CN114410664A - Tetrahydropyrimidine biosynthesis gene and application thereof - Google Patents
Tetrahydropyrimidine biosynthesis gene and application thereof Download PDFInfo
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- CN114410664A CN114410664A CN202111563038.6A CN202111563038A CN114410664A CN 114410664 A CN114410664 A CN 114410664A CN 202111563038 A CN202111563038 A CN 202111563038A CN 114410664 A CN114410664 A CN 114410664A
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- tetrahydropyrimidine
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- diaminobutyric acid
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- OTPDWCMLUKMQNO-UHFFFAOYSA-N 1,2,3,4-tetrahydropyrimidine Chemical compound C1NCC=CN1 OTPDWCMLUKMQNO-UHFFFAOYSA-N 0.000 title claims abstract description 53
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
A tetrahydropyrimidine biosynthesis gene and application thereof, relating to a tetrahydropyrimidine synthesis gene cluster. Ectoin synthesis gene ectABC is found in Halomonas secmenti QX-2 of Halomonas deeplyTThe nucleotide sequence is shown as 2244 base sequences of SEQ ID NO. 1. Comprises 3 genes which are different from the sequences of the currently known tetrahydropyrimidine synthesis gene cluster and belong to new gene sequences. Through cloning and expressing the strain by genetic engineering technology, the strain is found to have less difficulty in distinguishing after basic fermentationIsolated secondary metabolites and high yield of tetrahydropyrimidine. Meets the requirements of scientific research and application of tetrahydropyrimidine in the cosmetic industry as a sun-screening agent, a humectant and an antioxidant.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a deep sea waterHalomonas secmenti QX-2 of the sourceTA tetrahydropyrimidine-producing biosynthesis gene cluster and application thereof.
Background
Tetrahydropyrimidine (Ectoine), 1,4,5, 6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, was first found in the genus Ectothiorhodospira (Ectothiorhodiospira) and is therefore named. Tetrahydropyrimidines are cyclized amino acids, a class of compatible solutes that are widely present in bacteria, especially in the extreme microbial halophiles or halodurans. The extreme microorganisms form a unique environment adaptation mechanism due to the special living environment of the extreme microorganisms; therefore, a plurality of secondary metabolites are generated in order to adapt to the environment, the extreme high-salt environment has high environmental osmotic pressure, and cells are generally cracked and killed due to the difficulty in bearing the extreme osmotic pressure. The halophilic bacteria can generate substances such as secondary metabolite tetrahydropyrimidine and the like, and can maintain osmotic pressure and cell stability, so that halophilic microorganisms can survive in extremely high-concentration salt.
Tetrahydropyrimidine is a main osmotic pressure regulator in cells, has a certain stabilizing effect on macromolecules besides regulating the osmotic pressure of the cells, and has multiple biological functions, so that the tetrahydropyrimidine is one of biological engineering preparations adopted by high-grade cosmetics: 1) as a moisture binding agent, the tetrahydropyrimidine can keep moisture for a long time (7 days), and helps to moisten the skin and recombine skin cell membranes; 2) resisting oxidation (preventing aging, improving wrinkle), delaying skin premature aging, etc.; 3) the tetrahydropyrimidine can resist the damage of ultraviolet rays to the skin, repair the cell DNA damage caused by the ultraviolet rays, reduce sunburn of the skin caused by the ultraviolet irradiation and inhibit the generation of melanin; 4) stress resistance and protection, and can relieve skin aging caused by various stresses and dry environment, skin deterioration caused by surfactant, etc. The advantage of tetrahydropyrimidine in the field of cosmetics is that it is compatible with the metabolism in the cells and does not affect the biomacromolecule function or the physiological processes of the cells. It is not only an important osmotic pressure compensation solute, but also has good protection effect on cells and biological macromolecules under the stimulation of adverse environments such as high temperature, high salt, freezing, drying, radiation and the like. Therefore, the method plays an important role in gene protection and excavation of tetrahydropyrimidine.
