CN108359666B

CN108359666B - A kind ofnudCGene and application thereof in preparation of nicotinic acid

Info

Publication number: CN108359666B
Application number: CN201810023635.1A
Authority: CN
Inventors: 王绪德; 周亚凤; 刘雪宾
Original assignee: Shanghai Gene Optimal Biotechnology Co ltd
Current assignee: Shanghai Gene Optimal Biotechnology Co ltd
Priority date: 2018-01-10
Filing date: 2018-01-10
Publication date: 2021-04-20
Anticipated expiration: 2038-01-10
Also published as: CN108359666A

Abstract

The invention discloses anudCGene and application thereof in the aspect of preparing nicotinic acid. The above-mentionednudCThe nucleotide sequence of the gene is shown in SEQ ID No.1, and the amino acid sequence of the encoded protein nudC protein is shown in SEQ ID No. 2. Discovered by the inventionnudCThe genetically constructed engineering bacteria can directly obtain the nicotinic acid in one step, so that various defects in the existing chemical synthesis process of the nicotinic acid are avoided, the production condition is simple, pollution is avoided, the method is simple, convenient and easy to implement, easy to amplify and low in cost, and the method is suitable for large-scale industrial production and application and has great popularization value.

Description

A kind ofnudCGene and application thereof in preparation of nicotinic acid

Technical Field

The invention relates to the technical field of synthesis of nicotinic acid, and more specifically relates to a nicotinic acid synthesis methodnudCGene and application thereof in the aspect of preparing nicotinic acid.

Background

Nicotinic acid, 3-picolinic acid, also known as vitamin B3, belongs to the B vitamin family, is a water-soluble vitamin, and is one of the 13 vitamins essential to the human body. Nicotinic acid is an important factor for resisting a hypecopathy and a hydrogen carrier in body tissues, and has the effects of maintaining the health of skin and nerves and promoting digestion. In the absence of it, pellagra may occur, manifested as dermatitis, glossitis, oropharynx, diarrhea, dysphoria, insomnia, and paresthesia. Nicotinamide, collectively known as vitamin PP, is used for combating pellagra and also as a vasodilator. As a medical intermediate, is used for producing isoniazid, nicotinamide, nicousamide, inositol nicotinate and the like. Nicotinic acid is also used in food products, meat additives and feed additives to prevent pellagra. Especially, the feed added with nicotinic acid can improve the disease resistance of livestock and poultry, accelerate the growth, improve the utilization rate of the feed, save a large amount of feed and reduce the feeding cost. Compared with the control group, the milk yield of the dairy cows fed by the feed added with the nicotinic acid in the United states can be improved by 15 to 20 percent. In addition, nicotinic acid can also be used as a biochemical hormone for forming activated sludge, and a deodorant for air and exhaust gas. Nicotinic acid also has certain application in the dye industry, the photosensitive material industry, hair dyeing auxiliaries, detergents and the like.

At present, the chemical synthesis method is mainly used for producing the nicotinic acid industrially, the synthesis method mainly comprises a liquid phase method (a potassium permanganate oxidation method and a nitric acid oxidation method) and a gas phase method (an ozone oxidation method, an ammonia oxidation method and an air oxidation method), and the used raw materials mainly comprise 3-methylpyridine, 2-methyl-5-ethylpyridine, 3-cyanopyridine, aldine and the like. The most common method at present is to synthesize nicotinic acid by air oxidation method by using 3-methylpyridine as a raw material. 3-methylpyridine, air and ammonia gas are proportionally introduced into a fluidized bed reactor, and the temperature is 290-360 ℃ and V₂O₅Reacting under catalysis to generate nicotinonitrile; hydrolyzing in sodium hydroxide water solution at 160 deg.C under high pressure to obtain sodium nicotinate, and acidifying with hydrochloric acid to obtain nicotinic acid. In the synthesis process, the intermediate product nicotinonitrile is needed, the conditions of high temperature, high pressure, strong acid and strong alkali are needed for generating the nicotinic acid by the nicotinonitrile through chemical reaction, certain requirements on equipment are met, the equipment can be corroded, and the synthetic method is not environment-friendly. Patent CN114288A of qinghua violet corporation reports that 3-methylpyridine is used to generate nicotinic acid in one step, the process is to react 3-methylpyridine with sulfuric acid to generate methylpyridine sulfate, then to generate nicotinic acid sulfate by oxidation under the action of nitric acid as oxidant, the nicotinic acid sulfate is added into alkali solution to obtain nicotinic acid, and the yield of raw material is about 90%. In the process, strong acid and strong alkali substances such as sulfuric acid, nitric acid, alkali solution and the like are needed, and the pollution is serious.

