Method for enhancing yield of N-acetyl-5-hydroxytryptamine

文档序号：1793995 发布日期：2021-11-05 浏览：36次中文

阅读说明：本技术 一种增强n-乙酰基-5-羟基色胺产量的方法 (Method for enhancing yield of N-acetyl-5-hydroxytryptamine ) 是由李迪李扬凡冯斌朱敏姜黎于 2021-06-30 设计创作，主要内容包括：本发明涉及一种增强N-乙酰基-5-羟基色胺产量的方法。由包含催化5-羟色胺生成N-乙酰基-5-羟基色胺相关功能基因的重组细胞转化底物5-羟色胺得到N-乙酰基-5-羟基色胺,所述的催化5-羟色胺生成N-乙酰基-5-羟基色胺相关功能基因的重组细胞表达N-乙酰基-5-羟基色胺转移酶基因,或表达N-乙酰基-5-羟基色胺转移酶基因和增强N-乙酰基-5-羟基色胺产量的乙酰辅酶A合成酶基因,产N-乙酰基-5-羟基色胺转移酶和乙酰辅酶A合成酶。本发明通过过表达AANTA基因和ACS基因,增强AANAT催化底物5-羟色胺生成N-乙酰基-5-羟基色胺,明显提升了N-乙酰基-5-羟基色胺的合成产量。(The invention relates to a method for enhancing the yield of N-acetyl-5-hydroxytryptamine. Converting a substrate 5-hydroxytryptamine by a recombinant cell containing a functional gene related to catalyzing 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine to obtain N-acetyl-5-hydroxytryptamine, and expressing an N-acetyl-5-hydroxytryptamine transferase gene or an acetyl coenzyme A synthetase gene for enhancing the yield of N-acetyl-5-hydroxytryptamine by the recombinant cell containing the functional gene related to catalyzing 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine, so as to produce N-acetyl-5-hydroxytryptamine transferase and acetyl coenzyme A synthetase. According to the invention, through overexpression of the AANTA gene and the ACS gene, the generation of N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine serving as an AANAT catalytic substrate is enhanced, and the synthetic yield of the N-acetyl-5-hydroxytryptamine is obviously improved.)

1. A method for enhancing N-acetyl-5-hydroxytryptamine production, comprising: converting a substrate 5-hydroxytryptamine by recombinant cells containing functional genes related to catalyzing the production of the N-acetyl-5-hydroxytryptamine by the 5-hydroxytryptamine to obtain the N-acetyl-5-hydroxytryptamine, the recombinant cell catalyzing 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine related functional genes expresses N-acetyl-5-hydroxytryptamine transferase gene, or the recombinant cell catalyzing 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine related functional genes expresses N-acetyl-5-hydroxytryptamine transferase genes and acetyl coenzyme A synthetase genes for enhancing the yield of N-acetyl-5-hydroxytryptamine, and produces N-acetyl-5-hydroxytryptamine transferase and acetyl coenzyme A synthetase.

2. The method of claim 1, wherein: the amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is as follows:

a) the amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is represented by SEQ ID NO: 002, SEQ ID NO: 004, SEQ ID NO: 006, SEQ ID NO: 008 or any one of the sequences shown in (008);

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

c) or an amino acid sequence formed by adding, replacing or deleting 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, and most preferably 1 to 3 amino acid residues from the N-terminal and/or C-terminal of the amino acid sequence described in a) and having a function of (a) catalyzing the production of N-acetyl-5-hydroxytryptase from 5 hydroxytryptamine;

the amino acid sequence of the acetyl coenzyme A synthetase is as follows:

a) the amino acid sequence of the enzyme for enhancing the yield of the N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO: 010, SEQ ID NO: 012 or SEQ ID NO: 014;

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

c) or an amino acid sequence formed by adding, replacing or deleting 1 to 30, more preferably 1 to 10, still more preferably 1 to 6, most preferably 1 to 3 amino acid residues to the N-terminal and/or C-terminal of the amino acid sequence described in (a) and having the function described in (a).

3. The method of claim 1, wherein: the N-acetyl-5-hydroxytryptamine transferase gene sequence is shown as SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005 or SEQ ID NO: 007 is shown in the specification; the acetyl coenzyme A synthetase gene sequence is shown as SEQ ID NO: 009, SEQ ID NO: 011 or SEQ ID NO: 013.

4. The method of claim 1, wherein: it also comprises the further step of using the synthesized N-acetyl-5-hydroxytryptamine in the production of subsequent tryptophan derivatives such as N-acetyl-5-methoxytryptamine.

5. A recombinant vector comprising a nucleic acid sequence as set forth in SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005 or SEQ ID NO: 007 of any one of said genes for N-acetyl-5-hydroxytryptamine transferase,

or comprises the amino acid sequence as set forth in SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005, SEQ ID NO: 007 and a polypeptide sequence as set forth in any one of SEQ ID NOs: 009, SEQ ID NO: 011 or SEQ ID NO: 013 any one of the acetyl-CoA synthetase (ACS) genes.

6. A host cell comprising the recombinant vector of claim 5.

7. A host cell integrating an N-acetyl-5-hydroxytryptamine transferase gene and an acetyl-CoA synthetase (ACS) gene in a genome, wherein the N-acetyl-5-hydroxytryptamine transferase gene has a sequence shown in SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005 or SEQ ID NO: 007 is shown in the specification; the acetyl coenzyme A synthetase gene sequence is shown as SEQ ID NO: 009, SEQ ID NO: 011 or SEQ ID NO: 013.

8. An N-acetyl-5-hydroxytryptamine transferase, comprising: the amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is as follows:

a) the amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is represented by SEQ ID NO: 002, SEQ ID NO:

004, SEQ ID NO: 006 or SEQ ID NO: 008 or any one of the sequences shown in (008);

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

c) or an amino acid sequence formed by adding, replacing or deleting 1-30, more preferably 1-10, still more preferably 1-6, most preferably 1-3 amino acid residues at the N-terminal and/or C-terminal of the amino acid sequence described in a) and having the function of catalyzing the formation of N-acetyl-5-hydroxytryptamine from 5 hydroxytryptamine described in (a).

