A strain for producing equol and its application

Abstract

The present invention discloses a strain for producing equol and its application, belonging to the technical fields of genetic engineering and bioengineering. The present invention provides new uses of the gene pfkA of 6-phosphofructokinase, the gene sthA of NAD(P)<supgt;+< / supgt; transhydrogenase and / or the gene elaA of GNAT family N-acetyltransferase. By knocking out one or more of the genes pfkA, sthA, and elaA endogenous to the Escherichia coli genome, the production capacity of equol is improved, and the yield of equol at the shake flask level is increased from 90 mg / L to 110.6 - 224.1 mg / L.

Description

Technical Field

[0001] The invention relates to a strain for producing equol and application thereof, belonging to the technical field of genetic engineering and bioengineering. Background Art

[0002] Isoflavones are important functional substances in leguminous plants, playing an important role in plant resistance to diseases and pests, and also have a positive effect on human health. Daidzein is widely present in leguminous plants such as soybeans and can be easily extracted from leguminous plants. However, its main effect on the human body is reflected in its reduced form - equol. The functionality of equol is several times stronger than that of daidzein, and it has many effects that daidzein does not have, which makes equol have great application potential in the fields of medicine, cosmetics and food industry.

[0003] Because equol is generated by intestinal flora conversion of daidzein, it cannot be extracted from plants. At present, the production of equol mainly utilizes traditional chemical methods, by carrying out a series of reductions to daidzein, protection and deprotection reactions and preparation. For the synthesis of chiral compounds, there are usually problems such as high reaction energy consumption, large separation difficulty, and expensive catalysts, and usually more by-products are produced, resulting in a low yield of the target product. And the production of equol by the mode of microbial transformation can well avoid these problems, and the equol generated by microbial transformation is all S-type, which is just what we need. Therefore, for the limitations of chemically synthesized equol in product stability, quality safety and price, green, safe, and single microbial transformation methods of products provide feasible ideas for the synthesis of equol. Summary of the invention

[0004] The present invention provides a gene pfkA of 6-phosphofructokinase, NAD(P) + Application of the transhydrogenase gene sthA and / or the GNAT family N-acetyltransferase gene elaA in improving the equol production of Escherichia coli.

[0005] In one embodiment, the application is to inhibit the gene pfkA of 6-phosphofructokinase, NAD(P) + Expression of one or more genes in the transhydrogenase gene sthA, the GNAT family N-acetyltransferase gene elaA.

[0006] In one embodiment, the nucleotide sequence of the gene pfkA is shown as Gene ID: 948412; the nucleotide sequence of the gene elaA is shown as Gene ID: 946750; and the nucleotide sequence of the gene sthA is shown as Gene ID: 948461.

[0007] The present invention also provides a recombinant Escherichia coli for producing equol, wherein the Escherichia coli is used as a starting strain, and the gene expression of one or more of the following genes is inhibited: the gene pfkA encoding 6-phosphofructokinase, NAD(P) + Transhydrogenase gene sthA, GNAT family N-acetyltransferase gene.

[0008] In one embodiment, the recombinant E. coli lacks or silences the endogenous genes pfkA, NAD(P) encoding 6-phosphofructokinase in E. coli. + One or more of the transhydrogenase gene sthA and the GNAT family N-acetyltransferase gene elaA.

[0009] In one embodiment, the recombinant E. coli has knocked out the endogenous genes pfkA, NAD(P) + One or more of the transhydrogenase gene sthA and the GNAT family N-acetyltransferase gene elaA.

[0010] In one embodiment, the recombinant Escherichia coli lacks or silences the 6-phosphofructokinase gene pfkA and NAD(P) + Transhydrogenase gene sthA.

[0011] In one embodiment, the recombinant Escherichia coli lacks or silences the 6-phosphofructokinase gene pfkA and the GNAT family N-acetyltransferase gene elaA.

[0012] In one embodiment, the recombinant E. coli lacks or silences NAD(P) + transhydrogenase gene sthA and GNAT family N-acetyltransferase gene elaA.