In the chemical synthesis process of the nicotinic acid, a specific high-temperature and high-pressure environment is required or strong acid, strong base or a chemical catalyst is adopted for treatment, the reaction selectivity is not high, a large number of byproducts are generated, the product yield is not high, and the environmental pollution is large. In contrast, the biological method for preparing nicotinic acid has the characteristics of high substrate selectivity, high catalytic efficiency, mild reaction conditions, small environmental pollution and the like. In addition, the biological method is easy to amplify, has low cost and is suitable for large-scale industrial production and application. The biocatalytic production of nicotinic acid from 3-cyanopyridine has been reported using the microorganisms Bacillus subtilis, Rhodococcus rhodochrous, Nocardia, Fusarium solani and Pseudomonas putida for fermentation. However, the biocatalytic method for preparing nicotinic acid mainly relies on microbial fermentation to produce nitrilase, and the nitrilase catalyzes the conversion of 3-cyanopyridine into nicotinic acid by the nitrilase, and the raw material 3-cyanopyridine still needs to be added.

Disclosure of Invention

The invention aims to overcome the defects of the prior nicotinic acid preparation technology and provides a method for preparing nicotinic acidnudCThe new application of the gene, and the engineering bacteria constructed by the gene can directly produce the nicotinic acid.

The first purpose of the invention is to provide anudCA gene.

It is a second object of the present invention to provide a nucc protein.

It is a third object of the present invention to provide a composition comprisingnudCRecombinant vectors of genes.

It is a fourth object of the present invention to provide a container comprisingnudCGenetically engineered bacterium

It is a fifth object of the present invention to providenudCApplication of the gene in preparation of nicotinic acid.

The sixth purpose of the invention is to provide the application of the nucC protein in the preparation of nicotinic acid.

The seventh purpose of the invention is to provide the application of the recombinant vector in the preparation of nicotinic acid.

The eighth purpose of the invention is to provide the application of the recombinant engineering bacteria in the preparation of nicotinic acid.

An eighth object of the present invention is to provide a method for producing nicotinic acid.

In order to achieve the purpose, the invention is realized by the following technical scheme:

the present invention finds overexpressionnudCNicotinic acid could be detected separately from the culture filtrate of the gene strain, indicating thatnudCThe gene can be used for preparing nicotinic acid. Therefore, the following schemes are all within the protection scope of the invention:

a kind ofnudCThe nucleotide sequence of the gene is shown in SEQ ID No. 1.

A nudC protein, the amino acid sequence of which is shown in SEQ.ID.NO. 2.

A recombinant vector having said vector linked theretonudCA gene.

Preferably, the vector is pMV361 or pMV 261.

A recombinant engineering bacterium is obtained by recombining a sequence shown in SEQ.ID.NO.1 into a receptor bacterium, and also belongs to the protection scope of the invention.

The recipient bacterium is mycobacterium smegmatis, and the receptor bacterium can be normally expressed after recombining a sequence shown in SEQ.ID.NO. 1.

SaidnudCThe application of the gene in the preparation of nicotinic acid also belongs to the protection scope of the invention.

The application of the nucC protein in the preparation of nicotinic acid also belongs to the protection scope of the invention.

The application of the recombinant vector in the preparation of the nicotinic acid also belongs to the protection scope of the invention.

The application of the recombinant engineering bacteria in the preparation of the nicotinic acid also belongs to the protection scope of the invention.

A method for producing nicotinic acid comprises culturing the recombinant engineering bacteria, and collecting purified nicotinic acid from the culture solution.

Compared with the prior art, the invention has the following beneficial effects:

the invention discoversnudCThe new application of the gene, the recombinant plasmid and the engineering bacterium constructed by the gene can directly obtain the nicotinic acid in one step without depending on the addition of 3-cyanopyridine, not only avoids various defects in the chemical synthesis process of the prior nicotinic acid, but also has the advantages of simple production condition, no pollution, simplicity, convenience, easy implementation, easy amplification and low cost, and is suitable for large-scale industrial production and application.

Drawings

FIG. 1 shows the nucleic acid gel electrophoresis of pMV361-NudCms plasmid after restriction with EcoRI and HindIII restriction enzymes, M: DNA marker; 1-3: and (3) positive plasmids.

FIG. 2 is a schematic diagram of the pMV361-NudCms plasmid

FIG. 3 shows the nucleic acid gel electrophoresis of the PCR amplification product; m: DNA marker; 1-3: positive clones.

