CN110607335B - A kind of nicotinamide adenine dinucleotide compound biosynthesis method - Google Patents

Abstract

Translated fromChinese

本发明公开了一种烟酰胺腺嘌呤二核苷酸类化合物生物合成方法。本发明提供的合成路径以分支酸为起点，在PhzD蛋白的作用下经过转氨重排反应生成氨基脱氧异分支酸，氨基脱氧异分支酸在PhzE蛋白的催化下脱去丙酮酸部分生成2,3‑二氢‑3‑羟基邻氨基苯甲酸，采用特定酶将2,3‑二氢‑3‑羟基邻氨基苯甲酸催化生成3‑羟基邻氨基苯甲酸，在nabC蛋白的作用下3‑羟基邻氨基苯甲酸通过氧化开环重排反应生成喹啉酸，随后进入NAD补救途径，完成NAD的合成。本发明解除了NAD合成与色氨酸或天冬氨酸合成的耦联，同时，分支酸广泛存在于各种生命形式中，且对其他生命必需代谢途径的影响较小。The invention discloses a biosynthesis method of nicotinamide adenine dinucleotide compounds. The synthetic route provided by the present invention takes chorismate as the starting point, and undergoes a transamination rearrangement reaction under the action of PhzD protein to generate aminodeoxyisochorismic acid, and aminodeoxyisochorismate is catalyzed by PhzE protein to remove the pyruvate part to generate 2, 3-dihydro-3-hydroxyanthranilic acid, 2,3-dihydro-3-hydroxyanthranilic acid is catalyzed to generate 3-hydroxyanthranilic acid by a specific enzyme, and under the action of nabC protein, 3-hydroxyl Anthranilic acid generates quinolinic acid through oxidative ring-opening rearrangement reaction, and then enters the NAD salvage pathway to complete the synthesis of NAD. The present invention releases the coupling between NAD synthesis and tryptophan or aspartic acid synthesis, and meanwhile, chorismate widely exists in various life forms, and has little influence on other essential metabolic pathways of life.

Description

Biosynthesis method of nicotinamide adenine dinucleotide compound

Technical Field

The invention relates to a biosynthesis method of nicotinamide adenine dinucleotide compounds.

Background

Nicotinamide Adenine Dinucleotide (NAD)⁺Coenzyme I) and its corresponding reduced form NADH are among the coenzymes essential for all life activities and they are involved in redox reactions in various organisms as acceptors or donors of protons. In addition, NAD is involved in non-redox life processes such as cell growth, differentiation, regulation and disease⁺Also plays very important roles, such as participating in histone deacetylation reaction related to aging, poly (adenosine diphosphate ribose) reaction playing an important role in DNA repair process, and adenosine diphosphate ribose cyclization reaction for regulating calcium ion channelAnd the like. In recent years, more and more studies have shown that a decrease in NAD in vivo is an important cause of aging. In some fermentation industries and biotransformations, NAD⁺The addition of (NADH) can increase the conversion efficiency of the product.

In vivo, biosynthesis of NAD includes de novo and salvage pathways. The salvage pathway of NAD is widely present in various organisms, and mainly refers to the process of synthesizing NAD by directly taking it (such as animals taking it from food, bacteria taking it from culture medium, etc.) and using nicotinic acid and nicotinamide as precursors. There are two pathways for de novo NAD synthesis known in biology: a. the aspartate pathway; b. the tryptophan-kynurenic acid pathway. In general, de novo synthesis of NAD in bacteria and plants (including part of archaea) utilizes the aspartate pathway: aspartic acid forms alpha-imine succinic acid under the action of aspartate oxidase, then the alpha-imine succinic acid is condensed with dihydroxyacetone phosphate under the action of quinolinate synthase to produce quinolinic acid, and then the quinolinic acid reacts with ribose pyrophosphate under the action of quinolinate phosphotransferase to generate Nicotinic Acid Mononucleotide (NAMN) which enters a salvage pathway to synthesize NAD. In eukaryotes such as yeast and mammals, NAD is generally synthesized de novo using tryptophan as a precursor: tryptophan is degraded to generate 3-hydroxy anthranilic acid through a kynurenic acid pathway, then 3, 4-bit dioxygen is rearranged and condensed to form quinolinic acid, and the quinolinic acid enters a NAD salvage synthesis pathway to synthesize the NAD compounds in a similar way as aspartic acid.

Organisms in the synthesis of NAD compounds via the two de novo synthetic pathways described above require the consumption of amino acids (tryptophan or aspartate) from the synthetic protein, which limits the concentration of such compounds in the organism to a certain extent, resulting in the current relatively expensive production of such compounds by yeast fermentation. In addition, the preparation of NAD by in vitro enzymatic conversion requires purification of the corresponding protease and consumption of expensive ATP precursor, which is also costly. No methods for the production of NAD by total chemical synthesis have been reported.

Disclosure of Invention

The invention aims to provide a biosynthesis method of a nicotinamide adenine dinucleotide compound.

The invention provides a method for synthesizing quinolinic acid (method A), which comprises the following steps:

(1) using branched acid as an initiator to generate amino deoxy isochorismate through transamination rearrangement reaction;

(2) removing the pyruvic acid part by amino-deoxy isochorismate to generate 2, 3-dihydro-3-hydroxy-anthranilic acid;

(3) dehydrogenating 2, 3-dihydro-3-hydroxy anthranilic acid to produce 3-hydroxy anthranilic acid;

(4) the 3-hydroxy anthranilic acid generates quinolinic acid through oxidation ring-opening rearrangement reaction.

The invention also provides a synthetic method of nicotinamide adenine dinucleotide (method B), which comprises the following steps:

(a) preparing quinolinic acid according to the method A;

(b) reacting quinolinic acid with ribose pyrophosphate under the action of quinolinic acid phosphotransferase to generate nicotinic acid mononucleotide;

(c) the preparation from nicotinic acid mononucleotide to nicotinamide adenine dinucleotide is completed by utilizing a salvage synthesis pathway of nicotinamide adenine dinucleotide.

The invention also provides a synthetic method of nicotinamide adenine dinucleotide phosphate (method C), which comprises the following steps:

(I) preparing nicotinamide adenine dinucleotide according to the method B;

(II) reacting nicotinamide adenine dinucleotide with ATP under the catalysis of nicotinamide adenine dinucleotide kinase to generate nicotinamide adenine dinucleotide phosphate.

The salvage synthesis pathway of any of the above nicotinamide adenine dinucleotides can be specifically that nicotinic acid mononucleotide reacts with ATP under the catalysis of the adenylyltransferase thereof to generate nicotinic acid adenine dinucleotides, and nicotinic acid adenine dinucleotides are catalyzed by nicotinamide adenine dinucleotide synthase to synthesize the nicotinamide adenine dinucleotides.

In the step (1), 2-amino-4-deoxychorismate synthase is adopted to catalyze the transamination rearrangement of chorismate to generate amino-deoxyisochorismate;

in the step (2), 2, 3-dihydro-3-hydroxy anthranilate synthase is adopted to catalyze the amino-deoxy isochorismate to remove the pyruvic acid part to generate 2, 3-dihydro-3-hydroxy anthranilic acid;

in the step (3), DHHA-2, 3-dehydrogenase is adopted tocatalyze 2, 3-dihydro-3-hydroxy anthranilic acid to dehydrogenate to generate 3-hydroxy anthranilic acid;

in the step (4), 3-hydroxy anthranilic acid-3, 4-dioxygenase is adopted to catalyze the oxidation ring-opening rearrangement reaction of 3-hydroxy anthranilic acid to generate quinolinic acid.

The invention also protects the application of any DHHA-2, 3-dehydrogenase in catalyzing the dehydrogenation of 2, 3-dihydro-3-hydroxy anthranilic acid to generate 3-hydroxy anthranilic acid.

The invention also protects the use of any of the above 2-amino-4-deoxychorismate synthase and/or 2, 3-dihydro-3-hydroxyanthranilate synthase and/or DHHA-2, 3-dehydrogenase and/or 3-hydroxyanthranilic acid-3, 4-dioxygenase in the synthesis of nicotinamide adenine dinucleotide compounds.

The invention also provides a method (method D) for synthesizing 3-hydroxy anthranilic acid, which comprises the following steps: the DHHA-2, 3-dehydrogenase is used for catalyzing the dehydrogenation of 2, 3-dihydro-3-hydroxy anthranilic acid to generate 3-hydroxy anthranilic acid.

A method for synthesizing nicotinamide adenine dinucleotide compound (method V) comprises the following steps: constructing a starting organism into a recombinant organism capable of realizing the synthetic pathway of the method A, and synthesizing the nicotinamide adenine dinucleotide compound by using the recombinant organism; the recombinant organism is capable of performing the following reactions:

(b1) reacting quinolinic acid with ribose pyrophosphate to generate nicotinic acid mononucleotide;

(b2) salvage synthesis of nicotinamide adenine dinucleotide;

(b3) reacting nicotinamide adenine dinucleotide with ATP to produce nicotinamide adenine dinucleotide phosphate.

The method comprises the following steps: introducing a coding gene of 2-amino-4-deoxychorismate synthase, a coding gene of 2, 3-dihydro-3-hydroxy anthranilate synthase, a coding gene of DHHA-2, 3-dehydrogenase, and a coding gene of 3-hydroxy anthranilate-3, 4-dioxygenase into a starting organism to obtain a recombinant organism, and synthesizing the nicotinamide adenine dinucleotide compound by using the recombinant organism.

The "introduction of a coding gene for 2-amino-4-deoxychorismate synthase, a coding gene for 2, 3-dihydro-3-hydroxyanthranilate synthase, a coding gene for DHHA-2, 3-dehydrogenase and a coding gene for 3-hydroxyanthranilic acid-3, 4-dioxygenase into a starting organism" can be carried out by introducing a coding gene for expressing 2-amino-4-deoxychorismate synthase into a living system containing a chorismate metabolic pathway, 2, 3-dihydro-3-hydroxy anthranilate synthase, DHHA-2, 3-dehydrogenase and 3-hydroxy anthranilate-3, 4-dioxygenase.

The recombinant expression vector can be pXB1a-QA, and the preparation method of the pXB1a-QA can be as follows: the double-stranded DNA molecule shown insequence 3 is used to replace the fragment between the Nco I and EcoRI cleavage sites of plasmid pXB1a to obtain plasmid pXB1 a-HAA. The double-stranded DNA molecule shown in the sequence 4 is inserted into the Nco I cleavage site of the plasmid pXB1a-HAA to obtain the recombinant plasmid pXB1 a-QA.

The invention also protects a recombinant organism capable of carrying out the synthetic pathway described in method a and which is capable of carrying out the following reaction:

(b1) reacting quinolinic acid with ribose pyrophosphate to generate nicotinic acid mononucleotide;

(b2) salvage synthesis of nicotinamide adenine dinucleotide;

(b3) reacting nicotinamide adenine dinucleotide with ATP to produce nicotinamide adenine dinucleotide phosphate.

The recombinant organism may be specifically one obtained by introducing a coding gene for 2-amino-4-deoxychorismate synthase, a coding gene for 2, 3-dihydro-3-hydroxyanthranilate synthase, a coding gene for DHHA-2, 3-dehydrogenase, and a coding gene for 3-hydroxyanthranilic acid-3, 4-dioxygenase into a starting organism.

