A recombinant Corynebacterium glutamicum, its construction method and application

Abstract

This invention belongs to the fields of biotechnology and pharmaceutical synthesis technology, specifically relating to a recombinant Corynebacterium glutamicum, its construction method, and its applications. This invention introduces NADH-dependent 7β-hydroxysteroid dehydrogenase (Cle7β-HSDH) into Corynebacterium glutamicum for the first time, combining it with NAD... + A three-enzyme co-expression system was constructed using 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase to ensure that the entire ursodeoxycholic acid synthesis pathway uses NAD+. + NADH, as a coenzyme, solves the technical problem of mismatch between NADH and NADPH coenzymes in existing technologies. It can efficiently drive catalytic reactions with only a single coenzyme regeneration system, without the need for expensive external coenzymes or the introduction of glucose dehydrogenase and its substrates, thus significantly reducing production costs. Furthermore, the molar conversion rate of the substrate CDCA (based on the generated UDCA) is as high as 83.89%, and it can tolerate high concentrations of substrate. The product is mainly secreted extracellularly, which facilitates separation and purification.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and pharmaceutical synthesis technology, specifically relating to a recombinant Corynebacterium glutamicum, its construction method, and its application. Background Technology

[0002] Ursodeoxycholic acid (UDCA) is a widely used drug for treating hepatobiliary diseases and also has adjuvant therapeutic effects on certain cancers and neurological disorders. Currently, the main methods for synthesizing UDCA include chemical methods and biotransformation methods. Chemical methods suffer from problems such as cumbersome reaction steps, use of toxic reagents, poor stereoselectivity, and severe environmental pollution. Biotransformation methods, due to their advantages of high efficiency and environmental friendliness, have become the mainstream research method, mainly divided into two pathways: free enzyme catalysis and whole-cell catalysis. Free enzyme catalysis requires cell disruption and enzyme purification, which is a cumbersome process, and the enzyme has poor extracellular stability, especially requiring the addition of expensive coenzymes (NAD). + The presence of NADPH (non-diethylaminopropyl di ...

[0003] However, existing whole-cell catalysis technologies still face many bottlenecks in practical applications. Regarding host selection, current technologies mostly use *E. coli* as the expression host. Although its genetic system is mature, it poses endotoxin safety risks, and there is still room for improvement in its tolerance to high substrate concentrations and transformation efficiency. In terms of multi-enzyme co-expression, the synthesis of UDCA involves two key enzymatic reactions: 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 7β-hydroxysteroid dehydrogenase (7β-HSDH), and requires coenzyme regeneration such as lactate dehydrogenase (LDH) and glucose dehydrogenase (GDH). Currently, efficiently and balancedly co-expressing these four enzymes in a single strain for whole-cell catalysis of high substrate concentrations remains a technical challenge. Most constructed four-enzyme co-expression strains suffer from enzyme activity mismatch, intermediate product accumulation, low conversion rates, or the need for additional large amounts of coenzymes.

[0004] Furthermore, the UDCA enzymatic synthesis route inherently presents the challenge of coenzyme system mismatch. Specifically, the reaction catalyzed by 7α-HSDH to produce 7-ketolithocholic acid (7K-LCA) from chenodeoxycholic acid (CDCA) typically uses NAD+. +As a coenzyme, NADH is generated; however, most reported 7β-HSDH (e.g., those derived from Ruminococcus truncatula, Olsenella, Collins, etc.) are strictly NADPH-dependent, and their catalytic reduction of 7K-LCA to UDCA requires the consumption of NADPH. This results in two coenzymes, NADH and NADPH, operating in parallel within the same catalytic system, creating a "mismatch" in metabolic pathways. This mismatch presents several technical obstacles: First, to maintain the NADH and NADPH cycles simultaneously, two independent coenzyme regeneration systems must be introduced (e.g., LDH regenerating NADH, and GDH regenerating NADPH), significantly increasing the difficulty of strain construction and metabolic burden. Second, the regeneration efficiency of NADPH is often lower than that of NADH, and the substrate glucose for GDH is expensive, making it difficult to completely eliminate dependence on additional coenzymes or their precursors in industrial production. Third, industrial hosts such as Corynebacterium glutamicum and Escherichia coli have high requirements for NADPH during growth and product synthesis (e.g., Corynebacterium glutamicum is used for lysine synthesis), and the additional introduction of NADPH consumption pathways will further intensify intracellular reducing power competition, affecting cell growth and catalytic stability.