[0013] In one embodiment, the recombinant E. coli lacks or silences the endogenous 6-phosphofructokinase genes pfkA, NAD(P) + transhydrogenase gene sthA and GNAT family N-acetyltransferase gene elaA.

[0014] In one embodiment, the engineered Escherichia coli bacteria is further improved by at least one of the following:

[0015] (1) The daidzein reductase Ac_DZNR from Asaccharobacter celatus was expressed;

[0016] (2) expressed dihydrodaidzein racemase Lg_DDRC from Lactococcus garvieae;

[0017] (3) Dihydrodaidzein reductase Si_DHDR and tetrahydrodaidzein reductase Si_THDR from Slackia isoflavoniconvertens were expressed.

[0018] In one embodiment, the engineered Escherichia coli bacteria uses Escherichia coli BL21 (DE3) as a starting strain.

[0019] In one embodiment, the amino acid sequence of the soybean aglycone reductase Ac_DZNR is shown as SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.1.

[0020] In one embodiment, the amino acid sequence of the dihydrodaidzein racemase Lg_DDRC is shown as SEQ ID NO.6, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.2.

[0021] In one embodiment, the amino acid sequence of the dihydrodaidzein reductase Si_DHDR is shown as SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.3.

[0022] In one embodiment, the amino acid sequence of the tetrahydrodaidzein reductase Si_THDR is shown as SEQ ID NO.8, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO.4.

[0023] In one embodiment, pRSFDuet-1, pCDFDuet-1, or pETDuet-1 is used as an expression vector to express the daidzein reductase Ac_DZNR, dihydrodaidzein racemase Lg_DDRC, dihydrodaidzein reductase Si_DHDR, or tetrahydrodaidzein reductase Si_THDR.

[0024] The present invention also provides a method for producing equol by converting soybean aglycone through microorganisms. The method comprises using the Escherichia coli as a fermentation strain and fermenting in a medium containing soybean aglycone at 25-37°C for at least 24 hours.

[0025] In one embodiment, the method further employs IPTG induction.

[0026] In one embodiment, the fermentation is carried out in stages, and the stages include:

[0027] Phase 1: Control the temperature at 35-37°C and the dissolved oxygen concentration at 35-45% to promote cell growth;

[0028] The second stage: add the inducer with a final concentration of 0.1-0.2 mM, control the temperature at 23-28°C, and the oxygen concentration at 25-35%, and add daidzein after 5-7 hours of induction;

[0029] The third stage: control the temperature at 23-28℃ and ferment for at least 23-26 hours.

[0030] In one embodiment, the method comprises continuing to ferment glucose at a final concentration of 20 g / L at 30° C. and 150 rpm for 24 hours.

[0031] In one embodiment, the method is to inoculate the recombinant E. coli in LB medium and culture it for 8 to 12 hours as a seed solution, inoculate it in TB medium at an inoculum of 1% to 4%, and culture it at 35 to 37°C until the OD 600 =0.6~1.0, add IPTG with a final concentration of 0.1~0.5mM and induce by lowering the temperature for 8-12h, add daidzein at 22~28℃, and add glucose with a final concentration of 20g / L to continue fermentation for 12~48h.

[0032] In one embodiment, the method is to inoculate the E. coli seed solution into TB medium and culture at 37°C until the OD 600 =0.8, add IPTG with a final concentration of 0.1 mM and induce by lowering the temperature for 10 h, then add daidzein and glucose to the fermentation medium, and continue fermenting at 28-30°C and 120-180 rpm for at least 24 h.

[0033] In one embodiment, the seed solution is obtained by inoculating the Escherichia coli into LB medium and culturing for 10 hours.

[0034] The present invention also claims to protect the use of the Escherichia coli in producing products containing equol.

[0035] Beneficial effects:

[0036] (1) The present invention screened and obtained soybean aglycone reductase from Asaccharobacter celatus, and the conversion rate of the enzyme was as high as 58%.

[0037] (2) The present invention expresses the soybean aglycone reductase of Asaccharobacter celatus in Escherichia coli, and expresses Lg_DDRC, Si_DHDR, and Si_THDR, thereby realizing the synthesis of equol in Escherichia coli and providing a good basis for the synthesis of equol.