Several studies have shown. The synthesis approach of tetrahydropyrimidine in the moderately halophilic bacteria takes aspartic Acid Semialdehyde (ASA) as a precursor, and the steps are totally divided into 3 steps; the first step is to synthesize L-Diaminobutyric acid (DABA) by catalysis of L-Diaminobutyric Acid Transaminase (DAT), the second step is to synthesize N-acetyl-L-2, 4-Diaminobutyric acid (ADABA) by catalysis of L-Diaminobutyric Acid Acetyltransferase (DAA), and the third step is to synthesize tetrahydropyrimidine by catalysis of tetrahydropyrimidine synthase (ES), and the synthetic genes of DAT, DAA and ES of related enzyme systems are respectively ectoB, ectoA and ectoC. Because tetrahydropyrimidine has a chiral molecule and is difficult to chemically synthesize, no research result indicates that ectoine is chemically synthesized, so that only biosynthesis can be carried out.
Disclosure of Invention
The first purpose of the invention is to provide a tetrahydropyrimidine biosynthesis gene cluster.
Another object of the present invention is to provide an application of the tetrahydropyrimidine biosynthesis gene cluster.
The invention separates a Halomonas sp microorganism in a deep sea environment sample, and the identified strain belongs to Halomonas segment QX-2T. The strain is published and preserved as a model strain in the marine microorganism management and preservation center (MCCC) and Korean culture Collection center (KCTC), with the preservation numbers of MCCC 1A17876T，KCTC 82199T。
The ectoine biosynthetic gene cluster is gene ectABC which is derived from Halomonas segmenti QX-2TThe strain redirected by the escherichia coli can express a large amount of ectABC, the expression level is high, the nucleotide sequence of the biosynthesis gene cluster is shown as 2244 base sequences of SEQ ID NO.1, the biosynthesis gene cluster comprises 3 genes, and the method specifically comprises the following steps:
(1) a diaminobutyric acid acetyltransferase gene (L-2,4-diaminobutyric acid acetyltransferase) which is responsible for catalyzing L-diaminobutyric acid (DABA) to synthesize N-acetyl-L-2, 4 diaminobutyric acid (ADABA), namely ectA, is positioned at the 1 st to 579 th positions of the nucleotide sequence SEQ ID NO.1, has 579 basic groups in total and codes 192 amino acids.
(2) The diaminobutyric acid-2-oxoglutarate aminotransferase gene (diaminobutyrate-2-oxoglutarate aminotransferase), i.e., ectB, responsible for catalyzing the aspartic Acid Semialdehyde (ASA), which is a precursor substance for synthesizing tetrahydropyrimidine, to generate L-diaminobutyric acid (DABA) is located at position 580-1851 of the nucleotide sequence SEQ ID NO.1 for 1272 bases and encodes 423 amino acids.
(3) The ectoine synthase gene (ectoine synthase), i.e., the ectoC, responsible for catalyzing the formation of ectoine (ectoine) as a target product from N-acetyl-L-2, 4-diaminobutyric acid (ADABA), is 393 bases in total and encodes 130 amino acids at position 1852-2244 of the nucleotide sequence SEQ ID NO. 1.
Applications of the tetrahydropyrimidine biosynthesis gene cluster include, but are not limited to:
1) the synthetic product of gene ectABC is applied to the preparation of cosmetic antioxidants and the like.
2) The gene ectABC obtains the recombinant protein thereof through a genetic engineering technology, and the protein coded by the sequence is applied to the preparation of tetrahydropyrimidine.
3) Application of gene ectoA in preparation of N-acetyl-L-2, 4 diaminobutyric acid (ADABA).
4) Application of gene ectoB in preparation of L-diaminobutyric acid (DABA).
5) Application of gene ectoC in preparation of ectoine (ectoine).
6) The application of the tetrahydropyrimidine biosynthesis gene cluster in preparing an antioxidant biological agent.
