Genetically engineered bacteria and their applications

Abstract

This invention discloses genetically engineered bacteria and their applications, belonging to the field of bioengineering technology. By constructing a system that enhances the expression of polyphosphate kinase 2-I, polyphosphate kinase 2-II, and peptide bond synthase, this invention establishes an ATP recycling system, effectively ensuring the continuous execution of enzyme-catalyzed reactions. Using ursodeoxycholic acid or glycine and taurine as substrates, the genetically engineered bacteria expressing these three enzymes generate tauroursodeoxycholic acid or glycoursodeoxycholic acid, increasing the yield of these enzymes and achieving efficient biosynthesis of tauroursodeoxycholic acid or glycoursodeoxycholic acid, demonstrating promising prospects for industrial application.

Description

Technical Field

[0001] This invention relates to genetically engineered bacteria and their applications, belonging to the field of bioengineering technology. Background Technology

[0002] Tauroursodeoxycholic acid (TUDCA) is the main active component of bile acids contained in bear bile. Its chemical name is 3α,7β-dihydroxy-cholanyl-N-taurine, which is a product formed by the combination of ursodeoxycholic acid (UDCA) and taurine. The main function of TUDCA is to dissolve cholesterol stones and it is used for non-surgical treatment of gallstones. Studies have shown that TUDCA is suitable for treating common diseases such as cholesterol gallstones, primary sclerosing cholangitis, primary biliary cirrhosis, and chronic hepatitis C. (Crosignani A, Setchell KD, Invernizzi P, et al. Clinic alpharmacokinetics of therapeutic bileacids[J]. Clin Pharmacokinet, 1996, 30(5):333-358). In 2007, Rivard et al. discovered that TUDCA could inhibit induced apoptosis in a myocardial infarction model and improve cardiac function by regulating the Bcl-2 family and related signaling proteins. Drack et al. also reported that TUDCA could alleviate retinal degeneration and improve obesity in Barbie II syndrome mice. Cha et al. found that TUDCA could regulate integrin 5 (ITGA5), which is associated with the extracellular signal-regulated kinase bypass pathway, thereby inhibiting cell apoptosis and immune responses and promoting bone regeneration. Recent studies have found that TUDCA can reduce neuronal loss induced by chronic neurodegenerative diseases and exhibits anti-inflammatory effects in neuroinflammatory models. It has also been found that TUDCA can inhibit the activation of cochlear apoptosis proteases in mice, prevent hair cell apoptosis, and reduce hearing loss in mice, showing potential therapeutic effects for autosomal recessive deafness in humans. Therefore, the clinical role of TUDCA extends beyond its traditional cholelithiasis-dissolving effects; it exerts unique pharmacological effects in areas such as cell apoptosis and tissue repair. With further research, its clinical applications will continue to expand, playing an even greater role.

[0003] Currently, the main method for producing ursodeoxycholic acid (TUDCA) from taurine is the chemical semi-synthesis method. This method is a mainstream industrial method for synthesizing TUDCA and is relatively mature, but it also has certain problems. For example, it suffers from poor stereoselectivity, and the starting substrate must be UDCA of a certain purity, which is difficult to achieve in the bile acids of various poultry. The reaction process uses organic solvents and reagents, posing potential threats to the environment and human health, and meeting international standards for organic solvent residues in TUDCA is challenging. Therefore, it is necessary to find more efficient and environmentally friendly methods for the synthesis and conversion of TUDCA. Green and sustainable biocatalytic methods are receiving increasing attention.

[0004] Biocatalysis mainly includes enzyme catalysis and microbial catalysis. In 1986, Riva et al. utilized the epimerization function of NADP-dependent hydroxysteroid dehydrogenases (HSDH) to oxidize hydroxyl groups to ketones or reduce ketone groups to α or β hydroxyl groups in the presence of coenzyme I / II, thereby achieving the purpose of biotransformation of the target product. (Riva S, Bovara R, Pasta P, et al. Preparative-scale regio-andstereospecific oxidoreduction of cholic acid and dehydrocholicacid catalyzed by hydroxysteroid dehydrogenases[J]. J Org Chem, 1986, 51(5):2902-2906.) Ji et al. simultaneously immobilized two enzymes, 7α-HSDH and 7β-HSDH, on chitosan microspheres and used this immobilized enzyme to catalyze the bound state of TCDCA, generating the bound state of TUDCA under the action of coenzyme. (Ji Q, Tan J, Zhu L, et al. Preparing tauroursodeoxycholic acid (TUDCA) using a double-enzyme-coupled system[J]. BiochemEng J, 2016, 105:1-9.) Enzymatic catalysis requires the prior isolation and purification of the corresponding enzyme or immobilization to improve enzyme stability. For hydroxysteroid dehydrogenases that rely on NADP+ / NADPH cofactors, additional cofactors or cofactor regeneration systems must be added, further increasing costs. Sun et al. constructed recombinant plasmids expressing exogenous 3α-HSDH and 7β-HSDH genes using different combinations. The results showed that the transforming bacteria obtained by constructing a single plasmid containing the 3α-HSDH and 7β-HSDH genes and a gene carrying glucose dehydrogenase, and using a one-pot whole-cell catalysis method, achieved the highest transformation rate (Sun B, Kantzow C, Bresch S, et al. Multi-enzymatic one-pot reduction of dehydrocholic acid to 12-keto-ursodeoxycholic acid with whole-cell biocatalysts).