[0038] (3) The present invention knocks out the endogenous gene pfkA encoding 6-phosphofructokinase in Escherichia coli, NAD(P) +One or more genes in the transhydrogenase gene sthA and the N-acetyltransferase gene elaA of the GNAT family increased the production of equol from 91.9 mg / L to 110.6-224.1 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Diagram for the construction of the expression vector carrying daidzein reductase.

[0040] Figure 2 The chromatogram is that of whole-cell catalysis.

[0041] Figure 3 The mass spectrum of the conversion product dihydrodaidzein.

[0042] Figure 4 Comparison of yields for whole-cell catalysis to verify catalytic capacity.

[0043] Figure 5 This is the mass spectrum of equol.

[0044] Figure 6 This is a graph showing the shake flask level production of equol by the engineered strain under transformation conditions. DETAILED DESCRIPTION

[0045] (I) Culture medium

[0046] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 15g / L agar powder to prepare LB solid medium.

[0047] TB medium: peptone 12g / L, yeast powder 24g / L, 4mL glycerol, KH 2 PO 4 2.31g / L, K 2 HPO 4 12.54g / L.

[0048] Fermentation medium: peptone 12g / L, yeast powder 24g / L, glucose 5g / L, KH 2 PO 4 2.31g / L, K 2 HPO 4 12.54g / L.

[0049] Supplementary medium: D-glucose 500g / L, MgSO 4 7H 2 O 7.5g / L, yeast extract 10g / L.

[0050] 200g / L glucose mother solution: weigh 200g of anhydrous glucose and dissolve it in 500mL of deionized water. After it is completely dissolved, dilute to 1L.

[0051] 100mM DMSO stock solution of daidzein: weigh 100mmol daidzein and dissolve it in 500mL DMSO. After it is completely dissolved, add DMSO to make up to 1L.

[0052] 200mM DMSO stock solution of daidzein: weigh 200mmol daidzein and dissolve it in 500mL DMSO. After it is completely dissolved, add DMSO to make up to 1L.

[0053] (II) Preparation of chemical transformation competent cells of Escherichia coli:

[0054] Streak E. coli BL21 (DE3) on LB solid plates and culture for 12 hours. Pick a single colony and inoculate it into 5 mL of liquid LB medium and culture it for 8-10 hours. Inoculate it into 50 mL of LB medium at a 1.5-2.5% inoculum and culture it until OD 600 =0.6-1.0.

[0055] The competent state kit used was a high-efficiency competent state preparation kit from TAKRA. The competent state preparation process was performed according to its instructions.

[0056] (III) Preparation of competent cells for electroporation of E. coli:

[0057] Streak E. coli BL21 (DE3) on LB solid plates and culture for 12 hours. Pick a single colony and inoculate it into 5 mL of liquid LB medium and culture it for 8-10 hours. Inoculate it into 50 mL of LB medium (add arabinose at a final concentration of 1 mM for gene knockout) at a 2% inoculum and culture it until OD 600 =0.6-1.0. Cool the cells on ice for 30 min, collect the cells at 4000 rpm and 4°C, resuspend and wash the cells with 10% pre-cooled glycerol solution, repeat the washing once, resuspend the cells with 10% pre-cooled glycerol solution and dispense into sterile EP tubes to obtain E. coli electroporation competent cells.

[0058] (IV) Chemical transformation of Escherichia coli:

[0059] 1) Take the prepared competent cells and place them on ice for freeze-thaw, add the corresponding constructed expression vector, and place them on ice for 30 minutes.

[0060] 2) The competent cells containing the target vector were heat-shocked in a 42°C water bath for 90 seconds, and then incubated in a 37°C shaker for about 40 minutes.

[0061] 3) After low-speed centrifugation of the competent cells, remove part of the supernatant, aspirate the suspended cells, spread them on LB plates containing corresponding resistance, perform resistance screening, and perform colony PCR and sequencing to verify the recombinant transformants.

