A sam-dependent o-methyltransferase mutant and applications thereof

Abstract

The application discloses a SAM-dependent O-methyltransferase mutant and application thereof, and belongs to the technical field of biotechnology and enzyme engineering.The SAM-dependent O-methyltransferase mutant of the application takes the SAM-dependent O-methyltransferase with the amino acid sequence shown in SEQ ID NO2 as a parent, and is subjected to any one or more of the following mutations: the 20th alanine is mutated into proline; the 277th serine is mutated into alanine; the 278th alanine is mutated into glycine; the 279th glycine is mutated into alanine; the 281th threonine is mutated into alanine; and the 282th threonine is mutated into alanine.The SAM-dependent O-methyltransferase mutant of the application can be used for the biocatalytic preparation of 5-methoxy-L-tryptophan and / or melatonin, and can be increased by 271% at most relative to a starting strain, thereby providing a new enzymatic catalysis scheme for the preparation of O-methylated hydroxyindole compounds.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and enzyme engineering, and in particular to a SAM-dependent O-methyltransferase mutant and its applications. Background Technology

[0002] Hydroxyindole compounds and their O-methylated products have significant applications in the pharmaceutical, functional food, and fine chemical industries. Among them, 5-hydroxy-L-tryptophan (5-HTP) is an important precursor in serotonin synthesis, and N-acetylserotonin (NAS) is a direct precursor in the melatonin biosynthesis pathway. Through O-methylation, 5-HTP can be converted to 5-methoxy-L-tryptophan, and NAS can be converted to melatonin. Therefore, developing enzyme catalytic elements capable of efficiently catalyzing the O-methylation of hydroxyindole substrates is of great significance for constructing green and milder routes for the preparation of indole compounds.

[0003] Existing methods for preparing O-methylated hydroxyindole compounds mainly include chemical synthesis and biocatalysis. Chemical synthesis typically requires methylating agents, and some reactions involve steps such as protection / deprotection, regioselectivity control, and product purification. For hydroxyindole substrates such as 5-hydroxy-L-tryptophan and N-acetyl-5-hydroxytryptamine, their molecules contain structural units such as indole rings, phenolic hydroxyl groups, amino groups, carboxyl groups, or amide groups. Side reactions are easily generated during chemical methylation, affecting the yield and purification efficiency of the target product. For example, EP0197390A2 discloses a method for preparing N-acetylserotonin and melatonin, which involves acetylation of serotonin to generate N,O-diacetyl-5-hydroxytryptamine, followed by selective hydrolysis to obtain N-acetyl-5-hydroxytryptamine, and then methylation with dimethyl sulfate under alkaline conditions to prepare melatonin. Although this type of method can obtain the target product, the reaction involves steps such as acetylation, selective hydrolysis, methylation under strongly alkaline conditions, extraction, and recrystallization, making the process relatively complex.

[0004] Biocatalysis can achieve selective O-methylation of hydroxyindole substrates under mild conditions, representing a potential green route for the preparation of O-methylated hydroxyindole compounds. SAM-dependent O-methyltransferases can catalyze the O-methylation of substrate hydroxyl groups using S-adenosyl-L-methionine as a methyl donor. Existing technologies disclose some O-methyltransferases capable of catalyzing the conversion of N-acetyl-5-hydroxytryptamine to melatonin, or catalyzing the O-methylation of 5-hydroxy-L-tryptamine, indicating that SAM-dependent O-methyltransferases can serve as enzyme catalytic elements for the O-methylation preparation of hydroxyindole compounds. US10883127B2 discloses an ASMT mutant, the nucleic acid encoding the mutant, an expression vector, and a recombinant host cell, with the aim of enhancing the conversion of N-acetyl-5-hydroxytryptamine to melatonin. The patent preferably uses human ASMT as the parent enzyme and performs mutations on the corresponding A258–T272, P241–Y248, D259–H271 and / or T307–Q310 fragments, with preferred mutations including A258E, G260D and T272A.

