Construction method and application of genetically engineered corynebacterium glutamicum for producing mycosporine-glycine

Abstract

The application provides a construction method and application of a genetically engineered Corynebacterium glutamicum for producing mycosporine-glycine (M-Gly), and belongs to the field of microbial genetic engineering. The application takes Corynebacterium glutamicum ATCC 13032 as a starting strain, knocks out competitive pathway genes, multi-copy key enzyme genes, and acid production pathway genes on the genome of the strain, introduces xylose isomerase gene xylA and xylulose kinase gene xylB, D-xylose:H(+) transport protein gene xylE from Escherichia coli K-12 MG1655, optimizes the carbon source ratio and genome integration, and simultaneously constructs a double-plasmid expression system, so that high-efficiency synthesis of M-Gly is realized. In 72 h of shake flask fermentation, the genetically engineered strain can produce M-Gly up to 2.47 g / L, and in a 50 L fermenter, M-Gly can be accumulated to 15.8 g / L.

Description

Technical Field

[0001] This invention belongs to the field of microbial genetic engineering and synthetic biology, specifically relating to a method for constructing and applying a genetically engineered Corynebacterium glutamicum that produces the mycosporine-Glycine-like amino acid. Background Technology

[0002] Mycosporine-like amino acids (MAAs) are a large class of small-molecule secondary metabolites widely found in marine and terrestrial microorganisms, algae, and some fungi. Their core characteristic is the presence of a substituted cyclohexanone or imidazolinone structure, enabling them to exhibit strong ultraviolet absorption in the 310–360 nm region. Furthermore, after absorbing ultraviolet light, they dissipate the light energy in a non-toxic, non-reactive oxygen-forming form, thus physiologically exhibiting a highly efficient "natural sunscreen" function. Mycosporine-Glycine (M-Gly) is the most structurally typical member of the MAAs family, with a small molecular weight, good water solubility, and a maximum ultraviolet absorption peak typically located around 310 nm. It possesses strong UVA / UVB absorption capabilities and good photostability. Due to its combined properties of ultraviolet protection, antioxidant activity, and mild safety, M-Gly has broad potential applications in cosmetic sunscreens, medical skin protection, functional food additives, antioxidants, protection against photosensitive diseases, and photosensitivity in agricultural plants. Therefore, developing an industrialized production system for safe, efficient, and scalable M-Gly production has significant industrial prospects and practical implications.

[0003] However, the content of M-Gly in nature is generally low, and it mainly exists in some marine algae, cyanobacteria, and fungi. Obtaining natural resources is constrained by the difficulty of species cultivation, growth cycle, seasonal environmental fluctuations, and complex separation and extraction processes. Traditional methods for extracting M-Gly from natural organisms are costly and have unstable yields, making it difficult to meet the needs of industrial applications. Therefore, constructing microbial cell factories capable of efficiently synthesizing M-Gly based on synthetic biology and metabolic genetic engineering techniques has become a research hotspot in recent years.

[0004] Recent studies through genomics and functional gene research have shown that the biosynthesis of MAAs usually originates from metabolic intermediates branching from the shikimate pathway. It is generally believed that intermediates of the 3-dehydropentaerythritol acid (DHQ) or pentose phosphate pathway can serve as precursors, which undergo a series of enzymatic reactions to form a mycosporine nucleus, which then condenses with amino acids (such as glycine, serine, etc.) to generate MAAs with different structures.

