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

Abstract

This invention discloses a genetically engineered Bacillus subtilis strain for producing Mycosporine-glycine, its construction method, and its applications. Starting with Bacillus subtilis WB600, this strain significantly enhances Mycosporine-glycine synthesis by knocking out the competing pathway gene ywjH and expressing the AvmysABC gene cluster from Anabaena polymorpha at multiple sites. Further knocking out the sporulation genes spo0A and spoIIIE and introducing the xylose metabolism gene clusters xylA-xylB, combined with a mixed carbon source fermentation strategy, significantly increased the yield. The engineered strain achieved a Mycosporine-glycine yield of 3.64 g / L in shake-flask fermentation and 15.8 g / L in a 50 L fed-batch fermenter.

Description

Technical Field

[0001] This invention belongs to the field of synthetic biology and metabolic engineering, specifically relating 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 products with excellent UV absorption capabilities. Mycosporine-glycine, in particular, is not only an important UV protection and antioxidant molecule but also a key precursor for the synthesis of various high-value MAAs derivatives, showing broad application prospects in high-end sunscreen cosmetics, anti-photoaging skincare products, and biomedicine. Currently, the commercial production of mycosporine-glycine mainly relies on extraction from macroalgae or cyanobacteria. This method is limited by the long growth cycle of raw materials, low content, complex extraction processes, high costs, and unstable yields, making it difficult to meet the growing market demand. Chemical synthesis routes, on the other hand, suffer from cumbersome steps, difficulty in chiral control, and environmental unfriendliness, hindering their large-scale application.

[0003] In recent years, constructing microbial "cell factories" using synthetic biology and metabolic engineering techniques to achieve heterologous biosynthesis of natural products such as Mycosporine-glycine has become a key direction for overcoming traditional production bottlenecks. Bacillus subtilis, due to its non-pathogenicity, GRAS safety certification, clear genetic background, rapid growth, strong protein secretion capacity, and mature genetic manipulation tools, is considered an ideal host for metabolic engineering. However, the yield of Mycosporine-glycine produced by existing Bacillus subtilis strains remains generally low, mainly due to factors such as insufficient heterologous pathway expression efficiency, interference from competitive metabolic flux within the host, and an imbalance between cell growth and product synthesis.

[0004] To address the above problems, this invention systematically modifies Bacillus subtilis through metabolic engineering, including knocking out competing pathway genes, achieving multi-copy genome integration, blocking spore development to concentrate metabolic resources, and introducing xylose utilization capabilities to expand carbon source adaptability. This results in the construction of a highly efficient, stable, and easily industrially scaled-up Mycosporine-glycine producing strain, providing a practical solution for the green biomanufacturing of Mycosporine-glycine. Summary of the Invention

[0005] In view of the shortcomings and problems of the existing technology, the present invention provides a method for constructing genetically engineered Bacillus subtilis to synthesize Mycosporine-glycine (MG) and its application. Through systematic metabolic engineering, a Mycosporine-glycine synthesis pathway was constructed in Bacillus subtilis WB600. The methyl-4-deoxycodone synthase gene AvmysA, O-methyltransferase gene AvmysB, and ATP-grasp ligase gene AvmysC from *Anabaena variabilis* were heterologously expressed. By knocking out competing pathway genes (aldolase gene ywjH) and sporulation genes (spo0A, sporulation regulatory protein gene, and sporulation septum DNA transport protein gene spoIIIE), and introducing xylose metabolism genes (xylA, xylose isomerase gene, and xylulose kinase gene xylB) from *Escherichia coli*, and optimizing the carbon source, the yield of Mycosporine-glycine in Bacillus subtilis cells was effectively increased. This solved the problems of low purity, high cost, and environmental pollution associated with traditional extraction methods.

