Mutant of coptis chinensis oxygen methyl transferase CcOMT1 as well as preparation method and application of mutant

Abstract

The invention discloses a mutant of coptis chinensis oxygen methyltransferase CcOMT1 as well as a preparation method and application of the mutant. The mutant is a mutant CcOMT1-M3 obtained by mutating serine at the 109th site of the coptis chinensis oxygen methyltransferase CcOMT1 into leucine, mutating cysteine at the 250th site of the coptis chinensis oxygen methyltransferase CcOMT1 into alanine and mutating leucine at the 300th site of the coptis chinensis oxygen methyltransferase CcOMT1 into alanine. According to the invention, the CcOMT1-M3 mutant is successfully constructed through mutation iteration, and the CcOMT1-M3 mutant can greatly improve the catalytic efficiency and the substrate conversion rate.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a mutant of berberine methyltransferase CcOMT1, its preparation method, and its application. Background Technology

[0002] Many natural products derived from plants, such as proberberine alkaloids (e.g., berberine and epiberberine) in Coptis chinensis, possess a variety of pharmacological activities and are of great value in drug development.

[0003] However, the extremely low content of the aforementioned proberberine alkaloids (especially epiberberine) in natural plants limits their further research and application. Therefore, to efficiently produce these proberberine alkaloids (especially epiberberine), it is necessary to target the key enzyme in its biosynthetic pathway—the enzyme responsible for corydaline (… S )-scoulerine) C2-methylated corydaline-2-oxomethyltransferase (( S )-scoulerine 2- O The function and activity of α-methyltransferase (S2OMT) were analyzed, and its catalytic efficiency was improved through enzyme engineering to meet the needs of synthetic biology and industrial production.

[0004] However, existing technologies for modifying S2OMT enzymes have the following problems:

[0005] 1. There is insufficient research on S2OMT enzymes, whose three-dimensional structure is unknown, and whose catalytic mechanism and specific 2-position methylation mechanism are unclear; 2. Enzymes in plants that have been identified as having S2OMT function (such as CyOMT5) have substrate contamination and low catalytic efficiency, which limits their application in metabolic engineering; 3. Existing modification strategies rely on the structural details of the enzyme, but S2OMT lacks reference structural data as a guide, leading to blind mutation design and limited improvement in catalytic efficiency. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a mutant of berberine methyltransferase CcOMT1, its preparation method, and its application. The CcOMT1-M3 mutant was successfully constructed through mutation iteration. This CcOMT1-M3 mutant can significantly improve catalytic efficiency and substrate conversion rate, which helps to increase the yield of protoberberine alkaloids (especially epiberberine).

[0007] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem:

[0008] In a first aspect, a mutant of berberine oxymethyltransferase CcOMT1 is provided, wherein the mutant is CcOMT1-M3 obtained by mutating serine at position 109 to leucine, cysteine ​​at position 250 to alanine, and leucine at position 300 to alanine.

[0009] Secondly, a coding gene for the above-mentioned mutant is provided, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0010] Thirdly, a recombinant expression vector containing the gene encoding the aforementioned mutant is provided.

[0011] Fourthly, an engineered bacterium for expressing the above-mentioned mutant is provided, wherein the engineered bacterium is obtained by transforming the above-mentioned recombinant expression vector into a host bacterium.

[0012] Fifthly, a method for preparing the above-mentioned mutant is provided.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention can determine the single mutation site of amino acids in the enzyme substrate binding pocket based on the crystal structure of CcOMT1, and successfully construct the CcOMT1-M3 mutant through mutation iteration. Compared with the wild-type CcOMT1 protein, the catalytic efficiency of the CcOMT1-M3 mutant is increased by about 5 times and the substrate conversion rate is increased by about 2 times, thereby increasing the yield of protoberberine alkaloids (especially epiberberine). Attached Figure Description

[0015] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The electrophoretic band representing the wild-type CcOMT1 gene;

[0017] Figure 2 Electrophoretic bands for wild-type CcOMT1 protein;

[0018] Figure 3 The results are HPLC and MS detection of wild-type CcOMT1 protein reacting with substrate.

[0019] Figure 4 The crystal structure is that of the CcOMT1 / SAH complex.

[0020] Figure 5 It consists of 22 substrate pocket amino acid residues of the wild-type CcOMT1 protein;

[0021] Figure 6 Relative activity represents the change in enzyme activity for each CcOMT1 single mutant protein.

