A genetically engineered bacillus subtilis for synthesizing mycosporine-glycine, a construction method and application thereof

Abstract

The application discloses a genetically engineered bacillus subtilis for synthesizing mycosporine-glycine (MG), and construction and application thereof, and belongs to the field of microbial metabolic engineering and synthetic biology. The genetically engineered bacillus subtilis is obtained by knocking out a competitive pathway gene and integrating a gene cluster AvmysABC from an anabaena variabilis into a starting bacillus subtilis strain. veg The expression elements are systematically optimized, including using a strong constitutive promoter P43, replacing the original promoter, introducing an optimized 5' untranslated region (UTR) to enhance translation efficiency, and finally integrating the optimized expression cassette into the genome to obtain a genetically stable integrated strain. The yield of MG in the fermentation of the engineered strain in a flask reaches 1.25 g / L, and the yield is further improved to 5.61 g / L under the fed-batch culture condition in a fermenter.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and metabolic engineering, and relates to a genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine, its construction method, and its application. Background Technology

[0002] Mycosporine-like amino acids (MAAs) are a class of natural ultraviolet-absorbing compounds synthesized by microorganisms such as cyanobacteria, microalgae, and some fungi. Their molecular core is a cyclohexeneimine or cyclohexene ketone structure, exhibiting strong absorption properties in the 310-365 nm ultraviolet band. Mycosporine-glycine (MG), as a key basic compound and precursor molecule in the MAAs family, not only possesses excellent ultraviolet protection and antioxidant activity, but is also an essential intermediate in the synthesis of various high-value derivatives such as Shinorine and Porphyra-334, demonstrating enormous application potential in high-end sunscreens, anti-photoaging functional cosmetics, and biomedical materials.

[0003] Currently, the production of commercial MG and its derivatives still heavily relies on direct extraction from macroalgae (such as laver and ulna) or cyanobacteria. However, this natural extraction method has a series of insurmountable limitations: First, algae have long growth cycles and are significantly affected by seasons and geographical environments, leading to unstable raw material supply and large batch-to-batch variations; second, the content of the target product in organisms is extremely low, usually less than 0.1% of cell dry weight, making the extraction process complex, energy-intensive, and yielding, and difficult to control product purity; finally, from the perspective of resource sustainability, large-scale algae cultivation may face problems such as land use, water consumption, and ecological impact. Therefore, relying on natural extraction is not only costly but also difficult to meet the growing market demand. On the other hand, the total chemical synthesis route of MG involves cumbersome steps, multiple chiral control steps, harsh reaction conditions, and often uses toxic reagents, making it neither economically viable nor environmentally friendly, and difficult to achieve green industrialization.

[0004] In recent years, constructing microbial "cell factories" using synthetic biology and metabolic engineering technologies to achieve heterologous biosynthesis of MAAs has become the most promising strategic direction for breaking through the bottlenecks of traditional production models. Among numerous microbial hosts, Bacillus subtilis is considered an ideal chassis cell for producing high-value-added natural products due to its outstanding advantages, including non-pathogenicity, FDA-recognized GRAS (Generally Recognized As Safe) status, clear genetic background, low culture cost, rapid growth, strong protein secretion capacity, and mature genetic manipulation tools. Summary of the Invention

[0005] To address the lack of efficient and safe Mycosporine-glycine (MG) production strains in existing technologies, this invention provides a method for constructing a genetically engineered Bacillus subtilis strain for MG synthesis and its applications. Through genetic engineering, an efficient Mycosporine-glycine synthesis pathway was constructed in the protease-deficient Bacillus subtilis WB600. The methyl-4-deoxycodone synthase gene (AvmysA), O-methyltransferase gene (AvmysB), and ATP-grasp ligase gene (AvmysC) from the algae Anabaena variabilis were heterologously expressed. Furthermore, four promoters of different strengths (P...) were screened on the expression vector pP43NMK. veg P spoVG P yvyd P 43 The plasmids were then transformed into Bacillus subtilis WB600 cells containing the ywjH gene, which had been knocked out, to construct different genetically engineered bacteria. Furthermore, the gene cluster AvmysABC from Anabaenavariabilis was integrated into the genome, and the promoter P was selected. veg In addition to UTR4 expression, it effectively increased the yield of MG in Bacillus subtilis.