[0005] [J]. Biotechnol Bioeng, 2013, 110(1):68-77.). Lee et al. summarized the existing methods using 7α- and 7β-HSDH transformation and proposed that whole-cell catalysis is a more feasible method (Lee JY, Arai H, Nakamura Y, et al. Contribution of the 7β-hydroxysteroid dehydrogenase from Ruminococcus gnavus N53toursodeoxycholic acid formation in the human colon[J]. J LipidRes, 2013, 54(11):3062-3069.). However, current research is mostly in the initial stage of development. Finding relevant catalytically active genes, constructing efficient microbial expression systems, rationally utilizing existing animal bile acids as substrates, and exploring simple methods for biosynthesizing TUDCA through biological metabolism present new opportunities and challenges.

[0006] Glycoursodeoxycholic acid (GUDCA) is the main active component of bile acids in bear bile, and it is a product formed by the combination of ursodeoxycholic acid (UDCA) and glycine. Similar to ursodeoxycholic acid and tauroursodeoxycholic acid, Glycoursodeoxycholic acid also has therapeutic effects in neurodegenerative models and related diseases. (Ana Rita Vaz & Carolina Cunha & Cátia Gomes. Glycoursodeoxycholic acid reduces matrix metalloproteinase-9 and caspase-9 activation in a cellular model of superoxide dismutase-1 neurodegeneration, 2015, 51(3):864-877). In 2000, Rodrigues CMP et al. found that GUDCA had a protective effect on mouse neural RN33B cells by inhibiting mitochondrial expansion and cytochrome c release. In 2001, Lazaridis KN et al. demonstrated that oral administration of UDCA resulted in a higher amount of glycoursodeoxycholic acid (GUDCA) than tauroursodeoxycholic acid (TAO), thus drawing increasing attention to GUDCA. The beneficial effects of GUDCA include preventing mitochondrial swelling and neurite extension, reducing extracellular glutamate levels, and anti-inflammatory effects, playing an important role in patients with muscular dystrophy. In 2007, Adelaide Fernandes et al. discovered that GUDCA and Interleukin-10 can regulate the activity of cortical glial cells in response to unconjugated bilirubin (UCB). In 2015, Ana Rita Vaz et al., using NSC-34 / hSOD1G93A cells, found that GUDCA significantly reduced physiological indicators of muscular dystrophy, including mitochondrial dysfunction, apoptosis, caspase-9 activation, NO production, and MMP-9 activation. Targeting caspase-9 and MMP-9, GUDCA represents a potentially effective therapy for muscular dystrophy. As researchers continue to study the biological activities and metabolic characteristics of GUDCA, more and more biological activities are being discovered. Therefore, further rational development and utilization of GUDCA for human use will become a research hotspot in the future.

[0007] Currently, GUDCA is mainly synthesized industrially using chemical methods. Two main methods for the chemical synthesis of ursodeoxycholic acid (UGCA) have been reported in the literature: 1) using ursodeoxycholic acid and glycine ethyl ester hydrochloride as raw materials, with diethyl pyrocarbonate (DEPC) as the condensing agent; 2) using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as the condensing agent, prepared under microwave conditions. While these two methods are simple to operate and relatively mature, they also have certain problems. For example, they suffer from poor stereoselectivity, requiring the starting substrate to be UGCA of a certain purity, which is difficult to achieve in the bile acids of various poultry. Although the yield exceeds 90%, the condensing agents EEDQ and DEPC are expensive, and the reaction process requires organic solvents and reagents, posing potential threats to the environment and human health. Meeting international standards for organic solvent residues in GUDCA is also challenging. Therefore, it is necessary to find more efficient and environmentally friendly methods for the synthesis and conversion of GUDCA. Green and sustainable biocatalysis methods are receiving increasing attention. Summary of the Invention

[0008] [Technical Issues]

[0009] The technical problem to be solved by this invention is to synthesize tauroursodeoxycholic acid using ursodeoxycholic acid as a substrate, thereby increasing the yield of tauroursodeoxycholic acid synthesized by biological or enzymatic methods.