[0005] However, the aforementioned biocatalytic schemes mainly revolve around the reaction of ASMT catalyzing the conversion of N-acetyl-5-hydroxytryptamine to melatonin, focusing on improving the known ASMT's ability to convert NAS to the melatonin terminal step, or incorporating this step into the melatonin biosynthetic pathway of engineered bacteria. For the direct O-methylation of 5-hydroxy-L-tryptophan to 5-methoxy-L-tryptophan, and for bacterial SAM-dependent O-methyltransferases capable of simultaneously adapting to 5-hydroxy-L-tryptophan and N-acetyl-5-hydroxytryptamine, current technologies still lack corresponding enzyme catalytic elements and mutants.

[0006] Furthermore, existing ASMT mutant protocols primarily involve activity-enhancing mutations in specific fragments of the existing melatonin synthase ASMT. No methods have been publicly disclosed to obtain SAM-dependent O-methyltransferase mutants that simultaneously enhance activity and restore function by exploiting differential sites between active and inactive homologous enzymes. For bacterial enzymes already possessing hydroxyindole O-methylation activity, further enhancement of their catalytic ability is still needed; for homologous enzymes without significant activity, specific site mutations are also required to restore their O-methylation function for target substrates.

[0007] Therefore, there is an urgent need to develop a SAM-dependent O-methyltransferase and its mutants that differ from the traditional ASMT melatonin production route, enabling it to catalyze the O-methylation reaction of 5-hydroxy-L-tryptophan and / or N-acetyl-5-hydroxytryptamine for the preparation of 5-methoxy-L-tryptophan and / or melatonin, and further enhance the catalytic activity of the enzyme by specific site mutations. Summary of the Invention

[0008] Existing chemical methods for preparing O-methylated hydroxyindole compounds typically involve problems such as the use of chemical methylating agents, multi-step conversions, selectivity control, and product purification. This invention provides a SAM-dependent O-methyltransferase mutant and its applications. This invention provides a SAM-dependent O-methyltransferase A0 derived from Actinomadura sp. KC06, which uses S-adenosyl-L-methionine as a methyl donor to catalyze the O-methylation reaction of 5-hydroxy-L-tryptophan and N-acetyl-5-hydroxytryptamine, respectively, to generate 5-methoxy-L-tryptophan and melatonin. In a whole-cell reaction system, A0 achieves conversion rates of 18.8% for 5-hydroxy-L-tryptophan and 22.4% for N-acetyl-5-hydroxytryptamine. The present invention further provides an activity-enhancing mutant of A0, which improves its O-methylation activity for N-acetyl-5-hydroxytryptamine by mutating the 20-23 region and / or the 276-282 region of A0. The relative conversion rates of the A20P and G279A mutants reach about 271% and 270% of that of the starting enzyme A0, respectively.

[0009] The first objective of this invention is to provide a SAM-dependent O-methyltransferase mutant, using the SAM-dependent O-methyltransferase with the amino acid sequence shown in SEQ ID NO.2 as the parent, and performing one or more of the following mutations:

[0010] (1) Mutate the 20th alanine to proline;

[0011] (2) Mutate serine at position 277 to alanine;

[0012] (3) Mutate alanine at position 278 to glycine;

[0013] (4) Mutate glycine at position 279 to alanine;

[0014] (5) Mutate threonine at position 281 to alanine;

[0015] (6) Mutate threonine at position 282 to alanine.

[0016] In one embodiment of the present invention, the nucleotide sequence is as shown in SEQ ID NO.1 and is derived from Actinomadura sp. KC06.

[0017] A second objective of this invention is to provide a gene encoding the SAM-dependent O-methyltransferase mutant.

[0018] A third objective of this invention is to provide an expression vector carrying the gene.

[0019] In one embodiment of the present invention, the expression vector is the pET-28a(+) plasmid.

[0020] A fourth objective of this invention is to provide a recombinant bacterium expressing the SAM-dependent O-methyltransferase mutant or carrying the expression vector.

[0021] In one embodiment of the present invention, the recombinant bacteria uses bacteria or Escherichia coli as the host cell.

[0022] In one embodiment of the present invention, the host of the Escherichia coli is E. coli BL21(DE3).

