Recombinant Escherichia coli and method for preparing high-purity ursodeoxycholic acid

Abstract

To provide production methods of recombinant Escherichia coli (E. coli) and high purity ursodeoxycholic acid in the biotechnology field.SOLUTION: The present invention constructs a novel double-enzyme co-expressing engineered bacterium, that is, a recombinant E. coli. The recombinant bacterium simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH). The recombinant bacterium is applicable to the production of high-purity UDCA. The yield of a target product is increased through the co-expression and application of 7β-HSDH and GDH. The production method of high-purity UDCA in the present invention is simple with less generation of impurities in the process. The production method is a green process that satisfies environmental protection requirements, and has significant industrial application value.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology technology, and in particular to recombinant Escherichia coli (E. coli) and a method for preparing high-purity ursodeoxycholic acid (UDCA). [Background technology]

[0002] As an active ingredient in traditional Chinese medicine, UDCA (3α,7β-dihydroxy-5β-cholan-24-oic acid) has been recognized for its very wide clinical application and has excellent pharmaceutical value. UDCA has excellent therapeutic effects in the treatment of cholelithiasis, liver transplant promotion, bile reflux gastritis, alcoholic liver disease, biliary cirrhosis, and drug-induced hepatitis, and therefore has a high demand in the market.

[0003] At present, UDCA is mainly prepared from natural sources and synthesis. The natural source is extracted from the gallbladder or bile of live bears protected by the Animal Protection Act, and the source of natural bear gallbladder is gradually decreasing due to limited extraction sources. In the synthetic method, UDCA is chemically synthesized using chenodeoxycholic acid (CDCA) extracted from the bile of cows and geese, which is easily available, and the 7-OH is structurally changed using an oxidation-reduction method. However, there are a series of problems, such as complicated reaction processes, low selectivity, harsh reaction conditions, high energy consumption, and serious pollution. In particular, toxic and dangerous reagents are required during protection and deprotection, which greatly limits the industrial application of chemical methods. At present, UDCA prepared using chemical methods occupies about 30% of the market share, and its purity is relatively low at about 80%, which is far from meeting the market's requirements for the uses and quality of UDCA.

[0004] Compared with chemical epimerization, the biological synthesis of UDCA is efficient and relatively environmentally friendly. Microbial conversion or biological enzyme catalysis mainly involves the deployment of 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH). To carry out the biological conversion of CDCA to UDCA, Clostridium limosum, Clostridium absonum, Clostridium pasteurianum, and Xanthomonas maltophilia, which produce 7α-HSDH and 7β-HSDH, have been used. However, high concentrations of CDCA inhibit the accumulation of cell biomass, making it difficult to recycle and purify the product. Previous studies have also shown that it is impossible to carry out industrial preparation, as the yield of intermediates increases and UDCA decreases with the cultivation time. In recent years, research has focused on the use of 7α-HSDH and 7β-HSDH in a two-step process to prepare UDCA using CDCA as a substrate. The overall conversion rate currently being studied is over 90%. However, the reaction volume is at the milliliter level, which is far from industrialization. The main limiting bottlenecks are as follows: 1) 7β-HSDH, the main enzyme in the two-step process using CDCA as a substrate, is extremely unstable. Although the enzyme is fully active, it quickly becomes inactive in the catalytic reaction system. 2) The reaction system in the two-step method has an excessively small amount of substrate (volume ratio 1%), and the system is unstable. 3) The two key enzymes in the two-step process face the problem of large-scale supply in industrial applications. 4) The cost of isolating and purifying enzyme proteins is too high.

[0005] (overview) In order to solve the problems of the prior art, the present invention provides a method for preparing high-purity UDCA, constructs an engineered bacterium that co-expresses multiple enzymes, and carries out efficient preparation of UDCA. The present invention provides a recombinant engineered bacterium that can prepare UDCA at low cost. The present invention also aims to solve the technical problems related to strain construction and application.

[0006] To achieve the above objective, the present invention adopts the following technical solutions:

[0007] The first objective of the present invention is to provide a recombinant E. coli capable of preparing UDCA at low cost. The recombinant E. coli can simultaneously express two enzymes, 7β-HSDH and glucose dehydrogenase (GDH), respectively. The recombinant E. coli was deposited at the China Center for Type Culture Collection on December 27, 2021. Its deposit number is CCTCC NO: M20211644.

