Engineering bacterium capable of producing Ectoin at high yield as well as preparation method and application of engineering bacterium

Abstract

The invention belongs to the technical field of genetic engineering, and discloses an engineering bacterium capable of producing Ectoine at high yield as well as a preparation method and application of the engineering bacterium. Escherichia coli W3110 is used as a chassis cell, and an Ectoin metabolic pathway is constructed by using synthetic biology. A constitutive promoter Pglgs of a transcription factor related gene in escherichia coli is preferably selected, an ectBAC for coding an Ectoine synthetic gene is preferably selected as a reporter gene, a constitutive plasmid for expressing the Ectoine synthetic gene is constructed, the expression level of the gene is improved, an engineering bacterium Escherichia coli ECT-W020 with high yield of Ectoine is obtained, and the preservation number of the engineering bacterium Escherichia coli ECT-W020 is GDMCC No: 65012. When the engineering bacterium is used for producing Ectoin, the process of adding an inducer is omitted, and the purpose of reducing the production cost is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of genetic engineering, and in particular to an engineering bacterium with high yield of ectoine and a preparation method and application thereof. Background Art

[0002] Ectoine (2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid, Ectoine) is a cyclic amino acid derivative of the aspartic acid family. Ectoine has strong solubility and hydration properties. After combining with water, it forms an Ectoine-hydrogen complex, which can enhance the binding force of hydrogen bonds in aqueous solutions and build a protective hydration layer on the cell surface, effectively resisting potential damage to cells from the external environment.

[0003] Ectoin was first discovered in the halophilic photosynthetic bacteria Ectothiorhodospira halochloris. Merck in Germany used Ectoin in the field of skin care products and launched Ectoine products are sold at a high price. A "cell milking" fermentation production process of "high salt induced synthesis-low salt release" of wild strains of Halomonas elongata was established to produce Ectoine. However, the high-salt culture medium used in the "bacterial milking" production process not only corrodes the equipment, but also increases the difficulty of downstream process treatment. In addition, the repeated impact of high and low salt inhibits the growth of bacteria and reduces the yield of Ectoine.

[0004] The main methods to solve this problem are: First, new ectoine production strains can be screened to achieve the production of ectoine in a low-salt environment, but the equipment requirements are high and the yield is low, which increases the difficulty of the subsequent ectoine extraction and purification process. For example, in the Chinese invention patent document CN115651943A, a fermentation preparation method for a small molecule amino acid derivative ectoine and its application, 50g / L-100g / L of sodium chloride is used to stimulate halophilic bacteria to synthesize intracellular ectoine. The fermentation time is long, the ectoine yield is low, and the production equipment requirements are high, which is not conducive to industrial production. Another method is to introduce the ectoine synthesis pathway in halophilic bacteria into non-halophilic microbial cell factories to achieve heterologous synthesis of ectoine. However, many recombinant strains require inducers such as isopropyl-β-D-thiogalactopyranoside (IPTG) to synthesize and produce ectoine. IPTG is expensive, which increases the fermentation production cost and complicates the fermentation process, making it difficult to purify ectoine in the later stage. For example, in Chinese invention patent documents CN117866865A, a genetically engineered bacterium for efficiently producing ectoine, its construction method and application, and CN118028204A, an ectoine synthetic strain, its construction method and application, the engineered strains need to add IPTG during the fermentation process of ectoine, which increases the complexity of the operation and prolongs the fermentation cycle, greatly pushing up the cost of industrial large-scale production of ectoine and posing a significant challenge to economic benefits. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an engineering bacterium with high yield of ectoine and a preparation method and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides an engineered bacterium with high ectoine production, wherein the engineered bacterium is Escherichiacoli ECT-W020, and its deposit number is GDMCC No: 65012.

[0008] The strain ECT-W020 is named Escherichia coli ECT-W020, and its Latin name is named Escherichia coli. It was deposited in Guangdong Microbial Culture Collection Center on August 15, 2024, with a deposit number of GDMCC No: 65012, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The present invention uses Escherichia coli W3110 as the chassis cell and applies synthetic biology to construct the ectoine metabolic pathway. By selecting the constitutive promoter P of the gene related to the transcription factor in Escherichia coliglgs , ectBAC encoding the ectoine synthesis gene was selected as the reporter gene, a constitutive plasmid expressing the ectoine synthesis gene was constructed and the expression level of the gene was improved, the process of adding inducers was omitted, and the purpose of reducing production costs was achieved.