9. A method for constructing the recombinant vector of claim 5, comprising: amplifying a target gene by using a gene amplification instrument, and connecting the target gene with an expression vector by using a seamless cloning or enzyme digestion and enzyme ligation mode, wherein primers for amplifying the target gene are as follows:

10. a host cell according to claim 7, wherein the host cell is any one of prokaryotic E.coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), Brevibacterium flavum (Brevibacterium flavum), Serratia mucilaginosa (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Yarrowia lipolytica (Yarrowia lipolytica) and Aspergillus niger (Aspergillus niger) cells.

The technical field is as follows:

the invention relates to the technical field of biology, in particular to a method for enhancing the yield of N-acetyl-5-hydroxytryptamine.

Background art:

n-acetyl-5-methoxytryptamine, also known as melatonine, melatonin, is a biological clock-regulating hormone involved in the regulation of the expression of neural and peripheral tissue cell clock genes. N-acetyl-5-methoxytryptamine is also a potent free radical of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) in a wide variety of complex physiological events. Meanwhile, the N-acetyl-5-methoxytryptamine is taken as an amphiphilic molecule and can easily enter all subcellular structural spaces including cell membrane structures, cytoplasm, cell nucleus and mitochondria. The physiological functions of the known N-acetyl-5-methoxytryptamine mainly comprise regulating circadian rhythm in organisms, relieving sleep disorder, resisting oxidation and the like; the FDA in the united states recognizes N-acetyl-5-methoxytryptamine as a common dietary supplement. The health department of China approves the production and sale of products containing N-acetyl-5-methoxytryptamine as health care products for improving sleep. The health care functions of N-acetyl-5-methoxytryptamine in the aspects of regulating immune function, resisting aging and the like show strong vitality.

The biosynthesis pathway of N-acetyl-5-methoxytryptamine is mainly from tryptophan, 5-hydroxytryptamine is generated through hydroxylation and decarboxylation, then N-acetyl-5-hydroxytryptamine is generated under the action of N-acetyl-5-hydroxytryptamine transferase, and then N-acetyl-5-methoxytryptamine is generated through the action of N-acetyl-5-hydroxytryptamine oxygen methyltransferase or caffeic acid oxygen methyltransferase. N-acetyl-5 hydroxytryptamine transferase has been shown to be a key rate-limiting enzyme in the biosynthesis of N-acetyl-5-methoxytryptamine. Blood N-acetyl-5-methoxytryptamine is mainly synthesized in an organ, namely pineal, 5-hydroxytryptamine (5-HT) is derived from tryptophan decarboxylation reaction in blood, the tryptophan decarboxylation reaction is converted into N-acetyl-5-hydroxytryptamine (Ac-HT) under the catalysis of N-acetyl-5-hydroxytryptamine transferase, and the N-acetyl-5-methoxytryptamine is generated through methoxytransferase, while the activity of the N-acetyl-5-hydroxytryptamine transferase in pineal is inhibited by light, the yield is unstable, the diurnal law is shown, and the production is not facilitated. With the development of synthetic biology, the biosynthesis genes of N-acetyl-5-methoxytryptamine are integrated into the genome of a model microorganism through genetic engineering, so that the N-acetyl-5-methoxytryptamine is highly produced, and the aim of green biological manufacturing is fulfilled.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a method for enhancing the yield of N-acetyl-5-hydroxytryptamine.

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

provides a method for enhancing the yield of N-acetyl-5-hydroxytryptamine, converting a substrate 5-hydroxytryptamine into N-acetyl-5-hydroxytryptamine by using a recombinant cell containing a functional gene related to the production of N-acetyl-5-hydroxytryptamine by catalyzing 5-hydroxytryptamine, wherein the recombinant cell for catalyzing the production of N-acetyl-5-hydroxytryptamine by catalyzing 5-hydroxytryptamine expresses a transferase gene of N-acetyl-5-hydroxytryptamine, or the recombinant cell for catalyzing the production of N-acetyl-5-hydroxytryptamine by catalyzing 5-hydroxytryptamine expresses N-acetyl-5-hydroxytryptamine transferase gene and acetyl-CoA synthetase gene for enhancing the yield of N-acetyl-5-hydroxytryptamine, produce N-acetyl-5-hydroxytryptamine transferase and acetyl coenzyme A synthetase.

According to the scheme, the amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is as follows:

a) consisting of SEQ ID NO: 002, SEQ ID NO: 004, SEQ ID NO: 006, SEQ ID NO: 008 or any one of the sequences shown in (008);

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

the amino acid sequence of the enzyme for enhancing the yield of the N-acetyl-5-hydroxytryptamine (acetyl coenzyme A synthetase) is as follows:

a) the amino acid sequence of the enzyme for enhancing the yield of the N-acetyl-5-hydroxytryptamine is shown as SEQ ID NO: 010, SEQ ID NO: 012 or SEQ ID NO: 014;

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

According to the scheme, the gene sequence of the N-acetyl-5-hydroxytryptamine transferase is shown as SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005 or SEQ ID NO: 007 is shown in the specification; the acetyl coenzyme A synthetase gene sequence is shown as SEQ ID NO: 009, SEQ ID NO: 011 or SEQ ID NO: 013.

According to the scheme, the method also comprises the step of further using the synthesized N-acetyl-5-hydroxytryptamine for the production of subsequent tryptophan derivatives such as N-acetyl-5-methoxytryptamine.

Providing a recombinant vector comprising a nucleotide sequence set forth as SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005 or SEQ ID NO: 007 of any one of said genes.

And further provides a host cell comprising the recombinant vector or a gene integrating N-acetyl-5-hydroxytryptamine transferase in the genome thereof.

Providing a recombinant vector comprising a nucleotide sequence set forth as SEQ ID NO: 001, SEQ ID NO: 003, SEQ ID NO: 005, SEQ ID NO: 007 and a polypeptide sequence as set forth in any one of SEQ ID NOs: 009, SEQ ID NO: 011 or SEQ ID NO: 013 any one of the acetyl-CoA synthetase (ACS) genes.

And further, a host cell comprising the above recombinant vector or having an N-acetyl-5-hydroxytryptamine transferase gene and an acetyl-CoA synthetase gene integrated into its genome is provided.

