Construction and application of a high activity and high thermal stability o-methyltransferase mutant

Abstract

The application discloses construction and application of a high-activity and high-thermal-stability O-methyltransferase mutant, and belongs to the technical field of bioengineering. Lp COMT is the female parent, and a combination mutant containing T32V, S52F, I316S and I316T is constructed, and the amino acid sequence of the obtained optimal mutant M2 is shown as SEQ ID NO: 3. Compared with the wild type, the specific activity of the optimal mutant of the application is increased by 8.1 times (from 2.58 U / mg to 20.84 U / mg), the optimal reaction temperature is increased from 40 DEG C to 45 DEG C, and about 50% of the activity is still retained after 6 hours of treatment at 40 DEG C. The mutant disclosed by the application can efficiently catalyze the methylation of N-acetylserotonin to generate 2.86 g / L of melatonin, the conversion rate is greater than 90%, and the mutant has an excellent industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the construction and application of a highly active and thermally stable O-methyltransferase mutant. Background Technology

[0002] O-methyltransferases (OMTs) are a class of S-adenosylmethionine (SAM)-dependent methyltransferases that catalyze the transfer of methyl groups from SAM to the hydroxyl or carboxyl groups of the substrate. They play a crucial role in plant secondary metabolism, lignin synthesis, and the biosynthesis of alkaloids and flavonoids. In recent years, the application of OMTs in heterologous biosynthesis has received widespread attention, especially in the microbial synthesis of high-value natural products such as melatonin, vanillin, and ferulic acid, where OMT-catalyzed methylation reactions are often the rate-limiting steps.

[0003] Melatonin (chemical name: N-acetyl-5-methoxytryptamine) is an indoleamine hormone widely found in animals and plants, possessing important physiological functions such as regulating circadian rhythms, antioxidation, immune regulation, and neuroprotection. Currently, industrial production mainly relies on chemical synthesis, which involves cumbersome steps and the use of hazardous reagents. Microbial synthesis of melatonin offers a green alternative.

[0004] In the microbial synthesis of melatonin, O-methyltransferases catalyze the 5-hydroxymethylation of N-acetylserotonin (NAS) to produce melatonin, and are the key rate-limiting enzymes in this pathway. Currently, the OMTs used for melatonin synthesis are mainly derived from plant caffeic acid O-methyltransferase (COMT) or animal acetylserotonin O-methyltransferase (ASMT). However, these naturally derived OMTs share common shortcomings: (1) low catalytic activity on the non-natural substrate NAS; (2) poor thermal stability, rapidly inactivating at fermentation temperatures (30-40℃), and unable to support long-term catalytic reactions; and (3) limited soluble expression levels. Therefore, developing an O-methyltransferase mutant with both high catalytic activity and excellent thermal stability has important application value for improving the microbial synthesis efficiency of methylated natural products such as melatonin. Summary of the Invention

[0005] This invention addresses the technical shortcomings of existing O-methyltransferases, namely low catalytic activity and poor thermal stability, by providing an O-methyltransferase mutant with both high catalytic activity and high thermal stability. Its specific enzyme activity is increased by 8.1 times, and its optimal temperature is increased from 40℃ (wild type) to 45℃. After treatment at 40℃ for 6 hours, it retains approximately 50% of its activity, while the wild type loses more than 50% of its activity after treatment at 40℃ for 30 minutes. When applied to the biocatalysis of melatonin, it can generate 2.86 g / L of melatonin.

[0006] Technical solution: The complete technical means and methods of this invention.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an O-methyltransferase mutant derived from perennial ryegrass ( Lolium perenne Caffeic acid O-methyltransferase Lp COMT (whose amino acid sequence is shown in SEQ ID NO:1 and nucleotide sequence is shown in SEQ ID NO:2) is the parent material, containing substitution mutations of at least one amino acid residue selected from the following sites: position 32, position 52, position 128, position 193, position 252, position 263, position 316, and position 321.

