Construction method of high-purity hydroxyl Ectoin producing strain

Abstract

The invention discloses a construction method of a high-purity hydroxyl Ectoine producing strain, which comprises the following steps: by taking corynebacterium glutamicum as an original strain, carrying out integration overexpression on multiple copy numbers of aspartate kinase lysC and hydroxyl Ectoine operon EctABCD on a corynebacterium glutamicum genome, and taking high salt as environmental selection pressure, so as to obtain the high-purity hydroxyl Ectoine producing strain. And knocking out the salt resistance related gene in corynebacterium glutamicum to obtain the production strain. The production strain obtained according to the construction method disclosed by the invention is cultured by using a high-salt fermentation culture medium, so that the hydroxyl Ectoine with the purity of more than 99% can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic biology, and specifically relates to a method for constructing a high-purity hydroxyectoine-producing bacterium. Background Art

[0002] Hydroxyectoine is a naturally occurring compound belonging to the "amino acid-like" substances, widely present in certain microorganisms, especially extremophiles (such as high-salt-tolerant, high-temperature-tolerant or drought-tolerant microorganisms). It is a derivative of ectoine, and ectoine itself is a natural osmoprotectant, also known as "protective amino acid", which helps microorganisms survive in extreme environments.

[0003] Both hydroxyectoine and ectoine belong to natural osmoprotectants, and they have many similar biological functions, such as protecting cells from the stress of extreme environments (such as high temperature, high salt, drought, etc.). However, hydroxyectoine exhibits additional advantages in some aspects compared to traditional ectoine:

[0004] 1. Hydroxyectoine has a stronger moisturizing effect compared to ectoine. It can more effectively maintain the hydration of the skin and prevent water loss. Research shows that hydroxyectoine has a stronger regulatory effect on the hydration of cells and can help the skin maintain a long-term moist state;

[0005] 2. Hydroxyectoine is more effective than ectoine in terms of antioxidant. It can better neutralize free radicals and reduce oxidative damage caused by environmental stress. This property is particularly important for slowing down the aging process and improving the anti-aging ability of the skin.

[0006] 3. Hydroxyectoine helps to enhance the skin barrier function, especially after being stimulated or damaged by the environment. It may perform better than ectoine in promoting skin repair and reducing inflammation, and can help the skin recover to a healthy state, reducing irritation and redness.

[0007] 4. Although ectoine is an excellent osmoprotectant, hydroxyectoine shows better tolerance and adaptability when facing more extreme environmental stresses (such as higher temperature, stronger salinity, strong ultraviolet radiation, etc.). Minor changes in its structure enable it to provide more stable protection in extreme environments.

[0008] However, the problem of the commercial production of hydroxyectoine has not been effectively solved: Since traditional chemical synthesis of hydroxyectoine has a pyrrolidine ring structure and nitrogen-containing functional groups, chemical synthesis of it requires a high degree of selectivity and relatively high chemical reaction control in the synthesis path, resulting in a very high cost of producing hydroxyectoine using this method.

[0009] In recent years, with the continuous development of synthetic biology technology, more and more scientists have begun to consider using microbial fermentation to produce high-value-added products with clear structures, such as ergothioneine, ectoine, and hydroxyectoine. Hydroxyectoine has been found to be synthesized in extremophilic archaea, but the yield is extremely low. The synthesis of hydroxyectoine using Corynebacterium glutamicum has gradually become a trend, but most of them are through a two-step method, that is, first using Corynebacterium glutamicum to produce ectoine, and then separating and purifying ectoine and converting it into hydroxyectoine by enzymatic catalysis. This method has two disadvantages. First, the cost is relatively high. Second, it is impossible to completely convert ectoine into the target product. The structures of ectoine and hydroxyectoine are similar, and it is very difficult to separate and purify them in the later stage, resulting in the inability to obtain pure hydroxyectoine.