[0006] Preferably, the present invention provides a genetically engineered bacterium for synthesizing Mycosporine-glycine, using Bacillus subtilis WB600 as the starting strain, and cloning the methyl-4-deoxycodone synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC from Anabaena variabilis into the plasmid p43NMK (generated by the strong promoter P).veg Expression was driven by an optimized 5' untranslated region (UTR4) to enhance translation efficiency. The transaldolase gene ywjH was knocked out, and the optimized expression cassette was integrated into the genome. The expression of the phosphoglycerate kinase gene pgk and the pyruvate kinase gene pyk was enhanced, ultimately yielding a host bacterium capable of synthesizing MG. This engineered bacterium achieved an MG yield of 1.25 g / L in shake-flask fermentation, which was further increased to 5.61 g / L under fed-batch fermenter conditions.

[0007] The first objective of this invention is to provide plasmids that initiate the expression of genes AvmysA, AvmysB, and AvmysC on the vector pP43NMK.

[0008] In one implementation, pP43NMK is used as the expression vector.

[0009] In one embodiment, the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC are expressed in the vector plasmid.

[0010] In one embodiment, the methyl-4-deoxycodone synthase gene AvmysA is derived from the variable anabaena variabilis, and its encoded amino acid sequence is shown in SEQ ID NO. 1, and its nucleotide sequence is shown in SEQ ID NO. 13;

[0011] In one embodiment, the O-methyltransferase AvmysB is derived from Anabaenavariabilis, whose encoded amino acid sequence is shown in SEQ ID NO. 2 and whose nucleotide sequence is shown in SEQ ID NO. 14;

[0012] In one embodiment, the ATP-grasp ligase gene AvmysC is derived from Anabaenavariabilis, and its encoded amino acid sequence is shown in SEQ ID NO. 3, and its nucleotide sequence is shown in SEQ ID NO. 15.

[0013] In one embodiment, the expression of the phosphoglycerate kinase gene pgk and the pyruvate kinase gene pyk is enhanced. The nucleotide sequence of the phosphoglycerate kinase gene pgk is shown in SEQ ID NO. 17, and the nucleotide sequence of the pyruvate kinase gene pyk is shown in SEQ ID NO. 18.

[0014] A second objective of this invention is to provide promoters for the key enzymes expressing Mycosporine-glycine synthesis at different intensities.

[0015] In one implementation, P43NMK is used as the expression vector.

[0016] In one embodiment, the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC are expressed in the vector plasmid.

[0017] In one implementation, AvmysABC is powered by promoter P. veg P spoVG P yvyd and P 43 The nucleotide sequences of these sequences are shown in SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7, respectively.

[0018] A third objective of this invention is to provide a key enzyme for the synthesis of Mycosporine-glycine expressed under different intensities of UTR.

[0019] In one implementation, P43NMK is used as the expression vector.

[0020] In one embodiment, the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC are expressed in the vector plasmid.

[0021] In one embodiment, AvmysABC is expressed by UTR3, UTR4, UTR7 and UTR8, the nucleotide sequences of which are shown in SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11, respectively.

[0022] A fourth objective of this invention is to provide a genetically engineered Bacillus subtilis strain for producing Mycosporine-glycine, wherein the genetically engineered Bacillus subtilis strain enhances MG production by knocking out the transaldolase gene ywjH, which competes with the pathway.

[0023] In one embodiment, the amino acid encoded by the transaldolase gene ywjH has the NCBI sequence number NP_391592.3, and its nucleotide sequence is shown in SEQ ID NO. 16.

[0024] In one embodiment, the Bacillus subtilis includes, but is not limited to, Bacillus subtilis WB600.

[0025] The fifth objective of this invention is to provide a genetically engineered Bacillus subtilis strain that synthesizes Mycosporine-glycine, wherein the genetically engineered Bacillus subtilis strain heterologously expresses the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC.

