CN116676323A - Biosynthesis gene of MAAs precursor substance 4-deoxygadusol, recombinant plasmid and application thereof - Google Patents
Biosynthesis gene of MAAs precursor substance 4-deoxygadusol, recombinant plasmid and application thereof Download PDFInfo
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- CN116676323A CN116676323A CN202310528274.7A CN202310528274A CN116676323A CN 116676323 A CN116676323 A CN 116676323A CN 202310528274 A CN202310528274 A CN 202310528274A CN 116676323 A CN116676323 A CN 116676323A
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- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/405—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from algae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
- C12P7/26—Ketones
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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Abstract
The invention discloses a biosynthesis gene of MAAs precursor 4-deoxygadusol, a recombinant plasmid and application thereof. The invention optimizes EEVS gene and OMT gene from Antarctic ice algae Phaeodactylum tricornutum ICE-H, constructs recombinant plasmid by utilizing the optimized genes, introduces the recombinant plasmid into escherichia coli to obtain recombinant strain simultaneously expressing two genes, and leads the escherichia coli to produce MAAs synthesis precursor substance 4-deoxygadusol in cells by inducing the strain, and the product obtained by the method is single, simple, feasible and efficient. The 4-deoxygadusol obtained by the invention has stronger in-vitro antioxidant activity, can obviously improve the resistance of skin melanocytes to ultraviolet radiation, and has important significance for the research of the skin ultraviolet injury protection field.
Description
Technical Field
The invention belongs to the field of biosynthesis, and relates to a biosynthesis gene of a MAAs precursor substance 4-deoxygadusol, a recombinant plasmid thereof and application thereof.
Background
During long-term evolution, organisms have developed several photoprotection mechanisms to survive high levels of uv radiation, including accumulation of photoprotective compounds with UVR absorption capacity, such as retinoid amino acids (MAAs). MAAs are found in a large number of aquatic species, including marine and freshwater organisms that are exposed to high levels of damaging ultraviolet radiation. MAAs are involved in many biological processes, including UV protection, osmotic regulation, and resistance to oxidative stress of organisms and their embryos. It has been found that over 40 MAAs with molecular weight less than 400Da are light stable, easy to dissolve in water, high in melting point, high in heat stability, good in acid-base stability, and not only has powerful ultraviolet light protecting effect, but also has antioxidant, antitumor, anti-inflammatory and other activities, and these excellent performances are of great interest in MAAs research in the industries of biological medicine and the like. 4-Deoxygadusol is considered to be a direct precursor of MAAs and also has very strong antioxidant properties. The antioxidant capacity of 4-deoxygadusol has been evaluated by a phosphatidylcholine peroxidation inhibition assay and found to produce more potent activity than mycosporine-glycone (one of the MAAs).
At present, commercial sun protection products mainly depend on organic materials such as oxybenzone and avobenzone, or inorganic materials such as titanium dioxide and zinc oxide, but the materials are easy to pollute the environment, and free radicals can be generated by human bodies when the materials are frequently used, so that skin injury, anaphylactic reaction, endocrine disturbance or other nervous system diseases are caused. Thus, there is an urgent need to develop more benign MAAs natural sunscreens. MAAs natural products are mainly derived from extraction of large algae, and the extraction rate is extremely low due to the very small content, the sources are extremely difficult and expensive, application bottlenecks are encountered, and the intermediate 4-deoxygadusol is difficult to obtain by a direct extraction method. On the other hand, the structural features of MAAs and 4-deoxygadusol make their chemical synthesis very difficult and difficult to prepare and obtain efficiently. Therefore, it is needed to realize heterologous efficient expression of MAAs by a synthetic biology technology method so as to solve the problem of MAAs sources.
In order to survive and reproduce in the extreme environment of the strong ultraviolet radiation of the antarctic ice algae, genetic materials of the antarctic ice algae are subjected to long-term adaptive evolution to form a special molecular genetic mechanism, and the antarctic ice algae have unique biological synthesis mechanism and functional characteristics of MAAs, which are key survival mechanisms suitable for the environment of the strong ultraviolet radiation. The gene of MAAs synthesis path is excavated from the Antarctic ice algae genome, and is constructed to a heterologous host, so that the MAAs can be prepared in a microbial fermentation mode, and the method has important application value.
Disclosure of Invention
The invention aims to provide a biosynthesis gene of MAAs precursor 4-deoxygadusol and a recombinant plasmid thereof, and application thereof in ultraviolet radiation damage protection and preparation of protective cream.