The amino acid sequence of the N-acetyl-5-hydroxytryptamine transferase is N-acetyl-5-hydroxytryptamine transferase

a) Consisting of SEQ ID NO: 002, SEQ ID NO: 004, SEQ ID NO: 006, SEQ ID NO: 008 or any one of the sequences shown in (008);

b) or an amino acid sequence having 75%, preferably 85%, more preferably 95% sequence identity to the amino acid sequence of (a) and having the function of the protein of (a);

the construction method of the recombinant vector comprises the following steps: and amplifying the target gene by using a gene amplification instrument, and connecting the target gene with an expression vector by using seamless cloning or enzyme digestion and enzyme ligation.

According to the scheme, the target gene is transferred into the pCDF empty plasmid to obtain a recombinant vector carrying the pCDF replicon, the spectinomycin resistance gene and the T7 promoter, and the recombinant vector is transferred into host cells to construct genetically engineered bacteria.

According to the scheme, the primers for amplifying the target gene are as follows:

the construction method of the genetic engineering bacteria is provided as follows: introducing the recombinant vector into a host cell to obtain the recombinant vector; or integrating the N-acetyl-5-hydroxytryptamine transferase gene and acetyl coenzyme A synthetase gene on the host cell genome.

According to the scheme, the host cell is any one of prokaryotic cells of Escherichia coli (Escherichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), Brevibacterium flavum (Brevibacterium flavum), Serratia mucilaginosa (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerealis), Yarrowia lipolytica (Yarrowia lipolytica) and Aspergillus niger (Aspergillus niger) cells. According to the scheme, the method for integrating the N-acetyl-5-hydroxytryptamine transferase gene and the acetyl-CoA synthetase gene in the genome comprises the following steps: integrating the cat-sacB gene to the TrpR site of escherichia coli by using homologous recombinase of the lamda bacteriophage, wherein the strain integrated with the cat-sacB gene can grow on a chloramphenicol plate; and (3) continuing to replace the cat-sacB gene of the escherichia coli with the T7 promoter, the N-acetyl-5-hydroxytryptamine transferase gene and the acetyl coenzyme A synthetase gene fragment by using the homologous recombinase of the lamda bacteriophage, so that the strain carrying the cat-sacB gene cannot grow on a salt-free sucrose plate, and thus a correct strain integrating the T7 promoter, the N-acetyl-5-hydroxytryptamine transferase gene and the acetyl coenzyme A synthetase gene is obtained.

Provides a method for synthesizing N-acetyl-5-hydroxytryptamine, which comprises the following steps: culturing the genetic engineering bacteria in a liquid LB culture medium overnight to obtain a seed solution; inoculating thalli in the seed solution into a fermentation culture medium for fermentation culture, adding a supplemented medium containing a substrate 5-hydroxytryptamine into the fermentation medium, adding IPTG (isopropyl-beta-thiogalactoside) after fermentation for induction culture to synthesize N-acetyl-5-hydroxytryptamine, and adding a supplemented medium containing a substrate 5-hydroxytryptamine into the induction culture medium.

According to the scheme, the LB culture medium and the fermentation culture medium contain spectinomycin.

N-acetyl-5-hydroxytryptamine transferase is used as a key enzyme in the biosynthesis process of N-acetyl-5-methoxytryptamine, and can convert 5-hydroxytryptamine into N-acetyl-5-hydroxytryptamine, but the yield of recombinant bacteria of N-acetyl-5-hydroxytryptamine reported at present is not higher than 2.5 g/l. Acetyl coenzyme A is taken as an important intermediate metabolic compound in a microbial metabolic pathway and is involved in the synthesis of various biological substances, and the production of N-acetyl-5-hydroxytryptamine with high yield by improving the acetyl coenzyme A is a new direction. The invention constructs the biosynthesis route of N-acetyl-5-hydroxytryptamine by expressing the high-activity AANTA gene in a host cell and further combining and expressing the ACS gene. The method achieves the effect of enhancing the capability of catalyzing substrate 5-hydroxytryptamine to generate N-acetyl-5-hydroxytryptamine by AANAT through overexpression of AANTA gene and ACS gene, obviously improves the synthetic yield of N-acetyl-5-hydroxytryptamine, and achieves excellent effect.

The invention has the beneficial effects that:

the invention constructs a new biosynthesis path of N-acetyl-5-hydroxytryptamine by expressing the high-activity AANTA gene in a host cell and further combining and expressing the ACS gene. The purposes of enhancing the generation of N-acetyl-5-hydroxytryptamine from 5-hydroxytryptamine as an AANAT catalytic substrate and obviously increasing the synthetic yield of N-acetyl-5-hydroxytryptamine by over-expressing the AANTA gene and the ACS gene are achieved, and excellent effects are obtained. By using N-acetyl-5-hydroxytryptamine transferase from different sources and combining the N-acetyl-5-hydroxytryptamine transferase with acetyl coenzyme A from different sources to synthesize functional enzymes, the yield of the N-acetyl-5-hydroxytryptamine transferase can reach 7.1g/L by simultaneously over-expressing the two genes, which is the highest level reported at present.

Drawings

FIG. 1 is a map of plasmid pCDF-T7-A2;

FIG. 2 shows the yield of N-acetyl-5-hydroxytryptamine produced by fermentation of strains carrying plasmids pCDF-T7-A1, A2, A3 and A4;

FIG. 3 is a plasmid map of pCDF-T7-A2-ACS 3;

FIG. 4 shows the yield of N-acetyl 5-hydroxytryptamine produced by fermentation of pCDF-T7-A2-ACS1/ACS2/ACS3 strain;

FIG. 5 shows the yield of N-acetyl 5-hydroxytryptamine produced by fermentation of pCDF-T7-A4-ACS1/ACS2/ACS3 strain.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and experiments can be obtained commercially without specific descriptions. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.

The host cell of the invention may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells. In still other embodiments, the strains include, but are not limited to: escherichia coli (Eshcerichia coli), Corynebacterium glutamicum (Corynebacterium glutamicum), Bacillus subtilis (Bacillus subtilis), Brevibacterium flavum (Brevibacterium flavum), Serratia marcescens (Serratia marcescens), Saccharomyces cerevisiae (Saccharomyces cerevisiae). In a preferred embodiment, the strain is Escherichia coli, and Escherichia coli is also exemplified in the present invention.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells capable of DNA uptake are harvested after the exponential growth phase and treated by electroporation using procedures well known in the art. Another method is to use MgCl₂If desired, the transformation may also be with CaCl₂The method of (1). When the host is a eukaryotic cell, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation liposome packaging, etc.

