Process for the biosynthesis of n-acetyl-5-methoxytryptamine using 5-hydroxy-beta-indolylalanine as substrate

Abstract

The application provides a method for biosynthesis of N-acetyl-5-methoxytryptamine with 5-hydroxy beta-indole propionic acid as a substrate. The application provides a high-yield strain of N-acetyl-5-methoxytryptamine, which is used in the method for biosynthesis of N-acetyl-5-methoxytryptamine with 5-hydroxy beta-indole propionic acid as a substrate, and the yield of the synthesis of N-acetyl-5-methoxytryptamine is high. Further screening of the key enzyme gene AANAT of the 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine pathway and the key enzyme gene COMT of the N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine pathway and combined screening significantly improve the yield of biosynthesis of N-acetyl-5-methoxytryptamine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a novel method for synthesizing N-acetyl-5-methoxytryptamine and application thereof. Background Art

[0002] N-acetyl-5-methoxytryptamine (N-ATM) is an important indole neurohormone secreted primarily by the mammalian pineal gland. Due to its unique chemical structure, indole is used in synthetic pharmaceuticals such as antihypertensives, antihistamines, and antipyretics. Many alkaloids, high-efficiency plant growth regulators, and dyes are indole derivatives. N-ATM can improve sleep quality in animals. As animals age, N-ATM secretion gradually decreases. A growing body of research indicates that N-AT, in addition to its potential to treat insomnia, also possesses numerous physiological functions, including antioxidant, anti-aging, immune regulation, and anti-cancer properties.

[0003] N-acetyl-5-methoxytryptamine is a natural pharmaceutical ingredient with minimal side effects. Compared to currently used sedatives and hypnotics, it offers significant advantages in terms of toxicity, dosage, and addiction prevention. Therefore, the efficacy and market value of N-acetyl-5-methoxytryptamine are crucial and irreplaceable.

[0004] Currently, N-acetyl-5-methoxytryptamine is obtained primarily through biological extraction and chemical synthesis. However, its industrial application is limited by its low naturally occurring content in plants and animals, the limited availability of raw materials for extraction, and the high cost of extraction.

[0005] Compared to chemical synthesis, biosynthesis offers advantages such as environmental friendliness, low energy consumption, and environmental friendliness. With the advancement of synthetic biology, an increasing number of compounds have achieved bio-friendly production. Providing engineered bacteria and synthetic methods capable of high-yield biosynthesis of N-acetyl-5-methoxytryptamine is of great significance. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a high-yielding strain of N-acetyl-5-methoxytryptamine, and to construct and biosynthesize N-acetyl-5-methoxytryptamine using 5-hydroxyβ-indolylalanine as a substrate.

[0007] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0008] Provided is a method for biosynthesizing N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as a substrate, comprising the following steps:

[0009] 1) Using the recombinant genetically engineered bacteria capable of expressing the gene encoding the protein for synthesizing N-acetyl-5-methoxytryptamine or the gene encoding the protein for synthesizing 5-hydroxy-β-indolylalanine, 5-hydroxy-β-indolylalanine is used as a substrate to bioferment and synthesize N-acetyl-5-methoxytryptamine, wherein the gene encoding the protein capable of expressing the protein for synthesizing N-acetyl-5-methoxytryptamine includes the key enzyme gene DDC in the pathway of 5-hydroxy-β-indolylalanine to produce 5-hydroxytryptamine; the key enzyme genes AANAT and ACS in the pathway of 5-hydroxytryptamine to produce N-acetyl-5-hydroxytryptamine; and the key enzyme genes COMT and MAT in the pathway of N-acetyl-5-hydroxytryptamine to produce N-acetyl-5-methoxytryptamine;

[0010] 2) N-acetyl-5-methoxytryptamine is separated from the system of 1).

[0011] According to the above scheme, the nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is shown in any one of SEQ ID NOs: 2-10; the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is shown in any one of SEQ ID NOs: 12-18.

[0012] According to the above scheme, the nucleotide sequence of the key enzyme gene DDC in the 5-hydroxyβ-indolylalanine-5-hydroxytryptamine production pathway is shown in SEQ ID NO: 1;

[0013] The nucleotide sequence of the key enzyme gene ACS in the 5-hydroxytryptamine production N-acetyl-5-hydroxytryptamine pathway is shown in SEQ ID NO: 11;

[0014] The nucleotide sequence of the MAT gene, a key enzyme gene in the pathway of N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine, is shown in SEQ ID NO: 19.

[0015] Preferably, the nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 10; the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is shown in SEQ ID NO: 13 or SEQ ID NO: 18.

[0016] More preferably, the nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is as shown in any sequence in SEQ ID NO: 10, and the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is as shown in SEQ ID NO: 13;

[0017] Or the nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is shown in any of the sequences in SEQ ID NO: 10, and the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is shown in SEQ ID NO: 18.

[0018] The present invention also provides a recombinant vector comprising all enzyme coding genes for synthesizing N-acetyl-5-methoxytryptamine.

[0019] The present invention also provides a recombinant genetically engineered bacterium, which is a recombinant genetically engineered bacterium comprising the above-mentioned recombinant vector, or a recombinant genetically engineered bacterium obtained by integrating some enzyme genes into the genome of a host cell for expression, and expressing the remaining enzyme genes in a plasmid and then transferring them into the aforementioned host cell.

[0020] According to the above scheme, the DDC gene, a key enzyme gene in the pathway of 5-hydroxyβ-indolylalanine to produce 5-hydroxytryptamine, is expressed on the genome of the host cell, and other target enzyme genes are expressed in the plasmid and then transferred into the host cell whose genome expresses the DDC gene, a key enzyme gene in the pathway of 5-hydroxyβ-indolylalanine to produce 5-hydroxytryptamine.

[0021] Provided is a method for constructing a recombinant genetically engineered bacterium:

[0022] Transforming a recombinant vector containing all enzyme encoding genes for synthesizing N-acetyl-5-methoxytryptamine into a host cell to obtain a recombinant genetically engineered bacterium;

[0023] Alternatively, a portion of the target enzyme gene is integrated into the host cell genome for expression, and the remaining target enzyme gene is expressed in a plasmid and then transferred into the aforementioned host cell to obtain a recombinant genetically engineered bacterium.

[0024] Specifically, the method for constructing the recombinant genetically engineered bacteria of the present invention comprises the following steps:

[0025] The key enzyme genes AANAT and ACS in the 5-hydroxytryptamine-to-N-acetyl-5-hydroxytryptamine pathway are placed on the same plasmid for tandem expression; the key enzyme genes COMT and MAT in the N-acetyl-5-hydroxytryptamine-to-N-acetyl-5-methoxytryptamine pathway are placed on the same plasmid for tandem expression; the above plasmids are jointly transformed into a host cell expressing the DDC gene on its genome to obtain a recombinant genetically engineered bacterium.

[0026] Furthermore, the host cell is an Escherichia coli host cell, preferably a BL21 (DE3), ΔtrpR (DDC), ΔtnaA, ΔSPED Escherichia coli host cell; that is, BL21 (DE3) is used as the starting strain, the trpR gene on the genome is replaced with the DDC gene, and the tnaA gene and SPED gene are knocked out.

[0027] Beneficial effects of the present invention:

[0028] The present invention provides a high-yield strain of N-acetyl-5-methoxytryptamine, which is used to biosynthesize N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as a substrate. The yield of N-acetyl-5-methoxytryptamine is high. Furthermore, by screening the key enzyme gene AANAT (aromatic amine-N-acetyltransferase) in the 5-hydroxytryptamine-to-N-acetyl-5-hydroxytryptamine pathway and the key enzyme gene COMT (catechol-O-methyltransferase) in the N-acetyl-5-hydroxytryptamine-to-N-acetyl-5-methoxytryptamine pathway from different species, and by combined screening, the yield of biosynthesized N-acetyl-5-methoxytryptamine is significantly increased.

