CN114150027A

CN114150027A - Method for synthesizing N-acetyl-5-methoxytryptamine by using 5-hydroxy beta-indolylalanine as substrate through biological method

Info

Publication number: CN114150027A
Application number: CN202111466077.4A
Authority: CN
Inventors: 赵云现; 展全乐; 杨志彬; 崔金旺; 胡江林; 田皓博; 刘婵; 蒋硕生
Original assignee: Hebei Weidakang Biotechnology Co ltd
Current assignee: Hebei Weidakang Biotechnology Co ltd
Priority date: 2021-12-03
Filing date: 2021-12-03
Publication date: 2022-03-08

Abstract

The invention provides a method for synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate. The invention provides an N-acetyl-5-methoxytryptamine high-yield strain, which is used for a method for synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate, and the synthetic yield of the N-acetyl-5-methoxytryptamine is high. Further, the yield of the biosynthetic N-acetyl-5-methoxytryptamine is remarkably improved by screening the key enzyme gene AANAT in the pathway of producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine and screening and combined screening the key enzyme gene COMT in the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine.

Description

Method for synthesizing N-acetyl-5-methoxytryptamine by using 5-hydroxy beta-indolylalanine as substrate through biological method

Technical Field

The invention relates to the technical field of biology, in particular to a novel method for synthesizing N-acetyl-5-methoxytryptamine and application thereof.

Background

N-acetyl-5-methoxytryptamine is mainly an important indole neurohormone secreted by the pineal gland of mammals, indole has its unique chemical structure, and has its body shadow in chemosynthesis medicines such as antihypertensive drugs, antihistamine drugs, antipyretic analgesics and the like, and many fields such as alkaloid, high-efficiency plant growth regulator, dye and the like are indole derivatives. The N-acetyl-5-methoxytryptamine can improve the sleep quality of animal organisms. The secretion of N-acetyl-5-methoxytryptamine gradually decreases with the age of the animal. More and more researches show that the N-acetyl-5-methoxytryptamine not only can treat insomnia, but also has various physiological functions of resisting oxidation, resisting aging, regulating immunity, resisting cancer and the like.

The N-acetyl-5-methoxy primary color amine is a natural drug product component, generates little side effect, and has obvious advantages in the aspects of toxic and side effects, dosage and addiction cutoff compared with the existing widely used sedative hypnotic. Therefore, the effect and the curative effect and the market value of the N-acetyl-5-methoxytryptamine are important and cannot be replaced.

Currently, two main categories of biological extraction and chemical synthesis are mainly obtained from N-acetyl-5-methoxytryptamine. Because the content of the N-acetyl-5-methoxytryptamine naturally existing in animals and plants is very low, the extraction raw material source is limited, the extraction cost is high, and the industrial application is limited.

Compared with chemical synthesis, biosynthesis has the advantages of environmental friendliness, low energy consumption, environmental friendliness and the like. With the development of synthetic biology, more and more compounds are achieving biological green production. The engineering bacteria and the synthesis method for biologically synthesizing the N-acetyl-5-methoxytryptamine with high yield have great significance.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an N-acetyl-5-methoxytryptamine high-yield strain, a construction method and a method for synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

provides a method for synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate, which comprises the following steps:

1) the method comprises the steps of utilizing the recombinant genetic engineering bacteria for synthesizing the N-acetyl-5-methoxytryptamine protein coding gene or expressing and synthesizing the 5-hydroxy beta-indolylalanine protein coding gene to biologically ferment and synthesize the N-acetyl-5-methoxytryptamine by taking the 5-hydroxy beta-indolylalanine as a substrate, wherein the N-acetyl-5-methoxytryptamine protein coding gene capable of being expressed and synthesized comprises a 5-hydroxy beta-indolylalanine 5-hydroxytryptamine pathway key enzyme gene DDC; key enzyme genes AANAT and ACS of the process of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine; key enzyme genes COMT and MAT of a pathway for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine;

2) separating the N-acetyl-5-methoxytryptamine from the system in the step 1).