[0022] Figure 7 A schematic diagram of the construction route for the CcOMT1 multiple mutant protein;

[0023] Figure 8 The results show the enzymatic kinetics of CcOMT1-M3 mutant and wild-type CcOMT1 protein. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Unless otherwise specified, the experimental methods used in this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0026] Example 1

[0027] Coptis chinensis oxymethyltransferase CcOMT1 ( Coptis chinensis O- methyltransferase 1 Cloning and the construction of expression vectors

[0028] S1. Take four-year-old Coptis chinensis ( Coptis chinensis Franch.), after washing its rhizomes, used liquid nitrogen to flash freeze and grind them to obtain sample powder;

[0029] S2. Total RNA was extracted from the sample powder using a plant total RNA extraction kit (DP441, Tiangen Biotech Co., Ltd.), and the corresponding cDNA library was obtained using a reverse transcription kit (RK20429, Wuhan Aibote Biotechnology Co., Ltd.). This cDNA library was then used as the template for PCR amplification. Primer design tools such as Primer Premier 5 were used to obtain the sequences of the upstream primer CcOMT1-F and the downstream primer CcOMT1-R, as shown in Table 1.

[0030] Table 1. Primer sequences for CcOMT1 gene amplification

[0031]

[0032] S3. Gene cloning was completed based on the following amplification reaction system and PCR amplification program:

[0033] The amplification reaction system was as follows: 12.5 μl of 2×PhantaMax Buffer; 2 μl of cDNA from sample powder; 1 μl of dNTPMix; 1 μl each of upstream primer CcOMT1-F and downstream primer CcOMT1-R; 1 μl of high-fidelity DNA polymerase; and enzyme-free water to a final volume of 25 μl.

[0034] The PCR amplification program is as follows: 1. React at 95℃ for 5 min; 2. React at 95℃ for 15 s, at 58℃ for 15 s, and at 72℃ for 1 min, for a total of 25 cycles; 3. Extend at 72℃ for 10 min; 4. React at 15℃ for 10 min.

[0035] After the reaction, the PCR products were identified by agarose gel electrophoresis to obtain, for example... Figure 1 The electrophoretic band shown represents the wild-type CcOMT1 gene fragment, whose nucleotide sequence is shown in SEQ ID NO. 3.

[0036] S3. The above CcOMT1 gene fragment and pET28a plasmid were homologously recombinated using the Hieff Clone Plus Multi OneStep Cloning Kit to obtain the recombination product. The reaction system was: 2×Hieff Buffer 3μl; CcOMT1 gene fragment 1.5μl and recombination vector 1.5μl. The reaction was carried out at 37℃ for 60min to obtain the recombinant plasmid pET28a-(+)-CcOMT1, which was used as the CcOMT1 expression vector.

[0037] Expression and purification of berberine methyltransferase CcOMT1

[0038] S1. The recombinant plasmid pET28a-(+)-CcOMT1 was transformed into Rosetta (DE3) competent cells, and the transformed cells were seeded in 4L LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl), and 50μg / mL kanamycin was added. The cells were cultured at 37℃ and 220rpm with shaking until the OD600 reached 0.6.

[0039] Further addition of 0.4 mM isopropyl-β-D-thiogalactoside (IPTG) to induce protein expression, followed by continued culture at 16 °C for 19 h;

[0040] S2. After the culture is completed, the cells are centrifuged at 5000×g for 10 min to collect the cells. The cells are then resuspended in 120 mL of lysis buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0) and the cells are then lysed using a high-pressure homogenizer (AH-NANO, ATSEngineering Limited, China) at 800 bar.

[0041] S3. Centrifuge the lysis buffer containing the lysed cells at 12000×g for 60 min to remove cell debris, collect the supernatant, and load it onto a 5 mL Ni-NTA affinity resin (GenScript, Nanjing, China) gravity chromatography column that has been pre-equilibrated with lysis buffer.

[0042] The column was then washed with 10 column volumes of lysis buffer containing 20 mM imidazole, followed by 6 column volumes of lysis buffer containing 50 mM imidazole, and finally eluted with 4 column volumes of lysis buffer containing 300 mM imidazole to collect the His-tagged CcOMT1 protein.