Based on the teaching of the present invention and the prior art, those skilled in the art can also understand that the recombinant cells of the present invention can be made into immobilized cells and other forms of utilization.

In the present invention, the term "expression vector" refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors well known in the art. In general, any plasmid and vector can be used as long as they can replicate and stably express in a host. An important feature of expression vectors is the inclusion of an origin of replication, a promoter, a marker gene and translation control elements.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The invention specifically screens 4N-acetyl-5-hydroxytryptamine transferases and 3 enzymes for increasing the yield of N-acetyl-5-hydroxytryptamine. Based on the knowledge of the prior art, it will be readily appreciated by those skilled in the art that the 3 ACS enzymes involved in the present invention are not limited to the enzymes of a particular origin mentioned in the examples, and each enzyme may be replaced with a different enzyme of a different origin having the same or similar catalytic function, respectively, or a variant form of one or more of the enzymes of a particular origin in the examples may be used, which variant form has the same or similar function as the enzyme but whose amino acid sequence differs by a small amount from the amino acid sequence provided by the present invention. These variants include, but are not limited to: one or more (usually 1 to 30, preferably 1 to 10, more preferably 1 to 6, most preferably 1 to 3) amino acids, and one or more (usually less than 20, preferably less than 10, more preferably less than 6 or 3) amino acids are added at the C-terminal and/or N-terminal. For example, it is well known to those skilled in the art that substitutions with amino acids of similar or analogous properties, such as isoleucine and leucine, do not alter the function of the resulting protein. For another example, the addition of one or several amino acids to the C-terminus and/or N-terminus, such as protein tags added for ease of isolation, e.g., a 6xHis tag, will not generally alter the function of the resulting protein.

The invention specifically comprises the following embodiments:

example 1:

(1) synthesis of N-acetyl-5-hydroxytryptamine transferase gene AANAT

Synthesizing N-acetyl-5-hydroxytryptamine transferase A1 according to the gene sequence of sheep, wherein the nucleotide sequence is shown in SEQ ID NO.001, and the amino acid sequence is shown in SEQ ID NO. 002; synthesizing N-acetyl-5-hydroxytryptamine transferase A2 according to the gene sequence of Drosophila melanogaster, wherein the nucleotide sequence is shown in SEQ ID NO.003, and the amino acid sequence is shown in SEQ ID NO. 004; synthesizing N-acetyl-5-hydroxytryptamine transferase A3 according to the gene sequence of Streptomyces albus, wherein the nucleotide sequence is shown in SEQ ID NO.005, and the amino acid sequence is shown in SEQ ID NO. 006; according to the gene sequence of the streptomyces griseus, N-acetyl-5-hydroxytryptamine transferase A4 is synthesized, the nucleotide sequence is shown in SEQ ID NO.007, and the amino acid sequence is shown in SEQ ID NO. 008.

Designing an amplification primer of the N-acetyl-5-hydroxytryptamine transferase gene, wherein if an upstream primer of the A1 gene is F1: 5 '- -3' GAATTCCATGTCCACTCCGTCT, the downstream primer of the A1 gene is R15 '-3' AAGCTTTTAACGATCGCTGTTACG, and the A1 gene is used as a template to carry out PCR amplification to obtain a linear gene segment; in the same manner, linear gene fragments were obtained by PCR amplification of N-acetyl-5-hydroxytryptamine transferase A2, A3, A4 using the primers shown in the following Table:

(2) construction of recombinant vector of N-acetyl-5-hydroxytryptamine transferase gene and prokaryotic expression

Carrying out double enzyme digestion on the amplified A1, A2, A3 and A4 gene fragments and pCDF empty plasmids by using EcoR I and Hind III respectively, recovering products, connecting the products by using T4 DNA ligase to obtain a recombinant vector, transferring the recombinant vector into DH5 alpha competent cells, selecting positive clone for sequencing, and identifying and sequencing correctly, wherein the plasmid is named as pCDF-T7-A1 plasmid which carries pCDF replicon, spectinomycin resistance genes, T7 promoter and A1 gene. The plasmids pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 with correct sequencing results were obtained in the same manner.

Plasmids of pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 were extracted and transformed into a protein expression strain BL21(DE 3). Expression monoclonals carrying plasmids pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 are respectively picked and inoculated into 3ml of LB liquid medium (containing spectinomycin with the final concentration of 50 mug/ml) for overnight culture. 1ml of the strain was pipetted into 100ml of LB liquid (containing spectinomycin at a final concentration of 50. mu.g/ml, 1mM IPTG) and cultured with shaking at 30 ℃ for 8 hours on a shaker at 250 rpm. Transferring isopropyl-beta-D-thiogalactoside (IPTG) induced bacteria liquid into a 50ml centrifuge tube, centrifuging at 4 ℃ and 6000rpm for 5min to collect bacteria, discarding supernatant, and cleaning bacteria for 2 times by using PBS solution; and (3) placing the cleaned thallus on ice, and crushing the thallus by using an ultrasonic crusher, wherein the ultrasonic power is 200W, the ultrasonic time is 1.5s, the ultrasonic time is suspended for 3s, and the total ultrasonic time is 20 minutes until the solution in the centrifugal tube becomes clear and transparent. Sucking 1ml of the crushed solution into a 1.5ml clean EP tube, centrifuging at 4 ℃, 13000rpm for 20min, sucking the supernatant into a new 1.5ml EP tube, adding a proper amount of 5 Loading buffer into the crushed centrifuged supernatant, uniformly mixing, carrying out boiling water bath for 10min, then centrifuging at 12000rpm for 2min, sucking the supernatant and carrying out expression condition detection on the target protein. The results showed that the proteins of plasmids pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 were well expressed in the E.coli producing strain.

