CN115747240A

CN115747240A - Recombinant escherichia coli engineering strain for producing equol and application

Info

Publication number: CN115747240A
Application number: CN202210935167.1A
Authority: CN
Inventors: 周景文; 邓汉宁; 张天萌; 张伟平; 刘云鹏; 徐沙; 曾伟主
Original assignee: Jiangsu Huaxiyineng Biotechnology Co ltd; Jiangnan University; Bloomage Biotech Co Ltd
Current assignee: Jiangsu Huaxiyineng Biotechnology Co ltd; Jiangnan University; Bloomage Biotech Co Ltd
Priority date: 2022-08-05
Filing date: 2022-08-05
Publication date: 2023-03-07

Abstract

The invention discloses a recombinant escherichia coli engineering strain for producing equol and application thereof, belonging to the technical field of genetic engineering and biological engineering. According to the invention, one or more of Escherichia coli genomes are knocked out, and the synthesis of equol is realized by expressing the daidzein reductase Ac _ DZNR derived from Asaccharobacteriaceus, the dihydrodaidzein racemase Lg _ DDRC derived from Lactococcus garvieae, the dihydrodaidzein reductase Si _ DHDR derived from Slackia isovoronovertens and the tetrahydrodaidzein reductase Si _ THDR. By constructing NADPH regulation plasmid, introducing a soxR/soxS sensor system and a constitutive promoter to control the expression of related genes so as to enhance the production of NADPH, the capacity of producing equol by the strain is improved, and the yield of the equol fermented for 66 hours reaches 1.9g/L.

Description

Recombinant escherichia coli engineering strain for producing equol and application

Technical Field

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 biological engineering.

Background

Isoflavone substances are important functional substances in leguminous plants, play an important role in the aspects of plant disease and insect pest resistance and the like, and have positive effects on human health. Daidzein is widely present in legumes such as soybean and can be easily extracted from the legumes. But the main effect of the equol on human body is reflected by the reduced form of the equol, the functionality of the equol is several times stronger than that of the daidzein, and the equol has many effects which are not possessed by the daidzein, so that the equol has great application potential in the fields of medicine, cosmetics and food industry.

Since equol is produced by converting daidzein by intestinal flora, it cannot be extracted from plants. At present, equol is mainly produced by a series of reduction, protection and deprotection reactions of daidzein by using a traditional chemical method. For the synthesis of chiral compounds, there are usually problems of high reaction energy consumption, high separation difficulty, expensive catalyst, etc., and often more by-products are produced, resulting in low yield of the target product. The problems can be well avoided by producing the equol by a microbial transformation mode, and the equol produced by the microbial transformation is S-type, which is exactly needed by people. Therefore, aiming at the limitations of the chemical synthesis of equol in the aspects of product stability, quality safety, price and the like, the microbial transformation mode which is green and safe and has a single product provides a feasible idea for the synthesis of equol.

The Asaccharobacter celetus-derived daidzein reductase Ac _ DZNR has been proved to have a strong activity of reducing daidzein. Meanwhile, NADPH, which is a cofactor required for enzymes in the reaction pathway, affects the catalytic activity of the enzymes, and its supply amount is an important limiting factor in the conversion process. In addition, combinatorial optimization of gene expression levels and improvement of daidzein solubility also play important roles in equol production.

Disclosure of Invention

The present invention provides an NADPH-regulated plasmid for enhancing the supply of NADPH, which contains an NADH kinase gene pos5, an NAD + kinase gene nadK, a citrate synthase gene gltA, a glycerol-3-phosphate dehydrogenase gene gapA, a phosphopyruvate hydratase gene eno, a 6-phosphoglucose dehydrogenase gene zwf, and an isocitrate dehydrogenase gene icd.

In one embodiment, the NADPH-modulating plasmid has pACYCDuet-1 as backbone and P as backbone _sen Initiation of NADH kinase Gene pos5, NAD ⁺ Expression of the kinase gene nadK; by P _mdh Initiating the expression of a citrate synthase gene gltA, a glycerol-3-phosphate dehydrogenase gene gapA, and a phosphopyruvate hydratase gene eno; by P _rpmBG The expression of the glucose-6-phosphate dehydrogenase gene zwf, the isocitrate dehydrogenase gene icd is initiated.

In one embodiment, the NADPH-modulating plasmid has pACYCDuet-1 as backbone and P as backbone _sen Initial NADH kinase Gene pos5、NAD ⁺ Expression of the kinase gene nadK; by P _mdh The expression of an initial citrate synthase gene gltA, a glycerol-3-phosphate dehydrogenase gene gapA and a phosphopyruvate hydratase gene eno; by P _rpmBG The expression of the glucose-6-phosphate dehydrogenase gene zwf, the isocitrate dehydrogenase gene icd is initiated.