According to the scheme, the nucleotide sequence of the key enzyme gene AANAT of the pathway of producing N-acetyl-5-hydroxytryptamine by 5-hydroxyl is shown as any sequence in SEQ ID NO. 2-10; the nucleotide sequence of the key enzyme gene COMT of the N-acetyl-5-methoxytryptamine production pathway of the N-acetyl-5-hydroxytryptamine is shown as any sequence in SEQ ID NO 12-18.

According to the scheme, the nucleotide sequence of the 5-hydroxytryptamine pathway key enzyme gene DDC produced by 5-hydroxy beta-indolylalanine is shown as SEQ ID NO. 1;

the nucleotide sequence of ACS (cytochrome C) which is a key enzyme gene in the process of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine is shown as SEQ ID NO. 11;

the nucleotide sequence of MAT gene of key enzyme gene of the pathway for producing N-acetyl-5-methoxytryptamine by N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO: 19, respectively.

Preferably, the nucleotide sequence of the key enzyme gene AANAT of the pathway of producing N-acetyl-5-hydroxytryptamine by 5-hydroxyl is shown as any one sequence 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 of the pathway of producing N-acetyl-5-methoxytryptamine by N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO. 13 or SEQ ID NO. 18.

More preferably, the nucleotide sequence of the key enzyme gene AANAT in the pathway of producing N-acetyl-5-hydroxytryptamine from 5-hydroxyl is shown as any one of SEQ ID NO:10, and the nucleotide sequence of the key enzyme gene COMT in the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO: 13;

or the nucleotide sequence of the key enzyme gene AANAT in the pathway of producing N-acetyl-5-hydroxytryptamine from 5-hydroxyl is shown as any one sequence in SEQ ID NO. 10, and the nucleotide sequence of the key enzyme gene COMT in the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO. 18.

The invention also provides a recombinant vector containing the coding gene of all enzymes for synthesizing the N-acetyl-5-methoxytryptamine.

The invention also provides a recombinant gene engineering bacterium, which is a recombinant gene engineering bacterium comprising the recombinant vector, or a recombinant gene engineering bacterium obtained by integrating part of enzyme genes into the genome of a host cell for expression, and transferring the rest of the enzyme genes into the host cell after expression in a plasmid.

According to the scheme, 5-hydroxy beta-indole alanine 5-hydroxytryptamine pathway key enzyme gene DDC gene is expressed on the host cell genome, other target enzyme genes are transferred into the host cell with 5-hydroxy beta-indole alanine 5-hydroxytryptamine pathway key enzyme gene DDC gene expressed in the genome after being expressed in the plasmid.

Provides a construction method of recombinant gene engineering bacteria, which comprises the following steps:

transferring the recombinant vector comprising all enzyme coding genes for synthesizing the N-acetyl-5-methoxytryptamine into a host cell to obtain recombinant gene engineering bacteria;

or integrating a part of target enzyme genes into the genome of the host cell for expression, and transferring the rest target enzyme genes into the host cell after expression in a plasmid to obtain the recombinant gene engineering bacteria.

Specifically, the construction method of the recombinant gene engineering bacteria comprises the following steps:

putting key enzyme genes AANAT and ACS of a way of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine into the same plasmid for serial expression; key enzyme genes COMT and MAT of the pathway of producing the N-acetyl-5-methoxytryptamine by the N-acetyl-5-hydroxytryptamine are put into the same plasmid for serial expression; the plasmids are jointly transformed into a host cell with a genome expressing DDC genes to obtain the recombinant gene engineering bacteria.

Further, the host cell is an escherichia coli host cell, preferably BL21(DE3), Δ trpr (ddc), Δ tnaA, Δ SPED escherichia coli host cell; that is, BL21(DE3) was used as a starting strain, the trpR gene on the genome was replaced with the DDC gene, and the tnaA gene and the SPED gene were knocked out.