[0029] The present invention provides and optimizes a method for constructing a high-yielding strain of N-acetyl-5-methoxytryptamine. Partial target enzyme genes are integrated into the host cell genome for expression. Partial enzyme genes are expressed in plasmids and then transferred into the aforementioned host cells to optimize the construction of a high-yielding strain of N-acetyl-5-methoxytryptamine. This facilitates the expression of genes encoding proteins that synthesize N-acetyl-5-methoxytryptamine, resulting in a high-yielding strain of N-acetyl-5-methoxytryptamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0031] Figure 1 :5-HT producing N-acetyl-5-HT plasmid map

[0032] Figure 2 :N-acetyl-5-hydroxytryptamine produces N-acetyl-5-methoxytryptamine plasmid map DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] Escherichia coli is highly regarded as a host for exogenous gene expression due to its clear genetic background, simple technical operations, simple culture conditions, and economical large-scale fermentation. Currently, E. coli is the most widely used and successful expression system, often the preferred system for high-efficiency expression. The following describes how to modify E. coli to achieve efficient, large-scale, industrial production of N-acetyl-5-methoxytryptamine by fermentation.

[0039] Example 1 Construction of plasmid

[0040] Construction of a plasmid for producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine

[0041] Tandem expression of key enzyme genes AANAT and ACS in the 5-hydroxytryptamine production N-acetyl-5-hydroxytryptamine pathway.

[0042] Nine AANAT genes from different sources were selected, and A1-A9 were obtained by artificial synthesis after codon optimization, which were shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. A1-A9 and ACS gene were inserted into the NcoI and AflII sites of pACYCDuet plasmid to obtain pACYCDuet-A1, pACYCDuet-A2, pACYCDuet-A3, pACYCDuet-A4, pACYCDuet-A5, pACYCDuet-A6, pACYCDuet-A7, pACYCDuet-A8, pACYCDuet-A9, and pACYCDuet-ACS plasmids (as shown in Figure 1

[0043] The pACYCDuet plasmid vector containing ACS gene was cloned by using pACYCDuet-ACS-F and pACYCDuet-ACS-R primers and the pACYCDuet-ACS plasmid as a template; and the A1-A9 gene fragments were cloned by using A1-F, A1-R, A2-F, A2-R, A3-F, A3-R, A4-F, A4-R, A5-F, A5-R, A6-F, A6-R, A7-F, A7-R, A8-F, A8-R, A9-F, A9-R primers and the plasmids pACYCDuet-A1, pACYCDuet-A2, pACYCDuet-A3, pACYCDuet-A4, pACYCDuet-A5, pACYCDuet-A6, pACYCDuet-A7, pACYCDuet-A8, and pACYCDuet-A9 as templates, respectively.

[0044] The above A1-A9 gene fragments were connected with the pACYCDuet plasmid vector containing ACS gene by using a seamless cloning kit to form the plasmids pACYCDuet-A1-ACS, pACYCDuet-A2-ACS, pACYCDuet-A3-ACS, pACYCDuet-A4-ACS, pACYCDuet-A5-ACS, pACYCDuet-A6-ACS, pACYCDuet-A7-ACS, pACYCDuet-A8-ACS, and pACYCDuet-A9-ACS.

[0045] Table 1 primer sequence

[0046] ​

[0047]

[0048]

[0049] Construction of plasmid for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine

[0050] Tandem expression of COMT and MAT, key enzyme genes in the pathway of N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine.

[0051] COMT genes from different sources were codon-optimized and artificially synthesized to obtain C1-C7, whose nucleotide sequences are shown in SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. The C1-C7 and MAT genes were inserted between the NcoI and AflⅡ sites of the pETDuet plasmid to obtain the pETDuet-C1, pETDuet-C2, pETDuet-C3, pETDuet-C4, pETDuet-C5, pETDuet-C6, pETDuet-C7, and pETDuet-MAT plasmids.

[0052] The pETDuet plasmid vector containing the MAT gene was cloned using pETDuet-MAT-F and pETDuet-MAT-R as primers and the pETDuet-MAT plasmid as a template. The C1-C9 gene fragments were cloned using C1-F, C1-R, C2-F, C2-R, C3-F, C3-R, C4-F, C4-R, C5-F, C5-R, C6-F, C6-R, C7-F, and C7-R as primers and the plasmids pETDuet-C1, pETDuet-C2, pETDuet-C3, pETDuet-C4, pETDuet-C5, pETDuet-C6, and pETDuet-C7 as templates, respectively.

[0053] The C1-C9 gene fragments were ligated with the pETDuet plasmid vector containing the MAT gene using a seamless cloning kit to form plasmids pETDuet-C1-MAT, pETDuet-C2-MAT, pETDuet-C3-MAT, pETDuet-C4-MAT, pETDuet-C5-MAT, pETDuet-C6-MAT, and pETDuet-C7-MAT.

[0054] Table 2 Primer sequence 2

[0055]

[0056]

[0057]

[0058] Example 2 Transformation of host bacteria

[0059] Knockout of the tnaA gene, genomic expression of DDC, a key enzyme in the 5-hydroxyβ-indolylalanine-producing serotonin pathway, and knockout of the SPED gene.

[0060] Using primers tnaAup-F, tnaAup-R, tnaAdown-F, and tnaAdown-R, respectively, and the BL21(DE3) genome as a template, we obtained the upstream and downstream fragments of the tnaA gene. These fragments were ligated by fusion PCR using primers tnaAup-F and tnaAdown-R to generate the ΔtnaA fragment. This fragment was electroporated into BL21(DE3) competent cells, replacing the genomic tnaA gene. Positive clones were screened by PCR and designated HP114.

[0061] The DDC gene was codon-optimized, and its sequence was shown in SEQ ID No. 1, which was inserted between the NdeI and XhoI sites of the pET28a(+) plasmid to obtain the pET28a-DDC plasmid.

[0062] Using primers DDC / trpR-F and DDC / trpR-R and plasmid pET28a-DDC as a template, a DDC gene fragment containing upstream and downstream homology arms of trpR was obtained. This fragment was electroporated into HP114 competent cells, replacing the genomic trpR gene. Positive clones were screened by PCR and designated HP115.

[0063] Using primers SPEDup-F, SPEDup-R, SPEDdown-F, and SPEDdown-R, respectively, and the BL21(DE3) genome as a template, we obtained the upstream and downstream fragments of the SPED gene. These fragments were ligated by fusion PCR using primers SPEDup-F and SPEDdown-R to generate the ΔSPED fragment. This fragment was electroporated into HP115 competent cells, replacing the genomic tnaA gene. Positive clones were screened by PCR and designated HP116.

[0064] Table 3 Primer sequence 3

[0065]

[0066]

[0067] Example 3 Construction and screening of 5-hydroxy-β-indolylalanine producing strains

[0068] (1) Construction of engineered bacteria that produce N-acetyl-5-hydroxytryptamine and screening of the key gene AANAT: The plasmids pACYCDuet-A1-ACS, pACYCDuet-A2-ACS, pACYCDuet-A3-ACS, pACYCDuet-A4-ACS, pACYCDuet-A5-ACS, pACYCDuet-A6-ACS, pACYCDuet-A7-ACS, pACYCDuet-A8-ACS, and pACYCDuet-A9-ACS were electroporated into the competent strain HP116 to obtain engineered bacteria HP116-A1, HP116-A2, HP116-A3, HP116-A4, HP116-A5, HP116-A6, HP116-A7, HP116-A8, and HP116-A9. The engineered bacteria HP116-A1, HP116-A2, HP116-A3, HP116-A4, HP116-A5, HP116-A6, HP116-A7, HP116-A8 and HP116-A9 were cultured in a seed culture medium containing 34 μg / mL chloramphenicol antibiotics for 10 h to obtain seed solutions. The OD 600 =5, the seed culture medium (mass percentage) comprises 1% tryptone, 1% sodium chloride, and 0.5% yeast extract, and the seed solution is cultured at 37°C and 225 rpm; 2% of the seed solution is inoculated into a fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH2PO4, and 1.64% K2HPO4·3H2O for fermentation; after 2 h of fermentation, 1 mM IPTG is added for expression induction at 30°C. After 10 h of induction, fermentation is stopped, 5-hydroxytryptamine is added to a final concentration of 6 g / L, and conversion is carried out for 10 h. The yield of N-acetyl-5-hydroxytryptamine is detected by high performance liquid chromatography.

[0069] The results are shown in Table 4, where the ability of the engineered bacteria HP116-A1, HP116-A2, HP116-A4, HP116-A5, and HP116-A9 to produce N-acetyl-5-hydroxytryptamine was higher than that of the other strains, that is, the genes numbered A1, A2, A4, A5, and A9 had a higher ability to produce N-acetyl-5-hydroxytryptamine.