In one embodiment, the 5' end of the gene eno, the gene gapA, the gene icd, and the gene nadK has added thereto the SD sequence GGTATATCTCCTT.

In one embodiment, said P _sen The nucleotide sequence of (A) is shown as SEQ ID NO. 9.

In one embodiment, the nucleotide sequence of the truncated form of gene pos5 is shown in SEQ ID No. 10.

In one embodiment, the Gene nadK has a Gene ID of 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; the Gene ID of the Gene icd is 945702.

In one embodiment, the nucleotide sequence of the fragment CYCpart is as shown in SEQ ID No. 11.

In one embodiment, the segment P _mdh The nucleotide sequence of (A) is shown in SEQ ID NO. 12.

In one embodiment, the segment P _rpmBG The nucleotide sequence of (A) is shown in SEQ ID NO. 13.

The invention also provides application of the NADPH regulation plasmid in constructing recombinant microbial cells.

In one embodiment, the use is for enhancing a metabolic response in a microbial cell.

In one embodiment, the application includes, but is not limited to, introducing the NADPH-modulating plasmid into the microbial cell to increase the supply of coenzyme NADPH in the microbial cell and enhance the production capacity of metabolites requiring coenzyme NADPH to participate in production.

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

The invention also provides an escherichia coli engineering bacterium with improved ability of converting daidzein to produce equol, wherein the escherichia coli engineering bacterium contains the NADPH regulation plasmid and is subjected to at least one of the following improvements:

(1) Knockout of endogenous 6-phosphofructokinase gene pfkA, NAD (P) of Escherichia coli ⁺ One or more of catalase gene sthA, GNAT family N-acetyltransferase gene elaA;

(2) Expresses the daidzein reductase Ac _ DZNR derived from Asaccharobacter celetus;

(3) Expresses Lactococcus garvieae derived dihydrodaidzein racemase Lg _ DDRC;

(4) The dihydrodaidzein reductase Si _ DHDR and the tetrahydrodaidzein reductase Si _ THDR which are derived from the Slackia isovanins are expressed.

In one embodiment, the amino acid sequence of daidzein reductase Ac _ DZNR is shown as SEQ ID NO.5, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

In one embodiment, the amino acid sequence of the dihydrodaidzein racemase Lg _ DDRC is shown in SEQ ID No.6, and the nucleotide sequence of the coding gene is shown in SEQ ID No. 2.

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 coding gene is shown as SEQ ID No. 3.

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 coding gene is shown as SEQ ID No. 4.

In one embodiment, pRSFDuet-1, pCDFDuet-1, pETDuet-1 are used as expression vectors to express the daidzein reductase Ac _ DZNR, dihydrodaidzein racemase Lg _ DDRC, dihydrodaidzein reductase Si _ DHDR, or tetrahydrodaidzein reductase Si _ THDR.

In one embodiment, the 6-phosphofructokinase has Genbank accession number WP-014072359.1, and the NAD (P) ⁺ Transfer of hydrogenThe Genbank accession number of the enzyme is shown as NP-418397.2, and the Genbank accession number of the GNAT family N-acetyltransferase is shown as NP-416770.1.

In one embodiment, the nucleotide sequence of the Gene pfkA is as defined in Gene ID: 948412; the nucleotide sequence of the Gene elaA is as defined in Gene ID: 946750; the nucleotide sequence of sthA Gene is as follows from Gene ID: 948461.

The invention also provides a product for preparing equol, wherein the product contains the NADPH regulatory plasmid or the engineering bacterium of the Escherichia coli.

The invention also provides a method for producing equol by microbial transformation of daidzein, which takes the escherichia coli as a fermentation strain and ferments for at least 24 hours at 25-37 ℃ in a culture medium containing daidzein.

In one embodiment, the method is further induced using IPTG.

In one embodiment, the method comprises culturing the engineering bacteria of Escherichia coli in a culture medium without daidzein for a period of time, inducing with IPTG, and fermenting in the culture medium with daidzein at 25-37 deg.C for at least 24h.

In one embodiment, the fermentation is a staged fermentation, the staging comprising:

the first stage is as follows: controlling the temperature to be 35-37 ℃ and the dissolved oxygen concentration to be 35-45% to promote the growth of cells;

and a second stage: adding inducer with final concentration of 0.1-0.2mM, controlling temperature at 23-28 deg.C and oxygen concentration at 25-35%, and adding daidzein after inducing for 5-7 hr;

and a third stage: controlling the temperature to be 23-28 ℃ and fermenting for at least 23-26h.

In one embodiment, the process is to continue the fermentation at 150rpm at 30 ℃ for 24h with 20g/L final glucose concentration.