In order to solve the above-mentioned purpose, the invention adopts the following technical scheme to realize:
the invention provides a biosynthesis gene of MAAs precursor 4-deoxygadusol, which comprises an EEVS gene and an OMT gene, wherein the nucleotide sequence of the biosynthesis gene is optimized through the codon preference of escherichia coli; the nucleotide sequence of the EEVS gene is shown as SEQ ID NO. 3; the nucleotide sequence of the OMT gene is shown as SEQ ID NO. 4.
Furthermore, the original nucleotide sequences of the EEVS gene and the OMT gene are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the EEVS gene and OMT gene are derived from Antarctic ice algae Phaeodactylum tricornutum ICE-H.
The invention also provides a recombinant plasmid which contains the biosynthesis gene.
Furthermore, the recombinant plasmid contains both EEVS gene and OMT gene, and the nucleotide sequence is shown as SEQ ID NO. 9.
Further, the original plasmid of the recombinant plasmid is pacyclid 1, which comprises two insertion sites MCS1 and MCS2; the EEVS gene fragment is connected to the pACYCDuet1 MCS1 locus of the vector, the OMT gene fragment is connected to the pACYCDuet1 MCS2 locus of the vector, and each sequence has independent T7 promoter control.
The invention also provides a recombinant strain which contains the biosynthesis genes.
Furthermore, the recombinant strain can simultaneously express EEVS genes and OMT genes, and the construction steps are as follows:
(1) Performing double digestion on an expression vector pACYCDuet1, connecting with the EEVS gene, transforming escherichia coli, and screening positive transformants to obtain a strain containing recombinant plasmid pACYCDuet 1-EEVS;
(2) And (3) carrying out double enzyme digestion on the recombinant plasmid pACYCDuet1-EEVS to connect with the OMT gene, and screening positive transformants to obtain the recombinant strain containing the recombinant plasmid pACYCDuet 1-EEVS-OMT.
Furthermore, the MCS1 site of the expression vector pACYCDuet1 selects enzyme cutting sites of BamHI/HindIII, and the MCS2 site selects enzyme cutting sites of NdeI/XhoI.
The invention also provides the application of the biosynthesis genes, or the recombinant plasmids or the recombinant strains in the biosynthesis of MAAs precursor 4-deoxygadusol.
The invention also provides a method for biosynthesis of MAAs precursor 4-deoxygadusol, which comprises the following steps: after the recombinant strain was cultured, the strain was induced with isopropyl- β -D-thiogalactoside, and the cells were collected by centrifugation, dried and subjected to ice bath ultrasonication, and the supernatant was collected and eluted to give 4-deoxygadusol (fig. 1).
Further, the induction temperature is 16-20 ℃, the rotation speed of the shaking table is 150-180 rpm, and the induction time is 18-22 h.
Further, the ice bath ultrasonic crushing is carried out in pure water, the ultrasonic power is 20kHZ, the ultrasonic frequency is 5s of ultrasonic time for 2s, and the total crushing time is 10min.
The invention also provides an application of the 4-deoxygadusol synthesized by the method for synthesizing the MAAs precursor substance 4-deoxygadusol in preparing sunscreens, cosmetics or skin care products for protecting skin from ultraviolet injury.
Compared with the prior art, the invention has the advantages and beneficial technical effects that:
the invention optimizes 2-epi-5-epi-valiolone synthase (EEVS) and o-methyltransferase (OMT) genes from Antarctic ice algae Phaeodactylum tricornutum ICE-H, constructs recombinant plasmid by utilizing the optimized genes, introduces the recombinant plasmid into escherichia coli BL21 (DE 3) to obtain recombinant strain capable of simultaneously expressing two genes, and leads the escherichia coli to produce MAAs synthesis precursor substance 4-deoxygadusol in an intracellular way by inducing the strain, and the product obtained by the method is single, simple, feasible and efficient. The 4-deoxygadusol obtained by the invention has stronger in-vitro antioxidant activity and can obviously improve the resistance of skin melanocytes to ultraviolet radiation. In addition, the invention adopts a water extraction method to extract 4-deoxygadusol from escherichia coli thalli, avoids adopting organic reagents, is green and pollution-free, lays a technical foundation for the subsequent application of the escherichia coli thalli in ultraviolet protection products, and has important significance for the research of the escherichia coli thalli in the field of skin ultraviolet injury protection.