[0010] [Technical Solution]

[0011] This invention provides a method for synthesizing tauroursodeoxycholic acid or glycoursodeoxycholic acid using ursodeoxycholic acid or glycine as substrates. Ursodeoxycholic acid (or glycine) and taurine, catalyzed by an ATP-dependent peptide bond synthase, lose one molecule of water to generate tauroursodeoxycholic acid (or glycoursodeoxycholic acid). During this process, ATP is hydrolyzed to AMP. Polyphosphate kinase 2-II (PPK2-II) converts AMP to ADP, and ADP is further catalyzed by polyphosphate kinase 2-I (PPK2-I) to regenerate ATP.

[0012] This invention provides a recombinant cell expressing a peptide bond synthase, polyphosphokinase 2-I, and polyphosphokinase 2-II; the peptide bond synthase is derived from *Pontimonas salivibrio*, *Bradyrhizobium* sp. SUTN7-2, *Arabidopsis thaliana*, *Sparassis crispa*, *Bacillus amyloliquefaciens*, and *Variovorax* sp. WDL1; the polyphosphokinase 2-I is derived from *Sinorhizobium meliloti*; and the polyphosphokinase 2-II is derived from *Acinetobacter johnsonii*.

[0013] In one embodiment, the amino acid sequences corresponding to the peptide bond synthase have NCBI accession numbers of AVG23412.1, AFJ03901.1, NP_001117233.1, GBE79911.1, QEY93952.1, and ANS57223.1, respectively; the corresponding nucleotide sequences are shown in the NCBI accession numbers. The sequence numbered CP026923.1REGION:complement(384130..384603), JQ801439.1REGION(1..479), NM_001331546REGION(1..3378), XM_027755023REGION(1..525), CP044360.1REGION:complement(2214096..2215046), and KC146403.1REGION(2972..3574) is shown.

[0014] In one embodiment, the amino acid sequence of the polyphosphate kinase 2-I is shown as the sequence with accession number NP_384613.1 on NCBI, and the nucleotide sequence is shown as the sequence with accession number NC_003047REGION:complement(564142..565044) on NCBI.

[0015] In one embodiment, the polyphosphate kinase 2-II has an amino acid sequence as shown in GenBank:BAC76403.1; or, the nucleotide sequence of polyphosphate kinase 2-II is the sequence with accession number AB092983REGION(339..1766) on NCBI.

[0016] In one embodiment, Escherichia coli is used as the starting strain, and pACYCDuet-1, pETDuet-1, or pRSFDuet-1 is used as the expression vector.

[0017] In one implementation, each gene contains a T7 promoter and an RBS binding site before it, and a T7 promoter after it.

[0018] The present invention provides a recombinant cell composition, characterized in that the recombinant cell composition comprises one or more recombinant cells expressing peptide bond synthase, polyphosphokinase 2-II, and polyphosphokinase 2-I, respectively.

[0019] In one embodiment, recombinant cells are used to express peptide bond synthase, polyphosphate kinase 2-II, and polyphosphate kinase 2-I, respectively; or, polyphosphate kinase 2-II and polyphosphate kinase 2-I are expressed in the same recombinant cell, and peptide bond synthase is expressed in another recombinant cell.

[0020] In one embodiment, the peptide bond synthase is derived from *Pontimonas salivibrio*, *Bradyrhizobium* sp. SUTN7-2, *Arabidopsis thaliana*, *Sparassis crispa*, *Bacillus samyloliquefaciens*, and *Variovorax* sp. WDL1; the polyphosphoric acid kinase 2-I is derived from *Sinorhizobium meliloti*; and the polyphosphoric acid kinase 2-II is derived from *Acinetobacter johnsonii*.

[0021] In one embodiment, the amino acid sequences corresponding to the peptide bond synthase have NCBI accession numbers of AVG23412.1, AFJ03901.1, NP_001117233.1, GBE79911.1, QEY93952.1, and ANS57223.1, respectively; the corresponding nucleotide sequences are shown in the NCBI accession numbers. The sequence numbered CP026923.1REGION:complement(384130..384603), JQ801439.1REGION(1..479), NM_001331546REGION(1..3378), XM_027755023REGION(1..525), CP044360.1REGION:complement(2214096..2215046), and KC146403.1REGION(2972..3574) is shown.

[0022] In one embodiment, the amino acid sequence of the polyphosphate kinase 2-I is shown as the sequence with accession number NP_384613.1 on NCBI, and the nucleotide sequence is shown as the sequence with accession number NC_003047REGION:complement(564142..565044) on NCBI.