Experiments show that the tetrahydropyrimidine can effectively eliminate DPPH free radical activity and weaken the action of oxidative damage caused by oxidation, so that the tetrahydropyrimidine can be used as an antioxidant to be applied to cosmetics and has great application value and wide application prospect.
The research of the invention discovers that Halomonas segment QX-2TThe yield of the intracellular tetrahydropyrimidine is detected through simple fermentation, so that the yield is higher, and the yield is probably related to the fact that the yield is used as a deep sea moderate halophilic bacterium, has a certain new gene and a new structure, and the yield is higherGene ectABC of the invention encodes Halomonas segment QX-2TBiosynthesis of ectone of the strain, mass expression of ectoABC by using the strain reintroduced by escherichia coli, high expression level, convenience in purification due to single product and reduction of difficulty in purification of secondary metabolites. Has the characteristics of high-yield compounds, and the gene synthesis product has great application value and wide application prospect in the fields of pharmacy, food, cosmetics, biological agents, enzyme preparations, agriculture, chemical synthesis medicines, organic electronic materials and the like.
Drawings
FIG. 1 is an HPLC chart of the secondary metabolites of Halomonas.
FIG. 2 is an HPLC chart of secondary metabolites of the recombinant strain.
FIG. 3 is a graph of tetrahydropyrimidine on DPPH radical clearance.
Detailed Description
The invention is further illustrated by the following examples. The experimental procedures, for which specific conditions are not indicated in the following examples, can generally be carried out according to conventional conditions and methods, such as those described in molecular cloning, a laboratory manual written by J. Sambruk (Sambrook), et al, or according to conditions recommended by the manufacturer.
The ectoine biosynthetic gene cluster is ectABC which is a code Halomonas segmenti QX-2TThe DNA sequence and the amino acid sequence of ectoine synthetic gene cluster are shown in a sequence table SEQ ID NO. 1.
The sequencing comparison shows that the sequence of the gene ectABC has no sequence with the sequence of the ectoine synthetic gene cluster gene published in the prior art, has low similarity and is a new gene sequence.
EXAMPLE 1 clonal isolation of the ectABC Gene
By means of a pair of Halomonas segment QX-2TThe whole genome sequencing is carried out to obtain the ectone synthetic gene and the gene sequence thereof, wherein the ectone synthetic gene comprises an ectoABC gene and has a total length of 2452 bp. Biological software Primer5.0 was used to design an upstream primer (5 '-3') and a downstream primer (5 '-3') and to perform PCR reactions using genomic DNA as a template, the PCR reactions consisted of the following (50. mu.L reaction system): 2 μ L each of the upstream and downstream primers, DNA modelPlate 1. mu.L, 10 XBuffer 5. mu.L, dNTP Mix 5. mu.L, Ex Taq 0.5. mu.L, ddH2O34.5. mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 52.4 ℃ for 30s, extension at 72 ℃ for 1min and 20s, and final extension at 72 ℃ for 5min for 30 cycles. And after the reaction is finished, recovering the PCR product to obtain the ectoine synthetic gene cluster ectABC. Agarose gel electrophoresis, the size of which is in accordance with the length of the ectABC fragment.
Example 2: construction of Escherichia coli cloning vector pUC-ectABC
1. The EcoRI and Not I enzymes are used for carrying out double enzyme digestion on the ectABC vector and the pUC vector respectively, and the reaction system is as follows: pUC vector/ectABC 8. mu.L, EcoRI 1. mu.L, Not I1. mu.L, buffer 3.5. mu.L, ddH2O 6.5μL
2. Enzyme digestion is carried out for 3h at 37 ℃, two target fragments of 2.2kb and 5.6kb which are the same as the size of the ectABC/pUC vector fragment are respectively recovered after electrophoresis
3. Ligation was performed using T4 ligase, and the ligation system (10. mu.L) was as follows: 2 XBuffer 5. mu.L, 2.2kb DNA fragment 3. mu.L, 5.6kb pUC vector 1. mu.L, T4 ligase 1. mu.L, 20 ℃ overnight ligation. This gave a ligation solution, which was then transformed into E.coli DH 5. alpha.