[0070] Table 4: Comparison of 5-HT production by different AANAT strains

[0071]

[0072] (2) N-acetyl-5-hydroxy-tryptamine production N-acetyl-5-methoxy-tryptamine engineering bacteria construction and key gene COMT screening: plasmids pETDuet-C1-MAT, pETDuet-C2-MAT, pETDuet-C3-MAT, pETDuet-C4-MAT, pETDuet-C5-MAT, pETDuet-C6-MAT, pETDuet-C7-MAT were electrotransformed into the HP116 competent cells to obtain engineering bacteria HP116-C1, HP116-C2, HP116-C3, HP116-C4, HP116-C5, HP116-C6, HP116-C7. The above engineering bacteria were respectively cultured in seed culture medium containing 100 μg / mL ampicillin for 10 h to obtain seed liquid, the seed culture medium (mass percentage) was 1% tryptone, 1% sodium chloride and 0.5% yeast extract, the seed liquid culture condition was 37°C, 225 rpm; 2% of the above seed liquid was inoculated into fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH2PO4 and 1.64% K2HPO4·3H2O for fermentation culture; after 2 h of fermentation culture, 1 mM IPTG was added for induction expression, the induction expression temperature was 30°C, after 10 h of induction expression, the fermentation was stopped, and N-acetyl-5-hydroxy-tryptamine was added to a final concentration of 6 g / L, methionine was supplemented to keep the concentration of methionine at about 0.5 g / L, and after 10 h of conversion, N-acetyl-5-methoxy-tryptamine was detected by high performance liquid chromatography. 600 =5, the seed culture medium (mass percentage) was 1% tryptone, 1% sodium chloride and 0.5% yeast extract, the seed liquid culture condition was 37°C, 225 rpm; 2% of the above seed liquid was inoculated into fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH2PO4 and 1.64% K2HPO4·3H2O for fermentation culture; after 2 h of fermentation culture, 1 mM IPTG was added for induction expression, the induction expression temperature was 30°C, after 10 h of induction expression, the fermentation was stopped, and N-acetyl-5-hydroxy-tryptamine was added to a final concentration of 6 g / L, methionine was supplemented to keep the concentration of methionine at about 0.5 g / L, and after 10 h of conversion, N-acetyl-5-methoxy-tryptamine was detected by high performance liquid chromatography.

[0073] The results are shown in Table 5, wherein: the ability of engineering bacteria HP116-C2, HP116-C7 to produce N-acetyl-5-hydroxy-tryptamine is higher than that of other strains, that is, the genes numbered C2 and C7 have higher ability to produce N-acetyl-5-methoxy-tryptamine.

[0074] Table 5: Comparison of N-acetyl-5-hydroxy-tryptamine production N-acetyl-5-methoxy-tryptamine yield of different COMT strains

[0075]

[0076] (3) 5-hydroxy-tryptamine production N-acetyl-5-methoxy-tryptamine engineering bacteria construction and screening

[0077] The above excellent 5-hydroxytryptamine production N-acetyl-5-hydroxytryptamine key gene AANAT and N-acetyl-5-hydroxy-tryptamine production N-acetyl-5-methoxytryptamine key enzyme gene COMT were further combined to evaluate the effect of engineering bacteria with these different gene combinations in synthesizing N-acetyl-5-methoxytryptamine. Typically,

[0078] The plasmids pACYCDuet-A1-ACS, pACYCDuet-A2-ACS, pACYCDuet-A4-ACS, pACYCDuet-A5-ACS, and pACYCDuet-A9-ACS for producing N-acetyl-5-hydroxytryptamine from 5-hydroxyβ-indolylalanine and the plasmid for producing N-acetyl-5-methoxytryptamine from 5-hydroxytryptamine were electrotransformed into the competent strain HP116 according to the combination in Table 4 to obtain engineered bacteria HP116-A1C2, HP116-A2C2, HP116-A4C2, HP116-A5C2, HP116-A9C2, HP116-A1C7, HP116-A2C7, HP116-A4C7, HP116-A5C7, and HP116-A9C7. The engineered bacteria were cultured for 10 hours in a seed culture medium containing 34 μg / mL chloramphenicol and 100 μg / mL ampicillin to obtain a seed solution having an OD600 of 5. The seed culture medium (mass percentage) consisted of 1% tryptone, 1% sodium chloride, and 0.5% yeast extract, and the culture conditions for the seed solution were 37° C. and 225 rpm. 2% of the seed solution was inoculated into a fermentation medium (mass percentage) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH2PO4, and 1.64% K2HPO4·3H2O for fermentation culture. After 2 hours of fermentation culture, 1 mM IPTG was added for expression induction at a temperature of 30° C. Fermentation was stopped after 16 hours of induction, and 5-hydroxyβ-indolylalanine was added to a final concentration of 6 g / L. The reaction was carried out for 10 hours, and the yield of N-acetyl-5-methoxytryptamine was detected by high-performance liquid chromatography.

[0079] The results are shown in Table 6. The engineered bacteria provided by different gene combinations produced N-acetyl-5-methoxytryptamine at a yield of 2.41 g / L or higher. The best strain was HP116-A9C2, which had the highest yield of 4.5 g / L. This indicates that AANAT numbered A9 and COMT numbered C2 were the best at synthesizing N-acetyl-5-methoxytryptamine.

[0080] Table 6: Comparison of the yield of N-acetyl-5-methoxytryptamine produced by different strain combinations from 5-hydroxyβ-indolylalanine

[0081]

[0082] The present invention obtains an engineered strain for biosynthesizing N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as a substrate by combining and constructing a key enzyme gene DDC in the pathway of 5-hydroxy-β-indolylalanine to 5-hydroxytryptamine; key enzyme genes AANAT and ACS in the pathway of 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine; and key enzyme genes COMT and MAT in the pathway of N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine. The strain is used for biosynthesizing N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as a substrate, and the synthesis pathway is: 5-hydroxy-β-indolylalanine→5-hydroxytryptamine→N-acetyl-5-hydroxytryptamine→N-acetyl-5-methoxytryptamine.