[0006] Preferably, this invention provides a plasmid-type Mycosporine-glycine-producing genetically engineered bacterium. Using Bacillus subtilis WB600 as the starting strain, the methyl-4-deoxycodone alcohol synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC from *Anabaena variabilis* are cloned into plasmid p43NMK and expressed in multiple copies into the genome. By knocking out competing pathway genes (aldolase gene ywjH) and sporulation genes (spo0A, sporulation regulatory protein gene, and sporulation septum DNA transporter gene spoIIIE), and introducing xylose metabolism genes (xylA, xylose isomerase gene, and xylulose kinase gene xylB), carbon source optimization is performed, ultimately obtaining a host bacterium capable of synthesizing Mycosporine-glycine. After the above modifications, the engineered bacteria achieved a Mycosporine-glycine yield of 3.64 g / L in shake-flask fermentation and a yield of 15.8 g / L in a 50 L fermenter with fed-batch culture, providing an efficient technical solution for the green and low-cost industrial production of Mycosporine-glycine.

[0007] The first objective of this invention is to provide a solution by a strong promoter P veg A plasmid that initiates the expression of genes AvmysA, AvmysB, and AvmysC, and whose translation rate is enhanced by UTR4.

[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. 8;

[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. 9;

[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. 10.

[0013] In one implementation, AvmysABC is powered by promoter P. veg And UTR4 expression.

[0014] The second 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 Mycosporine-glycine production by knocking out the competing transaldolase gene ywjH, the spore formation regulatory protein gene (spo0A), and the spore septum DNA transport protein gene (spoIIIE).

[0015] 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.11;

[0016] In one embodiment, the NCBI sequence number of the amino acid encoded by the spo0A gene, which regulates spore formation, is NP_390302.1.

[0017] In one embodiment, the NCBI sequence number of the amino acid encoded by the spoIIIE gene of the spore septum DNA transporter is NP_389562.3.

[0018] In one embodiment, the Bacillus subtilis includes, but is not limited to, Bacillus subtilis WB600.

[0019] A third 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).

[0020] 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 Express;

[0021] 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 Initiate expression;

[0022] 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 Start the expression.

[0023] A third objective of this invention is to provide a method for culturing genetically engineered Bacillus subtilis using xylose.

[0024] In one embodiment, the genetically engineered bacteria introduces a xylose metabolic pathway, integrating a promoter P at a genomic site. 43 Driven by the xylose isomerase gene xylA and the xylulose kinase gene xylB;

[0025] In one embodiment, the xylose isomerase gene xylA is derived from Escherichia coli, and its encoded amino acid sequence is shown in SEQ ID NO. 4, and its nucleotide sequence is shown in SEQ ID NO. 12;

[0026] In one embodiment, the xyl kinase gene xylB is derived from Escherichia coli, and its encoded amino acid sequence is shown in SEQ ID NO. 5, and its nucleotide sequence is shown in SEQ ID NO. 13;

[0027] In one embodiment, the method uses a mixed carbon source culture medium containing glucose and xylose for fermentation.

[0028] A fourth objective of this invention is to provide a method for producing Mycosporine-glycine, wherein the method utilizes the genetically engineered Bacillus subtilis to ferment and produce Mycosporine-glycine.

[0029] In one embodiment, the seed culture of the genetically engineered Bacillus subtilis is added to a fermentation system containing 15 g / L glucose and 15 g / L xylose, and cultured at 35-38 ºC and 180-220 rpm for no less than 72 h.

[0030] 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.

[0031] 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, 15 g / L glucose, and 15 g / L xylose.

[0032] 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.

[0033] In one implementation, the strain ferments and grows to OD. 600At 30-40°C, begin adding yeast extract to provide sufficient nitrogen source to promote cell growth and the synthesis of Mycosporine-glycine. This process is done by adding the extract at a low flow rate.

[0034] 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, 15 g / L glucose, and 15 g / L xylose.

[0035] The fifth objective of this invention is to provide the application of the genetically engineered Bacillus subtilis in the preparation of Mycosporine-glycine-containing products.

[0036] The sixth objective of this invention is to provide the application of the genetically engineered Bacillus subtilis in the fields of medicine and cosmetics.