Example 3: construction of Escherichia coli recombinant strain DH5 alpha-PUC-ectABC
100 μ L of thawed competent Escherichia coli DH5 α and 10 μ L of the ligation mixture were added to a 1.5mL Eppendorf tube, ice-cooled for 30min, heat-shocked at 42 ℃ for 90s, ice-cooled for 30min, LB medium 900 μ L was added, shaking-cultured at 37 ℃ for 1h, the bacterial solution was spread on LB plates containing 4 μ L of IPTG, 100 μ g/mL of Amp and 40 μ g/mL of X-gal, and cultured overnight at 37 ℃. White single colony is selected for PCR detection, and the colony presenting a 2.2kb special band in agarose gel electrophoresis is a positive transformation colony, namely the colony of Escherichia coli containing pUC-ectABC.
Example 6: preparation of recombinant ectoine
Selecting a monoclonal of the recombinant Escherichia coli strain, performing shake culture at 37 deg.C for 12h in 5mL LB liquid medium containing 100. mu.g/mL ampicillin, inoculating 1mL of the bacterial solution into 100mL of the same LB liquid medium, performing amplification culture, performing shake culture at 37 deg.C and 180rpm for 24h, and measuring OD600Then 10mL of the solution is extracted into a centrifuge tube and separated at 12000r/minRemoving supernatant after heart for 5 min; adding 10mL of pure water, crushing for 7min by using an ultrasonic cell crusher, centrifuging for 5min at 12000r/min, and filtering supernatant by using a 0.22um water system microporous filter to obtain the recombinant strain tetrahydropyrimidine extracting solution.
Wild type strain Halomonas segmenti QX-2TThe preparation method of the tetrahydropyrimidine extract is the same as that of the tetrahydropyrimidine extract.
Example 7: detection of tetrahydropyrimidine content by high performance liquid chromatography (HPIC)
A1.0 mg/ml stock solution of a tetrahydropyrimidine standard was prepared and a standard curve (peak area and concentration) was established. HPLC detection conditions: the mobile phase is water: acetonitrile (V/V, 20: 80), the detection wavelength is 210nm, the flow rate is 1.0mL/min, the column pressure is 3.486-4.761MPa, the column temperature is 30 ℃, and the loading amount is 10 mu L. The concentration of tetrahydropyrimidine in the sample was checked according to the conditions and methods, and 10. mu.L of each of the tetrahydropyrimidine extracts of the wild type and the recombinant strain obtained in example 6 was used and tested under the same conditions as those of the tetrahydropyrimidine standard. The results show that the HPLC (figure 1) of the wild strain shows that the variety of the secondary metabolites of the wild strain is more, compared with the HPLC (figure 2) of the recombinant strain, the variety of the secondary metabolites of the recombinant strain is obviously reduced, and the significant improvement of the yield of the peritecte is found. This suggests that the separation and purification of the tetrahydropyrimidine compound in the next step will reduce the difficulty of other secondary metabolites that are difficult to separate and give a tetrahydropyrimidine compound with higher yield and better purity. So as to be further used for subsequent scientific research and industrialized research and development.
Example 8: determination of DPPH free radical scavenging Activity by tetrahydropyrimidine
(1) Preparation of DPPH solution: DPPH4mg was weighed out and prepared into 0.004% concentration of DPPH in absolute ethanol.