FIG. 4 is a HPLC separation chromatogram of control strain, over-expression strain culture solution filtrate and nicotinic acid standard; a: control Strain Mycobacterium smegmatis mc²155 culture solution filtrate; b: mycobacterium smegmatis mc overexpressing the NudC gene²155-pMV261-NudC_msA culture solution filtrate; c: and (4) a nicotinic acid standard substance.

FIG. 5 shows pMV261-NudC_msThe plasmid was digested with BamHI and HindIII restriction enzymes followed by nucleic acid gel electrophoresis, M: DNA marker; 1-3: and (3) positive plasmids.

FIG. 6 is pMV261-NudC_msSchematic representation of the plasmid.

FIG. 7 shows nucleic acid gel electrophoresis of PCR amplification products; m: DNA marker; 1-3: positive clones.

FIG. 8 is a HPLC separation chromatogram of control strain, over-expression strain culture solution filtrate and nicotinic acid standard; a is control strain Mycobacterium smegmatis mc²155 culture solution filtrate; b: mycobacterium smegmatis mc overexpressing the NudC gene²155-pMV261-NudC_msA culture solution filtrate; c: and (4) a nicotinic acid standard substance.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1nudCGene amplification and construction of plasmid pMV361-NudC_ms

1、nudCAmplification of genes

Using Mycobacterium smegmatis mc²155 genomic DNA as template, amplified using primer pair 5'-ATCGGAATTCATGAGCGAACACCGCACGT-3'/5'-TGCAAAGCTTTCAGTCGAGTGCGGCCCAGG-3', PCRnudCGene ORF sequence. Separating target DNA fragment from PCR product by nucleic acid gel electrophoresis, recovering target DNA fragment with Omega gel recovery kit, and sequencing to obtainnudCThe gene ORF sequence is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2. the recovered DNAs were digested with EcoRI and HindIII restriction enzymes and recovered by Omega's Cycle-pure recovery kit for use.

2. Enzyme digestion

The Escherichia coli DH5 alpha strain containing pMV361 plasmid was cultured in LB, the plasmid was extracted using Omega plasmid extraction kit, the pMV361 plasmid was digested with EcoRI and Hind III restriction enzymes, nucleic acid gel electrophoresis was performed, and the linearized plasmid was recovered using Omega gel recovery kit.

3. Connection of

The DNA fragment recovered by the digestion and the linearized plasmid pMV361 recovered by the same digestion were reacted with T4 DNA ligase at 16 ℃ overnight, the ligation product was transformed into E.coli DH 5. alpha. competent cells and spread on LB solid plate (containing 100. mu.g/ml kanamycin sulfate), and the plate was incubated in a 37 ℃ incubator overnight.

4. Screening for Positive plasmids

The monoclonal colonies growing on the plates were picked and inoculated into LB liquid medium and cultured overnight with shaking in a shaker at 200rpm at 37 ℃. Then extracting the plasmid by using a plasmid extraction kit of Omega company, carrying out nucleic acid gel electrophoresis detection and verification on the obtained plasmid after the plasmid is cut by using EcoRI and Hind III restriction enzymes,nudCthe gene ORF sequence was ligated into the pMV361 vector (see FIG. 1). Verification of the correct plasmid pMV361-NudC at the same time_msThe sequencing of the Scenario corporation verifies that the sequence has no mutation. Successfully constructed pMV361-NudC_msThe plasmid map is schematically shown in FIG. 2.

Example 2 construction of NudC overexpressing Mycobacterium smegmatis strains

1. Preparation of Mycobacterium smegmatis mc²155 competent cell

Preparation of Mycobacterium smegmatis mc²155 competent cells, picking fresh M.smegmatis mc²155 single colonies were inoculated in 5ml of 7H9 liquid medium, and cultured at 37 ℃ with shaking at 200rpm to logarithmic growth phase (OD 0.5-1.0); inoculating the culture into fresh 100 ml 7H9 liquid culture medium at a ratio of 1:100, culturing at 37 deg.C overnight to OD600 to about 0.6, centrifuging at 4 deg.C and 5000 rpm for 10 min to collect thallus, washing thallus with precooled 10% sterile glycerol at least twice, adding 10 ml (appropriate amount) precooled 10% glycerol, blowing to homogenize thallus, and freezing at-80 deg.C.