[0083] Further screening of the key enzyme genes AANAT in the 5-hydroxytryptamine-to-N-acetyl-5-hydroxytryptamine pathway, and COMT in the N-acetyl-5-hydroxytryptamine-to-N-acetyl-5-methoxytryptamine pathway, and their combined screening, significantly increased the yield of biosynthesized N-acetyl-5-methoxytryptamine, paving the way for efficient, large-scale industrial production of N-acetyl-5-methoxytryptamine. SEQUENCE LISTING <110> Hebei Vidakang Biotechnology Co., Ltd. <120> Method for biosynthesizing N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as substrate and its applications <130> 1 <160> 19 <170> PatentIn version 3.3 <210> 1 <211> 1428 <212> DNA <213> Artificial sequence <400> 1 atggaagcca accagtttaa agattttgcc aaagagatga tcgattacgt tagcggctat 60 ctggaaaata ttcgtgatcg tcgtgttctg ccgaccgttg aaccgggtta tctgcgtccg 120 ctgattccgg caaccgcacc gcagaaaccg gataaatggg aagatgttat ggcagatatt 180 gaacgcgtta tttgcctgg tgttacccat tggcatagtc cgcgttttca tgcatatttt 240 ccgaccgcaa atagctatcc ggcaattgtt gcagatattc tgagcggtgc aattgcctgt 300 attggtttta gctggattgc aagtccggca tgtaccgaac tggaagttgt tatgctggat 360 tggctgggta aaatgattgg tctgccggaa gattttctgg catgtagcgg tggtaaaggt 420 ggtggtgtta ttcagggcac cgcaagcgaa gcaaccctgg ttgcactgct gggtgcaaaa 480 gcacgtatga ttgatcgtgt gaaaaaagaa aaccggaaa tgagcgatag cgaaattgtt 540 gccaaactgg tggcatatac cagcgcacag agccatagca gcgttgaacg tgcaggtctg 600 ttaggtggtg tgaaaatgcg tggtctgcag ccggatgata ataatcgtct gcgtggtgaa 660 accctggaag tggcaattaa agaagatcgc gaagcaggtc tgattccgtt ttatgttgtt 720 gcgaccctgg gtacaaccag cagctgtacc tttgataatc tggaagaact gggtcctgtt 780 tgcaacagca ataacatttg gctgcatgtt gatgcagcct atgcaggtag cagctttatt 840 tgtccggaat ttcgttatct gatgaaaggt attgatcgcg cagatagctt taactttaat 900 ccgcataaat ggctgctggt gaattttgat tgtagcacca tgtggctgaa agatccgagc 960 tggctggtta atgcatttaa tgttgatccg ctgtatctga aacatgaaca gcagggtgca 1020 gcaccggatt atcgtcattg gcagattccg ctgggtcgtc gttttcgtgc actgaaactg 1080 tggtttgttc tgcgtctgta tggtattgaa aatctgcagg cctttattcg caaacatgtt 1140 gaactggccc attattttga aagcctggtt cgtggtgatg aacgctttga aattaccgaa 1200 gaagttgttc tgggtttagt ttgctttcgt ctgaaagcca gcaacgaaat taatgaagca 1260 ctgctgaaac gtctgaatgg tcgtggtgtg attcatctgg ttccgagcaa aattcgtgat 1320 gtgtattttc tgcgcctggc aatttgtagc cgttttaccg aaaaagccga tattgacatt 1380 agctggaaag aagttaaaga agcagcagac gaggtcctga aaaaataa 1428 <210> 2 <211> 624 <212> DNA <213> 人工序列 <400> 2 atgtccactc cgtctgttca ctgcctgaaa ccgagcccgc tgcacctgcc gtccggcatc 60 ccaggctctc cgggtcgtca gcgtcgtcac accctgccgg cgaacgaatt tcgttgcctg 120 accccggaag atgcggcggg tgttttcgaa atcgaacgtg aagcgttcat ctctgtttcc 180 ggtaactgcc cgctgaacct ggatgaagtt cagcacttcc tgaccctgtg cccggaactg 240 agcctgggct ggttcgttga aggccgtctg gttgcgttca tcatcggctc actgtgggat 300 gaagaacgtc tgacccagga atcactggcg ctgcaccgtc cgcgtggcca cagcgcgcac 360 ctgcacgcgc tggcggttca ccgtagcttc cgtcagcagg gcaaaggcag cgttctgctg 420 tggcgttacc tgcaccacgt tggcgcgcag ccggcggttc gtcgtgcggt tctgatgtgc 480 gaagatgcgc tggttccgtt ctatcagcgt ttcggcttcc acccggctgg cccgtgcgcg 540 atcgttgttg gtagcctgac cttcaccgaa atgcactgca gcctgcgtgg ccacgcggcg 600 ctgcgtcgta acagcgatcg ttaa 624 <210> 3 <211> 624 <212> DNA <213> Artificial sequence <400> 3 atgagcaccc agagcaccca cccgctgaaa ccggaagcgc cgcgtctgcc gccgggcatc 60 ccggaatctc cgtcttgcca gcgtcgtcac accctgccgg cgtctgagtt ccgttgcctg 120 accccggaag atgcggttag cgcgttcgaa atcgaacgtg aagcgttcat cagcgttctg 180 ggtgtttgcc cgctgtacct ggatgaaatc cgtcacttcc tgaccctgtg cccggaactg 240 agcctgggct ggttcgaaga aggctgcctg gttgcgttca tcatcggcag cctgtgggat 300 aaagaacgtc tgatgcagga aagcctgacc ctgcaccgta gcggcggcca catcgcgcac 360 ctgcacgttc tggcggttca ccgtgcgttc cgtcagcagg gtcgtggccc gatcctgctg 420 tggcgttacc tgcaccacct gggctcccag ccggcggttc gtcgtgcggc gctgatgtgc 480 gaagatgcgc tggttccgtt ctacgaacgt ttcagcttcc acgcggttgg tccgtgcgcg 540 atcaccgttg gcagcctgac cttcatggaa ctgcactgca gcctgcgtgg ccacccgttc 600 ctgcgtcgta atagcggttg ctaa 624 <210> 4 <400> 4 <211> 618 <212> DNA <213> 人工序列 atgacccagc aggttagcgg tagcccgttc ttcaaaccgt tcttcctgaa aaccccggtt 60 agcctgctgc gtcagcgtcg tcacaccctg ccggcgagcg aatttcgtaa cctgaccccg 120 CAGGATGCAT CAGCGTTTTC GAAATCGAAC GTGAAGCGTT CGTTAGCGTT AGCGGTGAA 180 TGCCCGCTGA CCCTGGATGA AGTTCTGAAC TTCCTGGGCC AGTGCCCGGA ACTGAGCCTG 240 GGCTGGTTCG AAGAAGGCCA GCTGGTTGCG TTATATATCG GGTAGCGGCT GGGGTAAGAA 300 CAGCTGAGCC AGGAAGCGAT GACCCAGCAC GTGCCGGATT CTCCGGCGGT GCACATCCAC 360 GTTCTGAGCG TTCACCCTCA CTGCCGTCAG CAGGGCAAAG GCAGCATCCT GCTGTGGCGT 420 TTCCTGCAGT ACCTGCCTTG CATCCCAGGC CTGCCTCGTG CGCTGCTGAT CTGCCTGAAA 480 TATCTGGTTC CTTCTACCAG AAAGCGGGCT TCAAAGAAAA AGGTCCGAGC GCGATCAGC 540 ATCTCTAACAT GCAGTTCCAG GAAATGGAA TACACCATCG GTGGCCAGGC GTACACCCGT 600 CGTAAC TCTGGTTGCTAA 618 <210> 5 <211> 723 <212> DNA <213> Artificial Sequence <400> 5 ATGG AAGATGC GCTGACCCT AGCGGCAAA CCGGCGGCGT CCCGGTTGAT CAGGATTGC 60 CCGTACACC ATCGAATTGA TCCAGCCGGA AGATGGCGAA GCGGTTATCG CGATGCTGAA 120 accttcttct tcaaagatga accgctgaac accttcctgg atctgggcga atgcaaagaa 180 ctggaaaaat acagcctgaa accgctgccg gataactgca gctacaaagc ggttaacaaa 240 aaaggtgaaa tcatcggcgt tttcctgaac ggcctgatgc gtcgtccgtc cccggatgat 300 gttccggaaa aagcggcgga ttcttgcgaa cacccgaaat tcaagaaaat cctgagcctg 360 atggatcacg ttgaagaaca gttcaacatc ttcgatgttt acccggatga agaactgatc 420 ctggatggta aaatcctgag cgttgatacc aactaccgtg gtctgggcat cgctggtcgt 480 ctgaccgaac gtgcgtacga atacatgcgt gaaaacggta tcaacgttta ccacgttctg 540 tgctctctc actactctgc gcgtgttatg gaaaaactgg gcttccacga agttttccgt 600 atgcagttcg cggattacaa accgcagggt gaagttgttt tcaaaccggc ggcgccgcac 660 gttggcatcc aggttatggc gaaagaagtt ggcccggcga aagcggcgca gaccaaactg 720 city ​​723 <210> 6 <211> 549 <212> DNA <213>人工序列 <400> 6 atgaacacct tccgtaccgc gaccgcgcgt gatctgccgg atgttgcggc gaccctgacc 60 gaagcgttcg cggcggatcc gccgacccag tgggttttcc