[0008] Preferably, the mutation comprises a threonine (T) mutation at position 32 to valine (V) (T32V).

[0009] Preferably, the mutation comprises a mutation of serine (S) at position 52 to phenylalanine (F) (S52F).

[0010] Preferably, the mutation comprises a mutation of histidine (H) at position 193 to lysine (K) (H193K).

[0011] Preferably, the mutation comprises a mutation of glutamic acid (E) at position 252 to phenylalanine (F) (E252F).

[0012] Preferably, the mutation includes a mutation of tryptophan (W) at position 263 to glycine (G) (W263G), alanine (A) (W263A), or leucine (L) (W263L).

[0013] Preferably, the mutation includes a mutation of isoleucine (I) at position 316 to serine (S) (I316S) or threonine (T) (I316T).

[0014] Preferably, the mutation includes a mutation at position 321 of asparagine (N) to phenylalanine (F) (N321F), threonine (T) (N321T), glutamine (Q) (N321Q), serine (S) (N321S), or tyrosine (Y) (N321Y).

[0015] More preferably, the mutant contains a combination of T32V and S52F mutations.

[0016] More preferably, the mutant contains a combined mutation of T32V and H193K.

[0017] More preferably, the mutant contains a combination of T32V and N321F mutations.

[0018] More preferably, the mutant comprises a combined mutation of S52F and N321F.

[0019] More preferably, the mutant contains a combined mutation of H193K and N321F.

[0020] More preferably, the mutant contains a combined mutation of T32V, S52F and N321F (named M1).

[0021] More preferably, the mutant comprises a combination of mutations of T32V, S52F, I316T and N321T.

[0022] More preferably, the mutant comprises a combination of mutations of T32V, S52F, I316T and N321S.

[0023] More preferably, the mutant comprises a combination of mutations of T32V, S52F, I316T and N321Y.

[0024] Most preferably, the mutant comprises a combined mutation of T32V, S52F, I316S and N321T (named M2), the amino acid sequence of which is shown in SEQ ID NO:3.

[0025] The present invention also provides a DNA molecule encoding the O-methyltransferase mutant.

[0026] Preferably, the nucleotide sequence of the DNA molecule is shown in SEQ ID NO:4.

[0027] The present invention also provides a recombinant expression vector comprising the DNA molecule.

[0028] In one embodiment, the expression vector includes, but is not limited to, pET-28a, pET-Duet, pACYC-Duet, pCDF-Duet, and pRSF-Duet vectors.

[0029] The present invention also provides a host cell comprising the recombinant expression vector.

[0030] Preferably, the host cell is *Escherichia coli* (E. coli). Escherichia coli ), more preferably BL21(DE3).

[0031] This invention also provides the application technology of the O-methyltransferase mutant in the preparation of melatonin.

[0032] In one embodiment, the application technique includes expressing the O-methyltransferase mutant in recombinant Escherichia coli and using the recombinant bacteria to catalyze the synthesis of melatonin using N-acetylserotonin as a substrate.

[0033] In one embodiment, the application technique includes inducing recombinant bacteria at 16-30°C for 10-16 h, more preferably 12 h.

[0034] In one embodiment, the application technology includes a catalytic temperature of 25-35°C, more preferably 25°C.