(2) DPPH free radical scavenging ability, namely precisely weighing 128mg tetrahydropyrimidine, dissolving the 128mg tetrahydropyrimidine in 10mL distilled water to obtain sample solution with the concentration of 12.8mg/mL, then diluting the sample solution in a gradient manner to respectively obtain sample solutions with the concentrations of 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4mg/mL (the distilled water is used as a blank control, three samples in each group are arranged in parallel), adding 1.0mL DPPH (0.004%) solution under the condition of keeping out of the sun, shaking up, reacting for 30min in the absence of the light, centrifuging, and taking supernatant. BHT was a positive control in determining absorbance values, and percent clearance was calculated:
percent clearance (%) [ (A0-A1)/A0 ]. times.100%
Wherein a1 represents the absorbance value of a sample (or positive control);
a0 represents the absorbance value of the blank set. The greater the clearance rate, the stronger the oxidation resistance.
The experimental result shows that compared with the clearance rate of BHT of the positive control group, when the sample concentration is 2mg/mL, the clearance capacity of DPPH free radicals is weaker than that of the positive control group, when the sample concentration is more than or equal to 4mg/mL, the clearance capacity of the free radicals is stronger than that of the positive control group, and the clearance capacity of the free radicals of the tetrahydropyrimidine is gradually weakened along with the reduction of the concentration. (FIG. 3). This indicates that tetrahydropyrimidine has a good ability to scavenge DPPH radicals within a certain concentration range, thereby indicating that tetrahydropyrimidine has a good antioxidant effect and efficacy.
Sequence listing
<110> third oceanographic institute of natural resources department; china ocean mineral resources research and development association (China ocean affairs administration)
<120> tetrahydropyrimidine biosynthesis gene and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2244
<212> DNA
<213> Halomonas sedimenti QX-2T(2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
atgagtacgc cgacacaacc ttttaccccc tctgctgacc ttgctaggcc aacggtggct 60
gatgctgttg tgggtcatgc ggagatgccg ctgtttattc gcaaacccaa tgcagatgat 120
ggctggggag tttacgagct tatcaaagcc tgtccgccgc tggatgtgaa ttcagcctat 180
gcctacctac tgcttgctac tcagtttcgt gacacctgcg cagtggcgac caatgaagac 240
ggcgaagtgg tggggtttgt gtctggctat gttaaggaca atgcccccga cacctatttt 300
ctgtggcaag tggctgtcgg tgaaaaggca cgcggcacag gcctagcgcg tcgcttggtt 360
gaggcgatca tgtcgcgtcc cgagcttgat gatgtgcatc accttgaaac taccatcacg 420
cctgacaatc tagcctcttg gggcttattt cgtcgcctgg ctgcgcgctg gcatgcgccg 480
ctaaacagcc gcgaatactt ctctaccgaa cagctcggag gagagcatga cccggaaaac 540
ctggttcgta tcggcccgtt ccaaacagac cgcatctaaa tgcagaccca gacgcttgaa 600
cgcatggaat ccaatgtacg tacttattct cgttcgtttc cggtggtgtt taccaaagcg 660
caaaatgcgc gcctaaccga tgagaatggc cgtgagtaca ttgatttcct cgccggcgcg 720
ggcacgctga actacggcca caataatccg cacatgaagc aggcgatgat tgattacctg 780
tcgactgacg gtgttgttca cggtttggat atgtggacca atgccaagcg cgattatctt 840
gaaacgctgg aagaagttat tttcaagcca cgcgggctgg attataaagt gcatctgcct 900
ggcccaaccg gtaccaatgc cgtagaggcc gctatccgct tggctcgtgt ggctaaaggc 960
cgtcacaaca ttgtcacctt caccaatggt ttccacggcg tcaccatggg ggctttggcg 1020
accacaggta atcgtaaatt ccgtgaagcc acgggtggca tacccaccca gggcgctagc 1080
