CN114395053A - DHQS-OMT fusion gene from zooxanthella and application thereof in high-efficiency synthesis of MAAs - Google Patents

DHQS-OMT fusion gene from zooxanthella and application thereof in high-efficiency synthesis of MAAs Download PDF

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CN114395053A
CN114395053A CN202210299777.7A CN202210299777A CN114395053A CN 114395053 A CN114395053 A CN 114395053A CN 202210299777 A CN202210299777 A CN 202210299777A CN 114395053 A CN114395053 A CN 114395053A
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张扬
喻子牛
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South China Sea Institute of Oceanology of CAS
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Abstract

The invention discloses a DHQS-OMT fusion gene from zooxanthella and application thereof in high-efficiency synthesis of MAAs, belonging to the field of biotechnology. The amino acid sequence of the DHQS-OMT fusion protein is shown in SEQ ID NO. 2. In the invention, a DHQS-OMT fusion gene is found in a zooxanthella transcriptome, and the DHQS-OMT fusion gene has two structural domains of a DHQS homologous gene and methyltransferase OMT, has dual catalytic reaction capability and can efficiently catalyze and generate a precursor molecule 4-deoxygadosol of MAA. The invention synthesizes a zooxanthellae MAAs biosynthesis gene for the first time, relates to generation of an ultraviolet absorption substance, successfully clones the ultraviolet absorption substance to a prokaryotic expression vector to obtain protein, and can be applied to the fields of biological medicines such as cosmetics, sunscreen cream and the like.

Description

DHQS-OMT fusion gene from zooxanthella and application thereof in high-efficiency synthesis of MAAs
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a DHQS-OMT fusion gene derived from zooxanthella and application thereof in high-efficiency synthesis of MAAs.
Background
Mycosporine-like amino acids (MAAs) are water-soluble secondary metabolites with small molecular weights (typically less than 400 Da) that accumulate primarily from marine organisms exposed to high light intensities. MAAs are a class of ultraviolet absorbing substances with absorption wavelengths between 310 and 365 nm. They are mainly composed of cyclohexenone as a core skeleton, and side chains formed by conjugation with amino acid residues or imino alcohols thereof. Gadusol and 4-deoxygadusol (4-DG) are precursor substances, and the synthesis of other MAAs needs to be modified on the basis of the precursor substances. Meanwhile, the absorption spectra of different MAAs are different due to the difference in their structures, i.e., the types of amino acid substituents bound to the side chains. MAAs can be used for a variety of purposes in organisms. Most studies have focused on their photoprotective and antioxidant properties, while MAAs can protect cells from salinity stress, desiccation and heat stress. Thus, a number of MAAs have been studied for their biological and cosmeceutical properties in humans, focusing primarily on the prevention of the harmful effects of ultraviolet radiation on the human body. Sunscreen is the most important function that MAAs play in nature.
At present, MAAs are generally considered to have 2 synthetic pathways: the shikimate pathway and the pentose phosphate pathway. The shikimic acid pathway comprises four major key enzymes, DHQS-like, O-MT, ATP-gradp and NRPS-like; DHQS is 3-dehydroquinic acid synthase, O-MT is methyltransferase, ATP-grapp is a protein with conserved regions of the adenosine triphosphate grapp superfamily, NRPS is a nonribosomal peptide synthase. Among them, DHQS-like and O-MT synthesize the cyclohexenone core skeleton of MAAs, while ATP-grapp and NRPS-like are mainly related to the assembly and synthesis of side chain amino acids. DHQS-like and O-MT are essential for the synthesis of all MAAs, and can synthesize the MAAs precursor substance 4-deoxygadosol (4-DG). Another synthetic pathway is the pentose phosphate pathway, including EEVS, MT-OX. EEVS and DDGS (DHQS-like homologous gene) are highly similar in sequence and both use sedoheptulose-7-phosphate (SH-7-P) as a substrate; MT-OX has two functional domains, methyltransferase and oxidoreductase. SH-7-P is catalyzed by these two gene products to produce Gadusol, another precursor of MAAs.