[0005] Corynebacterium glutamicum, a recognized safe (GRAS) industrial strain, is widely used in the production of high-value-added products such as amino acids and terpenoids due to its non-endotoxin production, rapid growth, and suitability for high-density fermentation. The production of amino acids (MAAs) using Corynebacterium glutamicum is currently at a mature industrial level, exhibiting strong metabolic plasticity, convenient gene editing, and food safety characteristics. As a high-yield amino acid bacterium, Corynebacterium glutamicum can simultaneously provide sufficient amino acid precursors such as glycine and serine. Related synthetic genes have been identified from naturally occurring M-Gly algae or microorganisms and their effective expression has been achieved in Corynebacterium glutamicum. Furthermore, efficient production of MAAs can be achieved through strategies such as pre-supply and carbon flow redirection. Summary of the Invention

[0006] To address the problems of difficulty, low yield, and environmental pollution associated with obtaining M-Gly naturally, this invention provides a method for constructing and applying a genetically engineered *Corynebacterium glutamicum* strain for synthesizing M-Gly. Through genetic engineering, competing pathway genes are knocked out, key enzyme genes are copied multiple times, and genes involved in the acid-producing pathway are knocked out. The xylose isomerase gene *xylA*, xylulose kinase gene *xylB*, and D-xylose:H(+) transporter gene *xylE* from *Escherichia coli* K-12 MG1655 are introduced. The carbon source ratio and genome integration are optimized. Simultaneously, a dual-plasmid expression system is constructed. The xylose isomerase gene *xylA*, xylulose kinase gene *xylB*, and D-xylose:H(+) transporter gene *xylE* from *E. coli* K-12 MG1655 are heterologously expressed on the pXMJ19 plasmid; while the xylose from *Anabaena variabilis* ATCC is heterologously expressed on the pEC-XK99E plasmid. The 3-dehydroquinanate synthase gene AvmysA, O-methyltransferase gene AvmysB, and ATP-grasp ligase gene AvmysC of 29413 were synthesized, and the carbon source ratio of the genetically engineered bacteria was optimized and xylose utilization-related genes were integrated into the genome. This effectively increased the yield of M-Gly in Corynebacterium glutamicum cells, which is an efficient and green method.

[0007] Preferably, this invention provides a dual-plasmid M-Gly production genetically engineered strain, in which the xylose isomerase gene xylA, xylulose kinase gene xylB, and D-xylose:H(+) transporter gene xylE from *Escherichia coli* K-12 MG1655 are cloned into plasmid pXMJ19 for expression. The 3-dehydroquinanate synthase gene AvmysA, O-methyltransferase gene AvmysB, and ATP-grasp ligase gene (AvmysC) from *Anabaena variabilis* ATCC29413 are cloned into plasmid pEC-XK99E for expression. A strong constitutive promoter P is used. tuf The xylose utilization pathway genes (xylose isomerase gene xylA, xylulose kinase gene xylB, and D-xylose:H(+) transporter gene xylE) were expressed and integrated into the genome, ultimately yielding a genetically engineered strain capable of synthesizing M-Gly. After this modification, under suitable carbon source ratios, the genetically engineered strain produced 2.47 g / L of M-Gly through 72 h of shake-flask fermentation, accumulating up to 15.8 g / L in a 50 L fermenter.

[0008] The first objective of this invention is to provide plasmids that can efficiently express the genes xylA, xylB and xylE.

[0009] In one embodiment, the pXMJ19 plasmid is used as an expression vector;

[0010] In one embodiment, genes xylA, xylB and xylE are linked into a gene cluster via RBS and constructed onto the pXMJ19 plasmid to obtain the recombinant plasmid pXMJ19-xylABE.

[0011] In one embodiment, the xylose isomerase gene xylA is derived from Escherichia coli K-12 MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.1, and its nucleotide sequence is shown in SEQ ID NO.12;

[0012] In one embodiment, the xyl kinase gene xylB is derived from Escherichia coli K-12 MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.2, and its nucleotide sequence is shown in SEQ ID NO.13.

[0013] In one embodiment, the D-xylose:H(+) transporter gene xylE is derived from Escherichia coli K-12MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.3, and its nucleotide sequence is shown in SEQ ID NO.14.

[0014] In one embodiment, the map of the high-copy expression plasmid pXMJ19-xylA-RBS-xylB-RBS-xylE is as follows: Figure 1 As shown.