[0043] S4. The CcOMT1 protein with the His tag was incubated with thrombin overnight at 4°C to remove the His tag. Then, the CcOMT1 protein with the His tag removed was reloaded onto Ni-NTA affinity resin and eluted sequentially with lysis buffer containing 10 mM imidazole and 20 mM imidazole (elution volume of 3 column volumes).

[0044] S5. Size exclusion chromatography (HiLoad 16 / 600 Superdex 75 column, GE Healthcare) was used to purify the His-tagged CcOMT1 protein using a 20mM Tris-HCl and 150mM NaCl (pH 8.0) buffer containing 1mM dithiothreitol. The target protein (i.e., wild-type CcOMT1 protein) fraction was collected and detected by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to obtain the following results: Figure 2 The CcOMT1 protein band shown (i.e., wild-type CcOMT1) was further determined by sequencing as shown in SEQ ID NO. 1;

[0045] S6. Protein was concentrated using Amicon Ultra-30kDa ultrafiltration centrifuge tubes (Millipore) and replaced with storage buffer (20mM Tris-HCl, 300mM NaCl, 10% glycerol, pH 8.0), flash-frozen in liquid nitrogen, and stored at –80°C. The absorbance at 280 nm was measured using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, USA), and the protein concentration was calculated, thus achieving heterologous expression of wild-type CcOMT1 protein in *E. coli*.

[0046] Functional identification of berberine oxymethyltransferase CcOMT1

[0047] S1. Prepare the enzyme activity assay reaction system. The reaction system is 100µL and contains 50mM sodium phosphate buffer (pH 8.0), 20µg wild-type CcOMT1 protein, 1.2mM corydaline, 2.4mM S-adenosylmethionine (SAM), and enzyme-free water to make up to 100µL.

[0048] S2. After incubating the above reaction system at 25°C for 6 hours, add 100µL of methanol to terminate the reaction.

[0049] S3. The reaction products were centrifuged and filtered through a 0.22µm filter membrane, and then analyzed by high performance liquid chromatography at a detection wavelength of 283nm.

[0050] like Figure 3 As shown, the detection results of high performance liquid chromatography (HPLC) and mass spectrometry (MS) both show clear product signal peaks, indicating that the wild-type CcOMT1 protein obtained by heterologous expression in Escherichia coli can catalyze the substrate (i.e., corydaline) to generate the corresponding product.

[0051] Site-directed mutagenesis and functional study of CcOMT1, an enzyme in Coptis chinensis.

[0052] S1. Based on the crystal structure of wild-type CcOMT1 protein (e.g. Figure 4(As shown) The amino acids W15, Y19, M106, S109, A112, I113, I145, W146, F159, A162, N165, D166, S167, L169, C250, I283, M296, L300, D303, M304, N307, and V388, which constitute the substrate binding pocket of the CcOMT1 enzyme, were selected as potential mutation sites, corresponding to tryptophan at position 15, tyrosine at position 19, methionine at position 106, serine at position 109, and so on. Alanine at position 112, isoleucine at position 113, isoleucine at position 145, tryptophan at position 146, phenylalanine at position 159, alanine at position 162, asparagine at position 165, aspartic acid at position 166, serine at position 167, leucine at position 169, cysteine ​​at position 250, isoleucine at position 283, methionine at position 296, leucine at position 300, aspartic acid at position 303, methionine at position 304, asparagine at position 307, valine at position 388;

[0053] Amplification primer sequences were designed to mutate each potential mutation site of the wild-type CcOMT1 protein. Mutation of each potential mutation site in the wild-type CcOMT1 protein includes: if the current potential mutation site is not any of alanine ("A"), leucine ("L"), or phenylalanine ("F"), then the current potential mutation site is mutated to alanine, leucine, or phenylalanine respectively; if the current potential mutation site is any of alanine, leucine, or phenylalanine, then the current potential mutation site is mutated to the other two amino acids respectively. For example, for potential mutation site A112, it only needs to be mutated to leucine and phenylalanine. Similarly, F159, A162, L169, and L300 are also subject to similar mutation treatment based on this principle.

[0054] Furthermore, the amplification primer sequences for each potential mutation site of the above-mentioned wild-type CcOMT1 protein are shown in Table 2, wherein the "target amino acid" refers to the amino acid obtained by mutation at the potential mutation site, namely alanine, leucine, or phenylalanine.