(3) Biological fermentation test of N-acetyl-5-hydroxytryptamine

A strain fermentation experiment for producing N-acetyl-5-hydroxytryptamine by a shake flask test comprises the following steps: 1) preparing a seed solution: respectively selecting BL21 strains integrated with plasmids pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 in the step (2), inoculating the strains into 20ml of LB culture medium containing spectinomycin (with the final concentration of 50 mu g/ml), and culturing at 37 ℃ and 250rpm for 12 h; then, 20ml of the culture was transferred to 300ml of LB medium containing spectinomycin (final concentration: 50. mu.g/ml), and cultured at 37 ℃ for 12 hours with shaking at 250rpm, to obtain a seed solution. 2) And (3) culturing thalli: inoculating the seed liquid into 1L of fermentation medium containing spectinomycin (final concentration 50 mug/ml) with the inoculation amount of 10%, fermenting by adopting a 5L fermentation tank, controlling the temperature at 37 ℃ in the culture process, stirring at the speed of 500-.

Wherein the fermentation medium is a universal mediumThe specific components of each 1L of fermentation liquor are as follows: glucose 10g, (NH)₄)₂PO₄，8g，KH₂PO₄ 13.3g，MgSO₄*7H₂O, 1.2g, citric acid 1.7g and trace salt solution 10ml, wherein the volume is adjusted to 1L by water, and the pH value is adjusted to 7.0 by NaOH. Composition per 1L feed medium: glucose 400g, MgSO₄*7H₂O, 10g, trace salt solution 10ml and 5-hydroxytryptamine 10 g. Preparing 1L of trace salt solution; FeSO₄*7H₂O，10g，ZnSO₄*7H₂O，2.25g，CuSO₄*5H₂O 1g，MnSO₄*5H₂O 5g，Na₂B₄O₇*10H₂O 0.23g，CaCl₂*2H₂O，2g,(NH₄)₆Mo₇O₂₄0.1g, pH is adjusted with 5M hydrochloric acid and the volume is adjusted to 1L.

And (3) induction culture: reducing the temperature of the fermentation liquor after the thalli are cultured to 30 ℃, adding IPTG (isopropyl-beta-thiogalactoside) to enable the final concentration of the IPTG to be 1mM, carrying out induction culture, feeding a supplemented medium in the induction process, and adjusting the feeding speed of the supplemented medium to 20 ml/h; fed to the cell density OD₆₀₀And reaching 250, finishing the induction culture process, and measuring the yield of the N-acetyl-5 hydroxytryptamine.

Respectively measuring the fermentation liquor yield of the strains carrying pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A3 and pCDF-T7-A4 plasmids, detecting the concentration of N-acetyl-5-hydroxytryptamine every 24 hours, finally fermenting for 96 hours, wherein the yield of the N-acetyl-5-hydroxytryptamine is 3.8-5.6g/L, and the yield of the strains carrying the pCDF-T7-A2 plasmids is obviously higher than that of the strains carrying the pCDF-T7-A1, pCDF-T7-A2, pCDF-T7-A4 plasmids and the yield of the N-acetyl-5-hydroxytryptamine reaches 5.6g/L, as shown in figure 2.

Method for detecting yield of N-acetyl-5-hydroxytryptamine by HPLC

Taking the detection solution, centrifuging at 5000rpm for 15min, sucking the supernatant, and performing centrifugation according to the following steps: water: methanol volume ratio of 1: 7.5: 1.5 preparing a sample to be detected, and filtering and removing insoluble substances by a 0.22um organic filter to obtain the HPLC detection sample to be detected. The HPLC detector was an Agilent 1260 definition LV, and the detection column was an Agilent ZOBAX C18 column. The ultraviolet detection wavelength of the N-acetyl-5-hydroxytryptamine is 275nm, the flow rate of mobile phase 15% (v/v) methanol is 1.0ml/min, the sample injection amount is 10 mu l, and the quantification is carried out by adopting an external standard method according to the peak area.

Example 2:

(1) construction of tandem expression plasmid for acetyl-CoA synthetase

Synthesizing an enzyme ACS1 with acetyl coenzyme A synthesis function according to the gene sequence of the Pantoea agglomerans, wherein the nucleotide sequence is shown in SEQ ID NO.009, and the amino acid sequence is shown in SEQ ID NO. 010; synthesizing an enzyme ACS2 with acetyl coenzyme A synthesis function according to a gene sequence of escherichia coli, wherein a nucleotide sequence is shown in SEQ ID NO.011, and an amino acid sequence is shown in SEQ ID NO. 012; according to the gene sequence of Shigella sonnei, an enzyme ACS3 with acetyl coenzyme A synthesis function is synthesized, the nucleotide sequence is shown in SEQ ID NO.013, and the amino acid sequence is shown in SEQ ID NO. 014.

The plasmids pCDF-T7-A2 are used as vectors, and the ACS1 of Pantoea agglomerans, the ACS2 of Escherichia coli and the ACS3 of Shigella sonnei are expressed in series with N-acetyl-5-hydroxytryptamine transferase on the plasmids by a seamless cloning and splicing method to construct pCDF-T7-A2-ACS1, pCDF-T7-A2-ACS2 and pCDF-T7-A2-ACS3 plasmids, and the pCDF-T7-A2-ACS2 is shown in figure 2. The DH5 alpha strain was transformed with the plasmid according to the method of example 1, and the positive clones of LB plate (containing spectinomycin at a final concentration of 50. mu.g/ml) were verified by sequencing, and the sequencing results were consistent with the sequences of SEQ ID 009, SEQ ID 011, and SEQ ID 013, demonstrating the successful construction of plasmids pCDF-T7-A2-ACS1, pCDF-T7-A2-ACS2, and pCDF-T7-A2-ACS 3.

According to the method, pCDF-T7-A4 plasmid with better N-acetyl 5-hydroxytryptamine yield is used as a vector to construct pCDF-T7-A4-ACS1, pCDF-T7-A4-ACS2 and pCDF-T7-A4-ACS3 plasmids.

(2) Biological fermentation test of N-acetyl-5-hydroxytryptamine

The plasmids pCDF-T7-A2-ACS1, pCDF-T7-A2-ACS2 and pCDF-T7-A2-ACS3 constructed as described above were transferred into BL21(DE3) strain, and the fermentation evaluation of the plasmids expressed in tandem was carried out according to the method of example 1. The fermentation result shows that the strain carrying the pCDF-T7-A2-ACS3 plasmid has the best result of N-acetyl-5-hydroxytryptamine, the yield reaches 7.1g/L, and the strains carrying the pCDF-T7-A2-ACS2 and the pCDF-T7-A2-ACS1 plasmids have the yields of 5.8g/L and 6.4 g/L.

