A mutant of 7β-HSDH derived from Ruminococcus gnavus strain that improves the conversion rate from 7-keto-LCA to ursodeoxycholic acid and a method for producing ursodeoxycholic acid using the same

Abstract

This invention relates to a mutant of 7β-HSDH (7β-hydroxysteroid) derived from Ruminococcus gnavus strain that improves the conversion rate of 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA), and a method for producing ursodeoxycholic acid using the mutant. In this invention, the conversion efficiency of 7-keto-LCA to UDCA has been improved through the production of mutants by enzyme improvement and the development of recombinant protein production, and it is expected that this can be widely used for improving UDCA biosynthesis and biosynthetic efficiency.

Description

[Technical field]

[0001] This invention relates to a mutant of 7β-HSDH (7β-hydroxysteroid) derived from a Ruminococcus gnavus strain that improves the conversion rate of 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA), and a method for producing ursodeoxycholic acid using the mutant. [Background technology]

[0002] Bile acids are the main components of bile and are biomolecules necessary for the digestion and absorption of fats, fatty acids, and hydrophobic vitamins. They are stable substances that are poorly metabolized, and various hydroxy groups are located at different carbon positions in the center of the cholesterol ring, which causes differences in biochemical function and solubility. Among the various types of bile acids, ursodeoxycholic acid (UDCA) is one of the bile acids found in small amounts. UDCA has recently been shown to have therapeutic functions in dissolving cholesterol-containing gallstones. Such compounds are industrially produced in tonne quantities by chemical or enzymatic steps. An important precursor for the synthesis of UDCA is 7-keto-LCA (7-keto lithocholic acid), which can be converted to UDCA by reacting 7-keto-LCA with n-butanol using sodium as a catalyst. In the case of the enzymatic method, the conversion to ursodeoxycholic acid (UDCA) is carried out using the enzyme catalysis catalyzed by 7β-HSDH (7β-hydroxysteroid), which can stereoselectively reduce the 7-keto group. Such a step requires the coenzyme NAD(P)H in the reaction using the enzyme catalysis catalyzed by 7β-HSDH (7β-hydroxysteroid). However, in the case of the conversion reaction from 7-Keto-LCA (7-keto lithocholic acid) to UDCA using the conventional 7β-HSDH (7β-hydroxysteroid) derived from Ruminococcus gnavus strain, when 30 g / L or more of 7-Keto-LCA (7-keto lithocholic acid) is present as a substrate, there is a problem that the conversion ability decreases due to inhibition by the substrate concentration.

[0003] Therefore, research is needed to alleviate the effect of the decrease in 7β-HSDH (7β-hydroxysteroid) enzyme activity that occurs with increasing substrate concentration by changing the amino acid sequence of the substrate binding site, or to mass-produce the improved enzyme using recombinant E. coli, but research in this field is still insufficient. Summary of the Invention [Problem to be solved by the invention]

[0004] One aspect is to provide a 7β-HSDH (7β-hydroxysteroid) mutant comprising an amino acid sequence mutation of 7β-HSDH derived from a Ruminococcus gnavus strain.

[0005] Another aspect is to provide a recombinant vector comprising said mutant.

[0006] Another aspect is to provide a cell transformed with said recombinant vector.

[0007] Another embodiment includes a step of converting 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA) using 7β-HSDH (7β-hydroxysteroid) in which the substrate binding site of the enzyme is mutated.

[0008] The present invention provides a method for producing ursodeoxycholic acid (UDCA) that improves the conversion rate from 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA).

[0009] Another embodiment includes a step of converting 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA) by 7β-HSDH (7β-hydroxysteroid) in which the substrate binding site of the enzyme is mutated,

[0010] The present invention provides a method for improving the conversion rate of 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA).

[0011] One embodiment is a 7β-hydroxysteroid (7β-HSDH) mutant comprising an amino acid sequence mutation of 7β-hydroxysteroid (7β-HSDH) derived from a Ruminococcus gnavus strain,

[0012] The amino acid sequence mutation is 1) M207X 1 2) T210X 2 , and 3) Q212X 3 and X 1 represents any amino acid other than methionine (M), and X 2 represents any amino acid other than threonine (T), and X 3 provides 7β-HSDH (7β-hydroxysteroid) mutants that express amino acids other than glutamine (Q).

[0013] In one embodiment, the 7β-HSDH (7β-hydroxysteroid) mutant is 4) D252X 4 Additional mutations, X 4 can be an amino acid other than aspartic acid (D).

[0014] As used herein, the term "7β-hydroxysteroid dehydrogenase (7β-HSDH)" refers to the stereospecific reduction (hydrogenation) of the corresponding 7β-HSDH of a 7-ketosteroid, or to a 7β-HSDH corresponding to a ketosteroid that contains a keto group at the 7-position and one or more additional keto groups on the steroid structure.