[0035] This invention provides an O-methyltransferase mutant, wherein the O-methyltransferase mutant is, The asparagine at position 321 of the O-methyltransferase, whose amino acid sequence is shown in SEQ ID NO:1, is mutated to phenylalanine and named N321F. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, and the asparagine at position 321 may be mutated to phenylalanine, and named T32V / N321F. Alternatively, the serine at position 52 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to phenylalanine, and the asparagine at position 321 can be mutated to phenylalanine, named S32F / N321F. Alternatively, the threonine at position 32 of the O-methyltransferase shown in SEQ ID NO:1 may be mutated to valine, the serine at position 52 to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine, threonine, serine, or tyrosine, and named T32V / S52F / N321F, T32V / S52F / N321T, T32V / S52F / N321S, and T32V / S52F / N321Y; Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the tryptophan at position 263 to glycine, and named T32V / S52F / N321F / W263G. Alternatively, the threonine at position 32 of the O-methyltransferase shown in SEQ ID NO:1 may be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the isoleucine at position 316 to either serine or threonine, and named T32V / S52F / N321F / I316S and T32V / S52F / N321F / I316T; Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the isoleucine at position 316 to serine, and the asparagine at position 321 to either threonine or serine, and named T32V / S52F / I316S / N321T or T32V / S52F / I316S / N321S. Alternatively, the amino acid sequence of the O-methyltransferase shown in SEQ ID NO:1 may be modified by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to threonine, and asparagine at position 321 to threonine, and named T32V / S52F / I316T / N321T.

[0036] The present invention also provides a nucleic acid that encodes the above-mentioned O-methyltransferase mutant.

[0037] The present invention also provides an expression cassette carrying the above-mentioned nucleic acid or mutant.

[0038] The present invention also provides a recombinant vector carrying the above-mentioned nucleic acid or mutant.

[0039] In one embodiment of the present invention, the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors.

[0040] In one embodiment of the present invention, the carrier is pET-28a, pET-Duet, pACYC-Duet, pCDF-Duet, or pRSF-Duet.

[0041] The present invention also provides recombinant cells expressing the above-mentioned O-methyltransferase mutant or carrying the above-mentioned nucleic acid or carrying the recombinant vector.

[0042] In one embodiment of the present invention, the recombinant cells are expressed using prokaryotic or eukaryotic microorganisms as the expression host.

[0043] In one embodiment of the present invention, the eukaryotic microorganism is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces martensii.

[0044] In one embodiment of the present invention, the prokaryotic microorganism is selected from one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella and Streptomyces.

[0045] The present invention also provides a recombinant Escherichia coli expressing the above-mentioned O-methyltransferase mutant.

[0046] The present invention also provides a recombinase catalyst containing the above-mentioned O-methyltransferase mutant, wherein the recombinase catalyst is any one of the following forms: (1) Culture recombinant expression transformants containing the O-methyltransferase mutant and isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme; (2) Culture recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, and break the transformant cells containing the recombinant O-methyltransferase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, break the transformant cells containing the recombinant O-methyltransferase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant O-methyltransferase mutant enzyme to obtain lyophilized powder. (4) Cultivate recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, break the transformant cells containing the recombinant O-methyltransferase mutant enzyme, obtain cell lysate, and purify the cell lysate of the recombinant O-methyltransferase mutant enzyme to obtain pure enzyme solution.

[0047] The present invention also provides a method for improving the enzyme activity or thermostability of wild-type O-methyltransferase, wherein the method comprises mutating asparagine at position 321 of the O-methyltransferase, as shown in SEQ ID NO:1, to phenylalanine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, and the asparagine at position 321 may be mutated to phenylalanine. Alternatively, the serine at position 52 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine, threonine, serine, or tyrosine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the tryptophan at position 263 to glycine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the isoleucine at position 316 to either serine or threonine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, the isoleucine at position 316 to serine, and the asparagine at position 321 to either threonine or serine. Alternatively, the amino acid sequence of the O-methyltransferase shown in SEQ ID NO:1 may be modified by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to threonine, and asparagine at position 321 to threonine.

[0048] This invention also provides a method for preparing melatonin, the method comprising: reacting a substrate N with a substrate N... Acetylserotonin and S adenosine L Melatonin is prepared by adding the above-mentioned O-methyltransferase mutant or the above-mentioned recombinant cells or the above-mentioned recombinant enzyme catalyst to the methionine reaction system.

[0049] In one embodiment of the present invention, the reaction conditions are: temperature of 25-35°C and time of 48-60h.