[0008] Preferably, the 7β-HSDH is derived from Clostridium absonum and its GenBank login number is JN191345.1.

[0009] Preferably, the GDH is derived from Bacillus subtilis and its GenBank login number is NC-000964.

[0010] Preferably, the 7β-HSDH and GDH genes are codon-optimized, and the nucleotide sequences of the 7β-HSDH and GDH genes are encoded as shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0011] Preferably, in the case of recombinant bacteria, the gene encoding the 7β-HSDH and the gene encoding the GDH are both ligated into a plasmid to construct a two-gene co-expression recombinant plasmid, and the recombinant plasmid is transformed into a corresponding strain to obtain a recombinantly engineered bacterium.

[0012] Preferably, the recombinant E. coli is specifically produced by the following method: (1) performing codon optimization on the 7β-HSDH gene and the GDH gene, respectively, and synthesizing the entire genes; (2) ligating the synthesized 7β-HSDH gene into vector pETDuet1 to obtain recombinant plasmid pETDuet1-7β-HSDH; (3) transferring the pETDuet1-7β-HSDH into E. coli DH5α competent cells to obtain E. coli DH5α-pETDuet1-7β-HSDH carrying the recombinant plasmid; (4) spreading the E. coli DH5α-pETDuet1-7β-HSDH on an antibiotic solid plate and screening to obtain positive transformants (the purpose of step 3 and step 4 is to screen using the resistance gene carried by the plasmid to obtain a recombinant plasmid that has been successfully ligated); (5) ligating the synthesized GDH gene into vector pETDuet1-7β-HSDH to obtain recombinant plasmid pETDuet1-GDH-7β-HSDH; (6) transferring the pETDuet1-GDH-7β-HSDH into E. coli BL21(DE3) competent cells to obtain E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH carrying the recombinant plasmid; and (7) spreading the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH on an antibiotic solid plate and screening to obtain a positive transformant, thereby obtaining the recombinant E. coli.

[0013] Preferably, the recombinant E. coli is constructed using E. coli BL21(DE3) as a host.

[0014] A second object of the present invention is to provide a method for preparing UDCA, wherein the preparation is carried out by using any of the recombinant E. coli of the present invention.

[0015] Preferably, said method for preparing UDCA is to perform a complete cell transformation production.

[0016] Preferably, the whole cell conversion preparation system contains cells with a wet weight of 1 g / L to 100 g / L, 7-oxo-lithocholic acid with a concentration of 1 g / L to 200 g / L, and glucose with a concentration of 1 g / L to 100 g / L, the pH is 7.0 to 9.0, and the reaction is carried out at 1°C to 30°C for 1 hour to 48 hours.

[0017] (Beneficial Effects) The present invention discloses a method for preparing high-purity UDCA. The present invention constructs a novel dual enzyme co-expression genetically engineered bacterium, which is applicable to the preparation of high-purity UDCA. The combined expression and application of 7β-HSDH and GDH increases the yield of the target preparation. The method of the present invention is simple, generates little impurities in the process, is an environmentally friendly process that meets environmental protection requirements, and has important industrial application value. [Brief description of the drawings]

[0018] [Figure 1] 1 is a profile of the recombinant plasmid pETDuet1-GDH-7β-HSDH. [Diagram 2] This is a liquid phase detection result profile of the reaction of preparing UDCA using 7-oxo-lithocholic acid under the catalysis of genetically engineered bacteria for UDCA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] (Description of the embodiment) The present invention will be described in detail below. In the description, the terms used in the present specification and the appended claims should not be interpreted limited to their ordinary meaning and dictionary meaning, but should be interpreted according to the meaning and concept corresponding to the technical aspects of the present invention based on the principle that the inventor can appropriately define the terms to make the best interpretation. Therefore, the description given in the present specification is merely a preferred example for explanation, and is not intended to limit the scope of the present invention, and it should be understood that other equivalent methods or improvements can be obtained therefrom without departing from the spirit and scope of the present invention.

[0020] It will be understood by those skilled in the art that the following embodiments are merely examples of the present invention, are not intended to limit the present invention, and modifications that do not deviate from the gist and concept of the present invention are within the scope of the present invention. Note that all reagents and instruments used in the following embodiments are commercially available unless otherwise specified.