[0005] To address the aforementioned coenzyme mismatch issue, a few studies have attempted to modify the naturally occurring NADPH-dependent 7β-HSDH to a NADH-dependent form through protein engineering. However, directed evolution or rational design processes are complex, have long screening cycles, and mutants often exhibit significant decreases in catalytic activity or stability, making them unsuitable for industrial applications. Furthermore, when using multiple single-enzyme or dual-enzyme engineered bacteria for catalysis, intermediate products need to shuttle between different cells, leading to high mass transfer resistance and low conversion efficiency. Therefore, existing technologies still require further improvement. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a recombinant Corynebacterium glutamicum, its construction method, and its application.

[0007] To achieve the above objectives, the present invention can adopt the following technical solutions: The present invention provides a recombinant Corynebacterium glutamicum, which expresses the following three enzymes: 7α-hydroxysteroid dehydrogenase, NADH-dependent 7β-hydroxysteroid dehydrogenase, and lactate dehydrogenase.

[0008] Preferably, in the above-mentioned recombinant Corynebacterium glutamicum, the amino acid sequence of the 7α-hydroxysteroid dehydrogenase is as shown in SEQ ID NO:1, or is an enzyme with more than 90% sequence identity with SEQ ID NO:1 and having 7α-hydroxysteroid dehydrogenase activity; the amino acid sequence of the NADH-dependent 7β-hydroxysteroid dehydrogenase is as shown in SEQ ID NO:2, or is an enzyme with more than 90% sequence identity with SEQ ID NO:2 and having NADH-dependent 7β-hydroxysteroid dehydrogenase activity; and the amino acid sequence of the lactate dehydrogenase is as shown in SEQ ID NO:3, or is an enzyme with more than 90% sequence identity with SEQ ID NO:3 and having lactate dehydrogenase activity.

[0009] Preferably, in the above-mentioned recombinant Corynebacterium glutamicum, the genes encoding the three enzymes are introduced into the Corynebacterium glutamicum via one or more recombinant expression vectors, or integrated into the chromosome of the Corynebacterium glutamicum.

[0010] More preferably, in the above-mentioned recombinant Corynebacterium glutamicum, the genes encoding the three enzymes are constructed on the same recombinant plasmid and expressed under the control of their respective independent promoters.

[0011] Another aspect of the present invention provides a method for constructing the above-mentioned recombinant Corynebacterium glutamicum, the method comprising: introducing genes encoding 7α-hydroxysteroid dehydrogenase, NADH-dependent 7β-hydroxysteroid dehydrogenase and lactate dehydrogenase into Corynebacterium glutamicum to obtain a recombinant strain co-expressing the three enzymes.

[0012] In another aspect, the present invention provides a method for the whole-cell catalytic production of ursodeoxycholic acid, the method comprising: using the above-mentioned recombinant Corynebacterium glutamicum as a whole-cell catalyst and chenodeoxycholic acid as a substrate, in a reaction system containing a carbon source, to generate ursodeoxycholic acid through a whole-cell catalytic reaction.

[0013] Preferably, in the above method, the reaction system contains coenzyme NAD. + and / or NADH, the coenzyme NAD + The amount of NADH added should not exceed 0.01 mM.

[0014] Preferably, in the above method, the carbon source includes glucose and / or sodium pyruvate; and / or the reaction system contains a buffer solution with a pH of 7.5-8.5.

[0015] Preferably, in the above method, the conditions for the whole-cell catalytic reaction include: a substrate chenodeoxycholic acid concentration of 50mM-150mM; and / or an addition amount of 5g / L-100g / L of recombinant Corynebacterium glutamicum; and / or a reaction temperature of 30℃-40℃ and a reaction time of 1-24 hours.

[0016] More preferably, in the above method, the conditions for the whole-cell catalytic reaction include: a substrate chenodeoxycholic acid concentration of 100 mM, an addition amount of recombinant Corynebacterium glutamicum cells of 5 g / L-10 g / L, a carbon source of glucose with a glucose concentration of 20 g / L, a reaction temperature of 30℃-35℃, a reaction time of 4 hours, and a reaction system pH of 7.5-8.0.

