Technical Field

[0001] This invention relates to a soybean aglycone reductase mutant and its application in the efficient synthesis of (S)-estrol, belonging to the fields of enzyme engineering, genetic engineering, metabolic engineering and fermentation engineering technology. Background Technology

[0002] (S)-Estrol is a chiral isoflavone metabolite formed from the soybean isoflavone daidzein through intestinal microbial metabolism, and it has applications in food, medicine, and health products. Its biosynthesis typically occurs sequentially via daidzein reductase DZNR, dihydrodaidzein racemic enzyme DDRC, dihydrodaidzein reductase DHDR, and tetrahydrodaidzein reductase THDR. DZNR catalyzes the first step of this pathway, converting daidzein to dihydrodaidzein, and serves as the entry point for the substrate into subsequent cascade reactions. Insufficient DZNR catalytic activity results in a large amount of daidzein remaining at the beginning of the pathway, limiting both the throughput and the final titer. Therefore, targeted protein engineering of DZNR is crucial for improving the efficiency of (S)-Estrol synthesis.

[0003] However, the modification of DZNR is highly dependent on the selection of mutation sites. DZNR and its cascade partners are mostly flavin-dependent oxidoreductases, and the contributions of different residues in their coenzyme / cofactor binding and substrate binding regions to catalytic performance vary significantly: not all site substitutions are beneficial, and mutations at some sites can lead to a significant decrease in activity; there is no simple additive effect between single-point mutations, and advantageous combinations must be experimentally verified. Furthermore, DZNR in engineered bacteria is not a free enzyme acting alone, but rather exists within a whole-cell cascade system deeply coupled with DDRC, DHDR, THDR, and intracellular cofactor metabolism. This reaction chain includes multiple reduction steps dependent on NAD(P)H and flavin cofactors. Therefore, the final yield of engineered bacteria depends not only on the transient activity of a single enzyme but also on cofactor supply, carbon metabolism flux, cell state, and the degree of multi-enzyme compatibility. In industrial scenarios with high substrate loading and long-term fed-batch conversion, insufficient cofactor supply often replaces enzyme activity itself, becoming the main factor limiting the final titer and conversion persistence. Although the strain can support short-cycle conversion with the existing reducing equivalent within the cell, if there is a lack of continuous carbon source supply and cofactor regeneration capacity in the tens of hours after the addition of daidzein in batches, the reducing power will gradually decline, and the cascade reaction will be difficult to advance to a high conversion rate.

[0004] Therefore, there is an urgent need to develop new soybean aglycone reductases and strategies to improve the insufficient supply of cofactors in order to enhance the ability of recombinant strains to produce (S)-estrol. Summary of the Invention

[0005] This invention constructs a DZNR mutant of daidzein reductase by site-specific modification of the NADPH-binding and substrate-binding regions of the daidzein reductase DZNR, and improves the estrol synthesis capacity of the engineered strain by combining a cofactor enhancement strategy.

[0006] This invention provides a soybean aglycone reductase mutant with mutations of L356A and / or M73C based on the amino acid sequence shown in SEQ ID NO:1.

[0007] In one embodiment, the mutation is to replace methionine at position 73 of the amino acid sequence shown in SEQ ID NO:1 with cysteine.

[0008] In one embodiment, the mutation is to replace leucine at position 356 of the amino acid sequence shown in SEQ ID NO:1 with alanine.

[0009] In one embodiment, the mutation is to replace leucine at position 356 of the amino acid sequence shown in SEQ ID NO:1 with alanine and methionine at position 73 with cysteine.

[0010] In one embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO:5. The present invention also provides a gene encoding the mutant.

[0011] In one embodiment, the nucleotide sequence of the gene is shown in SEQ ID NO:3 or SEQ ID NO:4.

[0012] The present invention also provides a recombinant plasmid containing the said gene.

[0013] In one embodiment, the plasmid uses pCDFDuet-1 as a plasmid backbone to express the mutant.