The invention has the beneficial effects that:

the invention provides an N-acetyl-5-methoxytryptamine high-yield strain which is used for synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate, and the synthetic yield of the N-acetyl-5-methoxytryptamine is high. Further, the yield of the biosynthetic N-acetyl-5-methoxytryptamine is remarkably improved by screening a key enzyme gene AANAT (aromatic amine-N-acetyltransferase) in the process of producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine and screening isozymes from different species of a key enzyme gene COMT (catechol-O-methyltransferase) in the process of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine and combining and screening.

The invention provides and optimizes a construction method of an N-acetyl-5-methoxytryptamine high-producing strain. Part of the target enzyme gene is integrated into the genome of a host cell for expression, and the partial enzyme gene is transferred into the host cell after being expressed in a plasmid to optimize and construct the N-acetyl-5-methoxytryptamine high-yield strain, which is beneficial to synthesizing the expression of the N-acetyl-5-methoxytryptamine protein coding gene and obtaining the high-yield N-acetyl-5-methoxytryptamine strain.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1: plasmid map for producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine

FIG. 2: plasmid map for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

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

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Escherichia coli is used as a host for exogenous gene expression, the genetic background is clear, the technical operation is simple, the culture condition is simple, and large-scale fermentation is economical and is emphasized. At present, Escherichia coli is the most widely and successfully applied expression system and is often the first choice for high-efficiency expression. The method starts from escherichia coli, and realizes the high-efficiency large-scale industrial production of the N-acetyl-5-methoxytryptamine by a fermentation method through transforming the escherichia coli.

EXAMPLE 1 construction of plasmid

Construction of plasmid for producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine

Tandem expression of key enzyme genes AANAT and ACS of a pathway for producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine.

Selecting 9 AANAT genes from different sources, and artificially synthesizing the genes through codon optimization to obtain A1-A9 which are respectively and sequentially shown as 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. A1-A9 and ACS gene are inserted between 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 plasmid (shown in figure 1).

Cloning pACYCDuet-ACS-F primers, pACYCDuet-ACS-R primers and pACYCDuet-ACS plasmids serving as templates to obtain pACYCDuet plasmid vectors containing ACS genes; A1-A9 gene segments are obtained by cloning with primers 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 and A9-F, A9-R as templates and plasmids pACYCDuet-A1, pACYCDuet-A2, pACYCDuet-A3, pACYCDuet-A4, pACYCDuet-A5, pACYCDuet-A6, pACYCDuet-A7, pACYCDuet-A8 and pACYCDuet-A9, respectively.

The A1-A9 gene fragment is respectively connected with pACYCDuet plasmid vector fragments containing ACS genes through a seamless cloning kit to form 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.

TABLE 1 primer sequences 1

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

The tandem expression of key enzyme genes COMT and MAT of the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine.

Selecting COMT genes from different sources, artificially synthesizing by codon optimization to obtain C1-C7, wherein the nucleotide sequences are respectively shown as 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 in sequence. The C1-C7 and MAT genes were inserted between the NcoI and AflII sites of plasmid pETDuet to obtain plasmids pETDuet-C1, pETDuet-C2, pETDuet-C3, pETDuet-C4, pETDuet-C5, pETDuet-C6, pETDuet-C7 and pETDuet-MAT.

pETDuet-MAT-F, pETDuet-MAT-R is used as a primer, and pETDuet-MAT plasmid is used as a template to clone and obtain pETDuet plasmid vector containing MAT gene; C1-F, C1-R, C2-F, C2-R, C3-F, C3-R, C4-F, C4-R, C5-F, C5-R, C6-F, C6-R and C7-F, C7-R are used as primers, and plasmids pETDuet-C1, pETDuet-C2, pETDuet-C3, pETDuet-C4, pETDuet-C5, pETDuet-C6 and pETDuet-C7 are used as templates to clone and obtain a C1-C9 gene fragment.

C1-C9 gene fragment and pETDuet plasmid vector containing MAT gene are connected together through seamless cloning kit to form plasmid pETDuet-C1-MAT, pETDuet-C2-MAT, pETDuet-C3-MAT, pETDuet-C4-MAT, pETDuet-C5-MAT, pETDuet-C6-MAT and pETDuet-C7-MAT.

