A recombinant Escherichia coli engineering strain for producing equol and its application

Abstract

The invention discloses a recombinant Escherichia coli engineering strain for producing equol and its application, and belongs to the technical field of genetic engineering and bioengineering. The invention knocks out one or more in the Escherichia coli genome, and expresses daidzein reductase Ac_DZNR from Asaccharobactercelatus, dihydrodaidzein racemase Lg_DDRC from Lactococcus garvieae, dihydrodaidzein reductase Si_DHDR and tetrahydrodaidzein reductase Si_THDR from Slackia isoflavoniconvertens to achieve the synthesis of equol. By constructing an NADPH regulating plasmid, introducing a soxR / soxS sensor system and a constitutive promoter to control the expression of related genes to enhance the production of NADPH, the ability of the strain to produce equol is improved, and the equol yield after fermentation for 66 hours reaches 1.9 g / L.

Description

Technical Field

[0001] The invention relates to a recombinant Escherichia coli engineering 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.

[0004] Ac_DZNR, a daidzein reductase from Asaccharobacter celatus, has been shown to have strong activity in reducing daidzein. At the same time, NADPH, as a cofactor required by the enzyme in the reaction pathway, affects the catalytic activity of the enzyme, and its supply is an important limiting factor in the conversion process. In addition, the combined optimization of gene expression and the improvement of daidzein solubility also play an important role in the production of equol. Summary of the invention

[0005] The invention provides a NADPH regulating plasmid for strengthening NADPH supply. The NADPH regulating plasmid contains NADH kinase gene pos5, NAD+ kinase gene nadK, citrate synthase gene gltA, glycerol-3-phosphate dehydrogenase gene gapA, phosphopyruvate hydratase gene eno, 6-phosphoglucose dehydrogenase gene zwf, and isocitrate dehydrogenase gene icd.

[0006] In one embodiment, the NADPH control plasmid is based on pACYCDuet-1 as the backbone. sen Initiation of NADH kinase gene pos5, NAD + The expression of kinase gene nadK was detected by P mdh The expression of the citrate synthase gene gltA, glycerol-3-phosphate dehydrogenase gene gapA, and phosphopyruvate hydratase gene eno was initiated; P rpmBG Initialize the expression of 6-phosphate glucose dehydrogenase gene zwf and isocitrate dehydrogenase gene icd.

[0007] In one embodiment, the NADPH control plasmid is based on pACYCDuet-1 as the backbone. sen Initiation of NADH kinase gene pos5, NAD + The expression of kinase gene nadK was detected by P mdh The expression of the citrate synthase gene gltA, glycerol-3-phosphate dehydrogenase gene gapA, and phosphopyruvate hydratase gene eno was initiated; P rpmBG Initiate the expression of 6-phosphate glucose dehydrogenase gene zwf and isocitrate dehydrogenase gene icd.

[0008] In one embodiment, the SD sequence GGTATATCTCCTT is added to the 5' end of the gene eno, gene gapA, gene icd, and gene nadK.

[0009] In one embodiment, the P sen The nucleotide sequence is shown in SEQ ID NO.9.

[0010] In one embodiment, the nucleotide sequence of the truncated form of gene pos5 is shown as SEQ ID NO.10.

[0011] In one embodiment, the Gene ID of the gene nadK is 947092; the Gene ID of the gene gltA is 945323; the Gene ID of the gene gapA is 947679; the Gene ID of the gene eno is 945032; the Gene ID of the gene zwf is 946370; and the Gene ID of the gene icd is 945702.

[0012] In one embodiment, the nucleotide sequence of the fragment CYCpart is shown as SEQ ID NO.11.

[0013] In one embodiment, the fragment P mdh The nucleotide sequence is shown in SEQ ID NO.12.

[0014] In one embodiment, the fragment P rpmBG The nucleotide sequence is shown in SEQ ID NO.13.

[0015] The present invention also provides the use of the NADPH regulating plasmid in constructing recombinant microbial cells.

[0016] In one embodiment, the application is to enhance metabolic reactions within microbial cells.

[0017] In one embodiment, the application includes but is not limited to introducing the NADPH regulatory plasmid into the microbial cell to increase the supply of coenzyme NADPH in the microbial cell and enhance the production capacity of metabolites that require the coenzyme NADPH to participate in production.

[0018] The present invention also provides recombinant microbial cells containing the NADPH regulating plasmid, including but not limited to bacterial cells or fungal cells.