Zooxanthellae (zoxanthellae) is a symbiotic alga and can form a stable symbiotic relationship with a host (coral and tridacna), so that the zooxanthellae (zoxanthellae) is particularly important in coral reef ecosystems. The host provides shelter for the zooxanthella, and inorganic nutrients such as carbon, nitrogen and the like, and the symbiotic zooxanthella provides photosynthetic metabolites for the host. Rapid changes in the environment, such as global warming, ultraviolet radiation, and marine acidification, can disrupt the symbiotic relationship between the host and the symbiont. The zooxanthella algae, in addition to providing photosynthetic metabolites to the host, also have the ability to resist damage from sunlight ultraviolet radiation.
Disclosure of Invention
In the invention, a DHQS-OMT fusion gene is found in a zooxanthella transcriptome, and the DHQS-OMT fusion gene has two structural domains of a DHQS homologous gene and methyltransferase OMT, has dual catalytic reaction capability and can efficiently catalyze and generate a precursor molecule 4-deoxygadosol of MAA.
The first purpose of the invention is to provide DHQS-OMT fusion protein, the amino acid sequence of which is shown as SEQ ID NO.2 or has more than 95% homology with the sequence shown as SEQ ID NO. 2.
It is a second object of the present invention to provide a DHQS-OMT fusion gene encoding the above DHQS-OMT fusion protein.
Preferably, the nucleotide sequence of the DHQS-OMT fusion gene is shown in SEQ ID NO.1, or has homology of more than 95 percent with the DHQS-OMT fusion gene.
It is a third object of the present invention to provide an expression vector comprising a DHQS-OMT fusion gene.
Preferably, the expression vector is pET-28 a.
It is a fourth object of the present invention to provide a host cell comprising the above expression vector.
Preferably, the host cell may be E.coli BL21(DE 3).
The fifth purpose of the invention is to provide the application of the fusion gene, the protein, the expression vector and the host cell in synthesizing MAAs, and further, the MAAs can be applied to resisting ultraviolet rays; on the other hand, it can also be applied to protect cells from salinity stress, desiccation and heat stress.
The sixth purpose of the invention is to provide a method for synthesizing MAAs, which is to express the DHQS-OMT fusion gene in host cells to biosynthesize MAAs.
Preferably, the host cell is Escherichia coli BL21(DE 3).
Preferably, the DHQS-OMT fusion gene is expressed in a host cell by transferring the DHQS-OMT fusion gene into an expression vector such as pET-28a, and then transforming into a host cell for expression.
The seventh purpose of the invention is to provide the application of the synthesized mycosporine-like amino acid in preparing cosmetics for absorbing ultraviolet rays.
Compared with the prior art, the invention has the following beneficial effects:
the invention synthesizes a zooxanthellae MAAs biosynthesis gene for the first time, which relates to the generation of an ultraviolet absorbing substance and successfully clones the ultraviolet absorbing substance to a prokaryotic expression vector to obtain DHQS-OMT fusion protein. Through expression and MAAs extraction analysis, the substance with ultraviolet absorption function can be generated, and the substance can be applied to the fields of cosmetics, sunscreen cream and other biological medicines, can protect the organism from being damaged by absorbing ultraviolet radiation, and has important economic effect.
Drawings
FIG. 1 is a diagram of domain function prediction of the DHQS-OMT fusion gene of Coccinia tebuformis.
FIG. 2 is a gel diagram showing the expression and purification of MAAs biosynthesis genes, wherein protein A expresses protein B and protein purification and protein C is concentrated; m represents protein marker, and the arrow points to the recombinant protein of interest.
FIG. 3 is a scanning spectrum of an extract of Escherichia coli strain.
FIG. 4 is an HPLC analysis of E.coli expression host extracts.
FIG. 5 is a mass spectrum of the MAA precursor molecule 4-deoxygadosol in positive ion mode.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The following examples are given without specifying the particular experimental conditions and methods, and the technical means employed are generally conventional means well known to those skilled in the art.