[0015] In one embodiment, the RBS used to link the three key enzyme genes on the plasmid is the universal RBS (CK1) of Corynebacterium glutamicum, and the CK1 gene sequence is shown in SEQ ID NO.8.

[0016] The second objective of this invention is to provide plasmids that can efficiently express the genes AvmysA, AvmysB, and AvmysC.

[0017] In one implementation, the pEC-XK99E plasmid is used as the expression vector;

[0018] In one embodiment, genes AvmysA, AvmysB, and AvmysC are linked together into a gene cluster via RBS and constructed onto the pEC-XK99E plasmid to obtain the recombinant plasmid pEC-XK99E-AvmysABC.

[0019] In one embodiment, the 3-dehydroquinanate synthase gene AvmysA is derived from Anabaenavariabilis ATCC 29413, and its encoded amino acid sequence is shown in SEQ ID NO.4, and its nucleotide sequence is shown in SEQ ID NO.9.

[0020] In one embodiment, the O-methyltransferase gene AvmysB is derived from Anabaenavariabilis ATCC 29413, and its encoded amino acid sequence is shown in SEQ ID NO.5, and its nucleotide sequence is shown in SEQ ID NO.10.

[0021] In one embodiment, the ATP-Grasp ligase gene AvmysC is derived from Anabaenavariabilis ATCC 29413, and its encoded amino acid sequence is shown in SEQ ID NO.6, and its nucleotide sequence is shown in SEQ ID NO.11.

[0022] In one embodiment, the map of the high-copy expression plasmid pEC-XK99E-AvmysABC is as follows: Figure 2 As shown.

[0023] In one embodiment, the RBS used to link the three key enzyme genes on the plasmid is the universal RBS (CK1) of Corynebacterium glutamicum, and the CK1 gene sequence is shown in SEQ ID NO.8.

[0024] The third objective of this invention is to provide a genetically engineered Corynebacterium glutamicum that synthesizes M-Gly. This genetically engineered Corynebacterium glutamicum integrates xylA (xylose isomerase), xylB (xylose kinase), and xylE (D-xylose:H(+) transporter) genes from Escherichia coli K-12 MG1655 into the genomic rph site, thereby enhancing the pentose phosphate pathway and achieving efficient synthesis of M-Gly.

[0025] In one embodiment, the xylose isomerase gene xylA is derived from *Escherichia coli* K-12 MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.1, with NCBI accession number NP_418022; the xylose isomerase gene xylA is generated by a strong constitutive promoter P. tuf Initiate expression;

[0026] In one embodiment, the xylose kinase gene xylB is derived from *Escherichia coli* K-12 MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.2, with NCBI accession number NP_418021; the xylose kinase gene xylB is generated by a strong constitutive promoter P. tuf Initiate expression;

[0027] In one embodiment, the D-xylose:H(+) transporter gene xylE is derived from *Escherichia coli* K-12MG1655, and its encoded amino acid sequence is shown in SEQ ID NO.3, with NCBI accession number NP_418455; the xylose kinase gene xylE is generated by a strong constitutive promoter P. tuf Initiate expression;

[0028] In one embodiment, the Corynebacterium glutamicum includes, but is not limited to, Corynebacterium glutamicum ATCC 13032.

[0029] A fourth objective of this invention is to provide an optimal carbon source ratio for producing the spore-like amino acid M-Gly.

[0030] In one embodiment, the carbon source ratio in the culture medium is optimized, with the glucose to xylose ratio set to 1:0, 3:2, 1:1, 2:3, and 0:1.

[0031] The fifth objective of this invention is to provide a method for producing the spore-like amino acid M-Gly, wherein the method utilizes the genetically engineered Corynebacterium glutamicum to produce the spore-like amino acid M-Gly through fermentation.

[0032] In one embodiment, Corynebacterium glutamicum is routinely cultured in BHIS medium at 30 °C and 220 rpm for 16–18 h.