[0019] The present invention also provides an engineered Escherichia coli bacteria with improved ability to transform daidzein to produce equol, wherein the engineered Escherichia coli bacteria contains the NADPH regulating plasmid and has at least one of the following improvements:

[0020] (1) The endogenous genes pfkA and NAD(P) encoding 6-phosphofructokinase in Escherichia coli were knocked out. + transhydrogenase gene sthA, one or more of the GNAT family N-acetyltransferase gene elaA;

[0021] (2) expressed the soybean aglycone reductase Ac_DZNR from Asaccharobacter celatus;

[0022] (3) expressed dihydrodaidzein racemase Lg_DDRC from Lactococcus garvieae;

[0023] (4) Dihydrodaidzein reductase Si_DHDR and tetrahydrodaidzein reductase Si_THDR from Slackia isoflavoniconvertens were expressed.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] In one embodiment, the Genbank accession number of the 6-phosphofructokinase is as shown in WP_014072359.1, and the NAD(P) + The Genbank accession number of the transhydrogenase is shown as NP_418397.2, and the Genbank accession number of the GNAT family N-acetyltransferase is shown as NP_416770.1.

[0030] 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.

[0031] The present invention also provides a product for preparing equol, wherein the product contains the NADPH regulating plasmid or the engineered Escherichia coli bacteria.

[0032] 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.

[0033] In one embodiment, the method further employs IPTG induction.

[0034] In one embodiment, the method comprises culturing the engineered Escherichia coli in a medium without daidzein for a period of time, inducing with IPTG, and then fermenting in a medium containing daidzein at 25-37° C. for at least 24 hours.

[0035] In one embodiment, the fermentation is carried out in stages, and the stages include:

[0036] Phase 1: Control the temperature at 35-37°C and the dissolved oxygen concentration at 35-45% to promote cell growth;

[0037] 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;

[0038] The third stage: control the temperature at 23-28℃ and ferment for at least 23-26 hours.

[0039] 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.

[0040] 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 at a final concentration of 0.1~0.5mM and induce by lowering the temperature for 8-12h, then add daidzein and glucose, and continue fermentation at 22~28℃ for at least 24h.

[0041] 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 24-48 h.

[0042] In one embodiment, the seed solution is obtained by inoculating the Escherichia coli into LB medium and culturing for 10 hours.

[0043] The present invention also claims to protect the use of the Escherichia coli in producing products containing equol.

[0044] Beneficial effects:

[0045] (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%;

[0046] (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;

[0047] (3) The present invention also increases the production of equol by knocking out genes related to the endogenous NADPH supply of Escherichia coli;

[0048] (4) The present invention provides a NADPH regulating plasmid, which improves the supply of NADPH in the engineered bacteria. Introducing the NADPH into the cells of the equol-producing strain can further increase the yield of equol. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Diagram for the construction of the expression vector carrying daidzein reductase.

[0050] Figure 2 Chromatogram of whole-cell catalysis.

[0051] Figure 3 The mass spectrum of the conversion product dihydrodaidzein.

[0052] Figure 4 Comparison of yields for whole-cell catalysis to verify catalytic capacity.

[0053] Figure 5 Plasmid construction map for the NADPH regulatory plasmid.

[0054] Figure 6 is the intracellular NADPH content of the constructed strain.

[0055] Figure 7 This is the mass spectrum of equol.

[0056] Figure 8 This is a graph showing the shake flask level production of equol by the engineered strain under transformation conditions.

[0057] Fig. 9 This is a diagram of the equol production in the fermentation tank of the engineered strain under transformation conditions. DETAILED DESCRIPTION

[0058] (I) Culture medium

[0059] LB medium: peptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L. Add 15g / L agar powder to prepare LB solid medium.

[0060] TB medium: peptone 12 g / L, yeast powder 24 g / L, glycerol 4 mL / L, KH2PO4 2.31 g / L, K2HPO4 12.54 g / L.

[0061] Fermentation medium: peptone 12g / L, yeast powder 24g / L, glucose 5g / L, KH2PO4 2.31g / L, K2HPO4 12.54g / L.

[0062] Supplement medium: D-glucose 500 g / L, MgSO4·7H2O 7.5 g / L, yeast extract 10 g / L.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] (II) Preparation of chemical transformation competent cells of Escherichia coli:

[0067] 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.

[0068] 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.

[0069] (III) Preparation of competent cells for electroporation of E. coli:

[0070] 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.

[0071] (IV) Chemical transformation of Escherichia coli:

[0072] 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.

[0073] 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.

[0074] 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.