Drawings
FIG. 1 is a schematic representation of the MAAs synthesis pathway;
FIG. 2 is a schematic diagram of the functional domain prediction of EEVS and OMT proteins;
FIG. 3 is a graph of codon versus frequency radar; wherein A is an EEVS gene original sequence radar chart, B is an EEVS gene optimized sequence radar chart, C is an OMT gene original sequence radar chart, and D is an OMT gene optimized sequence radar chart;
FIG. 4 is a schematic diagram of recombinant plasmid pACYCDuet 1-EEVS-OMT;
FIG. 5 is a schematic diagram of PCR detection bands of recombinant plasmid pACYCDuet 1-EEVS-OMT;
FIG. 6 is a schematic illustration of a 4-deoxygadusol UV full-wavelength scan;
FIG. 7 is a graph of the results of a 4-deoxygadusol LC-MS/MS assay;
FIG. 8 is a graph showing the results of in vitro antioxidant activity of 4-deoxygadusol;
FIG. 9 is a graph showing the result of evaluation of cytotoxicity of 4-deoxygadusol;
FIG. 10 is a graph showing the UV protective activity of 4-deoxygadusol on skin melanocytes.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
In the following examples, plasmid pACYCDuet1 was purchased from Bio-wind Inc., E.coli DH 5. Alpha. And BL21 (DE 3) were purchased from Beijing full gold, and the optimized EEVS and OMT sequences were synthesized by Shanghai BioInd.
EXAMPLE 1 construction of plasmid for the synthetic pathway of Botrytis antarctica 4-deoxygadusol
In this example, the EEVS and OMT original gene sequences were obtained by local BLAST on the Anemone antarctica Phaeodactylum tricornutum ICE-H transcriptome (sequences shown in SEQ ID NO.1 and SEQ ID NO.2, respectively). Blastp alignment of the reference sequence with the predicted protein sequence of the Antarctic ice algae Phaeodactylum tricornutum ICE-H transcriptome, parameter E-value=1e -5 The method comprises the steps of carrying out a first treatment on the surface of the Then the solid analysis is carried out to extract identity>＝10，align_ratio>Protein sequence=10%; the PfamScan was used to search whether the protein sequence contained a complete domain, and the gene containing the complete domain was used as a candidate gene. Finally, the screened gene sequences are compared and analyzed, and the synthetic route of MAAs in the Antarctic ice algae Phaeodactylum tricornutum ICE-H is presumed. EEVS and OMT protein functional domain prediction schemes are shown in FIGS. 2A and 2B, respectively.
To facilitate expression of the recombinant gene in the host organism, the EEVS and OMT gene sequences were sequence optimized according to the host e.coli codon bias. FIG. 3 is a graph of codon versus frequency radar showing the frequency of each individual codon versus codon frequency distribution, showing the applicability of the codon usage profile between the optimization sequence (red display) and the host (blue display). The optimized EEVS and OMT gene sequences are shown as SEQ ID NO.3 and SEQ ID NO.4 respectively, and are synthesized by Shanghai biological limited company.
The construction method of the plasmid pACYCDuet1-EEVS-OMT is as follows:
(1) And selecting proper enzyme cutting sites according to the vector sequence and the target gene sequence. The restriction enzyme site selected by the vector pACYCDuet1 MCS1 site is BamHI/HindIII, and the restriction enzyme site selected by the MCS2 site is NdeI/XhoI. The desired fragment EEVS was first ligated to MCS1 site, and vector pACYCDuet1 was double digested with BamHI and HindIII enzymes, digested for 15 minutes at 37℃and stopped at a final concentration of 1X by adding 10X DNA Loading Buffer. Recovery was performed using 1.0% agarose, and the linearized vector recovered and the target fragment were prepared according to a ratio of 5:2, and the directional cloning is completed under the catalysis of homologous recombinase Exnase. Optimal cloning vector usage (ng): 0.02 Xcloning vector base pair number; optimal insert usage (ng): 0.04 x base pair of insert. The ligation system was as follows (total reaction system 20. Mu.L): linearization vector 2.5. Mu.L, target fragment EEVS 1. Mu.L, 5 XCE II Buffer 4. Mu.L, exnase II 2. Mu.L, ddH 2 O10.5. Mu.L. After gently stirring and mixing, the mixture was reacted in a PCR instrument at 37℃for 30min and cooled on ice.