Example 1
1. Synthesis of Dinghuang algae DHQS-OMT gene sequence
The DHQS-OMT fusion gene was obtained from chrysophyceae transcriptome data and its nucleotide sequence is shown below:
atgtctaccaccatcactacgaccattaaaatcgcgcgcgatgtgctgcagcctagcaatcgtctgctgtttgatgtctacaagccgctgggtcgttgcgtggctgttgttgatgacaaagtagaggcacactttggcgcggacctggaaaaatacttcgaaatcaacggcatcgaattcgttaaactggttttctccggtaacgaagtggacaaaaacctgtccaacgtagagcagattctgctggcgctgaaagaaaactggcaggctcgtcacgagccggtgctgattgttggtggtggcgttatcgcggatgtggcaggtatggcctgcgccctgtactctcgtaacaccccgtacgtcatgctgtgtacttctctggtagctggcatcgacgctggcccgtcccctcgcgtatgctgtaatggttttagctacaaaaacctgtacggcgcctatcatccgccggctctgactatcaccgatcgtggtttctggcgcaccatgcacccgggtcagctgcgtcacggcgtggctgaaatcatcaaagttgcggctatggcggatctgcacctgtttgaactgatggaaaagtggggcaaagagaccgtggcgactaactttggtaccctgggcggcctggatgatccggcattccaagaagtatgcgacctgattatcggtaaggctatggaaggttatgtgcgtgcagaatacggtaacctgtgggaaacccaccaatgtcgtccgcatgcgtacggccacacttggtccccgggttatgaactgccgtctggtatgctgcatggccacgctgttggcacttgtatgggtttcggtgcctacctgtcttacaaagaaggttggctgagcgaagtggacatgcaccgcgtgctgaaggtgattagcgattgcgaactggcgctgtggcacccggttatggaggataccgtaagcgtatggaaagctcagattggcatcgttgaaaaacgtggcggtaacctgtgcgcaccgatcccacgtgctctgggtgagtctggttatctgaacagcctgccggaacgtctgctgcaccagcgcatgcacgagtatcaggaactgtgcagcaaatacccgcgtgaaggccgtggtgtgcaggaacactgtgtggacgtcggtctggaagacccgatgtccaagaaagactctattcgtgatgcagaccgttgcgtcgtaactccgatggatcgtgttgtcgttcagctgggcgcagcaagccagtccacctctggtgcgggcgctcgtgctatcggcgaggccgctcgcatcgttgacggtatcgacgaaatggtgaccaaatactcctctcagccgtccgaagcgctgaagaacgtgagcaaacgcaccgccatgactaacccgctgtgggcggaactgatgggtcaaggcaacacctctcgcctgatggaagccgaaatgatctccggtcaggctgaggcccagctgctgcagctgctgctgaagctgtctggcggcaaagacgtactggacatcggtaccttcacgggttactcttctctggctatggcagaagctatcccggaagatggtaccgtggttacgctggaacgtgagcaggaagcgaaagacatggccaaagctaactggatcggctctccgcacgcgtctaaaattgaatctctggtgggcgaggcacatgatctgctgatctgtctggcggctgaagacaagtctttcgacctggtgtttctggatgttgacaaaccgggctacttcggcctgtatcagatgctgatggataaaaatctgctgaaagtgaaaggcctgctggttgtcgataacgttatgtataaaggcgaagaactgtctggtaccgagctgaccaaaaacggccagggcgcgcaggcgctgaaccagggtctgctggacgacccgcgcgttcagcaggtcatgctgccactgcgtgacggtgtcaccctggtatatcgtgagtcctga (SEQ ID NO. 1), the amino acid sequence of the protein is shown in SEQ ID NO. 2. The ability to catalyze the formation of 4-deoxygadosol from sedoheptulose-7-phosphate (SH-7-P) in one step was predicted based on the domain of the Coccinum DHQS-OMT fusion gene (FIG. 1). According to the gene base sequence, the gene is sent to Tianyihui company for artificial synthesis.