[0023] The fifth objective of this invention is to provide the application of the SAM-dependent O-methyltransferase mutant or the recombinant bacteria in the preparation of O-methylated hydroxyindole compounds;

[0024] The O-methylated hydroxyindole compounds include 5-methoxy-L-tryptophan and / or melatonin.

[0025] In one embodiment of the present invention, in the pure enzyme reaction system, the substrate concentration is 0.1 mmol / L to 20 mmol / L, the SAM concentration is 0.1 mmol / L to 20 mmol / L, and the enzyme concentration is 1 μmol / L to 100 μmol / L.

[0026] The sixth objective of this invention is to provide a method for synthesizing O-methylated hydroxyindole compounds, using hydroxyindole compounds as substrates, S-adenosyl-L-methionine as a methyl donor, and the SAM-dependent O-methyltransferase mutant or the whole cells of the recombinant bacteria as catalysts to catalyze the synthesis of O-methylated hydroxyindole compounds.

[0027] The hydroxyindole compounds include 5-hydroxy-L-tryptophan and / or N-acetyl-5-hydroxytryptophan;

[0028] The O-methylated hydroxyindole compounds include 5-methoxy-L-tryptophan and / or melatonin.

[0029] In one embodiment of the present invention, the 5-hydroxy-L-tryptophan is converted to 5-methoxy-L-tryptophan by an O-methylation reaction.

[0030] In one embodiment of the present invention, the N-acetyl-5-hydroxytryptamine is converted into melatonin via an O-methylation reaction.

[0031] In one embodiment of the present invention, the concentration of the substrate is 0.1 mmol / L to 20 mmol / L.

[0032] In one embodiment of the present invention, the concentration of the S-adenosyl-L-methionine is 0.1 mmol / L to 20 mmol / L.

[0033] In one embodiment of the present invention, the concentration of the whole cells is 1 mg / mL to 200 mg / mL.

[0034] In one embodiment of the present invention, the catalytic conditions are: catalysis at 20℃-50℃ for 1 h to 48 h.

[0035] The SAM-dependent O-methyltransferase mutants of this invention, compared to the starting enzyme A0, exhibit increased O-methylation activity for N-acetyl-5-hydroxytryptamine. The A20P mutant achieves approximately 271% of the relative conversion of N-acetyl-5-hydroxytryptamine compared to the starting enzyme A0. The G279A mutant achieves approximately 270% of the relative conversion of N-acetyl-5-hydroxytryptamine compared to the starting enzyme A0. The S277A, A278G, T281A, and T282A mutants achieve approximately 233%, 243%, 264%, and 264% of the relative conversion of N-acetyl-5-hydroxytryptamine compared to the starting enzyme A0, respectively.

[0036] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0037] This invention provides a SAM-dependent O-methyltransferase mutant and its applications. Existing ASMT mutant schemes mainly focus on the conversion of N-acetyl-5-hydroxytryptamine to melatonin. However, the A0 provided by this invention can catalyze not only the conversion of N-acetyl-5-hydroxytryptamine to melatonin, but also the conversion of 5-hydroxy-L-tryptamine to 5-methoxy-L-tryptamine, thus expanding the application scope of SAM-dependent O-methyltransferases in the O-methylation preparation of hydroxyindole substrates. Experimental results show that in a whole-cell reaction system, A0 achieves a conversion rate of 18.8% for 5-hydroxy-L-tryptamine and 22.4% for N-acetyl-5-hydroxytryptamine, indicating that A0 can serve as an enzyme catalytic element simultaneously adaptable to two types of hydroxyindole substrates.

[0038] This invention provides activity-enhancing mutants of A0. Unlike traditional methods that modify only known ASMT activity pockets or SAM binding sites, this invention obtains multiple mutants with significantly enhanced activity for N-acetyl-5-hydroxytryptamine O-methylation by mutating regions 20-23 and 276-282 of A0. Specifically, the A20P mutant achieves a relative conversion rate of 271% of the starting enzyme A0; within the 276-282 region, the relative conversion rates of S277A, A278G, G279A, T281A, and T282A reach 233%, 243%, 270%, 264%, and 264%, respectively. These results indicate that regions 20-23 and 276-282 are effective mutation regions capable of improving the catalytic performance of A0, rather than isolated single-site effects.