[0014] The present invention also provides recombinant microorganisms containing the recombinant plasmid.

[0015] In one embodiment, the microorganisms include, but are not limited to, Escherichia coli.

[0016] In one embodiment, the mutant is expressed using Escherichia coli BL21(DE3) as a host, and dihydrogenin racemic enzyme DDRC, dihydrogenin reductase DHDR, and tetrahydrogenin reductase THDR are expressed.

[0017] In one embodiment, the *E. coli* contains a recombinant plasmid expressing DZNR.

[0018] In one embodiment, the *E. coli* contains a recombinant plasmid expressing dihydrogenin reductase DHDR, dihydrogenin racemic enzyme DDRC, and / or tetrahydrogenin reductase THDR.

[0019] In one embodiment, pCDFDuet-1 is used as a backbone to express the mutant gene and the DDRC gene shown in SEQ ID NO:6.

[0020] In one embodiment, pRSFDuet-1 is used as a vector to express the DHDR gene shown in SEQ ID NO:7 and the THDR gene shown in SEQ ID NO:8.

[0021] In one embodiment, the *E. coli* also contains a pCoenzy plasmid for enhancing NADPH supply and regeneration.

[0022] In one embodiment, the *E. coli* also expresses the GTP cyclase II gene ribA and the glucose-specific phosphotransferase system enzyme IIA gene crr; the nucleotide sequence of the ribA gene is shown in SEQ ID NO:9; the nucleotide sequence of the crr gene is shown in SEQ ID NO:10.

[0023] In one implementation, the DHDR gene, THDR gene, ribA gene, and crr gene are expressed using pRSFDuet-1 as a vector.

[0024] The present invention also provides a method for preparing (S)-estrol by fermentation of the recombinant Escherichia coli, wherein the recombinant Escherichia coli is cultured in a fermentation medium to an OD600 of 0.8 to 1.0 and then IPTG is added for induction.

[0025] In one embodiment, the glucose concentration is maintained at 1–3 g / L during fermentation.

[0026] In one embodiment, the induction temperature is 18–30°C, the final concentration of isopropyl-β-D-thiogalactoside is 0.05–0.20 mmol / L, and the inoculum size is 2%–8%.

[0027] In one embodiment, the induction temperature is controlled at 25°C and the final IPTG concentration is 0.1 mmol / L.

[0028] In one implementation, the fermentation process also involves the addition of daidzein in batches and the control of dissolved oxygen at a low level.

[0029] In one embodiment, dissolved oxygen is controlled to be no higher than 5% during the daidzein feeding stage; preferably, it is controlled to be close to 0%.

[0030] In one embodiment, fermentation continues for 48–72 hours after the addition of daidzein.

[0031] In one embodiment, the fermentation system also contains polyvinylpyrrolidone (PVP-40K).

[0032] The present invention also provides the use of the recombinant Escherichia coli or the method thereof in the preparation of (S)-estrol or products containing (S)-estrol.

[0033] Beneficial effects (1) This invention provides a soybean aglycone reductase DZNR mutant, which improves the (S)-estrol synthesis capacity of engineered strains under shake-flask screening conditions. According to Examples 2 and 3, the endpoint concentration of the engineered parent Ec-WT was 1.000 g / L, and that of the L356A mutant Ec-M1 was 1.442 g / L, an increase of 44.2%; after further introducing M73C into the L356A background, the endpoint concentration of the double mutant Ec-M2 was 1.538 g / L, an increase of 53.8% relative to the engineered parent, and the yield was 1.54 times that of the parent.

[0034] (2) This invention constructed strain Ec01-7, which additionally expresses ribA in the L356A-M73C double mutant background, and strain Ec01-22, which additionally expresses ribA and crr. The plasmid composition and construction method are described in Example 4. Using Ec01-7, culture conditions were screened, and the endpoint concentration of (S)-equadolol of 1405.2 mg / L was obtained by shake-flask verification; further, under the fed-batch conversion conditions of the 5L fermenter described in Example 7, the endpoint concentration of 8.0 g / L was obtained after 70 h. Attached Figure Description

[0035] Figure 1 This is a graph showing the changes in cell growth, dissolved oxygen, and substrate and product concentrations during fermentation.