2. Transformation of Mycobacterium smegmatis mc²155

Taking a positive plasmid pMV361-NudC with correct construction_msAdding 200 mu l of Mycobacterium smegmatis mc into the DNA²155 in the electric transfer competent cells, incubating for 10 min on ice, then transferring into a 2 mm BTX electric transfer cup, wiping off water on the outer wall of the electric transfer cup, and then using a BTX ECM630 electric converter to shock, wherein the shock parameters are as follows: the voltage is 2.5 kV, the resistance is 1000 Ω, and the capacitance is 25 μ F. After electric shock, 1 ml of 7H9 liquid medium was immediately added, incubated overnight in an incubator at 37 ℃ and then an appropriate amount of the culture was spread on a 7H10 solid plate (containing 50. mu.g/ml kanamycin sulfate), and the plate was incubated in an incubator at 37 ℃ for 3 to 5 days.

3. Screening positive recombinant bacteria

The monoclonal grown on the plate was picked, inoculated into 5mL of 7H9 liquid medium and cultured for 2-3 days to logarithmic phase, the cells were collected by centrifugation and the genomic DNA was extracted using the microbial genome extraction kit (Omega), and PCR was performed using primer set 5'-GTGGCAGCGAGGACAACTTG-3', 5'-GATGCCTGGCAGTCGATCGTAC-3' to verify pMV361-NudC using the extracted genomic DNA as a template_msThe plasmid was transferred into M.smegmatis (PCR results are shown in FIG. 3). Will verify the correct NudC_msThe overexpressed strain was named Mycobacterium smegmatis mc²155-pMV361-NudC_ms。

Example 3 Mycobacterium smegmatis mc²155-pMV361-NudC_msSecretion of nicotinic acid

1. M. smegmatis mc²155-pMV361-NudC_msAfter inoculating 7H9 liquid medium and culturing at 37 ℃ to a logarithmic growth phase, the culture solution was filtered using a Millipore 0.22 μm sterile filter and the filtered filtrate was collected, and then macromolecules such as proteins in the filtrate were removed using a Millipore 3-kDa ultrafiltration concentration tube and the filtrate after filtration was collected. The obtained filtrate was separated and analyzed for nicotinic acid using High Performance Liquid Chromatography (HPLC).

Specific conditions of HPLC:

the equipment model is as follows: thermo Fisher Scientific UltiMate 3000 ultra-high-performance chromatography

A chromatographic column: thermo Fisher, Hypersil Gold aQ, 150X 4.6 mm, 3 μm, column temperature: 30 ℃;

mobile phase: water phase: deionized water (containing 0.1% formic acid);

organic phase: methanol;

gradient elution: at 0 min, 95% aqueous phase +5% organic phase;

at 30 min, 5% aqueous phase +95% organic phase;

flow rate: 0.3 mL/min;

sample introduction amount: 10 mu L of the solution;

wavelength: 284 nm.

2. HPLC results show M.smegmatis mc²155-pMV361-NudC_msThe culture medium contains nicotinic acid (see FIG. 4).

EXAMPLE 4 construction of plasmid pMV261-NudC_ms

Plasmid pMV261-NudC was performed according to the method of example 1_msAnd (4) constructing. The vector was replaced with the pMV261 plasmid. The obtained plasmid is cut by using BamHI and HindIII restriction enzymes and verified by nucleic acid gel electrophoresis detection,nudCthe gene ORF sequence was ligated into the pMV261 vector (see FIG. 5). Verification of the correct plasmid pMV261-NudC_msThe sequencing of the Oncorhynchus company verifies that the sequence has no mutation, and successfully constructs the pMV261-NudC_msThe plasmid map is shown in FIG. 6.

Example 5 construction of NudC overexpressing Mycobacterium smegmatis strains

NudC over-expressing M.smegmatis strains were constructed according to the method of example 2, PCR-verified pMV261-NudC_msThe plasmid was transferred into M.smegmatis (PCR results are shown in FIG. 7). Will verify the correct NudC_msThe overexpressed strain was named Mycobacterium smegmatis mc²155-pMV261-NudC_ms。

Example 6 recombinant Mycobacterium smegmatis mc²155-pMV261-NudC_msSecretion of nicotinic acid

M. smegmatis mc²155-pMV261-NudC_msThe recombinant Mycobacterium smegmatis was cultured by inoculating 7H9 liquid medium according to the method of example 3 and nicotinic acid was isolated and analyzed by HPLC. HPLC results show M.smegmatis mc²155-pMV261-NudC_msThe culture medium contained nicotinic acid (see FIG. 8).