TABLE 2 primer sequences 2

EXAMPLE 2 engineering of host bacteria

the tNAA gene is knocked out, the genome expression of a key enzyme DDC of a 5-hydroxytryptamine pathway produced by 5-hydroxy beta-indolylalanine and the knocking out of an SPED gene.

The upstream fragment of the tnaA gene and the downstream fragment of the tnaA gene are obtained by respectively using tnaAup-F, tnaAup-R, tnaAdown-F and tnaAdown-R as primers and BL21(DE3) genome as a template. The two fragments are connected by fusion PCR with tnaAup-F, tnaAdown-R as primers to obtain a delta tnaA fragment. The fragment is electrically transferred into BL21(DE3) competence to replace the gene tnaA of the genome, and positive clones are screened by PCR. Designated HP 114.

The DDC gene is codon-optimized, has a sequence shown as SEQ ID No.1, and is inserted between NdeI and XhoI sites of pET28a (+) plasmid to obtain pET28a-DDC plasmid.

DDC/trpR-F and DDC/trpR-R are used as primers, and plasmid pET28a-DDC is used as a template to obtain a DDC gene fragment with trpR upstream and downstream homologous arms. The fragment was electroporated into HP114 competence to replace the genomic trpR gene, and positive clones were PCR-selected. Designated HP 115.

SPEDup-F, SPEDup-R, SPEDdown-F and SPEDdown-R are used as primers and BL21(DE3) genome is used as a template to obtain an upstream fragment and a downstream fragment of the SPED gene. The two fragments were ligated by fusion PCR using SPEDup-F, SPEDdown-R as a primer to obtain a.DELTA.SPED fragment. The fragment is electrically transferred into HP115 competence to replace the gene tnaA of the genome, and positive clones are screened out by PCR. Designated as HP 116.

TABLE 3 primer sequences 3

EXAMPLE 3 construction and screening of 5-Hydroxybeta-Indolylalanine-producing strains

(1) The construction of engineering bacteria producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine and the screening of key gene AANAT: 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 are electrically transformed into strains HP116 to obtain engineering bacteria HP116-A1, HP116-A2, HP116-A3, HP116-A4, HP116-A5, HP116-A6, HP116-A7, HP116-A8 and HP 116-A9. Respectively culturing engineering bacteria HP116-A1, HP116-A2, HP116-A3, HP116-A4, HP116-A5, HP116-A6, HP116-A7, HP116-A8 and HP116-A9 in a seed culture medium containing 34 mu g/mL chloramphenicol antibiotic to obtain a seed solution, wherein OD of the seed solution is₆₀₀The seed culture medium (mass percent) is 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract, and the culture conditions of the seed liquid are 37 ℃ and 225 rpm; inoculating 2% of the above seed solution to a solution containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, and 0.231% KH₂PO₄And 1.64% K₂HPO₄·3H₂Performing fermentation culture in a fermentation culture medium (in percentage by mass) of O; fermenting and culturing for 2h, adding 1mM IPTG for induction expression at 30 deg.C for 10h, stopping fermentation, and adding final concentrationThe conversion time is 10 hours when the content is 6g/L of 5-hydroxytryptamine, and the yield of the N-acetyl-5-hydroxytryptamine is detected by utilizing high performance liquid chromatography.

The results are shown in table 4, where: the engineering bacteria HP116-A1, HP116-A2, HP116-A4, HP116-A5 and HP116-A9 have higher N-acetyl-5-hydroxytryptamine producing capability than other strains, namely genes with numbers of A1, A2, A4, A5 and A9.