[0021] The functional core of the genetically engineered bacteria in the present invention is that it can simultaneously express two enzymes, 7β-HSDH and GDH. The principle of the engineered bacteria is to dehydrogenate glucose in the complete cells of the genetically engineered bacteria using GDH with NADP as a coenzyme to produce gluconic acid and NADPH. The 7β-HSDH converts 7-oxo-lithocholic acid to UDCA using NADPH produced in the process of dehydrogenating glucose, while simultaneously regenerating the coenzyme NADP.

[0022] 1. Strains and plasmids used in the present invention For the genes of 7β-HSDH and GDH, the gene sequences are codon optimized and the whole genes are synthesized according to the codon bias of E. coli by Sangon Biotech (Shanghai) Co., Ltd. The E. coli BL21 (DE3) strain is purchased from Sangon Biotech (Shanghai) Co., Ltd. The plasmid pETDuet1 is purchased from Shanghai Linyuan Biotechnology Co., Ltd.

[0023] 2. The E. coli co-expressing 7β-HSDH and GDH was prepared by the following method: (1) performing codon optimization on the 7β-HSDH gene and the GDH gene, respectively, and performing total gene synthesis, and the nucleotide sequences of the 7β-HSDH and GDH genes are encoded as shown in SEQ ID NO:1 and SEQ ID NO:2, respectively; (2) sequentially ligating the two synthesized genes into the vector pETDuet1 to obtain a recombinant plasmid pETDuet1-GDH-7β-HSDH that co-expresses the 7β-HSDH and the GDH; (3) transferring the pETDuet1-GDH-7β-HSDH into E. coli BL21(DE3) competent cells to obtain an engineered bacterium BL21(DE3)-pETDuet1-GDH-7β-HSDH carrying the recombinant plasmid; and (4) A step of culturing the engineered bacterium, i.e., a step of inoculating the engineered bacterium into LB liquid medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L) and culturing it to obtain E. coli that co-expresses the 7β-HSDH and the GDH.

[0024] 3. The culture of the engineered bacteria comprises the following steps: (1) The engineered bacteria is inoculated onto LB solid plate medium (tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, and agar 15.0 g / L) for activation, cultured at 37°C for 16 to 20 hours, and a single colony is picked from the LB solid plate medium. The single colony is then incubated with 100 mg / L ampicillin (Amp + ) into an Erlenmeyer flask containing LB liquid medium containing the strain, and then culturing with shaking at 200 rpm / min at 37°C for 16 to 20 hours; (2) The culture in step (1) was diluted with 100 mg / L ampicillin (Amp + ) in a ratio of 1:100, and cultured at 200 rpm / min at 37°C for 4 to 6 hours with shaking, adding 0.4 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG), and continuing culture at 25°C for 10 to 16 hours to obtain engineered bacteria, which are E. coli that co-express 7β-HSDH and GDH; and (3) After the end of the induced expression, the cells are harvested by centrifugation at 8,000 rpm at 4° C. for 10 minutes.

[0025] 4. Preparation of UDCA by Whole Cell Transformation The whole cell conversion preparation system contains cells with a wet weight of 1g / L-100g / L, 7-oxo-lithocholic acid with a concentration of 1g / L-200g / L, and glucose with a concentration of 1g / L-100g / L, pH is 7.0-9.0, and the reaction is carried out at 1°C-30°C for 1-48 hours. After the conversion is completed, the yield and conformation of UDCA are measured using liquid chromatography.

[0026] 5. Sample Detection and Analysis The chromatographic conditions were as follows: use of a RID detector, a mobile phase of acetonitrile (50)-water (0.0125% phosphoric acid) (50), a flow rate of 1 mL / min, a column temperature of 35° C., and a sample size of 10 μL.

[0027] In order to more clearly understand the technical problems, technical solutions and beneficial effects solved by the present invention, the present invention is described in detail below in conjunction with the embodiments. It should be noted that the specific embodiments described herein are only used to illustrate the present invention and are not intended to limit the present invention.