[0026] In one embodiment, the methyl-4-deoxycodone synthase gene AvmysA is derived from the variable anabaena variabilis, and its encoded amino acid sequence is shown in SEQ ID NO. 1, NCBI sequence number ABA23463.1; the methyl-4-deoxycodone synthase gene AvmysA is generated by the strong promoter P veg and UTR4 expression;

[0027] In one embodiment, the O-methyltransferase AvmysB is derived from *Anabaenavariabilis*, whose encoded amino acid sequence is shown in SEQ ID NO. 2 and NCBI sequence number NP_391592.3; the O-methyltransferase AvmysB is generated by the strong promoter P. veg and UTR4 expression;

[0028] In one embodiment, the ATP-grasp ligase gene AvmysC is derived from *Anabaenavariabilis*, and its encoded amino acid sequence is shown in SEQ ID NO. 3, NCBI sequence number ABA23461.1; the ATP-grasp ligase gene AvmysC is generated by the strong promoter P. veg And UTR4 expression.

[0029] In one embodiment, the expression of the phosphoglycerate kinase gene pgk and the pyruvate kinase gene pyk is enhanced. The nucleotide sequence of the phosphoglycerate kinase gene pgk is shown in SEQ ID NO. 17, and the nucleotide sequence of the pyruvate kinase gene pyk is shown in SEQ ID NO. 18.

[0030] The sixth object of the present invention is to provide a method for producing Mycosporine-glycine, the method being to produce Mycosporine-glycine by fermentation using the genetically engineered Bacillus subtilis.

[0031] In one embodiment, the seed culture of the genetically engineered Bacillus subtilis is added to a fermentation system containing 30 g / L glucose and cultured at 35-38 ºC and 180-220 rpm for no less than 72 h.

[0032] In one embodiment, the fermentation system further contains 24 g / L yeast extract, 12 g / L peptone, 12.54 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, 6 g / L ammonium sulfate, 3 g / L magnesium sulfate heptahydrate, 3 g / L urea, 0.1 g / L calcium chloride, and 30 g / L glucose.

[0033] In one embodiment, the seed culture of the genetically engineered Bacillus subtilis is inoculated into a fermenter system containing 30 g / L glucose. The fermentation temperature is 35-38 ºC, the stirring speed is 200-800 r / min, the aeration rate is 2-8 vvm, and the pH is controlled at 7.0±0.2. Fermentation continues until the OD reaches its maximum. 600 Add yeast extract at 30-40°C and incubate for at least 50 hours.

[0034] In one implementation, the strain ferments and grows to OD. 600 At 30-40, yeast extract is added simultaneously to provide sufficient nitrogen source to promote cell growth and the synthesis of Mycosporine-glycine. This process is done at a low flow rate.

[0035] In one embodiment, the fermentation system or fermenter system further contains 24 g / L yeast extract, 12 g / L peptone, 12.54 g / L dipotassium hydrogen phosphate, 2.31 g / L potassium dihydrogen phosphate, 6 g / L ammonium sulfate, 3 g / L magnesium sulfate heptahydrate, 3 g / L urea, 0.1 g / L calcium chloride, and 30 g / L glucose.

[0036] A seventh object of the present invention is to provide the use of the genetically engineered Bacillus subtilis in the preparation of Mycosporine-glycine or products containing Mycosporine-glycine.

[0037] The eighth objective of this invention is to provide the application of the genetically engineered Bacillus subtilis in the fields of medicine and cosmetics.

[0038] Beneficial effects:

[0039] The genetically engineered Bacillus subtilis constructed in this invention has significant beneficial effects: First, the selected food-grade (GRAS) host, Bacillus subtilis WB600, is free of endotoxins and exhibits strong environmental tolerance, ensuring the safety of downstream applications and simplifying post-processing procedures. Second, through systematic metabolic engineering modifications, including knocking out the competing gene ywjH and screening for and employing the strong promoter P, vegBy combining with a highly efficient UTR4 gene and ultimately stably integrating the optimized AvmysABC gene cluster into the genome, the expression of the phosphoglycerate kinase gene pgk and the pyruvate kinase gene pyk was enhanced, improving the ATP regeneration capacity of the strain and achieving efficient and stable expression of the Mycosporine-glycine biosynthetic pathway. This strategy increased the shake-flask yield of the engineered strain to 1.25 g / L, and further increased the yield to 5.61 g / L in a 50 L fermenter using high-density fed-batch fermentation, demonstrating excellent potential for industrial production. The entire fermentation process requires no antibiotics or inducers, significantly reducing production costs and providing a safe, reliable, and highly competitive core technology solution for the green, low-cost, and large-scale biomanufacturing of Mycosporine-glycine. Attached Figure Description

[0040] Figure 1 A schematic diagram of the recombinant plasmid p43NMK-Pveg-UTR4-AvmysABC. Detailed Implementation

[0041] The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples were all commercially available products, and the specific operations were performed according to the kit instructions. Routine procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to *Molecular Cloning: A Laboratory Manual (Fourth Edition)*. Sequencing of the plasmids and DNA products was performed by Genewiz (Suzhou).