cggatggcgc ggcggcggtt 120 agccgtttct tcttcggcgt tgctgatcgt gcgcgtgaag cgggtggcat cgttgaactg 180 ctgccgggta ccgcggcgat gatcgcgctg ccgccgcacg ttcgtctgcc ggatgcgccg 240 gcgtgcggcc gccaggcgga aatgcagcgt cgcctgggcg aacgtcgtcc gcgtaccccg 300 cactactacc tgctgttcta cggcgttcgt accgcgcacc agagcagcgg cctgggtggt 360 cgtatgctga gcgatctgat cagcttggcg gatcgtgatc gtgttggcac ctacaccgaa 420 gcgagcacct ggcgtggtgc gcgtctgatg ctgcgtcacg gcttccacac cgcgcagccg 480 ctgcgtctgc cgcacggtcc tccgatgttc ccgctgtggc gtgacccgat tcatgatcat 540 tgtgattaa 549 <210> 7 <211> 984 <212> DNA <213>人工序列 <400> 7 atggaatctg aagatgatct gaccttccag ctggttgcgg ctgatcagat ctctagcgcg 60 cacgaaattg aagttaaatc tttcccgccg gatgaagcgg gcagcctgga agcgttccgt 120 gaacgtcagc gtcagtgccc gagcctgttc ctgggcgcgt tcaccaaaag cgatagcgcg 180 ctgatcggct acatctgcgc tacccgtagc agcgcggaaa gcctggacca cgattccatg 240 agcaccaacg acccgaccgg ccgtagcgtt tgcatccacg cggttgcggt ttctccgccg 300 ttccgtaaac gcggcgttgc gagcgcgctg atgcgtaact acgttgaacg tatgcagacc 360 gaaccggatg ttgatcgtct gctgctgatc tgccatgacg acctggtgca gttctacgaa 420 cagtgcggct tcaaatacgt tggtaaaagc cacgttgttc acggtgcgcg tgcgtggttc 480 gaaatgtgcc tggaaatctc tacctctacc gcgccgcagt ctatctcccc ggaagttttc 540 gcggcgctga aaaaaccggc gccgcagcac ccgctgcgtt acctggatag cttcagcagc 600 ctgagcgcgg ttcgtgacag ctccggtctg aacgcacacg acctgatctg cccgcgtgtg 660 ggttgcggct ctatcatcct gaaatccggc gttgcggacc tggcgatccc gagcccggaa 720 ccgcagctgc cgccggaact gccgcgtctg ccggatccgt ggaccggtct gaatgctaac 780 catgaagaat ggtggctgat taccccgagc ccgatgagct ttgaaaacgt tagcttcagc 840 aaaccgaccc aatctcaggg tggttctagc accccgatca aatatctggg ctgtgctgaa 900 tgcgatctgg gcccgctgggg ttggtgtaaa gcaaccggtg gtgaattttg gctggcaccg 960 agccgtgttg gctacaaagt ttaa 984 <210> 8 <211> 501 <212> DNA <213> Artificial sequence <400> 8 atgcacagca gcctgacctt ccgtagcgcg accccgagcg ataccgatcg ttgcttccag 60 atcgaacagg aaggctacgc gggcgatgaa gcggcgaccc gtgaaaaaat ccagcagcgt 120 atcgaaacct acccggaagg cttcctggtt ctggaaaaag aaagccagat catcggcttc 180 atcaactgcg gcgcgtgctt cgatgttagc ctgagcgatg aagaatttaa agaactgatc 240 ggccacgatc cgatcggccc gaacctggtt gttatgagcg ttgttgttca cccggacttc 300 cagcaccagg gctacgcgac cgcgctgatg catgaattta tcgctatgat gcaggctatg 360 cagaaatctg cgatgtacct gatctgccag gaagaactgg ttggcttcta ccagcagttc 420 ggtttcgttg atgatggtac cagcgaatct agccacggtg gcctgcgttg gaacgatatg 480 cacctggttc tgagcaacta a 501 <210> 9 <211> 1029 <212> DNA <213>人工序列 <400> 9 atgaacacct tccgtaccgc gaccgcgcgt gatatcccgg atgttgcggc gaccctgacc 60 gaagcgttcg cgaccgatcc gccgacccag tgggttttcc cggatggtac cgcggcggtt 120 agccgcttct tcacccacgt tgcggatcgt gttcacaccg cgggtggcat cgttgaactg 180 ctgccggacc gtgcggctat gatcgcactg ccgccgcacg ttcgtctgcc gggtgaagcg 240 gcggatggcc gtcaggcgga aatccagcgc cgtctggctg accgtcaccc gctgaccccg 300 cactactacc tgctgttcta cggcgttcgt accgcgcacc agggtagcgg cctgggcggc 360 cgtatgctgg cgcgcctgac cagccgtgcg gatcgtgatc gtgttggtac ctacaccgaa 420 gcgtccacct ggcgtggcgc gcgtctgatg ctgcgtcacg gcttccacgc gacccgtccg 480 ctgcgtctgc cggatggccc gagcatgttc ccgttatggc gtgatccgat ccacgatcac 540 agcgattaa 549 <210> 10 <211> 549 <212> DNA <213> Artificial Sequence <400> 10 atgagcacca ccagcggcgt tttcttcgat ggtctgaccg aagaagaagt tctggcggct 60 caccgtatcg aaaccgaagg cttcccggcg gatgaagcgg gcagcctgga ggctttccag 120 taccgtcaca cccacgcgcc ggaactgttt ctgggcggct tcgaaccggc gagcgctccg 180 gacgcgggtc gtaccctgat cgcttacgtt aacgctaccc gttctacctc tgacgcgctg 240 acccacgcgt ctatgtccac ccacgaaccg ggtggccgta gcgcgtgcat tcacgcggtt 300 tgtgtgcgtg gcgatcacaa acgtaaaggc atcgcaagcg cgctgctgaa agaatacctg 360 gcgcgcctgg cagcaaccaa cgcggttgat cgcgcgctgc tgatcaccca tgaagaactg 420 cgtccgttct atgaaggcac cggcttccag tggatcggtc cgtctgccgt tcagcatggc 480 tctcgcccgt ggttcgaaat gcgctgggat gcgccgacct ccgcgctggc gccgtctggc 540 ccgtcccagc agcagatctt cgaagcactg caggcgtcgt cccgcaaacc gcgtgcgacc 600 ggccagctgt tgagcgaaat gaacggcggc atcgctgaag cgagcctggt ggatgaaaaa 660 tctggccgta cccgcaacgc gcatgacctg ctgtgcccgc gttctggctg tggctctgtg 720 atcctgcgta aaaacaccgc gtccctggaa cagcgtgaag cggttgatct ggacccgccg 780 accggtaaaa gcccggatct ggctgcgctg ccatctccgc cggcgtctgc agattggtgg 840 ctggttgaac cgagcccgat ggaatttgaa aacatcggtt tctcccgtcc ggtggctccg 900 accgcggaag gtaaaaaaca gctgaaactg ctgatctgcg cggattgcga tctgggtccg 960 ctgggttaca gcgaagttgg tggcacccag ttctggctgg cggcgaaccg tatccgttac 1020 cgtgcgtaa 1029 <210> 11 <211> 1959 <212> DNA <213> 人工序列 <400> 11 atgagccaga ttcataaaca taccattccg gcgaacattg cggatcgctg cctgattaac 60 ccgcagcagt atgaagcgat gtatcagcag agcattaacg tgccggatac cttttggggc 120 gaacagggca aaattctgga ttggattaaa ccgtatcaga aagtgaaaaa caccagcttt 180 gcgccgggca acgtgagcat taaatggtat gaagatggca ccctgaacct ggcggcgaac 240 tgcctggatc gccatctgca ggaaaacggc gatcgcaccg cgattatttg ggaaggcgat 300 gatgcgagcc agagcaaaca tattagctat aaagaactgc atcgcgatgt gtgccgcttt 360 gcgaacaccc tgctgggaact gggcattaaa aaaggcgatg tggtggcgat ttatatgccg 420 atggtgccgg aagcggcggt ggcgatgctg gcgtgcgcgc gcattggcgc ggtgcatagc 480 gtgatttttg gcggctttag cccggaagcg gtggcgggcc gcattattga tagcaacagc 540 cgcctggtga ttaccagcga tgaaggcgtg cgcgcgggcc gcagcattcc gctgaaaaaa 600 aacgtggatg atgcgctgaa aaacccgaac gtgaccagcg tggaacatgt ggtggtgctg 660 aaacgcaccg gcggcaaaat tgattggcag gaaggccgcg atctgtggtg gcatgatctg 720 gtggaacagg cgagcgatca gcatcaggcg gaaaaaatga acgcggaaga tccgctgttt 780 attctgtata ccagcggcag caccggcaaa ccgaaaggcg tgctgcatac caccggcggc 840 tatctggtgt atgcggcgct gacctttaaa tatgtgtttg attatcatcc gggcgatatt 900 tattggtgca ccgcggatgt gggctgggtg accggccata gctatctgct gtatggcccg 960 ctgacctgcg gcgcgaccac cctgatgttt gaaggcgtgc cgaactggcc gaccccggcg 1020 cgcatggcgc aggtggtgga taaacatcag gtgaacattc tgtataccgc gccgaccgcg 1080 attcgcgcgc tgatggcgga aggcgataaa gcgattgaag gcaccgatcg cagcagcctg 1140 cgcattctgg gcagcgtggg cgaaccgatt aacccggaag cgtgggaatg gtattggaaa 1200 