[0017] The beneficial effects of this invention include at least the following: This invention introduces, for the first time, a natural NADH-dependent 7β-hydroxysteroid dehydrogenase (Cle7β-HSDH) derived from the chicken intestinal metagenomics into Corynebacterium glutamicum, which, together with NAD... + A three-enzyme co-expression system was constructed using 7α-hydroxysteroid dehydrogenase and lactate dehydrogenase to ensure that the entire ursodeoxycholic acid synthesis pathway uses NAD+. + Using NADH as a coenzyme solves the technical problem of NADH / NADPH dual coenzyme mismatch in existing technologies. It can efficiently drive the catalytic reaction with only a single coenzyme regeneration system, without the need for expensive external coenzymes or the introduction of glucose dehydrogenase and its substrate, thus significantly reducing production costs. Furthermore, the molar conversion rate of the substrate CDCA (based on the generated UDCA) is as high as 83.89%, and it can tolerate high concentrations of substrate. The product is mainly secreted extracellularly, which facilitates separation and purification. At the same time, the use of food-grade Corynebacterium glutamicum as a host avoids the risk of endotoxins. The process is green and environmentally friendly, the difficulty of balancing multi-enzyme co-expression is low, the metabolic burden is light, and it has significant prospects for industrial application. Attached Figure Description

[0018] Figure 1 For recombinant plasmid pXMJ19- 7α-hsdh A schematic diagram of its construction; Figure 2 For recombinant plasmid pXMJ19- 7β-hsdh A schematic diagram of its construction; Figure 3 For recombinant plasmid pXMJ19- ldh A schematic diagram of its construction; Figure 4 To verify the recombinant Corynebacterium glutamicum by bacterial culture PCR ( 7α-hsdh Electrophoresis diagram of gene amplification; where lane 1 is the marker, lane 2 is the negative control, and lanes 3-5 are positive transformants; Figure 5To verify the recombinant Corynebacterium glutamicum by bacterial culture PCR ( 7β-hsdh Electrophoresis diagram of gene amplification; where lane 1 is the marker, lane 2 is the negative control, and lanes 3-5 are positive transformants; Figure 6 To verify the recombinant Corynebacterium glutamicum by bacterial culture PCR ( ldh Electrophoresis diagram of gene amplification; where lane 1 is the marker, lane 2 is the negative control, and lanes 3-5 are positive transformants; Figure 7 SDS-PAGE image of recombinant Corynebacterium glutamicum expressing the target protein (7α-HSDH); lane 1 is the marker, lane 2 is the sample before induction, and lanes 3 and thereafter are the samples after induction. Figure 8 SDS-PAGE image of recombinant Corynebacterium glutamicum expressing the target protein (Cle7β-HSDH); lane 1 is the marker, lane 2 is the sample before induction, and lanes 3 and thereafter are the samples after induction. Figure 9 SDS-PAGE image of recombinant Corynebacterium glutamicum expressing the target protein (LDH); lane 1 is the marker, lane 2 is the sample before induction, and lanes 3 and thereafter are the samples after induction. Figure 10 The images show the thin-layer chromatography (TLC) results of the enzyme-catalyzed reaction products. In lane A, 7α-HSDH catalyzes the formation of 7K-LCA from CDCA; lanes 1 and 2 are standards for CDCA and 7K-LCA, respectively; lane 3 is the blank control; lane 4 shows the crude enzyme reaction product before induction; and the remaining lanes show the crude enzyme reaction product after induction. In lane B, 7β-HSDH catalyzes the formation of UDCA from 7-KLCA; lanes 1 and 2 are standards for 7-KLCA and UDCA, respectively; lane 3 is the blank control; lane 4 shows the crude enzyme reaction product before induction; and the remaining lanes show the crude enzyme reaction product after induction. Figure 11 High-performance liquid chromatography (HPLC) of the standard and the whole-cell catalytic products of the fermentation broth. Figure 12 This is a thin-layer chromatography (TLC) image of the whole-cell catalytic products of the fermentation broth; lanes 1 and 2 are CDCA and UDCA standards, respectively, lane 3 is the supernatant of the fermentation broth, and lane 4 is the intracellular extract of the bacteria. Detailed Implementation

[0019] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0020] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0021] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0022] In the following examples, the chenodeoxycholic acid (CDCA) used is from Yuan Ye (product number: S25542, specification: 25g, purity ≥98%), and the ursodeoxycholic acid (UDCA) used is from Aladdin (product number: U110695, specification: 5g, purity ≥99%).