[0042] (a) Culture medium

[0043] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0044] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0045] (3) Fermentation medium: yeast extract 24 g / L, peptone 12 g / L, dipotassium hydrogen phosphate 12.54 g / L, potassium dihydrogen phosphate 2.31 g / L, ammonium sulfate 6 g / L, magnesium sulfate heptahydrate 3 g / L, urea 3 g / L, calcium chloride 0.1 g / L, glucose 30 g / L.

[0046] (4) Feeding solution for batch fermentation: 600 g / L glucose, 200 g / L yeast extract, pH adjustment: 14% ammonia (v / v).

[0047] (5) Preparation of competent cells of Bacillus subtilis: The strain preserved in the laboratory was streaked on an agar plate and incubated at 37 °C for 10-12 h. Single colonies were picked and placed in 1 mL of LB medium into 50 mL centrifuge tubes and incubated at 37 °C for about 8 h. 4 mL of LB medium diluted five times was added, along with 300 μL of 50% xylose, and incubated for 2 h. Finally, 1.25 mL of 50% glycerol was added and the culture was frozen at -80 °C.

[0048] (II) Shake-flask fermentation of Mycosporine-glycine

[0049] (1) Mycosporine-glycine shake flask fermentation process: The constructed strain was inoculated into LB liquid medium and cultured overnight at 37ºC and 220 rpm for 12 h to obtain seed liquid. 1 mL of seed liquid was inoculated into 25 mL of fermentation medium and cultured at 37ºC and 220 rpm for 72 h.

[0050] (2) Mycosporine-glycine fed-batch fermentation process: Colonies of the strain were picked from the plate and inoculated into 4 mL of LB medium supplemented with the corresponding antibiotic. The culture was carried out overnight at 37 ºC and 220 rpm for 12 h to obtain the primary seed culture. 4 mL of the primary seed culture was inoculated into 100 mL of fermentation medium and cultured at 37 ºC and 220 rpm until the OD reached the target value. 600 The OD value was 3-4, yielding a secondary seed culture. 200 mL of this secondary seed culture was inoculated into a 50L fermenter containing 2 L of fermenter medium for fermentation culture, maintained at 37 ºC. After the initial 30 g / L glucose in the medium was completely depleted, a fed-batch carbon source was started to meet cell growth requirements. When the OD... 600 When the pH reaches 30-40, add 200 g / L yeast extract at a low flow rate. Maintain the pH at 7.0±0.2 throughout the process, and control foaming by adding an antifoaming agent. Control dissolved oxygen by adjusting the stirring speed (200-800 rpm) and aeration rate (2-8 vvm).

[0051] (III) Mycosporine-glycine detection:

[0052] Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, collect the supernatant, and use it for HPLC determination.

[0053] HPLC detection conditions: High performance liquid chromatography (HPLC) system (Agilent); Column: ZORBAX Eclipse Plus C18; Detector: Agilent UV detector; Mobile phase: 0.25% formic acid in water; Flow rate: 0.65 mL / min; Column temperature: 30 ºC; Injection volume: 10 μL.

[0054] (iv) strains

[0055] The dual-plasmid gene editing system has been published in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825.

[0056] Table 1. Strains involved in the following examples

[0057]

[0058]

[0059] (v) Primers

[0060] Table 2 Primers required in the following examples

[0061]

[0062]

[0063]

[0064]

[0065]

[0066] Example 1: Construction of Mycosporine-glycine synthase plasmid

[0067] 1. Construction of p43NMK-P43-AvmysABC vector

[0068] Using pP43NMK as a template, the primers in Table 2 were used for amplification and ligation. The resulting cells were transformed into E. coli DH5α competent cells, plated on LB plates, cultured at 37°C, and the plasmids were extracted and sequenced. The sequencing results were successful, yielding p43NMK-P43-AvmysABC.