[0015] As used herein, the term "amino acid" includes any of the following amino acids: alanine (A; Alanine, A), cysteine ​​(C; Cysteine, Cys), aspartic acid (D; Aspartic acid, Asp), glutamic acid (E; Glu), phenylalanine (F; Phenylalanine, Phe), glycine (G; Glycine, G), histidine (H; Histidine, His), isoleucine (I; Isoleucine, Ile), lysine (K; Lysine, Lys), leucine (L; Leucine, Leu), methionine (M; Methionine, Met), asparagine (N; Asparagine, Asn), proline (P; Proline, Pro), glutamine (Q; Glutamine, ... The amino acid sequence may be, but is not limited to, valine (V; Valine, Val), isoleucine (I; Isoleucine, Ile), leucine (L; Leucine, Leu) or glutamic acid (E; Glu), and may be, but is not limited to, valine (V; Valine, Val), isoleucine (I; Isoleucine, Ile), leucine (L; Leucine, Leu), or glutamic acid (E; Glutamic acid, Glu).

[0016] As used herein, the term "amino acid sequence variation" refers to one or more amino acid sequence variations, with M207X representing one mutation. 1 , T210X 2 , Q212X 3 and D252X 4where X may be one selected from the group consisting of 1 ~X 4 can be selected from valine (V; Valine, Val), isoleucine (I; Isoleucine, Ile), leucine (L; Leucine, Leu) or glutamic acid (E; Glu), and specifically, X 1 Valine (V; Valine, Val), X 2 isoleucine (I;Ile), X 3 is leucine (L; Leu), and X is 4 can be glutamic acid (E; Glu).

[0017] In addition, when there are multiple amino acid sequence mutations (M207X 1 and T210X 2 ), (M207X 1 , and Q212X 3 ), (M207X 1 and D252X 4 ), (T210X 2 and Q212X 3 ), (T210X 2 and D252X 4 ), (M207X 1 , Q212X 3 and D252X 4 ), (M207X 1 , T210X 2 and Q212X 3 ), (M207X 1 , T210X 2 , and D252X 4 ), (T210X 2 , Q212X 3 and D252X 4 ) or (M207X 1 , T210X 2 , Q212X 3 and D252X 4 ), and X may be selected from 1 ~X 4can be selected from valine (V; Valine, Val), isoleucine (I; Isoleucine, Ile), leucine (L; Leucine, Leu) or glutamic acid (E; Glu), and specifically, X 1 Valine (V; Valine, Val), X 2 isoleucine (I;Ile), X 3 is leucine (L; Leu), and X is 4 can be glutamic acid (E; Glu).

[0018] In one embodiment, the 7β-HSDH (7β-hydroxysteroid) mutant may be a 7β-HSDH (7β-hydroxysteroid) mutant additionally having at least one mutation in a sequence motif corresponding to an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1, and the mutation may be, but is not limited to, VMKIAL in the sequence motif 207 to 212 of SEQ ID NO: 1.

[0019] The term "variant" as used herein refers to a polypeptide in which one or more amino acids have been conservatively substituted and / or modified, and which differs from the amino acid sequence of the variant before the mutation, but maintains its functions or properties. Such variants can generally be identified by modifying one or more amino acids in the amino acid sequence of the polypeptide and evaluating the characteristics of the modified polypeptide. That is, the ability of the variant may be increased, unchanged, or decreased compared to the polypeptide before the mutation. Some variants may also include variants in which one or more portions, such as an N-terminal leader sequence or a transmembrane domain, have been removed. Other variants may include variants in which a portion has been removed from the N-terminus and / or C-terminus of a mature protein. The term "variant" may be used in combination with terms such as mutant type, modification, modified polypeptide, modified protein, mutation, and variant (in English, modification, modified polypeptide, modified protein, mutant, mutein, divergent, etc.), and is not limited to these terms as long as they are used to mean mutated. For purposes of this application, the variant may be the polypeptide set forth in SEQ ID NO: 1 in which the 210th amino acid, threonine, of the amino acid sequence of SEQ ID NO: 3 has been replaced with isoleucine, and the 212th amino acid, glutamine, has been replaced with leucine.

[0020] As used herein, the term "stereoselectivity" refers to the property of selectively producing one stereochemical structure during a chemical reaction.

[0021] In one embodiment, the 7β-HSDH mutant may additionally have at least one mutation in a sequence motif corresponding to an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1. The mutant may consist of an amino acid sequence having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 1, and the mutation may be VMKIAL, a sequence motif of 207 to 212 of SEQ ID NO: 1.

[0022] The term "sequence motif" as used herein means the sequence of 207 to 212 of SEQ ID NO: 1, which is the substrate binding site in the amino acid sequence of 7β-HSDH derived from a R. gnavus strain, as a partial component of the sequence.

[0023] In another embodiment, the 7β-HSDH derived from the Ruminococcus gnavus strain can recognize 7-keto lithocholic acid (7-keto-LCA) as a substrate and induce the catalysis of stereospecific enzymatic reduction at the seventh carbon.