[0028] Example 1

[0029] Construction of engineered bacteria 1. Cultures of E. coli containing the plasmid PUC57-7β-HSDH and E. coli containing the vector pETDuet1 were grown. 10 μl of sample was taken and cultured in LB (Amp + ) was added to 5 mL of medium. The culture was carried out for 12 to 16 hours in a shaker at 37°C, with the shaker rotating at 200 rpm / min.

[0030] 2. The cultured plasmid E. coli was extracted using a column plasmid mini-preps kit purchased from Sangon Biotech (Shanghai) Co., Ltd. The operation was carried out according to the operating instructions of the kit.

[0031] 3. Plasmid PUC57-7β-HSDH and vector pETDuet1 were double digested with EcoRV and XhoI, respectively, and extracted. The digestion system is shown in Table 1 below: [Table 1]

[0032] The digestion was carried out for 3 to 6 hours at 37°C. The target segment and the linear vector were recycled and purified using a column DNA gel extraction kit purchased from Sangon Biotech (Shanghai) Co, Ltd.

[0033] 4. The recycled target gene segment 7β-HSDH was ligated with the linear vector pETDuet1 using T4 DNA ligase. The system is shown in Table 2 below: [Table 2]

[0034] Ligation was carried out at 22°C for 30 to 60 minutes.

[0035] 5. Transform E. coli DH5α competent cells with the ligation system. On a super-clean bench, 10 μL of the system was transferred to chemically competent E. coli DH5α prepared according to the standard solution. The mixture was gently mixed evenly and left on ice for 30 minutes.

[0036] A heat shock was performed at 42° C. for 60 seconds, and the mixture was allowed to stand on ice for 2 minutes. 700 μL of sterilized LB medium was added on the super clean bench.

[0037] The mixture was placed in a shaker at 37°C and 200 rpm for activation for 40 to 60 minutes, and incubated with LB (Amp + ) was spread onto solid plate medium.

[0038] The plate on which the cells were applied was placed in an incubator at 37° C. Inverted culture was carried out for 12 to 16 hours.

[0039] 6. Colony PCR Detection of Positive Clones The forward and reverse primers for amplifying 7β-HSDH were DuetUP2 and T7t, respectively.

[0040] The PCR reaction system is shown in Table 3 below. [Table 3]

[0041] Dominant colonies were picked using a sterile inoculation ring on a super clean bench and dipped into PCR tubes for use as templates.

[0042] The PCR amplification conditions were as follows: 94°C for 3 min, (94°C for 30 s, 60°C for 30 s, 72°C for 1 min) × 32 cycles, 72°C for 10 min, and storage at 4°C.

[0043] 7. The PCR product was detected by electrophoresis. The positive colony DH5a-pETDuet1-7β-HSDH carrying the target segment was selected.

[0044] The positive colonies were collected on a super clean bench. + ) 5 mL of liquid medium was added. The mixture was shaken in a shaker at 37°C and 200 rpm. Cultivation was carried out for 12 to 16 hours.

[0045] 8. A culture of E. coli containing the plasmid PUC57-GDH was grown in parallel. A 10 μL sample was taken. + ) 5 mL of medium was added. This mixture was cultured in a shaker at 37°C for 12 to 16 hours. The shaker speed was 200 rpm / min.

[0046] The cultured positive bacteria plasmid and plasmid PUC57-GDH were extracted using a column plasmid miniprep kit from Sangon Biotech (Shanghai) Co., Ltd., and the plasmid PUC57-GDH and vector pETDuet1-7β-HSDH were extracted using double digestion with EcoR1 and HindIII. The digestion system was the same as in item 3 above. Gel recycling was performed for the target gene segment and linear vector.

[0047] 9. The recycled GDH target gene and the linear vector pETDuet1-7β-HSDH were ligated. The ligation system was the same as that in item 4 above.

[0048] 10. The ligated system was transformed into E. coli DH5α competent cells by the same procedure as in item 5 above.

[0049] 11. Colony PCR detection of positive clones: GDH was amplified using forward and reverse primers pET Upstream and DuetDOWN1. The PCR product was detected by electrophoresis. Through screening, a positive colony carrying the target segment, pETDuet1-GDH-7β-HSDH, was obtained. The PCR reaction conditions and cycle conditions were the same as those in item 6 above.

