Method for efficiently synthesizing porphyran amino acid porphyra-334

Abstract

The application discloses a method for efficiently synthesizing a mycosporine-like amino acid Porphyra-334, and comprises the following steps: transforming a biosynthesis gene of the mycosporine-like amino acid Porphyra-334 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 culture of the recombinant strain for induction and continuing to culture, then collecting bacterial bodies, and harvesting the mycosporine-like amino acid Porphyra-334 from the bacterial bodies. The application makes the Escherichia coli produce the MAA substance Porphyra-334 in cells, and the method can obtain the MAA product with Porphyra-334 as a main component, and is simple, feasible and efficient, and lays a technical foundation for subsequent scale extraction, purification and production of Porphyra-334.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for the efficient synthesis of the spore-like amino acid Porphyra-334. Background Technology

[0002] Mycospore-like amino acids (MAAs) are highly promising natural sunscreen materials with an absorption spectrum ranging from 310 to 365 nm, capable of simultaneously covering both UV-A (315–400 nm) and UV-B (280–315 nm). They are primarily produced by cyanobacteria, algae, phytoplankton, corals, and numerous other marine organisms exposed to sunlight. As natural products, MAAs are widely available and environmentally friendly. In marine ecosystems, organisms subjected to prolonged exposure to intense sunlight have evolved the ability to synthesize MAAs for survival. This natural sunscreen not only absorbs ultraviolet rays, reducing their damage to cells, but also exerts anti-inflammatory effects at the cellular level, alleviating inflammatory responses caused by ultraviolet radiation. Simultaneously, its antioxidant function can scavenge free radicals, preventing further damage to skin cells, thus protecting skin health in multiple ways. It plays a significant role in preventing skin diseases and delaying skin aging, and has broad application prospects in cosmetics and pharmaceutical research and development. Currently, MAAs obtained through biosynthesis are usually in the form of mixtures, and the synthesis of single-component MAAs, especially single Porphyra-334, remains challenging. This situation significantly increases the technical difficulty of subsequent extraction, separation, and purification processes of Porphyra-334 products. To address this issue, this technology provides a method for synthesizing Porphyra-334. 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 objective of this invention is to provide a method for the efficient synthesis of the spore-like amino acid Porphyra-334.

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

[0006] Firstly, the biosynthetic protein of the spore-like amino acid 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 linckia*.

[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%, 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 the sequences in b).

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

[0011] Preferably, the base sequence of the biosynthesis gene of the spore-like amino acid Porphyra-334 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 the biosynthesis gene of the spore-like amino acid Porphyra-334 and a regulatory sequence for expression operatively linked to the biosynthesis gene of the spore-like amino acid 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 the spore-like amino acid 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 spore-like amino acid Porphyra-334.

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

[0016] 1) The biosynthesis gene of the spore-like amino acid Porphyra-334 was transformed into an Escherichia coli expression strain to obtain a recombinant strain that can simultaneously express MysA, MysB, MysC and MysD proteins; the amino acid sequences of MysA, MysB, MysC and MysD proteins are shown in SEQ ID NO: 1, 2, 3 and 4, respectively.

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

[0018] Preferably, in the method for efficiently synthesizing the spore-like amino acid Porphyra-334, the biosynthetic gene of the spore-like amino acid Porphyra-334 includes the nucleotide sequences shown in SEQ ID NO: 5, 6, 7, 8; in step 2), the induction temperature is 16℃-20℃, the shaking speed is 150rpm-200rpm, and the induction time is 18h-30h.

[0019] Preferably, in the method for efficiently synthesizing the spore-like amino acid Porphyra-334, the collected bacterial cells are subjected to ice bath ultrasonic disruption, and the extract is collected. The spore-like amino acid Porphyra-334 is 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 3.0 s pause and 2.5 s pause, and a total disruption time of 10 min.

[0020] Sixthly, the application of a polypeptide in the preparation of the spore-like amino acid Porphyra-334, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO:4, and preferably, the substrate for preparing the spore-like amino acid Porphyra-334 is Escherichia coli.