[0039] The mutation scheme provided by this invention has good scalability. The regions at positions 20-23 and 276-282 of A0 can serve as activity-enhancing regions. By determining the equivalent positions of these regions or sites in homologous SAM-dependent O-methyltransferases, this invention can be used to obtain homologous enzyme mutants with improved activity, thereby avoiding the problem of narrow applicability caused by using only a single enzyme or a single mutation point.

[0040] This invention provides genes, recombinant plasmids, and host cells encoding the aforementioned SAM-dependent O-methyltransferase or its mutants, enabling the enzyme and its mutants to be obtained through recombinant expression and used in whole-cell or pure enzyme reactions. The reaction system can use 5-hydroxy-L-tryptophan and / or N-acetyl-5-hydroxytryptamine as substrates, with SAM as a methyl donor, to prepare 5-methoxy-L-tryptophan and / or melatonin under mild conditions.

[0041] Therefore, compared to chemical methylation routes, this invention avoids reliance on chemical methylating agents such as dimethyl sulfate and multi-step protection, deprotection, or selective hydrolysis processes. Compared to existing ASMT melatonin production methods, this invention provides bacterial SAM-dependent O-methyltransferases A0 and A0 activity-enhanced mutants, which are applicable not only to the O-methylation reaction of N-acetyl-5-hydroxytryptamine to melatonin but also to the O-methylation reaction of 5-hydroxy-L-tryptophan to 5-methoxy-L-tryptophan, providing a new enzyme catalytic scheme for the biocatalytic preparation of O-methylated hydroxyindole compounds. Attached Figure Description

[0042] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0043] Figure 1The reaction formula is for the O-methylation of hydroxyindole compounds catalyzed by SAM-dependent O-methyltransferase;

[0044] Figure 2 The results of expression and purification analysis of SAM-dependent O-methyltransferase A0;

[0045] Figure 3 Mass spectrometry analysis results of SAM-dependent O-methyltransferase A0 catalyzing the formation of O-methylated products from hydroxyindole substrates using product standards;

[0046] Figure 4 The results of mass spectrometry analysis of the SAM-dependent O-methyltransferase A0 catalyzing the formation of O-methylated products from hydroxyindole substrates using 5-HTP as a substrate are shown.

[0047] Figure 5 The results of mass spectrometry analysis of the SAM-dependent O-methyltransferase A0 catalyzing the formation of O-methylated products from hydroxyindole substrates using NAS as a substrate.

[0048] Figure 6 The results show the comparison of the O-methylation reactions of different hydroxyindole substrates catalyzed by SAM-dependent O-methyltransferase A0 in pure enzyme reaction systems and whole-cell reaction systems;

[0049] Figure 7 This is the verification result of the A0 activity-enhanced mutant catalyzing the O-methylation reaction of N-acetyl-5-hydroxytryptamine. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0051] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0052] Product detection was performed using an Agilent 1260 HPLC system equipped with a UV detector. The chromatographic column was a Diamonsil C18 column, 250 mm × 4.6 mm, 5 μm. Mobile phase A was water, mobile phase B was acetonitrile, and mobile phase C was methanol. The flow rate was 0.6 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The gradient elution program was: 0–12 min, 95%–41% A; 12–13 min, 41%–5% A; 13–14 min, 5%–95% A; 14–19 min, 95% A.

[0053] The product structure was further confirmed using LC-MS / MS. LC-MS / MS analysis was performed using a TSQ Quantum UltraEMR triple quadrupole LC-MS / MS system equipped with an H-ESI II ion source connected to an UltiMate 3000 UHPLC system. Separation was performed using a Waters ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm) at a flow rate of 0.2 mL / min. The spray voltage was 3200 V in positive ion mode and 2800 V in negative ion mode, with a capillary temperature of 350 °C.