The "introduction of a coding gene for 2-amino-4-deoxychorismate synthase, a coding gene for 2, 3-dihydro-3-hydroxyanthranilate synthase, a coding gene for DHHA-2, 3-dehydrogenase and a coding gene for 3-hydroxyanthranilic acid-3, 4-dioxygenase into a starting organism" can be carried out by introducing a coding gene for expressing 2-amino-4-deoxychorismate synthase into a living system containing a chorismate metabolic pathway, 2, 3-dihydro-3-hydroxy anthranilate synthase, DHHA-2, 3-dehydrogenase and 3-hydroxy anthranilate-3, 4-dioxygenase.

The recombinant expression vector can be pXB1a-QA, and the preparation method of the pXB1a-QA can be as follows: the double-stranded DNA molecule shown insequence 3 is used to replace the fragment between the Nco I and EcoRI cleavage sites of plasmid pXB1a to obtain plasmid pXB1 a-HAA. The double-stranded DNA molecule shown in the sequence 4 is inserted into the Nco I cleavage site of the plasmid pXB1a-HAA to obtain the recombinant plasmid pXB1 a-QA.

Any of the recombinant organisms described above is an organism that contains or is capable of synthesizing chorismic acid in vivo.

Any of the above starting organisms may specifically be a prokaryote, a plant or a fungus, more specifically an escherichia coli.

The Escherichia coli may be specifically Escherichia coli in which nadA gene and nadB gene are eliminated.

The invention also protects the application of any recombinant organism in synthesizing the nicotinamide adenine dinucleotide compound.

The nicotinamide adenine dinucleotide compound can be Nicotinamide Adenine Dinucleotide (NAD) or Nicotinamide Adenine Dinucleotide Phosphate (NADP), and also comprises reduced forms of NADH and NADPH of the nicotinamide adenine dinucleotide compound.

Any one of the branched acids is a compound shown as a formula (I).

Any one of the amino deoxy isochorismates described above is a compound represented by formula (II).

Any one of the above 2, 3-dihydro-3-hydroxyanthranilic acids is a compound represented by formula (III).

Any one of the above 3-hydroxyanthranilic acids is a compound represented by formula (IV).

Any one of the quinolinic acids is a compound shown as a formula (V).

Any of the above DHHA-2, 3-dehydrogenases may be an enzyme that catalyzes the production of 3-hydroxyanthranilic acid from 2, 3-dihydro-3-hydroxyanthranilic acid by a specific enzymatic reaction.

Included in the enzymatic reaction isNAD⁺2, 3-dihydro-3-hydroxy anthranilic acid and enzyme solution to be detected.

The enzymatic reaction system may specifically comprise 50mM PBS (pH7.4), 2mM NAD⁺2mM DHHA and 10. mu.M enzyme solution. The enzymatic reaction condition can be specifically water bath at 30 ℃ for 1 h.

Specifically, whether 3-hydroxyanthranilic acid is produced or not can be detected by HPLC.

Any of the above DHHA-2, 3-dehydrogenases is NAD⁺Can be used as a cofactor for catalyzing the dehydrogenation of 2, 3-dihydro-3-hydroxy anthranilic acid to generate 3-hydroxy anthranilic acid.

The DHHA-2, 3-dehydrogenase is dehydrogenase A, dehydrogenase B, dehydrogenase C, dehydrogenase D, dehydrogenase E, dehydrogenase F or dehydrogenase G.

The dehydrogenase A is (a1) or (a2) as follows:

(a1) a protein consisting of an amino acid sequence shown in asequence 2 in a sequence table;

(a2) and (b) the protein which is derived from thesequence 2 and has the same function by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of thesequence 2.

The dehydrogenase B is (a3) or (a4) as follows:

(a3) a protein consisting of an amino acid sequence shown in a sequence 11 in a sequence table;

(a4) and (b) a protein which is derived from the sequence 11 and has the same function, wherein the amino acid sequence of the sequence 11 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

The dehydrogenase C is (a5) or (a6) as follows:

(a5) a protein consisting of an amino acid sequence shown as asequence 12 in a sequence table;

(a6) protein derived from thesequence 12 by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of thesequence 12 and having the same function.

The dehydrogenase D is (a7) or (a8) as follows:

(a7) a protein consisting of an amino acid sequence shown as a sequence 13 in a sequence table;

(a8) and (b) a protein which is derived from the sequence 13 and has the same function, wherein the amino acid sequence of the sequence 13 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

The dehydrogenase E is (a9) or (a10) as follows:

(a9) a protein consisting of an amino acid sequence shown as a sequence 14 in a sequence table;

(a10) and (b) a protein which is derived from the sequence 14, is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 14, and has the same function.

The dehydrogenase F is (a11) or (a12) as follows:

(a11) a protein consisting of an amino acid sequence shown as a sequence 15 in a sequence table;

(a12) and (b) the protein which is derived from the sequence 15 and has the same function, wherein the amino acid sequence of the sequence 15 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

The dehydrogenase G is (a13) or (a14) as follows:

(a13) a protein consisting of an amino acid sequence shown as a sequence 16 in a sequence table;

(a14) and (b) a protein which is derived from the sequence 16 and has the same function by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 16.

Any of the above 2-amino-4-deoxychorismate synthases may specifically be PhzD proteins. The PhzD protein is as follows (b1) or (b 2):

(b1) a protein consisting of an amino acid sequence shown as a sequence 17 in a sequence table;

(b2) and (b) the protein which is derived from the sequence 17 and has the same function and is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the sequence 17.

Any of the above 2, 3-dihydro-3-hydroxyanthranilate synthases may specifically be a PhzE protein. The PhzE protein is as follows (c1) or (c 2):

(c1) a protein consisting of an amino acid sequence shown as a sequence 18 in a sequence table;

(c2) and (b) a protein which is derived from the sequence 18 and has the same function, wherein the amino acid sequence of the sequence 18 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues.

The 3-hydroxy anthranilic acid-3, 4-dioxygenase may be NabC. The NabC protein is as follows (d1) or (d 2):

(d1) a protein consisting of an amino acid sequence shown as a sequence 19 in a sequence table;

(d2) and (b) the protein which is derived from the sequence 19 and has the same function by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the sequence 19.

The gene encoding 2-amino-4-deoxychorismate synthase described above may specifically be (e1) or (e2) or (e3) or (e 4):

(e1) the coding region is shown as DNA molecule from 818-1841 site of 5' end ofsequence 3 in the sequence table;

(e2) DNA molecule shown in the 818-1841 site of the 5' end of thesequence 3;

(e3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (e1) or (e2) and encodes a protein having the same function;

(e4) and (e) a DNA molecule which has more than 90% homology with the DNA sequence defined in (e1), (e2) or (e3) and encodes a protein with the same function.

The coding gene of any one of the above 2, 3-dihydro-3-hydroxyanthranilic acid synthases may specifically be (f1) or (f2) or (f3) or (f 4):

(f1) the coding region is DNA molecule shown as 1467 th to 3350 th site of 5' end ofsequence 3 in the sequence table;

(f2) DNA molecule shown in 1467 th to 3350 th ofsequence 3 from 5' end;

(f3) a DNA molecule which hybridizes with the DNA sequence defined in (f1) or (f2) under stringent conditions and encodes a protein having the same function;

(f4) and (f) a DNA molecule which has 90% or more homology with the DNA sequence defined in (f1), (f2) or (f3) and encodes a protein having the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (g1), (g2), (g3) or (g 4):

(g1) the coding region of the DNA molecule is shown as asequence 1 in a sequence table;

(g2) a DNA molecule shown as asequence 1;

(g3) a DNA molecule which hybridizes with the DNA sequence defined in (g1) or (g2) under stringent conditions and encodes a protein having the same function;

(g4) and (c) a DNA molecule which has more than 90% homology with the DNA sequence defined in (g1), (g2) or (g3) and encodes a protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (h1), (h2), (h3) or (h 4):

(h1) the coding region of the DNA molecule is shown as asequence 5 in a sequence table;

(h2) a DNA molecule shown as asequence 5;

(h3) a DNA molecule which hybridizes with the DNA sequence defined in (h1) or (h2) under stringent conditions and encodes a protein having the same function;

(h4) and (c) a DNA molecule which has more than 90% of homology with the DNA sequence limited by (h1), (h2) or (h3) and encodes a protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (i1), (i2), (i3) or (i 4):

(i1) the coding region is a DNA molecule shown as a sequence 6 in a sequence table;

(i2) a DNA molecule shown as a sequence 6;

(i3) a DNA molecule which hybridizes with the DNA sequence defined in (i1) or (i2) under stringent conditions and encodes a protein having the same function;

(i4) and (c) a DNA molecule which has more than 90% of homology with the DNA sequence limited by (i1) or (i2) or (i3) and encodes a protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (j1), (j2), (j3) or (j 4):

(j1) the coding region of the DNA molecule is shown as a sequence 7 in a sequence table;

(j2) a DNA molecule shown as a sequence 7;

(j3) a DNA molecule which hybridizes with the DNA sequence defined in (j1) or (j2) under stringent conditions and encodes a protein having the same function;

(j4) and (j) a DNA molecule which has more than 90% homology with the DNA sequence defined in (j1), (j2) or (j3) and encodes a protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (k1), (k2), (k3) or (k 4):

(k1) the coding region of the DNA molecule is shown as a sequence 7 in a sequence table;

(k2) a DNA molecule shown as a sequence 7;

(k3) a DNA molecule which hybridizes with the DNA sequence defined in (k1) or (k2) under stringent conditions and encodes a protein having the same function;

(k4) and (c) a DNA molecule which has more than 90% homology with the DNA sequence defined by (k1), (k2) or (k3) and encodes a protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (l1), (l2), (l3) or (l 4):

(l1) a DNA molecule whose coding region is shown as sequence 8 in the sequence table;

(l2) the DNA molecule shown in sequence 8;

(l3) a DNA molecule which hybridizes under stringent conditions with the DNA sequence defined in (l1) or (l2) and encodes a protein having the same function;

(l4) and (l1), (l2) or (l3) limit the DNA sequence has more than 90% of homology and coding protein with the same function DNA molecule.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (m1), (m2), (m3) or (m 4):

(m1) the coding region is a DNA molecule shown as a sequence 9 in the sequence table;

(m2) the DNA molecule shown in sequence 9;

(m3) a DNA molecule which hybridizes with the DNA sequence defined in (m1) or (m2) under stringent conditions and encodes a protein having the same function;

(m4) is a DNA molecule which has more than 90% of homology with the DNA sequence limited by (m1), (m2) or (m3) and encodes protein with the same function.

The encoding gene of any of the above DHHA-2, 3-dehydrogenases may be (n1), (n2), (n3) or (n 4):

(n1) the coding region is a DNA molecule shown as asequence 10 in the sequence table;

(n2) the DNA molecule shown in thesequence 10;

(n3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (n1) or (n2) and encodes a protein having the same function;

(n4) is a DNA molecule which has more than 90% homology with the DNA sequence limited by (n1), (n2) or (n3) and encodes a protein with the same function.

The coding gene of any one of the above 3-hydroxyanthranilic acid-3, 4-dioxygenase can be specifically (o1) or (o2) or (o3) or (o 4):

(o1) a DNA molecule whose coding region is represented by sequence 4 of the sequence listing;

(o2) the DNA molecule shown in sequence 4;

(o3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence defined in (o1) or (o2) and which encodes a protein having the same function;

(o4) has more than 90% homology with the DNA sequence limited by (o1), (o2) or (o3) and encodes a protein with the same function.

The invention has the following advantages:

1. the present invention decouples the synthesis of NAD from the synthesis of tryptophan or aspartate.