[0050] In one embodiment of the present invention, the amount of recombinant cells added is 1~10 g / L.

[0051] In one embodiment of the present invention, the N The dosage of serotonin added is 1~5 g / L, S adenosine L The amount of methionine added is 1~3 g / L.

[0052] The present invention also provides the use of the above-mentioned =O-methyltransferase mutant, or the above-mentioned nucleic acid, or the above-mentioned recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinase catalyst in the preparation of melatonin or products containing melatonin.

[0053] Beneficial effects Compared with existing technologies, this invention significantly improves the catalytic activity and thermostability of O-methyltransferases. The optimal mutant M2 (T32V-S52F-I316S-F321T) described in this invention exhibits a specific activity of 20.84 U / mg, which is 8.10 times that of the wild-type enzyme (2.58 U / mg). This makes it one of the most active O-methyltransferase mutants in the prior art, significantly improving the methylation efficiency of the non-natural substrate NAS. cat / K m Compared to the wild type, the activity was increased by 5.50 times. Simultaneously, the optimal reaction temperature increased from 40℃ for the wild type to 45℃. After treatment at 40℃ for 6 hours, approximately 50% of the activity was retained, while the wild type lost more than 50% of its activity after treatment at 40℃ for 30 minutes. This improved thermostability makes this mutant suitable for long-term industrial production. Using this mutant as a precursor of N-acetylserotonin, 2.86 g / L melatonin can be generated with a conversion rate >90%, demonstrating excellent prospects for industrial application. Attached Figure Description

[0054] Figure 1 This is a comparison of the relative activities of the mutants of the present invention.

[0055] Figure 2 Comparison of relative activities between the M1 mutant and the mutant with substrate pocket polar microenvironment remodeling.

[0056] Figure 3 The relative activity curves of wild-type LpCOMT and mutants M1 and M2 at different temperatures are shown.

[0057] Figure 4 Thermostability curves (40℃ treatment) of wild-type LpCOMT and mutants M1 and M2. Detailed Implementation

[0058] The present invention will be further illustrated below with specific examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0059] The detection methods involved in the following embodiments are as follows: Detection of melatonin production: HPLC detection method: The reaction solution was diluted 5 times with 5% methanol, centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm membrane. Chromatographic column: Dimosoil C18 (5 μL, 250 mm × 4.6 mm), mobile phase: A: 0.1% formic acid aqueous solution, B: 100% methanol, detector: UV detector, detection wavelength: 276 nm, column temperature: 30℃, injection volume: 10 μL, flow rate: 1 mL / min.

[0060] Example 1: Design, preparation and verification of O-methyltransferase mutants The specific steps are as follows: 1. Obtaining the target gene The maternal gene used in this invention is derived from perennial ryegrass ( Lolium perenne Caffeic acid O-methyltransferase gene Lp COMT (GenBank accession number: NC_067247.2). This gene was synthesized by a commercial company after codon optimization in E. coli. The nucleotide sequence is shown in SEQ ID NO:2, and the encoded amino acid sequence is shown in SEQ ID NO:1.

[0061] Caffeic acid O-methyltransferase amino acid sequence (SEQ ID NO.1): MGSTAADMAASADEEACMFALQLASSSILPMTLKNAIELGLLEILVAAGGKSLTPTEVAAKLPSAANPEAPDMVDRMLRLLASYNVVSCLVEEGKDGRLSRSYGAAPVCKFLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMSAFEYHGTDPRFNRVFNEGMKNH SIIITKKLLELYHGFQGLGTLVDVGGGVGATVAAITAHYPAIKGVNFDLPHVISEAPPFPGVTHVGGDMFKEVPSGDAILMKWILHDWSDQHCATLLKNCYDALPAHGKVVLVECILPVNPEAKPSSQGVFHVDMIMLAHNPGGRERYEREFEALARGAGFTGVKSTYIYANAWAIEFTK The nucleotide sequence encoding caffeic acid O-methyltransferase (SEQ ID NO.2): 2. Design of mutation sites Candidate mutation sites were identified using a combination of free energy calculations and evolutionarily conserved sequence analysis. Based on the activity screening results, T32V, S52F, and N321F were identified as key mutations for enhancing activity. Further, polar microenvironment remodeling of substrate binding pocket residues was performed on the M1 (T32V / S52F / N321F) model to screen for the optimal combination of I316S and F321T, ultimately yielding the optimal mutant M2 (T32V / S52F / I316S / N321T).