[0024] As used herein, the term "stereospecificity" means that certain stereoisomeric reactants in a reaction will produce one specific stereoisomeric (or enantiomeric pair) product, and means that the stereochemical outcome produced by a given reactant can be specified with certainty.

[0025] The term "enzymatic reduction" as used herein refers to a method using a reductase derived from a microorganism, and the enzymatic reduction method involves isolating a reductase gene from a novel microorganism isolated from nature or a microorganism known in a paper or patent, transferring the isolated gene to a plasmid and transforming Escherichia coli, cultivating the resulting Escherichia coli, disrupting and centrifuging the cells, and using the resulting cells as is for the reaction, or alternatively, disrupting and centrifuging the Escherichia coli to recover the supernatant for the reaction. The catalytic action of the enzymatic reduction can be induced using, but is not limited to, a Ruminococcus gnavus strain.

[0026] Another embodiment provides a recombinant vector comprising a gene for a 7β-HSDH (7β-hydroxysteroid) mutant.

[0027] In one embodiment, the mutant contained in the recombinant vector may consist of an amino acid sequence having sequence identity of 80% or more, more specifically 90% or more, 95%, 96%, 97%, 98%, 99% or more, to SEQ ID NO:1.

[0028] In one embodiment, the mutant may consist of the base sequence of SEQ ID NO:2.

[0029] The term "gene" as used herein means a structural unit that determines genetic information, and includes structural genes having information that determines the amino acid sequence of a protein or the base sequence of functional RNA (tRNA, rRNA, etc.), and / or regulatory genes (e.g., promoters, repressors, operators, etc.) that control the expression of structural genes, and is understood to mean a single strand containing a nucleotide sequence that is transcribed to produce a gene product.

[0030] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that contain one or more free ends, nucleic acid molecules that do not contain free ends (e.g., circular); or nucleic acid molecules that contain DNA, RNA, or both; and a variety of other polynucleotides known in the art.

[0031] The term "recombinant vector" as used herein means an expression vector for a polypeptide of interest that can be expressed with high efficiency in a suitable host cell when a gene encoding the polypeptide of interest (nucleic acid) to be expressed is operably linked thereto, and the recombinant vector can be expressed in the host cell. The host cell may be a prokaryotic cell, and expression regulatory sequences such as promoters, terminators, and enhancers, sequences for membrane targeting or secretion, etc. may be appropriately selected depending on the type of host cell, and various combinations may be used depending on the purpose.

[0032] In one embodiment, the recombinant vector may contain a Tac promoter and a kanamycin resistance gene.

[0033] The term "promoter" as used herein refers to a base sequence that determines when, where and to what extent a gene is expressed, i.e., a type of regulatory region that has a function of regulating gene expression. As a synthetic DNA promoter generated by combining the promoters of the trp and lac operons, the Tac promoter, which is generally used for protein production in E. coli, can be used.

[0034] As used herein, the term "kanamycin resistance gene" refers to a gene that confers drug resistance that enables a recombinant microorganism to survive exposure to the antibiotic.

[0035] Another aspect provides a cell transformed with said recombinant vector.

[0036] The term "transformation" as used herein may refer to the change in the genetic properties of an organism by exogenous DNA, i.e., when DNA, a type of nucleic acid extracted from cells of one lineage of an organism, is introduced into a living cell of another lineage, the DNA enters the cell and changes its genetic characteristics.

[0037] In one embodiment, the transformed cell may be a cell transformed by introducing a recombinant vector into E. coli, and the transformed cell may be the cell deposited under accession number KCTC 14715 BP.

[0038] In one embodiment, the E. coli may be cultured in a fermentation medium containing an LB medium at 30 to 40° C. for 4 to 48 hours, and the culture may be performed under aerobic conditions using a baffled flask, but is not limited thereto.

[0039] As used herein, the term "expression" refers to introducing an exogenous gene into an E. coli strain to artificially express an enzyme that the E. coli strain cannot express by itself, and the term "overexpression" refers to an E. coli strain that has a gene encoding the enzyme and can express it by itself, but artificially increasing the expression level of the enzyme and overexpressing it in order to increase the expression level for the purpose of mass production.

[0040] The term "primer" as used herein means a short nucleic acid sequence having a free 3' hydroxyl group that can form a base pair with a complementary nucleic acid template and serve as an initiation point for strand copying of the nucleic acid template. A primer is capable of initiating DNA synthesis in the presence of reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) and four different nucleotide triphosphates at an appropriate buffer solution and temperature.

[0041] When designing the primers, various constraints must be met, such as the A, G, C, and T content ratio of the primers, prevention of primer dimer formation, and prohibition of repeating the same base sequence more than three times. In addition, the amount of template DNA, primer concentration, dNTP concentration, and Mg concentration must be considered in the PCR reaction conditions. 2+ The reaction conditions, such as the concentration of, reaction temperature, and reaction time, must be appropriate.

