Process for the efficient synthesis of the bacteriochlorin amino acids shinorine and porphyra-334 compositions

Abstract

The application discloses a method for efficiently synthesizing a mycosporine-like amino acid Shinorine and Porphyra-334 composition, and comprises the following steps: transforming biosynthesis genes of the mycosporine-like amino acid Shinorine and Porphyra-334 composition into an Escherichia coli expression strain to obtain a recombinant strain capable of simultaneously expressing MysA, MysB, MysC and MysD genes; adding isopropyl-beta-D-thiogalactoside to the recombinant strain for induction and continuing to culture, then collecting bacterial bodies, and harvesting the mycosporine-like amino acid Shinorine and Porphyra-334 from the bacterial bodies. The application makes the Escherichia coli produce two MAAs substances of Shinorine and Porphyra-334 in cells, can obtain a combined type MAA product, is simple, feasible and efficient, and lays a technical foundation for subsequent application of scale production of MAAs.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for efficiently synthesizing a composition of spore-like amino acids Shinorine and Porphyra-334. Background Technology

[0002] Mycospore-like amino acids (MAAs) are a class of natural UV protectants with significant application value. Their characteristic absorption spectrum of 310-365 nm fully covers the UV-B (280-315 nm) and UV-A (315-400 nm) bands. MAA complexes, represented by Shinorine and Porphyra-334, not only exhibit significant UV absorption capabilities but also achieve a dual skin protection effect by inhibiting collagen and elastin degradation—acting as photoaging inhibitors to delay skin aging and as photodamage protectants to reduce the risk of skin diseases. Therefore, they are of great value in the development of functional skincare products and medical photoprotective agents. Current research on the heterologous biosynthesis of MAAs has been carried out in multiple microbial chassis systems, with the *E. coli* system being the preferred platform due to its mature genetic manipulation, short fermentation cycle, and low cost. However, current research on heterologous synthesis of MAAs in E. coli faces two major technical bottlenecks: first, all exogenous genes are derived from a single donor species, limiting the optimization space for metabolic pathways; second, existing studies have achieved qualitative detection of MAAs but lack yield enhancement. Developing superior E. coli engineered strains that produce a combination of Shinorine and Porphyra-334 is of great significance for the biotechnology-based production of MAAs. Summary of the Invention

[0003] One object of the embodiments of the present invention is to solve at least the above-mentioned problems and / or defects, and to provide at least the advantages described below.

[0004] Another object of the present invention is to provide a method for efficiently synthesizing a composition of spore-like amino acids Shinorine and Porphyra-334.

[0005] To achieve the above-mentioned objectives, the present invention employs the following technical solution:

[0006] Firstly, the biosynthetic protein of the combination of the spore-like amino acids Shinorine and Porphyra-334 is a protein as follows a) or b) or c):

[0007] a) The biosynthetic proteins include MysA, MysB, MysC, and MysD, wherein MysA, MysB, and MysC are derived from *Anabaena variabilis*, and MysD is derived from *Nostoc punctiforme*.

[0008] b) Includes the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 4,

[0009] c) A protein derived from b) by substitution, deletion or addition of one or more amino acids of the amino acid sequence in b), or an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the sequences in b).

[0010] Secondly, the biosynthetic gene of the spore-like amino acid Shinorine and Porphyra-334 composition encodes the biosynthetic protein of the spore-like amino acid Shinorine and Porphyra-334 composition.

[0011] Preferably, the base sequence of the biosynthetic gene of the spore-like amino acid Shinorine and Porphyra-334 composition includes nucleotide sequences as shown in SEQ ID NO: 5, 6, 7, 8, or nucleotide sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of these sequences.

[0012] Thirdly, a recombinant vector containing a biosynthetic gene of the spore-forming amino acid combination Shinorine and Porphyra-334 and a regulatory sequence for expression operatively linked to the biosynthetic gene of the spore-forming amino acid combination Shinorine and Porphyra-334.

[0013] Fourthly, a recombinant host cell comprising the said nucleotide sequence or the said recombinant vector.