[0021] Compared with the prior art, the advantages and beneficial technical effects of the present invention are:

[0022] This invention optimizes the genes of 3-dehydroquinatesynthase (MysA), o-methyltransferase (MysB), and ATP-grasp enzyme (MysC) from *Anabaena variabilis*, and D-Ala-D-Ala ligase (MysD) from *Nostoc linckia*. Recombinant plasmids were constructed using these 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 the MAAs substance Porphyra-334 intracellularly. This method yields MAAs products with Porphyra-334 as the main component, and is simple, feasible, and efficient, laying a technical foundation for the subsequent large-scale production of MAAs.

[0023] 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

[0024] Figure 1 This is a schematic diagram of the synthesis pathway of Porphyra-334, the MAAs in this invention.

[0025] Figure 2 This is a schematic diagram of the recombinant plasmid pET-29b(+)-MAAs in this invention.

[0026] Figure 3 This is a sequencing result diagram of the recombinant plasmid pET-29b(+)-MAAs in this invention.

[0027] Figure 4 This is a schematic diagram of the Porphyra-334 ultraviolet full-wavelength scan in this invention.

[0028] Figure 5 This is a chromatogram of the HPLC detection results of the recombinant Escherichia coli extract in this invention.

[0029] Figure 6 The image shows the HPLC results of the extract of recombinant Escherichia coli using the MysDv gene. Detailed Implementation

[0030] 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.

[0031] 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.

[0032] 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.

[0033] This invention provides a biosynthetic gene for the MAAs substance 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.

[0034] 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.

[0035] Furthermore, the MysA, MysB, and MysC genes are derived from *Anabaena variabilis*, and the MysD gene is derived from *Nostoc linckia*.

[0036] This invention also provides a recombinant plasmid (such as...) Figure 2 As shown), it contains the aforementioned biosynthetic gene.

[0037] Furthermore, the recombinant plasmid contains MysA, MysB, MysC, and MysD genes simultaneously.

[0038] Furthermore, the original plasmid of the recombinant plasmid is pET-29b(+), and the MAAs synthesis gene cluster is controlled by the T7 promoter.

[0039] The present invention also provides a recombinant strain containing the aforementioned biosynthetic gene.

[0040] Furthermore, the recombinant strain can simultaneously express the MysA, MysB, MysC and MysD genes.

[0041] Furthermore, the recombinant strain was obtained by transforming the above-mentioned recombinant plasmid into Escherichia coli BL21(DE3).

[0042] The present invention also provides a method for biosynthesizing the MAAs substance 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.

[0043] Furthermore, the induction temperature is 16℃-20℃, the shaking speed is 150rpm-200rpm, and the induction time is 18h-24h.

[0044] 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.

[0045] This invention identifies the MAAs substance Porphyra-334 by detecting the extract using ultraviolet full-wavelength scanning, liquid chromatography, and mass spectrometry.

[0046] 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 the Porphyra-334 synthetic pathway plasmid.

[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 linckia*. 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] mpvlrilhlvgsaqddfycdlsrlyaqdclaamaelpydsaiayitpdgqwrfprslsrediaqakpmpvseaiefiaaqnidivlpqmfcipgmtyyralfdlleipyigntpdlmaitahkartkaiveaagvkvprgevlrrgdvptitppvvikpvssdnslgvtlvkda aeyeaalekafehgdeaivetfiegrevrcgiivkdgeliglpleeylidsqekpirtyadklkktddgslgfaakgnnkswildpndpitqkvqevakkchqalgcrhyslfdfridsqgqpwfleaglycsfapksvissmakavgiplnelltiaiaetlgsnkysdrisvveinepsktprkerelsqmi

[0058] SEQ ID NO:5:

[0059]

[0060] SEQ ID NO:6:

[0061] Ctaacaaatgttatagtacaacccactgctcgcccggttaccccgcttggcatcctgacaaagcaactggaggcgatcgtgcaggaggtgaaacaacatccggacctgccgggtgagctgattgctaacattcatcaggcatggcgtctagctgcgggtatcgacccgtacctggaagagtgcactacgccggagtccccggagctggcggccctggctaaaaccaccgcaacggaagcgtggggtgaacactttcacggcggcaccaccgttcgtccgttggagcaagaaatgctgagcggtcatattgaaggtcagaccctgaaaatgttcgtgcacatgaccaaggcgaagaaggtgttggaaattggcatgttcaccggttacagcgcactcgctatggcagaagcgttgccagaagatggcctgttggtcgcctgcgaagtagacccgtacgcagctgagatcggccagaaagcgtttcaacagtccccgcatggtggcaaaatccgcgtggaactcgacgcggcgctggcgacgctggataagctggcggaggccggcgagtcgttcgatctggtgtttatcgatgcggataagaaagaatatgttgcgtatttccacaaactgttgggtagctctctgttggctccggacggttttatttgtgttgacaacaccttgttacaaggtgaggtctatttgccagcagaggaacgtagcgttaatggtgaggcgatcgcccaattcaaccacaccgtggccatcgaccctcgtgttgaacaggttctgctgccgctgcgtgatggcctgaccattatccgccgtattcagccgtaa

[0062] SEQ ID NO:7:

[0063]

[0064] SEQ ID NO:8:

[0065]

[0066] (2) Transformation: Add 5 μL of plasmid to 200 μL of competent E. coli BL21(DE3) 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, incubate at 37℃ and 250 rpm for 1 h, centrifuge (5000 rpm, 1 min), discard 400 μL of supernatant, reselect the remaining bacterial culture and spread it evenly on LB medium plates supplemented with kanamycin, and incubate the plates upside down at 37℃ for 15 h. Pick 5 single colonies from the plate and add them to 500 μL of LB liquid medium supplemented with kanamycin, incubate at 37℃ and 200 rpm for 6 h with shaking. Send the bacterial culture to Tianjin Qingke Biotechnology Co., Ltd. for sequencing. The sequencing primers are:

[0067] MAAs-MysA-F: catgagcccgaagtggcgag (SEQ ID NO: 11);

[0068] MAAs-MysD-F:caaattccgctgttgttgtt (SEQ ID NO: 12);

[0069] 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 at -80°C.

[0070] Example 2

[0071] This embodiment describes the preparation of Porphyra-334 from recombinant Escherichia coli.

[0072] (1) Preparation of extract: The *E. coli* strain containing the expression plasmid obtained in Example 1 was cultured overnight in LB medium supplemented with kanamycin and in a control strain containing the empty pET-29b(+) vector. The *E. coli* culture containing the expression plasmid was inoculated at a rate of 1% into 100 mL of LB medium supplemented with 50 μg / mL kanamycin. The culture was cultured until the cell density reached OD200. 600 After inoculation with 480 μM isopropyl-β-D-thiogalactopyranoside at 0.5°C for 24 hours at 200 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).

[0073] (2) Purification of Porphyra-334

[0074] The 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 the supernatant was collected by centrifugation. Porphyra-334 dissolved in methanol 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 Porphyra-334 product peak was collected and freeze-dried to obtain the pure product.

[0075] (3) Ultraviolet full-wavelength scanning detection:

[0076] The pure Porphyra-334 was dissolved in water and then subjected to a full-wavelength ultraviolet scan at 200–600 nm, with water as a control. The scanning results for Porphyra-334 are as follows. Figure 4 As shown, there is a significant absorption peak at 334 nm, which is consistent with the ultraviolet absorption characteristics of Porphyra-334.

[0077] (4) 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, ending at 40.01 min. Results are as follows. Figure 5 As shown, the extract exhibited a distinct chromatographic peak at 10.282 min, consistent with the elution time of the Porphyra-334 standard. The extract contained no impurities with large peak areas; therefore, recombinant E. coli can be used for the synthesis of high-purity Porphyra-334. Based on calculations using the standard, the content of Porphyra-334 was determined to be 2.5 mg / L.