[0023] In one embodiment, the amino acid sequence of the polyphosphate kinase 2-II is as shown in GenBank:BAC76403.1; or, the nucleotide sequence of the polyphosphate kinase 2-II is as shown in the sequence with accession number AB092983 REGION (339..1766) on NCBI.

[0024] In one implementation, pACYCDuet-1, pETDuet-1, and / or pRSFDuet-1 are used as expression vectors.

[0025] This invention provides a method for producing taurine deoxycholic acid or glycoursodeoxycholic acid. The method uses ursodeoxycholic acid and taurine as raw materials and recombinant cells or recombinant cell compositions to prepare taurine deoxycholic acid; and uses glycine and ursodeoxycholic acid as raw materials and recombinant cells or recombinant cell compositions to prepare glycoursodeoxycholic acid.

[0026] In one embodiment, the recombinant cells or recombinant cell composition are seeded into LB medium, and when the cell OD... 600 After reaching a concentration of 0.6–0.8, IPTG with a final concentration of 0.2–0.5 mM was added, and the cells were induced to express and cultured at 20°C for 8 h. The cells were then collected and added to a reaction system containing ursodeoxycholic acid or glycine, taurine, ATP, and sodium hexametaphosphate.

[0027] In one embodiment, the wet cell concentration in the reaction system is 1–200 g / L, the ursodeoxycholic acid or glycine concentration is 1–100 g / L, the taurine concentration is 1–100 g / L, the sodium hexapolyphosphate concentration is 3–300 g / L, and the ATP concentration is 0–1 g / L.

[0028] In one embodiment, the reaction is carried out at pH 5–9 and 5–40°C for 1–48 hours.

[0029] The present invention provides the use of the recombinant cells or the composition in the production of tauroursodeoxycholic acid, products containing tauroursodeoxycholic acid, substances with tauroursodeoxycholic acid as a precursor, glycouroursodeoxycholic acid, products containing glycouroursodeoxycholic acid and / or substances with glycouroursodeoxycholic acid as a precursor.

[0030] [Beneficial Effects]

[0031] This invention constructs an ATP recycling system by enhancing the expression of polyphosphate kinase 2-I derived from *Sinorhizobium meliloti*, polyphosphate kinase 2-II derived from *Acinetobacter johnsonii*, and peptide bond synthases from different sources, effectively ensuring the continuous execution of enzyme-catalyzed reactions. Using ursodeoxycholic acid or glycine and taurine as substrates, genetically engineered bacteria expressing these three enzymes generate tauroursodeoxycholic acid or glycoursodeoxycholic acid, thereby increasing the yield of tauroursodeoxycholic acid or glycoursodeoxycholic acid and achieving efficient biological synthesis of tauroursodeoxycholic acid or glycoursodeoxycholic acid. Attached Figure Description

[0032] Figure 1 A schematic diagram illustrating the catalytic principle of the tauroursodeoxycholic acid synthesis reaction;

[0033] Figure 2 This is a schematic diagram illustrating the catalytic principle of the synthesis reaction of glycoursodeoxycholic acid. Detailed Implementation

[0034] 1. The strains and plasmids involved in this invention

[0035] pRSFDuet-1, pETDuet-1 plasmids and Escherichia coli BL21(DE3) were purchased from Novagen.

[0036] 2. Construction of a multi-gene co-expression system and cell culture

[0037] The construction method of recombinant *E. coli* was based on the method described in Liu Xianglei's "Synthetic Biology Technology to Modify *E. coli* for the Production of Shikimic Acid and Resveratrol," 2016, Shanghai Institute of Pharmaceutical Industry. In the following examples, when multiple genes were co-expressed, each gene contained a T7 promoter and an RBS binding site before it, and a T7 terminator after it. Theoretically, because each gene has a T7 promoter and an RBS site before it, the gene expression intensity is not significantly affected by the order of the genes on the plasmid. The constructed plasmid was heat-transformed into competent *E. coli* cells and plated on monoclonal antibody or mixed antibiotic solid plates. Positive transformants were screened to obtain recombinant *E. coli*.

[0038] Cell culture: Following the classic recombinant E. coli culture and induction expression protocol, recombinant E. coli were transferred to LB fermentation medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) at a volume ratio of 2%. When the cell OD... 600 Once the concentration reached 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 8 hours. After induction, cells were collected by centrifugation at 4°C, 8000 rpm for 20 minutes.

[0039] 3. Selection of relevant enzymes

[0040] (1) Polyphosphate kinase 2-I

[0041] The gene smpkk, which encodes polyphosphate kinase 2-I and is derived from Sinorhizobium meliloti, was selected. The accession number of the gene smpkk on NCBI is NC_003047 REGION:complement(564142..565044), and the corresponding amino acid sequence is NP_384613.1.