2. Prokaryotic expression and purification
(1) The artificially synthesized sequence was constructed in pET28a vector (restriction site: NdeI/XhoI), and transformedE.coli BL21(DE3) and the transformants were cultured overnight at 37 ℃ on LB plates containing 50. mu.g/mL of kanamycin.
(2) A single colony was inoculated into 10mL of LB liquid medium containing kanamycin, cultured overnight at 37 ℃ and, on the following day, as 1: 100 portions of the cells were inoculated into 200mL of LB liquid medium containing kanamycin and cultured at 37 ℃ to OD600To 0.6.
(3) After 10min on ice, IPTG was added to a final concentration of 0.1mM, and the cells were cultured at 16 ℃ for 18 hours to induce expression of the N-staged protein with 6 His tags.
(4) Collecting thalli, centrifuging at 12000g and 4 ℃ for 10min, and mixing the thalli according to the weight ratio of 1: 20 proportions were resuspended in PBS buffer and then sonicated on ice.
(5) The sonicated solution was centrifuged at 12000g at 4 ℃ for 20min, the supernatant and the pellet were separated, and the pellet was resuspended in an equal amount of PBS solution to the supernatant.
(6) Recombinant proteins were purified using a Ni column, concentrated and the purified product was analyzed on a 12% SDS-PAGE gel, which is shown in FIG. 2.
Example 2 extraction and analysis of MAAs in microbial cells
(1) Selecting induced expression conditions of the engineering bacteria: inoculation of DHQS-OMT fusion gene-transfected E.coli BL21(DE3) in 10mL LBIn the culture medium, shake the bacteria to OD overnight600=1.5 or more, and the next day 1mL of sterilized LB medium (containing 1%)13C-Glucose), note that each plasmid (pET28a-DHQS-OMT recombinant vector, pET28a empty vector) was inoculated into two flasks of medium, one of which was used for control. The culture medium is incubated at 37 ℃ for about 3-4 h at 220rpm until OD600= 0.6. One of the flasks was selected and added with 20. mu.l of prepared 1M IPTG to a final concentration of 0.1mM IPTG. The reaction mixture was incubated at 16 ℃ for 20 h at 200rpm with a control without IPTG. And (4) centrifuging at 6000rpm for 10min to collect thalli, removing supernate as much as possible, and collecting for later use.
(2) Extraction and purification of MAAs
Adding 10mL of pure methanol into the collected thalli, blowing and uniformly mixing, crushing on ice for 20min, centrifuging at 4 ℃ and 10000rpm for 20min, collecting supernatant, and performing rotary evaporation to dryness. The dried residue was resuspended in 200. mu.L of ultrapure water and 12000g, centrifuged at 4 ℃ for 10min, and the supernatant was filtered through a 0.22 μm filter to give crude MAAs product for subsequent spectral scanning and HPLC analysis.
(3) Spectral scanning of MAAs crude extracts
The absorption spectrum at 200-800nm was scanned using an ultraviolet spectrophotometer. The maximum absorption peak of the IPTG induction product of the strain can be judged by scanning induction and non-induction. As shown in FIG. 3, the IPTG induced product absorption peak was around 300nm compared to that of the uninduced.
(4) HPLC analysis and mass spectrometric identification of MAAs crude extracts
And detecting the crude extract by using HPLC, and setting the wavelength according to the maximum absorption wavelength of the scanning spectrum. Using an advanced bio AAA C18 chromatography column (4.6 × 100 mm, 2.7 μm), 20 μ L was injected, mobile phase a was 0.2% TFA and ammonium hydroxide solution (ph 3.15), mobile phase B was 0.2% TFA + ammonium hydroxide (ph 2.20) methanol: acetonitrile = 80: 10: 10 (v: v: v) and the flow rate was set at 1 ml/min. As shown in FIG. 4, IPTG-induced E.coli extract had 2 distinct peaks compared to non-induction, with peak-off times of 2.5 min and 5min, respectively, indicating the formation of new product. Subsequently, a Q-exact mass spectrometry system is used for identifying the two separation peaks, ion information is analyzed by combining a full mass + ddMS2 (primary full scan + automatic trigger secondary) acquisition mode based on a positive and negative ion separation scanning mode, and the set conditions are electrospray voltage 3800/3200V (+/-), capillary temperature 300 ℃, sheath gas flow rate 40Arb and atomizer temperature 350 ℃. The final results are shown in FIG. 5, in which the isolated peak at 5min is C13 labeled 4-deoxygadosol, demonstrating the ability of the Coccomys chrysosporium DHQS-OMT gene to catalyze the production of 4-deoxygadosol in one step.