[0010] Based on this, it is necessary to explore a method for constructing a high-purity hydroxyectoine-producing strain. Summary of the Invention

[0011] The inventors studied the biosynthetic route of hydroxyectoine in the reported Corynebacterium glutamicum, developed a new metabolic pathway, used Corynebacterium glutamicum as the starting strain, integrated and overexpressed multiple copies of aspartokinase LysC and the hydroxyectoine operon EctABCD on the Corynebacterium glutamicum genome, and used high salt as the environmental selection pressure to knockout the salt resistance-related genes in Corynebacterium glutamicum to obtain an engineered strain for synthesizing high-purity hydroxyectoine.

[0012] Specifically, the present invention includes the following technical solutions.

[0013] In a first aspect, the present invention provides a method for constructing a high-purity hydroxyectoine-producing strain, and the construction method includes the following steps:

[0014] (1) Using Corynebacterium glutamicum ATCC13032LysCfbr that produces lysine as the starting strain, enhancing the expression of the gene lysC encoding aspartokinase in the strain genome to obtain a strain with overexpressed aspartokinase;

[0015] (2) On the basis of the strain overexpressing LysC, continue to overexpress the EctABC gene cluster from different sources to obtain ectoine-producing bacteria; the sources of the EctABC gene cluster are Halomonas elongata, Streptomyces avermitilis, Candidatus Nitrosopumilus sediminis, Vibrio natriegens NBRC or Pseudomonas stutzeri;

[0016] (3) On the basis of the above-obtained ectoine-producing bacteria, overexpress the EctD gene from different sources to obtain hydroxyectoine-producing bacteria; the sources of the EctD gene are Halomonas elongata, Mycobacterium smegmatis or Pseudomonas stutzeri;

[0017] (4) On the basis of the above-obtained hydroxyectoine-producing bacteria, separately or combinatorially knockout the key genes treY, osmC and / or glnA related to the osmoregulation of the strain.

[0018] In the present invention, the steps of (1), (2), (3) and (4) above are not limited, and they can be operated crosswise and reversely, as long as each step can achieve its respective function and complete the directional change of the host cell genotype. Those skilled in the art can adjust each step according to actual needs or actual plans. For example, Corynebacterium glutamicum capable of producing lysine can be used as the starting strain, first overexpress the gene EctABCD, then overexpress the gene lysC, and finally knockout the key genes related to the osmoregulation of the strain; or first knockout the key genes related to the osmoregulation of the strain, then overexpress the genes lysC and EctABCD; or first overexpress the gene lysC, then knockout the key genes related to the osmoregulation of the strain, and finally overexpress the gene EctABCD. Those skilled in the art can expect that the final production strains obtained according to different construction steps are not substantially different, including the growth of the strains and the fermentation products.

[0019] In the present invention, the source of the lysC gene encoding aspartokinase in step (1) is not limited, as long as the lysC gene with the same function can be applied to the present invention. As an example, the source of the lysC gene can be Corynebacterium glutamicum ATCC13032.

[0020] In the present invention, in step (4), key genes related to the osmotic pressure regulation of the strain are knocked out, and the knockout method is not limited. Those skilled in the art can select different knockout means according to the prior art and actual situations. For example, the following means / techniques can be used to knock out the relevant genes: homologous double exchange, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, MUCICAT or MuGENT (multiplex genome editing by natural transformation).

[0021] In one or more embodiments, the copy number of the gene lysC is 1-5, the copy number of each gene in the EctABC gene cluster is 1-3, and the copy number of the EctD gene is 1-3.

[0022] In the present invention, the proportional limitation of the copy number of each gene in the EctABC gene cluster, such as the copy number ratio of genes EctA, EctB, and EctC being 1:3:2, can be 1:1:1, 1:1:2, 1:2:1, 2:1:1, 2:2:1, 2:1:2, 1:2:2, 1:3:1, and 1:3:2.

[0023] Preferably, the source of the EctABC gene cluster can be Vibrio natriegens NBRC, in which the copy number ratio of genes EctA, EctB, and EctC is 1:3:2.

[0024] Preferably, the source of the EctD gene is Mycobacterium smegmatis, and its copy number is 2.