[0023] In the following examples, the 7α-hydroxysteroid dehydrogenase gene (7α-hsdh), the NADH-dependent 7β-hydroxysteroid dehydrogenase gene derived from the chicken intestinal metagenomics (Cle7β-HSDH, NCBI accession number: HIX02396.1), and the lactate dehydrogenase gene (ldh) derived from Corynebacterium glutamicum have all undergone codon optimization for Corynebacterium glutamicum. Their nucleotide sequences and encoded amino acid sequences are shown below: (1) 7α-hsdh gene nucleotide sequence (SEQ ID NO:4) ATGAACCGCTTCGAAAACAAGATCATCATCATCACCGGCGCAGCAGGCGGCATCGGTGCATCCACCACCCGCCGCATTGTGTCCGAGGGTGGCAAGGTTGTTATCGCAGATTACTCTCGCGAAAAAGCAGATCAGTTCGCAGCTGAGCTGTCCAACTCCGGCGCAGATGTGCGCCCAGTCTACTTCTCCGCTACCGAACTGAAGTCCTGCAAGGAACTGATCACCTTCACCATGAAGGAATACGGCCAGATCGATGTTCTGGTGAACAACGTGGGCGGCACCAACCCACGCCGCGATACCAATATTGAAACACTTGATATGGACTATTTTGATGAAGCATTCCACCTGAACCTGTCCTGCACCATGTACCTGTCCCAGCTGGTGATCCCAATCATGTCCACCCAGGGCGGGGGCAACATCGTTAACGTGGCATCCATCTCCGGCATCACCGCTGATTCCAACGGCACCCTGTACGGCGCATCCAAGGCTGGCGTGATCAACCTGACCAAGTACATCGCAACCCAGACCGGTAAGAAGAACATCCGCTGCAACGCAGTCGCACCAGGCCTGATCCTGACCCCAGCAGCTCTCAACAACCTGAACGAAGAAGTCCGCAAGATCTTCCTGGGCCAGTGCGCTACCCCATACCTGGGCGAACCACAGGATGTGGCTGCAACCATCGCATTCCTGGCTTCCGAAGATGCACGCTACATCACCGGCCAGACCATCGTGGTAGATGGCGGCCTGACCATCCATAACCCAACCATCAACCTGGTG。

[0024] 7α - HSDH amino acid sequence (SEQ ID NO:1) MNRFENKIIIITGAAGGIGASTTRRIVSEGGKVVIADYSREKADQFAAELSNSGADVRPVYFSATELKSCKELITFTMKEYGQIDVLVNNVGGTNPRRDTNIETLDMDYFDEAFHLNLSCTMYLSQLVIPIMSTQGGGNIVNVASISGITADSNGTLYGASKAGVINLTKYIATQTGKKNIRCNAVAPGLILTPAALNNLNEEVRKIFLGQCATPYLGEPQDVAATIAFLASEDARYITGQTIVVDGGLTIHNPTINLV。

[0025] (2)Nucleotide sequence of Cle 7β-hsdh gene (SEQ ID NO:5) ATGACCCTGTCCGATTTCCAGGCAAAGTACGGTAAATACGCAGTTCTGTTCGGCGGCGCAGATGGCCTGGGCGCTGAAACCGCAAAGAAGCTGGCTGAAAAGGGCCTGTCCATCATCTGCGTTGATTACTCCCAGGAAAAGCTGGATCAGTTCGAAGCAGAATTTCGCAAGATCTACTCCGTTGATTTCATCCCAGTGAAGATCGATCTGTCCGAAGAAAACGCAGTTCTTGATGTTTTCGATGTTACCGATCGTCTGGATGTTGGTTTCGTTTCCTACATCGCAGCACTGCACAAGTTCGGTAAGATCCAGGATATCTCCTGGGATGATTACATGAAGATGTTCAACGTTAACATCCTGAACTTCACCAAGGCAATGAAGCACTACATGGGCATCTTCGTTGAACAGGGCCACGGTGGCATCATGAACTACTCCTCCCTGACCGCACTGACCTCCTCCCCATACAACGTTGAATACGGTGCAGGCAAGGCATACATCAAGTCCTTCACCCAGGCAATGGCATACGAAGGCGAAAAGGAAGGCGTGGATGTTATGGTTGCAACCCTGGGTGCAACCGCAACCCCAACCGAACTTAAGGCTCAGCCACAGGGTGATCTGGGTGCAAAGATCCAGTCCATGGCATTGACCCCAGAAGATACCGTTAACGAAATCTTCGATAACTTCGGCAAGGTGCACTCCTACTACGTGGGTGAACACCCAAAGTCCCAGGTGAAGAAGTGGCGCATCGAAAACGATGATGATGGCCTGGCAGAATACATGGGTAAGTTCTACGAATAA。