[0009] In a second aspect, the present invention provides a method for preparing an engineered bacterium with high yield of ectoine, comprising the following steps:

[0010] (1) Knockout of EIIA in the starting strain Glc the coenzyme gene crr, the aspartate kinase / homoserine dehydrogenase gene thrA, the pyruvate formate lyase gene pflB, the lactate dehydrogenase gene ldhA, and the bifunctional aldol dehydrogenase gene adhE;

[0011] (2) knocking out the pyruvate kinase gene pykF of the starting strain and integrating the lysC-asd-aspC gene cluster;

[0012] The lysC-asd-aspC gene cluster sequentially includes the expression elements aspartate kinase gene lysC, aspartate semialdehyde dehydrogenase gene asd and aspartate aminotransferase gene aspC;

[0013] (3) Exogenous introduction of ectBAC gene cluster;

[0014] The ectBAC gene cluster sequentially includes the expression elements 2-aminobutyrate aminotransferase gene ectB, 2-aminobutyrate acetyltransferase gene ectA, and ectoine synthase gene ectC.

[0015] The present invention obtains an engineered bacterium with high yield of ectoine by transforming and constructing the following aspects:

[0016] (1) Constitutive promoter P glgs Application: The present invention is preferably related to the constitutive promoter P of the transcription factor-related gene glgs , a constitutive plasmid expressing the synthetic gene of Ecdoin was constructed. This promoter can increase the expression level of the gene, while eliminating the process of adding inducers, which helps to reduce production costs.

[0017] (2) Optimization of ectBAC reporter gene: The sequence of ectBAC encoding the ectoine synthetic gene was optimized.

[0018] (3) Application of CRISPR / Cas9 technology: Using CRISPR / Cas9 technology to knock out EIIA in the phosphotransferase system (PTS) GlcThe coenzyme crr reduces the consumption of PEP in glucose transport. In addition, aspartate kinase / homoserine dehydrogenase thrA, pyruvate formate lyase pflB, lactate dehydrogenase ldhA, and bifunctional aldol dehydrogenase adhE were knocked out to select the best knockout combination to reduce the consumption of side chain byproducts on the central carbon flow.

[0019] (4) Optimization of metabolic pathways: By knocking out pyruvate kinase pykF and integrating the trc-lysC-asd-aspC module, the conversion of PEP to pyruvate was inhibited, increasing the intracellular accumulation of oxaloacetate. trc Under the control of, the expression levels of aspartate kinase (lysC), aspartate semialdehyde dehydrogenase (asd) and aspartate aminotransferase (aspC) were increased, the carbon flux from glucose to oxaloacetate was increased, and the biosynthetic metabolic accumulation of ectoine was promoted.

[0020] As a preferred embodiment of the method for preparing the high-yield ectoine-producing engineered bacteria of the present invention, in step (1): CRISPR / Cas9 is used to knock out the gene; and the starting strain is Escherichia coli W3110.

[0021] As a preferred embodiment of the method for preparing the high-yield ectoine engineering bacteria of the present invention, in step (2): the trc-lysC-asd-aspC gene cluster is driven by the promoter P trc Start transcription; the promoter P trc The nucleotide sequence is shown in SEQ ID No.13.

[0022] As a preferred embodiment of the method for preparing the high-yield ectoine-producing engineered bacteria of the present invention, in step (2): the nucleotide sequence of the aspartate kinase gene lysC is shown in SEQ ID No.7; the nucleotide sequence of the aspartate semialdehyde dehydrogenase gene asd is shown in SEQ ID No.9; the nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID No.11.