[0079] Example 3

[0080] The *E. coli* strain containing the expression plasmid obtained in Example 1 was cultured overnight in LB medium supplemented with kanamycin and in a control strain containing the empty pET-29b(+) vector. The *E. coli* culture containing the expression plasmid was inoculated at a rate of 1.2% into 1000 mL of LB medium supplemented with 50 μg / mL kanamycin. Cell density was measured until OD200 reached... 600After inoculation with 520 μM isopropyl-β-D-thiogalactopyranoside at a concentration of 0.6, the cells were induced at 20°C and 180 rpm for 30 hours. After induction, the cells were collected by centrifugation at 4°C and 6500 rpm. Extraction analysis revealed that the content of Porphyra-334 was 3 mg / L, and the content of shinorine was 0.2 mg / L. The content of Porphyra-334 accounted for more than 93% of the MAAs, and this high proportion of Porphyra-334 facilitated subsequent extraction, separation, and purification.

[0081] Example 4

[0082] The *E. coli* strain containing the expression plasmid obtained in Example 1 was cultured overnight in LB medium supplemented with kanamycin. Then, the bacterial culture was inoculated at a rate of 1.2% into 2000 mL of LB medium supplemented with 50 μg / mL kanamycin. The culture was cultured until the cell density reached OD0.05. 600 After inoculation with 500 μM isopropyl-β-D-thiogalactopyranoside at a concentration of 0.5, the cells were induced at 16°C and 150 rpm for 18 hours. After induction, the cells were collected by centrifugation at 4°C and 6500 rpm. Extraction analysis revealed that the content of Porphyra-334 was 2.9 mg / L, and the content of shinorine was 0.19 mg / L. The content of Porphyra-334 accounted for more than 93.9% of the MAAs.

[0083] Example 5

[0084] The *E. coli* strain containing the expression plasmid obtained in Example 1 was cultured overnight in LB medium supplemented with kanamycin. Then, the bacterial culture was inoculated at a rate of 1.2% into 5000 mL of LB medium supplemented with 50 μg / mL kanamycin. The culture was cultured until the cell density reached OD200. 600 After inoculation with 490 μM isopropyl-β-D-thiogalactopyranoside at a concentration of 0.6, the cells were induced at 16°C and 190 rpm for 24 hours. After induction, the cells were collected by centrifugation at 4°C and 6500 rpm. Extraction analysis revealed that Porphyra-334 content was 3.2 mg / L and shinorine content was 0.20 mg / L. The content of Porphyra-334 accounted for more than 94.1% of the MAAs.

[0085] Comparative Example 1

[0086] In Example 1, the MysD gene sequence was replaced with MysDv (SEQ ID NO: 9) from *Anabaena variabilis*. Other steps were the same as in Example 3. *E. coli* was transformed to induce the expression of MAAs. The MAAs composition was extracted and detected to be shinorine, with a concentration of 2.3 mg / L (see liquid chromatography results). Figure 6 Porphyra-334 was not detected. The amino acid sequence encoded by MysDv is shown in SEQ ID NO: 10.

[0087] SEQ ID NO: 9:

[0088]

[0089] SEQ ID NO:10:

[0090] mqtidfnirkllvewnathrdydlsqslhelivaqvertpeaiavtfdkqqltyqelnhkanqlghylqtlgvqpetlvgvclersl emvicllgilkaggayvpidpeypqeriaymledsqvkvlltqekllnqiphhqaqticvdrewekistqantnpksniktdnlayviytsgstgkpkgamnthkgicnrllwmqeayqidstdsilqktpfsfdvsvweffwtlltgarlviakpgghkdsaylidlitqeqittlhfvpsmlqvflqnrhvskcsslkrvicsgealsidlqnrffqhlqcelhnlygpteaaidvtfwqcrkdsnlksvpigrpiantqiyildadlqpvnigvtgeiyiggvgvargylnkeeltkekfiinpfpnsefkrlyktgdlarylpdgnieylgrtdyqvkirgyrieigeienvlsshpqvreavviarddnaqekqiiayitynsikpqldnlrdflkarlpdfmipaafvmlehlpltpsgkvdrkalpkpdlfnysehnsyvaprneveeklvqiwsnilhlpkvgvtenffaiggnslkalhlisqieelfakeislatlltnpviadlakviqannqihnsplvpiqpqgkqqpffcihpagghvlcyfklaqyigtdqpfyglqaqgfygdeapltrvedmaslyvktirefqpqgpyrvggwsfggvvayevaqqlhrqgqevsllaildsyvpilldkqkpiddvylvgvlsrvfggmfgqdnlvtpeeienltveekinyiidkarsarifppgverqnnrrildvlvgtlkatysyirqpypgkvtvfrarekhimapdptlvwvelfsvmaaqeikiidvpgnhysfvlephvqvlaqrlqdclenns