[0042] The primer can incorporate additional features that do not change the basic properties. That is, the nucleic acid sequence can be modified using many means known in the art. Examples of such modifications include methylation, capping, substitution of one or more homologues of nucleotides, and modification of nucleotides to uncharged conjugates such as phosphonates, phosphotriesters, phosphoramidates or carbamates, or to charged conjugates such as phosphorothioates or phosphorodithioates. In addition, the nucleic acid can have one or more additional covalently attached residues such as nucleases, toxins, antibodies, signal peptides, proteins such as poly-L-lysine, intercalators such as acridine or psoralen, chelators such as metals, radioactive metals, iron oxide metals, and alkylators.

[0043] In one embodiment, the transformed cell may be one that has been transformed by introducing an expression vector into which the gene has been inserted into E. coli (E. coli MG1655) to express an enzyme in which four amino acids of the 7β-HSDH enzyme have been changed (SEQ ID NO: 1), and the recombinant E. coli (E. coli ABP-5 (Accession No. KCTC 14715 BP)) can obtain the improved 7β-HSDH enzyme mutant ABP-5 through a protein expression process using the strain derived from the R. gnavus strain.

[0044] Another embodiment includes a step of converting 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA) by 7β-HSDH (7β-hydroxysteroid) in which the substrate binding site of the enzyme is mutated,

[0045] Provided is a method for producing ursodeoxycholic acid (UDCA) that improves the conversion rate from 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA).

[0046] Another embodiment includes a step of converting 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA) by 7β-HSDH (7β-hydroxysteroid) in which the substrate binding site of the enzyme is mutated,

[0047] Provided is a method for improving the conversion rate of 7-keto lithocholic acid (7-keto-LCA) to ursodeoxycholic acid (UDCA).

[0048] In one embodiment, the substrate binding site of the enzyme may be, but is not limited to, a motif of the amino acid sequence 200 to 215 of 7β-HSDH.

[0049] In one embodiment, the mutation is 1) M207X 1 2) T210X 2 , and 3) Q212X 3 and X 1 represents any amino acid other than methionine (M), and X 2 represents any amino acid other than threonine (T), and X 3 can represent an amino acid other than glutamine (Q), but is not limited to this.

[0050] As used herein, the term "7-keto-LCA (7-keto lithocholic acid)" refers to an intermediate formed from chenodeoxycholic acid as an intermediate in the synthesis of the secondary bile acid ursodeoxycholic acid (UDCA) by human intestinal bacteria.

[0051] The term "conversion rate" as used herein means the percentage of a specific original substance (raw material) that is converted into another substance through a chemical reaction, based on the number of moles of the original specific substance (raw material). Effect of the Invention

[0052] In the present invention, the conversion efficiency from 7-keto-LCA (7-keto lithocholic acid) to ursodeoxycholic acid (UDCA) has been improved by producing mutants through enzyme improvement and developing recombinant protein production, and it is expected that this invention can be widely used for improving the biosynthesis and biosynthetic efficiency of ursodeoxycholic acid (UDCA). [Brief description of the drawings]

[0053] [Figure 1]FIG. 1 shows the results of a comparison between 7β-HSDH (7β-hydroxysteroid) (SEQ ID NO: 3) derived from an R. gnavus strain and the amino acid sequence of ABP-5 improved in the present invention (SEQ ID NO: 1). [Diagram 2] FIG. 2 shows the pCDFDuet-1-Tac-Kan vector (Tac promoter, kanamycin resistance) used to express the improved 7β-HSDH enzyme, ABP-5. [Diagram 3] Figure 3 shows the results of SDS-PAGE analysis of the expression of 7β-HSDH derived from R. gnavus strain and 7β-HSDH (ABP-5) enzyme derived from improved R. gnavus strain using E. coli. (A: 7β-HSDH enzyme derived from R. gnavus strain (29.32 kDa), B: ABP-5 (29.3 kDa)). [Figure 4] FIG. 4 is a schematic diagram of the conversion of 7-keto-LCA (7-keto lithocholic acid) to UDCA using 7β-HSDH enzyme including a coenzyme regeneration system. [Diagram 5] Figure 5 shows the results of comparing the conversion rate of 7-Keto-LCA to UDCA by each enzyme at various concentrations. (○: 7β-HSDH derived from R. gnavus strain, ■: ABP-5) [Figure 6] FIG. 6 shows the results of comparing the conversion rate of 7-Keto-LCA to UDCA by each enzyme at various concentrations by adjusting the pH. (○: ABP-5 with pH adjustment ■: ABP-5 without pH adjustment) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0054] The present invention will be described in more detail with reference to the following examples. It will be apparent to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. EXAMPLES

[0055] Example 1: Development of ABP-5 expression vector and expression strain It is known that the region around positions 200 to 215 of the amino acid sequence of 7β-HSDH derived from R. gnavus strain is involved in substrate binding, and the region around positions 240 to 259 is involved in substrate stereoselectivity. It was confirmed that activity was maintained even when the substrate concentration was increased when methionine at position 207, threonine at position 210, and glutamine at position 212, which are amino acids involved in substrate binding, and glutamic acid at position 252, which is an amino acid involved in substrate stereoselectivity, were replaced.