[0062] (V) Electrotransformation of Escherichia coli:

[0063] 1) Take the prepared competent cells and place them on ice for freeze-thaw, add the corresponding constructed vector or fragment, mix well, and place on ice for 5-15 minutes.

[0064] 2) Pipette the competent cell containing the target vector or fragment into a pre-cooled 0.2 mm electroporation cuvette and electroporate at 1.25 kV / mm, 25 μF, and 200 Ω.

[0065] 3) Immediately aspirate 1 mL of LB medium to resuspend the electroporated E. coli, transfer to a sterile EP tube, and culture at 37°C in a shaker for about 1-2 hours.

[0066] 4) After low-speed centrifugation of the competent cells, remove part of the supernatant, aspirate the suspended cells, spread them on LB plates containing corresponding resistance, perform resistance screening, and perform colony PCR and sequencing to verify the recombinant transformants.

[0067] (VI) HPLC determination of equol:

[0068] Shimadzu high performance liquid chromatography was used for detection, and the liquid detection conditions were as follows: CAPCELL PAK UG120 250 mm × 4.6 mm column (particle size 5 μm); mobile phase A, ultrapure water containing 1‰ formic acid; mobile phase B, methanol containing 1‰ formic acid; mobile phase ratio conditions, 0-2 min, 5% B, 2-13 min, 5-100% B, 13-15 min, 100% B, 15-18 min, 100-5% B, 18-20 min, 5% B; flow rate: 1 mL / min; column temperature: 40°C; injection volume: 10 μL; detector wavelength: 280 nm.

[0069] (VII) The strain information is shown in Table 1.

[0070] Table 1 Strains involved in the examples

[0071]

[0072]

[0073] Example 1 Single knockout and combined knockout of endogenous genes in Escherichia coli

[0074] In order to improve the ability of chassis cells to produce equol, the CRISPR / Cas9 system was used to knock out the relevant endogenous genes of E. coli BL21 (DE3). The plasmid pEcgRNA was amplified with primers sg-pfkA-F / sg-pfkA-R, sg-sthA-F / sg-sthA-R, and sg-elaA-F / sg-elaA-R, respectively, and transferred into E. coli BL21 (DE3) by heat shock method, and then spread on a resistance plate containing streptomycin to screen positive transformants. A single colony was picked and inoculated in 5 mL of liquid LB containing the corresponding resistance, and the plasmid was extracted to obtain plasmids pEcgRNA-pfkA, pEcgRNA-elaA, and pEcgRNA-sth A; primers pfkA-armup-F / pfkA-armup-R and pfkA-armdown-F / pfkA-armdown-R were used to amplify the gene pfkA to obtain the upper and lower homologous arms pfkA-armup and pfkA-armdown, and then pfkA-armup and pfkA-armdown were used as templates to perform fusion PCR with primers pfkA-armup-F / pfkA-armdown-R to obtain the fragment pfkA-arm; primers elaA- The gene elaA was amplified by armup-F / elaA-armup-R and elaA-armdown-F / elaA-armdown-R to obtain the upper and lower homologous arms elaA-armup and elaA-armdown, respectively. Then, using elaA-armup and elaA-armdown as templates, fusion PCR was performed with primers elaA-armup-F / elaA-armdown-R to obtain the fragment elaA-arm; the gene sthA was amplified by primers sthA-armup-F / sthA-armup-R and sthA-armdown-F / sthA-armdown-R to obtain the upper and lower homologous arms sthA-armup and sthA-armdown, respectively. Then, using sthA-armup and sthA-armdown as templates, fusion PCR was performed with primers sthA-armup-F / sthA-armdown-R to obtain the fragment sthA-arm; the PCR product sthA-arm was recovered by a kit and sequenced for verification.

[0075] 100 ng of pEcCas plasmid was transformed into chemical competent cells of Escherichia coli BL21 (DE3) to obtain strain BL21 (DE3)-Cas, and strain BL21 (DE3)-Cas was used to prepare electroporation competent cells. 100 ng of pEcgRNA-pfkA and 400 ng of pfkA-arm were electroporated into BL21 (DE3)-Cas competent cells to knock out the pfkA gene, and the plasmid pEcgRNA-pfkA was eliminated from the knockout strain to obtain strain BL21 (DE3) ΔpfkA-Cas, and the plasmid pCas plasmid was further eliminated to obtain strain EqC_No1.