[0075] (V) Electrotransformation of Escherichia coli:

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] (VI) HPLC determination of equol:

[0081] 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.

[0082] (VII) The strain information is shown in Table 1.

[0083] Table 1 Strains involved in the examples

[0084]

[0085] Example 1: Expression vector construction and expression

[0086] 1) Expression vector construction

[0087] 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 restriction digestion. Codon optimization, gene synthesis, and vector construction were all completed by Shanghai Sangon Biotechnology Co., Ltd.

[0088] 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.

[0089] 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.

[0090] 2) Protein expression

[0091] 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.

[0092] 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°C, 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.

[0093] Example 2: Whole-cell catalysis to verify the catalytic ability of the enzyme

[0094] The bacterial suspensions of the recombinant strains E. coli BL21(DE3) / pET28a(+)-1 to E. coli BL21(DE3) / pET28a(+)-10 in Example 1 were centrifuged at 4000 rpm and 4°C to collect the bacterial cells, and the bacterial cells were washed twice with PBS solution. 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.

[0095] 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%.

[0096] 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.

[0097] 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.

[0098] Example 3 Knockout of endogenous genes in Escherichia coli

[0099] 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 transformed 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-s thA; 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 The elaA gene was amplified with primers elaA-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 sthA gene was amplified with 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 products were recovered by a kit and verified by sequencing. 100 ng of pEcCas plasmid was transformed into chemically competent cells of Escherichia coli BL21 (DE3) to obtain strain BL21 (DE3)-Cas, and strain BL21 (DE3)-Cas was used to prepare electrocompetent cells.100ng pEcgRNA-pfkA and 400ng pfkA-arm were electroporated into BL21(DE3)-Cas competent medium to knock out the pfkA gene. The plasmid pEcgRNA-pfkA was eliminated from the knockout strain to obtain the strain BL21(DE3)ΔpfkA-Cas. The plasmid pCas was further eliminated to obtain the strain EqC_No1. Using the same strategy as above, 100ng pEcgRNA-sthA and 400ng sthA-arm were electroporated into BL21(DE3)-Cas competent medium to perform Gene ID The sthA gene was knocked out as shown in 948461, and the plasmid pEcgRNA-sthA was eliminated from the knockout strain to obtain the strain BL21(DE3)ΔsthA-Cas, and the plasmid pCas was further eliminated to obtain the strain EqC_No2; the electroporation competent medium was prepared in the same way, 100 ng pEcgRNA-sthA and 400 ng sthA-arm were electroporated into the BL21(DE3)ΔpfkA-Cas competent medium to perform double knockout of the pfkA and sthA genes, and the plasmid pEcgRNA-sthA was eliminated from the knockout strain to obtain the strain BL21(DE3)ΔpfkAΔsthA-Cas, and the plasmid pEcCas was further eliminated to obtain the strain EqC_No3. The same method was used to prepare the electroporation competent medium. 100 ng of pEcgRNA-elaA and 400 ng of elaA-arm were electroporated into the BL21(DE3)ΔpfkAΔsthA-Cas competent medium to perform triple knockout of the pfkA, sthA and elaA genes. After eliminating the plasmid pEcgRNA-elaA from the successfully knocked-out strain, the strain BL21(DE3)ΔpfkAΔsthAΔelaA-Cas was obtained, and the plasmid pEcCas was further eliminated to obtain the strain EqC_No4.

[0100] The primer sequences used are listed in Table 2.

[0101] Table 2 Primers and sequences

[0102]

[0103]

[0104] Example 4 Construction of an engineered strain of Escherichia coli for converting daidzein to produce equol

[0105] 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, 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 by Gibson assembly to obtain the plasmid pRSF-dt. Primers, gene synthesis, and sequencing were all completed by Shanghai Shenggong Biotechnology Co., Ltd. The plasmid pCDF-dt and plasmid pET-cz were constructed by the same method, and the plasmid pCDF-dt and plasmid pET-cz were transferred into E. coli BL21 (DE3) to obtain the strain WT_EqC containing only the pathway for the conversion of soybean aglycone into equol. Plasmid pCDF-dt and plasmid pET-cz were transferred into Ecq_No1 to Ecq_No4, respectively, and the obtained strains were named Ecq_No5, Ecq_No6, Ecq_No7, and Ecq_No8, respectively.

[0106] All primers and gene sequences are listed in Table 3.