[0036] Figure 2 This is a schematic diagram comparing the fermentation processes of strains Ec-M2 (L356A-M73C double mutant) and Ec01.

[0037] Figure 3 A schematic diagram comparing the fermentation processes of strains Ec01-7 (L356A-M73C double mutant with additional ribA expression) and Ec-M2. Detailed Implementation

[0038] The following examples illustrate the present invention. Each experiment was independently repeated at least three times, and the data are expressed as mean ± standard deviation, and one-way ANOVA was used. Strains without additional ribA or crr expression modules retained the corresponding chromosomal genes of the host.

[0039] (a) Culture medium LB medium: 5 g yeast extract, 10 g peptone, 10 g sodium chloride, bring to a final volume of 1 L with deionized water, and sterilize at 121°C for 20 min.

[0040] TB medium: 24 g yeast extract, 12 g peptone, 2.31 g KH2PO4, 12.54 g K2HPO4, 4 mL glycerol, bring the volume to 1 L with deionized water, and sterilize at 121℃ for 20 min.

[0041] (II) Reagents and Materials Appropriate antibiotics were added based on plasmid resistance. E. coli JM109 was used for plasmid construction and preservation, while E. coli BL21(DE3) was used for protein expression and fermentation transformation.

[0042] E. coli JM109 was used for plasmid construction and preservation, and E. coli BL21(DE3) was used for protein expression and fermentation. Both were purchased from Shanghai Sangon Biotech. The expression host genotype was F1. 、ompT、hsdSB(rB mB The culture medium contained gal, dcm(DE3), and pCDFDuet-1, a streptomycin resistance vector with dual T7 promoters and CloDF13 replication origin; pRSFDuet-1, a kanamycin resistance vector with dual T7 promoters and RSF replication origin; and pET22b, an ampicillin resistance vector. The culture system used ampicillin, kanamycin, chloramphenicol, and streptomycin, each at a final concentration of 50 mg / L. Amplification was performed using 2×Phanta MaxMaster Mix, and assembly was performed using the ClonExpress Ultra One Step Cloning Kit, both purchased from Novizan; DpnI was purchased from Beyotime, and PVP-40K was purchased from Solarbio.

[0043] (III) Detection Methods Fermentation products were detected using an Agilent 1260 Infinity II liquid chromatograph. Before sampling, the fermentation broth was thoroughly mixed and immediately diluted with methanol, ensuring complete dissolution of low-solubility products to minimize underestimation due to crystallization. The mixture was centrifuged at 12000 × g for 5 min, and the supernatant was filtered through a 0.22 μm nylon filter. A C18 column (4.6 mm × 250 mm, 5 μm) was used at 40 °C, with an injection volume of 10 μL and a detection wavelength of 290 nm. Phase A consisted of ultrapure water containing 0.1% formic acid, and phase B consisted of methanol containing 0.1% formic acid. The elution gradient was as follows: 0–10 min, phase B elution increased from 10% to 60%; 10–20 min, from 60% to 80%; 20–22 min, from 80% to 10%; 22–25 min, maintained at 10%. Quantification was performed using standards, and the fermentation broth concentration was calculated based on the actual dilution factor.

[0044] In this invention, "yield" or "endpoint concentration" refers to the volume of fermentation broth, in units of g / L or mg / L; "percentage increase" is calculated as (experimental group - control group) / control group × 100%, and "multiple of control" is calculated as experimental group / control group. Endpoint concentrations under different culture scales or conditions are reported separately; the relative increase is based on the control under the corresponding experimental conditions.