[0054] The target product 5-methoxy-L-tryptophan was confirmed by comparing its retention time with that of the 5-methoxy-DL-tryptophan standard, and the target product melatonin was confirmed by comparing its retention time with that of the melatonin standard. The O-methylated product of 5-hydroxy-L-tryptophan was further confirmed by LC-MS / MS detection of a mass-to-charge ratio signal consistent with that of 5-methoxy-L-tryptophan; the O-methylated product of N-acetyl-5-hydroxytryptophan was further confirmed by LC-MS / MS detection of a mass-to-charge ratio signal consistent with that of melatonin. In this invention, conversion rate refers to the percentage of the target product generated relative to the initial substrate feed amount, as determined by HPLC analysis; relative conversion rate refers to the percentage of the mutant conversion rate relative to the conversion rate of the starting enzyme A0. Unless otherwise specified, all reaction data are the average of three independent parallel experiments.

[0055] Example 1: Construction, expression, and purification of SAM-dependent O-methyltransferase A0 mutants

[0056] The pET-28a(+) plasmid was used as the expression vector. The gene encoding the SAM-dependent O-methyltransferase A0 (Uniprot ID: A0A0I9TIW5) with the nucleotide sequence shown in SEQ ID NO.1 was inserted between the NdeI and XhoI restriction sites of the pET-28a(+) vector to obtain the recombinant plasmid pET28a-A0.

[0057] The recombinant bacteria were inoculated into LB medium containing kanamycin (10.0 g / L tryptone). –1 Yeast extract 5.0 g·L –1 Sodium chloride 10.0 g·L –1The culture medium (pH 7.2, solid medium with 2% agar added) was cultured at 37℃ and 220 rpm. 2 mL of the overnight culture was inoculated into 50 mL of LB medium containing kanamycin and cultured for another 2.5 h at 37℃ and 220 rpm. The culture was then cooled in an ice bath for 20 min, and IPTG was added to a final concentration of 0.5 mM for induction. Induction was performed at 16℃ and 130 rpm for 14 h. After induction, the cells were collected by centrifugation at 4℃ and 4000 g for 3 min.

[0058] The collected bacterial cells were resuspended in 50 mM phosphate buffer containing 300 mM NaCl, the pH was adjusted to 7.4, and the concentration of the bacterial suspension was adjusted to 100 mg / mL. The resulting bacterial suspension could be used directly for whole-cell catalytic reactions; for pure enzyme reactions, the bacterial cells were lysed, centrifuged, and the supernatant was collected and purified to obtain A0 and its mutant enzyme solutions. The purified protein was analyzed by SDS-PAGE. Figure 2 The target protein band was consistent with the theoretical molecular weight, indicating that a SAM-dependent O-methyltransferase and its mutants that can be used for catalytic reactions were obtained.

[0059] Example 2: Heme protein mutant catalyzes L-tryptophan hydroxylation reaction

[0060] Using 5-hydroxy-L-tryptophan and N-acetyl-5-hydroxytryptamine as substrates, the ability of A0 obtained in Example 1 to catalyze the O-methylation reaction of hydroxyindole compounds was tested.

[0061] The total volume of the whole-cell reaction system was 200 μL, containing 50 μL of SAM stock solution, 50 μL of substrate stock solution, and 100 μL of whole-cell suspension. The concentrations of both the SAM and substrate stock solutions were 12 mM, and the substrate was either 5-hydroxy-L-tryptophan or N-acetyl-5-hydroxytryptamine. The final concentrations of SAM and substrate in the reaction system were 3 mM and 50 mg / mL, respectively. The reaction was carried out at 40 °C and 800 rpm for 24 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction. The precipitate was removed by centrifugation, and the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis.

[0062] HPLC results showed that the A0 whole-cell system could catalyze the conversion of 5-hydroxy-L-tryptophan to 5-methoxy-L-tryptophan and N-acetyl-5-hydroxytryptophan to melatonin. To eliminate the influence of host cell background reactions, spontaneous substrate methylation, substrate loss, and methyl donor loss on the detection results, empty vector whole-cell control, cell-free system control, substrate-free system control, and SAM-free system control were set up. No corresponding target product peaks were detected in the above control groups. Based on an initial substrate concentration of 3 mM, the conversion rate of 5-hydroxy-L-tryptophan in the A0 whole-cell system was 18.8%, and the conversion rate of N-acetyl-5-hydroxytryptophan was 22.4%. These results indicate that A0 can catalyze the O-methylation reaction of hydroxyindole compounds with SAM as a methyl donor and can serve as an enzyme catalytic element for the preparation of 5-methoxy-L-tryptophan and melatonin.