2. The starting point of the heterologous metabolic pathway designed by the invention is chorismate, which is widely existed in various life forms (such as bacteria, fungi, archaea, plants and the like), and the chorismate is a natural metabolic branch point, which theoretically has little influence on other vital metabolic pathways.

3. Compared with the traditional yeast fermentation method, the reconstructed NAD synthesis path can theoretically obtain higher yield of the target product, and the target compound can be obtained only by a simple inorganic salt-glucose culture medium without additionally adding a NAD synthesis precursor such as tryptophan.

4. The strain adopted by the embodiment of the invention is escherichia coli, and compared with other strains, the escherichia coli has the advantages of clear genetic background, simple and convenient genetic operation, mature fermentation process, high growth speed and the like; however, the pathway is not limited to coliform strains nor bacteria and can be extended to all life forms and non-life forms containing chorismate.

5. The invention can be further optimized by means of traditional fermentation, metabolic engineering and the like, theoretically, higher yield can be obtained, and the production cost is further reduced.

Drawings

FIG. 1 is an artificially designed NAD anabolic pathway.

FIG. 2 shows the Pau20 enzymatic activity assay.

FIG. 3 shows the detection of ClaB3, DhbX and StnN enzymatic activities.

FIG. 4 shows the enzymatic activity assays of BomO, CbxG and NatDB.

FIG. 5 shows the Asp-NAD pathway knock-out in E.coli.

FIG. 6 is a validation of the new NAD synthesis pathway in E.coli.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 establishment of synthetic route

Through extensive research studies, a new route for NAD synthesis was established (FIG. 1).

Taking chorismate as a starting point, generating Amino Deoxy Isochorismate (ADIC) through transamination rearrangement reaction under the action of PhzD protein, removing pyruvate part to generate 2, 3-dihydro-3-hydroxy anthranilic acid (DHHA) under the catalysis of PhzE protein, catalyzing 2, 3-dihydro-3-hydroxy anthranilic acid to generate 3-hydroxy anthranilic acid (3-HAA) by using specific enzyme (DHHA-2, 3-dehydrogenase), generating Quinolinic Acid (QA) through oxidation ring-opening rearrangement reaction of 3-hydroxy anthranilic acid under the action of NabC protein, reacting quinolinic acid in organism with ribose pyrophosphate under the action of quinolinate phosphotransferase to generate nicotinic acid mononucleotide, and then entering NAD remediation pathway to complete the synthesis of NAD.

In vivo, NAD can react with ATP to generate Nicotinamide Adenine Dinucleotide Phosphate (NADP) under the catalysis of nicotinamide adenine dinucleotide kinase.

NAD can accept protons to become reduced form NADH.

NADP can accept the proton to become NADPH in a reduced form.

Example 2 identification of DHHA-2, 3-dehydrogenase

Identification of Pau20

1. One enzyme which possibly has DHHA-2, 3-dehydrogenase activity is screened from a large number of candidate enzymes and named as Pau20, the coding gene of the enzyme is shown as asequence 1 in a sequence table, and the protein sequence is shown as asequence 2 in the sequence table.

2. Preparation and purification of Pau20

(1) A small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced by a double-stranded DNA molecule shown insequence 1 of the sequence Listing to obtain recombinant plasmid pET28a:: pau20 (sequencing verified).

(2) The recombinant plasmid pET28a: pau20 is introduced into E.coli BL21(DE3) to obtain recombinant strain BL21(DE3) pET28a-pau 20.

(3) The recombinant strain BL21(DE3) pET28a-pau20 was cultured in LB liquid medium containing 50. mu.g/mL kanamycin to a bacterial solution OD_600nmThe value is about 0.4, the mixture is placed in 4 ℃ to be cooled for about 15min, IPTG with the final concentration of 0.05mM is added, and the protein expression is induced at the temperature of 16 ℃ and 180rpm for 12-16 h.

(4) After the completion of step (3), the induction system was centrifuged at 5000rpm for 10min at 4 ℃ to collect the pellet, which was then washed once with an appropriate Binding buffer (0.5M NaCl, 20mM Tris-HCl, 5mM imidazole, pH 7.9) and centrifuged to discard the supernatant. Adding 10mL of Binding buffer for re-suspending, carrying out ice bath, and crushing the thalli by using ultrasonic waves (the ultrasonic power is set to 300W, the ultrasonic program is ultrasonic for 5s, the pause is 9.9s, and the total time is 20 min). After the completion of the sonication, the cells were centrifuged at high speed (13000 rpm at 4 ℃) for 45min to remove cell lysis debris, and the cell lysate supernatant was collected.

(5) And (3) performing His-tag affinity chromatography purification and desalination on the cell lysate supernatant obtained in the step (4) to obtain a target protein Pau20 solution:

1) 1mL of nickel NTA agarose gel pre-packed column is taken, 10mL of LBinding buffer is used for balancing, then 10mL of sample of cell lysate supernatant is taken for sampling, and effluent liquid is collected.

2) Washing with 15mL binding buffer to remove impurities at a flow rate of 1-2mL/min, and collecting the effluent.

3) Non-specifically adsorbed hetero-proteins were washed off with 15mL of shaking Buffer (0.5M NaC, 20mM Tris-HCl, 20mM imidazole, pH 7.9) at a flow rate of 1-2mL/min, and the effluent was collected.

4) Specifically adsorbed target protein was washed off with 5mL of Elution Buffer (0.5M NaCl, 20mM Tris-HCl, 500mM imidazole, pH 7.9), and 500. mu.L of each tube was collected for analysis.

5) The column was then re-packed with 10mL of dilution Buffer, 10mL of deionized water, 10mL of 20% ethanol, and finally the column was stored in 20% ethanol and blocked for further use.

6) And detecting the distribution and purification condition of the target protein by SDS-PAGE electrophoresis.

7) Desalting the target protein with commercial PD-10 desalting column, and the specific steps are shown in the manufacturer's instruction.

8) The desalted target protein was concentrated and purified by an ultrafiltration membrane, stored in a phosphate buffer (pH7.4) containing 20% glycerol, dispensed, and stored at-80 ℃.

3. The DHHA-2, 3-dehydrogenase activity of Pau20 was tested by the following steps:

(1) preparing an enzyme activity detection system (100 mu L): 50mM PBS (pH7.4), 2Mm NAD⁺(Sigma Co.), 2mM DH A (Apollo Scientific Co.), 10. mu.M Pau20 protein fromstep 2.

(2) Carrying out enzyme activation reaction, carrying out water bath at 30 ℃ for 1h, adding 100 mu L of chloroform to stop the reaction after the reaction is finished, centrifuging at 13000rpm for 10min, and taking 20 mu L of supernatant for HPLC detection.

3 groups of enzyme activity reactions are set in each group, and a Pau20 inactivation group (Pau 20 is boiled and inactivated) is set as a control.

HPLC detection conditions: agilent SB-Aq analytical column (5 μm, 4.6X 250mm, Aglient, Santa Clara, CA, USA), flow rate 0.8mL/min, mobile phase A: 1 per mill trifluoroacetic acid aqueous solution; mobile phase B: acetonitrile (Merck kGaA). PDA detector, detection wavelength set 320nm and 254 nm. Elution conditions: the concentration of the acetonitrile is increased from 1 percent to 50 percent in a gradient way within 0-30 min; 30min-35min, and the acetonitrile concentration is increased from 50 percent to 100 percent in a gradient way; 35-40 min, and maintaining the concentration of acetonitrile at 100%; 40min-45min, and the acetonitrile concentration is reduced to 1% from 100%; 45-55 min, and the concentration of acetonitrile is maintained at 1%.

The results are shown in FIG. 2. In FIG. 2, A is SDS-PAGE chromatogram of purified Pau20, and B is HPLC detection chromatogram of Pau20 in vitro enzymatic reaction product, wherein, Pau20+ is experimental group, and Pau 20-is control group.

Product standard 3-HAA was purchased from Sigma, USA.

The results show that Pau20 converts NAD⁺Can be used as a cofactor for catalyzing the dehydrogenation of 2, 3-dihydro-3-hydroxy anthranilic acid to generate 3-hydroxy anthranilic acid.

Identification of other DHHA-2, 3-dehydrogenases

1. Through further screening, other 6 proteins which may have DHHA-2, 3-dehydrogenase activity were screened, in order ClaB3(AEH42481), DhbX (CDG76955.1), StnN (AFW04566.1), BomO (ALE27507), CbxG (KDQ70111) and NatDB (CEK 42820). The sequence numbers of the proteins at NCBI are in parentheses.

2. The following recombinant plasmids were constructed, respectively:

(1) recombinant plasmid pET28a: ClaB 3: a small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced by a double-stranded DNA molecule shown insequence 5 of the sequence Listing to obtain recombinant plasmid pET28a:: ClaB3 (sequencing verified). The double-stranded DNA molecule shown in thesequence 5 encodes a protein shown in the sequence 11.

(2) Recombinant plasmid pET28a: DhbX: a small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced by a double-stranded DNA molecule shown in sequence 6 of the sequence Listing to give a recombinant plasmid pET28a:: DhbX (sequence verified). The double-stranded DNA molecule shown in sequence 6 encodes a protein shown insequence 12.

(3) Recombinant plasmid pET28a StnN: a small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced with a double-stranded DNA molecule shown in sequence 7 of the sequence Listing to give a recombinant plasmid pET28a:: StnN (sequence verified). The double-stranded DNA molecule shown in sequence 7 encodes a protein shown in sequence 13.

(4) Recombinant plasmid pET28a: BomO: a small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced by a double-stranded DNA molecule shown in sequence 8 of the sequence Listing to give a recombinant plasmid pET28a:: BomO (sequencing verified). The double-stranded DNA molecule shown in sequence 8 encodes a protein shown in sequence 14.

(5) Recombinant plasmid pET28a: CbxG: the double-stranded DNA molecule shown in sequence 9 of the sequence table is used for replacing a small fragment between Nde I and BamH I enzyme cutting sites of pET28a plasmid (Novagen company) to obtain a recombinant plasmid pET28a:: CbxG (sequence verified). The double-stranded DNA molecule shown in sequence 9 encodes a protein shown in sequence 15.

(6) Recombinant plasmid pET28a NatDB: a small fragment between Nde I and BamH I cleavage sites of pET28a plasmid (Novagen) was replaced with a double-stranded DNA molecule shown insequence 10 of the sequence Listing to give a recombinant plasmid pET28a:: NatDB (sequence verified). The double-stranded DNA molecule shown insequence 10 encodes a protein shown in sequence 16.

3. Six candidate proteins were tested for DHHA-2, 3-dehydrogenase activity according tostep 2 andstep 3 of step one (recombinant plasmid pET28a:: pau20 was replaced with a recombinant plasmid expressing the candidate proteins).

The results of ClaB3, DhbX, StnN are shown in FIG. 3. The results show that the three proteins have good reaction activity under the experimental conditions, and obvious 3-HAA generation can be found by HPLC.

BomO, CbxG and NatDB have slightly low reaction activity under the experimental conditions, generation of NADH can be found by HPLC, a suspected 3-HAA small peak is generated, and the generation is confirmed by LC-MS (figure 4) (an LC-MS analyzer is an Agilent 1260/6460 Triple-Quadrupole LC/MS system, and the working mode is an electrospray mode).