The same plasmids pCDF-T7-A4-ACS1, pCDF-T7-A4-ACS2 and pCDF-T7-A4-ACS3 were fermented according to the above procedure, and the results of the fermentation are shown in FIG. 5.

The results show that: through the tandem expression of the acetyl coenzyme A synthetase, the synthetic yield of the N-acetyl-5-hydroxytryptamine can be further improved, wherein the yield of the N-acetyl-5-hydroxytryptamine is obviously improved by the shigella sonnei-derived enzyme with the acetyl coenzyme A synthetic function, and the best acetyl coenzyme A expression enhancing effect of the ACS enzyme of the shigella sonnei is achieved.

Example 3:

(1) construction of N-acetyl-5-hydroxytryptamine recombinant bacteria

The expression of key genes on plasmids is less stable compared to chromosomes, and thus this example obtained an integrative N-acetyl-5-hydroxytryptamine synthetic strain BL-S16 by integrating the N-acetyl-5-hydroxytryptamine transferase gene A2 and the acetyl-CoA synthetase gene ACS3 into the E.coli chromosome.

The first step, obtaining a TrpR-knocked-out first-step homologous recombination fragment I, and carrying out PCR amplification by using a pXZ-SC plasmid (Tan et al,2013AEM) as a template and primers TrpR-F and TrpR-R to obtain a PCR product of about 3kb, namely the first-step homologous recombination fragment DNA containing cat and sacB genes and an upstream and downstream homology arm of 50 bp.

Secondly, the TrpR gene knockout DNA fragment is used for the first step of homologous recombination: the pTKred (Cox et al, 2010 nucleic acid Res) tool enzyme plasmid was first transformed into BL21 strain by means of electrotransformation, and then DNA fragment 1 was electrotransferred into BL21 strain carrying pTKRed plasmid.

And (3) electrotransfer conditions: preparing competent cells of E.coli BL21 carrying pTKRed plasmid (Dower et al 1998nucleic acid Res); 50. mu.l of competent cells were placed on ice, 50ng of DNA fragment I was added, and transferred to a 1mm Bio-Rad electric rotor. After electric shock using a Micro Pulser (Bio-Rad) electroporator, competent cells were transferred to 1ml LB liquid medium, incubated at 30 ℃ for 3h at 75rpm, plated on plates containing spectinomycin (final concentration 50. mu.g/ml) and chloramphenicol (34. mu.g/ml), incubated overnight at 37 ℃, and verified using cat-F and sacB-R primers to select a correct single clone, designated BL-TrpRC.

And thirdly, obtaining a second step fragment II of homologous recombination, and amplifying to obtain a homologous linear fragment containing 50bp of the upstream of the TrpR gene, a T7 promoter, an A2 gene, an ACS3 gene and 50bp of the downstream of the TrpR gene by using the pCDF-T7-A2-ACS3 plasmid as a template and T7-A2/ACS3-down as a primer. The amplified fragment was electroporated into BL-TrpRC strain.

And (3) electrotransfer conditions: preparing an electrotransformation competent cell of BL21-TrpRC carrying pTKRed plasmid (see Dower et al 1988 for preparation); 50. mu.l of competent cells were placed on ice, 50ng of DNA fragment was added, and transferred to a 1mm electric rotor. After electric shock by using a Micro Pulser (Bio-Rad) electroporator, competent cells were transferred to 1ml of LB liquid medium, incubated at 30 ℃ for 4 hours at 75rpm, transferred to a salt-free LB liquid medium containing 10% sucrose (containing spectinomycin at a final concentration of 50. mu.g/ml), and subjected to shaking culture at 30 ℃ for 24 hours at 220rpm, followed by streaking on a salt-free LB medium containing 6% sucrose (containing spectinomycin at a final concentration of 50. mu.g/ml). Single clones were verified using T7-A2/ACS3-down primers, and positive clones were designated BL-S16.

Primers integrating a2 and ACS genes are shown in the table below:

(2) fermentation of recombinant bacterium BLR-S16

When fermentation experiments were carried out on the strain BLR-S1 in the manner described in example 1, the yield of N-acetyl-5-hydroxytryptamine was detected to be 7.0g/L by HPLC, which is a desirable level.

In conclusion, the plasmid is used for expressing the N-acetyl-5-hydroxytryptamine transferase, and the enhanced expression tandem expression of the N-acetyl-5-hydroxytryptamine is further performed, so that the yield of the obtained target strain N-acetyl-5-hydroxytryptamine reaches 7.1g/L, and the highest level is reported at present.