[0050] 12. Positive clone bacteria were collected. + ) 5 mL of liquid medium was added. The mixture was shaken at 37°C and 200 rpm in a shaker. Cultivation was carried out for 12 to 16 hours, and the plasmid was extracted. Digestion and verification were performed to confirm that the vector was correctly constructed. The vector profile is shown in Figure 1.

[0051] 13. The recombinant plasmid was transferred into E. coli BL21(DE3) expression strain to obtain the engineered bacteria BL21(DE3)-pETDuet1-GDH-7β-HSDH.

[0052] 14. Cultivation of Engineered Bacteria The genetically engineered bacteria were inoculated onto LB solid plate medium for activation. Activation and cultivation were carried out at 37°C for 16 to 20 hours. Single colonies were picked from the LB solid plate medium and incubated with ampicillin (Amp + The bacteria was inoculated into an Erlenmeyer flask containing LB liquid medium containing 100 mg / L of the bacteria, and cultured with shaking at 200 rpm / min at 37° C. for 16 to 20 hours.

[0053] The culture was diluted with 100 mg / L ampicillin (Amp +The culture was then transferred to TB liquid medium containing 1:100 of 1:10 ...

[0054] The LB solid medium contains the following components: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, and agar 15.0 g / L.

[0055] The LB liquid medium contains the following components: 10 g / L tryptone, 5 g / L yeast extract, and 10 g / L sodium chloride (NaCl).

[0056] The TB liquid medium contains the following components: tryptone 12 g / L, yeast extract 24 g / L, glycerol 5 g / L, dipotassium phosphate (K2HPO4) 9.4 g / L, and monopotassium phosphate (KH2PO4) 2.2 g / L.

[0057] Example 2

[0058] After completion of induction expression according to the induction expression method of Example 1, the cells were harvested. In a 100 mL reaction system, the wet cell weight was 3 g / L, glucose was 4 g / L, 7-oxo-lithocholic acid was 7 g / L, pH=8.0, and temperature was 10° C. Transformation was carried out for 10 hours, during which the pH was controlled at 8.0±0.2 using 1 M NaOH.

[0059] Figure 2 shows the liquid-phase detection profile of the reaction of UDCA prepared from 7-oxo-lithocholic acid under the catalysis of bacteria genetically engineered for UDCA. UDCA was prepared by conversion of 7-oxo-lithocholic acid with a liquid chromatography detection rate of over 99%.

[0060] The enzymes mentioned above, and the transformation and construction of genetically engineered bacteria co-expressing the enzymes, the medium components and culture methods of the cells, and the biological transformation of the whole cells are merely preferred examples of the present invention and are not used to limit the present invention. In theory, other bacteria, filamentous fungi, actinomycetes, and animal cells can all be subjected to genome transformation and used in the catalysis of multi-gene co-expressing whole cells. Any modifications and equivalent substitutions can be made within the principle and spirit of the present invention.

Claims

[Claim 1] A composition for preparing ursodeoxycholic acid, comprising: The composition comprises a recombinant Escherichia coli, 7-oxo-lithocholic acid and glucose as substrates, and the coenzyme NADP, wherein the recombinant Escherichia coli simultaneously expresses 7β-hydroxysteroid dehydrogenase (7β-HSDH) and glucose dehydrogenase (GDH); The 7β-HSDH and the GDH are co-expressed by the vector pETDuet1; the host bacterium for the recombinant E. coli is E. coli BL21(DE3); The recombinant E. coli is produced by the following method: performing codon optimization for the 7β-HSDH gene and the GDH gene, respectively, and synthesizing the entire genes; The synthesized 7β-HSDH gene is first ligated into a vector pETDuet1 to obtain a recombinant plasmid pETDuet1-7β-HSDH, and then the GDH gene is ligated into the plasmid pETDuet1-7β-HSDH to obtain a recombinant plasmid pETDuet1-GDH-7β-HSDH that co-expresses the 7β-HSDH and the GDH; Transforming the pETDuet1-GDH-7β-HSDH into E. coli BL21(DE3) competent cells to obtain E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH carrying the recombinant plasmid; and Cultivating the E. coli BL21(DE3)-pETDuet1-GDH-7β-HSDH to obtain the recombinant E. coli; Here, the composition increases the yield of ursodeoxycholic acid to 99% or more by expressing and applying 7β-HSDH and GDH in combination.

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