阅读说明：本技术 一种烟酰胺腺嘌呤二核苷酸类化合物生物合成方法 (Biosynthesis method of nicotinamide adenine dinucleotide compound ) 是由 陈义华 丁勇 于 2018-06-14 设计创作，主要内容包括：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 synthesis path provided by the invention takes chorismate as a starting point, amino-deoxy-isochorismate is generated through transamination rearrangement reaction under the action of PhzD protein, the amino-deoxy-isochorismate removes part of pyruvic acid under the catalysis of PhzE protein to generate 2, 3-dihydro-3-hydroxy-anthranilic acid, 2, 3-dihydro-3-hydroxy-anthranilic acid is catalyzed by specific enzyme to generate 3-hydroxy-anthranilic acid, the 3-hydroxy-anthranilic acid generates quinolinic acid through oxidation ring-opening rearrangement reaction under the action of nabC protein, and then the quinolinic acid enters NAD remediation pathway to complete the synthesis of NAD. The invention decouples NAD synthesis from tryptophan or aspartate synthesis, and meanwhile, the branched acid is widely present in various life forms and has small influence on other vital metabolic pathways.)

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) the preparation from the nicotinic acid mononucleotide to the amide adenine dinucleotide is completed by utilizing a salvage synthesis path of the 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) the 3-hydroxy anthranilic acid generates quinolinic acid through oxidation ring-opening rearrangement reaction.

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

4. A method according to any of claims 1 to 3, characterized by:

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.

5. The method of claim 4, wherein: 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 (a1) or (a2) as follows:

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

(a2) a protein which is derived from the sequence 2 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 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) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the sequence 11, has the same function and is derived from the sequence 11;

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

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

(a6) a protein which is derived from the sequence 12 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 12;

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) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the sequence 13, has the same function and is derived from the sequence 13;

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

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) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the sequence 15, has the same function and is derived from the sequence 15;

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.

6. Use of a DHHA-2, 3-dehydrogenase as claimed in claim 5 for catalyzing the dehydrogenation of 2, 3-dihydro-3-hydroxyanthranilic acid to 3-hydroxyanthranilic acid.

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

8. A method for synthesizing 3-hydroxy anthranilic acid comprises the following steps: use of the DHHA-2, 3-dehydrogenase of claim 5 to catalyze the dehydrogenation of 2, 3-dihydro-3-hydroxyanthranilic acid to 3-hydroxyanthranilic acid.

9. 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 realizing the synthetic pathway of 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 produce nicotinamide adenine dinucleotide phosphate.

10. 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;

or the like, or, alternatively,

the recombinant organism is applied to synthesizing nicotinamide adenine dinucleotide compounds.

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 essential for all life activitiesAs an acceptor or donor of protons, they participate in redox reactions in various organisms. 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 participation in histone deacetylation reaction related to aging, poly (adenosine diphosphate ribose) reaction playing an important role in DNA repair process, adenosine diphosphate ribose cyclization reaction regulating calcium ion channels and 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 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.

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 in sequence 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 in sequence 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 is NAD⁺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 a sequence 2 in a sequence table;

(a2) and (b) the protein which is derived from the sequence 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 the sequence 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 a sequence 12 in a sequence table;

(a6) protein derived from the sequence 12 by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence of the sequence 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 of sequence 3 in the sequence table;

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

(f2) DNA molecule shown in 1467 th to 3350 th of sequence 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 a sequence 1 in a sequence table;

(g2) a DNA molecule shown as a sequence 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 a sequence 5 in a sequence table;

(h2) a DNA molecule shown as a sequence 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 a sequence 10 in the sequence table;

(n2) the DNA molecule shown in the sequence 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.