[0025] In the present invention, the construction method is not limited to only including the steps (1), (2), (3), and (4). Those skilled in the art can further modify the strain obtained in each step according to the prior art to improve the final yield of hydroxyectoine. For example, gene editing can be performed on sites that are beneficial to increasing the yield of ectoine in step (2), or gene editing can be performed on sites that are beneficial to increasing the yield of ectoine in step (3). The inventors found that after gene editing of the above remaining steps, the finally obtained strain can still produce high-purity hydroxyectoine.

[0026] In one or more embodiments, the ectoine-producing strain obtained in step (2) is Corynebacterium glutamicum, which is deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 22733 and can be constructed according to the steps disclosed in Patent CN113481233B.

[0027] In one or more embodiments, the enhanced expression of gene lysC, gene cluster EctABC, and gene EctD is achieved by placing the genes downstream of the sod promoter.

[0028] In one or more embodiments, the expression of gene lysC, gene cluster EctABC, and gene EctD is obtained by integrating the genes into plasmid pK18mobSacB, introducing them into host cells, and performing SacB sucrose counter-selection to obtain positive clones.

[0029] In a second aspect, the present invention provides a bacterium for producing high-purity hydroxyectoine, which is obtained by the construction method described in any one of the above.

[0030] In a third aspect, the present invention provides the application of the bacterium in the production of high-purity hydroxyectoine.

[0031] In a fourth aspect, the present invention provides a method for producing high-purity hydroxyectoine, which includes culturing the bacterium obtained by the construction method described in any one of the present invention in a high-salt fermentation medium, wherein the concentration of NaCl in the high-salt fermentation medium is 40 - 60 mM.

[0032] In the present invention, the bacterium for producing high-purity hydroxyectoine obtained by construction can be first activated and subjected to shake-flask fermentation, and then the bacterial liquid can be added to a fermenter for large-scale fermentation; or the bacterium can be directly inoculated into a fermenter for fermentation.

[0033] In the present invention, the composition of the seed medium for shake-flask fermentation can be: peptone 10 g / L, beef extract 5 g / L, yeast powder 5 g / L, sodium chloride 2.5 g / L, urea 2 g / L, glucose 10 g / L. The medium needs to be sterilized at 115 °C for 15 min. The temperature during fermentation of the bacterium is preferably about 30 °C.

[0034] In the present invention, the medium for the fermenter can be:

[0035]

[0036] Description of the Drawings

[0037] Figure 1 It is the plasmid map of pK18mobSacBΔaroB Psod lysC in the examples.

[0038] Figure 2 It is the plasmid map of K18mobSacBΔhisD Psod lysC in the examples.

[0039] Figure 3 It is the plasmid map of pK18mobSacBΔpheA Psod lysC in the examples.

[0040] Figure 4 It is the plasmid map of pK18mobSacBΔsodA Psod lysC in the examples.

[0041] Figure 5 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(111) in the examples.

[0042] Figure 6 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(112) in the examples.

[0043] Figure 7 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(121) in the examples.

[0044] Figure 8 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(112) in the examples.

[0045] Figure 9 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(131) in the examples.

[0046] Figure 10 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(132) in the examples.

[0047] Figure 11 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(211) in the examples.

[0048] Figure 12 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(212) in the examples.

[0049] Figure 13 It is the plasmid map of pK18mobSacB-ΔmetB-ECTABC(221) in the examples.

[0050] Figure 14 It is the plasmid map of pK18mobSacB-Δhpt-ECTD in the embodiment.

[0051] Figure 15 It is the plasmid map of the plasmid with treY knocked out in the embodiment.

[0052] Figure 16 It is the plasmid map of the plasmid with osmC knocked out in the embodiment.

[0053] Figure 17 It is the plasmid map of the plasmid with glnA knocked out in the embodiment.

[0054] Figure 18 It is the product concentration graph of hydroxyectoine at different fermentation times in Example 3. Detailed implementation manners

[0055] The inventors studied the biosynthetic route of hydroxyectoine in the reported Corynebacterium glutamicum, developed a new metabolic pathway. Using Corynebacterium glutamicum as the starting strain, by integrating and overexpressing multiple copies of aspartokinase LysC and the hydroxyectoine operon EctABCD on the Corynebacterium glutamicum genome, and using high salt as the environmental selection pressure, knocking out the salt resistance-related genes in Corynebacterium glutamicum, making the high expression of hydroxyectoine the main way for the strain to resist high salt osmotic pressure, and obtaining an engineered strain for highly efficient synthesis of hydroxyectoine.