[0055] Table 2. Primer sequences for amplification of single mutation sites

[0056]

[0057]

[0058]

[0059]

[0060] Furthermore, the crystal structure of the wild-type CcOMT1 protein was obtained through the following steps:

[0061] Purified wild-type CcOMT1 protein (concentration 10.0 mg / mL) was incubated with S-adenosyl homocysteine ​​(SAH, 5 mM) on ice for 30 minutes. Then, using the hanging drop gas-phase diffusion method, 0.7 μL of the protein incubation solution was mixed with 0.7 μL of stock solution (tris(hydroxymethyl)amino) base / hydrochloric acid pH 8.5, 500 mM sodium chloride, 8% (w / v) PEG 8000, 30% (v / v) 2-methyl-2,4-pentanediol). After culturing at 18°C ​​for 3-4 days, CcOMT1 / SAH complex crystals were obtained and frozen for storage.

[0062] Crystallographic data of the CcOMT1 / SAH composite crystal were acquired using the BL19U1 beamline of the Shanghai Synchrotron Radiation Facility (SSRF), and diffraction images were processed using XDS software.

[0063] Furthermore, the Phaser module from the CCP4 package was used, with the CcOMT1 structure predicted by AlphaFold2 as the search model. The initial model was automatically built using AutoBuild, manually corrected using Coot, and iteratively refined and structurally validated using Phenix software. The final result is as follows: Figure 4 The crystal structure of the CcOMT1 / SAH complex is shown below;

[0064] Further searches of the enzyme substrate pocket amino acid residues were conducted using PyMOL software to determine, for example... Figure 5 The 22 potential mutation sites shown;

[0065] S2. Using the recombinant plasmid pET28a-(+)-CcOMT1 as a template, polymerase chain reaction (PCR) amplification was performed using a reaction system containing the above-mentioned single mutation site amplification primers and KOD One™ PCR premixed reagents. Each potential mutation site of the wild-type CcOMT1 protein was mutated to the corresponding amino acid (i.e., one of alanine, leucine, or phenylalanine) to obtain 61 CcOMT1 single mutant gene fragments. Each CcOMT1 single mutant gene fragment contains only one single mutation site, thus completing a single-point mutation of the wild-type CcOMT1 protein.

[0066] Each potential mutation site is mutated to any one of alanine, leucine, or phenylalanine, and is recorded as a single mutation site. For example, if the leucine L300 at position 300 of the wild-type CcOMT1 protein is mutated to alanine ("A"), leucine ("L"), or phenylalanine ("F"), it is recorded as a single mutation site L300A, a single mutation site L300L, and a single mutation site L300F, respectively.

[0067] S3. Each CcOMT1 single mutant gene fragment was digested with restriction endonuclease DpnI at 37°C for 2 hours to remove methylated template DNA. The CcOMT1 mutant gene fragments with demethylated template DNA were then transformed into E. coli DH5α competent cells. The nucleotide and amino acid sequences of 61 CcOMT1 single mutant gene fragments were obtained by sequencing to determine whether the target gene fragment was obtained.

[0068] S4. Construct expression vectors for 61 CcOMT1 single mutant gene fragments (the expression vectors are any one of plasmids (such as pET28a plasmid), bacteriophages, or viral vectors containing CcOMT1 single mutant gene fragments), and express and purify the corresponding CcOMT1 single mutant gene fragments based on each expression vector to obtain 61 CcOMT1 single mutant proteins after single-point mutation of wild-type CcOMT1 protein, and each CcOMT1 single mutant protein contains only one corresponding amino acid single mutation site, such as "L300A";

[0069] The construction of the expression vector for the CcOMT1 single mutant gene fragment can be referenced from " Coptis chinensis oxymethyltransferase Cloning and Construction of Expression Vectors for CcOMT1 The process of expressing and purifying the CcOMT1 single mutant gene fragment can be performed according to step S3 in the document. Expression and purification of berberine methyltransferase CcOMT1 "The expression vector can be obtained through other existing technologies, or CcOMT1 expression and purification can be performed, which will not be elaborated further;

[0070] S5. Refer to " Functional identification of berberine oxymethyltransferase CcOMT1 Steps S1-S3 in the procedure involve analyzing the catalytic reaction products of 61 CcOMT1 single mutant proteins and calculating the conversion rate based on the following formula as the enzyme activity:

[0071] Conversion rate (%) = (Product peak area / (Product peak area + Substrate peak area)) × 100%

[0072] The change in enzyme activity of each CcOMT1 single mutant protein relative to the wild-type CcOMT1 protein was calculated using the following formula and denoted as the relative activity:

[0073] Relative activity (%) = CcOMT1 single mutant enzyme activity / wild-type CcOMT1 enzyme activity

[0074] Among them, the wild-type CcOMT1 protease activity was calculated at 100%, and the statistical results of the relative activity changes of each CcOMT1 monomutant protein are as follows: Figure 6 As shown;

[0075] S6. The enzyme activities (all can be measured by the relative changes in enzyme activity as described above) of 61 CcOMT1 single mutant proteins are arranged in descending order. Among the top N CcOMT1 single mutant proteins with the largest enzyme activity, the single mutation site of the CcOMT1 single mutant protein with the largest enzyme activity is recorded as the major mutation site, and the single mutation sites of the other CcOMT1 single mutant proteins are recorded as minor mutation sites.

[0076] Furthermore, the CcOMT1 single mutant protein before the N position of enzyme activity is combined with mutation to obtain several corresponding CcOMT1 multiple mutant proteins, and each CcOMT1 multiple mutant protein contains a major mutation site and at least one minor mutation site. Preferably, at least one CcOMT1 multiple mutant protein contains a major mutation site and at least two minor mutation sites.

[0077] Specifically, the CcOMT1 single mutant protein with the N positions preceding enzyme activity is subjected to combinatorial mutations to obtain the corresponding CcOMT1 multiple mutant protein, including the following steps:

[0078] S61. Mutate the CcOMT1 single mutant protein containing only the major mutation site to obtain several (e.g., Each CcOMT1 multimutant protein contains a major mutation site and a minor mutation site, and the minor mutation sites of each CcOMT1 multimutant protein are different. Therefore, each CcOMT1 multimutant protein is a CcOMT1 double mutant protein.

[0079] S62. Mutate the CcOMT1 multi-mutant protein with the highest enzyme activity obtained in the previous step to obtain several (e.g., Each CcOMT1 multimutant protein contains all the single mutation sites (i.e., the major mutation site and one secondary mutation site) of the CcOMT1 multimutant protein with the largest enzyme activity obtained in the previous step, as well as one secondary mutation site. Furthermore, the secondary mutation sites of each CcOMT1 multimutant protein obtained in this step are different. Therefore, each CcOMT1 multimutant protein obtained in this step is a CcOMT1 triple mutant protein.

[0080] S63. Repeat step S62 to obtain several CcOMT1 multiple mutant proteins.

[0081] S7. Among all the CcOMT1 multimutant proteins obtained in steps S61-S63, the CcOMT1 multimutant protein with the highest enzyme activity is determined as the final CcOMT1 mutant.

[0082] Therefore, this embodiment can mutate the amino acids in the substrate pocket based on the Focused Rational Iterative Site-Specific Mutagenesis (FRISM) strategy, guided by catalytic activity, to obtain a series of candidate mutants, and finally obtain the optimal mutant through iterative combination of mutants.

[0083] For example, in this embodiment, such as Figure 6 As shown, the single mutation sites of the top 6 CcOMT1 single mutant proteins with the largest relative changes in enzyme activity are L300A (L300 is the single mutation site with the largest relative change in enzyme activity when the wild-type CcOMT1 protein is mutated), Y19L, S109L, N165A, C250A, and M106F.

[0084] like Figure 7 As shown, five CcOMT1 double mutant proteins were constructed sequentially, denoted as Y19L / L300A double mutant (indicating that the double mutant contains single mutation sites Y19L and L300A; the meanings of other double mutants and subsequent multiple mutants are similar), S109L / L300A double mutant, N165A / L300A double mutant, C250A / L300A double mutant, and M106 / L300A. The relative change in enzyme activity of each CcOMT1 double mutant relative to the wild-type CcOMT1 enzyme activity was determined by in vitro enzyme activity assay.