[0014] Fifthly, a method for efficiently synthesizing a composition of spore-like amino acids Shinorine and Porphyra-334, utilizing the aforementioned biosynthetic protein, or the aforementioned biosynthetic gene, or the aforementioned recombinant vector, or the aforementioned recombinant host cell to synthesize the composition of spore-like amino acids Shinorine and Porphyra-334.

[0015] Preferably, the method for efficiently synthesizing the spore-like amino acid combination Shinorine and Porphyra-334 includes the following steps:

[0016] 1) The biosynthesis gene of the combination of spore-like amino acids Shinorine and Porphyra-334 was transformed into an Escherichia coli expression strain to obtain a recombinant strain that can simultaneously express the MysA, MysB, MysC and MysD genes.

[0017] 2) The recombinant strain was cultured to OD200. 600 When the concentration is 0.5-0.6, isopropyl-β-D-thiogalactoside is added to achieve a final concentration of 480-520 μM for induction and continued culture. Then, the bacterial cells are collected, and the spore-like amino acids Shinorine and Porphyra-334 are harvested from the bacterial cells.

[0018] Preferably, in the method for efficiently synthesizing the spore-like amino acid combination Shinorine and Porphyra-334, the biosynthetic gene of the spore-like amino acid combination Shinorine and Porphyra-334 includes the nucleotide sequences shown in SEQ ID NO: 5, 6, 7, and 8.

[0019] In step 2), the induction temperature is 18℃-22℃, the shaking speed is 180rpm-200rpm, and the induction time is 24h-36h.

[0020] Preferably, in the method for efficiently synthesizing the spore-like amino acid Shinorine and Porphyra-334 composition, the collected bacterial cells are subjected to ice bath ultrasonic disruption, and the extract is collected. The spore-like amino acid Shinorine and Porphyra-334 are present in the extract. The ice bath ultrasonic disruption is carried out in water, with an ultrasonic power of 20 kHz, an ultrasonic frequency of 5 seconds of ultrasonic stimulation followed by a 2-second pause, and a total disruption time of 10 minutes.

[0021] Sixthly, the use of a polypeptide in the preparation of a composition of spore-like amino acids Shinorine and Porphyra-334, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:4, and preferably, the substrate for preparing the composition of spore-like amino acids Shinorine and Porphyra-334 is Escherichia coli.

[0022] Compared with the prior art, the advantages and beneficial technical effects of the present invention are:

[0023] This invention optimizes the genes of 3-dehydroquinatesynthase (MysA), o-methyltransferase (MysB), and ATP-grasp enzyme (MysC) from *Anabaena variabilis*, and the gene of D-Ala-D-Ala ligase homolog (MysD) from *Nostoc punctiforme*. A recombinant plasmid was constructed using the optimized genes and introduced into *Escherichia coli* BL21(DE3) to obtain a recombinant strain simultaneously expressing all four genes. By inducing this strain, *E. coli* produced two MAAs substances, Shinorine and Porphyra-334. This method yields a combined MAAs product that is simple, feasible, and efficient, laying a technical foundation for the subsequent large-scale production of MAAs.

[0024] Other advantages, objectives, and features of the embodiments of the present invention will be apparent in part from the following description, and in part will be understood by those skilled in the art through study and practice of the embodiments of the present invention. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the synthesis pathways of the MAAs substances Shinorine and Porphyra-334 in this invention.

[0026] Figure 2 This is a schematic diagram of the recombinant plasmid pET-29b(+)-MAAs in this invention.

[0027] Figure 3 This is a sequencing result diagram of the recombinant plasmid pET-29b(+)-MAAs in this invention.

[0028] Figure 4 This is a schematic diagram of the ultraviolet full-wavelength scan of the recombinant Escherichia coli extract in this invention.

[0029] Figure 5 This is a chromatogram of the HPLC detection results of the recombinant Escherichia coli extract in this invention.

[0030] Figure 6 This is a graph showing the results of Shinorine mass spectrometry detection in this invention. Detailed Implementation

[0031] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, further explains the invention. The specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0032] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0033] Using modern bioengineering techniques to perform heterologous biosynthesis of MAAs in rapidly growing and easily cultured microorganisms has become a practical and promising solution to address the continued growth in global demand for sunscreens.