[0045] (iv) Plasmid and strain information The NADPH-enhancing plasmid pCoenzy was published in the paper "High Titer of (S)". The plasmid "Equol Synthesis from Daidzein in Escherichia coli" was used in conjunction with pCDF and pRSF-derived plasmids to express the host.

[0046] Strain Ec00: Contains plasmids pCoenzy and pCDF-czM2 in E. coli BL21(DE3); Strain Ec01: E. coli BL21(DE3) contains plasmids pCoenzy, pCDF-czM2, and pRSF-dt; Strain Ec01-7 (also named Ec-M2-R): contains plasmids pCoenzy, pCDF-czM2 and pRSF-dt-ribA in E. coli BL21(DE3). This strain is based on Ec01 and additionally expresses ribA.

[0047] Strain Ec01-22 (also named Ec-M2-RC): contains plasmids pCoenzy, pCDF-czM2 and pRSF-dt-ribA-crr in E. coli BL21(DE3). This strain is based on strain Ec01 and additionally expresses ribA and crr.

[0048] Example 1: Construction of engineered parental sequence, expression plasmid, and control strain (1) Construction of pCDF-cz Using pCDFDuet-1 as a backbone, the backbone fragment outside the first expression region was amplified using pCDF-cz1-F / pCDF-cz1-R, and the DDRC coding fragment from *Lactococcus sp. 20-92* was amplified using pCDF-DDRC-F / pCDF-DDRC-R. The resulting intermediate plasmid pCDF-c was assembled. Then, using pCDF-c as a template, the backbone outside the second expression region was amplified using pCDF-cz2-F / pCDF-cz2-R, and the engineered parental DZNR expression fragment was inserted to obtain pCDF-cz. DDRC is located in the first expression region, and DZNR is located in the second expression region, both driven by the vector T7 promoter. The nucleotide sequence of DDRC is shown in SEQ ID NO:6; the nucleotide sequence of DZNR is shown in SEQ ID NO:2.

[0049] (2) Construction of pRSF-dt Using pRSFDuet-1 as a backbone, the backbone was amplified with pRSF-dt1-F / pRSF-dt1-R and assembled with the DHDR fragment amplified by pRSF-DHDR-F / pRSF-DHDR-R to obtain pRSF-d. This intermediate plasmid backbone was then amplified with pRSF-dt2-F / pRSF-dt2-R and assembled with the THDR fragment amplified by pRSF-THDR-F / pRSF-THDR-R to obtain pRSF-dt. Both DHDR and THDR are derived from *Slackia isoflavoniconvertens*, located in the first and second T7 expression regions, respectively, with nucleotide sequences as shown in SEQ ID NO:7 and SEQ ID NO:8, respectively.

[0050] Each fragment was amplified using high-fidelity PCR, purified using a kit, assembled using the ClonExpress Ultra One Step Cloning Kit, and transformed. E. coli JM109 was constructed and saved, and after screening, the target fragment and linker region were identified by sequencing.

[0051] (3) Construction of DZNR mutant plasmid Using pCDF-cz as a template, site-directed mutagenesis was performed using L356A-F / L356A-R to replace CTG at positions 1066-1068 of the DZNR coding sequence with GCG, resulting in plasmid pCDF-czM1 containing the L356A-DZNR mutant shown in SEQ ID NO.3.

[0052] Using the previously constructed plasmid pCDF-czM1 as a template, positions 217-219 of the ATG were replaced with TGC using M73C-F / M73C-R to obtain the recombinant plasmid pCDF-czM2 containing the mutant L356A-M73C-DZNR shown in SEQ ID NO.4. Each plasmid retains the DDRC expression region.

[0053] PCR was performed using 2×Phanta Max Master Mix. After amplification, the template was digested with DpnI, the product was purified, processed according to the ClonExpress assembly system, and transformed into JM109. The mutation site and target coding region were confirmed by sequencing. The basic construction and site-directed mutagenesis primers are shown in Tables 1 and 2.