aaaattggca acgaaaaatg cccggtggtg gatacctggt ggcagaccga aaccggcggc 1260 tttatgatta ccccgctgcc gggcgcgacc gaactgaaag cgggcagcgc gacccgcccg 1320 ttttttggcg tgcagccggc gctggtggat aacgaaggca acccgctgga aggcgcgacc 1380 gaaggcagcc tggtgattac cgatagctgg ccgggccagg cgcgcaccct gtttggcgat 1440 catgaacgct ttgaacagac ctattttagc acctttaaaa acatgtattt tagcggcgat 1500 ggcgcgcgcc gcgatgaaga tggctattat tggattaccg gccgcgtgga tgatgtgctg 1560 aacgtgagcg gccatcgcct gggcaccgcg gaaattgaaa gcgcgctggt ggcgcatccg 1620 aaaattgcgg aagcggcggt ggtgggcatt ccgcataaca ttaaaggcca ggcgatttat 1680 gcgtatgtga ccctgaacca tggcgaagaa ccgagcccgg aactgtatgc ggaagtgcgc 1740 aactgggtgc gcaaagaaat tggcccgctg gcgaccccgg atgtgctgca ttggaccgat 1800 agcctgccga aaacccgcag cggcaaaatt atgcgccgca ttctgcgcaa aattgcggcg 1860 ggcgatacca gcaacctggg cgataccagc accctggcgg atccgggcgt ggtggaaaaa 1920 ctgctggaag aaaaacaggc gattgcgatg ccgagctaa 1959 <210> 12 <211> 1083 <212> DNA <213> 人工序列 <400> 12 atgggtagca ccgcggaaac ccagctgacc ccggttcagg ttaccgatga tgaagcggcg 60 ctgttcgcga tgcagctggc tagcgcgagc gttctgccga tggctctgaa aagcgcgctg 120 gaactggatc tgctggaaat catggcgaaa aacggctctc cgatgagccc gaccgaaatc 180 gcgagcaaac tgccgaccaa aaacccggaa gcaccggtta tgctggatcg tatcctgcgt 240 ctgctgacca gctatagcgt tctgacctgc tctaaccgta aactgtctgg tgatggcgtt 300 gaacgtatct acggtctggg cccggtgtgc aaatacctga ccaaaaacga agatggtgtt 360 tccatcgctg cgctgtgcct gatgaaccag gacaaagttc tgatggaatc ctggtaccac 420 ctgaaagatg caattctgga cggtggcatt ccgttcaaca aagcatacgg tatgtctgcg 480 ttcgaatacc acggcaccga tccgcgtttc aacaaagttt ttaacaacgg tatgagcaac 540 cactctacca tcaccatgaa gaaaatcctg gaaacctaca aaggcttcga aggcctgacc 600 agcctggttg acgttggtgg tggtatcggc gcgaccctga aaatgatcgt ttctaaatac 660 ccgaacctga aaggtatcaa ctttgatctg ccgcacgtta tcgaagatgc gccgagccac 720 ccaggtatcg aacacgtggg cggcgatatg ttcgttagcg ttccgaaagg tgatgcgatc 780 ttcatgaaat ggatctgcca cgactggagc gatgaacact gcgttaaatt cctgaaaaac 840 tgctacgaaa gcctgccgga agatggtaaa gttattctgg cggagttcat cctgccggaa 900 accccggata gcagcctgag caccaaactg gttgtgcaca ccgattgcat catgctggcg 960 cacaatccgg gtggtaaaga acgtaccgaa aaagaatttg aagcgctggc gaaagcatct 1020 ggcttcaaag gcatcaaagt tgtttgcgat gcgttcggcg ttaacctgat cgaactgctg 1080 aaaaaactgt aa 1092 <210> 13 <211> 1092 <212> DNA <213>人工序列 <400> 13 atgggtagca ccgcggcgga tatggcggcg tctgcggatg aagaagcgtg catgttcgcg 60 ctgcagctgg cgagctccag cattctgccg atgaccctga aaaacgcgat cgaactgggc 120 ctgctggaaa tcctggttgc ggcgggcggt aaaagcctga ccccgaccga agttgcggcg 180 aaactgccga gcgcggcaaa cccggaagcg ccggatatgg ttgaccgcat gctgcgtctg 240 ctggcaagct acaacgttgt gtcctgcctg gtggaagaag gtaaagacgg tcgtctgagc 300 cgtagctacg gtgcagcgcc ggtttgcaaa ttcctgaccc cgaacgaaga tggtgtgtct 360 atggcggcgc tggcgctgat gaaccaggac aaagttctga tggaatcttg gtactacctg 420 aaagatgcgg ttctggacgg tggcatcccg ttcaacaaag cgtacggtat gtctgcgttc 480 gaataccacg gtaccgatcc gcgtttcaac cgtgtgttca acgaaggcat gaaaaaccac 540 agcatcatca tcaccaaaaa actgctggaa ctgtaccacg gcttccaggg cctgggcacc 600 ctggtggatg ttggcggcgg cgttggcgct actgttgctg cgatcaccgc gcactacccg 660 gcgatcaaag gtgttaactt tgacctgccg cacgttatct ctgaagcgcc gccgtttccg 720 ggcgttaccc acgttggtgg cgatatgttc aaagaagttc cgtctggtga tgcgattctg 780 atgaaatgga tcctgcacga ttggtctgat cagcactgtg cgaccctgct gaaaaactgc 840 tatgatgcgc tgccggccca cggtaaagtt gttctggttg aatgcatcct gccggttaac 900 ccggaagcga aaccgtcctc tcagggtgtt ttccacgttg atatgatcat gctggcgcac 960 aacccaggtg gtcgtgaacg ttacgaacgt gaatttgaag cgctggcgcg tggcgcgggc 1020 tttaccggcg ttaaatctac ctacatctac gcgaacgcgt gggcgattga gttcaccaaa 1080 taa 1083 <210> 14 <211> 1095 <212> DNA <213> 人工序列 <400> 14 atgggtagca ccggtgaaac ccagatgtct ccggcgcaga tcctggatga agaagcgaac 60 ttcgcgctgc agctgatctc tagcagcgtt ctgccgatgg ttctgaaaac cgcgatcgaa 120 ctggatctgc tggaaatcat ggcgaaagcg ggtccgggcg cgctgctgcc gccgagcgat 180 atcgcgagcc acctgccgac caaaaacccg aacgcgccgg ttatgctgga tcgtatcctg 240 cgtctgctgg cgagctactc tatcctgatc tgcagcctgc gtgatctgcc ggatggtaaa 300 gttgaacgtc tgtacggtct ggctagcgtt tgcaaattcc tgacccgtaa cgaagatggt 360 gtttctgtta gcccgctgtg cctgatgaac caggataaag ttctgatgga aagctggtac 420 cacctgaaag atgcgatcct ggaaggcggc atcccgttca acaaagcata cggcatgacc 480 gcgttcgaat accacggtac cgatccgcgt ttcaacaaag tgttcaacaa aggtatgagc 540 gttcacagca aaatggcgat gaaaaagatc ctggaaacct acaaaggctt cgaaggtctg 600 gcgagcctgg ttgatgttgg cggtggcacc ggtgcagttg tgtctaccat cgtttctaaa 660 tacccgagca tcaaaggtat caacttcgat ctgccgcacg tgatcgctga tgcgccggct 720 ttccctggtg ttgaaaacgt tggtggcgat atgttcgtta gcgttccgaa agctgatgcg 780 gttttcatga aatggatctg ccacgattgg agcgatgaac actgcctgac cttcctgaaa 840 aactgctacg atgcgctgcc ggaaaacggt aaagttatcc tggttgaatg catcctgccg 900 gttgcgccgg acacctctct ggctaccaaa ggtgttatgc acgttgatgt tatcatgctg 960 gcgcacaacc ctggcggtaa agaacgtacc gatcgtgaat ttgaaagcct ggcgcgtggc 1020 gcgggcttca aaggtttcga agttatgtgc tgcgcgttca acacccacgt tatcgaattt 1080 cgtaaaaaag cgtaa 1095 <210> 15 <211> 1095 <212> DNA <213> Artificial Sequence <400> 15 atgggcagca ccgcggctga tatggcggcg gttgcggatg aagaagcgtg catgtacgcg 60 ctgcagctgg cgagcagcag catcctgccg atgaccctga aaaacgctat cgaactgggc 120 ctgctggatg ttctggaagc ggcgcgtaaa tctgcggccg cttctctggc gccggaagaa 180 gttgttgctc gtctgccggt tgcgccgcgt aacccggatg cgccggttat ggtggatcgt 240 atgctgcgtc tgctggcgtc ttacgaaatc gttaaatgcg aaatggaaga aggcaaagat 300 ggcaaatact ctcgccgtta cgcggcggcg ccggtttgca aatggctgac cccgaacgaa 360 gatggcgtta gcatggcggc gctggcgctg atgaaccagg ataaagttct gatggaaagc 420 tggtactacc tgaaagatgc ggttctggat ggtggcatcc cgttcaacaa agcgtacggt 480 atgaccgcgt tcgaatacca cggtaccgac ccgcgtttca accgtgtttt caacgaaggc 540 atgaaaaacc acagcgtgat catcaccaaa aaactgctgg agttctacac cggcttcgaa 600 ggtatcggta ccctggttga tgttggcggc ggcgttggcg cgaccctgca cgcgatcacc 660 agccaccacc cgcagatcaa aggtgttaac ttcgatctgc cgcacgttat cagcgaagcg 720 