[0091] A comparison of Comparative Example 1 and Examples 2-5 shows that when using the biosynthetic gene of the present invention (i.e., containing the amino acid sequence shown in SEQ ID NO:4) with *E. coli* as the substrate, the yield of the spore-like amino acid Porphyra-334 can reach over 93% of the produced spore-like amino acid. This high proportion of Porphyra-334 facilitates subsequent extraction, separation, purification, and application. However, when using MysDv (SEQ ID NO: 9) from *Anabaena variabilis*, only shinorine can be produced, and Porphyra-334 cannot be obtained. This indicates that differences in gene sequences significantly affect the types of biosynthesized products. The following reasons for this phenomenon can be further analyzed from the above:

[0092] The biosynthetic gene of this invention contains the amino acid sequence shown in SEQ ID NO:4. Its specific gene sequence determines that it can direct *E. coli* to synthesize the spore-like amino acid Porphyra-334, achieving a yield of over 93% of the produced spore-like amino acid. However, the gene sequence of *MysDv* (SEQ ID NO: 9) from *Anabaena variabilis* differs from the biosynthetic gene of this invention. This difference leads to its functional specificity, meaning it can only direct the production of shinorine and cannot synthesize Porphyra-334. This difference may be because the difference in gene sequence significantly affects the type of biosynthesized product. *E. coli*, as a chassis cell, exhibits good adaptability and compatibility with the biosynthetic gene of this invention. This gene can be normally expressed and function in *E. coli*, thereby efficiently synthesizing Porphyra-334. However, for the MysDv (SEQ ID NO: 9) gene, possibly due to evolutionary differences between its source organism and *E. coli*, the expression and function of this gene in *E. coli* are restricted, preventing the initiation of the relevant metabolic pathway for Porphyra-334 synthesis and resulting in the synthesis of only shinorine. This may be because the gene cannot activate the key enzymes or metabolic steps required for Porphyra-334 synthesis in *E. coli*, or because certain regulatory mechanisms inhibit the activation of the Porphyra-334 synthesis pathway, thus resulting in only shinorine as a product. This demonstrates the decisive role of differences in metabolic pathways regulated by different genes in the final product and reflects the significant impact of the compatibility between chassis cells and different genes on product synthesis. This invention has significant implications for subsequent scientific research, and the method of this invention yields MAAs products with Porphyra-334 as the main component, and is simple, feasible, and efficient, laying a technical foundation for the subsequent large-scale production of MAAs.

[0093] 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 the spore-like amino acid Porphyra-334, characterized in that, Proteins that are a), b), or c) below: 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 linckia*. 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%, 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 the sequences in b).

2. The biosynthesis gene of the spore-like amino acid Porphyra-334, characterized in that, It encodes the biosynthetic protein of the spore-like amino acid Porphyra-334 as described in claim 1.

3. The biosynthesis gene for the spore-like amino acid Porphyra-334 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 Porphyra-334 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 Porphyra-334.

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 the spore-like amino acid 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 of claim 5, can be used to synthesize the spore-like amino acid Porphyra-334.

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

8. The method for efficiently synthesizing the spore-like amino acid Porphyra-334 as described in claim 7, characterized in that, The biosynthesis gene of the spore-like amino acid Porphyra-334 includes the nucleotide sequences shown in SEQ ID NO: 5, 6, 7, 8; in step 2), the induction temperature is 16℃-20℃, the shaking speed is 150rpm-200rpm, and the induction time is 18h-30h.

9. The method for efficiently synthesizing the spore-like amino acid 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 acid Porphyra-334 was 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 3.0 s for 2.5 s pause. The total disruption time was 10 min.

10. The application of a polypeptide in the preparation of the spore-like amino acid 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 Porphyra-334 is Escherichia coli.

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