[0054] For DZNR mutant activity assays, DZNR or its mutant fragments were amplified using DZNR-F / DZNR-R, and the pET22b backbone was amplified using pET22b-DZNR-F / pET22b-DZNR-R. After purification, the plasmids were assembled using ClonExpress to obtain pET22b-DZNR and the corresponding mutant expression plasmids, respectively. These plasmids were then transformed into BL21(DE3) and cultured at 37°C until OD600. 600 =0.6, add IPTG to 0.1 mmol / L and induce at 18℃ for 12 h. The pET22b system was used for enzymatic characterization.

[0055] (4) Construction and nomenclature of expression strains The plasmids pCoenzy, pCDF-cz, and pRSF-dt constructed above were co-introduced into E. coli BL21(DE3), and the resulting strain was designated Ec-WT. Following the same method, plasmids pCoenzy and pCDF-czM2 were co-introduced into E. coli BL21(DE3), and the resulting strain was named Ec00; the strain co-introduced into E. coli BL21(DE3) with plasmids pCoenzy, pCDF-czM1, and pRSF-dt was named Ec-M1; and the strain co-introduced into E. coli BL21(DE3) with plasmids pCoenzy, pCDF-czM2, and pRSF-dt was named Ec-M2 (also denoted as Ec01). Among them, Ec-WT, Ec-M1 and Ec-M2 all contain pCoenzy and none of them introduce additional ribA or crr expression modules; Ec-M1 and Ec-M2 are used to evaluate the effects of L356A single mutation and L356A-M73C double mutation, respectively.

[0056] Table 1 Key plasmid construction and site-directed mutagenesis primers

[0057] Table 2 Primers for DZNR single-point mutation

[0058] Example 2: DZNR single-point mutation screening Using the recombinant plasmid pCDF-cz constructed in Example 1 as a template, single-point mutant plasmids of N64A, M73A, F113A, E128A, I129A, N132A, C174A, P232A, T261A, H262A, V263A, T264A, N266A, S268A, I355A, L356A, P543A, G545A, Q547A, and I550A were constructed using the primers corresponding to those in Table 2. Amplification, DpnI treatment, assembly, and sequencing were performed according to the method in Example 1. The resulting plasmid was named pCDF-cz (mut), where mut represents a specific substitution.

[0059] The mutant-containing plasmid pCDF-cz(mut) was co-transformed with plasmids pCoenzy and pRSF-dt into [a specific gene / organization]. E. coliBL21(DE3) was used to construct recombinant bacteria expressing different single mutants. Using the recombinant bacteria Ec-WT constructed in Example 1 as a control, the recombinant bacteria were inoculated into TB medium at a 3% inoculum size. After culturing at 37℃ for 3 h, IPTG was added to a final concentration of 0.2 mM, and daidzein was added as a substrate to a final concentration of 3 g / L. Fermentation was continued at 120 rpm and 25℃ for 38 h. The catalytic performance was evaluated by the daidzein conversion rate, and the results are shown in Table 3. Strains expressing mutants N64A, I129A, N132A, C174A, P232A, T261A, V263A, T264A, and L356A (…) S The levels of 1,000 g / L for 1,000 g / L were higher in L356A than in the engineered parent, with L356A having the highest level at 1,442 g / L, representing a 44.2% increase.

[0060] Table 3. (S)-Emperorphenol endpoint concentrations corresponding to DZNR single-point mutants

[0061] Example 3: Replacement screening of M73, G74 and F113 sites Using pCDF-czM1 constructed in Example 1 as a template, replacements were introduced at sites M73, G74, and F113 using the primers listed in Table 4, respectively, to construct pCDF-cz (L356A-mut). The L356A-M73C plasmid is also known as pCDF-czM2, and its coding sequence is SEQ ID NO:4. PCR, DpnI digestion, ClonExpress assembly, JM109 preservation, and sequencing verification were performed as in Example 1.