[0085] Four CcOMT1 triple mutant proteins were constructed based on the CcOMT1 double mutant with the largest relative change in enzyme activity. For example, if the S109L / L300A double mutant has the largest relative change in enzyme activity, then four more CcOMT1 triple mutants were constructed based on this CcOMT1 double mutant, denoted as S109L / M106F / L300A triple mutant, S109L / C250A / L300A triple mutant, S109L / N165A / L300A triple mutant, and Y19L / C250A / L300A triple mutant (not shown). The relative change in enzyme activity of each CcOMT1 triple mutant relative to the wild-type CcOMT1 enzyme activity was determined by in vitro enzyme activity assay.

[0086] Based on the CcOMT1 triple mutant with the largest relative change in enzyme activity, CcOMT1 quad mutants are constructed. For example, if the S109L / C250A / L300A triple mutant has the largest relative change in enzyme activity, then based on this CcOMT1 triple mutant, three more CcOMT1 quad mutants are constructed, including the M106F / S109L / C250A / L300A quad mutant, the S109L / / N165A / C250A / L300A quad mutant, and the S109L / / N165A / Y19L / L300A quad mutant (not shown). The relative change in enzyme activity of each CcOMT1 quad mutant relative to the wild-type CcOMT1 enzyme activity is determined by in vitro enzyme activity detection.

[0087] Two CcOMT1 pentamutators were constructed based on the CcOMT1 quadrat with the largest relative activity change, for example, the M106F / S109L / N165A / C250A / L300A pentamutator and the M106F / Y19L / N165A / C250A / L300A pentamutator (not shown). The relative activity change of the CcOMT1 pentamutators relative to the wild-type CcOMT1 enzyme activity was determined by in vitro enzyme activity assay.

[0088] Finally, based on the CcOMT1 pentamutator with the largest relative activity, a CcOMT1 hexamutator was constructed, for example, the M106F / S109L / N165A / C250A / Y19L / L300A hexamutator (not shown).

[0089] The CcOMT1 multimutant with the largest relative change in enzyme activity among all the aforementioned CcOMT1 multimutants (including all CcOMT1 double mutants, CcOMT1 triple mutants, CcOMT1 quadruple mutants, CcOMT1 pentamutants, and CcOMT1 hexamutants) was determined to be the optimal CcOMT1 multimutant. For example, in this embodiment, the S109L / C250A / L300A triple mutant showed the largest relative change in enzyme activity. The highest activity (4.88) indicates that the enzyme activity of the S109L / C250A / L300A triple mutant is 4.88 times higher than that of the wild-type CcOMT1 protein, which can significantly enhance the substrate catalytic activity. Therefore, the S109L / C250A / L300A triple mutant can be used as the final CcOMT1 mutant for subsequent applications, and is designated as CcOMT1-M3 mutant. Further sequencing can yield the nucleotide sequence (as shown in SEQ ID NO. 2) and amino acid sequence (as shown in SEQ ID NO. 4) of the CcOMT1-M3 mutant.

[0090] To further verify the optimal catalytic efficiency of the CcOMT1 multiple mutants, this example tested the catalytic efficiency of wild-type CcOMT1 protein and CcOMT1-M3 mutant protein (i.e., Figure 8 Enzyme kinetics were detected using "M3" in the enzyme, and the results are as follows: Figure 8 As shown, the CcOMT1-M3 mutant, compared to the wild-type CcOMT1 protein (i.e., Figure 8 Regarding the “WT” in the figure, it refers to the maximum catalytic rate for the substrate (i.e., corydaline). V max and enzyme conversion efficiency k cat Both increased by more than two times, confirming that the CcOMT1-M3 mutant can effectively modify CcOMT1 and significantly improve the biosynthetic efficiency of active ingredients such as berberine alkaloids (especially epiberberine) in Coptis chinensis.

[0091] Example 2:

[0092] This embodiment provides a recombinant expression vector containing the CcOMT1-M3 mutant nucleotide sequence as shown in SEQ ID NO. 3, wherein the recombinant expression vector is any one of a plasmid (such as the pET-28a plasmid), a bacteriophage, or a viral vector.

[0093] We also provide an engineered bacterium for expressing the CcOMT1-M3 mutant described in Example 1, which is obtained by transforming the above-described recombinant expression vector into a host bacterium.

[0094] In summary, this invention first obtains the crystal structure of CcOMT1 by analysis, and then determines the single mutation site of amino acids in the enzyme substrate binding pocket based on the crystal structure. After mutation iteration, the CcOMT1-M3 mutant is successfully constructed. Compared with the wild-type CcOMT1 protein, the catalytic efficiency of the CcOMT1-M3 mutant is increased by about 5 times and the substrate conversion rate is increased by about 2 times.

