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Biosynthesis method of nicotinamide adenine dinucleotide compound

A nicotinamide adenine and dinucleotide technology, applied in the field of biosynthesis of nicotinamide adenine dinucleotide compounds, can solve the problems of expensive consumption, limited concentration, high production cost, etc.

Active Publication Date: 2019-12-24
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] When organisms synthesize NAD compounds through the above two de novo synthesis pathways, they need to consume amino acids (tryptophan or aspartic acid) for protein synthesis, which limits the concentration of such compounds in organisms to a certain extent, leading to the current Production of such compounds by yeast fermentation is costly
In addition, the preparation of NAD by in vitro enzymatic conversion requires the purification of the corresponding protease and the consumption of expensive precursor ATP, and the production cost is also high.
The method of producing NAD by chemical total synthesis has not been reported yet

Method used

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Experimental program
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Effect test

Embodiment 1

[0159] Embodiment 1, establishment of synthetic route

[0160] Through a large number of investigations and studies, a new NAD synthesis route was established ( figure 1 ).

[0161] With chorismic acid as the starting point, aminodeoxyisochorismic acid (ADIC) is generated through transamination rearrangement under the action of PhzD protein, and aminodeoxyisochorismic acid is catalyzed by PhzE protein to remove the pyruvate part to generate 2,3-di Hydrogen-3-hydroxyanthranilic acid (DHHA), catalyzed by a specific enzyme (DHHA-2,3-dehydrogenase) from 2,3-dihydro-3-hydroxyanthranilic acid to 3-hydroxyanthranilic acid Formic acid (3-HAA), under the action of NabC protein, 3-hydroxyanthranilic acid undergoes an oxidative ring-opening rearrangement reaction to generate quinolinic acid (QA), and quinolinic acid in vivo is under the action of quinolinic acid phosphotransferase React with pyrophosphate ribose to generate nicotinic acid mononucleotide, and then enter the NAD salvage ...

Embodiment 2

[0165] Embodiment 2, the identification of DHHA-2,3-dehydrogenase

[0166] 1. Identification of Pau20

[0167] 1. An enzyme that may have DHHA-2,3-dehydrogenase activity was screened out from a large number of candidate enzymes, and it was named Pau20. Its coding gene is shown in sequence 1 of the sequence listing, and its protein sequence is shown in sequence listing Sequence 2 is shown.

[0168] 2. Preparation and purification of Pau20

[0169] (1) The small fragment between the NdeI and BamH I restriction sites of the pET28a plasmid (Novagen) is replaced by the double-stranded DNA molecule shown in Sequence 1 of the sequence listing to obtain the recombinant plasmid pET28a::pau20 (sequencing verification) .

[0170] (2) The recombinant plasmid pET28a::pau20 was introduced into Escherichia coli E. coli BL21(DE3) to obtain the recombinant bacterium BL21(DE3)::pET28a-pau20.

[0171] (3) Cultivate the recombinant bacteria BL21(DE3)::pET28a-pau20 in LB liquid medium containi...

Embodiment 3

[0202] Embodiment 3, the construction of NAD synthesis pathway plasmid

[0203] The gene sequences of phzD, phzE, nabC and pau20 were optimized according to the codon preference of the heterologous expression strain Escherichia coli, and then connected to the plasmid pXB1a (references: Cui Q, Zhou F, Liu W, et al.Avermectin biosynthesis:stable functional expression of branched chainα-keto acid dehydrogenase complex from Streptomyces avermitilis,in Escherichiacoli,by selectively regulating individual subunit gene expression[J].Biotechnology Letters,2017,39(10):1-8.; the public can get from China obtained from the Institute of Microbiology, Chinese Academy of Sciences) to obtain recombinant plasmids.

[0204] The double-stranded DNA molecule shown in Sequence 3 was used to replace the fragment between NcoI and EcoRI restriction sites of plasmid pXB1a to obtain plasmid pXB1a-HAA (sequenced and verified).

[0205] In sequence 3 of the sequence listing, the 1st to 786th from the 5...

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Abstract

The invention discloses a biosynthesis method of a nicotinamide adenine dinucleotide compound. A synthesis path provided by the invention takes chorismic acid as a starting point, under the action ofPhzD protein, a transamination rearrangement reaction is carried out to generate amino deoxy isobranched acid, pyruvic acid is removed from amino deoxy isobranched acid under the catalysis of the PhzEprotein to generate 2,3-dihydro-3-hydroxyanthranilicacid, 2,3-dihydro-3- hydroxyanthranilicacid is catalyzed to generate 3-hydroxyanthranilicacid by adopting a specific enzyme, quinolinic acid is generated from 3-hydroxyanthranilicacid through an oxidation ring-opening rearrangement reaction under the action of nabC protein, and then enters an NAD remediation way to finish NAD synthesis. According to the present invention, the coupling between the NAD synthesis and the tryptophan or aspartic acid synthesis is removed, and the chorismic acid widely exists in various life forms, and has less influence on other life essential metabolic pathways.

Description

technical field [0001] The invention relates to a biosynthetic method of nicotinamide adenine dinucleotide compounds. Background technique [0002] Nicotinamide adenine dinucleotide (NAD + , Coenzyme I) and its corresponding reduced form NADH are one of the essential coenzymes for all life activities, and they participate in redox reactions in various organisms as proton acceptors or donors. In addition, in non-redox life processes such as cell growth, differentiation, regulation and disease, NAD + It also plays a very important role, such as participating in the aging-related histone deacetylation reaction, the polyadenosine diphosphate-ribose reaction that plays an important role in the DNA repair process, and the adenosine diphosphate-ribose cyclization reaction that regulates calcium ion channels, etc. . In recent years, more and more studies have shown that the reduction of NAD in the body is an important reason for aging. In some fermentation industries and biotran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/36C12P17/12C12P13/04C12R1/19
CPCC12P13/04C12P17/12C12P19/36
Inventor 陈义华丁勇
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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