Table 4: comparison of yields of different AANAT strains producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine

(2) The construction of the engineering bacteria producing the N-acetyl-5-methoxytryptamine by the N-acetyl-5-hydroxy-tryptamine and the screening of the key gene COMT: plasmids pETDuet-C1-MAT, pETDuet-C2-MAT, pETDuet-C3-MAT, pETDuet-C4-MAT, pETDuet-C5-MAT, pETDuet-C6-MAT and pETDuet-C7-MAT are electrically transformed into strain HP116 competence to obtain engineering bacteria HP116-C1, HP116-C2, HP116-C3, HP116-C4, HP116-C5, HP116-C6 and HP 116-C7. Culturing the engineering bacteria in seed culture medium containing 100 μ g/mL ampicillin for 10 hr to obtain seed solution, OD of the seed solution₆₀₀The seed culture medium (mass percent) is 1% of tryptone, 1% of sodium chloride and 0.5% of yeast extract, and the culture conditions of the seed liquid are 37 ℃ and 225 rpm; inoculating 2% of the above seed solution to a solution containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, and 0.231% KH₂PO₄And 1.64% K₂HPO₄·3H₂Performing fermentation culture in a fermentation culture medium (in percentage by mass) of O; after 2h of fermentation culture, adding 1mM IPTG for induction expression, wherein the temperature of the induction expression is 30 ℃, stopping the fermentation after 10h of the induction expression, adding N-acetyl-5-hydroxy-tryptamine with the final concentration of 6g/L, simultaneously supplementing methionine, keeping the concentration of the methionine at about 0.5g/L all the time, converting for 10h, and detecting the N-acetyl-5-methoxytryptamine by using high performance liquid chromatography.

The results are shown in table 5, where: the engineering bacteria HP116-C2 and HP116-C7 have higher capability of producing N-acetyl-5-hydroxytryptamine than other strains, namely genes with numbers of C2 and C7 have higher capability of producing N-acetyl-5-methoxytryptamine.

Table 5: comparison of yields of N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine with respect to COMT strains

(3) Construction and screening of engineering bacteria producing N-acetyl-5-methoxytryptamine from 5-hydroxytryptamine

Further, the excellent key gene AANAT for producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine and the key gene COMT for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine are combined, the effect of synthesizing N-acetyl-5-methoxytryptamine by screening engineering bacteria of different gene combinations is evaluated, typically,

plasmids pACYCDuet-A1-ACS, pACYCDuet-A2-ACS, pACYCDuet-A4-ACS, pACYCDuet-A5-ACS and pACYCDuet-A9-ACS which produce N-acetyl-5-hydroxytryptamine from 5-hydroxy beta-indolylalanine and plasmids which produce N-acetyl-5-methoxytryptamine from 5-hydroxytryptamine are combined and electrically transformed into strains HP116 competence according to the table 4 to obtain the engineering bacteria HP116-A1C2, HP116-A2C2, HP116-A4C2, HP116-A5C2, HP116-A9C2, HP116-A1C7, HP116-A2C7, HP116-A4C7, HP116-A5C7 and HP116-A9C 7. Culturing the engineering bacteria in a seed culture medium containing 34 mu g/mL chloramphenicol and 100 mu g/mL ampicillin for 10h respectively to obtain a seed solution, wherein the OD600 of the seed solution is 5, the seed culture medium (in percentage by mass) comprises 1% tryptone, 1% sodium chloride and 0.5% yeast extract, and the culture conditions of the seed solution are 37 ℃ and 225 rpm; inoculating 2% of the above seed solution into a fermentation medium (mass percent) containing 1.2% tryptone, 2.4% yeast extract, 0.5% glycerol, 0.231% KH2PO4 and 1.64% K2HPO 4.3H2O, and performing fermentation culture; and after 2h of fermentation culture, adding 1mM IPTG (isopropyl-beta-D-thiogalactoside) for induction expression, wherein the temperature of induction expression is 30 ℃, stopping fermentation after 16h of induction expression, adding 5-hydroxy beta-indolylalanine with the final concentration of 6g/L, converting for 10h, and detecting the yield of the N-acetyl-5-methoxytryptamine by using high performance liquid chromatography.

As shown in Table 6, the yield of N-acetyl-5-methoxytryptamine synthesized by engineering bacteria provided by different genome combinations is more than 2.41(g/L), and most preferably, the yield is the highest as 4.5g/L for the strain HP116-A9C2, namely, the effect of synthesizing N-acetyl-5-methoxytryptamine by AANAT with the number of A9 and COMT with the number of C2 is the best.