[0076] Using the same strategy as above, 100 ng of pEcgRNA-sthA and 400 ng of sthA-arm were electroporated into the BL21(DE3)-Cas competent cell to knock out the sthA gene shown in Gene ID 948461. The plasmid pEcgRNA-sthA was eliminated from the knockout strain to obtain the strain BL21(DE3)ΔsthA-Cas. After further elimination of the plasmid pEcCas, the strain EqC_No2 was obtained.

[0077] Using the same strategy as above, 100 ng of pEcgRNA-elaA and 400 ng of elaA-arm were electroporated into the BL21 (DE3) -Cas competent cell to knock out the elaA gene shown in Gene ID 946750. After the plasmids pEcgRNA-elaA and pEcCas were eliminated from the knockout strain, strain EqC_No2-1 was obtained.

[0078] The primer sequences used are listed in Table 2.

[0079] Table 2 Primers and sequences

[0080]

[0081]

[0082] Example 2 Double knockout of endogenous genes in Escherichia coli

[0083] The same strategy as in Example 1 was followed, except that 100 ng of pEcgRNA-sthA and 400 ng of sthA-arm were electroporated into BL21 (DE3) ΔpfkA-Cas (EqC_No1 containing pCas plasmid) to knock out the sthA gene shown in Gene ID 948461, and the plasmid pEcgRNA-sthA was eliminated from the successfully knocked-out strain to obtain the strain BL21 (DE3) ΔpfkA ΔsthA-Cas. After further elimination of pEcCas, a strain with simultaneous knockout of pfkA and sthA was obtained, named EqC_No3.

[0084] The same strategy as above was used, except that 100 ng of pEcgRNA-elaA and 400 ng of elaA-arm were electroporated into BL21 (DE3) ΔpfkA-Cas (EqC_No1 containing pCas plasmid) to knock out the elaA gene shown in Gene ID 946750. After the plasmids pEcgRNA-elaA and pEcCas were eliminated from the knockout strain, the pfkA and elaA knockout strain was obtained and named EqC_No3-1.

[0085] The same strategy as above was used, except that 100 ng pEcgRNA-elaA and 400 ng elaA-arm were electroporated into BL21 (DE3) ΔsthA-Cas competent cells (EqC_No2 containing pCas plasmid) to knock out the elaA gene shown in Gene ID 948461. After the plasmids pEcgRNA-elaA and pEcCas were eliminated from the knockout strain, the elaA and sthA knockout strain was obtained and named EqC_No3-2.

[0086] Example 3 Triple knockout of endogenous genes in Escherichia coli

[0087] The same strategy as in Example 1 was followed, except that 100 ng of pEcgRNA-elaA and 400 ng of elaA-arm were electroporated into the BL21 (DE3) ΔpfkA ΔsthA-Cas competent medium to knock out the elaA gene shown in Gene ID 946750, and the plasmid pEcgRNA-elaA was eliminated from the successfully knocked-out strain to obtain the strain BL21 (DE3) ΔpfkA ΔsthA ΔelaA-Cas. After further eliminating pEcCas, a strain in which the three genes pfkA, sthA and elaA were simultaneously knocked out was obtained, and was named EqC_No4.

[0088] Example 4 Construction of plasmid expressing daidzein reductase and expression of enzyme

[0089] 1) Expression vector construction

[0090] The amino acid sequence of soybean aglycone reductase Si_DZNR from Slackia isoflavoniconvertens is shown in Genbank accession number WP_123220034.1. After codon optimization, the gene fragment encoding Si_DZNR was obtained and constructed in the multiple cloning site of pET28a(+) expression vector to obtain the recombinant plasmid pET28a(+)-1. The plasmid map is shown in Figure 1 As shown, the His tag and thrombin site were retained at the N-terminus and constructed by double enzyme digestion. Codon optimization, gene synthesis, and vector construction were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0091] The enzyme derived from Adlercreutzia celatus with an amino acid sequence as shown in SEQ ID NO.5 was codon optimized to obtain a gene fragment with a nucleotide sequence as shown in SEQ ID NO.1, and the gene fragment was constructed into a pET28a(+) expression vector using the same method as the recombinant plasmid pET28a(+)-1 to obtain a recombinant plasmid pET28a(+)-2.