[0042] (2) Polyphosphate kinase 2-II

[0043] The gene ajpkk, which encodes polyphosphate kinase 2-II, derived from Acinetobacter johnsonii, was selected. The sequence of gene ajpkk with accession number AB092983 REGION:339..1766 on NCBI and the corresponding amino acid sequence is shown in GenBank:BAC76403.1.

[0044] (3) Peptide bond synthase

[0045] See Example 1.

[0046] 4. Sample detection and analysis

[0047] The determination method for tauroursodeoxycholic acid content was based on the literature: Method development and validation of ursodiol and its major metabolites in human plasma by HPLC-tandem mass spectrometry,[J]. Clin Pharmacol,2019,11:1-13.

[0048] The enzyme activity of peptide bond synthase was determined according to the literature: Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver, [J]. J Biol Chem, 1991, 5: 10227-10233.

[0049] Enzyme activity (U mg) -1 Enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of product per mg of enzyme.

[0050] Polyphosphate kinase 2-I specific enzyme activity assay: The assay was performed according to the literature Biochemical and Biophysical Research Communications 281, 821–826 (2001).

[0051] Polyphosphate kinase 2-II specific enzyme activity assay: determined according to the literature Journal Of Bacteriology, 1991, 6484 6488.

[0052] Example 1: Screening and expression of peptide bond synthases

[0053] Peptide synthases are widely distributed in various organisms. Based on the peptide synthase information of Pontimonas salivibrio, Bradyrhizobium sp. SUTN7-2, Arabidopsis thaliana, Sprassis crispa, Bacillus samyloliquefaciens, and Variovorax sp. WDL1, the corresponding amino acid sequences have accession NOs of AVG23412.1, AFJ03901.1, NP_001117233.1, GBE79911.1, QEY93952.1, and ANS57223.1 on NCBI, respectively. The peptide synthase genes psat, bsat, atat, scat, baat, and vwat were synthesized according to the sequence information. The synthesized genes were ligated into the pETDuet-1 vector and induced to express in Escherichia coli BL21(DE3).

[0054] Induction method: Recombinant Escherichia coli was transferred to LB fermentation medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl) at a volume ratio of 2%. When the cell OD... 600After reaching a concentration of 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20°C for 8 h. After induction, cells were collected by centrifugation at 4°C, 8000 rpm for 20 minutes. After cell lysis, the enzyme was purified using the Histag tagging method, and its activity was measured after obtaining the purified enzyme.

[0055] When ursodeoxycholic acid and taurine are used as substrates, the specific enzyme activities expressed by the peptide bond synthase genes psat, bsat, atat, scat, baat, and vwat are 152, 143, 161, 179, 202, and 174 U / mg, respectively.

[0056] Example 2: Construction of recombinant Escherichia coli expressing three enzymes simultaneously

[0057] Construction of recombinant E. coli:

[0058] As shown in Table 1, three plasmids, pACYCDuet-1, pETDuet-1, and pRSFDuet-1, were selected. The genes encoding the three enzymes were ligated to the same plasmid, two plasmids (each expressing two genes), or three plasmids (each expressing one gene). Each gene contained a T7 promoter and an RBS binding site, and each gene was followed by a T7 terminator. The constructed recombinant plasmids were transformed into *Escherichia coli* BL21, and positive transformants were obtained using mixed antibiotic plates, thus yielding recombinant *E. coli* expressing the three genes.

[0059] Recombinant Escherichia coli was induced to express its contents by transferring 2% (v / v) of the recombinant E. coli into LB fermentation medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl). When the cell OD... 600 After reaching a concentration of 0.6-0.8, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 20℃ for 8 h. After induction, cells were collected by centrifugation at 4℃, 8000 rpm for 20 minutes. The collected cells were placed in a 100 ml reaction system with a cell wet weight of 200 g / L, ursodeoxycholic acid 20 g / L, taurine 20 g / L, ATP 1 g / L, sodium hexapolyphosphate 60 g / L, and pH 8. The reaction was carried out at 30℃ for 24 hours. The results are shown in Table 1.

[0060] The collected bacterial cells were placed in a 100 ml reaction system with a cell wet weight of 200 g / L, ursodeoxycholic acid 20 g / L, glycine 20 g / L, ATP 1 g / L, sodium hexapolyphosphate 60 g / L, and pH 8. The reaction was carried out at 30 °C for 24 hours. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] Example 3: In vitro synthesis of tauroursodeoxycholic acid using three enzymes

[0064] The smpkk, ajpkk, and baat genes were ligated into the pETDuet-1 vector to obtain three recombinant vectors. These vectors were then transformed into *Escherichia coli* BL21 to obtain recombinant *E. coli* strains expressing the three enzymes, respectively. After expression and purification using the same method as in Example 1, three pure enzymes were obtained, and their specific activities were measured. The specific activity of smpkk was 13.7 U / mg, that of ajpkk was 40 U / mg, and that of baat was 202 U / mg.