ccgccgttcc caggcgttga acacgttggc ggtgatatgt tcaaatctgt gccgtctggt 780 gatgcgatcc tgatgaaatg gatcctgcac gattggtctg atgcgcactg cgcgaccctg 840 ctgaaaaact gctacgatgc gctgccggcg cacggcaaag tgatcgttgt tgaatgcatc 900 ctgccggttg atccggaagc gaccccgaaa gcgcagggcg tgttccacgt tgatatgatc 960 atgctggctc acaatccggg tggcaaagaa cgttacgaaa aagaatttga agatctggcg 1020 cgtggcgcgg gtttcgcggg cgttaaagcg acctacatct acgcgaacgc gtgggcgatc 1080 gagtcacca aataa 1095 <210> 16 <211> 1095 <212> DNA <213>人工序列 <400> 16 atgggtagca ccgcgggtga tgttgcggcg gtggttgatg aagaagcgtg catgtacgcg 60 atgcagctgg cgagctctag catcctgccg atgaccctga aaaacgcgat cgaactgggc 120 ctgctggaag ttctgcagaa agaagcgggt ggtggtaaag cggcgctggc gccggaagaa 180 gtggttgcgc gtatgccggc tgcgccgtct gatccgaccg ctgcggcggt tatggttgat 240 cgtatgctgc gtctgctggc gtcttacgat gttgttcgct gccagatgga agatcgtgat 300 ggtcgttacg aacgtcgtta ctctgcggcg ccggtgtgca aatggctgac cccgaacgaa 360 gatggtgtga gcatggctgc gctggcgctg atgaaccagg ataaagttct gatggaaagc 420 tggtactacc tgaaagatgc ggttctggat ggcggtatcc cgttcaacaa agcgtacggt 480 atgaccgcgt tcgaatacca cggtaccgat ccgcgtttca accgtgtttt caacgaaggc 540 atgaaaacc actctgtgat catcaccaaa aaactgctgg atttctacac cggttcgaa 600 ggcgttagca ccctggttga tgttggcggt ggtgtgggtg ctaccctgca cgcgatcacc 660 tcccgccacc cgcacatttc tggtgttaac ttcgatctgc cgcacgttat cagcgaaacc 720 ccgccgtttc cgggtgttcg tcacgttggc ggtgatatgt tcgcgagcgt gccggcgggt 780 gatgcgatcc tggttaaatg gatcctgcac gattggagcg acgcgcactg cgcgaccctg 840 ctgaaaaact gctacgatgc gctgccggaa aacggtaaag ttattgttgt tgaatgcgtt 900 ctgccggtta acaccgaagc gaccccgaaa gcgcagggcg tgttccacgt tgatatgatc 960 atgctggcgc acaacccagg cggcaaagaa cgttacgaac gtgaatttcg tgaactggcg 1020 aaaggtgcgg gtttcagcgg tttcaaagcg acctacatct acgcgaacgc gtgggcgatc 1080 gaatttatta aataa 1095 <210> 17 <211> 1083 <212> DNA <213> 人工序列 <400> 17 atgggtagca ccgcggcgga tatggcggcg tctgcggatg aagaagcgtg catgttcgcg 60 ctgcagctgg cgagctccag cattctgccg atgaccctga aaaacgcgat cgaactgggc 120 ctgctggaaa tcctggttgc ggcgggcggt aaaagcctga ccccgaccga agttgcggcg 180 aaactgccga gcgcggcaaa cccggaagcg ccggatatgg ttgaccgcat gctgcgtctg 240 ctggcaagct acaacgttgt gtcctgcctg gtggaagaag gtaaagacgg tcgtctgagc 300 cgtagctacg gtgcagcgcc ggtttgcaaa ttcctgaccc cgaacgaaga tggtgtgtct 360 atggcggcgc tggcgctgat gaaccaggac aaagttctga tggaatcttg gtactacctg 420 aaagatgcgg ttctggacgg tggcatcccg ttcaacaaag cgtacggtat gtctgcgttc 480 gaataccacg gtaccgatcc gcgtttcaac cgtgtgttca acgaaggcat gaaaaaccac 540 agcatcatca tcaccaaaaa actgctggaa ctgtaccacg gcttccaggg cctgggcacc 600 ctggtggatg ttggcggcgg cgttggcgct actgttgctg cgatcaccgc gcactacccg 660 gcgatcaaag gtgttaactt tgacctgccg cacgttatct ctgaagcgcc gccgtttccg 720 ggcgttaccc acgttggtgg cgatatgttc aaagaagttc cgtctggtga tgcgattctg 780 atgaaatgga tcctgcacga ttggtctgat cagcactgtg cgaccctgct gaaaaactgc 840 tatgatgcgc tgccggccca cggtaaagtt gttctggttg aatgcatcct gccggttaac 900 ccggaagcga aaccgtcctc tcagggtgtt ttccacgttg atatgatcat gctggcgcac 960 aacccaggtg gtcgtgaacg ttacgaacgt gaatttgaag cgctggcgcg tggcgcggc 1020 tttaccggcg ttaaatctac ctacatctac gcgaacgcgt gggcgattga gttcaccaaa 1080 city ​​1083 <210> 18 <211> 1038 <212> DNA <213>人工序列 <400> 18 atgggtagca gcgaagatca ggcgtaccgt ctgctgaacg attacgcgaa cggtttcatg 60 gttagccagg ttctgttcgc ggcgtgcgaa ctgggtgttt tcgatctgct ggcggaagcg 120 ccgggtccgc tggatgttgc ggcggttgcg gcgggcgttc gtgcgtccgc tcacggcacc 180 gaactgctgc tggatatctg cgtgagcctg aaactgctga aagttgaaac ccgtggcggt 240 aaagcgttct accgtaacac cgaactgagc tctgattacc tgaccaccgt ttctccgacc 300 agccagtgca gcatgctgaa atacatgggc cgtaccagct accgttgctg gggccacctg 360 gcggatgcgg ttcgtgaagg ccgtaaccag tacctggaaa ccttcggcgt tccggctgaa 420 gaactgttca ccgcaatcta ccgcagcgaa ggtgaacgtc tgcagttcat gcaggcgctg 480 caggaagttt ggtctgttaa cggccgtagc gttctgaccg cgttcgacct gagcgttttc 540 ccgctgatgt gcgatctggg cggtggcgcg ggtgcgctgg cgaaagaatg catgagcctg 600 tatccgggct gcaaaatcac cgttttcgat atcccggaag ttgtttggac cgcgaaacag 660 cacttcagct tccaggaaga agaacagatc gatttccagg aaggtgattt cttcaaagat 720 ccgctgccgg aagcggatct gtacatcctg gcgcgtgttc tgcacgattg ggaagatggc 780 aaatgcagcc acctgctgga acgtatctac cacacctgca aaccgggtgg tggtatcctg 840 gttatcgaat ctctgctgga tgaagatcgt cgtggtccgc tgctgaccca gctgtactcc 900 ctgaacatgc tggttcagac cgaaggccag gaacgtaccc cgacccacta ccacatgctg 960 ctgtctagcg cgggtttccg tgatttccag ttcaagaaaa ccggcgcgat ctacgatgcg 1020 atcctggcgc gtaaataa 1038 <210> 19 <211> 1179 <212> DNA <213> 人工序列 <400> 19 atggaaacct ttctattcac atccgagtca gtgaacgagg gccaccccga caaactatgc 60 gatcagatct ctgatgcggt gctcgatgcc tgccttgagc aggacccaga cagcaaggtt 120 gcttgcgaga catgtacaaa gaccaacatg gtcatggtct ttggagagat caccaccaag 180 ggcaagatag actatgaaaa gattgttcgt gacacatgcc gtaacattgg atttatttct 240 gatgatgttg gtcttgatgc tgacaagtgc aaagtcttgg ttaacattga gcagcagagc 300 cctgatattg ctcagggtgt ccacggtcac tttaccaagc ggccagagga gattggtgct 360 ggtgaccagg gccatatgtt tggttatgcc accgatgaga cccctgagta tatgcctttg 420 agccatgtac ttgccaccaa gctcggggct cgcctcactg aagttaggaa gaatggcacc 480 tgcccttggc taagacctga tggcaagact caggttactg ttgaatacta caatgacaac 540 ggtgcaatgg tccctgtccg tgtccacact gttctcatct ccactcagca tgatgagact 600 gtcacaaatg atgcaattgc tgctgatcta aaggagcatg tcatcaagcc tgtcatccct 660 gagaagtacc ttgatgagaa aactatcttc cacctaaacc catctggccg ttttgttatt 720 ggtggccctc atggtgatgc aggtctcact ggacgcaaga tcattattga cacctacggt 780 ggctggggag cccatggtgg tggtgctttc tcagggaagg acccaactaa ggtggataga 840 agtggtgctt acattgttag gcaggctgcc aagagcatcg tagcaaatgg tcttgctcgt 900 aggtgcattg tgcaagtctc ctatgctatt ggtgtacccg agcctttgtc tgtctttgtg 960 gacacctacg gcactggaaa aattcctgac aaggagattc ttaagattgt gaaggagaac 1020 tttgacttta ggcctggaat gatgaccatc aacctggatc tcaagagggg tggcaatagg 1080 ttcttgaaga cagccgcata cggacatttt ggaagggatg acccagactt cacctgggag 1140 gttgtcaagc ccctcaaatg ggagaagccc caagcttaa 1179