[0107] Table 3 Primers and sequences

[0108]

[0109]

[0110] Example 5 Construction of NADPH-regulated plasmid

[0111] The eno genes shown in Gene ID 945032, gapA shown in Gene ID 947679, icd shown in Gene ID 945702, and nadK genes shown in Gene ID 947092 were amplified from the Escherichia coli genome using primer pairs eno-F / eno-R, gapA-F / gapA-R, icd-F / icd-R, and nadK-F / nadK-R, respectively, to add the SD sequence (GGTATATCTCCTT) to the 5' end of each gene to obtain fragments eno1, gapA1, icd1, and nadK1. The primer pairs eno-fwd / eno-rev, gapA-fwd / gapA-rev, icd-fwd / icd-rev, nadK-fwd / nadK-rev were used to amplify fragments eno1, gapA1, icd1, nadK1, and the restriction site BsaI (GGTCTCn^nnnn_) was added to the above fragments to obtain fragments eno2, gapA2, icd2, nadK2. The CYCpart element of plasmid pACYCDuet-1 (as shown in SEQ ID NO.11) was amplified with primers pACYC-fwd / pACYC-rev; the promoter P shown in SEQ ID NO.12 was amplified from the Escherichia coli genome with primers pmdh-fwd / pmdh-rev and primers prpmBG-fwd / prpmBG-rev. mdh and P as shown in SEQ ID NO.13 rpmBG ; The truncated form of pos5 gene pos5 (the signal peptide sequence was removed from the original pos5, and the nucleotide sequence is shown in SEQ ID NO.10) was amplified from the Saccharomyces cerevisiae genome using primers pos5-fwd / pos5-rev; the SoxR / SoxS sensor part on the Escherichia coli BL21 (DE3) genome was amplified using primers psen1-fwd / psen1-rev and primers psen2-fwd / psen2-rev to obtain fragment P sen(The nucleotide sequence is shown in SEQ ID NO.9); the fragment gltA was amplified from the genome of Escherichia coli BL21 (DE3) using primers gltA-fwd / gltA-rev; the fragment zwf was amplified from the genome of Escherichia coli BL21 (DE3) using primers zwf-fwd / zwf-rev; the above fragments were added with BsaI restriction sites at the corresponding positions. The obtained fragments were purified and recovered by the kit, and then assembled by Golden Gate sequence. The assembled fragments were transformed into Escherichia coli JM09, and positive clones were selected and sequenced to obtain the plasmid NADPH regulation plasmid ( Figure 5 ). The NADPH regulatory plasmid was introduced into E. coli BL21 (DE3), WT_EqC, EqC_No4, and Eqc_No8 competent cells, respectively, to obtain strains EqC_napdh, EqC_No9, EqC_No10, and EqC_No11, respectively. The plasmids pRSF-dt and pCDF-cz constructed in Example 3 were introduced into EqC_No10 together to obtain strain EqC_No12.

[0112] The primer sequences used are listed in Table 4.

[0113] Table 4 Primers and sequences

[0114]

[0115]

[0116] Example 6 Determination of intracellular NADPH in engineered strains

[0117] The NADPH / NADP+ detection kit (Cat. no. S0179, Bio-Tech, Shanghai, China) was used to determine the intracellular NADPH content of the starting bacteria E. coli BL21 (DE3), the engineered bacteria EqC_nadph, EqC_No1, EqC_No2, EqC_No3, EqC_No4 and EqC_No10. The above-mentioned engineered bacteria were cultured in LB medium at 37°C and 220rpm until the logarithmic phase, and the cells were collected to determine the intracellular NADPH content. The determination method was carried out according to the instructions of the kit. The results are shown in the figure. Figure 6 .

[0118] Example 7 Shake flask production of E. coli engineered strains for conversion of daidzein to equol

[0119] The strains WT_Eqc, Ecq_No5-EqC_No9, Ecq_No11-EqC_No12 constructed in Example 5 were streaked on LB plates containing streptomycin, ampicillin, and chloramphenicol and cultured overnight at 37°C for 12h. The colonies were picked and inoculated in LB liquid containing streptomycin, ampicillin, and chloramphenicol, and cultured at 37°C, 220rpm for 8-10h as seed liquid, and inoculated in 25mL TB medium (containing streptomycin, ampicillin, and chloramphenicol) containing 5% (w / v) polyvinyl pyrrolidone (PVP40) at an inoculum size of 1.5-2.5%, and cultured for 1.5-3h. 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 7 , Figure 8 ), the equol production of strain Ecq_No12 was 784.9 mg / L, and the production of other strains were Ecq_No5: 55.4 mg / L, Ecq_No6: 110.6 mg / L, Ecq_No7: 224.2 mg / L, Ecq_No8: 201.6 mg / L, Ecq_No9: 129.4 mg / L, and Ecq_No11: 539.1 mg / L.