[0069] The specific steps are as follows:

[0070] (1) PCR system (100 μl as an example): H2O: 45 μL; template: 0.5-1 μL; primer 1: 2 μL; primer 2: 2 μL; PrimerSTAR Max: 50 μl.

[0071] (2) Based on the PCR system in (1), the gene AvmysA was amplified using primers AvmysA-F / R, the gene AvmysBC was amplified using primers AvmysBC-F / R, and the vector fragment was amplified using p43NMK-F / R with p43NMK as a template.

[0072] (3) Verify the fragments amplified in (2) by nucleic acid electrophoresis and recover them using a kit.

[0073] (4) The fragments obtained in (3) are assembled using seamless cloning. The formula for calculating the amount of each fragment added is V = (0.02 × fragment length) / fragment concentration. The reaction is carried out at 50°C for 5 minutes for ligation.

[0074] (5) The assembled plasmid is transferred into the cloning host, plated on the corresponding resistant LB plate and cultured overnight at 37°C. Then, a single colony is picked and the plasmid is extracted for Sanger sequencing. Successful sequencing completes the construction of the recombinant plasmid p43NMK-P43-AvmysABC.

[0075] 2. Construction of p43NMK-Pveg-AvmysABC vector

[0076] Using p43NMK-P43-AvmysABC as a template, amplification was performed using the primers in Table 2. After removing the template DNA with DpnI, the PCR product was transformed into E. coli DH5α competent cells, plated on LB plates, cultured at 37°C, and the plasmid was extracted and sequenced. Successful sequencing yielded p43NMK-Pveg-AvmysABC.

[0077] The specific steps are as follows:

[0078] (1) PCR system (100 μl as an example): H2O: 45 μL; template: 0.5-1 μL; primer 1: 2 μL; primer 2: 2 μL; PrimerSTAR Max: 50 μl.

[0079] (2) Based on the PCR system in (1), the plasmid p43NMK-P43-AvmysABC was amplified using primers Pveg-F / R, while the promoter was replaced.

[0080] (3) The PCR product was digested at 37ºC for 1 h after removing the template DNA with DpnI.

[0081] (4) Transplant into the cloning host, plate onto the corresponding LB plate and incubate overnight at 37°C. Then pick a single colony and extract the plasmid for Sanger sequencing. Successful sequencing completes the construction of the recombinant plasmid p43NMK-Pveg-AvmysABC.

[0082] The plasmid construction process for p43NMK-PspoVG-AvmysABC, p43NMK-Pyvyd-AvmysABC, p43NMK-Pveg-UTR3-AvmysABC, p43NMK-Pveg-UTR4-AvmysABC, p43NMK-Pveg-UTR7-AvmysABC, p43NMK-Pveg-UTR8-AvmysABC, p43NMK-Pveg-UTR12-AvmysABC, and p43NMK-Pveg-UTR13-AvmysABC is as described above.

[0083] Example 2: Knockout of competitive pathways in chassis strains

[0084] The dual-plasmid gene editing system used in this invention has been disclosed in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825. The specific operation is as follows:

[0085] The gene editing system of Bacillus subtilis consists of two plasmids, pHT-XCR2 and pcrF11. pHT-XCR2 is a Cpf1 expression vector, and pcrF11 is a crRNA expression vector, used to express crRNA and insert homology repair template.

[0086] (1) A 23 bp specific targeting sequence (N23) was designed targeting the knockout / integration site ywjH on the Bacillus subtilis WB600 genome. Using pcrF11 plasmid as a template, primers were designed to replace the original N23 sequence on the plasmid with the N23 sequence targeting ywjH through reverse PCR or site-directed mutagenesis. The PCR product was digested with DpnI enzyme to remove the template plasmid, purified, and transformed into Escherichia coli DH5α competent cells. After successful sequencing, the plasmid pcrF11-ywjH was obtained.

[0087] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the ywjH gene locus, the downstream homologous arm (DH, approximately 1000 bp) of the sacB gene locus, and the linearized backbone of the vector pcrF11-ywjH. The PCR products were subjected to gel electrophoresis and purified. Using a seamless cloning (or Gibson Assembly) kit, the fragments were mixed in proportion and homologous recombination was performed to obtain the plasmid pcrF11-ywjH-Δ.