[0033] In one embodiment, Corynebacterium glutamicum is fermented in a modified CGXII culture medium at 30 °C, containing 20 g / L glucose, 20 g / L xylose, 5 g / L yeast extract, 5 g / L corn steep liquor, 20 g / L ammonium sulfate, 10 g / L urea, 1 g / L potassium dihydrogen phosphate, 1.0 g / L dipotassium hydrogen phosphate, 42 g / L 3-morpholinopropanesulfonic acid, and trace metal elements. The trace metal elements include: 250 mg / L magnesium sulfate heptahydrate, 10 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 1 mg / L zinc sulfate heptahydrate, 0.2 g / L anhydrous copper sulfate, 0.02 mg / L nickel chloride hexahydrate, 0.2 mg / L biotin, 10 mg / L calcium chloride, 30 mg / L protocatechuic acid, and 0.1 mg / L vitamin B1.

[0034] Shake flask culture to OD 600 =1.0±0.1, add IPTG to a final concentration of 0.8~1.2 mM, and continue induction culture at 30 °C and 220 rpm for no less than 72 h;

[0035] The fermenter was operated at 30 °C, with a stirring speed of 200-800 r / min, an aeration rate of 2-6 vvm, and a pH of 7.0±0.2, until fermentation reached OD. 600 When the concentration is 15±0.5, add IPTG to a final concentration of 0.8~1.2mM and induce culture at 30 °C for no less than 72 h. If necessary, add antibiotics of appropriate concentration.

[0036] More preferably, the induction culture in the fermenter system shall be no less than 72 h.

[0037] In one embodiment, during the reaction, a mother liquor concentration of 500 g / L glucose or 500 g / L xylose is added to maintain its final concentration at 10-15 g / L, while 50% ammonia is added to bring the pH of the entire system to 7.0, thereby maintaining the production of the product M-Gly.

[0038] Preferably, when the concentration of glucose or xylose in the reaction system is less than 10 g / L, glucose or xylose is added at once to a final concentration of 10 g / L to ensure sufficient energy and normal cell growth, thereby promoting the continuous production of M-Gly energy.

[0039] A sixth object of the present invention is to provide the use of the genetically engineered Corynebacterium glutamicum in the preparation of M-Gly or products containing M-Gly.

[0040] The seventh objective of this invention is to provide the application of the genetically engineered Corynebacterium glutamicum in the fields of food, cosmetics, pharmaceutical development, and biomaterials.

[0041] The beneficial effects of this invention are:

[0042] This invention uses Corynebacterium glutamicum ATCC 13032 as the starting strain. By expressing a foreign gene from Anabaena variabilis ATCC 29413 and a xylose utilization gene from Escherichia coli MG155, respectively, on plasmids pEC-XK99E and pXMJ19, the resulting Corynebacterium glutamicum produced a maximum M-Gly yield of 2.47 g / L under 72 h fermentation conditions in shake flasks. When this Corynebacterium glutamicum was fermented in a fermenter for 72 h, the M-Gly yield reached 15.8 g / L, representing an approximately 6.4-fold increase in yield from shake flasks to a scale-up fermenter system. Attached Figure Description

[0043] Figure 1 A schematic diagram of plasmid pEC-XK99E-AvmysABC.

[0044] Figure 2 A schematic diagram of plasmid pXMJ19-xylABE. Detailed Implementation

[0045] 1. The plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products. The specific operations should be performed in accordance with the kit instructions.

[0046] 2. Routine procedures such as colony PCR, nucleic acid agarose gel electrophoresis, heat shock transformation, preparation of competent cells, and extraction and preservation of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0047] 3. The sequencing of plasmids and DNA products was completed by Shanghai Sangon Biotech Co., Ltd.

[0048] 4. Preparation of competent Escherichia coli cells: TAKARA kit.

[0049] 5. Reference for competent cells of Corynebacterium glutamicum: Jiang, Y., Qian, F., Yang, J. et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun8, 15179 (2017).