tttctgcctt acgatggtta catgggtgag cataccgata ctctggatta ttttgaaaag 1140
ctgctcagcg acaaatctgg cggtttggat attccagcgg gcgttatcat tgaaaccgtg 1200
caaggcgagg gcggtattaa cgttgcaggc ttggagtggt tgaagcgtct cgaagggatt 1260
tgccgtgccc acgatattct gctgattatc gatgatattc aggcaggctg tggccgtaca 1320
ggtaagttct ttagcttcga acatgcgggt attacacctg acattattac caactcgaaa 1380
tcgctctccg gttttggcct gccatttgcc catgtgttga tgcgccctga gctggacaaa 1440
tggaaaccgg gtcaatataa cggcactttc cgtggtttca gcctggcgat ggtgaccgcc 1500
acagcggcgt tgaaaaaata ctggactaat gacacctttg agcgtgatgt tcagcgcaaa 1560
gggcgtattg tagaagagcg ctttcagaag ttagcagcgt tgttaacgga acacggcatg 1620
cctgccactg aacgtggtcg tggtttgatg cgcggtattg acgtcgtgtc gggtgatatt 1680
gccgataaaa ttaccagtaa agcctttgag catggtctgg taattgaaac cagtggtcag 1740
gatggtgaag tggttaagtg tctatgcccg ctaaccatta gcgatgatga tctgctggaa 1800
ggtttggata ttctcgaaat gtgcgttaaa gctgttgttg ctagcgaata aatgatcgtt 1860
cgtaatcttg atgaagcgcg taaaacagac cgtctagtga ccgctgaaaa tggtaactgg 1920
gacagcacgc gccttgttct ggccaacgat aatgcaggtt tttcgttcca tattacccgc 1980
atttttccag gcactgaaac gcatatccac tacaaaaatc actacgaagc agtgttttgc 2040
tacgaaggtg aaggcgaagt tgaaaccctg gctgacggta aaatttggcc gatcaaagca 2100
ggcgacattt atctactcga ccagcatgat gaacacttgc tgcgcggcaa agagaagggc 2160
atgacggttg cctgtgtatt tactccgccg atcacaggta atgaggttca ccaggaagat 2220
ggctcgtacg cagccccaga gtaa 2244
Claims (8)
1. The tetrahydropyrimidine biosynthesis gene cluster is characterized in that the nucleotide sequence is shown as 2244 base sequences of SEQ ID NO. 1.
2. The tetrahydropyrimidine biosynthesis gene cluster according to claim 1 characterized in that it comprises the following 3 genes:
(1) the diaminobutyric acid acetyltransferase gene which is responsible for catalyzing L-diaminobutyric acid to synthesize N-acetyl-L-2, 4 diaminobutyric acid, namely ectA, is positioned at the 1 st to 579 th positions of the nucleotide sequence SEQ ID NO.1, has 579 basic groups in total and codes 192 amino acids;
(2) the diaminobutyric acid-2-oxoglutarate aminotransferase gene which is responsible for catalyzing aspartate semialdehyde which is a precursor substance for synthesizing tetrahydropyrimidine to generate L-diaminobutyric acid, namely ectB, is positioned at the 580-1851 site of the nucleotide sequence SEQ ID NO.1 and has 1272 basic groups in total, and encodes 423 amino acids;
(3) the ectoine synthase gene, namely, the ectC, which is responsible for catalyzing the N-acetyl-L-2, 4-diaminobutyric acid to generate the target product ectoine is positioned at 1852-2244 site of the nucleotide sequence SEQ ID NO.1, has 393 basic groups in total and codes 130 amino acids.
3. Use of the tetrahydropyrimidine biosynthesis gene cluster according to claim 1 as antioxidant in the preparation of cosmetics.
4. The use of the tetrahydropyrimidine biosynthesis gene cluster according to claim 1, wherein the encoded protein catalyzes the production of tetrahydropyrimidine.
5. The use of the gene ectoA of claim 2 for the catalytic production of N-acetyl-L-2, 4 diaminobutyric acid.
6. The use of the gene ectB as defined in claim 2 for the catalytic preparation of L-diaminobutyric acid.
7. The use of the gene ecc of claim 2 for the catalytic preparation of tetrahydropyrimidine.
8. Use of the tetrahydropyrimidine biosynthesis gene cluster according to claim 1 for preparing an antioxidant biological agent.
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