[0056] In order to produce high-purity hydroxyectoine, various genes to be knocked out, weakened, integrated, overexpressed, and strongly expressed in the present invention need to be cloned on plasmid vectors respectively. Then they are transferred into Corynebacterium glutamicum (C. glutamicum) competent cells separately, successively or simultaneously; or two or more genes can be cloned on one plasmid, and then transferred into Corynebacterium glutamicum competent cells separately, successively or simultaneously. Among them, available plasmid vectors include pK18mobsacB, etc., but are not limited thereto.

[0057] The following illustrates the implementation manners of the present invention through specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than for limiting the protection scope of the present invention.

[0059] When the embodiments give a numerical range, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0060] For those technical or conditions not specified in the embodiments, they are carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. For reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through regular channels.

[0061] Regarding the addition amounts, contents and concentrations of various substances involved in the embodiments, the percentage content, unless otherwise specified, all refers to the mass percentage content.

[0062] Example

[0063] Materials and Methods

[0064] The whole gene synthesis, primer synthesis and sequencing in the embodiments were commissioned to General Biosystems (Anhui) Co., Ltd.

[0065] The molecular biology experiments in the embodiments, including plasmid construction, restriction digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., were mainly carried out with reference to the third edition of "Molecular Cloning: A Laboratory Manual" (edited by J. Sambrook, D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0066] The PCR amplification experiment was carried out according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions, and could be adjusted through simple experiments when necessary.

[0067] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride, pH 7.2. (20 g / L agar powder was added to the LB solid medium.)

[0068] BHIS medium: 37 g / L BHI, 91 g / L sorbitol.

[0069] BHIS-suc medium: 37 g / L BHI, 91 g / L sorbitol, 200 g / L sucrose, 10 g / L glucose.

[0070] BHI medium: 37 g of BHI powder was added to 1 L of pure water and sterilized at 115 °C for 15 min.

[0071] LB-SUC100: 100 g of sucrose was added to 1 L of LB solid medium.

[0072] LB-SUC100-K25: 100 g of sucrose and 25 mg of kanamycin were added to 1 L of LB solid medium.

[0073] 20X electroporation stock solution: 80 g / L glycine, 2% Tween 80.

[0074] The medium for the 5 L fermenter was:

[0075]

[0076] In the following examples, when using a medium containing kanamycin (kan), the final concentration of kanamycin in the medium was 50 μg / ml; when using a medium containing chloramphenicol (cm), the final concentration of chloramphenicol in the medium was 30 μg / ml; when using a medium containing apramycin resistance (Apr), the final concentration of apramycin in the medium was 50 μg / ml; when using IPTG for induction, the final concentration of IPTG in the medium was 0.2 mM.

[0077] In the following examples, the HPLC detection method for hydroxyectoine and ectoine was as follows: Agilent 1260 high performance liquid chromatograph; the chromatographic column was Agilent AQ-C18, the detector was VWD detector, the detection wavelength was 210 nm, the mobile phase was 10 mM potassium dihydrogen phosphate (pH 3.5): acetonitrile = 99:1, the flow rate was 0.7 ml / min, the column oven temperature was 20 °C; the RT of ectoine was 5.4 min, and the RT of hydroxyectoine was 2.8 min.

[0078] In the following examples, the starting strain used was Corynebacterium glutamicum ATCC13032 LysC fbr , which was constructed by Huarui Biotechnology (Chuzhou) Co., Ltd. according to the method reported in Biotechnol. J. 2019, 14, 1800417.