[0063] Example 3: Comparison of catalytic performance of A0 in pure enzyme system and whole cell system

[0064] To further evaluate the reactivity of A0 under different catalytic forms, the O-methylation activity of A0 for 5-hydroxy-L-tryptophan, N-acetyl-5-hydroxytryptamine, and serotonin was detected using both pure enzyme and whole-cell systems. Figure 6 ).

[0065] The total volume of the pure enzyme reaction system was 200 μL, containing 50 μL of SAM stock solution, 50 μL of substrate stock solution, and 100 μL of enzyme solution. The concentrations of the SAM stock solution and substrate stock solution were 12 mM and 30 μM, respectively. The final concentrations of SAM, substrate, and enzyme in the reaction system were 3 mM and 15 μM, respectively. The reaction was carried out at 40℃ and 800 rpm for 24 h. After the reaction was completed, an equal volume of methanol was added to terminate the reaction. After mixing, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis.

[0066] The results showed that, under pure enzyme conditions, A0 exhibited detectable O-methylation activity for N-acetyl-5-hydroxytryptamine (N-acetyl-5-hydroxytryptamine) with a conversion rate of 6.9%; while no significant conversion of 5-hydroxy-L-tryptamine and serotonin was detected under the same pure enzyme conditions. In contrast, in the whole-cell reaction system, A0 catalyzed the O-methylation of 5-hydroxy-L-tryptamine and N-acetyl-5-hydroxytryptamine, with conversion rates reaching 18.8% and 22.4%, respectively, while no significant conversion of serotonin was detected. These results indicate that A0 possesses catalytic ability for both 5-hydroxy-L-tryptamine and N-acetyl-5-hydroxytryptamine in the whole-cell system; and in the pure enzyme system, A0 exhibits clear O-methylation activity for N-acetyl-5-hydroxytryptamine. Therefore, further activity enhancement was verified using N-acetyl-5-hydroxytryptamine as a substrate.

[0067] Example 4: A0 activity-enhanced mutant catalyzes the O-methylation of N-acetyl-5-hydroxytryptamine

[0068] To enhance the O-methylation activity of A0 for N-acetyl-5-hydroxytryptamine, mutations were made in the 20-23 and 276-282 regions of A0 to obtain A0 activity-enhancing mutants such as A20P, S277A, A278G, G279A, T281A, and T282A.

[0069] Using recombinant plasmid pET28a-A0 as a template, site-directed mutagenesis was performed using whole-plasmid PCR. Mutations were made in the 20-23 and 276-282 regions of A0 to construct expression plasmids for A0 activity-enhancing mutants. Specifically, recombinant plasmids containing mutations A20P, S277A, A278G, G279A, T281A, and T282A were constructed.

[0070] The primers used in the study are shown in Table 1 below:

[0071] Table 1 Primer Table

[0072]

[0073] PCR amplification products were digested with DpnI and transformed into *E. coli* BL21(DE3). Positive single clones were selected for sequencing verification, yielding recombinant strains expressing A0 and A0 activity-enhancing mutants, respectively. The A0 activity-enhancing mutants included A20P, S277A, A278G, G279A, T281A, and T282A.

[0074] The ability of the pure enzyme reaction system described in Example 3 to catalyze the production of melatonin from N-acetyl-5-hydroxytryptamine was tested. Figure 7 ).

[0075] The relative conversion rates of each mutant were calculated using the conversion rate of the starting enzyme A0 as 100%. The results showed that the relative conversion rate of the A20P mutant reached 271%, indicating that the mutation at position 20 can significantly improve the O-methylation activity of A0 for N-acetyl-5-hydroxytryptamine.