[0056] Therefore, ABP-5, a 7β-HSDH mutant, contains the M207V, T210I, Q212L, and D252E mutations in the amino acids of existing 7β-HSDH; more specifically, it is a mutant in which the 207th amino acid, methionine, is replaced by valine, the 210th amino acid, threonine, is replaced by isoleucine, the 212th amino acid, glutamine, is replaced by leucine, and the 252nd amino acid, aspartic acid, is replaced by glutamic acid (Figure 1).

[0057] In order to insert the gene for ABP-5, a recombinant enzyme improved from 7β-HSDH enzyme derived from Ruminococcus gnarvus strain, into E. coli MG1655 strain, codon optimization was performed using the amino acid sequence of ABP-5 using the Codon Optimization Tool provided on the Integrated DNA Technologies, Inc. website, and DNA synthesis was requested to Macrogen Co., Ltd. (Seoul, Republic of Korea).

[0058] Specifically, in order to obtain the codon-optimized ABP-5 gene (sequence number 2) in this experiment and the 7β-HSDH (sequence number 4) derived from the R. gnavus strain for comparison, gene amplification was performed by PCR using primers ABP-5_F (sequence number 5), ABP-5_R (sequence number 6), 7β-HSDH-F (sequence number 7), and 7β-HSDH-R (sequence number 8).

[0059] To prepare a vector for expression, pCDFDuet-1, an expression vector for E. coli, was used. A hangover PCR method was used to replace the T7 promoter present in the vector with a Tac promoter, and the primers used, F_Tac_overhang and R_Tac_overhang, were prepared with the sequences shown in SEQ ID NOs: 2 and 3. To prepare the vector, DNA polymerase (Q5 (registered trademark) High-Fidelity DNA Polymerase, NEB) was used under the conditions of 98°C for 10 seconds, 60°C for 30 seconds, and 72°C for 30 seconds per 1 kb of gene size, and the above cycle (98°C for 10 seconds, 50°C for 30 seconds, and 72°C for 30 seconds per 1 kb of gene size) was repeated 30 times, and finally, a reverse PCR reaction was performed on the entire vector under the conditions of 72°C for 5 minutes. Then, to remove the template vector pCDFDuet-1, the vector was treated with DpnI (NEB) at 37°C for 1 hour, and then inserted into the cloning host E. coli DH5α, after which the prepared vector was amplified and used.

[0060] In addition, to replace the ampicillin cassette of the pCDFduet-1 vector with a kanamycin cassette, DNA polymerase (Q5 (registered trademark) High-Fidelity DNA Polymerase, NEB) was used with all vector-F and R (all vector-F, R) primers (SEQ ID NOs: 9, 10). The reaction was set under the conditions of 98°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds per 1 kb of gene size, and the above cycle (98°C for 10 seconds, 50°C for 30 seconds, and 72°C for 30 seconds per 1 kb of gene size) was repeated 30 times, and finally, a reverse PCR reaction was performed on the entire vector under the conditions of reacting at 72°C for 5 minutes. In order to remove the remaining template vector, this was reacted with DpnI (NEB) at 37 ° C for 1 hour, and the kanamycin cassette was reacted with kanamycin F and R (Kanamycin-F, R) primers (SEQ ID NO: 11, 12) using GXL DNA polymerase (Takara Bio, Japan) at 98 ° C for 30 seconds, and the reaction was set at 98 ° C for 10 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds per gene size of 1 kb, and the above cycle (98 ° C for 10 seconds, 50 ° C for 30 seconds, and 72 ° C for 30 seconds per gene size of 1 kb) was performed 30 times, and finally reacted under the condition of 72 ° C for 5 minutes. The vector and kanamycin cassette reacted by reverse PCR were cloned by the in-fusion method using In Fusion HD Enzyme (Takara Bio, Japan), and the finally prepared expression vector was named pCDFDuet-1-Tac-Kan vector.

[0061] Then, the ABP-5 enzyme and the 7β-HSDH gene derived from the R. gnavus strain were amplified after treating the pCDFDuet-1-Tac-Kan vector (Figure 2) with NcoI and BamHI restriction enzymes, and inserted by the in-fusion method using In Fusion HD Enzyme (Takara Bio, Japan), and finally the recombinant vector was inserted into the E. coli MG1655 strain.