In one embodiment, the method comprises the steps of inoculating the recombinant escherichia coli into an LB culture medium to be cultured for 8-12h as a seed solution, inoculating the recombinant escherichia coli into a TB culture medium with the inoculation amount of 1% -4%, and culturing the recombinant escherichia coli to OD at 35-37 DEG C ₆₀₀ 0.1 to 0.5 percent of addition of 0.6 to 1.0Cooling and inducing for 8-12h by using IPTG with mM final concentration, supplementing daidzein and glucose, and continuously fermenting for at least 24h at 22-28 ℃.

In one embodiment, the method comprises inoculating the E.coli seed solution into TB medium, and culturing at 37 deg.C to OD ₆₀₀ =0.8, adding IPTG with final concentration of 0.1mM, cooling and inducing for 10h, supplementing daidzein and glucose into the fermentation medium, and continuing fermenting at 28-30 ℃ and 120-180rpm for 24-48 h.

In one embodiment, the seed solution is obtained by inoculating the escherichia coli into LB medium and culturing for 10 h.

The invention also claims the application of the escherichia coli in the production of equol-containing products.

Has the advantages that:

(1) The invention obtains the daidzein reductase from Asaccharobacter celetus by screening, and the conversion rate of the daidzein reductase reaches up to 58 percent;

(2) The daidzein reductase of Asaccharobacter celetus is expressed in escherichia coli, and Lg _ DDRC, si _ DHDR and Si _ THDR are expressed, so that the synthesis of equol in the escherichia coli is realized, and a good basis is provided for the synthesis of equol;

(3) The invention also improves the yield of equol by knocking out genes related to the endogenous NADPH supply of the escherichia coli;

(4) The invention provides an NADPH regulation plasmid, which improves the supply of NADPH in engineering bacteria cells, and can further improve the yield of equol by introducing the NADPH into equol production strain cells.

Drawings

FIG. 1 is a construction diagram of an expression vector carrying daidzein reductase.

FIG. 2 is a chromatogram of whole cell catalysis.

FIG. 3 is a mass spectrum of the converted product dihydrodaidzein.

FIG. 4 is a graph comparing the yields of whole-cell catalytic validation of catalytic ability.

FIG. 5 is a diagram of construction of NADPH-regulated plasmid plasmids.

FIG. 6 shows intracellular NADPH content of the constructed strain.

Figure 7 is a mass spectrum histogram of equol.

FIG. 8 is a graph of the horizontal shake flask yield of equol under the transformation conditions of the engineering strains.

FIG. 9 is a graph of equol fermentor yield for the engineered strain under transformation conditions.

Detailed Description

(I) culture Medium

LB culture medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. 15g/L agar powder was added to prepare an LB solid medium.

TB culture medium: peptone 12g/L, yeast powder 24g/L, glycerin 4mL/L, KH ₂ PO ₄ 2.31g/L、K ₂ HPO ₄ 12.54g/L。

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。

Supplementing a culture medium: d-glucose 500g/L, mgSO ₄ ·7H ₂ O7.5 g/L and yeast extract 10g/L.

200g/L glucose mother liquor: 200g of anhydrous glucose is weighed and dissolved in 500mL of deionized water, and the volume is adjusted to 1L after the anhydrous glucose is completely dissolved.

DMSO stock solution of 100mM daidzein: weighing 100mmoL daidzein, dissolving in 500mL DMSO, and diluting to 1L with DMSO after completely dissolving.

200mM daidzein in DMSO stock: weighing 200mmoL daidzein, dissolving in 500mL DMSO, and diluting to 1L with DMSO after completely dissolving.

(II) preparing chemical transformation competence of Escherichia coli:

coli BL21 (DE 3) was streaked on LB solid plates for 12h. Selecting single colony, inoculating in 5mL liquid LB culture medium, culturing for 8-10 hr, inoculating in 50mL LB culture medium at 1.5-2.5% inoculum size, and culturing to OD ₆₀₀ ＝0.6-1.0。

The competence kit adopts a high-efficiency competence preparation kit of TAKRA company. The competent preparation was carried out according to the instructions.

(III) preparing the electrotransformation competence of the escherichia coli:

coli BL21 (DE 3) was streaked on LB solid plates for 12h. Single colonies were picked and inoculated into 5mL liquid LB medium for 8-10h, inoculated into 50mL LB medium at 2% (for gene knockout, 1mM arabinose was added to the final concentration), and cultured to OD ₆₀₀ And (5) =0.6-1.0. Cooling the thalli on ice for 30min,4000rpm, collecting the thalli at 4 ℃, re-suspending and washing the thalli by using a 10% precooled glycerol solution, repeatedly washing once, re-suspending the thalli by using a 10% precooled glycerol solution, and subpackaging the thalli into a sterile EP tube to obtain the electro-transformation competence of the escherichia coli.

(IV) chemical transformation of E.coli:

1) And (3) placing the prepared competent cells on ice for freeze thawing, adding the correspondingly constructed expression vector, and placing on ice for 30min.

2) And (3) thermally heating the competence containing the target vector in a water bath kettle at 42 ℃ for 90s, and then placing the competence in a shaking table at 37 ℃ for about 40 min.