[0092] According to the same strategy as above, the amino acid sequences from Adlercreutzia mucosicola (amino acid sequence as shown in Genbank accession number: WP_160344852.1), Sharpea porci (amino acid sequence as shown in Genbank accession number: WP_154515424.1), Sharpea azabuensis (amino acid sequence as shown in Genbank accession number: WP_074782075.1), Traorella massiliensis (amino acid sequence as shown in Genbank accession number: WP_071441835.1), Catenisphaera adipataccumulans (amino acid sequence as shown in Genbank accession number: WP_183327643.1), Clostridium saccharogumia (amino acid sequence as shown in Genbank accession number: WP_027090791.1), Intestinibaculum porci (amino acid sequence as shown in Genbank accession number: WP_179951177.1) and Holdemania massiliensis (amino acid sequence as shown in Genbank accession number: WP_020223358.1) were codon optimized and constructed into the pET28a(+) expression vector using the same strategy as the recombinant plasmid pET28a(+)-1 to obtain recombinant plasmids pET28a(+)-3 to pET28a(+)-10, respectively.

[0093] 2) Protein expression

[0094] The recombinant plasmids pET28a(+)-1 to pET28a(+)-10 constructed in step (1) were transformed into competent Escherichia coli cells, and the transformation solution was spread on an LB plate and cultured overnight at 37°C. The transformants were verified by colony PCR to obtain recombinant strains E. coli BL21(DE3) / pET28a(+)-1 to E. coli BL21(DE3) / pET28a(+)-10.

[0095] The verified transformants were transferred to 5 mL LB medium containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C to prepare seed solution. The cultured seed solution was transferred to 50 mL TB medium containing 50 mg / L kanamycin sulfate in a certain proportion to control the initial OD 600 =0.02~0.04, 37℃, 220rpm, culture until the bacterial concentration reaches OD 600=0.8, the temperature was lowered to 22-28°C, and isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.1 mM to induce expression for 8-12 h to obtain a bacterial solution.

[0096] Example 5 Whole cell catalysis to verify the catalytic ability of the enzyme

[0097] The bacterial suspensions of the recombinant strains E. coli BL21(DE3) / pET28a(+)-1 to E. coli BL21(DE3) / pET28a(+)-10 in Example 4 were centrifuged at 4000 rpm and 4°C to collect the bacterial cells, and the bacterial cells were washed twice with PBS solution, and the collected cells were resuspended in KPB (pH=8.0) solution, and the bacterial cell amount of the cell resuspended solution was controlled to be OD 600 =10, then, 100 mM daidzein DMSO stock solution with a volume fraction of 6‰ was added to the cell resuspension, mixed evenly, and placed under the reaction conditions of 25°C and 150 rpm for whole-cell catalysis.

[0098] The whole cell catalyzes daidzein to compare and verify the daidzein reductase activity of Si_DZNR with amino acid sequence as shown in Genbank accession number WP_123220034.1 and screened in Example 1, and samples were taken regularly for liquid phase detection of product generation and substrate consumption. The conversion rate (%) was calculated according to the following formula: the actual number of moles of dihydrodaidzein generated / theoretical number of moles of dihydrodaidzein generated by complete conversion × 100%.

[0099] The results are as follows Figure 2 to Figure 4 As shown, the enzymes derived from Adlercreutzia celatus, Adlercreutziamucosicola, Sharpea porci, Sharpea azabuensis, and Traorella massiliensis have daidzein reductase activity and can reduce daidzein to dihydrodaidzein. However, the enzymes derived from Catenisphaeraadipataccumulans, Clostridium saccharogumia, Intestinibaculum porci, and Holdemaniamassiliensis do not have the ability to catalyze the reduction of daidzein to dihydrodaidzein, and the conversion rate is 0.