[0079] In the following examples, the primers used are shown in the following table:

[0080]

[0081]

[0082]

[0083] It should be noted that for the convenience of description, in the embodiments, the strain number, plasmid number, enzyme number, and enzyme-encoding gene number can share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0084] Example 1: Construction of Plasmid for Gene Modification Target

[0085] 1.1 Construction of LysC Expression Plasmid

[0086] a. Amplify the LysC sequence, with the DNA template being Corynebacterium glutamicum ATCC13032;

[0087] The primers are LysC_F and LysC_R;

[0088] PCR conditions: 95°C for 5 min; 92°C for 30 s, 58°C for 30 s, 65°C for 35 s, 30 cycles; 65°C for 10 min.

[0089] b. Amplify the upstream sequence of aroB, with the DNA template being Corynebacterium glutamicum ATCC13032

[0090] The primers are aroBup_F and aroBup_R;

[0091] PCR conditions: 95°C for 5 min; 92°C for 30 s, 58°C for 30 s, 65°C for 35 s, 30 cycles; 65°C for 10 min.

[0092] c. Amplify the downstream sequence of aroB, with the DNA template being Corynebacterium glutamicum ATCC13032;

[0093] The primers are aroBdn_F and aroBdn_R;

[0094] PCR conditions: 95°C for 5 min; 92°C for 30 s, 58°C for 30 s, 65°C for 35 s, 30 cycles; 65°C for 10 min.

[0095] d. Amplify the plasmid backbone, with the DNA template being the pK18mobSacB-ddh-ECT plasmid (the plasmid constructed in Example 1 of Patent CN113481233B);

[0096] The primers are backbone_F1 and backbone_R1;

[0097] PCR conditions: 95°C for 5 min; 92°C for 30 s, 58°C for 30 s, 65°C for 35 s, 30 cycles; 65°C for 10 min.

[0098] Using the method of fusion PCR, the four PCR products amplified above were fused to obtain a new plasmid pK18mobSacBΔaroB Psod lysC, and its plasmid map is as Figure 1 shown.

[0099] Plasmids pK18mobSacBΔhisD Psod lysC, pK18mobSacBΔpheAPsod lysC, and pK18mobSacBΔsodAPsod lysC were constructed according to the above method, and their plasmid maps are as Figures 2 - 4 shown. The other primer pairs used include: hisDup_F and hisDup_R, hisDdn_F and hisDdn_R, backbone_F2 and backbone_R2, pheAup_F and pheAup_R, pheAdn_F and pheAdn_R, backbone_F3 and backbone_R3, sodaup_F and sodaup_R, sodadn_F and sodadn_R, backbone_F4 and backbone_R4.

[0100] 1.2 Construction of EctABC expression plasmid

[0101] General Biosystems (Anhui) Co., Ltd. was entrusted to synthesize the genes of ECTABC with different sources and copy ratios, and then the synthesized genes were used as DNA templates; the sources include Halomonas elongata, Streptomycesavermitilis, Candidatus Nitrosopumilus sediminis, and Vibrio natriegens NBRC.

[0102] The EctABC from the above different sources was amplified using primers respectively, and plasmids pK18mobSacB-ΔmetB-ECTABC (111, representing the copy number ratio of genes EctA, EctB, and EctC as 1:1:1) were constructed by the method of fusion PCR respectively, and their plasmid maps are as Figure 5 shown.

[0103] Among them, the primer pairs related to the source of Halomonas elongata include: EctA_F1 and EctA_R1, EctB_F1 and EctB_R1, EctC_F1 and EctC_R1, MetBup_F1 and MetBup_R1, MetBdn_F1 and MetBdn_R1, backboneEctABC_F1 and backboneEctABC_R1.

[0104] Among them, the primer pairs derived from Streptomyces avermitilis include: EctA_F2 and EctA_R2, EctB_F2 and EctB_R2, EctC_F2 and EctC_R2, MetBup_F2 and MetBup_R2, MetBdn_F2 and MetBdn_R2, backboneEctABC_F2 and backboneEctABC_R2.

[0105] Among them, the primer pairs derived from Candidatus Nitrosopumilus sediminis include: EctA_F3EctA_R3, EctB_F3 and EctB_R3, EctC_F3 EctC_R3, MetBup_F3 and MetBup_R3, MetBdn_F3 and MetBdn_R3, backboneEctABC_F3 and backboneEctABC_R3.