3) And (3) removing part of supernatant after low-speed centrifugation, blowing and sucking suspension cells, coating the suspension cells on an LB (Langmuir-Blodgett) plate containing corresponding resistance, carrying out resistance screening, and carrying out colony PCR (polymerase chain reaction) and sequencing verification on recombinant transformants.

(V) electrotransformation of Escherichia coli:

1) And (3) placing the prepared competent cells on ice for freezing and thawing, adding the correspondingly constructed vector or fragment, uniformly mixing, and placing on ice for 5-15min.

2) The competence containing the target vector or fragment was pipetted into a pre-cooled 0.2mm cuvette and electrically stimulated at 1.25kV/mm, 25. Mu.F, 200. Omega.

3) Immediately sucking 1mL LB culture medium to resuspend the electro-excited Escherichia coli, transferring to a sterile EP tube, placing in a shaker at 37 ℃ and culturing for about 1-2 h.

4) And (3) removing part of supernatant after low-speed centrifugation, blowing and sucking suspension cells, coating the suspension cells on an LB (Langmuir-Blodgett) plate containing corresponding resistance, carrying out resistance screening, and carrying out colony PCR (polymerase chain reaction) and sequencing verification on recombinant transformants.

(VI) HPLC determination of equol:

detecting by using Shimadzu high performance liquid chromatography under the following liquid phase detection conditions: large Cao chromatographic column CAPCELL PAK UG120 mm × 4.6mm column (particulate size 5 μm); mobile phase A, ultrapure water containing 1 ‰ formic acid; mobile phase B, methanol containing 1% formic acid; mobile phase conditions, 0-2min,5% B,2-13min,5-100% B,13-15min,100% B,15-18min,100-5% B,18-20min,5% B; flow rate: 1mL/min; column temperature: 40 ℃; sample injection amount: 10 mu L of the solution; detector wavelength: 280nm.

(VII) Strain information is shown in Table 1.

TABLE 1 strains referred to in the examples

Example 1: expression vector construction and expression

1) Expression vector construction

The amino acid sequence of daidzein reductase Si _ DZNR derived from Slackia isovaninens is shown as Genbank accession number WP _123220034.1, a gene fragment for coding Si _ DZNR is obtained after codon optimization, and is constructed at a multiple cloning site of a pET28a (+) expression vector to obtain a recombinant plasmid pET28a (+) -1, the plasmid map is shown as figure 1, and the N end of the recombinant plasmid retains a His tag and a thrombin site and is constructed in a double enzyme digestion mode. Codon optimization, gene synthesis and vector construction are all completed by Shanghai Bioengineering Co., ltd.

A gene fragment with a nucleotide sequence shown as SEQ ID NO.1 is obtained by codon optimization of an enzyme with an amino acid sequence shown as SEQ ID NO.5 derived from Adlercutzia celetus, and is constructed into a pET28a (+) expression vector by adopting the same method as that of the recombinant plasmid pET28a (+) -1, so as to obtain the recombinant plasmid pET28a (+) -2.

According to the same strategy as described above, recombinant plasmids derived from Adlercreutzia mucositicola (amino acid sequence shown in Genbank accession No.: WP _ 160344852.1), sharpea poracus (amino acid sequence shown in Genbank accession No.: WP _ 154515424.1), sharpea azabunensis (amino acid sequence shown in Genbank accession No.: WP _ 0782075.1), trorella maliensis (amino acid sequence shown in Genbank accession No.: WP _ 071183445.1), catenisophera adipaculans (amino acid sequence shown in Genbank accession No.: WP _ 327643.1), clostridium saccharologlia (amino acid sequence shown in Genbank accession No.: WP _ 027090791.1), pEinibanum pora (amino acid sequence shown in Genbank accession No.: (+) -179957.1), and Holdria plasmid pE t-28 (amino acid sequence shown in Genbank accession No. (+) -9.7.1) were constructed using the same plasmid, and pE plasmid pE-copy T-28 (amino acid sequence shown in Genbank accession No. (+) -11-179957.1, (+) -plasmid T- (+) -codon-28. 1).

2) Protein expression

And (2) respectively transforming the recombinant plasmids pET28a (+) -1-pET 28a (+) -10 constructed in the step (1) into escherichia coli competent cells, coating the transformation solution on an LB (Luria Brookfield) flat plate, culturing overnight at 37 ℃, and verifying transformants by using colony PCR (polymerase chain reaction) to obtain recombinant strains E.coli BL21 (DE 3)/pET 28a (+) -1-E.coli BL21 (DE 3)/pET 28a (+) -10.