[0026] Cle 7β-HSDH amino acid sequence (SEQ ID NO:2) MTLSDFQAKYGKYAVLFGGADGLGAETAKKLAEKGLSIICVDYSQEKLDQFEAEFRKIYSVDFIPVKIDLSEENAVLDVFDVTDRLDVGFVSYIAALHKFGKIQDISWDDYMKMFNVNILNFTKAMKHYMGIFVEQGHGGIMNYSSLTALTSSPYNVEYGAGKAYIKSFTQAMAYEGEKEGVDVMVATLGATATPTELKAQPQGDLGAKIQSMALTPEDTVNEIFDNFGKVHSYYVGEHPKSQVKKWRIENDDDGLAEYMGKFYE。

[0027] (3) Nucleotide sequence of the ldh gene (SEQ ID NO: 6)

[0028] LDH amino acid sequence (SEQ ID NO:3) .

[0029] Preparation Example Example 1: Construction of Recombinant Corynebacterium glutamicum (1) Obtaining the target gene Three target genes were artificially synthesized after codon optimization of Corynebacterium glutamicum: 7α-hydroxysteroid dehydrogenase gene (7α-hsdh) from Bacteroides fragilis, NADH-dependent 7β-hydroxysteroid dehydrogenase gene (Cle7β-HSDH, NCBI accession number: HIX02396.1) from chicken intestinal metagenomics, and lactate dehydrogenase gene (ldh) from Corynebacterium glutamicum.

[0030] (2) Construction of recombinant plasmids 1) The three target genes synthesized above were ligated into the same digested shuttle expression vector pXMJ19 via double digestion (Hind III / Eco RⅠ) to construct three independent expression plasmids: pXMJ19-7α-hsdh, pXMJ19-Cle7β-HSDH, and pXMJ19-ldh (the construction diagrams of the above recombinant plasmids pXMJ19-7α-hsdh, pXMJ19-Cle7β-HSDH, and pXMJ19-ldh are shown in the figure below). Figure 1 , Figure 2 and Figure 3 (As shown).

[0031] 2) The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing chloramphenicol, and incubated overnight at 37°C. Single colonies were picked for colony PCR verification, and positive clones were sent for sequencing. Recombinant plasmids with correct sequencing results were used for subsequent experiments.

[0032] (3) Transformation of Corynebacterium glutamicum with recombinant plasmid The successfully constructed recombinant plasmids (pXMJ19-7α-hsdh, pXMJ19-Cle7β-HSDH, and pXMJ19-ldh) were electroporated into competent Corynebacterium glutamicum cells. The electroporation parameters were: voltage 1800 V, resistance 200 Ω, and duration 5 ms. After electroporation, the bacterial culture was heat-shocked at 46 °C for 6 min, followed by recovery culture at 30 °C for 2 h. The recovered bacterial culture was plated on LBHIS plates containing chloramphenicol and incubated at 30 °C for 48 h. Single colonies were then picked.

[0033] (4) Verification of positive transformants Using selected single colonies as templates, bacterial culture PCR was performed using primers specific to each gene for verification. The specific steps are as follows: 1) Preparation of bacterial culture PCR template Select a single, morphologically intact colony from a chloramphenicol-resistant plate and inoculate it into 5 mL of LBHIS liquid medium (containing 10 μg / mL chloramphenicol). Incubate at 30°C with shaking at 200 rpm until the medium becomes noticeably turbid. Use this bacterial culture directly as a PCR template (no need to extract genomic DNA) for PCR amplification. The PCR reaction system (total volume 50 μL), PCR amplification program, and amplification primers are shown in Tables 1, 2, and 3 below.

[0034] Table 1 PCR reaction system (total volume 50 μL)

[0035] Table 2 PCR amplification program

[0036] Table 3 Specific primer sequences

[0037] 2) Result determination Take 5 μL of PCR product and perform 1% agarose gel electrophoresis and UV imaging. If a single clear band appears at the expected size, and the negative control (empty bacteria) shows no band, it is considered a positive recombinant.

[0038] Agarose gel electrophoresis results are as follows Figure 4 , Figure 5 and Figure 6 shown. Specifically, Figure 4 The result is the amplification of the 7α-hsdh gene (777 bp). Figure 5 The result is the amplification of the Cle7β-HSDH gene (795 bp). Figure 6 The results show the amplification of the ldh gene (1715 bp). The results show that the experimental group showed a single clear band at the corresponding size position, while the negative control showed no band, proving that the three recombinant plasmids have been successfully transformed into Corynebacterium glutamicum.