[0100] The 10 enzymes screened in the example showed the maximum conversion rate after the substrate was added and reacted for 12 hours. Among them, the conversion rate of the enzyme with the amino acid sequence shown in SEQ ID NO.5 was 58%, which was about 3 times the conversion rate of Si_DZNR. The enzymes derived from Adlercreutzia mucosicola, Sharpea porci, Sharpea azabuensis, and Traorella massiliensis also had enzyme activity for catalyzing the reduction of daidzein to dihydrodaidzein, and the conversion rates were 6%, 3%, 8%, and 7%, respectively.

[0101] Example 6 Construction of an engineered strain of Escherichia coli for converting daidzein to produce equol

[0102] In order to construct an engineering strain for transforming soybean aglycone to synthesize equol, pCDFDuet-1 and pRSFDuet-1 plasmids were used as templates, and the other fragments of pCDFDuet-1 except the MCS1 site were amplified by primer pair pCDF-cz1-F / pCDF-cz1-R, which were named pcdf1; the DDRC encoding gene fragment ddrc shown in SEQ ID NO.2 was amplified by primer pair pCDF-DDRC-F / pCDF-DDRC-R; pcdf1 and ddrc were sequentially assembled by Gibson assembly, clones were verified by resistance screening, positive clones were picked out, plasmids were extracted and sent for sequencing, and the plasmid with correct sequencing was named pCDF-c. The primer pair pCDF-cz2-R / pCDF-cz2-F was used to amplify the other fragments of pCDF-c except the MCS2 site, which were named pcdf2; the primer pair pCDF-DZNR-F / pCDF-DZNR-R was used to amplify the coding gene fragment dznr of Ac_DZNR shown in SEQ ID NO.1; pcdf2 and dznr were assembled sequentially using Gibson assembly to obtain the plasmid pCDF-cz. According to the same strategy as above, for the construction of plasmid pRSFDuet-dt, the primer pair pRSF-dt1-F / pRSF-dt1-R was used to amplify the other fragments of pRSFDuet-1 except the MCS1 site, which were named pRSF1; the primer pair pRSF-DHDR-F / pRSF-DHDR-R was used to amplify the DHDR encoding gene fragment dhdr shown in SEQ ID NO.3; pRSF1 and dhdr were assembled sequentially using Gibson assembly, the clones were verified by resistance screening, the positive clones were picked out, the plasmids were extracted and sent for sequencing, and the plasmids with correct sequencing were named pRSF-d. The primer pair pRSF-dt2-R / pRSF-dt2-F was used to amplify the fragments of pRSF-d except the MCS2 site, which was named pRSF2; the primer pair pRSF-THDR-F / pRSF-THDR-R was used to amplify the coding gene fragment thdr of THDR as shown in SEQ ID NO.4; pRSF2 and thdr were assembled sequentially using Gibson assembly to obtain the plasmid pRSF-dt. Primers, gene synthesis, and sequencing were all completed by Shanghai Shenggong Biotechnology Co., Ltd. pCDF-dt and pET-cz were transformed into E.coli BL21 (DE3) competent cells to obtain strain WT_EqC. pCDF-dt and pET-cz were transferred to EqC_No1, EqC_No2, EqC_No3, and EqC_No4, respectively, and the obtained strains were named EqC_No5, EqC_No6, EqC_No7, and EqC_No8, respectively.

[0103] All primers and gene sequences are listed in Table 3.