The transformants confirmed to be correct were transferred to 5mL of LB medium containing 50mg/L kanamycin sulfate and cultured overnight at 37 ℃ to prepare a seed solution. Transferring the cultured seed solution into 50mL TB medium containing 50mg/L kanamycin sulfate at a certain ratio, and controlling initial OD ₆₀₀ Culturing at 37 deg.C and 220rpm until the thallus concentration reaches OD =0.02-0.04 ₆₀₀ After the concentration is 0.8, the temperature is reduced to 22-28 ℃, isopropyl-beta-D-thiogalactoside (IPTG) is added to the final concentration of 0.1mM, and induction expression is carried out for 8-12h, thus obtaining a bacterial solution.

Example 2: whole-cell catalytic verification of catalytic capability of enzyme

The bacterial liquid of the recombinant strains E.coli BL21 (DE 3)/pET 28a (+) -1-E.coli BL21 (DE 3)/pET 28a (+) -10 in example 1 was centrifuged at 4000rpm at 4 ℃ to collect the bacterial cells, the bacterial cells were washed with PBS solution for 2 times, the collected cells were resuspended in KPB (pH = 8.0) solution, and the bacterial amount of the cell resuspension was controlled to OD ₆₀₀ =10, then, 100mM daidzein DMSO stock solution with a volume fraction of 6 ‰ is added to the cell resuspension, and after mixing well, whole-cell catalysis is performed under reaction conditions of 25 ℃ and 150 rpm.

The amino acid sequences such as Si _ DZNR shown in Genbank accession No. WP _123220034.1 and the activity of the daidzein reductase screened in example 1 were compared and verified by whole-cell catalysis of daidzein, and samples were taken periodically for the formation of liquid phase assay products and substrate consumption. The conversion (%) was calculated as follows: the number of moles of the dihydrodaidzein actually produced/the number of moles of dihydrodaidzein theoretically produced by complete conversion is 100%.

As shown in FIGS. 2 to 4, the enzymes derived from Adlercutzia cellus, adlercutzia mucosiocola, sharpea porci, sharpea azabunsis and Trorella massiviensis have daidzein reductase activity, and can reduce daidzein to dihydrodaidzein. While enzymes derived from Caterpillar adipaccumulans, clostridium saccharogumia, intestinibulum porci, and Holdemania massilisensis do not have the ability to catalyze the reduction of daidzein to dihydrodaidzein, and the conversion rate is 0.

The 10 enzymes selected in the examples showed the maximum conversion rate after the reaction for 12 hours by adding the substrate, wherein the conversion rate of the enzyme represented by the amino acid sequence of SEQ ID NO.5 was 58% which was about 3 times the conversion rate of Si-DZNR, and the enzymes derived from Adlercreutzia mucositicola, sharpea porci, sharpea azabunsis and Trorella masseinsis also had the enzyme activity of catalyzing the reduction of daidzein to dihydrodaidzein, and the conversion rates were 6%, 3%, 8% and 7%, respectively.

Example 3 knockout of endogenous genes of E.coli

In order to improve the ability of the underpan cells to produce equol, the E.coli BL21 (DE 3) -associated endogenous gene is knocked out by using a CRISPR/Cas9 system. Amplifying plasmids pEcgRNA by using primers sg-pfkA-F/sg-pfkA-R, sg-sthA-F/sg-sthA-R and sg-elaA-F/sg-elaA-R respectively, transferring the plasmids pEcgRNA into escherichia coli BL21 (DE 3) by using a heat shock method, coating resistance plates containing streptomycin to screen positive transformants, selecting a single colony, inoculating the single colony into 5mL of liquid LB containing corresponding resistance, and extracting plasmids to obtain the plasmids pEcgRNA-pfkA, pEcgRNA-elaA and pEcgRNA-sthA; amplifying genes pfkA respectively by using primers pfkA-armup-F/pfkA-armup-R and pfkA-armdown-F/pfkA-armdown-R to obtain upper and lower homologous arms pfkA-armup and pfkA-armdown, then performing fusion PCR by using the primers pfkA-armup and pfkA-armdown as templates, and obtaining a fragment pfkA-arm; respectively amplifying genes elaA by using primers elaA-armup-F/elaA-armup-R and elaA-armdown-F/elaA-armdown-R to obtain upper and lower homologous arms elaA-armup and elaA-armdown, and then carrying out fusion PCR by using the primers elaA-armup and elaA-armdown as templates and using the primers elaA-armup-F/elaA-armdown-R to obtain a fragment elaA-arm; amplifying the gene sthA with primers sthA-armup-F/sthA-armup-R and sthA-armdown-F/sthA-armdown-R to obtain upper and lower homologous arms sthA-armup and sthA-armdown, and performing fusion PCR with primers sthA-armup-F/sthA-armdown-R to obtain a fragment sthA-arm; PCR products were recovered by kit and verified by sequencing. 100ng of pEcCas plasmid is transformed into chemically competent cells of escherichia coli BL21 (DE 3) to obtain a strain BL21 (DE 3) -Cas, and the strain BL21 (DE 3) -Cas is used for preparing electric transformation competence. Electrically transferring 100ng of pEcgRNA-pfkA and 400ng of pfkA-arm into BL21 (DE 3) -Cas electrotransfer competence to knock out the pfkA gene, eliminating plasmid pEcgRNA-pfkA from the successfully knocked-out strain to obtain a strain BL21 (DE 3) delta pfkA-Cas, further eliminating plasmid pCas, and obtaining a strain EqC _ No1; according to the same strategy, 100ng of pEcgRNA-sthA and 400ng of sthA-arm are electrically transferred into BL21 (DE 3) -Cas competence for the knock-out of sthA Gene shown by Gene ID 948461, the successfully knocked-out strain is subjected to plasmid pEcgRNA-sthA elimination to obtain a strain BL21 (DE 3) delta sthA-Cas, and the plasmid pCas is further eliminated to obtain a strain EqC _ No2; preparing electrotransformation competence by the same method, electrotransforming 100ng pEcgRNA-sthA and 400ng sthA-arm into BL21 (DE 3) delta pfkA-Cas competence to perform double knockout of pfkA and sthA genes, eliminating plasmid pEcgRNA-sthA from successfully knocked-out strains to obtain strains BL21 (DE 3) delta pfkA delta sthA-Cas, further eliminating plasmid pEcgAS to obtain strains EqC _ No3. Preparing electrotransformation competence by the same method, electrically transferring 100ng of pEcgRNA-ela A and 400ng of elaA-arm into BL21 (DE 3) delta pfkA delta sthA-Cas competence, carrying out three knockouts of pfkA, sthA and elaA genes, eliminating plasmid pEcgRNA-ela from successfully knocked-out strains to obtain strains BL21 (DE 3) delta pfkA delta sthA delta ela-Cas, further eliminating plasmid pEcgAS to obtain strains EqC _ No4.