[0095] CcOMT1 is an S2OMT enzyme that can serve as a key metabolic flux node in the biosynthetic pathway of proberberine alkaloids. Therefore, the CcOMT1-M3 mutant can increase the yield of proberberine alkaloids (especially epiberberine) in the efficient acquisition of trace components of Coptis chinensis by improving catalytic activity and conversion rate.

[0096] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A mutant of berberine methyltransferase CcOMT1, characterized in that, The mutant is CcOMT1-M3, obtained by mutating serine at position 109 of berberine oxymethyltransferase CcOMT1 to leucine, cysteine ​​at position 250 to alanine, and leucine at position 300 to alanine.

2. The mutant as described in claim 1, characterized in that, The amino acid sequence of the berberine methyltransferase CcOMT1 is shown in SEQ ID NO.

1.

3. A gene encoding the mutant as described in claim 1, characterized in that, The nucleotide sequence of the gene encoding the mutant CcOMT1-M3 is shown in SEQ ID NO.

2.

4. A recombinant expression vector comprising the coding gene of the mutant as described in claim 2.

5. The recombinant expression vector as described in claim 4, characterized in that, The recombinant expression vector is any one of plasmid, bacteriophage, or viral vector.

6. An engineered bacterium for expressing the mutant as described in claim 1 or 2, characterized in that, The engineered bacteria are obtained by transforming the recombinant expression vector of claim 4 or 5 into host bacteria.

7. A method for preparing a mutant as described in claim 1 or 2, characterized in that, Includes the following steps: Several amino acids of the wild-type CcOMT1 protein were selected as potential mutation sites. Design amplification primer sequence for each potential mutation site, wherein the mutation of each potential mutation site includes: if the current potential mutation site is not any one of alanine, leucine, or phenylalanine, then mutate the current potential mutation site to alanine, leucine, or phenylalanine respectively; if the current potential mutation site is any one of alanine, leucine, or phenylalanine, then mutate the current potential mutation site to the other two amino acids respectively. Based on the amplification primer sequence, each potential mutation site of the wild-type CcOMT1 protein is mutated to the corresponding amino acid to obtain a CcOMT1 single mutant gene fragment, and each CcOMT1 single mutant gene fragment contains only one single mutation site. Expression vectors for each CcOMT1 single mutant gene fragment were constructed, and the corresponding CcOMT1 single mutant gene fragment was expressed based on each expression vector to obtain CcOMT1 single mutant protein after single-point mutation of wild-type CcOMT1 protein. The enzyme activities of all CcOMT1 single mutant proteins were arranged in descending order. Among the top N CcOMT1 single mutant proteins with the highest enzyme activity, the single mutation site of the CcOMT1 single mutant protein with the highest enzyme activity was recorded as the major mutation site, and the single mutation sites of the other CcOMT1 single mutant proteins were recorded as minor mutation sites. Combination mutations were performed on the CcOMT1 single mutant protein before the N position of enzyme activity to obtain several corresponding CcOMT1 multiple mutant proteins, and each CcOMT1 multiple mutant protein contained a major mutation site and at least one minor mutation site. The CcOMT1 multimutant protein with the highest enzyme activity among all CcOMT1 multimutant proteins was identified as the final CcOMT1 mutant.

8. The preparation method according to claim 7, characterized in that, The potential mutation sites include tryptophan (W15) at position 15, tyrosine (Y19) at position 19, methionine (M106) at position 106, serine (S109) at position 109, alanine (A112) at position 112, isoleucine (I113) at position 113, isoleucine (I145) at position 145, tryptophan (W146) at position 146, phenylalanine (F159) at position 159, alanine (A162) at position 162, and phenanthrene (T) at position 165 in the wild-type CcOMT1 protein. Asparagine N165, aspartic acid at position 166 D166, serine at position 167 S167, leucine at position 169 L169, cysteine ​​at position 250 C250, isoleucine at position 283 I283, methionine at position 296 M296, leucine at position 300 L300, aspartic acid at position 303 D303, methionine at position 304 M304, asparagine at position 307 N307, and valine at position 388 V338.

9. The preparation method according to claim 8, characterized in that, At least one CcOMT1 multimutant protein contains a master mutation site and at least two secondary mutation sites.