[0062] 3. Preparation of recombinant plasmids carrying site-directed mutagenesis mutant enzymes and recombinant strains. (1) Construction of single-mutation site mutants 1) Construction of recombinant plasmids containing wild-type enzymes: The LpCOMT gene, with a chemically synthesized nucleotide sequence as shown in SEQ ID NO:2, was then ligated into the pET-28a plasmid. Bam HI、 Hin After dIII restriction site removal, the recombinant plasmid pET-28a-LpCOMT was prepared.

[0063] 2) Construction of recombinant vectors containing mutant enzymes: Using the pET-28a plasmid carrying the LpCOMT gene as a template, site-directed mutagenesis was introduced via inverse plasmid PCR. The primers shown in Table 1 were used for mutagenesis at the corresponding sites. The PCR reaction mixture was as follows: 1 μL upstream primer, 1 μL downstream primer, 1 μL template plasmid, 25 μL 2 × Phanta Max Master Mix, and 22 μL ddH2O. The pre-denaturation temperature was 95℃ for 3 min, followed by denaturation at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 2 min, and final extension at 72℃ for 5 min, for a total of 30 cycles.

[0064] Table 1: Primers for mutation sites

[0065] The PCR product was digested with Dpn I, self-ligated, and transformed into Escherichia coli JM109. Sequencing confirmed the correctness of the mutant sequence, and recombinant vectors expressing different mutants were prepared.

[0066] 3) Construction of recombinant strains The recombinant vectors containing wild-type enzymes and the recombinant vectors containing mutants obtained in steps 1) and 2) were respectively transformed into... E. coliRecombinant strains were prepared from BL21(DE3) competent cells.

[0067] (2) Construction of double-mutant, triple-mutant, and quadruple-mutant mutants Using the single mutant with enhanced activity as a template (using primers in Table 1), a double mutant was constructed according to the single mutant construction method described above. After verifying the catalytic activity, a triple mutant was constructed using the double mutant as a template (using primers in Table 1) according to the single mutant construction method described above. After verifying the activity, a quad mutant was constructed using the triple mutant as a template (using primers in Table 1) according to the single mutant construction method described above. (Regarding the mutation at position 321, the first round of mutation mutated N to F: N321F, and the second round of mutation was based on the first round, so it is represented as F321Q, F321S, F321T, and F321Y.)

[0068] Recombinant strains expressing double-mutant, triple-mutant, and quadruple-mutant mutants were prepared according to the above method.

[0069] 4. Recombinant expression of enzymes and determination of relative activity The recombinant strains prepared in the above steps were inoculated into 10 mL of liquid LB medium containing kanamycin (concentration: 50 μg / mL) and cultured at 37℃ for 12 h to prepare seed culture. The seed culture was then transferred to 50 mL of LB liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ until OD600 = 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and the culture was induced at 25℃ for 12 h to prepare fermentation broth. The fermentation broth was centrifuged, the bacterial cells were collected, and the mixture was sonicated. The supernatant was used as crude enzyme solution.

[0070] Reaction system (1 mL): 50 mM Tris-HCl (pH 7.4), 1 mM SAM (S adenosine L Methionine), 2 mMNAS (N Acetylserotonin), 100 μL crude enzyme solution. React at 37℃ for 10 min, then terminate the reaction with methanol. HPLC was used to quantitatively detect melatonin production.