[0062] The constructed recombinant plasmid pCDF-cz (L356A-mut) was co-transformed with plasmids pCoenzy and pRSF-dt into E. coli BL21 (DE3) to construct recombinant bacteria expressing different double mutants.

[0063] The strains were cultured according to the method in Example 2, and the endpoint concentration of (S)-equadolphin corresponding to each mutant combination was determined. The results are shown in Table 5. Based on the screening results, the L356A-M73C double mutant was selected for subsequent cofactor module construction and culture condition screening.

[0064] Table 4. Replacement primers for M73, G74, and F113 sites

[0065] Table 5. Equisetol production of different mutants

[0066] The results showed that strain Ec-M2, which further introduced M73C under the L356A background, had the highest (S)-equorol concentration at the shake-flask endpoint, at 1.538 g / L; the strain with introduced M73N had a concentration of 1.500 g / L, slightly higher than the L356A control of 1.442 g / L. M73Q, M73S, and M73T did not increase yield, and all G74 substitutions and F113P / F113H also decreased yield. While F113Y maintained a relatively high level, it was still lower than the control. Compared to the engineered parent Ec-WT (1.000 g / L), the L356A-M73C double mutant showed a 53.8% increase in yield, 1.54 times that of the parent. Therefore, this double mutant was selected for subsequent cofactor module construction and process optimization.

[0067] Example 4: Construction of a cofactor and carbon metabolism synergistic module (1) Construction of pRSF-dt-ribA Using the pRSF-dt constructed in Example 1 as the base backbone, the backbone was amplified using pRSF-FYZ-F / pRSF-FYZ-R. E. coli Using BL21(DE3)-derived ribA (nucleotide sequence shown in SEQ ID NO:9) as the target, a fragment with homologous linker sequences was obtained using ribA-F / ribA-R, and then assembled using ClonExpress to obtain pRSF-dt-ribA. This plasmid retains the DHDR and THDR expression modules, and adds a ribA expression module downstream of THDR.

[0068] (2) Construction of pRSF-dt-ribA-crr; Based on the previously constructed pRSF-dt-ribA, a crr fragment derived from E. coli BL21(DE3) (nucleotide sequence shown in SEQ ID NO:10) was introduced downstream of ribA. A crr fragment with homologous sequences was obtained using crr-F / crr-R, and after assembly, pRSF-dt-ribA-crr was obtained. This plasmid contains DHDR, THDR, ribA, and crr expression modules. The primers used are shown in Table 6.

[0069] pRSF-dt-ribA and pRSF-dt-ribA-crr were introduced into strain Ec00, which carried pCoenzy and pCDF-czM2, constructed in Example 1, to obtain Ec01-7 (also referred to as Ec-M2-R) and Ec01-22 (also referred to as Ec-M2-RC), respectively.

[0070] Table 6 Primers for constructing ribA and crr expression modules

[0071] Example 5: Optimization of inoculum size, induction temperature and IPTG concentration The strain Ec01-7 constructed in Example 4 was used for fermentation, and the fermentation process was optimized. The basic fermentation process was as follows: a seed culture was prepared by culturing in 5 mL LB medium for 8–10 h. The seed culture was inoculated at a rate of 2% into TB medium containing 5% polyvinylpyrrolidone (PVP-40K) and cultured for 1.5–3 h until OD600 = 0.8–1.0. IPTG was added to a final concentration of 0.1 mmol / L and the temperature was lowered to 25 °C for induction culture for 12 h. Then, 10% (v / v) 200 g / L glucose solution and 10% (v / v) 25 g / L daidzein solution were added, and the culture was continued for 48 h.

[0072] The inoculum size, induction temperature, and IPTG concentration were optimized. Inoculum sizes of 2%, 4%, and 8% were set; induction temperatures of 18℃, 25℃, and 30℃ were set; and final IPTG concentrations of 0.05, 0.10, and 0.20 mmol / L were set, resulting in 27 combinations. The screening results are shown in Table 7. In the 24-well plate condition screening, the optimal combination was 4%, 25℃, and 0.10 mmol / L, achieving a yield of 1111.79 mg / L; the yield in shake flask validation reached 1405.2 mg / L.