[0034] This invention provides a biosynthetic gene for the combination of MAAsShinorine and Porphyra-334, such as Figure 1 As shown, it includes MysA, MysB, MysC and MysD genes, whose nucleotide sequences have been optimized for E. coli codon preference; the nucleotide sequences of the MysA, MysB, MysC and MysD genes are shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0035] Furthermore, the amino acid sequences encoded by the MysA, MysB, MysC, and MysD genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively.

[0036] Furthermore, the MysA, MysB, and MysC genes are derived from *Anabaena variabilis*, and the MysD gene is derived from *Nostoc punctiforme*.

[0037] This invention also provides a recombinant plasmid (such as...) Figure 2 As shown), it contains the aforementioned biosynthetic gene.

[0038] Furthermore, the recombinant plasmid contains MysA, MysB, MysC, and MysD genes simultaneously.

[0039] Furthermore, the original plasmid of the recombinant plasmid is pET-29b(+), and the MAAs synthesis gene cluster is controlled by the T7 promoter.

[0040] The present invention also provides a recombinant strain containing the aforementioned biosynthetic gene.

[0041] Furthermore, the recombinant strain can simultaneously express the MysA, MysB, MysC and MysD genes.

[0042] Furthermore, the recombinant strain was obtained by transforming the above-mentioned recombinant plasmid into Escherichia coli BL21(DE3).

[0043] This invention also provides a method for biosynthesizing two MAAs, Shinorine and Porphyra-334, comprising the following steps: culturing the recombinant strain, inducing it with isopropyl-β-D-thiogalactoside, collecting the bacterial cells by centrifugation, performing ultrasonic disruption in an ice bath, and collecting the extract.

[0044] Furthermore, the induction temperature is 16℃-20℃, the shaking speed is 150rpm-200rpm, and the induction time is 18h-24h.

[0045] Furthermore, the ice bath ultrasonic disruption is carried out in pure water, with an ultrasonic power of 20 kHz, an ultrasonic frequency of 5 seconds of ultrasonic stimulation followed by a 2-second pause, and a total disruption time of 10 minutes.

[0046] This invention identifies two MAAs, Shinorine and Porphyra-334, by detecting the extract using ultraviolet full-wavelength scanning, liquid chromatography, and mass spectrometry. In the following examples, Escherichia coli BL21(DE3) was purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd., and the gene sequence was synthesized by Nanjing Genscript Biotech Co., Ltd.

[0047] Example 1

[0048] This embodiment describes the construction and transformation of plasmids for the MAAs synthesis pathway.

[0049] (1) Plasmid construction: In this embodiment, the original gene sequences of MysA, MysB, and MysC were derived from *Anabaena variabilis*, and the original gene sequence of MysD was derived from *Nostoc punctiforme*. The amino acid sequences encoded by the MysA, MysB, MysC, and MysD genes are shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. The codon-optimized gene sequences of MysA, MysB, MysC, and MysD (sequences shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively) were synthesized and inserted into the pET-29b(+) plasmid. The plasmid map is shown below. Figure 2 As shown.

[0050] SEQ ID NO: 1:

[0051] msivqakfeaketsfhvegyekieydlvyvdgifeiqnsaladvyqgfgrclaivdanvsrlygnqiqayfqyygielrlfpititepdktiqtfervidvfadfklvrkepvlvvggglitdvvgfacstyrrssnyiripttliglidasvaikvavnhrklknrlgayhasrkvfldfsllrtlptdqvrngmaelvkiavvahqevfellekygeellrthfgnidatpeikeiahrltykaihkmlelevpnlheldldrviayghtwsptlelaprlpmfhghavnvdmafsatiaarrgyitiaerdrilglmsrvglsldhpmldidilwrgtesitltrdgllraampkpigdcvfvndltreelaaaladhkelctsyprggegvdvypvyqkeligsvk

[0052] SEQ ID NO:2:

[0053] mtnvivqptarpvtplgiltkqleaivqevkqhpdlpgelianihqawrlaagidpyleecttpespelaalakttateawgehfhggttvrpleqemlsghiegqtlkmfvhmtkakkvleigmftgysalamaealpedgllvacevdpyaaeigqkafqqsphggkirveldaalatldklaeagesfdlvfidadkkeyvayfhkllgssllapdgficvdntllqgevylpaeersvngeaiaqfnhtvaidprveqvllplrdgltiirriqp

[0054] SEQ ID NO:3:

[0055] Maqslplssapatpslpsqtkiaaiiqnictlallllalpinativfisllvfrpqkvkaanpqtilisggkmtkalqlarsfhaaghrvvlvethkywltghrfsqavdkfytvpapqdnpqayiqalvdivkqenidvyipvtspvgsyydslakpelshycevfhfdaditqmlddkfaltqkarslglsvpksfkitspeqvinfdfsgetrkyilksipydsvrrldltklpcatpeetaafvrslpitpekpwimqefipgkefcthstvrngelrlhcccessafqvnyenvnnpqitewvqhfvkelkltgqisfdfiqaedgtvyaiecnprthsaittfydhpqvaeaylsqapttetiqplttskptywtyhevwrltgirsftqlqrwlgniwrgtdaiyqpddplpflmvhhwqipllllnnlrrlkgwtridfnigklvelggd

[0056] SEQ ID NO:4:

[0057] Mpvlnilhlvgsahdkfycdlsrlyaqdclaatadpslynfqiayitpdrqwrfpdslsredialtkpipvfdaiqfltgqnidmmlpqmfcipgmtqyralfdllkipyigntpdimaiaahkarakaiveaagvkvprgellrqgdiptitppavvkpvssdnslgvvlvk

[0058] dvteydaalkkafeyaseviveafielgrevrcgiivkdgeliglpleeylvdphdkpirnyadklqqtddgdlhltakdnikawildpn

[0059] dpitqkvqqvakrchqalgcrhyslfdfridpkgqpwfleaglycsfapksvissmakaagiplndllitainetlgsnkkvlqn

[0060] SEQ ID NO:5:

[0061]

[0062] SEQ ID NO:6:

[0063] Ctaacaaatgttatagtacaacccactgctcgcccggttaccccgcttggcatcctgacaaagcaactggaggcgatcgtgcaggaggtgaaacaacatccggacctgccgggtgagctgattgctaacattcatcaggcatggcgtctagctgcgggtatcgacccgtacctggaagagtgcactacgccggagtccccggagctggcggccctggctaaaaccaccgcaacggaagcgtggggtgaacactttcacggcggcaccaccgttcgtccgttggagcaagaaatgctgagcggtcatattgaaggtcagaccctgaaaatgttcgtgcacatgaccaaggcgaagaaggtgttggaaattggcatgttcaccggttacagcgcactcgctatggcagaagcgttgccagaagatggcctgttggtcgcctgcgaagtagacccgtacgcagctgagatcggccagaaagcgtttcaacagtccccgcatggtggcaaaatccgcgtggaactcgacgcggcgctggcgacgctggataagctggcggaggccggcgagtcgttcgatctggtgtttatcgatgcggataagaaagaatatgttgcgtatttccacaaactgttgggtagctctctgttggctccggacggttttatttgtgttgacaacaccttgttacaaggtgaggtctatttgccagcagaggaacgtagcgttaatggtgaggcgatcgcccaattcaaccacaccgtggccatcgaccctcgtgttgaacaggttctgctgccgctgcgtgatggcctgaccattatccgccgtattcagccgtaa

[0064] SEQ ID NO:7:

[0065]

[0066] SEQ ID NO:8:

[0067]