[0106] Among them, the primer pairs derived from Vibrio natriegens NBRC include: EctA_F4 and EctA_R4, EctB_F4 and EctB_R4EctC_F4 and EctC_R4, MetBup_F4 and MetBup_R4, MetBdn_F4 and MetBdn_R4, backboneEctABC_F4 and backboneEctABC_R4.

[0107] According to the above method, EctABC expression plasmids from different sources were constructed respectively. Subsequently, General Biosystems (Anhui) Co., Ltd. was commissioned to replicate the above plasmids with a copy number of 1, and separately replicate and integrate individual EctA / B / C into each expression plasmid to form plasmids with 112 / 121 / 122 / 131 / 132 / 211 / 212 / 221 (representing the copy number ratio of genes EctA, EctB, and EctC) replicons. Their plasmid maps are respectively as Figures 6 - 13 shown.

[0108] 1.3 Construction of EctD expression plasmid

[0109] General Biosystems (Anhui) Co., Ltd. was commissioned to synthesize the EctD gene from different sources, and then a single-copy expression vector pK18mobSacB-Δhpt-ECTD was formed by fusion PCR. Its plasmid map is as Figure 14 shown.

[0110] Among them, the primer pairs derived from Halomonas elongate include: EctD_F1 and EctD_R1, Δhptup_F1 and Δhptup_R1, Δhptdn_F1 and Δhptdn_R1, backboneectD_F1 and backboneectD_R1.

[0111] Among them, the primer pairs derived from Mycobacterium smegmatis include: EctD_F2 and EctD_R2, Δhptup_F2 and Δhptup_R2, Δhptdn_F2 and Δhptdn_R2, backboneectD_F2 and backboneectD_R2.

[0112] Among them, the primer pairs derived from Pseudomonas stutzeri include: EctD_F3 and EctD_R3, Δhptup_F3 and Δhptup_R3, Δhptdn_F3 and Δhptdn_R3, backboneectD_F3 and backboneectD_R3.

[0113] In summary, the plasmid carrying the EctD1 copy was successfully constructed. Then, General Biosystems (Anhui) Co., Ltd. was commissioned to integrate the multi-copy (2 / 3) EctD into the expression plasmid, and subsequently integrate it into the hpt gene locus.

[0114] Example 2: Construction of genetically engineered bacteria

[0115] 2.1 Pipette 3 μl (more than 500 ng) of the constructed plasmid for expressing lysC into Corynebacterium glutamicum ATCC13032 LysC fbrIn competent cells, after mixing, transfer to a 2-μm electroporation cuvette and perform electroporation at 2.5 kV for 5.3 ms. Immediately after electroporation, transfer into 800 μl of pre-warmed (46 °C) BHIS liquid medium and incubate in a 46 °C water bath for 6 min. Then place in a constant temperature shaker at 30 °C and 220 rpm for 1 h to allow the bacteria to recover. After recovery, take 100 μl of the bacteria and spread them on a BHIS plate containing kanamycin. Invert the plate and culture it in a 30 °C constant temperature incubator for 48 h. Pick the transformants on the BHIS plate containing kanamycin and inoculate them into a non-resistant BHIS test tube medium. Culture in a constant temperature shaker at 30 °C and 220 rpm for 24 h to allow double crossover to occur. Dilute the bacteria 1000-fold and spread them on a BHIS-suc plate containing 20% sucrose. Invert the plate and culture it in a 30 °C constant temperature incubator for 48 h. Pick the transformants on the BHIS-suc plate and spot them on a BHIS plate and a BHIS plate containing kanamycin respectively. Invert the plate and culture it in a 30 °C constant temperature incubator for 24 h. The transformants that can grow on the BHIS plate but cannot grow on the BHIS plate containing kanamycin are positive strains. Pick the positive transformants into a 4-ml BHIS test tube medium and culture in a constant temperature shaker at 30 °C and 220 rpm for 16 h. Preserve the bacteria using 20% glycerol to obtain the strain overexpressing aspartokinase.