[0050] 6. Fermentation process and detection of mycotoxin-like amino acid M-Gly:

[0051] (1) BHIS liquid culture medium: BHI 37 g / L, D-sorbitol 91 g / L.

[0052] (2) BHIS solid medium: BHI 37 g / L, D-sorbitol 91 g / L, 15 g / L agar powder.

[0053] (3) Fermentation medium: 20 g / L glucose, 20 g / L xylose, 5 g / L yeast extract, 5 g / L corn steep liquor, 20 g / L ammonium sulfate, 10 g / L urea, 1 g / L potassium dihydrogen phosphate, 1.0 g / L dipotassium hydrogen phosphate, 42 g / L 3-morpholinopropanesulfonic acid and trace metal elements; the trace metal elements include: 250 mg / L magnesium sulfate heptahydrate, 10 mg / L ferrous sulfate heptahydrate, 10 mg / L manganese sulfate monohydrate, 1 mg / L zinc sulfate heptahydrate, 0.2 g / L anhydrous copper sulfate, 0.02 mg / L nickel chloride hexahydrate, 0.2 mg / L biotin, 10 mg / L calcium chloride, 30 mg / L protocatechuic acid, 0.1 mg / L vitamin B1. When adding plasmids for expression, kanamycin or chloramphenicol at a final concentration of 25 μg / mL and IPTG at a final concentration of 0.9 mM should be added to induce plasmid activity.

[0054] (4) Fermentation process for producing the spore-like amino acid M-Gly: The constructed strain was inoculated into BHI liquid medium, and kanamycin or chloramphenicol was added to a final concentration of 25 μg / mL. The culture was carried out overnight at 30 °C and 220 rpm for 16-18 h to obtain the seed culture. 2 mL of the seed culture was inoculated into 50 mL of fermentation medium and cultured at 30 °C and 220 rpm until the OD reached the target concentration. 600 =1.0±0.1, add IPTG to a final concentration of 0.9 mM, and continue induction culture at 30 °C and 220 rpm for 72 h. Take 1 mL of fermentation broth, centrifuge at 10,000 rpm for 10 min, collect the supernatant, and use it for HPLC analysis.

[0055] (5) HPLC detection conditions: High performance liquid chromatography (HPLC) system (Agilent); Column: ZORBAX Eclipse Plus C18; Detector: Agilent UV detector; UV absorption wavelength: 310 nm; Mobile phase: 0.25% formic acid aqueous solution; Flow rate: 0.65 mL / min; Column temperature: 35 °C; Injection volume: 10 μL.

[0056] Example 1: Construction of pXMJ19-xylABE expression plasmid

[0057] Using the empty pXMJ19 vector as a template, a linear vector was amplified using primers pXMJ19-VF / R. Using the *E. coli* MG1655 genome as a template, gene fragment xylA was amplified using primers xylA-F / R, gene fragment xylB using primers xylB-F / R, and gene fragment xylE using primers xylE-F / R. Multiple fragments were assembled using homologous recombination at 50 °C for 30 min. After chemical transformation into DH5α, and successful culture, bacterial selection, plasmid extraction, and sequencing verification, the recombinant plasmid pXMJ19-xylABE was obtained.

[0058] Example 2: Construction of genetically engineered strains of Corynebacterium glutamicum

[0059] The specific steps for constructing gene-editing plasmids and genetically engineered strains are as follows (the primer sequences involved are shown in Table 1):

[0060] The exogenous gene xylABE was integrated into the rph site of Corynebacterium glutamicum ATCC 13032 using the SacB-assisted CRISPR-FnCpf1 gene editing system, and the strong constitutive promoter P was utilized. tuf For efficient expression, refer to the CRISPR / Cpf1 gene editing system. See Zhang, J., et al. (2020). De Novo Engineering of Corynebacterium glutamicum for l-Proline Production. ACS Synthetic Biology 9(7): 1897–1906.