[0037] Beneficial effects:

[0038] The genetically engineered Bacillus subtilis strain constructed in this invention exhibits significant beneficial effects: First, through systematic metabolic engineering modifications, including knocking out the competing pathway gene ywjH and integrating the optimized AvmysABC gene cluster at multiple sites in the genome, the Mycosporine-glycine synthesis pathway was enhanced. Building upon this, the sporulation genes spo0A and spoIIIE were further knocked out, effectively blocking cell differentiation and concentrating metabolic resources on product synthesis. Simultaneously, the xylose metabolism gene xylAB was introduced, and the carbon source composition was optimized, significantly improving the strain's substrate utilization efficiency and physiological adaptability. The synergistic effect of these strategies resulted in a Mycosporine-glycine yield of up to 3.64 g / L in shake-flask fermentation, further increased to 15.8 g / L under fed-batch culture conditions in a 50 L fermenter, demonstrating excellent high-yield performance and industrial scale-up potential. Attached Figure Description

[0039] Figure 1 A schematic diagram of the recombinant plasmid p43NMK-Pveg-UTR4-AvmysABC. Detailed Implementation

[0040] 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).

[0041] (a) Culture medium

[0042] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0043] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, 15 g / L agar powder.

[0044] (3) Fermentation medium 1: 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, 30 g / L glucose and sugar.

[0045] (4) Fermentation tank culture medium 2: 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 15 g / L and xylose 15 g / L.

[0046] (5) Feeding solution for batch fermentation: 600 g / L glucose, 500 g / L xylose, 200 g / L yeast extract, pH adjustment: 14% ammonia (v / v).

[0047] (6) Preparation of competent cells of Bacillus subtilis: The strain preserved in the laboratory was streaked on an agar plate and incubated at 37ºC for 10-12 h. Single colonies were picked and placed in 1 mL of LB medium into 50 mL centrifuge tubes and incubated at 37ºC 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 -80ºC.

[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 37ºC 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 37ºC 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 5 L fermenter containing 2 L of fermenter medium for fermentation culture, maintained at 37ºC. After the initial 30 g / L glucose in the medium was completely depleted, fed-batch feeding of carbon source and xylose was initiated to meet cell growth and provide substrate. When 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: 30ºC; 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: Knockout of competitive pathways in chassis strains

[0067] 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:

[0068] 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.

[0069] (1) A 23 bp specific targeting sequence (N23) was designed targeting the spo0A knockout / integration site on the genome of protease-deficient Bacillus subtilis WB600. Using pcrF11 plasmid as a template, primers were designed to replace the original N23 sequence on the plasmid with the N23 sequence targeting spo0A 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-spo0A was obtained.

[0070] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the spo0A gene locus, the downstream homologous arm (DH, approximately 1000 bp) of the spo0A gene locus, and the linearized backbone of the vector pcrF11-spo0A. 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-spo0A-Δ.

[0071] (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-spo0A-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.

[0072] (4) Perform colony PCR on the successfully screened single colonies to verify whether the target gene has been successfully knocked out. To ensure that the target gene on the genome has been knocked out, send the remaining PCR products to Sanger sequencing to finally obtain the correct genetically engineered strain.

[0073] The procedure for knocking out genes such as spoIIIE is the same as above.

[0074] Example 2: Integration and Modification of Key Genes

[0075] 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:

[0076] 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.

[0077] (1) A 23 bp specific targeting sequence (N23) was designed targeting the knockout / integration site ctc 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 ctc via 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-ctc was obtained.

[0078] (2) Three pairs of primers were designed and synthesized to amplify the upstream homologous arm (UH, approximately 1000 bp) of the ctc 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-ctc. The PCR products were subjected to gel electrophoresis and purified. Using a seamless cloning kit, the fragments were mixed in proportion and homologous recombination was performed to obtain the plasmid pcrF11-ctc-Pveg-AvmysABC.

[0079] (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-ctc-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.

[0080] (4) Perform colony PCR on the successfully screened single colonies to verify whether the target gene has been successfully knocked out. To ensure that the target gene on the genome has been knocked out, send the remaining PCR products to Sanger sequencing to finally obtain the correct genetically engineered strain.

[0081] Other integrations include yckB::P veg -AvmysABC、sacA::P veg -AvmysABC and eutD::P 43 The operation procedure for -xylAB is the same as above.

[0082] Example 3: Construction of Mycosporine-glycine production chassis strain

[0083] The engineered strain was obtained by transformation. The specific procedure is as follows: 1 μL of plasmid was injected into competent Bacillus subtilis cells, and the cells were placed on a shaker 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.