[0071] Enzyme activity unit (U) is defined as the amount of enzyme required to generate 1 μmol of melatonin per minute under the above conditions.

[0072] The enzyme activity and relative enzyme activity of different mutants were detected separately, and the results are as follows: Figures 1-2 As shown, where, Figure 1The optimal mutants are N321F, T32V-N321F, S52-N321F (S52F / N321F), and T32V-S52F-N321F; Figure 2 A list of mutants showing positive effects is given, and the results are shown in Table 2: Table 2: Some mutants that outperform wild-type

[0073] Example 2: O Enzymatic characterization of methyltransferase mutants The specific steps are as follows: 1. Protein expression and purification The recombinant strains containing wild-type and M1 (T32V / S52F / I316S / N321T) mutant enzymes, prepared in Example 1, were inoculated into 10 mL of liquid LB medium containing kanamycin (concentration: 50 μg / mL) and cultured at 37℃ for 12 h to prepare seed culture. The prepared seed culture was then transferred to 50 mL of LB liquid medium at an inoculation rate of 2% (v / v) and cultured at 37℃ until OD600 = 0.6-0.8. IPTG was added to a final concentration of 0.2 mM, and induction was performed at 25℃ for 12 h to prepare fermentation broth. The prepared fermentation broth was centrifuged, the cell pellet was collected, sonicated, and the target protein was purified by Ni-NTA affinity chromatography to prepare pure enzyme solutions of wild-type, M1 mutant, and M2 mutant enzymes, respectively.

[0074] 2. Determination of specific enzyme activity and kinetic parameters of wild-type enzymes and M1 and M2 mutant enzymes Reaction system (1 mL): 50 mM Tris-HCl (pH 7.4), 1 mM SAM (S adenosine L Methionine), 2 mMNAS (N Acetylserotonin), 100 μL of purified enzyme solution. The reaction was carried out at 37℃ for 10 min, and the reaction was terminated with methanol. Melatonin production was quantitatively detected by HPLC.

[0075] Enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of melatonin per minute under the above conditions. Specific enzyme activity refers to the activity per milligram of protein.

[0076] The measurement results are shown in Table 3 below.

[0077] Table 3: Determination of specific enzyme activity and its kinetic parameters

[0078] The above results indicate that the specific activity of the optimal mutant M2 in this invention is 8.10 times that of the wild type, k cat / K m It is 5.50 times higher than the wild type, which is significantly better than other O-methyltransferase mutants in the prior art.

[0079] SEQ ID NO.3 (M2): MGSTAADMAASADEEACMFALQLASSSILPMVLKNAIELGLLEILVAAGGKFLTPTEVAAKLPSAANPEAPDMVDRMLRLLASYNVVSCLVEEGKDGRLSRSYGAAPVCKFLTPNEDGVSMAALALMNQDKVLMESWYYLKDAVLDGGIPFNKAYGMSAFEYHGTDPRFNRVFNEGMKNH SIIITKKLLELYHGFQGLGTLVDVGGGVGATVAAITAHYPAIKGVNFDLPHVISEAPPFPGVTHVGGDMFKEVPSGDAILMKWILHDWSDQHCATLLKNCYDALPAHGKVVLVECILPVNPEAKPSSQGVFHVDMSMLAHTPGGRERYEREFEALARGAGFTGVKSTYIYANAWAIEFTK SEQ ID NO.4: 3. Optimal temperature and thermal stability of the best mutant M2 (1) Detection of optimal temperature The specific reaction system is as in step 2, except that the reaction is carried out at different temperatures (25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃) for 10 min, and the enzyme activities of the wild-type, M1 and M2 mutants prepared in Example 1 are measured to determine the optimal reaction temperature.