[0061] The specific steps are as follows:

[0062] (1) Constructing plasmid pK18mobsacB-spec-Δrph::P tuf -xylABE

[0063] Using the genome of Corynebacterium glutamicum ATCC 13032 as a template, the upstream and downstream fragments of the rph gene were amplified by PCR using rph-UH / DH-F / R primers (Table 1); the constitutive promoter P was amplified using ptuf-F / R. tuf Using pK18-F / R primers, the vector fragment was amplified using plasmid pK18mobsacB-spec as a template; using xylA-ptuf-F / xylE-rph-R primers, the gene fragment xylABE was amplified using plasmid pXMJ19-xylABE as a template. Homologous recombination was used to assemble multiple fragments at 50 °C for 30 min. After chemical transformation into DH5α, and successful verification through culture, bacterial selection, plasmid extraction, and sequencing, the recombinant gene editing plasmid pK18mobsacB-spec-Δrph::P was obtained. tuf -xylABE.

[0064] (2) Constructing a genetically engineered strain of Corynebacterium glutamicum

[0065] The gene-editing plasmid pK18mobsacB-spec-Δrph::P constructed above was applied... tuf -xylABE was transferred into *Corynebacterium glutamicum* via electroporation at a concentration of 800–1000 ng, operated at 25 μF, 200 Ω, and 2.5 kV. The transformed knockout strains were then plated on spectinomycin-resistant BHIS plates. Single-crossover mutants were selected, and individual colonies of each mutant were cultured in BHIS medium for 16–18 h. Once single colonies appeared, the strains were picked and cultured overnight to create competent cells. The constructed pJYS3-rph-N23 plasmid was then transferred into the competent cells via electroporation as described above. The cells were heat-shocked at 46 °C for 6 min, incubated on a shaker at 30 °C for 2 h, and plated on BHIS plates containing kanamycin and sucrose. The plates were then incubated at 30 °C until single colonies appeared. Successfully grown single colonies were identified as double-crossover mutants. Colony PCR was used to verify the correct editing of the strain, and the samples were then sent to a sequencing company for further confirmation of gene integration. The successfully validated strains were inoculated into test tubes containing BHIS medium and cultured overnight at 34 °C to remove plasmids. The plasmids were then streaked onto BHIG agar plates containing kanamycin to screen for plasmid-free strains. Finally, plasmid-free, antibiotic-free Corynebacterium glutamicum strains were obtained for use in the next round of editing or for fermentation validation.

[0066] (3) Constructing genetically engineered Corynebacterium glutamicum containing plasmids pEC-XK99E-AvmysABC and pXMJ19-xylABE

[0067] The genetically engineered strain with successfully edited genes was prepared as an electroporation competent state. pEC-XK99E-AvmysABC was transferred into the genetically engineered strain integrating the exogenous gene by electroporation, as described in (2). Then, it was plated on a BHIS agar plate containing kanamycin resistance. After colonies grew, a genetically engineered Corynebacterium glutamicum containing plasmid pEC-XK99E-AvmysABC was obtained. The competent state of this strain was then prepared to electroporate plasmid pXMJ19-xylABE, resulting in a genetically engineered strain containing two plasmids, which was used for fermentation verification.

[0068] Table 1 Primers used for plasmid and genome construction

[0069]

[0070] Example 3: Production of spore-like amino acid M-Gly by shake-flask fermentation