[0023] As a further preferred embodiment of the method for preparing the high-yield ectoine engineering bacteria of the present invention, in step (2): integrating the trc-lysC-asd-aspC gene cluster; the trc-lysC-asd-aspC gene cluster sequentially includes the expression element promoter P trc , aspartate kinase gene lysC, aspartate semialdehyde dehydrogenase gene asd and aspartate aminotransferase gene aspC; the promoter P trcThe nucleotide sequence of the aspartate kinase gene lysC is shown in SEQ ID No.13; the nucleotide sequence of the aspartate kinase gene lysC is shown in SEQ ID No.7; the nucleotide sequence of the aspartate semialdehyde dehydrogenase gene asd is shown in SEQ ID No.9; and the nucleotide sequence of the aspartate aminotransferase gene aspC is shown in SEQ ID No.11.

[0024] As a preferred embodiment of the method for preparing the high-yield ectoine-producing engineered bacteria of the present invention, in step (3): the ectBAC gene cluster is driven by the promoter P glgs Start transcription; the promoter P glgs The nucleotide sequence is shown in SEQ ID No.14.

[0025] As a preferred embodiment of the method for preparing the high-yield ectoine engineered bacteria described in the present invention, in step (3): the nucleotide sequence of the 2-aminobutyrate aminotransferase gene ectB is as shown in SEQ ID No.3; the nucleotide sequence of the 2-aminobutyrate acetyltransferase gene ectA is as shown in SEQ ID No.1; the nucleotide sequence of the ectoine synthase gene ectC is as shown in SEQ ID No.5.

[0026] As a preferred embodiment of the method for preparing the high-yield ectoine-producing engineered bacteria of the present invention, in step (3): the exogenous introduction uses a constitutive plasmid to express the ectBAC gene cluster.

[0027] Preferably, the constitutive plasmid includes pACYC-Duet-1 plasmid, pCDF, pET and the like.

[0028] In a third aspect, the present invention applies the engineered bacteria and the strain obtained by the preparation method to the production of ectoine.

[0029] In a fourth aspect, the present invention applies the engineered bacteria and the strain obtained by the preparation method to the production of food, medicine, health care products and / or cosmetics containing ectoine.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The high-yield edoin engineering bacteria of the present invention is produced by using a constitutive promoter P glgs, the ectoine synthesis gene can be continuously expressed at a high level without adding the inducer IPTG, which simplifies the production process and reduces the extra cost in production. The preparation method of the high-yield ectoine engineering bacteria of the present invention improves the carbon source utilization rate in the glucose transport process, reduces the generation of by-products in the metabolic process, increases the accumulation of ectoine precursor substances, expands the metabolic flux, and improves the ectoine production efficiency by optimizing the metabolic pathway method. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of the optimization of the metabolic pathway of ectoine of the present invention;

[0033] Figure 2 This is the standard curve of ectoine standard. DETAILED DESCRIPTION

[0034] The engineered bacteria of the present invention uses Escherichia coli W3110 as the base cell and applies synthetic biology to construct the metabolic pathway of ectoine. Figure 1 As shown, by optimizing the constitutive promoter P of the transcription factor-related gene in Escherichia coli glgs , ectBAC encoding the ectoine synthesis gene was selected as the reporter gene, a constitutive plasmid expressing the ectoine synthesis gene was constructed and the expression level of the gene was improved, the process of adding inducers was omitted, and the purpose of reducing production costs was achieved.

[0035] CRISPR / Cas9 technology was used to knock out EIIA in the phosphotransferase system (PTS) system. Glc The coenzyme crr was used to reduce the consumption of PEP in the process of glucose transport, and aspartate kinase / homoserine dehydrogenase thrA, pyruvate formate lyase pflB, lactate dehydrogenase ldhA, and bifunctional aldol dehydrogenase adhE were knocked out. The best knockout combination was selected to reduce the consumption of central carbon flow by side chain by-products such as ethanol, lactate and threonine, increase the intracellular accumulation of precursor substances such as oxaloacetate and aspartate and acetyl-CoA, and expand the metabolic flux of ectoine.

[0036] Further investigation of the metabolic pathway of oxaloacetate was performed by knocking out pyruvate kinase pykF and integrating it into the trc-lysC-asd-aspC module to inhibit the conversion of phosphoenolpyruvate (PEP) into pyruvate and increase the intracellular accumulation of oxaloacetate. trc Under the control of, the expression levels of aspartate kinase (lysC), aspartate semialdehyde dehydrogenase (asd) and aspartate aminotransferase (aspC) were increased, the carbon flux from glucose to oxaloacetyl was increased, the conversion of oxaloacetate to L-aspartate and then to L-aspartate-β-semialdehyde was promoted, the accumulation of ectoine biosynthesis metabolism was accelerated, and the synthesis of ectoine was increased.