Sequence listing
<110> Nanhai ocean institute of Chinese academy of sciences
<120> DHQS-OMT fusion gene from zooxanthella and application thereof in high-efficiency synthesis of MAAs
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1998
<212> DNA
<213> Chlorella zoonotic (Zooxantherae)
<400> 1
atgtctacca ccatcactac gaccattaaa atcgcgcgcg atgtgctgca gcctagcaat 60
cgtctgctgt ttgatgtcta caagccgctg ggtcgttgcg tggctgttgt tgatgacaaa 120
gtagaggcac actttggcgc ggacctggaa aaatacttcg aaatcaacgg catcgaattc 180
gttaaactgg ttttctccgg taacgaagtg gacaaaaacc tgtccaacgt agagcagatt 240
ctgctggcgc tgaaagaaaa ctggcaggct cgtcacgagc cggtgctgat tgttggtggt 300
ggcgttatcg cggatgtggc aggtatggcc tgcgccctgt actctcgtaa caccccgtac 360
gtcatgctgt gtacttctct ggtagctggc atcgacgctg gcccgtcccc tcgcgtatgc 420
tgtaatggtt ttagctacaa aaacctgtac ggcgcctatc atccgccggc tctgactatc 480
accgatcgtg gtttctggcg caccatgcac ccgggtcagc tgcgtcacgg cgtggctgaa 540
atcatcaaag ttgcggctat ggcggatctg cacctgtttg aactgatgga aaagtggggc 600
aaagagaccg tggcgactaa ctttggtacc ctgggcggcc tggatgatcc ggcattccaa 660
gaagtatgcg acctgattat cggtaaggct atggaaggtt atgtgcgtgc agaatacggt 720
aacctgtggg aaacccacca atgtcgtccg catgcgtacg gccacacttg gtccccgggt 780
tatgaactgc cgtctggtat gctgcatggc cacgctgttg gcacttgtat gggtttcggt 840
gcctacctgt cttacaaaga aggttggctg agcgaagtgg acatgcaccg cgtgctgaag 900
gtgattagcg attgcgaact ggcgctgtgg cacccggtta tggaggatac cgtaagcgta 960
tggaaagctc agattggcat cgttgaaaaa cgtggcggta acctgtgcgc accgatccca 1020
cgtgctctgg gtgagtctgg ttatctgaac agcctgccgg aacgtctgct gcaccagcgc 1080
atgcacgagt atcaggaact gtgcagcaaa tacccgcgtg aaggccgtgg tgtgcaggaa 1140
cactgtgtgg acgtcggtct ggaagacccg atgtccaaga aagactctat tcgtgatgca 1200
gaccgttgcg tcgtaactcc gatggatcgt gttgtcgttc agctgggcgc agcaagccag 1260
tccacctctg gtgcgggcgc tcgtgctatc ggcgaggccg ctcgcatcgt tgacggtatc 1320
gacgaaatgg tgaccaaata ctcctctcag ccgtccgaag cgctgaagaa cgtgagcaaa 1380
cgcaccgcca tgactaaccc gctgtgggcg gaactgatgg gtcaaggcaa cacctctcgc 1440
ctgatggaag ccgaaatgat ctccggtcag gctgaggccc agctgctgca gctgctgctg 1500
aagctgtctg gcggcaaaga cgtactggac atcggtacct tcacgggtta ctcttctctg 1560
gctatggcag aagctatccc ggaagatggt accgtggtta cgctggaacg tgagcaggaa 1620
gcgaaagaca tggccaaagc taactggatc ggctctccgc acgcgtctaa aattgaatct 1680
ctggtgggcg aggcacatga tctgctgatc tgtctggcgg ctgaagacaa gtctttcgac 1740
ctggtgtttc tggatgttga caaaccgggc tacttcggcc tgtatcagat gctgatggat 1800