[0073] Table 7. Screening results of 27 combinations of inoculum size, induction temperature, and IPTG concentration.

[0074] Example 6: Feed conversion and dissolved oxygen control in a 5 L fermenter The double mutant strain Ec-M2 (also denoted as Ec01) constructed in Example 1 was used for fed-batch transformation in a 5 L fermenter. This strain contains pCoenzy, pCDF-czM2, and pRSF-dt, and expresses L356A-M73C-DZNR, DDRC, DHDR, and THDR, without the additional introduction of ribA or crr expression modules. The initial working volume of the reactor was 2.5 L, and it was cultured at 37°C and pH 7.0. A 4% inoculum was added, and IPTG was added 8 h after inoculation to a final concentration of 0.1 mmol / L. Feedback feeding was performed using 700 g / L glucose solution to maintain the glucose concentration in the fermentation system at 1–3 g / L. 14–24 h after inoculation, 62.5 g / L daidzein DMSO stock solution was linearly fed at 20–40 mL / h, with a cumulative addition of 10 g / L. During the growth stage, the dissolved oxygen (DO) was controlled to be above 20% by agitation and ventilation at 200–700 rpm. 24 h after inoculation, the air intake was reduced to 0 L / min, and agitation was adjusted by DO control to bring the calibrated probe reading close to 0%. Here, 0% DO means that the probe signal is close to zero under oxygen-limited conditions, not that there is absolutely no oxygen.

[0075] Taking inoculation as the zero point, when fermentation reaches 70 h, the ( S The endpoint concentration of 5907.2 mg / L (5.9072 g / L) was estrol.

[0076] Example 7: Cultivation of Ec01-7 in a 5 L fermenter The strain Ec01-7 (also denoted as Ec-M2-R) constructed in Example 4 was used in an independent 5 L fermenter experiment according to the method in Example 6. Strain Ec01-7 contains pCoenzy, pCDF-czM2, and pRSF-dt-ribA, and additionally expresses ribA in the L356A-M73C double mutant background. It also expresses DDRC, DHDR, and THDR, without introducing an additional crr expression module. The endpoint concentration of (S)-equorol was measured to be 8.0 g / L in this experiment.

[0077] 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 soybean aglycone reductase mutant, characterized in that, Compared to the parent shown in SEQ ID NO:1, it contains amino acid substitutions of L356A and / or M73C.

2. The daidzein reductase mutant according to claim 1, characterized in that, The amino acid sequence is shown in SEQ ID NO:

5.

3. A nucleic acid molecule encoding the daidzein reductase mutant of claim 1 or 2.

4. A recombinant expression vector, characterized in that, It includes the nucleic acid molecule as described in claim 3.

5. Recombinant microbial cells, characterized in that, Expressing the mutant as described in claim 1 or 2 or containing the recombinant expression vector as described in claim 4.

6. Recombinant Escherichia coli, characterized in that, The mutant described in claim 1 or 2 is expressed and dihydrogenin racemic enzyme DDRC, dihydrogenin reductase DHDR, and tetrahydrogenin reductase THDR are expressed.

7. The recombinant Escherichia coli according to claim 6, characterized in that, It also contains the pCoenzy plasmid and overexpresses the ribA gene and / or the crr gene.

8. A method for preparing (S)-estrol, characterized in that, The recombinant Escherichia coli according to claim 6 or 7 is cultured in a medium containing glucose and daidzein.

9. The method according to claim 8, characterized in that, The addition of daidzein and whole-cell multi-enzyme cascade transformation under glucose-fed conditions.

10. The mutant of claim 1 or 2, the recombinant microbial cell of claim 5, the recombinant Escherichia coli of claim 6 or 7, or the method of any one of claims 8-9 in the preparation of ( S )-Estrol or containing ( S Application of )-estrol in products.

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