[0065] Then, 2 mg of each of the three pure enzymes, 20 g / L of ursodeoxycholic acid, 20 g / L of taurine, 1 g / L of ATP, 60 g / L of sodium hexapolyphosphate, and pH 8 were added to a 100 ml reaction system; the reaction was carried out at 30 °C for 5 hours, and the final concentration of tauroursodeoxycholic acid in the reaction solution was determined by liquid chromatography to be 30 g / L.

[0066] Example 4: Synthesis of tauroursodeoxycholic acid using three enzymes

[0067] The smpkk, ajpkk, and baat genes were ligated into the pETDuet-1 vector to obtain three recombinant vectors. These vectors were then transformed into *Escherichia coli* BL21 cells to obtain recombinant *E. coli* strains expressing one of the three enzymes. The enzyme expression in the recombinant *E. coli* was induced using the same method as in Example 1. Then, 20 g / L each of the three whole-cell enzymes, 20 g / L ursodeoxycholic acid, 20 g / L taurine, 1 g / L ATP, and 60 g / L sodium hexapolyphosphate were added to a 100 ml reaction system at pH 8. The reaction was carried out at 30°C for 5 hours. The final concentration of tauroursodeoxycholic acid in the reaction solution was determined by liquid chromatography to be 24 g / L.

[0068] Example 5: Synthesis of tauroursodeoxycholic acid using whole-cell catalysis by recombinant Escherichia coli

[0069] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E1) and Escherichia coli BL21(DE3) / pETDuet-1-baat (named E2).

[0070] According to the method described in Example 1, E1 and E2 cells were induced to express the cells, and then the cells were collected. In a 100 ml reaction system, the wet weight of E1 cells was 30 g / L, the wet weight of E2 cells was 50 g / L, ursodeoxycholic acid was 100 g / L, taurine was 10 g / L, sodium hexameric metaphosphate was 300 g / L, ATP was 1 g / L, and pH was 9. The reaction was carried out at 40°C for 48 hours. After transformation, the tauroursodeoxycholic acid content was determined by liquid chromatography to be 148 g / L.

[0071] Example 6: Synthesis of tauroursodeoxycholic acid using whole-cell catalysis by recombinant Escherichia coli

[0072] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E1) and Escherichia coli BL21(DE3) / pACYCDuet-1-scat (named E3).

[0073] According to the method described in Example 1, E1 and E3 cells were induced to express the cells, and then the cells were collected. In a 100 ml reaction system, the wet weight of E3 cells was 100 g / L, the wet weight of E4 cells was 100 g / L, ursodeoxycholic acid was 1 g / L, taurine was 1 g / L, ATP was 1 g / L, sodium hexameric metaphosphate was 3 g / L, and the pH was 5. The reaction was carried out at 15°C for 48 hours. After transformation, the tauroursodeoxycholic acid content was determined by liquid chromatography to be 1.8 g / L.

[0074] Example 7: Synthesis of tauroursodeoxycholic acid using whole-cell catalysis by recombinant Escherichia coli

[0075] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-baat (named E4) and Escherichia coli BL21(DE3) / pACYCDuet-1-smpkk-ajpkk (named E5).

[0076] According to the method described in Example 1, E4 and E5 cells were induced to express their contents, and then the cells were collected. In a 100 ml reaction system, the wet weight of E4 cells was 100 g / L, the wet weight of E5 cells was 100 g / L, ursodeoxycholic acid was 1 g / L, taurine was 1 g / L, sodium hexameric metaphosphate was 3 g / L, ATP was 0.5 g / L, and the pH was 7. The reaction was carried out at 15°C for 1 hour. After transformation, the tauroursodeoxycholic acid content was determined by liquid chromatography to be 4.8 g / L.

[0077] Example 8: Synthesis of tauroursodeoxycholic acid using whole-cell catalysis by recombinant Escherichia coli

[0078] Recombinant bacteria *Escherichia coli* BL21(DE3) / pRSFDuet-1-atat (named E6) and *Escherichia coli* BL21(DE3)pETDuet-1-smpkk-ajpkk (named E7) were constructed. Following the method described in Example 1, the recombinant bacteria were induced to express the bacteria, and then the cells were collected. In a 100 ml reaction system, the cell wet weight was 1 g / L, ursodeoxycholic acid 1 g / L, taurine 1 g / L, ATP 1 g / L, sodium hexapolyphosphate 20 g / L, and the pH was 8. The reaction was carried out at 40 °C for 48 hours. After transformation, the tauroursodeoxycholic acid content was determined by liquid chromatography to be 0.4 g / L.