Example 3 construction of plasmid for NAD Synthesis pathway

The gene sequences of phzD, phzE, nabC and pau20 were optimized for specificity according to the codon preference of the heterologous expression strain Escherichia coli, and then ligated to plasmid pXB1a (ref: Cui Q, Zhou F, Liu W, et al. Aero. biosynthesis: stable functional expression of branched chain alpha-keto acid dehydrogenase from Streptomyces avermitilis, in Escherichia coli, by selective differentiation and viral reduction gene expression [ J ]. Biotechnology Letters,2017,39(10): 1-8; publicly available from the institute of microorganisms of Chinese academy of sciences) to obtain recombinant plasmids.

The double-stranded DNA molecule shown insequence 3 was used to substitute the fragment between the Nco I and EcoRI cleavage sites of plasmid pXB1a to obtain plasmid pXB1a-HAA (which was verified by sequencing).

In thesequence 3 of the sequence table, thepositions 1 to 786 from the 5' end are pau20 gene, the positions 818-1841 are phzD gene (encoding the protein shown in the sequence 17 of the sequence table), and the positions 1467 to 3350 are phzE gene (encoding the protein shown in the sequence 18 of the sequence table).

Double-stranded DNA molecules (nabC gene, protein shown in sequence 19 of the coding sequence table) shown in sequence 4 are inserted into the Nco I enzyme cutting site of the plasmid pXB1a-HAA to obtain the recombinant plasmid pXB1a-QA (the sequencing verification is carried out).

In the recombinant plasmid pXB1a-QA, the promoter for induction of expression by arabinose initiates the expression of the target gene.

Example 4 knock-out of the E.coli de novo NAD pathway

BW 25113:. DELTA. nadA and BW 25113:. DELTA. nadB are nadA gene sterilization and nadB gene sterilization, respectively, and are described in the literature: baba T, Ara T, Hasegawa M, et al.construction of Escherichia coli, K-12 in-frame, single-gene knock out variants the Keio Molecular Systems Biology,2006,2(1): 2006.0008-2006.0008; the public is available from the institute for microorganisms of the Chinese academy of sciences.

A P1 phage is used for transduction to introduce BW 25113:. delta. nadB lysate into recipient bacteria BW 25113:. delta. nadA, so as to obtain a double knockout strain, and the specific steps are as follows:

1. the BW 25113. delta. nadB bacterial solution was inoculated into 5ml LB medium (50. mu.L of 20% glucose and 25. mu.L of 1M calcium chloride were added to the medium), and cultured at 37 ℃ until OD of bacterial solution was reached_600nm0.6-0.8, 100 μ L P1 phage stock solution (P1 phage titer 10) was added^9-10pfu/mL), continuously culturing for 2-3 hours, centrifuging at 4 deg.C and 1000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain BW 25113:. delta. nadB lysate for use.

2. The overnight cultured recipient bacterium BW25113 (1.5 mL. delta. nadA), was centrifuged at 6000rpm for 3min, the supernatant was removed, the pellet was resuspended in 700. mu.L of P1 salt solution (10mM calcium chloride and 5mM magnesium sulfate), and 100. mu.L of each tube was dispensed into sterile EP tubes.

3. 0/1/10/100. mu.L of BW 25113:. delta. nadA lysate was added to the prepared recipient bacteria BW 25113:. delta. nadA instep 2, and after standing at room temperature for 30min, 200. mu.L of 1M sodium citrate and 1mL of LB liquid medium were added, and the mixture was cultured at 37 ℃ and 220rpm for 1 hour.

4. After thestep 3 is completed, the bacterial liquid is centrifuged at 8000rpm for 2min to remove supernatant, 200 mu L LB is adopted to resuspend the bacterial sediment and then the bacterial sediment is coated on a kana resistant LB flat plate to obtain a bacterial strain BW25113, wherein delta nadA delta nadB is verified by respectively adopting a primer pair consisting of a primer nadA-F and a primer nadA-R and a primer pair consisting of a primer nadB-F and a primer nadB-R.

nadA-F：5’-TCAGGCATCCTCAATTTC-3’；

nadA-R：5’-GGCATACAGCTGAATCTG-3’；

nadB-F：5’-AACATCGCATTATCTGTG-3’；

nadB-F：5’-GCGTAGTGCTGCCAGAGC-3’。

The results are shown in FIG. 5. In the electrophoretogram of FIG. 5, the left side shows the result of amplification using a primer pair consisting of primer nadA-F and primer nadA-R, which demonstrates that nadA gene in Δ nadA Δ nadB of strain BW25113 is knocked out, and the right side shows the result of amplification using a primer pair consisting of primer nadB-F and primer nadB-R, which demonstrates that nadB gene in Δ nadA Δ nadB of strain BW25113 is knocked out. Taken together, strain BW 25113. DELTA. nadA. DELTA. nadB was knocked out of its de novo NAD pathway.

The wild type strain and the strain BW 25113. delta. nadA. delta. nadB were cultured in M9 medium, and the growth of the strain is shown in FIG. 5. Strain BW 25113:. DELTA.nadA. DELTA.nadB did not grow on M9 medium, but when quinolinic acid was exogenously added (final concentration 10mM), the strain recovered growth, indicating that the aspartate-NAD pathway in E.coli is essential under M9 medium.

Example 5 construction and validation of Escherichia coli containing New NAD Synthesis pathway

1. The recombinant plasmid pXB1a-QA is introduced into a strain BW 25113:. delta. nadA. delta. nadB, and the recombinant strain BW25113A-QA (containing a new NAD synthesis way) is obtained.

2. The plasmid pXB1a was introduced into strain BW 25113:. DELTA. nadA. DELTA. nadB, resulting in an empty vector strain.

3. The recombinant strain BW25113A-QA or the empty vector strain was inoculated into 3mL of liquid LB medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), and cultured at 37 ℃ and 220rpm for 12 hours. Adjusting concentration value of bacterial liquid to obtain bacterial liquid OD_600nmAfter 0.1, 2. mu.L of the suspension was spotted on M9 solid medium (divided into two groups, one group consisting of M9 solid medium containing 10mM arabinose and the other group consisting of M9 solid medium containing no arabinose) and cultured at 37 ℃ for 48 hours.

The results are shown in FIG. 6. A + is the growth of the empty vector-containing strain on the plate containing 10mM arabinose M9; A-QA + is the growth condition of the recombinant bacterium BW25113A-QA on a solid culture medium containing 10mM arabinose M9; A-QA-is the growth condition of the recombinant bacterium BW25113A-QA in M9 solid culture medium without arabinose.

The results show that under the action of arabinose induction, the strain BW25113A-QA restores the growth ability on M9 medium, indicating that the new NAD synthesis pathway can functionally replace the original pathway of Escherichia coli.

Example 6 determination of intracellular NAD levels in E.coli containing the novel NAD Synthesis pathway

The recombinant bacterium BW25113A-QA inoculated in example 5 was inoculated into 3mL of liquid LB medium (containing 50. mu.g/mL kanamycin and 50. mu.g/mL streptomycin), and cultured at 37 ℃ and 220rpm for 12 hours. Centrifuging the obtained bacterial solution (8000rpm, 5min), washing with sterile water twice, and diluting with sterile water to obtain bacterial solution OD_600nmAfter 1, 500 μ L was inoculated into 50mL M9 liquid medium (10mM arabinose-induced and blank, with the wild strain BW25113 as a control). Culturing at 37 deg.C for 4h, collecting thallus, diluting with precooled PBS buffer solution, cleaning thallus, and diluting bacterial liquid to OD_600nmAfter changing to 1, 1mL of bacterial liquid is taken and centrifuged (6000rpm, 10min), and then the bacterial body is left to detect the intracellular NAD⁺And the NADH content.

Kit method for detecting intracellular NAD⁺And the content of NADH:the kit used was ab65348(NAD/NADH Assaykit), and the detailed procedures were carried out according to the kit instructions. The results are shown in Table 1.

TABLE 1 intracellular NAD content of E.coli

Note: "-" indicates that the strain did not grow under the corresponding conditions, A-QA + was the strain BW25113A-QA 10mM arabinose-induced group, A-QA was the strain BW 25113A-QA-uninduced group; BW25113+ was a wild strain 10mM arabinose experimental group, BW25113 was an arabinose-free group.

Sequence listing

<110> institute of microbiology of Chinese academy of sciences

<120> biosynthesis method of nicotinamide adenine dinucleotide compound

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggcacag ccaattccga caaggtcgca ctggtgaccg gggccgccgg aggcatcggc 60

cgggcggtcg tgctcgcgct cgccggagcc ggcgccccgg tggccgccgt cgacatcgac 120

ggcgaggccc tcggggtgct ggagcggcag gcgcgggacg agggcctgga cgtcgccggc 180

ttcgccgcgg acgtcacctc ggccgcgcag acggaggcgg ccgtcgccgc cgccgagaac 240

cgcttcggcc caattcacca cctggtcaac acagccggcg tgctgtgctc gtccccggcc 300

ctggaactca ccgaggacga ctgggagcgg accttcgcgg tcaacaccac ggcggtcttc 360

agggtctccg ccaccgtcac ccgccggatg gtcgcccacg gcgtccgcgg cgcggtcgtc 420

accgtggcgt ccaacgcggc gaacgtgccc cggatgaaca tgtcggcgta cagcgcgtcg 480

aaggccgcgg ctgccgcctg gaccaagaac ctcggcctgg agctcgcggc gcacggcatt 540

cgctgcaacg tcgtgggtcc cgggtcgacc gacaccccga tgctgcgctc actgtggacc 600

gacgcgtccg ggccgtccgg ctccctcgac ggtgtgccgt cgcagtaccg gctcgggatt 660

ccgctcggca ggttcgccgc acccgccgac atcgccgacg ccgtcacatt cctgctctcc 720

gaccgcgcgg cccacatcac catgcacgac ctgatcgtcg acgggggcgc gaccctgggc 780

cgctag 786

<210> 2

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Gly Thr Ala Asn Ser Asp Lys Val Ala Leu Val Thr Gly Ala Ala