[0037] In order to better illustrate the purpose, technical scheme and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0038] The experimental materials involved in the embodiments are as follows:

[0039] Escherichia coli W3110 DSM5911, purchased from Guangzhou Qiyun Biotechnology Co., Ltd.; pTargetF plasmid purchased from Molecular Cloud Cat. No.: MC_0000012; pACYC-Duet-1 plasmid purchased from Novagen; tryptone and yeast powder purchased from Oxoid; DNA marker, Primer Star Max, purchased from Takara; 2×Phanta Max Master Mix, 2×Rapid Taq Master Mix, Phanta HS Super-Fidelity DNA Polymerase, Ultra One Step Cloning Kit was purchased from Nanjing Novozymes Biotech Co., Ltd.; agarose gel DNA recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; ectoine standard (chromatographic grade) was purchased from Tanmo Quality Inspection Standard Material Center; kanamycin and spectinomycin were purchased from Sangon Biotech (Shanghai) Co., Ltd.; other drugs or reagents were imported or domestically produced analytical grade or chromatographic grade.

[0040] The components of the culture medium and solution involved in the embodiment are as follows:

[0041] LB liquid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L;

[0042] LB solid medium: peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L, agar 15 g / L;

[0043] Antibiotic working concentration: chloramphenicol 34 mg / L, kanamycin 50 mg / L, spectinomycin 50 mg / L;

[0044] Basic culture medium: glucose 20g / L, yeast extract powder 10g / L, potassium dihydrogen phosphate 7g / L, dipotassium hydrogen phosphate 14g / L, magnesium sulfate 1g / L, citric acid monohydrate 1.1g / L, sterilized at 121℃ for 20min, chloramphenicol was added to the final concentration of 34μg / mL before use of the culture medium;

[0045] Basic fermentation medium: glucose 20g / L, yeast extract 10g / L, potassium dihydrogen phosphate 7g / L, dipotassium hydrogen phosphate 14g / L, magnesium sulfate 1g / L, citric acid monohydrate 1.1g / L, 0.1g / L vitamin B complex, trace elements 2mL / L (manganese sulfate 1.5g / L, ferrous sulfate 7g / L, cobalt chloride 0.8g / L, zinc chloride 4g / L, sodium molybdate 0.1g / L, calcium chloride 1.1g / L, dissolved in 5mol / L HCI solution);

[0046] Optimized fermentation medium: glucose 25g / L, peptone 21.5g / L, yeast extract 11g / L, ammonium sulfate 6g / L, sodium aspartate 100mM, potassium dihydrogen phosphate 7g / L, dipotassium hydrogen phosphate 14g / L, magnesium sulfate 1g / L, citric acid monohydrate 1.1g / L, 0.1g / L vitamin B complex, trace elements 2mL / L (manganese sulfate 1.5g / L, ferrous sulfate 7g / L, cobalt chloride 0.8g / L, zinc chloride 4g / L, sodium molybdate 0.1g / L, calcium chloride 1.1g / L, dissolved in 5mol / L HCI solution), initial pH 7.2;

[0047] Working concentration of inducer: isopropyl β-D-thiogalactoside 0.1mmol / L.

[0048] The synthesis of the gene sequence involved in the embodiment is as follows:

[0049] PCR primers are shown in Table 1 and were synthesized by Suzhou Genewise Biotechnology Co., Ltd.;

[0050] Table 1 List of primers used in the construction of recombinant expression vectors

[0051]

[0052]

[0053]

[0054] The 2-aminobutyrate acetyltransferase (ectA), 2-aminobutyrate transaminase (ectB), ectoine synthetase (ectC), aspartate kinase (lysC, derived from GeneID: 1021294), aspartate semialdehyde dehydrogenase (asd, derived from GeneID: 947939) and aspartate aminotransferase (aspC, derived from GeneID: 945553) used were all synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. (codon optimization was performed according to the codon preference of Escherichia coli).