[0120] Example 8 5L fermentation tank level production of Escherichia coli engineered strains for conversion of daidzein to equol

[0121] Seed culture: strain Ecq_No12 was cultured in LB medium at 37°C, 220rpm for 10h to prepare seed solution, and then transferred to a 5L fermenter containing 2.5L fermentation medium at a 4% inoculum. A mixture of D-glucose (500g / L), MgSO4-7H2O (7.5g / L), and yeast extract (10g / L) was used as a supplementary medium. When the glucose concentration was lower than 5g / L, feed was performed, and the concentration of D-glucose was maintained at 2-4g / L. The pH value was maintained at 7.0-8.0 using 5M NaOH solution. A three-stage fermentation scheme was adopted:

[0122] Phase 1: In the early stage of bacterial growth, the temperature was controlled at 37°C, the angular velocity (200-600 rpm) was used, and the dissolved oxygen concentration was set to 35-45% saturation;

[0123] The second stage: add the inducer with a final concentration of 0.1-0.2 mM, control the temperature at 22-28°C during the induction period, control the oxygen concentration at 25-35%, and add 150 mL of 200 mM daidzein mother solution at an average rate of 16 mL / h after 5-7 hours of induction;

[0124] Stage 3: After the addition of substrate, the temperature was controlled at 22-28°C and the reaction was carried out at a fixed speed of 200 rpm. Samples were taken at each time point for testing, such as Fig. 9 As shown, the yield after 33 hours of fermentation can reach more than 1 g / L, and the strain can produce 1.9 g / L (S)-equol after 66 hours of fermentation.

[0125] 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 NADPH regulating plasmid for enhancing NADPH supply, characterized in that, Psen was used to initiate the expression of NADH kinase gene pos5 and NAD+ kinase gene nadK; Pmdh was used to initiate the expression of citrate synthase gene gltA, glyceraldehyde-3-phosphate dehydrogenase gene gapA, and phosphopyruvate hydratase gene eno; and PrpmBG was used to initiate the expression of 6-phosphoglucose dehydrogenase gene zwf and isocitrate dehydrogenase gene icd.

2. The NADPH-regulated plasmid according to claim 1, characterized in that: The plasmid uses pACYCDuet-1 as the backbone.

3. Use of the NADPH-regulating plasmid according to claim 1 or 2 in constructing recombinant microbial cells.

4. A recombinant microbial cell containing the NADPH-regulating plasmid according to claim 1 or 2.

5. A recombinant Escherichia coli, characterized in that A method comprising: comprising: comprising: comprising: comprising: (1) The daidzein reductase Ac_DZNR from Asaccharobacter celatus was expressed; (2) expressed dihydrodaidzein racemase Lg_DDRC from Lactococcus garvieae; (3) expressed dihydrodaidzein reductase Si_DHDR and tetrahydrodaidzein reductase Si_THDR from Slackia isoflavoniconvertens; (4) Knockout of the endogenous 6-phosphofructokinase gene pfkA and NAD(P) + One or more of the transhydrogenase gene sthA and the GNAT family N-acetyltransferase gene elaA.

6. The recombinant Escherichia coli according to claim 5, characterized in that Escherichia coli BL21 (DE3) was used as the host.

7. A product for preparing equol, comprising the NADPH regulating plasmid according to claim 1 or 2, or the recombinant Escherichia coli according to claim 5 or 6.

8. A method for producing equol by converting daidzein into equol by microorganisms, characterized in that: The recombinant Escherichia coli according to claim 5 or 6 is used as a fermentation strain, and is first cultured in a medium without daidzein for a period of time, and then fermented in a medium containing daidzein.

9. The method according to claim 8, characterized in that The fermentation is carried out in stages, and the stages include: The first stage: control the temperature at 30-40°C and the dissolved oxygen concentration at 35-45% to promote cell growth; The second stage: add the inducer with a final concentration of 0.1-0.2 mM, control the temperature at 20-28°C, the dissolved oxygen concentration at 25-35%, and feed daidzein after 5-7 hours of induction; The third stage: control the temperature at 23-28℃ and ferment for at least 20-40 hours.

10. Use of the NADPH-regulating plasmid according to claim 1 or 2, or the recombinant Escherichia coli according to claim 5 or 6 in producing a product containing equol.

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