[0068] (2) Transformation: Add 5 μL of plasmid to 200 μL of BL21(DE3) competent cells, gently tap the tube wall to mix, incubate on ice for 45 min, and heat shock at 42℃ for 45 s. Immediately cool on ice for 2 min; add 500 μL of LB medium to the transformation product in a clean bench, and incubate at 37℃ and 250 rpm for 1 h. After centrifugation (5000 rpm, 1 min), discard 400 μL of supernatant. Reselect the remaining bacterial culture and spread it evenly on LB agar plates supplemented with kanamycin. Incubate the plates upside down at 37℃ for 15 h. Pick 5 single colonies from the plates and add them to 500 μL of LB liquid medium supplemented with kanamycin. Incubate at 37℃ and 200 rpm with shaking for 6 h. Send the bacterial culture to Tianjin Qingke Biotechnology Co., Ltd. for sequencing. The sequencing primers are:

[0069] MAAs-MysA-F: catgagcccgaagtggcgag (SEQ ID NO: 9);

[0070] MAAs-MysD-F:cgttgcggtattatcgttaag (SEQ ID NO: 10);

[0071] Sequencing results as follows Figure 3 As shown, the sequence is consistent with the optimized gene sequence. The correctly sequenced strain was preserved in glycerol and stored at -80°C.

[0072] Example 2

[0073] This embodiment describes the preparation of combinatorial MAAs from recombinant Escherichia coli and the detection of related components.

[0074] (1) Preparation of extract: *E. coli* containing the expression plasmid was cultured overnight in LB medium supplemented with kanamycin. 1 mL of the seed culture was inoculated into 100 mL of LB medium supplemented with kanamycin and cultured at 37°C and 250 rpm. The OD was collected... 600 After inoculation with 480 μM isopropyl-β-D-thiogalactopyranoside at 0.5°C for 24 hours at 180 rpm, the cells were collected by centrifugation at 6000 rpm at 4°C after induction. The cells were resuspended in pure water and then sonicated under ice bath conditions. The sonication power was 20 kHz, the sonication frequency was 3.0 s sonication followed by a 2.5 s pause, and the total sonication time was 10 min. The supernatant was collected by centrifugation at 4°C (12000 rpm, 30 min).

[0075] (2) Ultraviolet full-wavelength scanning detection:

[0076] The above-mentioned Escherichia coli extract was subjected to a full-wavelength ultraviolet scan from 200 to 600 nm. Using the bacterial cell extract with empty pET-29b(+) vector as a control, the scanning results of the recombinant Escherichia coli extract were compared. Figure 4 As shown, the extract has a significant absorption peak at 334 nm, which is consistent with the UV absorption characteristics of MAAs substances Shinorine and Porphyra-334.

[0077] (3) HPLC detection:

[0078] The above-mentioned *E. coli* extract was analyzed by HPLC (10 μL sample volume) at a detection wavelength of 330 nm, using a heated high-carbohydrate column (150 mm × 4.6 mm, 5 μm pore size) at a flow rate of 1.5 mL / min. The following gradient elution was used (mobile phase A was 0.3% ammonium formate aqueous solution, pH 9.0; mobile phase B was acetonitrile): 0 min 2% B, 20 min 15% B, 26 min 50% B, 27-33 min 90% B, 35-40 min 2% B, and 40.01 min (end). The results are shown in the figure below. Figure 5 As shown, the extract exhibited distinct chromatographic peaks at 10.282 min, 11.147 min, and 11.553 min. The elution times of the MAAs substances Shinorine, Porphyra-334, and the standards Mycosporine-glycine (MG) were consistent. MG, as a substrate for the formation of Shinorine and Porphyra-334, showed a lower concentration in the extract compared to the two products, indicating that MG was almost entirely converted into the two MAAs products, demonstrating the high catalytic efficiency of MysD. Based on calculations using the standards, the concentration of Shinorine in the culture medium was 1.5 mg / L, and the concentration of Porphyra-334 was 0.5 mg / L.

[0079] (4) Separation and purification of MAAs

[0080] The *E. coli* extract was frozen at -80°C for 12 hours, and then freeze-dried in a vacuum freeze dryer. The resulting lyophilized powder was resuspended in methanol and centrifuged to collect the supernatant. The supernatant was purified by preparative liquid chromatography (HPLC) with an injection volume of 1 mL, a detection wavelength of 330 nm, and an Agilent ODS-2 preparative HPLC column (250 mm × 21.2 mm, 10 μm pore size) at a flow rate of 10 mL / min. Isocratic elution was performed using 2% acetonitrile as the mobile phase. The single MAAs product peak was collected and freeze-dried to obtain the pure product.