Claims

1. A method for biosynthesizing N-acetyl-5-methoxytryptamine using 5-hydroxy-β-indolylalanine as a substrate, characterized in that: The following steps are involved: 1) Using a recombinant genetically engineered bacterium capable of expressing a gene encoding a protein for synthesizing N-acetyl-5-methoxytryptamine, 5-hydroxy-β-indolylalanine is used as a substrate to bioferment and synthesize N-acetyl-5-methoxytryptamine, wherein the gene encoding a protein capable of expressing a protein for synthesizing N-acetyl-5-methoxytryptamine comprises a key enzyme gene DDC in the pathway of 5-hydroxy-β-indolylalanine to produce 5-hydroxytryptamine; key enzyme genes AANAT and ACS in the pathway of 5-hydroxytryptamine to produce N-acetyl-5-hydroxytryptamine; and key enzyme genes COMT and MAT in the pathway of N-acetyl-5-hydroxytryptamine to produce N-acetyl-5-methoxytryptamine, wherein the nucleotide sequence of the key enzyme gene AANAT in the pathway of 5-hydroxytryptamine to produce N-acetyl-5-hydroxytryptamine is as shown in any one of SEQ ID NOs: 2-10; and the nucleotide sequence of the key enzyme gene COMT in the pathway of N-acetyl-5-hydroxytryptamine to produce N-acetyl-5-methoxytryptamine is as shown in SEQ ID NOs: The nucleotide sequence of the key enzyme gene DDC in the pathway of 5-hydroxyβ-indolylalanine to 5-hydroxytryptamine is shown in SEQ ID NO: 1; the nucleotide sequence of the key enzyme gene ACS in the pathway of 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine is shown in SEQ ID NO: 11; the nucleotide sequence of the key enzyme gene MAT in the pathway of N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine is shown in SEQ ID NO: 19; 2) N-acetyl-5-methoxytryptamine was isolated from the system of 1).

2. The method according to claim 1, wherein: The nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is shown in any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 10; the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is shown in SEQ ID NO: 13 or SEQ ID NO:

18.

3. The method according to claim 1, wherein: The nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxytryptamine production pathway for N-acetyl-5-hydroxytryptamine is shown in SEQ ID NO: 10, and the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway for N-acetyl-5-methoxytryptamine is shown in SEQ ID NO: 13; Or the nucleotide sequence of the key enzyme gene AANAT in the 5-hydroxy-N-acetyl-5-hydroxytryptamine production pathway is shown in SEQ ID NO: 10, and the nucleotide sequence of the key enzyme gene COMT in the N-acetyl-5-hydroxytryptamine production pathway is shown in SEQ ID NO:

18.

4. A recombinant vector comprising genes encoding all enzymes for synthesizing N-acetyl-5-methoxytryptamine, characterized in that: The genes encoding the synthetic N-acetyl-5-methoxytryptamine protein include the key enzyme gene DDC of the 5-hydroxyβ-indolylalanine-5-hydroxytryptamine pathway; the key enzyme genes AANAT and ACS of the 5-hydroxytryptamine-5-N-acetyl-5-hydroxytryptamine pathway; the key enzyme genes COMT and MAT of the N-acetyl-5-hydroxytryptamine-5-methoxytryptamine pathway; the nucleotide sequence of the key enzyme gene AANAT of the 5-hydroxytryptamine-5-N-acetyl-5-hydroxytryptamine pathway is shown in any one of SEQ ID NOs: 2-10; the nucleotide sequence of the key enzyme gene COMT of the N-acetyl-5-hydroxytryptamine-5-methoxytryptamine pathway is shown in any one of SEQ ID NOs: 12-18; the nucleotide sequence of the key enzyme gene DDC of the 5-hydroxyβ-indolylalanine-5-hydroxytryptamine pathway is shown in SEQ ID NO: 1; the nucleotide sequence of the key enzyme gene ACS of the 5-hydroxytryptamine-5-N-acetyl-5-hydroxytryptamine pathway is shown in SEQ ID NO: NO:11; the nucleotide sequence of the MAT gene, a key enzyme gene in the N-acetyl-5-hydroxytryptamine production pathway, is shown in SEQ ID NO:

19.

5. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria is a recombinant genetically engineered bacteria comprising the recombinant vector according to claim 4, Alternatively, some enzyme genes in the synthetic N-acetyl-5-methoxytryptamine protein encoding gene described in claim 4 are integrated into the host cell genome for expression, and the remaining enzyme genes are expressed in a plasmid and then transferred into the aforementioned host cell to obtain a recombinant genetically engineered bacterium, wherein the synthetic N-acetyl-5-methoxytryptamine protein encoding gene includes DDC, a key enzyme gene in the pathway of 5-hydroxyβ-indolylalanine to 5-hydroxytryptamine; AANAT and ACS, key enzyme genes in the pathway of 5-hydroxytryptamine to N-acetyl-5-hydroxytryptamine; and COMT and MAT, key enzyme genes in the pathway of N-acetyl-5-hydroxytryptamine to N-acetyl-5-methoxytryptamine.

6. The recombinant genetically engineered bacterium according to claim 5, characterized in that: The DDC gene, a key enzyme gene in the pathway of 5-hydroxyβ-indolylalanine producing 5-hydroxytryptamine, is expressed on the genome of the host cell. Other target enzyme genes are expressed in plasmids and then transferred into the host cell whose genome expresses the DDC gene, a key enzyme gene in the pathway of 5-hydroxyβ-indolylalanine producing 5-hydroxytryptamine.

7. The method for constructing the recombinant genetically engineered bacteria according to claim 5, characterized in that: Transforming the recombinant vector according to claim 4 into a host cell to obtain a recombinant genetically engineered bacterium; Alternatively, the key enzyme genes AANAT and ACS in the 5-hydroxytryptamine-to-N-acetyl-5-hydroxytryptamine pathway described in claim 4 are placed in the same plasmid for tandem expression; the key enzyme genes COMT and MAT in the N-acetyl-5-hydroxytryptamine-to-N-acetyl-5-methoxytryptamine pathway are placed in the same plasmid for tandem expression; and the above plasmids are co-transformed into a host cell expressing the DDC gene on its genome to obtain a recombinant genetically engineered bacterium.

8. The method for constructing a recombinant genetically engineered bacterium according to claim 7, wherein: The host cell is an Escherichia coli host cell.

9. The method for constructing a recombinant genetically engineered bacterium according to claim 8, wherein: The host cell is a BL21 (DE3), ΔtrpR (DDC), ΔtnaA, ΔSPED Escherichia coli host cell; that is, BL21 (DE3) is used as the starting strain, the trpR gene on the genome is replaced with the DDC gene, and the tnaA gene and the SPED gene are knocked out.

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