[0088] (3) The plasmid pHT-XCR2 containing the Cpf1 protein was first transformed into Bacillus subtilis WB600 competent cells to obtain an intermediate host with CRISPR-Cpf1 editing capability. Electroporation competent cells were prepared from this intermediate host. The donor plasmid pcrF11-ywjH-Δ constructed in step (2) was electroporated into the competent cells. After electroporation, resuscitation medium was added and the cells were revived at 37°C for 2 h. The revived bacterial culture was plated on double antibiotic plates (such as chloramphenicol and kanamycin) and incubated upside down at 37°C for 24-48 h to screen for transformants that underwent homologous recombination.

[0089] (4) The successfully screened single colonies were subjected to colony PCR to verify whether the target gene was successfully knocked out. To ensure that the target gene on the genome was knocked out, the remaining PCR products were sent to Sanger sequencing to obtain the correct recombinant strain.

[0090] Example 3: Integration and Modification of Key Genes

[0091] The dual-plasmid gene editing system used in this invention has been disclosed in the literature: Wu Y, Liu Y, Lv X, Li J, Du G, Liu L. CAMERS‐B: CRISPR / Cpf1 assisted multiple‐genes editing and regulation system for Bacillus subtilis. Biotechnology and Bioengineering. 2020;117:1817–1825. The specific operation is as follows:

[0092] The gene editing system of Bacillus subtilis consists of two plasmids, pHT-XCR2 and pcrF11. pHT-XCR2 is a Cpf1 expression vector, and pcrF11 is a crRNA expression vector, used to express crRNA and insert homology repair template.

[0093] (1) A 23 bp specific targeting sequence (N23) was designed targeting the sacB knockout / integration site on the Bacillus subtilis genome. Using pcrF11 plasmid as a template, primers were designed to replace the original N23 sequence on the plasmid with the N23 sequence targeting sacB through reverse PCR or site-directed mutagenesis. The PCR product was digested with DpnI enzyme to remove the template plasmid, purified, and transformed into Escherichia coli DH5α competent cells. After successful sequencing, the plasmid pcrF11-sacB was obtained.

[0094] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the sacB gene locus, the downstream homologous arm (DH, approximately 1000 bp) of the sacB gene locus, the key enzyme gene AvmysABC, and the linearized backbone of the vector pcrF11-sacB. The PCR products were subjected to gel electrophoresis and purified. Using a seamless cloning (or Gibson Assembly) kit, the fragments were mixed in proportion and homologous recombination was performed to obtain the plasmid pcrF11-sacB-Pveg-AvmysABC.

[0095] (3) The plasmid pHT-XCR2 containing the Cpf1 protein was first transformed into Bacillus subtilis WB600 competent cells to obtain an intermediate host with CRISPR-Cpf1 editing capability. Electroporation competent cells were prepared from this intermediate host. The donor plasmid pcrF11-sacB-Pveg-AvmysABC constructed in step (2) was electroporated into competent cells. After electroporation, resuscitation medium was added and the cells were revived at 37°C for 2 h. The revived bacterial culture was plated on double antibiotic plates (such as chloramphenicol and kanamycin) and incubated upside down at 37°C for 24-48 h to screen for transformants that underwent homologous recombination.

[0096] (4) The successfully screened single colonies were subjected to colony PCR to verify whether the target gene was successfully knocked out. To ensure that the target gene on the genome was knocked out, the remaining PCR products were sent to Sanger sequencing to obtain the correct recombinant strain.

[0097] Example 4: Construction of Mycosporine-glycine production chassis strain

[0098] The engineered strain was obtained by transformation. The specific procedure is as follows: 1 μL of the constructed plasmid was injected into competent Bacillus subtilis cells, and the cells were shaken at 37ºC for 2 h. The mixture was then spread onto LB agar plates containing the corresponding antibiotic concentration. The plates were incubated at 37ºC for 10–12 h, and the engineered strain containing the expression plasmid was obtained by picking bacteria.

[0099] Example 5: Shake Flask Fermentation Production

[0100] The engineered bacteria were inoculated into LB liquid medium containing the corresponding antibiotic and cultured overnight at 37 ºC and 220 rpm for 12 h to obtain a seed culture. 1 mL of the seed culture was inoculated into 25 mL of fermentation medium and cultured at 37 ºC and 220 rpm for 72 h. 1 mL of the fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis.