10. The preparation method according to claim 8, characterized in that, The amplification primers for mutating amino acid W15 to alanine, leucine and phenylalanine are W15A-F / W15A-R, W15L-F / W15L-R and W15F-F / W15F-R, respectively. And / or, the amplification primers for mutating amino acid Y19 to alanine, leucine, and phenylalanine are Y19A-F / Y19A-R, Y19L-F / Y19L-R, and Y19F-F / Y19F-R, respectively. And / or, the amplification primers for mutating amino acid M106 to alanine, leucine, and phenylalanine are M106A-F / M106A-R, M106L-F / M106L-R, and M106F-F / M106F-R, respectively. And / or, the amplification primers for mutating amino acid S109 to alanine, leucine, and phenylalanine are S109A-F / S109A-R, S109L-F / S109L-R, and S109F-F / S109F-R, respectively. And / or, the amplification primers for mutating amino acid A112 to leucine and phenylalanine, respectively, are A112L-F / A112L-R and A112F-F / A112F-R; And / or, the amplification primers for mutating amino acid I113 to alanine, leucine, and phenylalanine are I113A-F / I113A-R, I113L-F / I113L-R, and I113F-F / I113F-R, respectively. And / or, the amplification primers for mutating amino acid I145 to alanine, leucine, and phenylalanine are I145A-F / I145A-R, I145L-F / I145L-R, and I145F-F / I145F-R, respectively. And / or, the amplification primers for mutating amino acid W146 to alanine, leucine, and phenylalanine are W146A-F / W146A-R, W146L-F / W146L-R, and W146F-F / W146F-R, respectively. And / or, the amplification primers for mutating amino acid F159 to alanine and leucine, respectively, are F159A-F / F149A-R and F159L-F / F149L-R; And / or, the amplification primers for mutating amino acid A162 to leucine and phenylalanine, respectively, are A162L-F / A162L-R and A162F-F / A162F-R; And / or, the amplification primers for mutating amino acid N165 to alanine, leucine, and phenylalanine are N165A-F / N165A-R, N165L-F / N165L-R, and N165F-F / N165F-R, respectively. And / or, the amplification primers for mutating amino acid D166 to alanine, leucine, and phenylalanine are D166A-F / D166A-R, D166L-F / D166L-R, and D166F-F / D166F-R, respectively. And / or, the amplification primers for mutating amino acid S167 to alanine, leucine, and phenylalanine are S167A-F / S167A-R, S167L-F / S167L-R, and S167F-F / S167F-R, respectively. And / or, the amplification primers for mutating amino acid L169 to alanine and phenylalanine, respectively, are L169A-F / L169A-R and L169F-F / L169F-R; And / or, the amplification primers for mutating amino acid C250 to alanine, leucine, and phenylalanine are C250A-F / C250A-R, C250L-F / C250L-R, and C250F-F / C250-R, respectively. And / or, the amplification primers for mutating amino acid I283 to alanine, leucine, and phenylalanine are I283A-F / I283A-R, I283L-F / I283L-R, and I283F-F / I283F-R, respectively. And / or, the amplification primers for mutating amino acid M296 to alanine, leucine, and phenylalanine are M296A-F / M296A-R, M296L-F / M296L-R, and M296F-F / M296F-R, respectively. And / or, the amplification primers for mutating amino acid L300 to alanine and phenylalanine, respectively, are L300A-F / L300A-R and L300F-F / L300F-R; And / or, the amplification primers for mutating amino acid D303 to alanine, leucine, and phenylalanine are D303A-F / D303A-R, D303L-F / D303L-R, and D303F-F / D303F-R, respectively. And / or, the amplification primers for mutating amino acid M304 to alanine, leucine, and phenylalanine are M304A-F / M304A-R, M304L-F / M304L-R, and M304F-F / M304F-R, respectively. And / or, the amplification primers for mutating amino acid N307 to alanine, leucine, and phenylalanine are N307A-F / N307A-R, N307L-F / N307L-R, and N307F-F / N307F-R, respectively; And / or, the amplification primers for mutating amino acid V338 to alanine, leucine, and phenylalanine are V338A-F / V338A-R, V338L-F / V338L-R, and V338F-F / V338F-R, respectively; The sequences of each amplification primer are as follows: ； ； 。

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