1 5 10 15

Gly Gly Ile Gly Arg Ala Val Val Leu Ala Leu Ala Gly Ala Gly Ala

20 25 30

Pro Val Ala Ala Val Asp Ile Asp Gly Glu Ala Leu Gly Val Leu Glu

35 40 45

Arg Gln Ala Arg Asp Glu Gly Leu Asp Val Ala Gly Phe Ala Ala Asp

50 55 60

Val Thr Ser Ala Ala Gln Thr Glu Ala Ala Val Ala Ala Ala Glu Asn

65 70 75 80

Arg Phe Gly Pro Ile His His Leu Val Asn Thr Ala Gly Val Leu Cys

85 90 95

Ser Ser Pro Ala Leu Glu Leu Thr Glu Asp Asp Trp Glu Arg Thr Phe

100 105 110

Ala Val Asn Thr Thr Ala Val Phe Arg Val Ser Ala Thr Val Thr Arg

115 120 125

Arg Met Val Ala His Gly Val Arg Gly Ala Val Val Thr Val Ala Ser

130 135 140

Asn Ala Ala Asn Val Pro Arg Met Asn Met Ser Ala Tyr Ser Ala Ser

145 150 155 160

Lys Ala Ala Ala Ala Ala Trp Thr Lys Asn Leu Gly Leu Glu Leu Ala

165 170 175

Ala His Gly Ile Arg Cys Asn Val Val Gly Pro Gly Ser Thr Asp Thr

180 185 190

Pro Met Leu Arg Ser Leu Trp Thr Asp Ala Ser Gly Pro Ser Gly Ser

195 200 205

Leu Asp Gly Val Pro Ser Gln Tyr Arg Leu Gly Ile Pro Leu Gly Arg

210 215 220

Phe Ala Ala Pro Ala Asp Ile Ala Asp Ala Val Thr Phe Leu Leu Ser

225 230 235 240

Asp Arg Ala Ala His Ile Thr Met His Asp Leu Ile Val Asp Gly Gly

245 250 255

Ala Thr Leu Gly Arg

260

<210> 3

<211> 3350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggcacag ccaattccga caaggtcgca ctggtgaccg gggccgccgg aggcatcggc 60

cgggcggtcg tgctcgcgct cgccggagcc ggcgccccgg tggccgccgt cgacatcgac 120

ggcgaggccc tcggggtgct ggagcggcag gcgcgggacg agggcctgga cgtcgccggc 180

ttcgccgcgg acgtcacctc ggccgcgcag acggaggcgg ccgtcgccgc cgccgagaac 240

cgcttcggcc caattcacca cctggtcaac acagccggcg tgctgtgctc gtccccggcc 300

ctggaactca ccgaggacga ctgggagcgg accttcgcgg tcaacaccac ggcggtcttc 360

agggtctccg ccaccgtcac ccgccggatg gtcgcccacg gcgtccgcgg cgcggtcgtc 420

accgtggcgt ccaacgcggc gaacgtgccc cggatgaaca tgtcggcgta cagcgcgtcg 480

aaggccgcgg ctgccgcctg gaccaagaac ctcggcctgg agctcgcggc gcacggcatt 540

cgctgcaacg tcgtgggtcc cgggtcgacc gacaccccga tgctgcgctc actgtggacc 600

gacgcgtccg ggccgtccgg ctccctcgac ggtgtgccgt cgcagtaccg gctcgggatt 660

ccgctcggca ggttcgccgc acccgccgac atcgccgacg ccgtcacatt cctgctctcc 720

gaccgcgcgg cccacatcac catgcacgac ctgatcgtcg acgggggcgc gaccctgggc 780

cgctagcata tgtctagaga aagaggagaa atactagatg tcaggtatcc ctgaaatcac 840

cgcatatcca ttacctaccg cccagcagtt accggccaat ctggcacgct ggagcttaga 900

acctcgtcgc gccgttctgt tagttcatga tatgcagcgc tattttctgc gtccactgcc 960

ggaatcttta cgcgcaggtc tggttgctaa tgcggcccgc ttacgtcgtt ggtgtgttga 1020

acagggcgtt cagatcgcct atacggctca acccggctca atgaccgaag aacagcgtgg 1080

cttactgaaa gatttttggg gtccggggat gcgtgcaagt cctgcggatc gcgaagtggt 1140

ggaagaatta gctccgggtc cagatgattg gttattaacc aaatggcgct atagtgcctt 1200

ttttcatagc gatttattac agcgtatgcg cgcggcaggt cgtgatcagt tagttctgtg 1260

cggtgtttat gcacatgtgg gcgtgttaat ctctacagtg gatgcttatt ctaatgatat 1320

acagccgttt ctggttgctg atgccattgc cgattttagc gaagcacatc atcgtatggc 1380

cttagaatat gccgcatctc gctgtgcaat ggttgtgaca acggatgaag tgctggaatg 1440

atctagagaa agaggagaaa tactagatga atgccctgcc tacctcactg ttacagcgcc 1500

tgttagaacg tccagctccg tttgcgttac tgtatcgtcc ggaaagtaat ggccctggcc 1560

tgctggatgt gattcgcggc gaagccttag aattacatgg ccttgctgat ttaccattag 1620

atgaaccggg acctggttta ccacgccatg atttattagc cttaatcccg tatcgccaga 1680

ttgccgaacg cggctttgaa gccctggatg atggtacccc gctgttagca ttaaaagttc 1740

tggaacagga attactgcca ctggaacagg cattagcact gttacctaat caggccttag 1800

aactgagtga agaaggcttt gatctggatg atgaagccta cgcggaagtt gtgggtcgtg 1860

tgattgcgga tgaaatagga cgcggcgaag gcgccaattt tgttatcaaa cgccgctttc 1920

aggctcgcat cgatggctat gcaaccgcaa gcgcactgtc tttttttcgt cagctgttac 1980

tgcgtgaaaa aggtgcatat tggaccttta ttgttcatac gggcgaacgc accctggtgg 2040

gcgcctcacc agaacgccat atctcagttc gcgatggttt agcagttatg aatcctatct 2100

caggtacata tcgctatcct ccagccggtc ctaatttagc agaagttatg gaatttctgg 2160

ataatcgtaa agaagccgat gaactgtata tggttgttga tgaagaatta aaaatgatgg 2220

cacgtatttg cgaagatggt ggtcgtgtgc tgggtccgta tttaaaagaa atggcacatc 2280

tggcacatac ggaatatttt atcgaaggcc agacctctcg cgatgtgcgt gaagtgttac 2340

gtgaaacact gtttgcaccg acggtgacgg gttctccatt agaaagcgct tgtcgcgtta 2400

ttcgccgcta tgaaccacag ggtcgtggct attatagtgg tgtggcagcc ctgatcggcg 2460

gcgatggtca gggggggcgg acactggata gcgcaatctt aattcgtacg gcagaaattg 2520

aaggtgatgg tcgcttacgc atcggcgttg gtagtacaat tgtgcgtcat tcagatcctc 2580

tgggcgaagc tgccgaaagc cgtgccaaag caagcggttt aattgccgca ttaaaatcac 2640

aggctccaca gcgcttaggt agtcatccac atgttgttgc agccttagct agtcgtaatg 2700

ctcctattgc cgatttttgg ctgcgcggtg ctagtgaacg ccagcagtta caggccgatc 2760

tgagcggtcg cgaagtgctg attgtggatg cggaagatac ctttacgagt atgattgcta 2820

aacagttaaa aagtctgggc ctgacggtga ccgttcgtgg ttttcaggaa ccatatagct 2880

ttgatggcta tgatctggtt atcatggggc cgggtccggg taatccgacc gaaatcggtc 2940

agcctaaaat cggtcattta catttagcta ttcgtagttt actgtcggaa cgtcgtccgt 3000

ttctggcagt gtgtctgagt catcaggttc tgagcttatg tctgggcttg gacttgcaac 3060

gccgtcagga accaaatcag ggcgttcaga aacagatcga tctgtttggt gccgcagaac 3120

gggttggttt ttataatacc tttgctgctc gggctctaca agatcgcatc gaaatcccgg 3180

aagttggccc aattgaaatc tctcgcgatc gcgaaaccgg cgaagttcat gccctgcgcg 3240

gtccacgctt tgcctcaatg cagtttcatc cggaaagtgt gctgactaga gaaggtcctc 3300

gtatcattgc ggatttactg cgtcatgcat tagtggaacg tcgcccataa 3350

<210> 4

<211> 555

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgatgttta cctttggtaa accattaaat tttcagcgct ggttagatga tcatagcgat 60

ctgttacgtc ctccagtggg taatcagcag gtgtggcagg atagcgattt tattgtgacc 120

gttgtgggcg gccctaattt tcgtacagat tttcatgatg atcctatgga agaatttttt 180

tatcagttta aaggtaatgc ctatctgaat attatggatc gcggccagat ggatcgcgtg 240

gaactgaaag aaggcgacat ctttctgctg cctcctcatc tgcgtcatag tccacagcgt 300

ccggaagccg gctctcgctg tctggttatc gaacgccagc gcccgaaagg catgttagat 360

ggctttgaat ggtattgctt aagttgtaat ggcttagtgt atcgtgttga tgtgcagctg 420

aatagtattg tgacggattt accgccactg tttgatattt tttatggcaa tgttggcctg 480

cgcaaatgcc cacagtgcgg tcaggtgcat ccgggcaaag ccgcaatcga agcagttgca 540

cgcggtgatc agccg 555

<210> 5

<211> 813

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaatttc gtaccatgac ccgtaccacc gattctcgcg caatggaagg taaagtggca 60

ctggtgaccg gcgcagcagg cggtattggt gcagcagttg ttcgcgcaat tgcagaacgc 120

ggcggtgtgc tggcagcagt ggatgcaaat agtgtggcac tgaaagaaac caccgaagca 180

ctggcaggcg aaggcctgcg tgttgaaggc tttgttgcag atgtgacccg tccggatgaa 240

gttgaagcaa ccgttgcagc agttgaagca cgtctgggtc cggttgatca tctggttaat 300

gcagcaggta ttctgcgtct gggcgatgca cgtaccctgt cagataccga ttgggcagat 360

accattgcag ttaatgcaac gggcgttttt cacatgagtc gcgcagttgt taatcgtatg 420

gttccgcgtc gctcaggcgc actggtgacc gtggcatcta atgcagcagg gactccgcgc 480

acccagatgg cagcatacgc agcatcaaaa gcagcagcaa ccatgtttac caaatgtctg 540

ggcctggaag tggcagaata tggtattcgc tgtaatctgg ttgcaccggg tagtaccgat 600

accccgatgc tttcctctat gtggcatgat gatagcggtc gtgaagcaac cgttcgcggc 660

tctctggaaa cctttaaagt gggtattccg ctgcgtaaac tggcagcacc gcgtgatgtg 720

gcagatgcag ttgtgtttct gctgagcgaa gaagcaagtc atattaccct gcacgcactg 780

accgtggatg gcggcgcaac cctgggcgtt taa 813

<210> 6

<211> 786

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgacctttg ataaagatta tattgcagtt attaccggcg catgtggcgg tattggtgaa 60

agcgtggcac atgcactggc aaaagaaggt ctgtctctgg cactgctgga taataatgca 120

acccagctgg caaccctggt ggcaaccttg caggataatc atccgcagcc gattgcaggc 180

tttaccgtgg atgttgcaga tgatcgttgt gtggcagaag catttaccgc agtgggtcat 240

cagctgggtc cggtgggcta tctggtgaat ggcgcaggcg ttctgtgtca tgcaagtgtt 300

gcagaaaccc agccgcagga ttgggcaaaa acctttgcag ttaatgcaac cggcgtgttt 360

aatacctcac gtcatgcagc aaatctgatg atggcacagc gtaaaggttc aattgtgacc 420

attgcatcaa atgcagcgcg tgttccgcgc gcaacgatgg cagcatattg tgcatctaaa 480

gcagcagcac aggcatttac ctatgcactg ggcctggaag tggcaccgta tggtattcgc 540

tgtaatgttg ttgcaccggg tagtaccgat accccgatgc tgcgcggtat gtggcatagc 600

gaaagcgata aacagaatac cctgaatggt aatccgcagc agtttcgtat tggtattccg 660

ctgaataaag ttgcaaccgc agaagaaatt gcagcagcag tttgttttta tctgtgtgaa 720

gaatcaggtc agaccaccct gagtaccctg ctggttgatg gcggcgcagc actgggctct 780

tgttaa 786

<210> 7

<211> 861

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggttgcac gtatgagtac cgggcgtaca ggaacaggcc gtgcagaacc ggaacgtcgt 60