aaaaatctgc tgaaagtgaa aggcctgctg gttgtcgata acgttatgta taaaggcgaa 1860
gaactgtctg gtaccgagct gaccaaaaac ggccagggcg cgcaggcgct gaaccagggt 1920
ctgctggacg acccgcgcgt tcagcaggtc atgctgccac tgcgtgacgg tgtcaccctg 1980
gtatatcgtg agtcctga 1998
<210> 2
<211> 665
<212> PRT
<213> Chlorella zoonotic (Zooxantherae)
<400> 2
Met Ser Thr Thr Ile Thr Thr Thr Ile Leu Ile Ala Ala Ala Val Leu
1 5 10 15
Gly Pro Ser Ala Ala Leu Leu Pro Ala Val Thr Leu Pro Leu Gly Ala
20 25 30
Cys Val Ala Val Val Ala Ala Leu Val Gly Ala His Pro Gly Ala Ala
35 40 45
Leu Gly Leu Thr Pro Gly Ile Ala Gly Ile Gly Pro Val Leu Leu Val
50 55 60
Pro Ser Gly Ala Gly Val Ala Leu Ala Leu Ser Ala Val Gly Gly Ile
65 70 75 80
Leu Leu Ala Leu Leu Gly Ala Thr Gly Ala Ala His Gly Pro Val Leu
85 90 95
Ile Val Gly Gly Gly Val Ile Ala Ala Val Ala Gly Met Ala Cys Ala
100 105 110
Leu Thr Ser Ala Ala Thr Pro Thr Val Met Leu Cys Thr Ser Leu Val
115 120 125
Ala Gly Ile Ala Ala Gly Pro Ser Pro Ala Val Cys Cys Ala Gly Pro
130 135 140
Ser Thr Leu Ala Leu Thr Gly Ala Thr His Pro Pro Ala Leu Thr Ile
145 150 155 160
Thr Ala Ala Gly Pro Thr Ala Thr Met His Pro Gly Gly Leu Ala His
165 170 175
Gly Val Ala Gly Ile Ile Leu Val Ala Ala Met Ala Ala Leu His Leu
180 185 190
Pro Gly Leu Met Gly Leu Thr Gly Leu Gly Thr Val Ala Thr Ala Pro
195 200 205
Gly Thr Leu Gly Gly Leu Ala Ala Pro Ala Pro Gly Gly Val Cys Ala
210 215 220
Leu Ile Ile Gly Leu Ala Met Gly Gly Thr Val Ala Ala Gly Thr Gly
225 230 235 240
Ala Leu Thr Gly Thr His Gly Cys Ala Pro His Ala Thr Gly His Thr
245 250 255
Thr Ser Pro Gly Thr Gly Leu Pro Ser Gly Met Leu His Gly His Ala
260 265 270
Val Gly Thr Cys Met Gly Pro Gly Ala Thr Leu Ser Thr Leu Gly Gly
275 280 285
Thr Leu Ser Gly Val Ala Met His Ala Val Leu Leu Val Ile Ser Ala
290 295 300
Cys Gly Leu Ala Leu Thr His Pro Val Met Gly Ala Thr Val Ser Val
305 310 315 320
Thr Leu Ala Gly Ile Gly Ile Val Gly Leu Ala Gly Gly Ala Leu Cys
325 330 335
Ala Pro Ile Pro Ala Ala Leu Gly Gly Ser Gly Thr Leu Ala Ser Leu
340 345 350
Pro Gly Ala Leu Leu His Gly Ala Met His Gly Thr Gly Gly Leu Cys
355 360 365
Ser Leu Thr Pro Ala Gly Gly Ala Gly Val Gly Gly His Cys Val Ala
370 375 380
Val Gly Leu Gly Ala Pro Met Ser Leu Leu Ala Ser Ile Ala Ala Ala
385 390 395 400
Ala Ala Cys Val Val Thr Pro Met Ala Ala Val Val Val Gly Leu Gly
405 410 415
Ala Ala Ser Gly Ser Thr Ser Gly Ala Gly Ala Ala Ala Ile Gly Gly
420 425 430
Ala Ala Ala Ile Val Ala Gly Ile Ala Gly Met Val Thr Leu Thr Ser
435 440 445
Ser Gly Pro Ser Gly Ala Leu Leu Ala Val Ser Leu Ala Thr Ala Met