[0076] Furthermore, multiple mutants in the region of positions 276–282 exhibited significantly enhanced activity. Specifically, the relative conversion rates of S277A, A278G, G279A, T281A, and T282A reached 233%, 243%, 270%, 264%, and 264%, respectively. These results indicate that the region of positions 276–282 is a crucial region for regulating the catalytic performance of A0, and mutations in this region can significantly improve the O-methylation activity of A0.

[0077] The above results further demonstrate that the regions at positions 20-23 and 276-282 of A0 are not isolated single-point effects, but rather effective mutation regions that can be used to obtain activity-enhancing SAM-dependent O-methyltransferase mutants. Among them, A20P and G279A are preferred activity-enhancing mutations.

[0078] Example 5: Mass spectrometric confirmation of O-methylation products

[0079] To confirm the structure of the reaction products, the reaction solutions obtained in Examples 2-5 were analyzed by LC-MS / MS.

[0080] For the 5-hydroxy-L-tryptophan reaction system, HPLC analysis revealed a product peak with the same retention time as the 5-methoxy-L-tryptophan standard. Further LC-MS / MS analysis detected a mass-to-charge ratio signal consistent with 5-methoxy-L-tryptophan, confirming that A0 can catalyze the O-methylation reaction of 5-hydroxy-L-tryptophan to generate 5-methoxy-L-tryptophan (…). Figures 3-5 ).

[0081] For the N-acetyl-5-hydroxytryptamine reaction system, HPLC analysis revealed a product peak with the same retention time as the melatonin standard. Further LC-MS / MS analysis detected a mass-to-charge ratio signal consistent with melatonin, confirming that the A0 and A0 activity-enhanced mutants can catalyze the O-methylation reaction of N-acetyl-5-hydroxytryptamine to generate melatonin.

[0082] The above results indicate that the SAM-dependent O-methyltransferase and its mutants provided by this invention can catalyze the O-methylation reaction of hydroxyindole substrates to generate the corresponding O-methylated hydroxyindole products.

[0083] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A SAM-dependent O-methyltransferase mutant, characterized in that, Using the SAM-dependent O-methyltransferase with the amino acid sequence shown in SEQ ID NO.2 as the parent, perform one or more of the following mutations: (1) Mutate the 20th alanine to proline; (2) Mutate serine at position 277 to alanine; (3) Mutate alanine at position 278 to glycine; (4) Mutate glycine at position 279 to alanine; (5) Mutate threonine at position 281 to alanine; (6) Mutate threonine at position 282 to alanine.

2. A gene encoding the SAM-dependent O-methyltransferase mutant of claim 1.

3. An expression vector carrying the gene of claim 2.

4. A recombinant bacterium expressing the SAM-dependent O-methyltransferase mutant of claim 1 or carrying the expression vector of claim 3.

5. The application of the SAM-dependent O-methyltransferase mutant of claim 1 or the recombinant bacteria of claim 4 in the preparation of O-methylated hydroxyindole compounds; The O-methylated hydroxyindole compounds include 5-methoxy-L-tryptophan and / or melatonin.

6. A method for synthesizing O-methylated hydroxyindole compounds, characterized in that, Using hydroxyindole compounds as substrates and S-adenosyl-L-methionine as a methyl donor, and employing the whole cells of the SAM-dependent O-methyltransferase mutant as described in claim 1 or the recombinant bacteria as described in claim 4 as catalysts, O-methylated hydroxyindole compounds are obtained by catalysis. The hydroxyindole compounds include 5-hydroxy-L-tryptophan and / or N-acetyl-5-hydroxytryptophan; The O-methylated hydroxyindole compounds include 5-methoxy-L-tryptophan and / or melatonin.

7. The method according to claim 6, characterized in that, The concentration of the substrate is 0.1 mmol / L to 20 mmol / L.

8. The method according to claim 6, characterized in that, The concentration of the S-adenosyl-L-methionine is 0.1 mmol / L to 20 mmol / L.

9. The method according to claim 6, characterized in that, The concentration of the whole cells is 1 mg / mL to 200 mg / mL.

10. The method according to claim 6, characterized in that, The catalytic conditions are: catalysis at 20℃-50℃ for 1 h to 48 h.

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