Sequence listing

<110> Shanghai Jingnuo Biotech Co., Ltd

<120> nucC gene and application thereof in preparation of nicotinic acid

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 936

<212> DNA

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400> 1

atgagcgaac accgcacgtt cgggctccgt aacgtcccgc tgctgtcccg ggtcggcgcc 60

gatcgcgccg ataccttgcg caccgacgtc gacgccgccc tggcgggctg gcccgacgcg 120

ctggtgctac gcgtggaccg ccgcaaccag gtgctcatcg ccaacggtca ggtggtgctc 180

ggtgaggccg gcgcactcgg agaccggccg cccgagcacg cggtgttcct gggacgtctg 240

caggacggca ggcacgtatg gggtatccgg gcggatctgg aggcgcccga ggatgccgac 300

ctggggaccg aggtgctcga cctgcgccgg gccgggcaga tcttcgacga caccagcgcc 360

cagttggtgg cgaccgccac ggcgctgctc aactggcatg acaacgcgcg gcacagcgcg 420

atcgacgggg cgccgacccg gcccgccaag ggcggctggt cgcgcgtcaa cccgctgacc 480

ggccacgagg agttcccgcg gatcgacccc gccatcatct gcctggtgca cgacgggcat 540

gaccgggcgg tgctggcccg tcagacgctg tggccggagc ggttgttctc gatcctggcc 600

ggcttcgtcg aggccggcga gtcgttcgag acatgcgtgc agcgcgagat cgccgaggag 660

atcgggctca cggtcaccga cgtgcagtac ctcggcagtc agccgtggcc gttcccgcgc 720

tcgctcatgg tgggattcca cgcgatcggc gacccggagc agccgttctc ctacaacgac 780

ggcgagatcg ccgaggccgc gtggttcacc cgcgatgaga tccgcgcggc actcgatcag 840

ggggactgga acagcgactc gccgtcacgg ctcctgctgc caggctccat ctcgatcgcc 900

cgcgagatca tcgaatcctg ggccgcactc gactga 936

<210> 2

<211> 311

<212> PRT

<213> Mycobacterium smegmatis (Mycobacterium smegmatis)

<400> 2

Met Ser Glu His Arg Thr Phe Gly Leu Arg Asn Val Pro Leu Leu Ser

1 5 10 15

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

20 25 30

Ala Leu Ala Gly Trp Pro Asp Ala Leu Val Leu Arg Val Asp Arg Arg

35 40 45

Asn Gln Val Leu Ile Ala Asn Gly Gln Val Val Leu Gly Glu Ala Gly

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Leu Asn Trp His Asp Asn Ala Arg His Ser Ala Ile Asp Gly Ala

130 135 140

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

145 150 155 160

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

165 170 175

His Asp Gly His Asp Arg Ala Val Leu Ala Arg Gln Thr Leu Trp Pro

180 185 190

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

195 200 205

Phe Glu Thr Cys Val Gln Arg Glu Ile Ala Glu Glu Ile Gly Leu Thr

210 215 220

Val Thr Asp Val Gln Tyr Leu Gly Ser Gln Pro Trp Pro Phe Pro Arg

225 230 235 240

Ser Leu Met Val Gly Phe His Ala Ile Gly Asp Pro Glu Gln Pro Phe

245 250 255

Ser Tyr Asn Asp Gly Glu Ile Ala Glu Ala Ala Trp Phe Thr Arg Asp

260 265 270

Glu Ile Arg Ala Ala Leu Asp Gln Gly Asp Trp Asn Ser Asp Ser Pro

275 280 285

Ser Arg Leu Leu Leu Pro Gly Ser Ile Ser Ile Ala Arg Glu Ile Ile

290 295 300

Glu Ser Trp Ala Ala Leu Asp

305 310

Claims

The application of the nucC gene in preparing nicotinic acid in mycobacterium smegmatis is characterized in that the nucleotide sequence of the nucC gene is shown as SEQ ID NO: 1 is shown.
The application of nucC protein in preparing nicotinic acid in mycobacterium smegmatis is characterized in that the amino acid sequence of the nucC protein is shown as SEQ ID NO: 2, respectively.
3. The application of a recombinant vector in preparing nicotinic acid in mycobacterium smegmatis is characterized in that a nucd gene is connected in the vector, and the nucleotide sequence of the nucd gene is shown as SEQ ID NO: 1 is shown.
4. The use according to claim 3, wherein said vector is pMV361 or pMV 261.
5. The application of the recombinant engineering bacteria in the preparation of the nicotinic acid is characterized in that the recombinant engineering bacteria is prepared by carrying out the following steps of: 1 into Mycobacterium smegmatis.
6. A method for producing nicotinic acid, which comprises culturing the recombinant engineered bacterium of claim 5 and collecting purified nicotinic acid from the culture medium.