[0116] 2.2 Referring to the above method, transform the plasmid constructed for expressing EctABC into the above strain overexpressing aspartokinase by electroporation method, and then operate according to the sacB counter-selection method to obtain the ectoine-producing bacterium.

[0117] 2.3 Referring to the above method, transform the plasmid expressing EctD into the above ectoine-producing bacterium by electroporation method, and then operate according to the sacB counter-selection method to obtain the hydroxyectoine-producing bacterium.

[0118] 2.4 Transform the plasmid constructed for knocking out osmC and / or glnA into the above hydroxyectoine-producing bacterium by electroporation method, and then operate according to the sacB counter-selection method to obtain the high-purity hydroxyectoine-producing bacterium.

[0119] Example 3: Screening of the optimal high-purity hydroxyectoine-producing bacterium

[0120] 3.1 Effects of different copy numbers and sources of EctABC on the yield of ectoine

[0121] Ferment the ectoine-producing bacteria with different copy numbers and different sources of EctABC obtained in step 2.2 of Example 2 in a 5-L fermenter, and detect the concentration of the ectoine fermentation product at 78 h. The results are as follows:

[0122]

[0123]

[0124] As can be seen from the above data, the best ectoine high-yield strain with a copy number ratio of EctABC from Vibrio natriegens NBRC of 1:3:2.

[0125] 3.2 Different copy numbers of lysC fbr Effect on ectoine production

[0126] For the above-mentioned best ectoine high-yield strain, different copy numbers of genes were expressed in the best ectoine high-yield strain according to step 2.1 in Example 2, and the product concentration of ectoine was detected after fermenting the obtained strains in a 5L fermenter for 78h. The results are as follows:

[0127] Copy number Product concentration at 78h 1 copy 85g / l 2 copies 90g / l 3 copies 98g / l 4 copies 110g / l 5 copies 103g / l

[0128] As can be seen from the above data, 4copy of lysC fbr Is the best, and the yield of ectoine can reach 110g / l.

[0129] 3.3 Effect of different copy numbers and sources of EctD on the production of hydroxyectoine

[0130] For the above-mentioned best ectoine high-yield strain, the results of converting ectoine to hydroxyectoine using EctD from Halomonas elongate, Mycobacterium smegmatis or Pseudomonas stutzeri in a 5L fermenter are as follows:

[0131]

[0132] As can be seen from the above data, 2copy of EctD from Mycobacterium smegmatis has the best effect on converting to hydroxyectoine.

[0133] 3.4 Effect of knocking out genes related to osmotic regulation on the purity of hydroxyectoine product

[0134] According to step 2.4 in Example 2, one or more genes related to osmotic regulation, treY, osmC and glnA, were knocked out (the constructed knockout plasmids are respectively as Figures 15 - 17As shown in the figure, it was found that it was of no help to the production of hydroxyectoine. Subsequently, we cultured it using the following high-salt fermentation medium and found that the strain with osmC and glnA knocked out had a higher conversion rate of hydroxyectoine at 40 - 60 mM NaCl. When the NaCl concentration was 50 mM, after fermenting in a 5 L fermenter for 78 h, the concentration of hydroxyectoine was detected to be 102.5 g / l, and the concentration of ectoine was only 1 g / l. Therefore, the purity of the hydroxyectoine product was greater than 99%, and the product concentration of hydroxyectoine at different fermentation times was as Figure 18 shown. Based on this, we successfully constructed an engineered strain for super hydroxyectoine synthesis.

[0135] Example 4: Explore whether further gene editing of the strain will affect the concentration of hydroxyectoine product 4.1 Strain construction

[0136] Prepare the strain for producing ectoine according to the method described in Patent CN113481233B, which uses Corynebacterium glutamicum ATCC13032 LysC that produces lysine fbr as the starting strain, not only overexpresses the gene lysC encoding aspartokinase and integrates the ectABC gene from Pseudomonas stutzeri, but also further performs other gene editing treatments, including: knocking down the hom gene encoding homoserine kinase in the genome, knocking out the pck gene encoding phosphoenolpyruvate carboxykinase in the strain genome, and enhancing the expression of the genes tkt encoding transketolase tkt, tal encoding tyrosine ammonia-lyase tal, and zwf encoding 6-phosphogluconate dehydrogenase zwf in the strain genome.