The primer sequences used are listed in table 2.

TABLE 2 primers and sequences

EXAMPLE 4 construction of E.coli engineered Strain for conversion of daidzein to produce equol

In order to construct an engineering strain for transforming daidzein to synthesize equol, a primer pair pCDFDuet-1 and pRSFDuet-1 plasmid is used as a template to amplify other fragments of the pCDFDuet-1 except the MCS1 site by using the pCDF-cz1-F/pCDF-cz1-R, and the fragment is named as pCDF1; amplifying a DDRC encoding gene fragment DDRC shown in SEQ ID NO.2 by using a primer pair pCDF-DDRC-F/pCDF-DDRC-R; and sequentially assembling pCDF1 and ddrc by utilizing Gibson assembly, verifying clones by resistance screening, picking positive clones, extracting plasmids, sequencing, and naming the correctly sequenced plasmids as pCDF-c. Amplifying the other fragment of the pCDF-c except the MCS2 site by using a primer pair pCDF-cz2-R/pCDF-cz2-F, and naming the fragment as pCDF2; amplifying an encoding gene fragment DZNR of Ac _ DZNR shown in SEQ ID NO.1 by using a primer pair pCDF-DZNR-F/pCDF-DZNR-R; and assembling pCDF2 and dznr sequentially by utilizing Gibson assembly to obtain the plasmid pCDF-cz. According to the same strategy as above, for the construction of plasmid pRSFDuet-dt, a fragment of pRSFDuet-1 except the MCS1 site was amplified with the primer pair pRSF-dt1-F/pRSF-dt1-R and named pRSF1; amplifying a DHDR encoding gene fragment DHDR shown in SEQ ID NO.3 by using a primer pair pRSF-DHDR-F/pRSF-DHDR-R; and (3) sequentially assembling pRSF1 and dhdr by utilizing Gibson assembly, verifying clones by resistance screening, picking positive clones, extracting plasmids, sequencing, and naming the correctly sequenced plasmids as pRSF-d. Amplifying the other fragment of pRSF-d except the MCS2 site by using a primer pair pRSF-dt2-R/pRSF-dt2-F, and naming the fragment as pRSF2; amplifying a THdr encoding gene segment of the THDR shown in SEQ ID NO.4 by using a primer pair pRSF-THDR-F/pRSF-THDR-R; and (3) sequentially assembling pRSF2 and thdr by utilizing Gibson assembly to obtain the plasmid pRSF-dt. The primer, gene synthesis and sequencing are all completed by Shanghai Bioengineering Co., ltd. Plasmid pCDF-dt and plasmid pET-cz were constructed in the same manner, and the plasmid pCDF-dt and plasmid pET-cz were transferred to E.coli BL21 (DE 3), to obtain a strain WT _ EqC containing only daidzein for conversion into equol. The plasmids pCDF-dt and pET-cz were transformed into Ecq _ No1 to Ecq _ No4, and the resulting strains were named Ecq _ No5, ecq _ No6, ecq _ No7, and Ecq _ No8, respectively.