Table 6: comparison of yields of different combination strains of N-acetyl-5-methoxytryptamine from 5-hydroxy beta-indolylalanine

The invention produces 5-hydroxytryptamine pathway key enzyme gene DDC through 5-hydroxy beta-indolylalanine; key enzyme genes AANAT and ACS of the process of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine; the engineering strain for synthesizing the N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate is obtained by matching and constructing key enzyme genes COMT and MAT in the process of producing the N-acetyl-5-methoxytryptamine by the N-acetyl-5-hydroxytryptamine, is used for synthesizing the N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate, and has the following synthetic process: 5-hydroxy β -indolylalanine → 5-hydroxytryptamine → N-acetyl-5-methoxytryptamine.

Further, the yield of the biosynthetic N-acetyl-5-methoxytryptamine is remarkably improved by screening the key enzyme gene AANAT in the pathway of producing N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine and screening and combined screening the key enzyme gene COMT in the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine. Provides conditions for the efficient large-scale industrial production of the N-acetyl-5-methoxytryptamine.

SEQUENCE LISTING

<110> Hebei Weidakang Biotech Co., Ltd

<120> method for synthesizing N-acetyl-5-methoxytryptamine by using 5-hydroxy beta-indolylalanine as substrate through biological method

And uses thereof

<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 ttatgcctgg 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 aaaccggaaa 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> Artificial sequence

<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> Artificial sequence

atgacccagc aggttagcgg tagcccgttc ttcaaaccgt tcttcctgaa aaccccggtt 60

agcctgctgc gtcagcgtcg tcacaccctg ccggcgagcg aatttcgtaa cctgaccccg 120

caggatgcga tcagcgtttt cgaaatcgaa cgtgaagcgt tcgttagcgt tagcggtgaa 180

tgcccgctga ccctggatga agttctgaac ttcctgggcc agtgcccgga actgagcctg 240

ggctggttcg aagaaggcca gctggttgcg ttcatcatcg gtagcggctg gggtaaagaa 300

cgcctgagcc aggaagcgat gacccagcac gtgccggatt ctccggcggt gcacatccac 360

gttctgagcg ttcaccgtca ctgccgtcag cagggcaaag gcagcatcct gctgtggcgt 420

ttcctgcagt acctgcgttg catcccaggc ctgcgtcgtg cgctgctgat ctgcgaagaa 480

tatctggttc cgttctacca gaaagcgggc ttcaaagaaa aaggtccgag cgcgatcagc 540

atctctaaca tgcagttcca ggaaatggaa tacaccatcg gtggccaggc gtacacccgt 600

cgtaactctg gttgctaa 618

<210> 5

<211> 723

<212> DNA

<213> Artificial sequence

<400> 5

atggaagatg cgctgaccgt tagcggcaaa ccggcggcgt gcccggttga tcaggattgc 60

ccgtacacca tcgaactgat ccagccggaa gatggcgaag cggttatcgc gatgctgaaa 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

tgctcttctc actactctgc gcgtgttatg gaaaaactgg gcttccacga agttttccgt 600

atgcagttcg cggattacaa accgcagggt gaagttgttt tcaaaccggc ggcgccgcac 660

gttggcatcc aggttatggc gaaagaagtt ggcccggcga aagcggcgca gaccaaactg 720

taa 723

<210> 6

<211> 549

<212> DNA

<213> Artificial sequence

<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> Artificial sequence

<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 gcccgctggg 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> Artificial sequence

<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> Artificial sequence

<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 tgctggaact 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> Artificial sequence

<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> Artificial sequence

<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> Artificial sequence

<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

gagttcacca aataa 1095

<210> 16

<211> 1095

<212> DNA

<213> Artificial sequence

<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

atgaaaaacc actctgtgat catcaccaaa aaactgctgg atttctacac cggtttcgaa 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> Artificial sequence

<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 tggcgcgggc 1020

tttaccggcg ttaaatctac ctacatctac gcgaacgcgt gggcgattga gttcaccaaa 1080

taa 1083

<210> 18

<211> 1038

<212> DNA

<213> Artificial sequence

<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> Artificial sequence

<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 synthesizing N-acetyl-5-methoxytryptamine by a biological method by taking 5-hydroxy beta-indolylalanine as a substrate is characterized by comprising the following steps: the method comprises the following steps:

2) separating the N-acetyl-5-methoxytryptamine from the system in the step 1).