cgcgcacatc cggcaatgga agtgggtacg atggaagata aagtggcact ggtgaccggc 120

gcagcaggcg gtattggtgc agcagttgca cgcgcactgg cacgtcgcgg cgcacgtgtt 180

gcagcagtgg atctgcatac aggccgcctg accgaagaag ttggtaaact gaccgcagat 240

ggtctggcag ttgaagcatt tccggcagat gtgacccgtg cagcagcagt ggaagaactg 300

gttgaaggtg tggaaacccg cctgggcccg gttgatctgc tggttaatgc agcaggtgtt 360

ctgcgtctgg gtccggttca tcgcctgggc gatgaagatt gggcagcaac ctttgcagtt 420

aataccaccg gcgtttttct ggtgtctcgt gcagtggcag gccgtatgat gccgcgtagt 480

cgcggtgcaa ttgtgaccgt tgcaagtaat gcagcaggga ctccgcgtac cgaaatggca 540

gcctacgcag catctaaagc agcagcaacc atgtttacca aatgtctggc actggaagtg 600

gcaggtcatg gtattcgctg taatgttgtg gcaccgggct caaccgatac cgcaatgctg 660

cgctctatgt ggcaggatga atcaggcgca cgcgcaacca ttgaaggccg tccggaagca 720

tataaactgg gtattccgct gggtaaactg gcacgcccgg cagatattgc agatgcagtt 780

gtgtttctgc tgagcgatcg tgcaggtcat attaccatgc atgaactgac cgttgatggc 840

ggcgcaaccc tgggcgcata a 861

<210> 8

<211> 828

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atgacgcacg gtccggatca ggatggtctg ggcctggaag gtcgtgttgc agttgtgacc 60

ggcgcagcag gcggtattgg tgcagaaacc gttgcacagc tggttcgtca tcgcgcagca 120

gtggcactgg tggatcgtga tggtccggca ctggatgaac tggttgcacg cctgcgtaaa 180

gaagcagcag cagatccgcg tgcaccggaa ccgcgtctgc tggcagttcc ggcagatgtt 240

agtcatggcg gtgaagtgac cgcagtggtt gaacgtgtgg aatcagaact gggcccgatt 300

acccatctgg ttaatgcagc aggtattctg cgtccgggtc cggttgcagg cctgagcgaa 360

cgcgattggg atgataccct ggcagttaat gcaacggggg tttatctgat gtctcgtgca 420

gtggcaagtc gtatggcacc gcgcggcttt ggcgcaattg tgaccgttac ctctaatgca 480

gcacgtaccg cacgtactgg aatggcagcg tatgcagcaa gtaaagcagc agcacaggca 540

tttaccaaat gtctgggcct ggaactggca gggacaggta ttcgctgtaa tgttgttgca 600

ccgggctcaa ccgatacccc gatgctgacc tcactgtggg aaggcgaaga tgcaggtcgc 660

ccgtctattg aaggcgcacc ggaagcatat cgcgtgggta ttccgaccgg ccgcctggca 720

cgcccgtatg atgtggcaca ggcagttgtt tttctgctga gcgaacgcgc aggtcatatt 780

acccttcagg atctgaccgt tgatggcggc gcaaccctgg gtgtgtaa 828

<210> 9

<211> 771

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgaaagata ccattgcagt ggttaccggc gcagcaggcg gtattggtgc agcagtggca 60

gaagcactgg cagtgcgcgg cgcaagcgtg gcactgctgg atcgtgatgc agatcgcctg 120

gcagttgtgg caaaagaact gacctcggca ggtcatcgcg caaccgcatt tccggcagat 180

gtgacctcag gtcaggatgt ggaagcagtg attgaagcag ttgaagaacg cctgggtccg 240

gttgatcgtc tggttaatgc agcaggcgtg ctgcgtaccg gcccggcaca tgaatttgca 300

gatgaagatt gggaagcaac ctttgcagtt aataccacag gcgtttttca tgtgtctcgc 360

gcagttgttc gtcgtatgcg tacccgccgt cgcggtgcac tggtgaccat tgcaagtaat 420

gcagcaggct ctgcacgtac cgaaatggca gcttatgcag catctaaagc agcagcatct 480

atgtttacca aatgtctggg cctggaaaat gcagcgtacg gtattcgctg taatgttgtg 540

gcaccgggta gtaccgatac cccgatgctg accgcactgt gggatgatgc aagcgcagca 600

gatgcatcaa ttgcaggtgt tccggaagca tatcgcgttg gtattccgct ggcaaaactg 660

gcacgtccga aagatgtggc agatgcagtt ctgtttctgc tgagcgatca ggcaagtcat 720

attaccatgc atcatctgac cgttgatggc ggcgcaaccc tgggcgcata a 771

<210> 10

<211> 825

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgagcgata ccctgaccac caccggcaca accggcacag cagcagcagc aggcgcagaa 60

tggagcggta gcgcagttgt taccggcgca gcaggcggta ttggtgcaga aattgcacgc 120

gcactggcag cagcaggtgt gccggttgca ctgctggatc gtgaaccggg cgcactgcgt 180

gaactggcaa gcgaactgag cgcagcaggc ggaactgtgc tggcagtggc agcagatgtg 240

accgatagcg cagatgttga tgcagcagtg gcaaaagcag aagcagaact gggtccggtt 300

gcatatctgg ttaatggtgc aggcgttctg cattcaggtc cggcaggcga atttagtgat 360

gaagattggg atcataccct ggcagttaat gcaggtggtg tttttcgtgt tagtcgcgca 420

gtggcacgtc tgatggttcc gcgtggccgc ggctcaattg tgaccattgc atctaatgca 480

gcactgaccc cgcgtacctc tatggcagca tacgcagcaa gtaaagcagc aagtgcaatg 540

tttaccaaat gtctgggcct ggaactgggt cgtcatggta ttcgctgtaa tgttgtggca 600

ccgggctcaa cccgtacctc tatgctgacc gcacttcaag gcgatgcagc agttcgcgca 660

tcagtggatg gtgtgccgga tgcatatcgc gttggtattc cgctgggtcg tattgcagaa 720

ccggcacata ttgcagatgc agttctgttt ctgctgtcag atcgctctgc acatattacc 780

ttgcaggatc tgaccgttga tggcggcgca gcactgggtg tgtaa 825

<210> 11

<211> 270

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Met Glu Phe Arg Thr Met Thr Arg Thr Thr Asp Ser Arg Ala Met Glu

1 5 10 15

Gly Lys Val Ala Leu Val Thr Gly Ala Ala Gly Gly Ile Gly Ala Ala

20 25 30

Val Val Arg Ala Ile Ala Glu Arg Gly Gly Val Leu Ala Ala Val Asp

35 40 45

Ala Asn Ser Val Ala Leu Lys Glu Thr Thr Glu Ala Leu Ala Gly Glu

50 55 60

Gly Leu Arg Val Glu Gly Phe Val Ala Asp Val Thr Arg Pro Asp Glu

65 70 75 80

Val Glu Ala Thr Val Ala Ala Val Glu Ala Arg Leu Gly Pro Val Asp

85 90 95

His Leu Val Asn Ala Ala Gly Ile Leu Arg Leu Gly Asp Ala Arg Thr

100 105 110

Leu Ser Asp Thr Asp Trp Ala Asp Thr Ile Ala Val Asn Ala Thr Gly

115 120 125

Val Phe His Met Ser Arg Ala Val Val Asn Arg Met Val Pro Arg Arg

130 135 140

Ser Gly Ala Leu Val Thr Val Ala Ser Asn Ala Ala Gly Thr Pro Arg

145 150 155 160

Thr Gln Met Ala Ala Tyr Ala Ala Ser Lys Ala Ala Ala Thr Met Phe

165 170 175

Thr Lys Cys Leu Gly Leu Glu Val Ala Glu Tyr Gly Ile Arg Cys Asn

180 185 190

Leu Val Ala Pro Gly Ser Thr Asp Thr Pro Met Leu Ser Ser Met Trp

195 200 205

His Asp Asp Ser Gly Arg Glu Ala Thr Val Arg Gly Ser Leu Glu Thr

210 215 220

Phe Lys Val Gly Ile Pro Leu Arg Lys Leu Ala Ala Pro Arg Asp Val

225 230 235 240

Ala Asp Ala Val Val Phe Leu Leu Ser Glu Glu Ala Ser His Ile Thr

245 250 255

Leu His Ala Leu Thr Val Asp Gly Gly Ala Thr Leu Gly Val

260 265 270

<210> 12

<211> 261

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Met Thr Phe Asp Lys Asp Tyr Ile Ala Val Ile Thr Gly Ala Cys Gly

1 5 10 15

Gly Ile Gly Glu Ser Val Ala His Ala Leu Ala Lys Glu Gly Leu Ser

20 25 30

Leu Ala Leu Leu Asp Asn Asn Ala Thr Gln Leu Ala Thr Leu Val Ala

35 40 45

Thr Leu Gln Asp Asn His Pro Gln Pro Ile Ala Gly Phe Thr Val Asp

50 55 60

Val Ala Asp Asp Arg Cys Val Ala Glu Ala Phe Thr Ala Val Gly His

65 70 75 80

Gln Leu Gly Pro Val Gly Tyr Leu Val Asn Gly Ala Gly Val Leu Cys

85 90 95

His Ala Ser Val Ala Glu Thr Gln Pro Gln Asp Trp Ala Lys Thr Phe

100 105 110

Ala Val Asn Ala Thr Gly Val Phe Asn Thr Ser Arg His Ala Ala Asn

115 120 125

Leu Met Met Ala Gln Arg Lys Gly Ser Ile Val Thr Ile Ala Ser Asn

130 135 140

Ala Ala Arg Val Pro Arg Ala Thr Met Ala Ala Tyr Cys Ala Ser Lys

145 150 155 160

Ala Ala Ala Gln Ala Phe Thr Tyr Ala Leu Gly Leu Glu Val Ala Pro

165 170 175

Tyr Gly Ile Arg Cys Asn Val Val Ala Pro Gly Ser Thr Asp Thr Pro

180 185 190

Met Leu Arg Gly Met Trp His Ser Glu Ser Asp Lys Gln Asn Thr Leu

195 200 205

Asn Gly Asn Pro Gln Gln Phe Arg Ile Gly Ile Pro Leu Asn Lys Val

210 215 220

Ala Thr Ala Glu Glu Ile Ala Ala Ala Val Cys Phe Tyr Leu Cys Glu

225 230 235 240

Glu Ser Gly Gln Thr Thr Leu Ser Thr Leu Leu Val Asp Gly Gly Ala

245 250 255

Ala Leu Gly Ser Cys

260

<210> 13

<211> 285

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Met Ala Arg Met Ser Thr Gly Arg Thr Gly Thr Gly Arg Ala Glu Pro

1 5 10 15

Glu Arg Arg Arg Ala His Pro Ala Met Glu Val Gly Thr Met Glu Asp

20 25 30

Lys Val Ala Leu Val Thr Gly Ala Ala Gly Gly Ile Gly Ala Ala Val

35 40 45

Ala Arg Ala Leu Ala Arg Arg Gly Ala Arg Val Ala Ala Val Asp Leu

50 55 60

His Thr Gly Arg Leu Thr Glu Glu Val Gly Lys Leu Thr Ala Asp Gly

65 70 75 80

Leu Ala Val Glu Ala Phe Pro Ala Asp Val Thr Arg Ala Ala Ala Val

85 90 95

Glu Glu Leu Val Glu Gly Val Glu Thr Arg Leu Gly Pro Val Asp Leu

100 105 110

Leu Val Asn Ala Ala Gly Val Leu Arg Leu Gly Pro Val His Arg Leu

115 120 125

Gly Asp Glu Asp Trp Ala Ala Thr Phe Ala Val Asn Thr Thr Gly Val

130 135 140

Phe Leu Val Ser Arg Ala Val Ala Gly Arg Met Met Pro Arg Ser Arg

145 150 155 160

Gly Ala Ile Val Thr Val Ala Ser Asn Ala Ala Gly Thr Pro Arg Thr

165 170 175

Glu Met Ala Ala Tyr Ala Ala Ser Lys Ala Ala Ala Thr Met Phe Thr

180 185 190

Lys Cys Leu Ala Leu Glu Val Ala Gly His Gly Ile Arg Cys Asn Val

195 200 205

Val Ala Pro Gly Ser Thr Asp Thr Ala Met Leu Arg Ser Met Trp Gln

210 215 220

Asp Glu Ser Gly Ala Arg Ala Thr Ile Glu Gly Arg Pro Glu Ala Tyr

225 230 235 240

Lys Leu Gly Ile Pro Leu Gly Lys Leu Ala Arg Pro Ala Asp Ile Ala

245 250 255

Asp Ala Val Val Phe Leu Leu Ser Asp Arg Ala Gly His Ile Thr Met

260 265 270

His Glu Leu Thr Val Asp Gly Gly Ala Thr Leu Gly Ala

275 280 285

<210> 14

<211> 275

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Met Thr His Gly Pro Asp Gln Asp Gly Leu Gly Leu Glu Gly Arg Val