All primers and gene sequences are listed in table 3.

TABLE 3 primers and sequences

Example 5 construction of NADPH-regulated plasmids

The primer pairs eno-F/eno-R, gapA-F/gapA-R, icd-F/icd-R and nadK-F/nadK-R are used to amplify eno shown in Gene ID 945032, gapA shown in Gene ID 947679, icd shown in Gene ID 945702 and nadK Gene shown in Gene ID 947092 from the genome of E.coli to add SD sequences (GGTATATCTCCTT) at the 5' end of each Gene to obtain fragments eno1, gapA1, icd1 and nadK1. Fragments eno1, gapA1, icd1 and nadK1 are amplified respectively by primer pairs eno-fwd/eno-rev, gapA-fwd/gapA-rev, icd-fwd/icd-rev and nadK-fwd/nadK-rev, and enzyme cutting sites BsaI (GGTCTCTCn ^ nnn \) are added on the basis of the fragments to obtain fragments eno2, gapA2, icd2 and nadK2. Amplifying the CYCpart element of plasmid pACYCDuet-1 (shown as SEQ ID NO. 11) with primers pACYC-fwd/pACYC-rev; amplification of the promoter P shown in SEQ ID NO.12 from the E.coli genome with the primers pmdh-fwd/pmdh-rev and the primers prpBG-fwd/prpBG-rev _mdh And P as shown in SEQ ID NO.13 _rpmBG (ii) a Amplifying a truncated form pos5 of the pos5 gene from a saccharomyces cerevisiae genome by using a primer pos5-fwd/pos5-rev (a signal peptide sequence is removed on the basis of the original pos5, and a nucleotide sequence is shown as SEQ ID NO. 10); with primer psen1-fwd/psen1-rev and primer psen2-fwdThe SoxR/SoxS sensor part on the genome of Escherichia coli BL21 (DE 3) is amplified by/psen 2-rev to obtain a fragment P _sen (the nucleotide sequence is shown as SEQ ID NO. 9); amplifying a fragment gltA from the genome of the Escherichia coli BL21 (DE 3) by using a primer gltA-fwd/gltA-rev; amplifying a segment zwf from a genome of escherichia coli BL21 (DE 3) by using a primer zwf-fwd/zwf-rev; bsaI restriction sites were added to the above fragments at the corresponding positions. And purifying and recovering the obtained fragments by using a kit, sequentially assembling by using Golden Gate, transforming the assembled fragments into escherichia coli JM09, selecting positive clones, and performing sequencing verification to obtain the plasmid NADPH regulatory plasmid (shown in figure 5). NADPH-regulated plasmids were introduced into e.coli BL21 (DE 3), WT _ EqC, eqC _ No4, and EqC _ No8 competent cells, respectively, to obtain strains EqC _ napdh, eqC _ No9, eqC _ No10, and EqC _ No11, respectively. The plasmid pRSF-dt and pCDF-cz constructed in example 3 were introduced into EqC _ No10 together to obtain a strain EqC _ No12.

The primer sequences used are listed in table 4.

TABLE 4 primers and sequences

Example 6 intracellular NADPH assay of engineered strains

Intracellular NADPH contents of spawn running bacteria E.coli BL21 (DE 3), engineering bacteria EqC _ NADPH, eqC _ No1, eqC _ No2, eqC _ No3, eqC _ No4 and EqC _ No10 are measured by using an NADPH/NADP + detection kit (Cat. No. S0179, biyun sky, china, shanghai). The engineering bacteria are cultured in LB culture medium at 37 deg.C and 220rpm to logarithmic phase, the bacteria are collected to determine intracellular NADPH content, the determination method is carried out according to the kit instructions, and the results are shown in FIG. 6.

EXAMPLE 7 Shake flask level production of E.coli engineered strains transformed for daidzein production of equol

The strains WT _ Eqc and Ecq constructed in example 5 were separately culturedThe _No5-EqC _ No9 and the Ecq _ No 11-EqC _ No12 are streaked on LB plates containing streptomycin, ampicillin and chloramphenicol and cultured overnight at 37 ℃ for 12h, colonies are picked and inoculated in LB liquid containing streptomycin, ampicillin and chloramphenicol and cultured at 37 ℃ and 220rpm for 8-10h to serve as seed liquid, the inoculation amount of 1.5-2.5% is inoculated in 25mL TB culture medium (containing streptomycin, ampicillin and chloramphenicol) containing 5% (w/v) polyvinylpyrrolidone (PVP 40), the culture is carried out for 1.5-3h, and the OD of the thalli is obtained ₆₀₀ If the concentration is 0.8-1.0, adding IPTG with 0.1mM final concentration, cooling to 22-28 deg.C, inducing at 220rpm for 10-12h, adding 10% of glucose mother liquor with concentration of 200g/L, adding 5% of 100mM daidzein mother liquor, culturing at 25 deg.C and 120-180rpm for 24h, sampling, and detecting the equol content by HPLC. The analysis of the results of the liquid phase showed (FIGS. 7 and 8) that the yield of equol was 784.9mg/L for strain Ecq-No 12, and that the yields for the remaining strains were Ecq-No 5:55.4mg/L, ecq-No 6:110.6mg/L, ecq-No 7:224.2mg/L, ecq-No 8:201.6mg/L, ecq-No 9:129.4mg/L, ecq-No 11:539.1mg/L.