[0062] After transformation, in order to select pCDFDuet-1-Tac-Kan insertion strains containing the ABP-5 gene, the strains were smeared on LB medium containing 50 μg / mL kanamycin and cultured at 37°C for one day. Then, colony PCR was performed using BioFACT's 2X Taq PCR pre-Mix product and ABP-5_F and ABP-5_R primers or 7β-HSDH-F and 7β-HSDH-R primers, and finally pCDFDuet-1-Tac-Kan insertion strains containing the ABP-5 gene and the 7β-HSDH gene derived from R. gnavus were selected. E. coli (E. coli MG1655) carrying the pCDFDuet-1-Tac-Kan vector containing the ABP-5 gene was named 'E. coli ABP-5' and deposited at the Korea Institute of Bioscience and Biotechnology on September 17, 2021, and was assigned the accession number KCTC 14715 BP. In addition, the improved 7β-HSDH expressed in this strain was named ABP-5.

[0063] [Table 1]

[0064] <Example 2: Confirmation of ABP-5 enzyme expression and conversion rate of 7-Keto-LCA to UDCA> The recombinant Escherichia coli (E. coli ABP-5 (Accession No. KCTC 14715 BP)) prepared in Example 1 and transformed to overexpress the ABP-5 enzyme expression gene was used to confirm the expression of the recombinant protein and the conversion of 7-Keto-LCA to UDCA using the recombinant protein.

[0065] The growth of the strain (E. coli ABP-5 (deposition number KCTC 14715 BP)) was measured by measuring the optical density (OD value) at 600 nm using a UV-visible spectrophotometer (UV-1800, SHIMADZU). After cultivation, protein analysis was performed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Mini-PROTEIN Tetra system, Bio-Rad) using analytical polyacrylamide gels (Mini-PROTEAN TGX Precast Gels) and sample buffer (2× Laemmli Sample Buffer).

[0066] To confirm the conversion of 7-Keto-LCA to UDCA using recombinant protein ABP-5, a high performance liquid chromatograph (HPLC Chromaster) (Hitachi, Japan) and an ACCUCORE C30 2.6UM 250×2.1MM COLUMN were used to analyze the 3-HP in the culture medium and the UFLC (LC-20AD, SHIMADZU) and a refractive index detector (RID) were used to analyze the 3-HP in the culture medium. 2 HPO 4 2.2 mL of (pH 3.0), 370 mL of HPLC water, 300 mL of acetonitrile, and 400 mL of methanol were mixed and then filtered through a 0.2 μm pore size filter. The analysis was carried out under conditions of 40°C and 0.4 mL of solvent per minute.

[0067] 2-1. Expression of recombinant protein (ABP-5) and SDS-PAGE confirmation

[0068] The strain (E. coli ABP-5) was precultured in LB medium (Luria bertani medium) with the addition of 50 μg / L kanamycin, 0.5 g / L glucose, 5 g / L glycerol, and 2 g / L lactose. The strain used for inoculation was a strain stored at -80°C by the glycerol stock method, inoculated into LB medium at a concentration of 1%, and precultured at 180 rpm and 37°C. After preculture, the strain was harvested in the logarithmic phase, washed twice with sterilized distilled water, and used for main culture.

[0069] On the other hand, for the production of ABP-5 enzyme and 7β-HSDH derived from R. gnavus strain as a comparison group, 50 mL of LB_Kan medium was used in a 250 mL Erlenmeyer flask. Cultivation was performed in a baffled flask at a stirring speed of 180 rpm at a temperature of 37°C, during which E. coli culture and enzyme expression were carried out.

[0070] After each incubation period, the culture medium was collected and centrifuged to remove the supernatant. The collected E. coli was washed twice with sterile distilled water and resuspended in 50 mM phosphate buffer (pH 8.0), and then disrupted using an ultrasonic homogenizer and protein expression was confirmed (Figure 3). As a result, it was confirmed that the expression of ABP-5 enzyme and 7β-HSDH derived from the R. gnavus strain, which was used as a comparison group, proceeded smoothly after 2 hours.

[0071] 2-2. Measurement of 7β-HSDH and ABP-5 activities derived from R. gnavus strains

[0072] To measure the activity of ABP-5 enzyme, 7β-HSDH and ABP-5 enzymes derived from R. gnavus strains were expressed, and then the enzyme activity was measured using the conversion reaction of 7-Keto-LCA to UDCA.

[0073] The enzymatic conversion of 7-Keto-LCA to UDCA requires NADPH as a coenzyme, and therefore the enzyme activity was measured using the absorbance of NADPH at 340 nm wavelength, which was measured using a UV-visible spectrophotometer (UV-1800, SHIMADZU).

[0074] For the enzyme reaction, NADPH was added at a concentration of 0.5 mM, and 50 mM phosphate buffer (pH 8.0) was used as the reaction buffer. The substrate 7-Keto-LCA was added at a concentration of 10 g / L, and in the case of 7β-HSDH and ABP-5 enzymes derived from R. gnavus strains, 2 μL of the cell lysate was added, and the reaction was carried out in a final volume of 2 mL.

[0075] For activity measurement, after adding each enzyme, the change in absorbance at 340 nm was measured at 1-minute intervals, and the measured absorbance change value was substituted into the NADPH standard curve (340 nm, 0.01-0.1 mM NADPH) to measure the enzyme activity. The Bradford protein assay was used to measure the protein concentration of the cell lysate, and each measured protein concentration was measured.