[0039] Characterization test In the following tests, OD 600 =0.6-0.8 represents the optical density value of recombinant Corynebacterium glutamicum cultured in LBHIS liquid medium during the mid-logarithmic growth phase. At this stage, the bacteria are metabolically active and most suitable for IPTG-induced expression. This parameter is a process control indicator for the fermentation stage, used to determine the optimal induction time. A cell wet weight of 5g / L-10g / L represents the whole-cell catalytic reaction stage, where the bacterial cells collected after fermentation (after centrifugation and washing) are resuspended in the reaction system at their wet weight. This parameter is a feed control indicator for the catalytic reaction stage. These two stages belong to different operational phases (induction expression stage and whole-cell catalytic stage), and the culture medium composition and treatment methods (whether centrifugation and washing, and the type of buffer used for resuspension, etc.) of the bacteria are different; therefore, there is no direct numerical conversion relationship. This invention independently optimized the parameters for each stage, each representing the optimal implementation conditions for that stage.

[0040] (I) Induction of target protein expression and SDS-PAGE verification (1) Induced expression The recombinant Corynebacterium glutamicum positive transformants verified in Example 1 were inoculated into LBHIS liquid medium and cultured at 30°C with shaking at 200 rpm until OD500. 600 The concentration was 0.6-0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the cells were induced at 30°C for another 24 hours. Cells were collected before and after induction for subsequent analysis.

[0041] (2) Preparation of crude enzyme solution The collected bacterial cells were washed with PBS buffer (pH 7.5) and resuspended in an appropriate amount of PBS buffer. They were then disrupted by sonication or high-pressure homogenization. The mixture was centrifuged at 12,000 rpm for 15 min at 4°C, and the supernatant was collected to obtain the crude enzyme solution.

[0042] (3) SDS-PAGE detection Add the crude enzyme solution to the protein loading buffer, denature in a boiling water bath for 10 min, and then perform SDS-PAGE electrophoresis for detection.

[0043] The results are as follows Figure 7 , Figure 8 and Figure 9 shown, specifically, Figure 7 The expression results for 7α-HSDH Figure 8 The expression results for Cle7β-HSDH are as follows. Figure 9 The results show the expression of LDH. The results indicate that, compared to the pre-induction lanes, all sample lanes after induction showed distinct specific protein bands, the sizes of which corresponded to the theoretical molecular weights of the target proteins: 7α-HSDH between 25kDa and 35kDa, Cle7β-HSDH with a theoretical molecular weight of 28.5kDa (SDS-PAGE showed a molecular weight between 28kDa and 30kDa), and LDH between 55kDa and 70kDa. All three target proteins were successfully expressed, and no non-specific bands were detected.

[0044] (II) Enzyme Function Verification (1) Verification of α-HSDH enzyme activity Recombinant Corynebacterium glutamicum cells were collected after 24 hours of induction using the method described above, and crude enzyme solution was prepared. The reaction system (total volume 200 μL) contained: 100 μL 50 mM Tris-HCl buffer (pH=7.5), 16 μL 50 mM chenodeoxycholic acid (CDCA), and 4 μL 100 mM coenzyme NAD. + 80 μL of crude enzyme solution was added. The system containing the crude enzyme solution before induction and the system without crude enzyme solution were used as controls. The reaction system was incubated in a water bath at 35 °C in the dark for 1 hour. After the reaction was completed, an equal volume of ethyl acetate was added to terminate the reaction, and the product was extracted. The upper organic phase was then analyzed by thin-layer chromatography (TLC).

[0045] TLC test results are as follows Figure 10As shown in Figure A, lane 1 contains the CDCA standard, lane 2 contains the 7-ketolithocholic acid (7K-LCA) standard, lane 3 is the blank control (without crude enzyme solution), lane 4 contains the product of the crude enzyme solution reaction before induction, and the remaining lanes contain the product of the crude enzyme solution reaction after induction. The results show that the crude enzyme solution after induction can efficiently catalyze the conversion of CDCA to 7K-LCA, with obvious product spots appearing on the TLC plate corresponding to the 7K-LCA standard position; while no obvious product was generated in the crude enzyme solution before induction or the blank control, indicating that the recombinantly expressed 7α-HSDH has catalytic activity.