(2) Conversion: adding 10 μl of recombinant product into 100 μl DH5 α clone competence, mixing the light elastic tube wall, standing on ice for 30min, and heat-shock at 42deg.C for 45s. Immediately cooling on ice for 2-3min; 750. Mu.L of LB medium is added to the transformed product in a super clean bench, and the mixture is cultured for 1 hour at 37 ℃ and 200rpm, then 50. Mu.L of bacterial liquid is evenly coated on a LB medium plate added with chloramphenicol (preheated at 37 ℃), and the plate is cultured for 12-16 hours at 37 ℃ in an inverted mode. 5-6 single colonies are picked from a flat plate, cultured in 800 mu L of LB liquid medium added with chloramphenicol at 37 ℃ under shaking at 200rpm for 3-6 hours, and EEVS genes are amplified by using a primer with the bacterial liquid as a template, wherein the primer sequence is as follows:
EEVS-F:TCATCACCACAGCCAGGATCCGATGACCCTGAAATCTCTGCGTAA(SEQ ID NO.5)；
EEVS-R:GCATTATGCGGCCGCAAGCTTTTACGGTTCGAATTCTTTCCAAA(SEQ ID NO.6)；
the PCR system is as follows: 1. Mu.L of bacterial solution, 1. Mu.L of forward and reverse primers, 1X Phnata Max Master Mix. Mu.L of forward and reverse primers, and ddH 2 O7 μl; the PCR procedure was: 95 ℃ for 3min, then 35 cycles, each cycle comprising 95 ℃ for 15s,55 ℃ for 15s,72 ℃ for 90s, and finally 72 ℃ for 5min; the PCR detection bands are shown in FIG. 5.
(3) Performing double digestion on pACYCDuet1-EEVS plasmid inserted into EEVS fragment by using NdeI and XhoI endonuclease, wherein the digestion and glue recovery steps are the same as those described above; connecting OMT fragments with a linearized pACYCDuet1-EEVS vector, and connecting, transforming and bacterial liquid PCR steps are the same as those described above; the primers used were:
OMT-F:TAAGAAGGAGATATACATATGATGGAAGATGATCCGTTCGCG(SEQ ID NO.7)；
OMT-R:GGTTTCTTTACCAGACTCGAGTTACGGGTCCTGCAGGTACTG(SEQ ID NO.8)。
the PCR detection band is shown in FIG. 5, and recombinant plasmid pACYCDuet1-EEVS-OMT (FIG. 4) and recombinant escherichia coli containing the recombinant plasmid are obtained; the nucleotide sequence of the recombinant plasmid pACYCDuet1-EEVS-OMT is shown in SEQ ID NO. 9. The strain with the correct band size is sent to Shanghai biological limited company for sequencing, and the strain with the correct sequencing is preserved in glycerol and is preserved to-80 ℃.
EXAMPLE 2 preparation of 4-deoxygadusol by recombinant E.coli
At the same time activating the heavy material at 37 DEG CGroup E.coli and empty plasmid E.coli were inoculated in 600mL of LB liquid medium containing 15. Mu.g/mL chloramphenicol at an inoculum size of 1%, and left to OD 600 When the concentration reaches 0.8, IPTG with the final concentration of 0.1mM is added, the temperature is 18 ℃, the speed is 150rpm, the induction is carried out for 20 hours, and after the induction is finished, the bacterial cells are collected by centrifugation at 6000rpm at 4 ℃. Freeze drying thallus at-60deg.C, adding 10mL pure water into every 0.2g, ultrasonic crushing with ultrasonic power of 20kHZ for 5s, suspending for 2s, crushing total time of 10min, centrifuging at low temperature (10000 rpm,10 min), and collecting supernatant.
The recombinant escherichia coli extract is subjected to ultraviolet full-wave scanning at 200-400 nm, the scanning result is shown in figure 6, the extract has obvious absorption peak at 294nm, and the ultraviolet absorption characteristic of 4-deoxygadusol is met.
EXAMPLE 3 purification of crude 4-deoxygadusol sample Using gel column
The supernatant from example 2 was loaded onto a 2% column volume, separated by Sephadex LH-20 column using pure water as eluent, and the fractions containing the MS-identified product were combined and lyophilized. LC-MS/MS conditions were as follows: using Agilent qtof6550, run in positive ion mode, analytes were separated on Agilent C18 (2.1 mm x 100mm,1.7 μm), eluting with 1% methanol for 20 minutes, then gradient eluting from 1% to 95% methanol over 20 minutes, flow rate 0.3mL/min. The LC-MS/MS detection results of the recombinant strain extract are shown in FIG. 7, and the results show that the recombinant strain successfully synthesizes 4-deoxygadusol.
Example 4 4 Activity assay of Deoxygadusol
(1) In vitro antioxidant Activity detection procedure
The freeze-dried 4-deoxygadusol was prepared into three concentration gradients of 10mg/mL, 20mg/mL and 40mg/mL by using pure water, and the ABTS and hydroxyl radical scavenging activity of the sample were determined by using a Soxhlet kit, respectively, and three replicates were made for each concentration.