[0076] In order to compare the enzyme activity of 7β-HSDH and ABP-5 derived from R.gnavus strain, 10 colonies obtained during transformation were selected using recombinant E.coli ABP-5 and E.coli 7β-HSDH derived from R.gnavus strain, which were inserted with expression vectors containing 7β-HSDH and ABP-5 enzyme genes derived from R.gnavus strain, and expressed according to the expression method of Example 2.1, and the activity of 7β-HSDH and ABP-5 derived from R.gnavus strain, which are recombinant 7β-HSDH expressed in recombinant E.coli, was measured (Table 2). As a result, the 7β-HSDH derived from R.gnavus strain showed an average activity of 41.62±4.55U / mg, and the ABP-5 showed an activity of 49.13±4.72U / mg, which was confirmed to be about 18.10% higher than that of the existing enzyme.

[0077] [Table 2]

[0078] 2-3. Conversion of 7-Keto-LCA to UDCA using ABP-5 enzyme and 7β-HSDH derived from R. gnavus strain

[0079] The conversion of 7-Keto-LCA to UDCA was carried out using the expressed ABP-5 enzyme and 7β-HSDH from a control strain, R. gnavus.

[0080] 7β-HSDH, an enzyme used to convert 7-Keto-LCA to UDCA, requires NADPH as a coenzyme. However, since this reaction is an equivalent-to-equivalent reaction, it requires the same amount of NADPH as the 7-Keto-LCA to be converted. This can lead to an inefficient reaction that requires a large amount of NADPH. To solve this problem, a reaction system including a cofactor regeneration system (Co-factor regeneration) shown in Figure 4 was constructed and then the conversion was carried out.

[0081] For the conversion reaction, 50 mM phosphate buffer solution (pH 8.0) was used, and the reaction was carried out under the conditions of 7-Keto-LCA (10, 20, 30, 40, or 50 g / L), 1 mM NADPH, 200 mM glucose, 2 U / mL glucose dehydrogenase (GDH), and 4 U / mL ABP-5 enzyme or 7β-HSDH derived from R. gnavus strain, at 30°C and 150 rpm.

[0082] As a result of the reaction, it was confirmed that the conversion rate to UDCA was 100% for both enzymes up to a concentration of 20g / L of 7-Keto-LCA. However, in the case of conversion using 7β-HSDH derived from R. gnavus strain, the conversion rate decreased with increasing substrate concentration, to 55.12% at a substrate concentration of 30g / L, 23.53% at a substrate concentration of 40g / L, and 5.37% at a substrate concentration of 50g / L.

[0083] In contrast, when the conversion was carried out using the ABP-5 enzyme, the conversion rate decreased to 100% at a concentration of 30 g / L, 92.44% at a concentration of 40 g / L, and 79.82% at a concentration of 50 g / L, confirming that the decrease in conversion rate with increasing substrate concentration was significantly lower than that of 7β-HSDH derived from the R. gnavus strain (Figure 5). In other words, the improved enzyme mutant showed the same conversion efficiency not only at a 7-Keto-LCA (7-keto lithocholic acid) concentration of 20 g / L, which is the concentration at which the existing conversion activity is maintained, but also at a concentration of 30 g / L, confirming that conversion to UDCA is possible even at 7-Keto-LCA (7-keto lithocholic acid) concentrations of 40 g / L and 50 g / L.

[0084] Based on these results, it was confirmed that the 7β-HSDH enzyme mutant ABP-5 is more resistant to inhibition caused by increasing substrate concentration than existing enzymes, and it is believed that this advantage can be utilized to obtain various process benefits.

[0085] Example 3: Conversion of 7-Keto-LCA to UDCA using ABP-5 enzyme with pH adjustment As in Example 2, when the enzyme ABP-5 newly developed in the present invention is used, unlike the case of using 7β-HSDH derived from R. gnavus strain, it was confirmed that the inhibition of conversion to UDCA that occurs at a 7-Keto-LCA concentration of 40 g / L or more is alleviated to some extent. However, when the 7-Keto-LCA concentration is increased to 50 g / L or more, the conversion rate tends to decrease significantly to 79.82%.

[0086] In the case of GDH, which is used as a coenzyme regeneration system to convert 7-Keto-LCA to UDCA, glucose is converted to gluconic acid and NADP is used as the coenzyme. Therefore, as the reaction proceeds, the pH of the reaction solution decreases due to the accumulated gluconic acid, and if the reaction is started at an initial pH of 8.0, the pH tends to decrease to 7.2 at the end. This decrease in pH is thought to be the cause of activity inhibition, so the pH of the reaction solution was measured every hour and readjusted to 8.0 to confirm the conversion ability.