[0081] (5) Mass spectrometry detection of pure MAAs

[0082] The lyophilized pure MAAs were analyzed by time-of-flight mass spectrometry using a T3 column (100 mm × 2.1 mm, 1.8 μm pore size). Mobile phase A was 0.1% acetic acid, and mobile phase B was acetonitrile. The elution program was 0 min 1% B, 1.5 min 1% B, 15 min 99% B, 20 min 99% B, 21 min 1% B, and 30 min (end). The flow rate was 0.3 mL / min, and the injection volume was 1 μL. The detection results are as follows: Figure 6 As shown, the obtained pure product is Shinorine.

[0083] Example 3

[0084] E. coli containing the expression plasmid were cultured overnight in LB medium supplemented with kanamycin. 12 mL of the resulting seed culture was inoculated into 1000 mL of LB medium supplemented with kanamycin and cultured at 37°C with a rotation speed of 230 rpm. Cell density was measured until OD200 reached. 600 After inoculation with 520 μM isopropyl-β-D-thiogalactopyranoside at a concentration of 0.6, the cells were induced at 20°C and 200 rpm for 30 hours. After induction, the cells were collected by centrifugation at 4°C and 6500 rpm. Extraction analysis revealed that the concentration of Shinorine in the culture medium was 2 mg / L, and the concentration of Porphyra-334 was 0.4 mg / L.

[0085] Example 4

[0086] E. coli containing the expression plasmid were cultured overnight in LB medium supplemented with kanamycin. 24 mL of the resulting seed culture was inoculated into 2000 mL of LB medium supplemented with kanamycin and cultured at 37°C and 230 rpm. Cell density was measured until OD200 reached. 600 At a concentration of 0.6, the cells were inoculated with 510 μM isopropyl-β-D-thiogalactopyranoside for induction. Induction was performed at 18°C ​​and 200 rpm for 32 hours. After induction, the cells were collected by centrifugation at 4°C and 6500 rpm. Extraction analysis revealed that the concentration of Shinorine in the culture medium was 2.8 mg / L, and the concentration of Porphyra-334 was 0.55 mg / L.

[0087] Example 5

[0088] E. coli containing the expression plasmid were cultured overnight in LB medium supplemented with kanamycin. 60 mL of the resulting seed culture was inoculated into 5000 mL of LB medium supplemented with kanamycin and cultured at 37°C with a rotation speed of 230 rpm. Cell density was measured until OD200 reached... 600At a concentration of 0.6, the culture medium was inoculated with 500 μM isopropyl-β-D-thiogalactopyranoside for induction. Induction was performed at 22°C and 200 rpm for 36 hours. At the 12th and 24th hours, 1% of the total volume of supplemental medium (pH 8) was added, consisting of 50 g / L yeast extract, 100 g / L peptone, and 50 g / L NaCl. After induction, the cells were collected by centrifugation at 6500 rpm at 4°C. Extraction analysis revealed that the concentration of shinorine in the culture medium was 3.6 mg / L, and the concentration of porphyra-334 was 0.73 mg / L.

[0089] This invention optimizes the genes of 3-dehydroquinatesynthase (MysA), o-methyltransferase (MysB), and ATP-grasp enzyme (MysC) from *Anabaena variabilis*, and the gene of D-Ala-D-Ala ligase homolog (MysD) from *Nostoc punctiforme*. A recombinant plasmid was constructed using the optimized genes and introduced into *Escherichia coli* BL21(DE3) to obtain a recombinant strain simultaneously expressing all four genes. By inducing this strain, *E. coli* produced two MAAs substances, Shinorine and Porphyra-334, within the cells. This method yields a combined MAAs product that is simple, feasible, and efficient, laying a technical foundation for the subsequent large-scale production of MAAs.