1 5 10 15

Ala Val Val Thr Gly Ala Ala Gly Gly Ile Gly Ala Glu Thr Val Ala

20 25 30

Gln Leu Val Arg His Arg Ala Ala Val Ala Leu Val Asp Arg Asp Gly

35 40 45

Pro Ala Leu Asp Glu Leu Val Ala Arg Leu Arg Lys Glu Ala Ala Ala

50 55 60

Asp Pro Arg Ala Pro Glu Pro Arg Leu Leu Ala Val Pro Ala Asp Val

65 70 75 80

Ser His Gly Gly Glu Val Thr Ala Val Val Glu Arg Val Glu Ser Glu

85 90 95

Leu Gly Pro Ile Thr His Leu Val Asn Ala Ala Gly Ile Leu Arg Pro

100 105 110

Gly Pro Val Ala Gly Leu Ser Glu Arg Asp Trp Asp Asp Thr Leu Ala

115 120 125

Val Asn Ala Thr Gly Val Tyr Leu Met Ser Arg Ala Val Ala Ser Arg

130 135 140

Met Ala Pro Arg Gly Phe Gly Ala Ile Val Thr Val Thr Ser Asn Ala

145 150 155 160

Ala Arg Thr Ala Arg Thr Gly Met Ala Ala Tyr Ala Ala Ser Lys Ala

165 170 175

Ala Ala Gln Ala Phe Thr Lys Cys Leu Gly Leu Glu Leu Ala Gly Thr

180 185 190

Gly Ile Arg Cys Asn Val Val Ala Pro Gly Ser Thr Asp Thr Pro Met

195 200 205

Leu Thr Ser Leu Trp Glu Gly Glu Asp Ala Gly Arg Pro Ser Ile Glu

210 215 220

Gly Ala Pro Glu Ala Tyr Arg Val Gly Ile Pro Thr Gly Arg Leu Ala

225 230 235 240

Arg Pro Tyr Asp Val Ala Gln Ala Val Val Phe Leu Leu Ser Glu Arg

245 250 255

Ala Gly His Ile Thr Leu Gln Asp Leu Thr Val Asp Gly Gly Ala Thr

260 265 270

Leu Gly Val

275

<210> 15

<211> 256

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Met Lys Asp Thr Ile Ala Val Val Thr Gly Ala Ala Gly Gly Ile Gly

1 5 10 15

Ala Ala Val Ala Glu Ala Leu Ala Val Arg Gly Ala Ser Val Ala Leu

20 25 30

Leu Asp Arg Asp Ala Asp Arg Leu Ala Val Val Ala Lys Glu Leu Thr

35 40 45

Ser Ala Gly His Arg Ala Thr Ala Phe Pro Ala Asp Val Thr Ser Gly

50 55 60

Gln Asp Val Glu Ala Val Ile Glu Ala Val Glu Glu Arg Leu Gly Pro

65 70 75 80

Val Asp Arg Leu Val Asn Ala Ala Gly Val Leu Arg Thr Gly Pro Ala

85 90 95

His Glu Phe Ala Asp Glu Asp Trp Glu Ala Thr Phe Ala Val Asn Thr

100 105 110

Thr Gly Val Phe His Val Ser Arg Ala Val Val Arg Arg Met Arg Thr

115 120 125

Arg Arg Arg Gly Ala Leu Val Thr Ile Ala Ser Asn Ala Ala Gly Ser

130 135 140

Ala Arg Thr Glu Met Ala Ala Tyr Ala Ala Ser Lys Ala Ala Ala Ser

145 150 155 160

Met Phe Thr Lys Cys Leu Gly Leu Glu Asn Ala Ala Tyr Gly Ile Arg

165 170 175

Cys Asn Val Val Ala Pro Gly Ser Thr Asp Thr Pro Met Leu Thr Ala

180 185 190

Leu Trp Asp Asp Ala Ser Ala Ala Asp Ala Ser Ile Ala Gly Val Pro

195 200 205

Glu Ala Tyr Arg Val Gly Ile Pro Leu Ala Lys Leu Ala Arg Pro Lys

210 215 220

Asp Val Ala Asp Ala Val Leu Phe Leu Leu Ser Asp Gln Ala Ser His

225 230 235 240

Ile Thr Met His His Leu Thr Val Asp Gly Gly Ala Thr Leu Gly Ala

245 250 255

<210> 16

<211> 274

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Met Ser Asp Thr Leu Thr Thr Thr Gly Thr Thr Gly Thr Ala Ala Ala

1 5 10 15

Ala Gly Ala Glu Trp Ser Gly Ser Ala Val Val Thr Gly Ala Ala Gly

20 25 30

Gly Ile Gly Ala Glu Ile Ala Arg Ala Leu Ala Ala Ala Gly Val Pro

35 40 45

Val Ala Leu Leu Asp Arg Glu Pro Gly Ala Leu Arg Glu Leu Ala Ser

50 55 60

Glu Leu Ser Ala Ala Gly Gly Thr Val Leu Ala Val Ala Ala Asp Val

65 70 75 80

Thr Asp Ser Ala Asp Val Asp Ala Ala Val Ala Lys Ala Glu Ala Glu

85 90 95

Leu Gly Pro Val Ala Tyr Leu Val Asn Gly Ala Gly Val Leu His Ser

100 105 110

Gly Pro Ala Gly Glu Phe Ser Asp Glu Asp Trp Asp His Thr Leu Ala

115 120 125

Val Asn Ala Gly Gly Val Phe Arg Val Ser Arg Ala Val Ala Arg Leu

130 135 140

Met Val Pro Arg Gly Arg Gly Ser Ile Val Thr Ile Ala Ser Asn Ala

145 150 155 160

Ala Leu Thr Pro Arg Thr Ser Met Ala Ala Tyr Ala Ala Ser Lys Ala

165 170 175

Ala Ser Ala Met Phe Thr Lys Cys Leu Gly Leu Glu Leu Gly Arg His

180 185 190

Gly Ile Arg Cys Asn Val Val Ala Pro Gly Ser Thr Arg Thr Ser Met

195 200 205

Leu Thr Ala Leu Gln Gly Asp Ala Ala Val Arg Ala Ser Val Asp Gly

210 215 220

Val Pro Asp Ala Tyr Arg Val Gly Ile Pro Leu Gly Arg Ile Ala Glu

225 230 235 240

Pro Ala His Ile Ala Asp Ala Val Leu Phe Leu Leu Ser Asp Arg Ser

245 250 255

Ala His Ile Thr Leu Gln Asp Leu Thr Val Asp Gly Gly Ala Ala Leu

260 265 270

Gly Val

<210> 17

<211> 207

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Met Ser Gly Ile Pro Glu Ile Thr Ala Tyr Pro Leu Pro Thr Ala Gln

1 5 10 15

Gln Leu Pro Ala Asn Leu Ala Arg Trp Ser Leu Glu Pro Arg Arg Ala

20 25 30

Val Leu Leu Val His Asp Met Gln Arg Tyr Phe Leu Arg Pro Leu Pro

35 40 45

Glu Ser Leu Arg Ala Gly Leu Val Ala Asn Ala Ala Arg Leu Arg Arg

50 55 60

Trp Cys Val Glu Gln Gly Val Gln Ile Ala Tyr Thr Ala Gln Pro Gly

65 70 75 80

Ser Met Thr Glu Glu Gln Arg Gly Leu Leu Lys Asp Phe Trp Gly Pro

85 90 95

Gly Met Arg Ala Ser Pro Ala Asp Arg Glu Val Val Glu Glu Leu Ala

100 105 110

Pro Gly Pro Asp Asp Trp Leu Leu Thr Lys Trp Arg Tyr Ser Ala Phe

115 120 125

Phe His Ser Asp Leu Leu Gln Arg Met Arg Ala Ala Gly Arg Asp Gln

130 135 140

Leu Val Leu Cys Gly Val Tyr Ala His Val Gly Val Leu Ile Ser Thr

145 150 155 160

Val Asp Ala Tyr Ser Asn Asp Ile Gln Pro Phe Leu Val Ala Asp Ala

165 170 175

Ile Ala Asp Phe Ser Glu Ala His His Arg Met Ala Leu Glu Tyr Ala

180 185 190

Ala Ser Arg Cys Ala Met Val Val Thr Thr Asp Glu Val Leu Glu

195 200 205

<210> 18

<211> 627

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Met Asn Ala Leu Pro Thr Ser Leu Leu Gln Arg Leu Leu Glu Arg Pro