[0079] Example 9: Synthesis of tauroursodeoxycholic acid using whole-cell catalysis by recombinant Escherichia coli

[0080] The following two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E8) and Escherichia coli BL21(DE3) / pETDuet-1-vwat (named E9).

[0081] According to the method described in Example 1, E1 and E2 cells were induced to express the cells, and then the cells were collected. In a 100 ml reaction system, the wet weight of E1 cells was 30 g / L, the wet weight of E2 cells was 50 g / L, ursodeoxycholic acid was 100 g / L, taurine was 10 g / L, sodium hexapolyphosphate was 300 g / L, ATP was 0.1 g / L, and the pH was 9. The reaction was carried out at 40°C for 48 hours. After transformation, the tauroursodeoxycholic acid content was determined by liquid chromatography to be 113 g / L.

[0082] Example 10: In vitro synthesis of glycoursodeoxycholic acid using three enzymes

[0083] The smpkk, ajpkk, and baat genes were ligated into the pETDuet-1 vector to obtain three recombinant vectors. These vectors were then transformed into *Escherichia coli* BL21 to obtain recombinant *E. coli* strains expressing the three enzymes, respectively. After expression and purification using the same method as in Example 1, three pure enzymes were obtained, and their specific activities were measured: smpkk activity was 13.7 U / mg, ajpkk activity was 40 U / mg, and baat activity was 202 U / mg.

[0084] Then, 2 mg of each of the three pure enzymes, 20 g / L of ursodeoxycholic acid, 20 g / L of glycine, 1 g / L of ATP, and 60 g / L of sodium hexameric metaphosphate were added to a 100 ml reaction system, and the pH was 8. The reaction was carried out at 30 °C for 5 hours, and the final concentration of glycine-ursodeoxycholic acid in the reaction solution was determined by liquid chromatography to be 36 g / L.

[0085] Example 11: Cellular synthesis of glycoursodeoxycholic acid using three enzymes

[0086] The smpkk, ajpkk, and baat genes were ligated into the pETDuet-1 vector to obtain three recombinant vectors. These vectors were then transformed into *Escherichia coli* BL21 cells to obtain recombinant *E. coli* strains expressing one of the three enzymes. The enzyme expression in the recombinant *E. coli* was induced using the same method as in Example 1. Then, 20 g / L each of the three whole-cell enzymes, 20 g / L ursodeoxycholic acid, 20 g / L glycine, 1 g / L ATP, and 60 g / L sodium hexameric metaphosphate were added to a 100 ml reaction system at pH 8. The reaction was carried out at 30°C for 5 hours. The final concentration of ursodeoxycholic acid in the reaction solution was determined by liquid chromatography to be 23 g / L.

[0087] Example 12: Synthesis of glycoursodeoxycholic acid by whole-cell catalysis of recombinant Escherichia coli

[0088] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E1) and Escherichia coli BL21(DE3) / pETDuet-1-baat (named E2).

[0089] According to the method described in Example 1, E1 and E2 cells were induced to express the cells, and then the cells were collected. In a 100 ml reaction system, the wet weight of E1 cells was 30 g / L, the wet weight of E2 cells was 50 g / L, ursodeoxycholic acid was 100 g / L, glycine was 10 g / L, sodium hexameric metaphosphate was 300 g / L, ATP was 1 g / L, and pH was 9. The reaction was carried out at 40°C for 48 hours. After transformation, the glycine-ursodeoxycholic acid content was determined by liquid chromatography to be 162 g / L.

[0090] Example 13: Synthesis of glycoursodeoxycholic acid by whole-cell catalysis of recombinant Escherichia coli

[0091] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E1) and Escherichia coli BL21(DE3) / pACYCDuet-1-scat (named E3).

[0092] According to the method described in Example 1, E1 and E3 cells were induced to express their contents, and then the cells were collected. In a 100 ml reaction system, the wet weight of E1 cells was 100 g / L, the wet weight of E3 cells was 100 g / L, ursodeoxycholic acid was 1 g / L, glycine was 1 g / L, ATP was 1 g / L, sodium hexameric metaphosphate was 3 g / L, and the pH was 5. The reaction was carried out at 15°C for 48 hours. After transformation, the glycine-ursodeoxycholic acid content was determined by liquid chromatography to be 1.3 g / L.

[0093] Example 14: Synthesis of glycoursodeoxycholic acid by whole-cell catalysis of recombinant Escherichia coli

[0094] Two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-baat (named E4) and Escherichia coli BL21(DE3) / pACYCDuet-1-smpkk-ajpkk (named E5).