[0137] On the basis of the above ectoine-producing bacterium, obtain the hydroxyectoine-producing bacterium according to the method described in Step 2.3 of Example 2 (2 copies of EctD from Mycobacterium smegmatis), and knock out the genes osmC and glnA according to the method described in Step 2.4 of Example 2 to obtain a high-purity hydroxyectoine-producing bacterium.

[0138] 4.2 Verification of the purity of the hydroxyectoine product

[0139] Subsequently, we cultured it using a high-salt fermentation medium (where the NaCl concentration was 50 mM). After fermenting in a 5 L fermenter for 78 h, the concentration of hydroxyectoine was detected to be 93 g / l, and the concentration of ectoine was only 2 g / l. The purity of the hydroxyectoine product was close to 98%.

[0140] Therefore, based on the construction method described in the present invention, other gene editing treatments are performed on the strain that are beneficial to its production of hydroxyectoine, which has almost no effect on the purity of the final hydroxyectoine product, that is, the treated strain is still a producer that can produce high-purity hydroxyectoine.

[0141] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for constructing a high-purity hydroxyectoine-producing bacterium, characterized in that, The construction method includes the following steps: (1) Using Corynebacterium glutamicum ATCC13032LysCfbr, which produces lysine, as the starting strain, enhancing the expression of the gene lysC encoding aspartokinase in the strain genome to obtain a strain overexpressing aspartokinase; (2) On the basis of the strain overexpressing LysC, continuously overexpressing the EctABC gene cluster from different sources to obtain an ectoine-producing bacterium; the sources of the EctABC gene cluster are Halomonas elongata, Streptomyces avermitilis, Candidatus Nitrosopumilus sediminis, Vibrio natriegens NBRC, or Pseudomonas stutzeri; (3) On the basis of the above-obtained ectoine-producing bacterium, overexpressing the EctD gene from different sources to obtain a hydroxyectoine-producing bacterium; the sources of the EctD gene are Halomonas elongata, Mycobacterium smegmatis, or Pseudomonas stutzeri; (4) On the basis of the above-obtained hydroxyectoine-producing bacterium, separately or combinatorially knocking out the key genes osmC and / or glnA related to the osmoregulation of the strain.

2. The construction method according to claim 1, wherein The copy number of the gene lysC is 1-5, the copy number of each gene in the EctABC gene cluster is 1-3, and the copy number of the EctD gene is 1-3.

3. The construction method according to claim 1, characterized in that, The source of the EctABC gene cluster is Vibrio natriegens NBRC, and the copy number ratio of the genes EctA, EctB, and EctC is 1:3:

2.

4. The construction method according to claim 1, wherein The source of the EctD gene is Mycobacterium smegmatis, and its copy number is 2.

5. The construction method according to claim 1, wherein, The ectoine-producing strain obtained in step (2) is Corynebacterium glutamicum, which is deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 22733.

6. The construction method according to claim 1, characterized in that, The enhanced expression of the gene lysC, the gene cluster EctABC, and the gene EctD is achieved by placing the gene downstream of the sod promoter.

7. The construction method according to claim 1, wherein The expression of the gene lysC, the gene cluster EctABC, and the gene EctD is obtained by integrating the gene into the plasmid pK18mobSacB, introducing it into the host cell, and performing SacB sucrose counter-selection for positive clones.

8. A production bacterium of high-purity hydroxyectoine, characterized in that, Obtained by the construction method according to any one of claims 1-7.

9. Use of the production bacterium according to claim 8 in the production of high-purity hydroxyectoine.

10. A method for producing high-purity hydroxyectoine, characterized in that, The method includes the step of culturing the production bacterium obtained by the construction method according to any one of claims 1-7 in a high-salt fermentation medium, wherein the concentration of NaCl in the high-salt fermentation medium is 40-60 mM.

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