[0046] (2) Verification of Cle7β-HSDH enzyme activity Recombinant Corynebacterium glutamicum cells were collected after 24 hours of induction using the method described above, and crude enzyme solution was prepared. The reaction system (total volume 200 μL) contained: 100 μL 50 mM Tris-HCl buffer (pH=7.5), 16 μL 50 mM substrate 7K-LCA, 4 μL 100 mM coenzyme NADH, and 80 μL crude enzyme solution. The system before induction and the system without crude enzyme solution were used as controls. The reaction system was incubated in a 35°C water bath in the dark for 1 hour. After the reaction, an equal volume of ethyl acetate was added to terminate the reaction, and the product was extracted. The upper organic phase was analyzed by TLC.

[0047] TLC test results are as follows Figure 10 As shown in Figure B, lane 1 contains 7K-LCA standard, lane 2 contains ursodeoxycholic acid (UDCA) standard, lane 3 is the blank control (without crude enzyme solution), lane 4 contains the product of the crude enzyme solution reaction before induction, and the remaining lanes contain the product of the crude enzyme solution reaction after induction. The results showed that the crude enzyme solution after induction efficiently catalyzed the conversion of 7K-LCA to UDCA, with obvious product spots appearing on the TLC plate corresponding to the UDCA standard position; while no obvious product was generated in the crude enzyme solution before induction or the blank control, indicating that the recombinantly expressed Cle7β-HSDH possesses NADH-dependent 7β-hydroxysteroid dehydrogenase activity.

[0048] Application testing The HPLC chromatographic conditions for the following tests were as follows: Instrument: Agilent 1260 high performance liquid chromatograph; Column: Ultimate XB-C18 (4.6×150mm, 5μm); Column temperature: 40℃; Mobile phase: methanol:acetonitrile:water = 110:100:90 (volume ratio), pH adjusted to 2.60 with phosphoric acid; Flow rate: 1.0mL / min; Detector: refractive index detector (RID), temperature 40℃; Injection volume: 5μL; Sample preparation: After centrifugation of the fermentation broth, the supernatant was collected, filtered through a 0.22μm filter membrane, and injected directly.

[0049] In the following tests, the external standard method is used for quantification. Specifically, a standard curve is plotted with the concentration of the standard on the x-axis and the peak area on the y-axis. The concentration of each component in the test sample is calculated using the following formula: C sample =A sample / A standard ×C standard ; Where A is the peak area and C is the concentration.

[0050] (1) Whole-cell catalytic reaction The recombinant Corynebacterium glutamicum positive transformants verified in Example 1 were inoculated into LBHIS liquid medium and cultured at 30°C with shaking at 200 rpm until OD500. 600 The concentration was 0.6-0.8. IPTG (inducer isopropyl-β-D-thiogalactoside) was added to a final concentration of 0.5 mM for induction, while CDCA substrate was added to a final concentration of 100 mM. Glucose (final concentration 20 g / L) was then added as a carbon source for coenzyme regeneration. The mixture was cultured at 30°C and 200 rpm for 24 hours to obtain the fermentation broth.

[0051] (2) Product extraction and detection In the following tests, the TLC detection conditions included: the developing solvent was dichloromethane:acetone:acetic acid = 30:60:1 (volume ratio), and the colorimetric reaction was performed using 10% sulfuric acid methanol solution; the HPLC detection conditions included: a C18 reversed-phase column (4.6 × 250 mm, 5 μm), a mobile phase of acetonitrile:0.02% phosphoric acid aqueous solution = 45:55 (volume ratio), a flow rate of 1.0 mL / min, and a detection wavelength of 210 nm.

[0052] After the reaction was complete, 1 mL of fermentation broth was taken, an equal volume of ethyl acetate was added, the mixture was vigorously shaken and centrifuged, and the upper organic phase was concentrated and analyzed by TLC or HPLC. The results showed (see...). Figure 11 The presence of chromatographic peaks in the reaction solution sample with retention times consistent with those of the UDCA standard indicates that CDCA was successfully converted into UDCA.

[0053] In addition, the fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant and cell pellet were collected separately. The cell pellet was washed with PBS buffer and resuspended, and intracellular material was extracted by sonication. Then, the supernatant and intracellular extract were extracted with ethyl acetate and analyzed by TLC. The results showed (see...) Figure 12 A large amount of UDCA and a small amount of residual substrate CDCA were detected in the supernatant of the fermentation broth, while only a small amount of the intermediate product 7K-LCA was detected in the intracellular extract. This indicates that UDCA is efficiently secreted into the extracellular space, facilitating subsequent separation and purification.