[0101] Example 6: Fermentation tank fed-batch culture

[0102] A batch-feed fermentation experiment was conducted in a 50 L fermenter.

[0103] The optimal engineered bacteria were inoculated into 8 mL of LB medium containing the corresponding antibiotic and cultured overnight at 37ºC and 220 rpm for 10–12 h to obtain primary seed culture. 8 mL of the primary seed culture was then inoculated into 800 mL of fermentation medium containing the corresponding antibiotic and fermentation medium and cultured at 37ºC and 220 rpm until OD reached [value missing]. 600 The OD value was 3-4, yielding a secondary seed culture. 200 mL of this secondary seed culture was inoculated into a 50L fermenter containing 2L of fermentation medium with the corresponding antibiotic for fermentation culture. The fermentation temperature was maintained at 37ºC. After the initial 30 g / L glucose in the medium was completely depleted, glucose was added continuously. When the OD... 600 When the concentration reaches 30–40, yeast extract and 600 g / L glucose are added at a low flow rate (5 ml / h) to maintain a glucose concentration of 10–15 g / L. The pH is maintained at 7.0 ± 0.2 throughout the process, and foam is controlled by adding an antifoaming agent. Dissolved oxygen is controlled by adjusting the stirring speed (220 rpm) and aeration rate (2–8 vvm).

[0104] After 55 hours of fermentation in the upper tank, strain BsM-9 achieved a yield of 4.52 g / L and a maximum OD600 of 62.1; after 45 hours of fermentation, strain BsM-12 achieved a yield of 5.61 g / L and a maximum OD600 of 80.2. This demonstrates significant production potential for large-scale industrial applications.

[0105] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine, characterized in that: Using Bacillus subtilis WB600 as the starting strain, the modification method for the starting strain is as follows: 1) The transaldolase gene ywjH was knocked out of the genome; 2) Knock out the fructan sucrase gene sacB and replace it with one generated by a strong constitutive promoter P. veg The gene cluster that initiates expression is AvmysABC; 3) Knock out the phosphoenolpyruvate carboxykinase gene pckA and replace it with the strong promoter P. veg The phosphoglycerate kinase gene pgk is initiated for expression; 4) Knock out the glycosyltransferase gene gspA and replace it with the strong promoter P. veg The pyruvate kinase gene pyk is initiated for expression; The gene cluster AvmysABC consists of the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC, all derived from the variable fish algae Anabaena variabilis.

2. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 1, characterized in that, The modification method also includes: Import recombinant plasmid; The recombinant plasmid uses plasmid p43NMK as a vector to heterologously express the gene cluster AvmysABC.

3. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 2, characterized in that, The promoter of the recombinant plasmid is selected from the strong promoter P. 43 P veg P spoVG and P yvyd Any one of them.

4. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 2, characterized in that, The recombinant plasmid contains any one of the 5' untranslated regions UTR3, UTR4, UTR7, and UTR8.

5. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 4, characterized in that, The recombinant plasmid is selected from any one of p43NMK-P43-AvmysABC, p43NMK-Pveg-AvmysABC, p43NMK-Pyvyd-AvmysABC, p43NMK-Pveg-UTR3-AvmysABC, p43NMK-Pveg-UTR4-AvmysABC, p43NMK-Pveg-UTR7-AvmysABC, and p43NMK-Pveg-UTR8-AvmysABC.

6. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 5, characterized in that, The recombinant plasmid is p43NMK-Pveg-AvmysABC or p43NMK-Pveg-UTR4-AvmysABC.

7. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 6, characterized in that, The recombinant plasmid is p43NMK-Pveg-UTR4-AvmysABC.

8. A method for constructing a genetically engineered Bacillus subtilis strain that synthesizes Mycosporine-glycine, characterized in that, The construction method includes the modification method according to any one of claims 1-7.

9. The use of the genetically engineered Bacillus subtilis for synthesizing Mycosporine-glycine as described in any one of claims 1-7, or the genetically engineered Bacillus subtilis for synthesizing Mycosporine-glycine obtained by the construction method described in claim 8, in the production of Mycosporine-glycine.

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