[0055] Example 1: Cloning of target gene

[0056] The synthetic gene, laboratory preserved genome or empty vector was used as a template, and the target genes ectB, ectA, ectC were amplified by PCR reaction program according to the corresponding paired primers in Table 2, and the plasmid backbone pMB1-trc was amplified using the pTargetF (Molecular Cloud Cat. No.: MC_0000012) plasmid as a template, the backbone pACYC fragment was amplified from the pACYC-Duet-1 (Novagen) plasmid, and the other fragments lysC, asd and aspC and the promoter P lpp , P glgs and P trc .

[0057] Table 2. Strains used and plasmids constructed

[0058]

[0059]

[0060]

[0061] The PCR reaction conditions are shown in Table 3:

[0062] Table 3 PCR reaction system

[0063] Reagents Dosage (μL) Upstream primer 1μL Downstream primer 1μL Template DNA 0.5μL <![CDATA[ddH2O]]> 17.5μL 2×Phanta Max Master Mix(Dye Plus) 20μL Overall system 40μL

[0064] The PCR reaction program is shown in Table 4:

[0065] Table 4 PCR reaction program

[0066] PCR reaction steps Reaction parameters Step 1: Pre-denaturation 95℃；30s Step 2: Denaturation 95℃；10s Step 3: Annealing 55℃；10s Step 4: Extension 72℃; 2min Step 5: Repeat steps 2-4 A total of 30 cycles Step 6: Final Extension 72℃; 5min Step 7: Save Store at 4℃

[0067] After the PCR reaction was completed, the PCR reaction products of the promoter fragment, target gene fragment and vector fragment were subjected to 1% agarose gel nucleic acid electrophoresis. After verifying the success of the PCR reaction, the PCR reaction product was purified and the DNA was recovered and stored at 4°C for future use.

[0068] Example 2: Short fragment overlap extension

[0069] The PCR product of Example 1 was taken, and the three gene clusters etcA, etcB and etcC in the biosynthetic pathway of ectoine were connected in the order of etcABC and etcBAC respectively by overlapping extension PCR method, and the ectoine synthesis gene cluster P was obtained by gel cutting and recovery. etcABC (etcABC-F and etcABC-R) and P etcBAC The two target genes were linked to the promoter P lpp , P glgs Overlap extension PCR amplification verification, gel excision purification recovery to obtain P lpp -P etcABC (lpp-etcABC-F and etcABC-R), P glgs -P etcABC (glgs-F and etcABC-R), P lpp -P etcBAC (lpp-etcBAC-F and etcBAC-R) and P glgs -P etcBAC (glgs-F and etcBAC-R).

[0070] Similarly, overlap extension PCR was used to connect lysC, asd and aspC in sequence to obtain module P lysC -P asd -P aspC The template was extended and connected to the promoter Ptrc to obtain the product Ptrc-P lysC -P asd -P aspC PCR amplification of Ptrc-P using primers trc-lysC-asd-aspC-F and trc-lysC-asd-aspC-R lysC -P asd -P aspC The PCR reaction was confirmed to be successful by gel electrophoresis, and the DNA was purified and recovered by gel excision and stored at 4°C for future use.

[0071] The overlap extension PCR reaction system and procedure are shown in Table 5:

[0072] Table 5 Overlap extension PCR reaction system and procedure

[0073]

[0074] The PCR reaction program is shown in Table 6:

[0075] Table 6 PCR reaction system and procedure

[0076]

[0077] Example 3: Construction of recombinant plasmid

[0078] The plasmid backbone pACYC fragments amplified in Example 1 and Example 2, P lpp -P etcABC Fragment and P trc -P lysC -P asd -P aspC Fragment, The MultiS One Step Cloning Kit was used for connection and assembly. After the plasmid template was digested with Dpn1, the product was transformed into E. coli Turbo cells by heat stimulation. The strain was screened on a plate containing chloramphenicol resistance. After about 16 hours of culture, a single colony was picked for colony PCR verification. The verified bacteria were cultured, and the plasmids were extracted and sent to Shanghai Meiji Company for sequencing. The recombinant plasmids with correct sequencing were named pACYC-lpp-etcABC-trc- lysC-asd-aspC .