EXAMPLE 8 transformation of daidzein for Eschericol production engineering Strain production in 5L fermentor

Seed culture: the strain Ecq _ No12 was cultured in LB medium at 37 ℃ for 10 hours at 220rpm to prepare a seed solution, which was then transferred to a 5L fermentor containing 2.5L of fermentation medium at an inoculum size of 4%. Using D-glucose (500 g/L), mgSO ₄ -7H ₂ O (7.5 g/L) and yeast extract (10 g/L) were added to the medium at a glucose concentration of less than 5g/L to maintain the D-glucose concentration at 2-4 g/L and the pH at 7.0-8.0 with 5M NaOH. A three-stage fermentation protocol was employed:

the first stage is as follows: in the early stage of bacterial growth, the temperature is controlled at 37 ℃, the angular speed (200-600 rpm) is adopted, and the dissolved oxygen concentration is set to be 35-45% of saturation;

and a second stage: adding inducer with final concentration of 0.1-0.2mM, controlling temperature at 22-28 deg.C and oxygen concentration at 25-35% during induction period, and adding 150mL 200mM soybean aglycone mother liquor at average speed of 16mL/h after inducing for 5-7 h;

and a third stage: after the addition of the substrate was completed, the reaction was carried out at a fixed speed of 200rpm while controlling the temperature at 22 to 28 ℃. When samples were taken at each time point for testing, as shown in FIG. 9, the yield of the strain can reach more than 1g/L after fermentation for 33 hours, and the strain can produce 1.9g/L (S) -equol after fermentation for 66 hours.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. An NADPH-regulated plasmid for enhancing the supply of NADPH, which 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.

2. The NADPH-regulating plasmid according to claim 1, wherein pACYCDuet-1 is used as a backbone and Psen is used to initiate the expression of NADH kinase gene pos5 and NAD + kinase gene nadK; using Pmdh to start the expression of citrate synthase gene gltA, glycerol-3-phosphate dehydrogenase gene gapA and phosphopyruvate hydratase gene eno; prpmBG was used to initiate the expression of the glucose-6-phosphate dehydrogenase gene zwf, the isocitrate dehydrogenase gene icd.

3. Use of the NADPH modulating plasmid of claim 1 or 2 in the construction of recombinant microbial cells.

4. A recombinant microbial cell comprising the NADPH modulating plasmid of claim 1 or 2.

5. A recombinant escherichia coli comprising the NADPH-regulating plasmid of claim 1 or 2, modified with at least one of the following:

(1) Expresses the daidzein reductase Ac _ DZNR derived from Asaccharobacter celetus;

(2) Expresses Lactococcus garvieae derived dihydrodaidzein racemase Lg _ DDRC;

(3) The dihydrodaidzein reductase Si _ DHDR and the tetrahydrodaidzein reductase Si _ THDR which are derived from the Slackia isovaninens are expressed;

(4) The endogenous gene 6-phosphofructokinase gene pfkA, NAD (P) of Escherichia coli is knocked out ⁺ One or more of catalase gene sthA and GNAT family N-acetyltransferase gene elaA.

6. The recombinant Escherichia coli of claim 5, wherein Escherichia coli BL21 (DE 3) is used as a host.

7. A product for producing equol, which comprises the NADPH-modulating plasmid of claim 1 or 2, or the recombinant Escherichia coli of claim 5 or 6.

8. A method for producing equol by microbial conversion of daidzein, which comprises culturing the recombinant Escherichia coli of claim 4 or 5 as a fermentation strain in a daidzein-free medium for a certain period of time, and fermenting in a daidzein-containing medium.

9. The method of claim 8, wherein the fermentation is a staged fermentation, the staging comprising:

the first stage is as follows: controlling the temperature to be 30-40 ℃ and the dissolved oxygen concentration to be 35-45% to promote the growth of cells;

and a second stage: adding inducer with final concentration of 0.1-0.2mM, controlling temperature at 20-28 deg.C and dissolved oxygen concentration at 25-35%, and supplementing daidzein after inducing for 5-7 hr;

and a third stage: fermenting at 23-28 deg.C for at least 20-40h.

10. Use of the NADPH-modulating plasmid of claim 1 or 2, or the recombinant E.coli of claim 5 or 6, for the production of equol-containing products.