[0071] Based on Example 2, the genetically engineered strain Cg-09 was fermented in shake flasks. The strain was then transformed with plasmid pXMJ19-xylABE to obtain the genetically engineered strain Cg-10. Different glucose-to-xylose ratios were set: 1:0 (glucose 40 g / L), 3:2 (glucose 24 g / L, xylose 16 g / L), 1:1 (glucose 20 g / L, xylose 20 g / L), 2:3 (glucose 16 g / L, xylose 24 g / L), and 0:1 (xylose 40 g / L). This verified that the highest M-Gly production ratio could be obtained. The entire process was carried out at 30 °C and 220 rpm until OD (dose over time). 600=1.0±0.1, add IPTG to a final concentration of 0.9 mM. After 72 h of shake-flask fermentation, samples were taken. 1 mL of fermentation broth was centrifuged at 10,000 rpm for 10 min, and the supernatant was collected for HPLC analysis. The maximum M-Gly yield accumulated to 1.68 g / L after 72 h. Different carbon source ratios had different effects on M-Gly production, as shown in Table 2. Analysis of the data in the table shows that when only glucose was present in the culture medium, the yield was only 1.15 g / L. The yield increased further when xylose was introduced. Because *Corynebacterium glutamicum* does not contain genes for xylose utilization and transport, the introduction of exogenous xylose utilization genes allows *Corynebacterium glutamicum* to synthesize M-Gly more quickly via the pentose phosphate pathway. Analysis of the fermentation results indicates that *Corynebacterium glutamicum* utilizes glucose carbon flow more towards the glycolysis pathway. Therefore, when the strain can utilize xylose, excess carbon can flow towards the pentose phosphate pathway, thus flowing towards the M-Gly biosynthesis pathway. However, when only xylose is used as a carbon source in the culture medium, the yield of M-Gly will not reach its maximum. This may be because the strain needs more carbon source for growth and prefers to use glucose. Therefore, when the ratio of the two is 1:1, better growth and production can be achieved, and the maximum yield of M-Gly can be realized.

[0072] Table 2. Detailed information on shake-flask fermentation of genetically engineered bacteria

[0073]

[0074] Based on the above experimental results, the introduction of xylose utilization and transport genes into the strain, followed by optimization of the carbon source ratio, can achieve efficient product accumulation. The exogenous gene xylABE was integrated into the genome for stable expression, resulting in the genetically engineered strain Cg-11. This strain was then individually transfected with plasmid pEC-XK99E-AvmysABC and simultaneously transfected with the dual plasmids pEC-XK99E-AvmysABC and pXMJ19-xylABE, with a control group without plasmids included. Shake-flask fermentation was performed to verify the results, as shown in Table 2. The plasmid-infused strain accumulated 1.92 g / L and 2.47 g / L, respectively, after 72 h of shake-flask fermentation. Simultaneous overexpression of the exogenous enzyme gene with dual plasmids maximized M-Gly production.

[0075] Example 4: Synthesis of M-Gly by fed-batch culture in a fermenter

[0076] Fed-batch fermentation experiments were conducted on genetically engineered strains of M-Gly in a 50 L fermenter.

[0077] The genetically engineered Corynebacterium glutamicum, which performed best in Example 2, was inoculated into liquid BHIS medium containing kanamycin and chloramphenicol and cultured overnight at 30 °C and 220 rpm for 16–18 h to obtain a primary seed culture. This primary seed culture was then inoculated into a new fermentation system for further expansion culture at 30 °C and 220 rpm. 600 =1.0±0.1, to obtain secondary seed culture, which was then inoculated into a 50 L fermenter to achieve a final culture volume of 20 L for fermentation. Fermentation was carried out at 30 °C, with a stirring speed of 220 rpm, an aeration rate of 1~2 vvm, and a pH of 7.0±0.2, until OD reached. 600 When the concentration of the sample reached 15 ± 0.5, IPTG was added to a final concentration of 0.9 mM, and the mixture was induced and cultured at 30 °C for 72 h. Dissolved oxygen was controlled by adjusting the stirring speed (220 rpm) and aeration rate (2–6 vvm). During the reaction, a mother liquor concentration of 500 g / L glucose or 500 g / L xylose was added to maintain the final glucose and xylose concentrations at 10–15 g / L (with a final glucose to xylose ratio of 1:1). Simultaneously, 50% ammonia was added to maintain the pH of the entire system at 7.0, thereby ensuring the production of the product M-Gly.

[0078] After 72 h of fermentation, strain Cg-13 achieved an M-Gly yield of 15.8 g / L and an OD of [missing value]. 600 The highest value reached 112.