450 455 460
Thr Ala Pro Leu Thr Ala Gly Leu Met Gly Gly Gly Ala Thr Ser Ala
465 470 475 480
Leu Met Gly Ala Gly Met Ile Ser Gly Gly Ala Gly Ala Gly Leu Leu
485 490 495
Gly Leu Leu Leu Leu Leu Ser Gly Gly Leu Ala Val Leu Ala Ile Gly
500 505 510
Thr Pro Thr Gly Thr Ser Ser Leu Ala Met Ala Gly Ala Ile Pro Gly
515 520 525
Ala Gly Thr Val Val Thr Leu Gly Ala Gly Gly Gly Ala Leu Ala Met
530 535 540
Ala Leu Ala Ala Thr Ile Gly Ser Pro His Ala Ser Leu Ile Gly Ser
545 550 555 560
Leu Val Gly Gly Ala His Ala Leu Leu Ile Cys Leu Ala Ala Gly Ala
565 570 575
Leu Ser Pro Ala Leu Val Pro Leu Ala Val Ala Leu Pro Gly Thr Pro
580 585 590
Gly Leu Thr Gly Met Leu Met Ala Leu Ala Leu Leu Leu Val Leu Gly
595 600 605
Leu Leu Val Val Ala Ala Val Met Thr Leu Gly Gly Gly Leu Ser Gly
610 615 620
Thr Gly Leu Thr Leu Ala Gly Gly Gly Ala Gly Ala Leu Ala Gly Gly
625 630 635 640
Leu Leu Ala Ala Pro Ala Val Gly Gly Val Met Leu Pro Leu Ala Ala
645 650 655
Gly Val Thr Leu Val Thr Ala Gly Ser
660 665

Claims (10)

1. A DHQS-OMT fusion protein is characterized in that the amino acid sequence of the DHQS-OMT fusion protein is shown in SEQ ID NO. 2.
2. A DHQS-OMT fusion gene encoding the DHQS-OMT fusion protein of claim 1.
3. The DHQS-OMT fusion gene according to claim 2, having the nucleotide sequence shown in SEQ ID No. 1.
4. An expression vector comprising the DHQS-OMT fusion gene of claim 2 or 3.
5. A host cell comprising the expression vector of claim 4.
6. Use of the DHQS-OMT fusion protein according to claim 1, the DHQS-OMT fusion gene according to claim 2 or 3, the expression vector according to claim 4, or the host cell according to claim 5 for the synthesis of mycosporine-like amino acids.
7. A method for synthesizing mycosporine-like amino acids, comprising expressing the DHQS-OMT fusion gene of claim 2 or 3 in a host cell to biosynthesize the mycosporine-like amino acids.
8. The method of claim 7, wherein the host cell is Escherichia coli BL21(DE 3).
9. The method according to claim 7, wherein the expression of the DHQS-OMT fusion gene of claim 2 or 3 in a host cell is performed by transferring the DHQS-OMT fusion gene of claim 2 or 3 into an expression vector, and then transforming the expression vector into a host cell.
10. Use of the mycosporine-like amino acid synthesized by the method of claim 7 in the preparation of a UV-absorbing cosmetic.
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