[0087] The reaction was carried out at 30°C and 150 rpm using 50 mM phosphate buffer (pH 8.0) containing 7-Keto-LCA (10, 20, 30, 40, or 50 g / L), 1 mM NADPH, 200 mM glucose, 2 U / mL glucose dehydrogenase (GDH), and 4 U / mL ABP-5 enzyme or 7β-HSDH derived from R. gnavus strain. The pH of the reaction solution was measured every hour using a pH meter (FiveEasy, Mettler toledo), and the conversion was performed by titrating the pH of the reaction solution to 8.0 using 5N NaOH. As a result, it was confirmed that the conversion rate was improved by 4.2% (92.44 to 96.24%) at 40 g / L and 12.43% (79.82% to 92.45%) at 50 g / L compared to the conventional condition without pH adjustment (Figure 6).

[0088] The above description is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features. Therefore, it should be understood that the above-described embodiment is illustrative in all respects and is not limiting.

Claims

1. A 7β-HSDH (7β-hydroxysteroid) mutant comprising an amino acid sequence mutation of 7β-HSDH (7β-hydroxysteroid) derived from a Ruminococcus gnavus strain, The 7β-HSDH derived from the Ruminococcus gnavus strain has the amino acid sequence of SEQ ID NO: 3, The amino acid sequence mutation is 1) M207X １ 、 2) T210X ２ , and 3)Q212X ３ The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: X １ represents an amino acid other than methionine (M), X ２ represents an amino acid other than threonine (T) and asparagine (N), X ３ represents an amino acid other than glutamine (Q), 7β-HSDH (7β-hydroxysteroid) mutant.

2. The amino acid sequence mutation is such that the amino acid sequence at positions 207 to 212 of SEQ ID NO: 3 is replaced with VMKIAL. A 7β-HSDH (7β-hydroxysteroid) mutant according to claim 1.

3. The amino acid sequence mutation is 4) D252X ４ and X ４ represents an amino acid other than aspartic acid (D); A 7β-HSDH (7β-hydroxysteroid) mutant according to claim 1.

4. The 7β-HSDH mutant has the amino acid sequence of SEQ ID NO:

1. A 7β-HSDH (7β-hydroxysteroid) mutant according to claim 1.

5. The 7β-HSDH (7β-hydroxysteroid) derived from the Ruminococcus gnavus strain recognizes 7-keto-LCA (7-keto lithocholic acid) as a substrate and induces the catalytic activity of stereospecific enzymatic reduction at the seventh carbon of 7-keto-LCA. A 7β-HSDH (7β-hydroxysteroid) mutant according to claim 1.

6. A recombinant vector comprising a polynucleotide consisting of a base sequence encoding the 7β-HSDH (7β-hydroxysteroid) mutant according to any one of claims 1 to 5.

7. The recombinant vector according to claim 6 , wherein the polynucleotide consists of a base sequence encoding the amino acid sequence of SEQ ID NO:

1.

8. The recombinant vector according to claim 6 , wherein the polynucleotide consists of the base sequence of SEQ ID NO:

2.

9. The recombinant vector according to claim 6 , comprising a Tac promoter and a kanamycin resistance gene.

10. A cell transformed with the recombinant vector according to claim 6.

11. The cell of claim 10 , wherein the cell is Escherichia coli.

12. The cell described in claim 11, wherein the Escherichia coli is deposited under accession number KCTC 14715 BP.

13. The method includes a step of converting 7-keto-LCA (7-keto lithocholic acid) into ursodeoxycholic acid (UDCA) by a 7β-HSDH (7β-hydroxysteroid) mutant derived from Ruminococcus gnavus strain, The 7β-HSDH derived from the Ruminococcus gnavus strain has the amino acid sequence of SEQ ID NO: 3, The amino acid sequence mutation is 1) M207X1, 2) T210X2, and 3) Q212X3 The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: X 1 represents an amino acid other than methionine (M); X 2 represents an amino acid other than threonine (T) and asparagine (N); X 3 represents an amino acid other than glutamine (Q); A method for producing ursodeoxycholic acid (UDCA).

14. The amino acid sequence mutation is such that the amino acid sequence at positions 207 to 212 of SEQ ID NO: 3 is replaced with VMKIAL. A method for producing ursodeoxycholic acid (UDCA) according to claim 13.

15. The method includes a step of converting 7-keto-LCA (7-keto lithocholic acid) into ursodeoxycholic acid (UDCA) by a 7β-HSDH (7β-hydroxysteroid) mutant derived from a Ruminococcus gnavus strain, The 7β-HSDH derived from the Ruminococcus gnavus strain has the amino acid sequence of SEQ ID NO: 3, The amino acid sequence mutation is 1) M207X1, 2) T210X2, and 3) Q212X3 The present invention relates to a method for the preparation of a nucleic acid sequence comprising the steps of: X 1 represents an amino acid other than methionine (M); X 2 represents an amino acid other than threonine (T) and asparagine (N); X 3 represents an amino acid other than glutamine (Q); A method for improving the conversion rate of 7-keto lithocholic acid (7-keto-LCA) to ursodeoxycholic acid (UDCA).

Images