< 110 > Hebei Weidakang Biotech Ltd

Less than 120, method for enhancing yield of N-acetyl-5-hydroxytryptamine

＜160＞ 14

＜210＞ 1

＜211＞ 624bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 1

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＞ 2

＜211＞ 207

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 2

MSTPSVHCLK PSPLHLPSGI PGSPGRQRRH TLPANEFRCL TPEDAAGVFE IEREAFISVS 60

GNCPLNLDEV QHFLTLCPEL SLGWFVEGRL VAFIIGSLWD EERLTQESLA LHRPRGHSAH 120

LHALAVHRSF RQQGKGSVLL WRYLHHVGAQ PAVRRAVLMC EDALVPFYQR FGFHPAGPCA 180

IVVGSLTFTE MHCSLRGHAA LRRNSDR 207

＜210＞ 3

＜211＞ 723bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 3

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＞ 4

＜211＞ 240

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 2

MEDALTVSGK PAACPVDQDC PYTIELIQPE DGEAVIAMLK TFFFKDEPLN TFLDLGECKE 60

LEKYSLKPLP DNCSYKAVNK KGEIIGVFLN GLMRRPSPDD VPEKAADSCE HPKFKKILSL 120

MDHVEEQFNI FDVYPDEELI LDGKILSVDT NYRGLGIAGR LTERAYEYMR ENGINVYHVL 180

CSSHYSARVM EKLGFHEVFR MQFADYKPQG EVVFKPAAPH VGIQVMAKEV GPAKAAQTKL 240

＜210＞ 5

＜211＞ 549bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 3

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＞ 6

＜211＞ 182

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 6

MNTFRTATAR DLPDVAATLT EAFAADPPTQ WVFPDGAAAV SRFFFGVADR AREAGGIVEL 60

LPGTAAMIAL PPHVRLPDAP ACGRQAEMQR RLGERRPRTP HYYLLFYGVR TAHQSSGLGG 120

RMLSDLISLA DRDRVGTYTE ASTWRGARLM LRHGFHTAQP LRLPHGPPMF PLWRDPIHDH 180

CD 182

＜210＞ 7

＜211＞ 549bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 7

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＞ 8

＜211＞ 182

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 8

MNTFRTATAR DIPDVAATLT EAFATDPPTQ WVFPDGTAAV SRFFTHVADR VHTAGGIVEL 60

LPDRAAMIAL PPHVRLPGEA ADGRQAEIQR RLADRHPLTP HYYLLFYGVR TAHQGSGLGG 120

RMLARLTSRA DRDRVGTYTE ASTWRGARLM LRHGFHATRP LRLPDGPSMF PLWRDPIHDH 180

SD 182

＜210＞ 9

＜211＞ 1956bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 9

atgagccaaa tacacaaaca tcccgttcca gagaatattg cagcaaacac cctgattact 60

gcagaacagt atcaggcgat gtatcagcaa tctgtcgacg atccagacag tttctggggc 120

gaacagggca aaatccttga ctggatcaag ccttacagca aagtgaaaaa ctgctcgttt 180

gcaccaggta acattaacat tcgctggtac gaagatggca cgctcaacct ggcggcaaac 240

tgccttgacc gtcatttagc cacgcgcggc gatcacccgg cgattatctg ggaaggcgac 300

gacgccagcg aaagcaaaac cctgactttc cgccagctgc atgccgaagt ctgtcgcttc 360

gccaacgtgc tggaactgct ggaagtgaaa aaaggcgacg tggtggcgat ctatatgccg 420

atggtgccgg aagccgcagt agcgatgctg gcctgtgcgc gtatcggtgc cgttcactcg 480

gtgatctttg gcggtttctc accggaggcg gtcgcgggcc gcatcgtcga ttccagcgcc 540

tcgctggtaa tcacggccga tgaagggatc cgcgccggtc gcagcattcc gctgaagaaa 600

aatgtcgatg acgcgttaaa aaatcctaat gtcaccagcg tgaaaaacgt ggtggtgttc 660

cagcgcactg gtaaagatgt gggctgggtt gaaggccgcg atcagtggtg gcacgacctg 720

atgctgaacg cgagcaccaa ccacgctgcc gcagaagtcg gcgcggaaga tccgctgttt 780

atcctttata cctccggctc aaccggcaaa cccaaaggcg tgttgcactc caccggtggg 840

tatctggtct atgccgccac aacctttaag ttcgtctttg attaccatca ggatgatatc 900

tactggtgca ccgccgatgt tggctgggtg acgggccaca gctatctggt ttatggaccg 960

ctggcctgtg gcgccaccac actgatgttt gaaggggtgc cgaactggcc aacgccgagc 1020

cgtatggcgg aagtggtcga taaacataaa gtcacgctgc tctacaccgc gcccactgca 1080

atccgtgcgc tgatggcgga aggtgataag gcgattgccg gcaccgaccg cagcagcctg 1140

cgcattatgg gatcggtcgg tgagccaatt aacccggaag cctgggagtg gtactacaag 1200

aaaatcggca acagccgctg cccgattgtt gatacctggt ggcagaccga aaccggcggc 1260

ttcatgatca cgccgctgcc gggtgcgacc gaactgaaag cgggttccgc cacccgaccg 1320

ttctttggcg tgcagcccgc gctggtggat aacgaaggtc agccacagga aggtgcctgt 1380

gaaggcaatc tggtgatcac cgagtcgtgg ccgggtcagg cacgtaccct ctatggcgat 1440

cacgaccgtt tcgagcagac ctacttctct acctttaaaa atgtctactt cagtggtgat 1500

ggtgcccgtc gtgatgagga tggctactac tggatcaccg gacgcgtcga tgacgtgctc 1560

aatgtttccg gtcaccgact cggcaccgcc gaaattgaat cggcgctggt ctcgcaccca 1620

aaaattgccg aagcggcggt agtggggatt ccgcacagca tcaaaggcca ggcgatctat 1680

gcctatatca cgctgaatca tggcgaagag ccgtcgccgg agttgtacac cgaagtgcgc 1740

cagtgggtcc gtaaagagat tggcccgata gccacaccgg atgtgctgca ctggaccgac 1800

tcactgccga aaacccgctc cggcaaaatc atgcggcgta ttctgcgcaa aattgccacc 1860

ggggatacca gtaatcttgg tgatacttct acgctcgccg atcctggcgt agtagaaaaa 1920