[0079] 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 Corynebacterium glutamicum that produces the mycosporine-glycine-like amino acid, characterized in that, Starting with Corynebacterium glutamicum ATCC 13032, the competing pathway gene transaldolase gene tal was knocked out; the pyruvate dehydrogenase gene poxB and the phosphoacetyltransferase gene pta were knocked out; the xylose utilization gene cluster xylABE and the gene cluster AvmysABC were integrated into the genome; The gene cluster xylABE consists of xylose isomerase gene xylA, xylulose kinase gene xylB, and D-xylose:H(+) transporter gene xylE, all derived from Escherichia coli K-12 MG1655. The gene cluster AvmysABC consists of the 3-dehydroquinacid synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC, all derived from the algae Anabaena ATCC 29413.

2. The genetically engineered Corynebacterium glutamicum for producing the mycosporine-Glycine-like amino acid according to claim 1, characterized in that, It also contains plasmids pEC-XK99E-AvmysABC and pXMJ19-xylABE.

3. A genetically engineered Corynebacterium glutamicum that produces the mycosporine-glycine-like amino acid, characterized in that, The following methods were used to modify Corynebacterium glutamicum ATCC 13032, the starting strain. 1) Knock out the aldolase gene tal on the genome of Corynebacterium glutamicum; 2) Knock out the short-chain dehydrogenase gene Cgl1036 and replace it with one generated by the strong constitutive promoter P. tuf The gene cluster that initiates expression is AvmysABC; 3) Knock out the L-lactate dehydrogenase gene lldA and replace it with one generated by a strong constitutive promoter P. tuf The gene cluster that initiates expression is AvmysABC; 4) Knock out the glucosamine-6-phosphate deaminase gene nagB and replace it with one generated by a strong constitutive promoter P. tuf The gene cluster that initiates expression is AvmysABC; 5) Knock out the phosphoenolpyruvate carboxylase gene ppc and replace it with one generated by a strong constitutive promoter P. tuf The gene cluster that initiates expression is AvmysABC; 6) Knock out the pyruvate dehydrogenase gene poxB and the phosphoacetyltransferase gene pta; 7) Knock out the putative ribonuclease pH gene rph and replace it with the strong constitutive promoter P. tuf Xylose expression is initiated using the gene cluster xylABE; The gene cluster AvmysABC is composed of the 3-dehydroquinanate synthase gene AvmysA, the O-methyltransferase gene AvmysB, and the ATP-grasp ligase gene AvmysC, all derived from the algae Anabaena ATCC 29413; the gene cluster xylABE is composed of the xylose isomerase gene xylA, the xylulose kinase gene xylB, and the D-xylose:H(+) transporter gene xylE, all derived from Escherichia coli K-12 MG1655.

4. The genetically engineered Corynebacterium glutamicum for producing the mycosporine-glycine-like amino acid according to claim 3, characterized in that, It also contains plasmid pEC-XK99E-AvmysABC and / or plasmid pXMJ19-xylABE.

5. The genetically engineered Corynebacterium glutamicum for producing the mycosporine-glycine-like amino acid according to claims 1-4, characterized in that, 1) The amino acid sequence encoded by the xylA xylose isomerase gene is shown in SEQ ID NO.

1. 2) The amino acid sequence encoded by the xylB xyl kinase gene is shown in SEQ ID NO.

2. 3) The amino acid sequence encoded by the D-xylose:H(+) transporter gene xylE is shown in SEQ ID NO.

3. 4) The amino acid sequence encoded by the 3-dehydroquinanate synthase gene AvmysA is shown in SEQ ID NO.4; 5) The amino acid sequence encoded by the O-methyltransferase gene AvmysB is shown in SEQ ID NO.5; 6) The amino acid sequence encoded by the ATP-grasp ligase gene AvmysC is shown in SEQ ID NO.

6.

6. The use of the genetically engineered Corynebacterium glutamicum according to any one of claims 1-4 in the production of the spore-like amino acid M-Gly.

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