[0080] The results show: like Figure 3 As shown, the optimal temperature for the wild type is 40℃, while the optimal temperature for M1 (T32V / S52F / F321T) and M2 (T32V / S52F / I316S / N321T) is 45℃. (2) Thermal stability test The wild-type pure enzyme solution, M1 mutant enzyme pure enzyme solution, and M2 mutant enzyme pure enzyme solution prepared in Example 1 were incubated at 40°C for different times. After cooling in an ice bath, the residual enzyme activity was measured according to the method in step 1.

[0081] The results show: like Figure 4 As shown, after treatment at 40°C for 6 hours, M2 (T32V / S52F / I316S / N321T) still retained about 50% of its initial activity, while the wild type lost more than 50% of its activity after treatment at 40°C for 30 minutes and was completely inactivated after 60 minutes.

[0082] The above results show that the mutant of the present invention not only has higher catalytic activity, but also significantly improved thermal stability.

[0083] Example 3: Application of mutants in whole-cell melatonin synthesis The specific steps are as follows: 1. Preparation of whole-cell catalysts Recombinant bacteria carrying the wild-type LpCOMT or mutant M2 gene ( E. coli BL21(DE3) / pET-28a-LpCOMT、 E. coli BL21(DE3) / pET-28a-T32V / S52F / I316S / N321T were inoculated into 10 mL LB liquid medium containing 50 μg / mL kanamycin and cultured at 37℃ for 12 h to prepare seed culture. The prepared seed culture was transferred to TB medium at an inoculation rate of 2% and cultured until the OD600 reached 0.6-0.8. Then, 0.2 mM IPTG was added to induce culture at 25℃ for 20 h. After centrifugation, the cells were collected as whole-cell catalyst.

[0084] 2. Preparation of melatonin To a substrate containing 3 g / L NAS (N 1 g / L SAM (acetylserotonin) and 1 g / L SAM (S adenosine L In a system containing methionine, 8 g / L of the whole-cell catalyst prepared in step 1 was added, and catalysis was carried out at 30 °C for 48 h.

[0085] The liquid phase detection results show: Expression of wild-type LpCOMT strain ( E. coli The melatonin production of BL21(DE3) / pET-28a-LpCOMT was 455.63 mg / L, and the expression mutant strain M2 (( E. coli The melatonin production of BL21(DE3) / pET-28a-T32V / S52F / I316S / N321T was 2.86 g / L.

[0086] The above results indicate that the mutant of this invention also exhibits significantly better catalytic efficiency than the wild type in whole-cell catalytic systems, and has good potential for industrial application.

[0087] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. An O-methyltransferase mutant, characterized in that, The O-methyltransferase mutant is, The asparagine at position 321 of the O-methyltransferase, whose amino acid sequence is shown in SEQ ID NO:1, is mutated to phenylalanine and named N321F. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, and the asparagine at position 321 may be mutated to phenylalanine, and named T32V / N321F. Alternatively, the serine at position 52 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to phenylalanine, and the asparagine at position 321 can be mutated to phenylalanine, named S32F / N321F. Alternatively, the threonine at position 32 of the O-methyltransferase shown in SEQ ID NO:1 may be mutated to valine, the serine at position 52 to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine, threonine, serine, or tyrosine, and named T32V / S52F / N321F, T32V / S52F / N321T, T32V / S52F / N321S, and T32V / S52F / N321Y; Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the tryptophan at position 263 to glycine, and named T32V / S52F / N321F / W263G. Alternatively, the threonine at position 32 of the O-methyltransferase shown in SEQ ID NO:1 may be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the isoleucine at position 316 to either serine or threonine, and named T32V / S52F / N321F / I316S and T32V / S52F / N321F / I316T; Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the isoleucine at position 316 to serine, and the asparagine at position 321 to either threonine or serine, and named T32V / S52F / I316S / N321T or T32V / S52F / I316S / N321S. Alternatively, the amino acid sequence of the O-methyltransferase shown in SEQ ID NO:1 may be modified by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to threonine, and asparagine at position 321 to threonine, and named T32V / S52F / I316T / N321T.