1 5 10 15

Ala Pro Phe Ala Leu Leu Tyr Arg Pro Glu Ser Asn Gly Pro Gly Leu

20 25 30

Leu Asp Val Ile Arg Gly Glu Ala Leu Glu Leu His Gly Leu Ala Asp

35 40 45

Leu Pro Leu Asp Glu Pro Gly Pro Gly Leu Pro Arg His Asp Leu Leu

50 55 60

Ala Leu Ile Pro Tyr Arg Gln Ile Ala Glu Arg Gly Phe Glu Ala Leu

65 70 75 80

Asp Asp Gly Thr Pro Leu Leu Ala Leu Lys Val Leu Glu Gln Glu Leu

85 90 95

Leu Pro Leu Glu Gln Ala Leu Ala Leu Leu Pro Asn Gln Ala Leu Glu

100 105 110

Leu Ser Glu Glu Gly Phe Asp Leu Asp Asp Glu Ala Tyr Ala Glu Val

115 120 125

Val Gly Arg Val Ile Ala Asp Glu Ile Gly Arg Gly Glu Gly Ala Asn

130 135 140

Phe Val Ile Lys Arg Arg Phe Gln Ala Arg Ile Asp Gly Tyr Ala Thr

145 150 155 160

Ala Ser Ala Leu Ser Phe Phe Arg Gln Leu Leu Leu Arg Glu Lys Gly

165 170 175

Ala Tyr Trp Thr Phe Ile Val His Thr Gly Glu Arg Thr Leu Val Gly

180 185 190

Ala Ser Pro Glu Arg His Ile Ser Val Arg Asp Gly Leu Ala Val Met

195 200 205

Asn Pro Ile Ser Gly Thr Tyr Arg Tyr Pro Pro Ala Gly Pro Asn Leu

210 215 220

Ala Glu Val Met Glu Phe Leu Asp Asn Arg Lys Glu Ala Asp Glu Leu

225 230 235 240

Tyr Met Val Val Asp Glu Glu Leu Lys Met Met Ala Arg Ile Cys Glu

245 250 255

Asp Gly Gly Arg Val Leu Gly Pro Tyr Leu Lys Glu Met Ala His Leu

260 265 270

Ala His Thr Glu Tyr Phe Ile Glu Gly Gln Thr Ser Arg Asp Val Arg

275 280 285

Glu Val Leu Arg Glu Thr Leu Phe Ala Pro Thr Val Thr Gly Ser Pro

290 295 300

Leu Glu Ser Ala Cys Arg Val Ile Arg Arg Tyr Glu Pro Gln Gly Arg

305 310 315 320

Gly Tyr Tyr Ser Gly Val Ala Ala Leu Ile Gly Gly Asp Gly Gln Gly

325 330 335

Gly Arg Thr Leu Asp Ser Ala Ile Leu Ile Arg Thr Ala Glu Ile Glu

340 345 350

Gly Asp Gly Arg Leu Arg Ile Gly Val Gly Ser Thr Ile Val Arg His

355 360 365

Ser Asp Pro Leu Gly Glu Ala Ala Glu Ser Arg Ala Lys Ala Ser Gly

370 375 380

Leu Ile Ala Ala Leu Lys Ser Gln Ala Pro Gln Arg Leu Gly Ser His

385 390 395 400

Pro His Val Val Ala Ala Leu Ala Ser Arg Asn Ala Pro Ile Ala Asp

405 410 415

Phe Trp Leu Arg Gly Ala Ser Glu Arg Gln Gln Leu Gln Ala Asp Leu

420 425 430

Ser Gly Arg Glu Val Leu Ile Val Asp Ala Glu Asp Thr Phe Thr Ser

435 440 445

Met Ile Ala Lys Gln Leu Lys Ser Leu Gly Leu Thr Val Thr Val Arg

450 455 460

Gly Phe Gln Glu Pro Tyr Ser Phe Asp Gly Tyr Asp Leu Val Ile Met

465 470 475 480

Gly Pro Gly Pro Gly Asn Pro Thr Glu Ile Gly Gln Pro Lys Ile Gly

485 490 495

His Leu His Leu Ala Ile Arg Ser Leu Leu Ser Glu Arg Arg Pro Phe

500 505 510

Leu Ala Val Cys Leu Ser His Gln Val Leu Ser Leu Cys Leu Gly Leu

515 520 525

Asp Leu Gln Arg Arg Gln Glu Pro Asn Gln Gly Val Gln Lys Gln Ile

530 535 540

Asp Leu Phe Gly Ala Ala Glu Arg Val Gly Phe Tyr Asn Thr Phe Ala

545 550 555 560

Ala Arg Ala Leu Gln Asp Arg Ile Glu Ile Pro Glu Val Gly Pro Ile

565 570 575

Glu Ile Ser Arg Asp Arg Glu Thr Gly Glu Val His Ala Leu Arg Gly

580 585 590

Pro Arg Phe Ala Ser Met Gln Phe His Pro Glu Ser Val Leu Thr Arg

595 600 605

Glu Gly Pro Arg Ile Ile Ala Asp Leu Leu Arg His Ala Leu Val Glu

610 615 620

Arg Arg Pro

625

<210> 19

<211> 185

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Met Met Phe Thr Phe Gly Lys Pro Leu Asn Phe Gln Arg Trp Leu Asp

1 5 10 15

Asp His Ser Asp Leu Leu Arg Pro Pro Val Gly Asn Gln Gln Val Trp

20 25 30

Gln Asp Ser Asp Phe Ile Val Thr Val Val Gly Gly Pro Asn Phe Arg

35 40 45

Thr Asp Phe His Asp Asp Pro Met Glu Glu Phe Phe Tyr Gln Phe Lys

50 55 60

Gly Asn Ala Tyr Leu Asn Ile Met Asp Arg Gly Gln Met Asp Arg Val

65 70 75 80

Glu Leu Lys Glu Gly Asp Ile Phe Leu Leu Pro Pro His Leu Arg His

85 90 95

Ser Pro Gln Arg Pro Glu Ala Gly Ser Arg Cys Leu Val Ile Glu Arg

100 105 110

Gln Arg Pro Lys Gly Met Leu Asp Gly Phe Glu Trp Tyr Cys Leu Ser

115 120 125

Cys Asn Gly Leu Val Tyr Arg Val Asp Val Gln Leu Asn Ser Ile Val

130 135 140

Thr Asp Leu Pro Pro Leu Phe Asp Ile Phe Tyr Gly Asn Val Gly Leu

145 150 155 160

Arg Lys Cys Pro Gln Cys Gly Gln Val His Pro Gly Lys Ala Ala Ile

165 170 175

Glu Ala Val Ala Arg Gly Asp Gln Pro

180 185

Claims (8)

1. A method for synthesizing nicotinamide adenine dinucleotide comprises the following steps:

(a) preparing quinolinic acid;

(b) reacting quinolinic acid with ribose pyrophosphate under the action of quinolinic acid phosphotransferase to generate nicotinic acid mononucleotide;

(c) completing the preparation from nicotinic acid mononucleotide to amide adenine dinucleotide by utilizing a salvage synthesis pathway of nicotinamide adenine dinucleotide;

the preparation method of the quinolinic acid comprises the following steps:

(1) using branched acid as an initiator to generate amino deoxy isochorismate through transamination rearrangement reaction;

(2) removing the pyruvic acid part by amino-deoxy isochorismate to generate 2, 3-dihydro-3-hydroxy-anthranilic acid;

(3) dehydrogenating 2, 3-dihydro-3-hydroxy anthranilic acid to produce 3-hydroxy anthranilic acid;

(4) carrying out an oxidative ring-opening rearrangement reaction on the 3-hydroxy anthranilic acid to generate quinolinic acid;

in the step (1), 2-amino-4-deoxychorismate synthase is adopted to catalyze the transamination rearrangement of chorismate to generate amino-deoxyisochorismate;

in the step (2), 2, 3-dihydro-3-hydroxy anthranilate synthase is adopted to catalyze the amino-deoxy isochorismate to remove the pyruvic acid part to generate 2, 3-dihydro-3-hydroxy anthranilic acid;

in the step (3), DHHA-2, 3-dehydrogenase is adopted to catalyze 2, 3-dihydro-3-hydroxy anthranilic acid to dehydrogenate to generate 3-hydroxy anthranilic acid;

in the step (4), 3-hydroxy anthranilic acid-3, 4-dioxygenase is adopted to catalyze the oxidation ring-opening rearrangement reaction of 3-hydroxy anthranilic acid to generate quinolinic acid.

2. A synthetic method of nicotinamide adenine dinucleotide phosphate comprises the following steps:

(I) nicotinamide adenine dinucleotide prepared according to the method of claim 1;

(II) reacting nicotinamide adenine dinucleotide with ATP under the catalysis of nicotinamide adenine dinucleotide kinase to generate nicotinamide adenine dinucleotide phosphate.

3. A method for synthesizing quinolinic acid, comprising the steps of:

(1) using branched acid as an initiator to generate amino deoxy isochorismate through transamination rearrangement reaction;

(2) removing the pyruvic acid part by amino-deoxy isochorismate to generate 2, 3-dihydro-3-hydroxy-anthranilic acid;

(3) dehydrogenating 2, 3-dihydro-3-hydroxy anthranilic acid to produce 3-hydroxy anthranilic acid;

(4) carrying out an oxidative ring-opening rearrangement reaction on the 3-hydroxy anthranilic acid to generate quinolinic acid;

in the step (1), 2-amino-4-deoxychorismate synthase is adopted to catalyze the transamination rearrangement of chorismate to generate amino-deoxyisochorismate;

in the step (2), 2, 3-dihydro-3-hydroxy anthranilate synthase is adopted to catalyze the amino-deoxy isochorismate to remove the pyruvic acid part to generate 2, 3-dihydro-3-hydroxy anthranilic acid;

in the step (3), DHHA-2, 3-dehydrogenase is adopted to catalyze 2, 3-dihydro-3-hydroxy anthranilic acid to dehydrogenate to generate 3-hydroxy anthranilic acid;

in the step (4), 3-hydroxy anthranilic acid-3, 4-dioxygenase is adopted to catalyze the oxidation ring-opening rearrangement reaction of 3-hydroxy anthranilic acid to generate quinolinic acid.

4. A method according to any of claims 1-3, characterized by: the DHHA-2, 3-dehydrogenase is dehydrogenase A or dehydrogenase B or dehydrogenase C or dehydrogenase D or dehydrogenase E or dehydrogenase F or dehydrogenase G;

the dehydrogenase A is a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

the dehydrogenase B is a protein consisting of an amino acid sequence shown as a sequence 11 in a sequence table;

the dehydrogenase C is a protein consisting of an amino acid sequence shown as a sequence 12 in a sequence table;

the dehydrogenase D is a protein consisting of an amino acid sequence shown as a sequence 13 in a sequence table;

the dehydrogenase E is a protein consisting of an amino acid sequence shown as a sequence 14 in a sequence table;

the dehydrogenase F is a protein consisting of an amino acid sequence shown as a sequence 15 in a sequence table;

the dehydrogenase G is a protein consisting of an amino acid sequence shown as a sequence 16 in a sequence table.

5. Use of a 2-amino-4-deoxychorismate synthase, a2, 3-dihydro-3-hydroxyanthranilate synthase, a DHHA-2, 3-dehydrogenase and a 3-hydroxyanthranilic acid-3, 4-dioxygenase as claimed in claim 1 in the synthesis of nicotinamide adenine dinucleotide.

6. A method for synthesizing nicotinamide adenine dinucleotide compound is method A or method B;

the method A comprises the following steps: constructing a starting organism into a recombinant organism capable of carrying out the method according to claim 3, and synthesizing a nicotinamide adenine dinucleotide compound by using the recombinant organism; the recombinant organism is capable of performing the following reactions:

(b1) reacting quinolinic acid with ribose pyrophosphate to generate nicotinic acid mononucleotide;

(b2) salvage synthesis of nicotinamide adenine dinucleotide;

(b3) reacting nicotinamide adenine dinucleotide with ATP to generate nicotinamide adenine dinucleotide phosphate;

the method B comprises the following steps: introducing a coding gene of 2-amino-4-deoxychorismate synthase, a coding gene of 2, 3-dihydro-3-hydroxy anthranilate synthase, a coding gene of DHHA-2, 3-dehydrogenase and a coding gene of 3-hydroxy anthranilate-3, 4-dioxygenase into a starting organism to obtain a recombinant organism, and synthesizing to obtain a nicotinamide adenine dinucleotide compound by using the recombinant organism; the recombinant organism is capable of performing the following reactions:

(b1) reacting quinolinic acid with ribose pyrophosphate to generate nicotinic acid mononucleotide;

(b2) salvage synthesis of nicotinamide adenine dinucleotide;

(b3) reacting nicotinamide adenine dinucleotide with ATP to generate nicotinamide adenine dinucleotide phosphate;

the organism is a prokaryote, a plant or a fungus.

7. A recombinant organism capable of carrying out the synthetic pathway of claim 3 and capable of performing the following reaction:

(b1) reacting quinolinic acid with ribose pyrophosphate to generate nicotinic acid mononucleotide;

(b2) salvage synthesis of nicotinamide adenine dinucleotide;

(b3) reacting nicotinamide adenine dinucleotide with ATP to generate nicotinamide adenine dinucleotide phosphate;

the organism is a prokaryote, a plant or a fungus.

8. Use of the recombinant organism of claim 7 for the synthesis of nicotinamide adenine dinucleotide.

Images