[0084] Example 4: Shake Flask Fermentation Production

[0085] The engineered bacteria were inoculated into LB liquid medium containing the corresponding antibiotic and cultured overnight at 37ºC and 220 rpm for 12 h to obtain seed culture. 1 mL of seed culture was inoculated into 25 mL of fermentation medium 1 or fermentation medium 2 and cultured at 37ºC and 220 rpm for 72 h. 1 mL of fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis.

[0086] Example 5: Fermentation tank fed-batch culture

[0087] A batch-feed fermentation experiment was conducted in a 50 L fermenter.

[0088] 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 50 L fermenter containing 2 L of fermenter medium with the corresponding antibiotic for fermentation. 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... 600When the concentration reaches 30-40, add 600 g / L glucose and maintain the glucose concentration at 10-15 g / L, add 500 g / L xylose and maintain the xylose concentration at 10-15 g / L; add 200 g / L yeast extract at a rate of 5 ml / h; add 14% ammonia water, maintain the pH at 7.0±0.2 throughout, and control foaming by adding an antifoaming agent. Dissolved oxygen is controlled by adjusting the stirring speed (220 rpm) and aeration rate (2-8 vvm). In a 50 L fermenter, the yield of Mycosporine-glycine can reach 15.8 g / L after 60 h, OD 600 With a maximum value of 68.8, it demonstrates significant production potential in large-scale industrial applications.

[0089] 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 producing Mycosporine-glycine, characterized in that, Using Bacillus subtilis WB600 as the starting strain, the gene cluster AvmysABC was heterologously expressed using plasmid p43NMK as a vector. Knock out the transaldolase gene ywjH, the spore formation regulatory protein gene spo0A, and the spore septum DNA transport protein gene spoIIIE. The gene cluster AvmysABC was integrated and expressed in 4 copies in the genome of the originating bacterium; and xyl isomerase gene xylA and xylulose kinase gene xylB derived from Escherichia coli were integrated and expressed in the genome. 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. A genetically engineered Bacillus subtilis strain for producing Mycosporine-glycine, characterized in that, The following methods were used to modify Bacillus subtilis strain WB600 as the starting strain: 1) Knock out the transaldolase gene ywjH; 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 BL25 ribosomal protein gene ctc that binds to 5S ribosomal RNA and replace it with one generated by the strong promoter P. veg The gene cluster that initiates expression is AvmysABC; 4) Introduce the recombinant plasmid p43NMK-Pveg-UTR4-AvmysABC; 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.

3. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 2, characterized in that, The modification method also includes: The amino acid ABC transporter (binding lipoprotein subunit) gene yckB was knocked out and replaced with the gene cluster AvmysABC, which is expressed by the strong promoter Pveg.

4. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 3, characterized in that, The modification method also includes: Knock out the sacA gene of sucrose 6-phosphate hydrolase and replace it with one generated by the strong promoter P. veg The gene cluster AvmysABC is initiated for expression.

5. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 4, characterized in that, The modification method also includes: Knock out the gene spo0A, a protein that regulates spore formation, and the gene spoIIIE, a DNA transporter for spore septum.

6. The genetically engineered Bacillus subtilis strain for synthesizing Mycosporine-glycine according to claim 5, characterized in that, The modification method also includes: Knock out the phosphoryltransferase gene eutD and replace it with one generated by the strong promoter P veg The xylose isomerase gene xylA and the xylulose kinase gene xylB are initiated for expression.

7. 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 2-6.

8. A method for fermenting and producing Mycosporine-glycine, characterized in that, include: The genetically engineered Bacillus subtilis that synthesizes Mycosporine-glycine according to any one of claims 1-6 or the genetically engineered Bacillus subtilis that synthesizes Mycosporine-glycine obtained by the construction method of claim 7 is cultured in a culture medium.

9. The method according to claim 8, characterized in that, The culture medium contains glucose and xylose as a mixed carbon source, wherein the mass concentration ratio of glucose to xylose in the mixed carbon source is 1:

1.

10. The use of the genetically engineered Bacillus subtilis for synthesizing Mycosporine-glycine as described in any one of claims 1-6, or the genetically engineered Bacillus subtilis for synthesizing Mycosporine-glycine obtained by the construction method described in claim 7, in the production of Mycosporine-glycine.

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