ctgcttgagg agaagcagtc gatcaaaatg ccttaa 1956

＜210＞ 10

＜211＞ 651

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 10

MSQIHKHPVP ENIAANTLIT AEQYQAMYQQ SVDDPDSFWG EQGKILDWIK PYSKVKNCSF 60

APNNINIRWY EDGTLNLAAN CLDRHLATRG DHPAIIWEGD DASESKTLTF RQLHAEVCRF 120

ANVLELLEVK KGDVVAIYMP MVPEAAVAML ACARIGAVHS VIFGGFSPEA VAGRIVDSSA 180

SLVITADEGI RAGRSIPLKK NVDDALKNPN VTSVKNVVVF QRTGKDVGWV EGRDQWWHDL 240

MLNASTNHAA AEVGAEDPLF ILYTSGSTGK PKGVLHSTGG YLVYAATTFK FVFDYHQDDI 300

YWCTADVGWV TGHSYLVYGP LACGATTLMF EGVPNWPTPS RMAEVVDKHK VTLLYTAPTA 360

IRALMAEGDK AIAGTDRSSL RIMGSVGEPI NPEAWEWYYK KIGNSRCPIV DTWWQTETGG 420

FMITPLPGAT ELKAGSATRP FFGVQPALVD NEGQPQEGAC EGNLVITESW PGQARTLYGD 480

HDRFEQTYFS TFKNVYFSGD GARRDEDGYY WITGRVDDVL NVSGHRLGTA EIESALVSHP 540

KIAEAAVVGI PHSIKGQAIY AYITLNHGEE PSPELYTEVR QWVRKEIGPI ATPDVLHWTD 600

SLPKTRSGKI MRRILRKIAT GDTSNLGDTS TLADPGVVEK LLEEKQSIKM P 651

＜210＞ 11

＜211＞ 1959bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 11

atgagccaaa ttcacaaaca caccattcct gccaacatcg cagaccgttg cctgataaac 60

cctcagcagt acgaggcgat gtatcaacaa tctattaacg tacctgatac cttctggggc 120

gaacagggaa aaattcttga ctggatcaaa ccttaccaga aggtgaaaaa cacctccttt 180

gcccccggta atgtgtccat taaatggtac gaggacggca cgctgaatct ggcggcaaac 240

tgccttgacc gccatctgca agaaaacggc gatcgtaccg ccatcatctg ggaaggcgac 300

gacgccagcc agagcaaaca tatcagctat aaagagctgc accgcgacgt ctgccgcttc 360

gccaataccc tgctcgagct gggcattaaa aaaggtgatg tggtggcgat ttatatgccg 420

atggtgccgg aagccgcggt tgcgatgctg gcctgcgccc gcattggcgc ggtgcattcg 480

gtgattttcg gcggcttctc gccggaagcc gttgccgggc gcattattga ttccaactca 540

cgactggtga tcacttccga cgaaggtgtg cgtgccgggc gcagtattcc gctgaagaaa 600

aacgttgatg acgcgctgaa aaacccgaac gtcaccagcg tagagcatgt ggtggtactg 660

aagcgtactg gcgggaaaat tgactggcag gaagggcgcg acctgtggtg gcacgacctg 720

gttgagcaag cgagcgatca gcaccaggcg gaagagatga acgccgaaga tccgctgttt 780

attctctaca cctccggttc taccggtaag ccaaaaggtg tgctgcatac taccggcggt 840

tatctggtgt acgcggcgct gacctttaaa tatgtctttg attatcatcc gggtgatatc 900

tactggtgca ccgccgatgt gggctgggtg accggacaca gttacttgct gtacggcccg 960

ctggcctgcg gtgcgaccac gctgatgttt gaaggcgtac ccaactggcc gacgcctgcc 1020

cgtatggcgc aggtggtgga caagcatcag gtcaatattc tctataccgc acccacggcg 1080

atccgcgcgc tgatggcgga aggcgataaa gcgatcgaag gcaccgaccg ttcgtcgctg 1140

cgcattctcg gttccgtggg cgagccaatt aacccggaag cgtgggagtg gtactggaaa 1200

aaaatcggca acgagaaatg tccggtggtc gatacctggt ggcagaccga aaccggcggt 1260

ttcatgatca ccccgctgcc tggcgctacc gagctgaaag ccggttcggc aacacgtccg 1320

ttcttcggcg tgcaaccggc gctggtcgat aacgaaggta acccgctgga gggggccacc 1380

gaaggtagcc tggtaatcac cgactcctgg ccgggtcagg cgcgtacgct gtttggcgat 1440

cacgaacgtt ttgaacagac ctacttctcc accttcaaaa atatgtattt cagcggcgac 1500

ggcgcgcgtc gcgatgaaga tggctattac tggataaccg ggcgtgtgga cgacgtgctg 1560

aacgtctccg gtcaccgtct ggggacggca gagattgagt cggcgctggt ggcgcatccg 1620

aagattgccg aagccgccgt agtaggtatt ccgcacaata ttaaaggtca ggcgatctac 1680

gcctacgtca cgcttaatca cggggaggaa ccgtcaccag aactgtacgc agaagtccgc 1740

aactgggtgc gtaaagagat tggcccgctg gcgacgccag acgtgctgca ctggaccgac 1800

tccctgccta aaacccgctc cggcaaaatt atgcgccgta ttctgcgcaa aattgcggcg 1860

ggcgatacca gcaacctggg cgatacctcg acgcttgccg atcctggcgt agtcgagaag 1920

ctgcttgaag agaagcaggc tatcgcgatg ccatcgtaa 1959

＜210＞ 12

＜211＞ 652

＜212＞ PRT

< 213 > Artificial sequence

＜400＞ 12

MSQIHKHTIP ANIADRCLIN PQQYEAMYQQ SINVPDTFWG EQGKILDWIK PYQKVKNTSF 60

APGNVSIKWY EDGTLNLAAN CLDRHLQENG DRTAIIWEGD DASQSKHISY KELHRDVCRF 120

ANTLLELGIK KGDVVAIYMP MVPEAAVAML ACARIGAVHS VIFGGFSPEA VAGRIIDSNS 180

RLVITSDEGV RAGRSIPLKK NVDDALKNPN VTSVEHVVVL KRTGGKIDWQ EGRDLWWHDL 240

VEQASDQHQA EEMNAEDPLF ILYTSGSTGK PKGVLHTTGG YLVYAALTFK YVFDYHPGDI 300

YWCTADVGWV TGHSYLLYGP LACGATTLMF EGVPNWPTPA RMAQVVDKHQ VNILYTAPTA 360

IRALMAEGDK AIEGTDRSSL RILGSVGEPI NPEAWEWYWK KIGNEKCPVV DTWWQTETGG 420

FMITPLPGAT ELKAGSATRP FFGVQPALID NEGNPLEGAT EGSLVITDSW PGQARTLFGD 480

HERFEQTYFS TFKNMYFSGD GARRDEDGYY WITGRVDDVL NVSGHRLGTA EIESALVAHP 540

KIAEAAVVGI PHNIKGQAIY AYVTLNHGEE PSPELYAEVR NWVRKEIGPL ATPDVLHWTD 600

SLPKTRSGKI MRRILRKIAA GDTSNLGDTS TLADPGVVEK LLEEKQAIAM PS 652

＜210＞ 13

＜211＞ 1959 bp

＜212＞ DNA

< 213 > Artificial sequence

＜400＞ 13