[0095] According to the method described in Example 1, E5 and E6 cells were induced to express their contents, and then the cells were collected. In a 100 ml reaction system, the wet weight of E5 cells was 100 g / L, the wet weight of E6 cells was 100 g / L, ursodeoxycholic acid was 1 g / L, glycine was 1 g / L, sodium hexameric metaphosphate was 3 g / L, ATP was 0.5 g / L, and the pH was 7. The reaction was carried out at 15°C for 1 hour. After transformation, the glycine-ursodeoxycholic acid content was determined by liquid chromatography to be 1.5 g / L.

[0096] Example 15: Synthesis of glycoursodeoxycholic acid by whole-cell catalysis of recombinant Escherichia coli

[0097] Recombinant bacteria *Escherichia coli* BL21(DE3) / pRSFDuet-1-atat (named E6) and *Escherichia coli* BL21(DE3)pETDuet-1-smpkk-ajpkk (named E7) were constructed. Following the method described in Example 1, the recombinant bacteria were induced to express their contents, and the cells were then collected. In a 100 ml reaction system, the cell wet weight was 1 g / L, ursodeoxycholic acid was 1 g / L, glycine was 1 g / L, ATP was 1 g / L, sodium hexameric metaphosphate was 20 g / L, and the pH was 8. The reaction was carried out at 40 °C for 48 hours. After transformation, the glycine-ursodeoxycholic acid content was determined by liquid chromatography to be 1.8 g / L.

[0098] Example 16: Synthesis of glycoursodeoxycholic acid by whole-cell catalysis of recombinant Escherichia coli

[0099] The following two recombinant bacteria were constructed: Escherichia coli BL21(DE3) / pRSFDuet-1-smpkk-ajpkk (named E8) and Escherichia coli BL21(DE3) / pETDuet-1-vwat (named E9).

[0100] According to the method described in Example 1, E8 and E9 cells were induced to express their contents, and then the cells were collected. In a 100 ml reaction system, the wet weight of E8 cells was 30 g / L, the wet weight of E9 cells was 50 g / L, ursodeoxycholic acid was 100 g / L, glycine was 10 g / L, sodium hexameric metaphosphate was 300 g / L, ATP was 0.1 g / L, and pH was 9. The reaction was carried out at 40°C for 48 hours. After transformation, the glycine-ursodeoxycholic acid content was determined by liquid chromatography to be 146 g / L.

[0101] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A recombinant cell, characterized in that, The recombinant cells expressed peptide bond synthase, polyphosphokinase 2-I, and polyphosphokinase 2-II; the polyphosphokinase 2-I was derived from... Sinorhizobium_meliloti The polyphosphokinase 2-II is derived from Acinetobacter johnsonii The peptide bond synthase comes from Bacillus amyloliquefaciens The recombinant cells are Escherichia coli. Escherichia coli BL21(DE3) was the starting strain, and pETDuet-1 was used as the vector for expression. Bacillus amyloliquefaciens The peptide bond synthase derived from this source was co-expressed using pRSFDuet-1 as a vector. Sinorhizobium_meliloti Polyphosphokinase 2-I and its source Acinetobacter johnsonii Polyphosphokinase 2-II derived from this source; The NCBI accession number for the amino acid sequence of polyphosphokinase 2-I is NP_384613.1, the amino acid sequence of polyphosphokinase 2-II is shown in GenBank: BAC76403.1, and the NCBI accession number for the amino acid sequence of the peptide bond synthase is QEY93952.

1.

2. A method for producing taurodeoxycholic acid or glycoursodeoxycholic acid, characterized in that, Using ursodeoxycholic acid and taurine as raw materials, and utilizing the recombinant cells described in claim 1, taurodeoxycholic acid is prepared and produced; using glycine and ursodeoxycholic acid as raw materials, and utilizing the recombinant cells described in claim 1, glycine-ursodeoxycholic acid is prepared and produced.

3. The method according to claim 2, characterized in that, The recombinant cells were seeded into LB medium, and when the cell OD... 600 After reaching a concentration of 0.6-0.8, IPTG with a final concentration of 0.2-0.5 mM was added and the cells were induced to express for 8 h at 20 °C. The cells were then collected and added to a reaction system containing ursodeoxycholic acid or glycine, taurine, ATP, and sodium hexametaphosphate.

4. The method according to claim 3, characterized in that, React at pH 5-9 and 5-40℃ for 1-48 hours.

5. The use of the recombinant cells of claim 1 in the production of tauroursodeoxycholic acid, products containing tauroursodeoxycholic acid, substances using tauroursodeoxycholic acid as a precursor, glycouroursodeoxycholic acid, products containing glycouroursodeoxycholic acid, and / or substances using glycouroursodeoxycholic acid as a precursor.

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