As a result, as shown in FIG. 8, the maximum clearance of 4-deoxygadusol to ABTS was 92%, the maximum clearance to hydroxyl radicals was 78.2%, and there was a clear concentration dependence. A large amount of data show that the occurrence mechanism of various difficult and complicated diseases such as inflammation, tumor, aging, hematopathy, heart, liver, lung, skin and the like has a close relationship with the excessive generation of free radicals in the body or the reduced capability of scavenging the free radicals. The scavenging activity of 4-Deoxygadusol on free radicals shows that the compound has wide application potential in the field of medicines.
(2) UV protection of skin cells by 4-Deoxygadudosol
A. Evaluation of cytotoxicity of 4-deoxygadusol
The toxicity of 4-deoxygadusol to skin melanocytes was evaluated using CCK-8. 4-deoxygadusol was added to cells at a series of concentrations (25, 50, 75, 100, 125, 150 and 175. Mu.M), incubated in a cell incubator for 24 hours, removed, 10. Mu.L of CCK-8 was added to each well, and absorbance was measured at 450nm after further incubation for 2 hours. Cell viability formula: cell viability = [ (a) s －A b )/(A c －A b )]X 100%, where A s For experimental hole A c For control wells, A b Is a blank hole.
As shown in FIG. 9, after the treatment for 24 hours at different concentrations of 4-deoxygadusol, the cell activity was not significantly inhibited, indicating that 4-deoxygadusol has no toxic or side effect on skin melanocytes in this concentration range.
B. Irradiation of skin melanocytes with UVB lamp with irradiance of 30mJ/cm 2 After 24h treatment with 175. Mu.M 4-deoxygadusol, the cell activity was determined using CCK-8.
As shown in FIG. 10, the cell activity of the 4-deoxygadusol treated cells was significantly higher than that of the untreated group (p < 0.01), indicating that the 4-deoxygadusol had good protective effect on the cell viability of skin melanocytes after UVB irradiation.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (10)
1. A biosynthesis gene of MAAs precursor 4-deoxygadusol, characterized in that the biosynthesis gene comprises EEVS gene and OMT gene, and the nucleotide sequence is optimized through the codon preference of escherichia coli; the nucleotide sequence of the EEVS gene is shown as SEQ ID NO. 3; the nucleotide sequence of the OMT gene is shown as SEQ ID NO. 4.
2. A recombinant plasmid comprising the biosynthetic gene of claim 1.
3. The recombinant plasmid according to claim 2, wherein the recombinant plasmid contains both EEVS gene and OMT gene, and the nucleotide sequence is shown in SEQ ID No. 9.
4. A recombinant strain comprising the biosynthetic gene of claim 1.
5. The recombinant strain according to claim 4, wherein the recombinant strain is capable of simultaneously expressing EEVS gene and OMT gene, comprising the steps of:
(1) Performing double digestion on an expression vector pACYCDuet1, connecting with the EEVS gene, transforming escherichia coli, and screening positive transformants to obtain a strain containing recombinant plasmid pACYCDuet 1-EEVS;
(2) And (3) carrying out double enzyme digestion on the recombinant plasmid pACYCDuet1-EEVS to connect with the OMT gene, and screening positive transformants to obtain the recombinant strain containing the recombinant plasmid pACYCDuet 1-EEVS-OMT.
6. Use of the biosynthetic gene of claim 1, or the recombinant plasmid of claim 3, or the recombinant strain of claim 4, for the biosynthesis of the MAAs precursor 4-deoxygadusol.
7. A method for biosynthesis of MAAs precursor 4-deoxygadusol, comprising the steps of: after culturing the recombinant strain according to claim 4, the recombinant strain is induced by isopropyl-beta-D-thiogalactoside, and the cells are collected by centrifugation, dried and subjected to ice bath ultrasonication, and the supernatant is collected and eluted to obtain 4-deoxygadusol.
8. The use according to claim 7, wherein the induction temperature is 16-20 ℃, the rotation speed of the shaking table is 150-180 rpm, and the induction time is 18-22 h.
9. The use according to claim 7, wherein the ice bath ultrasonic crushing is carried out in pure water with an ultrasonic power of 20kHZ and an ultrasonic frequency of 5 seconds of pause for 2 seconds, the total crushing time being 10 minutes.
10. Use of 4-deoxygadusol synthesized by the method of biosynthesis of MAAs precursor 4-deoxygadusol according to claim 7 for the preparation of sunscreens, cosmetics or skin care products for protecting skin against uv damage.
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