[0054] In addition, using 100 mM CDCA as a substrate, and under optimal reaction conditions (10 g / L bacterial cell addition, 20 g / L glucose, 30℃, pH 7.5, reaction time 4 h), the whole-cell catalysis results are shown in Tables 4 and 5 below.

[0055] Table 4. Unconversion control (the test sample was a blank reaction solution without sterile addition)

[0056] Table 5. Test samples after whole-cell catalytic reaction

[0057] Conversion rate (in molar percentage of UDCA in total product): =C UDCA / (C UDCA +C 7K-LCA +C CDCA) ×100%=0.8242 / (0.8242+0.09406+0.06427)×100%≈83.89%, This result is consistent with TLC qualitative detection ( Figure 12 The high degree of consistency indicates that the recombinant strain of the present invention can achieve efficient conversion of CDCA to UDCA (conversion rate of 83.89%) without the addition of expensive coenzymes, and the product is mainly secreted into the extracellular space.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A recombinant Corynebacterium glutamicum, characterized in that, The recombinant Corynebacterium glutamicum expresses the following three enzymes: 7α-hydroxysteroid dehydrogenase, NADH-dependent 7β-hydroxysteroid dehydrogenase, and lactate dehydrogenase.

2. The recombinant Corynebacterium glutamicum according to claim 1, characterized in that, The amino acid sequence of the 7α-hydroxysteroid dehydrogenase is shown in SEQ ID NO:1, or it is an enzyme that has more than 90% sequence identity with SEQ ID NO:1 and has 7α-hydroxysteroid dehydrogenase activity. The amino acid sequence of the NADH-dependent 7β-hydroxysteroid dehydrogenase is shown in SEQ ID NO:2, or it is an enzyme that has more than 90% sequence identity with SEQ ID NO:2 and has NADH-dependent 7β-hydroxysteroid dehydrogenase activity. The amino acid sequence of the lactate dehydrogenase is shown in SEQ ID NO:3, or it is an enzyme that has more than 90% sequence identity with SEQ ID NO:3 and has lactate dehydrogenase activity.

3. The recombinant Corynebacterium glutamicum according to claim 1 or 2, characterized in that, The genes encoding the three enzymes are introduced into the *Corynebacterium glutamicum* via one or more recombinant expression vectors, or integrated into the chromosome of the *Corynebacterium glutamicum*.

4. The recombinant Corynebacterium glutamicum according to claim 3, characterized in that, The genes encoding the three enzymes were constructed on the same recombinant plasmid and expressed under the control of their respective independent promoters.

5. The method for constructing recombinant Corynebacterium glutamicum according to any one of claims 1 to 4, characterized in that, The construction method includes: introducing genes encoding 7α-hydroxysteroid dehydrogenase, NADH-dependent 7β-hydroxysteroid dehydrogenase and lactate dehydrogenase into Corynebacterium glutamicum to obtain a recombinant strain that co-expresses the three enzymes.

6. A method for the whole-cell catalytic production of ursodeoxycholic acid, characterized in that, The method includes: using the recombinant Corynebacterium glutamicum as described in any one of claims 1 to 4 as a whole-cell catalyst and chenodeoxycholic acid as a substrate, ursodeoxycholic acid is generated by whole-cell catalytic reaction in a reaction system containing a carbon source.

7. The method according to claim 6, characterized in that, The reaction system contains coenzyme NAD. + and / or NADH, the coenzyme NAD + The amount of NADH added should not exceed 0.01 mM.

8. The method according to claim 6 or 7, characterized in that, The carbon source includes glucose and / or sodium pyruvate; and / or The reaction system contains a buffer solution with a pH of 7.5-8.

5.

9. The method according to claim 6 or 7, characterized in that, The conditions for the whole-cell catalytic reaction include The substrate concentration of chenodeoxycholic acid is 50 mM-150 mM; and / or The addition amount of recombinant Corynebacterium glutamicum is 5 g / L-100 g / L; and / or The reaction temperature is 30℃-40℃, and the reaction time is 1-24 hours.

10. The method according to claim 9, characterized in that, The conditions for the whole-cell catalytic reaction include: The substrate chenodeoxycholic acid concentration was 100 mM, the amount of recombinant Corynebacterium glutamicum added was 5 g / L~10 g / L, the carbon source was glucose at a concentration of 20 g / L, the reaction temperature was 30℃~35℃, the reaction time was 4 hours, and the pH of the reaction system was 7.5~8.0.

Images