[0104] Table 3 Primers and sequences

[0105]

[0106]

[0107] Example 7 Shake flask production of E. coli engineered strains for conversion of daidzein to equol

[0108] The strains WT_EqC, EqC_No5 to EqC_No8 constructed in Example 6 were streaked on LB plates containing streptomycin, ampicillin, and chloramphenicol and cultured overnight at 37°C for 12 h. The colonies were picked and inoculated in LB liquid containing streptomycin, ampicillin, and chloramphenicol, and cultured at 37°C, 220 rpm for 8-10 h as seed liquid. The inoculum was inoculated with 1.5-2.5% in 25 mL TB medium (containing streptomycin, ampicillin, and chloramphenicol) containing 5% (w / v) polyvinyl pyrrolidone (PVP40) and cultured for 1.5-3 h. When the bacterial OD 600 =0.8-1.0, add IPTG with a final concentration of 0.1mM, cool to 22-28°C, induce at 220rpm, after 10-12h of induction, add 10% 200g / L glucose mother solution, and add 5% 100mM daidzein mother solution, continue to culture at 25°C, 120-180rpm for 24h, and take samples for HPLC detection of equol content. Liquid phase analysis results show ( Figure 5-6 ) The equol production of the control strain WT_EqC was 53.2 mg / L; the production of each modified strain was EqC_No5: 91.9 mg / L, EqC_No6: 110.6 mg / L, EqC_No7: 224.1 mg / L, and EqC_No8: 201.6 mg / L.

[0109] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A gene knockout for 6-phosphofructokinase pkq , or knockout NAD(P) + Transhydrogenase gene sthA , or knockout gene pkq and genes sthA , or knockout gene pkq and genes sthA and genes eLA Application in increasing the production of equol in Escherichia coli; in, The Escherichia coli has been improved as follows: (1) Expressed Asaccharobacter celatus Soybean aglycone reductase Ac_ DZNR; (2) Expressed Lactococcus garvieae Dihydrodaidzein racemase Lg_ DDRC; (3) expressed Slackia isoflavoniconvertens Dihydrodaidzein reductase Si_ DHDR and Tetrahydrodaidzein Reductase Si_ THDR; The gene pkq The nucleotide sequence of is shown in Gene ID: 948412; the gene eLA The nucleotide sequence of is shown in Gene ID: 946750; the gene sthA The nucleotide sequence is shown in Gene ID: 948461.

2. A recombinant Escherichia coli for producing equol, It is characterized in that This includes knocking out any of the genes in (a) to (d) of the starting strain: (a) 6-phosphofructokinase gene pkq ; (b) NAD(P) + Transhydrogenase gene sthA ; (c) 6-phosphofructokinase gene pkq and NAD(P) + Transhydrogenase gene sthA ; (d) 6-phosphofructokinase gene pkq 、NAD(P) + Transhydrogenase gene sthA and GNAT family N-acetyltransferase genes eLA ; The following improvements were also made to the recombinant Escherichia coli: (1) Expressed Asaccharobacter celatus Soybean aglycone reductase Ac_ DZNR; (2) Expressed Lactococcus garvieae Dihydrodaidzein racemase Lg_ DDRC; (3) expressed Slackia isoflavoniconvertens Dihydrodaidzein reductase Si_ DHDR and Tetrahydrodaidzein Reductase Si_ THDR.

3. The recombinant Escherichia coli according to claim 2, It is characterized in that The gene pkq The nucleotide sequence of is shown in Gene ID: 948412; the gene eLA The Gene ID is 946750; the gene sthA The Gene ID is 948461.

4. The recombinant Escherichia coli according to claim 3, It is characterized in that The daidzein reductase Ac_ DZNR contains the amino acid sequence shown in SEQ ID NO.5; the dihydrodaidzein racemase Lg_ DDRC contains the amino acid sequence shown in SEQ ID NO.6; the dihydrodaidzein reductase Si_ DHDR contains the amino acid sequence shown in SEQ ID NO.7; the tetrahydrodaidzein reductase Si_ THDR contains the amino acid sequence shown in SEQ ID NO.

8.

5. The recombinant Escherichia coli according to any one of claims 2 to 4, It is characterized in that Escherichia coli BL21 (DE3) was used as the host.

6. A method for producing equol by converting daidzein into equol by microorganisms, It is characterized in that The recombinant Escherichia coli according to any one of claims 2 to 5 is used as the fermentation strain.

7. Use of the recombinant Escherichia coli according to any one of claims 2 to 5 or the method according to claim 6 in producing a product containing equol.

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