[0090] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. It can be applied to various fields suitable for embodiments of the present invention. Other modifications can be readily implemented by those skilled in the art. Therefore, without departing from the general concept defined by the claims and their equivalents, embodiments of the present invention are not limited to the specific details and illustrations shown and described herein.

Claims

1. A biosynthetic protein of a composition of the spore-like amino acids Shinorine and Porphyra-334, characterized in that, Proteins that are a), b), or c) of the following: a) The biosynthetic proteins include MysA, MysB, MysC, and MysD, wherein MysA, MysB, and MysC are derived from *Anabaena variabilis*, and MysD is derived from *Nostoc punctiforme*. b) Includes the amino acid sequences shown in SEQ ID NO: 1, 2, 3, 4, c) A protein derived from b) by substitution, deletion or addition of one or more amino acids of the amino acid sequence in b), or an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the sequences in b).

2. A biosynthetic gene for a combination of the spore-like amino acids Shinorine and Porphyra-334, characterized in that, It encodes the biosynthetic protein of the composition of the mycotoxin amino acids Shinorine and Porphyra-334 as described in claim 1.

3. The biosynthetic gene of the spore-like amino acid Shinorine and Porphyra-334 composition as described in claim 2, characterized in that, Its base sequence includes nucleotide sequences as shown in SEQ ID NO: 5, 6, 7, 8, or nucleotide sequences having at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of these sequences.

4. A recombinant vector, characterized in that, It contains the biosynthesis gene of the spore-like amino acid Shinorine and Porphyra-334 composition as described in claim 2 or 3, and a regulatory sequence for expression operatively linked to the biosynthesis gene of the spore-like amino acid Shinorine and Porphyra-334 composition.

5. A recombinant host cell comprising the nucleotide sequence as described in claim 2 or 3 or the recombinant vector as described in claim 4.

6. A method for efficiently synthesizing a composition of the spore-like amino acids Shinorine and Porphyra-334, characterized in that, The biosynthetic protein of claim 1, or the biosynthetic gene of claim 2 or 3, or the recombinant vector of claim 4, or the recombinant host cell composition of the spore-like amino acid Shinorine and Porphyra-334 of claim 5 can be used.

7. The method for efficiently synthesizing the composition of the mycotoxin-like amino acids Shinorine and Porphyra-334 as described in claim 6, characterized in that, Includes the following steps: 1) The biosynthesis gene of the combination of spore-like amino acids Shinorine and Porphyra-334 was transformed into an Escherichia coli expression strain to obtain a recombinant strain that can simultaneously express the MysA, MysB, MysC and MysD genes. 2) The recombinant strain was cultured to OD200. 600 When the concentration is 0.5-0.6, isopropyl-β-D-thiogalactoside is added to achieve a final concentration of 480-520 μM for induction and continued culture. Then, the bacterial cells are collected, and the spore-like amino acids Shinorine and Porphyra-334 are harvested from the bacterial cells.

8. The method for efficiently synthesizing the composition of the mycotoxin-like amino acids Shinorine and Porphyra-334 as described in claim 7, characterized in that, The biosynthesis gene of the spore-like amino acid Shinorine and Porphyra-334 composition includes nucleotide sequences as shown in SEQ ID NO: 5, 6, 7, and 8. In step 2), the induction temperature is 18℃-22℃, the shaking speed is 180rpm-200rpm, and the induction time is 24h-36h.

9. The method for efficiently synthesizing the composition of the mycotoxin-like amino acids Shinorine and Porphyra-334 as described in claim 7, characterized in that, The collected bacterial cells were subjected to ice bath ultrasonic disruption, and the extract was collected. The spore-like amino acids Shinorine and Porphyra-334 were present in the extract. The ice bath ultrasonic disruption was carried out in water with an ultrasonic power of 20 kHz and an ultrasonic frequency of 5 seconds of ultrasonication followed by a 2-second pause. The total disruption time was 10 minutes.

10. The use of a polypeptide in the preparation of a composition of the spore-like amino acids Shinorine and Porphyra-334, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

4. Preferably, the substrate for preparing the spore-like amino acid Shinorine and Porphyra-334 composition is Escherichia coli.

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