2. The method of claim 1, wherein: the nucleotide sequence of 5-hydroxyl N-acetyl-5-hydroxytryptamine production pathway key enzyme gene AANAT is shown in any sequence of SEQ ID NO. 2-10; the nucleotide sequence of the key enzyme gene COMT of the N-acetyl-5-methoxytryptamine production pathway of the N-acetyl-5-hydroxytryptamine is shown as any sequence in SEQ ID NO 12-18.

3. The method of claim 1, wherein: the nucleotide sequence of 5-hydroxytryptamine pathway key enzyme gene DDC produced by 5-hydroxy beta-indolylalanine is shown in SEQ ID NO 1;

4. The method of claim 1, wherein: the nucleotide sequence of 5-hydroxyl N-acetyl-5-hydroxytryptamine production pathway key enzyme gene AANAT is shown as any one sequence 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 of the pathway of producing N-acetyl-5-methoxytryptamine by N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO. 13 or SEQ ID NO. 18.

5. The method of claim 1, wherein: the nucleotide sequence of a key enzyme gene AANAT in the pathway of producing N-acetyl-5-hydroxytryptamine from 5-hydroxyl is shown as any one of SEQ ID NO. 10, and the nucleotide sequence of a key enzyme gene COMT in the pathway of producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO. 13;

6. A recombinant vector comprising all enzyme encoding genes for synthesizing N-acetyl-5-methoxytryptamine, wherein the encoding genes for synthesizing N-acetyl-5-methoxytryptamine protein comprise 5-hydroxy beta-indolylalanine 5-hydroxytryptamine pathway key enzyme genes DDC; key enzyme genes AANAT and ACS of the process of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine; key enzyme genes COMT and MAT of the pathway for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine.

7. A recombinant gene engineering bacterium, the recombinant gene engineering bacterium comprises the recombinant vector of claim 6, or is a recombinant gene engineering bacterium obtained by integrating part of the enzyme genes in the synthetic N-acetyl-5-methoxytryptamine protein coding gene into the genome of a host cell for expression, and transferring the rest of the enzyme genes into the host cell after expression in a plasmid, wherein the synthetic N-acetyl-5-methoxytryptamine protein coding gene comprises a 5-hydroxy beta-indolylalanine 5-hydroxytryptamine pathway key enzyme gene DDC; key enzyme genes AANAT and ACS of the process of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine; key enzyme genes COMT and MAT of the pathway for producing N-acetyl-5-methoxytryptamine from N-acetyl-5-hydroxytryptamine.

8. The recombinant gene engineering bacteria of claim 7, wherein the 5-hydroxytryptamine pathway key enzyme gene DDC gene produced by 5-hydroxy beta-indolylalanine is expressed on the genome of the host cell, and other target enzyme genes are transferred into the host cell with the genome expressing the 5-hydroxytryptamine pathway key enzyme gene DDC gene produced by 5-hydroxy beta-indolylalanine after being expressed in the plasmid.

9. The method for constructing a recombinant genetically engineered bacterium according to claim 8, wherein:

or integrating a part of target enzyme genes into the genome of a host cell for expression, and transferring the rest target enzyme genes into the host cell after expression in a plasmid to obtain the recombinant gene engineering bacteria, wherein the recombinant gene engineering bacteria comprise the following steps: putting key enzyme genes AANAT and ACS of a way of producing N-acetyl-5-hydroxytryptamine by 5-hydroxytryptamine into the same plasmid for serial expression; key enzyme genes COMT and MAT of the pathway of producing the N-acetyl-5-methoxytryptamine by the N-acetyl-5-hydroxytryptamine are put into the same plasmid for serial expression; the plasmids are jointly transformed into a host cell with a genome expressing DDC genes to obtain the recombinant gene engineering bacteria.

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