2. A nucleic acid, characterized in that, The nucleic acid encodes the O-methyltransferase mutant of claim 1.

3. An expression cassette or recombinant vector carrying the nucleic acid described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, CRISPR / Cas9 vectors, or viral vectors; Preferably, the carrier is pET-28a, pET-Duet, pACYC-Duet, pCDF-Duet, or pRSF-Duet.

5. Recombinant cells expressing the O-methyltransferase mutant of claim 1, or carrying the nucleic acid of claim 2, or carrying the recombinant vector of claim 3 or 4.

6. The recombinant cell according to claim 5, characterized in that, The recombinant cells use prokaryotic or eukaryotic microorganisms as expression hosts; Preferably, the eukaryotic microorganism is selected from one or more of Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces martensii; Preferably, the prokaryotic microorganism is selected from one or more of Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, Salmonella, and Streptomyces.

7. A recombinant enzyme catalyst containing the O-methyltransferase mutant of claim 1, characterized in that, The recombinase catalyst is any one of the following forms: (1) Culture recombinant expression transformants containing the O-methyltransferase mutant and isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme; (2) Culture recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, and break the transformant cells containing the recombinant O-methyltransferase mutant enzyme to obtain cell lysate; (3) Cultivate recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, break the transformant cells containing the recombinant O-methyltransferase mutant enzyme, obtain cell lysate, and freeze-dry the cell lysate of the recombinant O-methyltransferase mutant enzyme to obtain lyophilized powder. (4) Cultivate recombinant expression transformants containing the O-methyltransferase mutant, isolate transformant cells containing the recombinant O-methyltransferase mutant enzyme, break the transformant cells containing the recombinant O-methyltransferase mutant enzyme, obtain cell lysate, and purify the cell lysate of the recombinant O-methyltransferase mutant enzyme to obtain pure enzyme solution.

8. A method for improving the activity or thermal stability of wild-type O-methyltransferase, characterized in that, The method involves mutating asparagine at position 321 of the O-methyltransferase, as shown in SEQ ID NO:1, to phenylalanine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, and the asparagine at position 321 may be mutated to phenylalanine. Alternatively, the serine at position 52 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, and the asparagine at position 321 may be mutated to phenylalanine, threonine, serine, or tyrosine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, can be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the tryptophan at position 263 to glycine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, the asparagine at position 321 to phenylalanine, and the isoleucine at position 316 to either serine or threonine. Alternatively, the threonine at position 32 of the O-methyltransferase, as shown in SEQ ID NO:1, may be mutated to valine, the serine at position 52 to phenylalanine, the isoleucine at position 316 to serine, and the asparagine at position 321 to either threonine or serine. Alternatively, the amino acid sequence of the O-methyltransferase shown in SEQ ID NO:1 may be modified by mutating threonine at position 32 to valine, serine at position 52 to phenylalanine, isoleucine at position 316 to threonine, and asparagine at position 321 to threonine.

9. A method for preparing melatonin, characterized in that, The method involves introducing substrate N into a container... Acetylserotonin and S adenosine L Melatonin is prepared by adding the O-methyltransferase mutant of claim 1, the recombinant cell of claim 5 or 6, or the recombinase catalyst of claim 7 to the methionine reaction system. Preferably, the reaction conditions are: temperature 25-35℃, time 48-60 h; Preferably, the amount of recombinant cells added is 1~10 g / L; Preferably, the N The dosage of serotonin added is 1~5 g / L, S adenosine L The amount of methionine added is 1~3 g / L.

10. The use of the O-methyltransferase mutant of claim 1, or the nucleic acid of claim 2, or the recombinant vector of claim 3 or 4, or the recombinant cell of claim 5 or 6, or the recombinase catalyst of claim 7 in the preparation of melatonin or products containing melatonin.

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