[0079] The amplified plasmid backbone pACYC fragment, P lpp -P etcBAC Fragment and P trc -P lysC -P asd -P aspC Fragment, The MultiS One Step Cloning Kit was used for ligation and assembly, and the operation was the same as above. The recombinant plasmid with correct sequencing was named pACYC-lpp-etcBAC-trc-lysC-asd-aspC .

[0080] The amplified plasmid backbone pACYC fragment, P glgs -P etcABC Fragment and P trc -P lysC -P asd -P aspC Fragment, The MultiS One Step Cloning Kit was used for ligation and assembly, and the operation was the same as above. The recombinant plasmid with correct sequencing was named pACYC-glgs-etcABC-trc-lysC-asd-aspC .

[0081] The amplified plasmid backbone pACYC fragment, P glgs -P etcBAC Fragment and P trc -P lysC -P asd -P aspC Fragment, The MultiS One Step Cloning Kit was used for ligation and assembly, and the operation was the same as above. The recombinant plasmid with correct sequencing was named pACYC-glgs-etcBAC-trc-lysC-asd-aspC .

[0082] Example 4: Construction of recombinant strains

[0083] The plasmids pACYC-lpp-etcABC-trc-lysC-asd-aspC, pACYC-lpp-etcBAC-trc-lysC-asd-aspC, pACYC-glgs-etcABC-trc-lysC-asd-aspC and pACYC-glgs-etcBAC-trc-lysC-asd-aspC constructed in Example 3 were transformed into Escherichia coli cloning competent cells W3110 by heat shock method.

[0084] Heat shock method: Take out the competent W3110 cells from the -80℃ refrigerator and thaw them on ice. Transfer the above plasmids into the competent W3110 cells and mix them by pipetting. Ice bath for 30 minutes, heat stimulate at 42℃ for 90 seconds, add 1mL of antibiotic-free LB medium, incubate at 37℃ for 20 minutes, centrifuge at 8000rpm for 1 minute, discard the supernatant, take 300μL of LB medium to resuspend and mix the bacterial solution, take 100μL of the bacterial solution and spread it on a chloramphenicol-resistant LB plate for screening, place the plate in a 37℃ incubator and culture for 16 hours, pick multiple single clones for gene identification by PCR, and streak on a chloramphenicol-resistant plate to continue passage to verify the stability of the plasmid.

[0085] Example 5: Optimization of Ecdoin Metabolic Pathway

[0086] (1) Construction of Escherichia coli knockout and interference gene plasmids

[0087] The CHOPCHOP website was used to design gene knockout and interference N20 sites. pTargetF-cadA was used as a template and primers thrA-CRISPR-F and thrA-CRISPR-R were used for reverse PCR to amplify the plasmid pTargetF-thrA sequence. After the plasmid template was digested with DpnΙ, pTargetF-thrA was transformed into competent cells Turbo by heat shock method. The strains were screened on plates containing spectinomycin resistance. After culturing for 16 hours, single colonies were picked for PCR identification. The successfully identified strains were cultured and sent for testing to extract the pTargetF-thrA plasmid.

[0088] The CRISPR-Cas9 gene editing system was used to delete gene fragments on the Escherichia coli genome. Using thrA-UP-F and thrA-UP-R as primers and the W3110 genome as a template, PCR amplification was performed to obtain the upstream homology arm of thrA-UP; similarly, thrA-DOWN-F and thrA-DOWN-R primers were used to obtain the downstream homology arm of thrA-DOWN. Subsequently, using thrA-UP-F and thrA-DOWN-R as primers and thrA-UP and thrA-DOWN as templates, overlapping amplification was performed to obtain the replacement homology arm fragment of the thrA gene deletion.

[0089] The pTargetF-thrA plasmid contains the N20 sequence targeting the thrA gene site. The fragment △thrA was introduced into the W3110 strain carrying the pCas plasmid by electroporation via the pTarget-thrA plasmid, spread on an LB resistance plate containing spectinomycin (50 mg / L), and cultured overnight at 30°C. Single colonies with successful thrA knockout were screened by colony PCR using primers thrA-CHECK-F and thrA-CHECK-R. After verifying the knockout of thrA, 0.1mmol / L IPTG was added to remove the pTarget-thrA plasmid, and the plasmid pCas was removed by shaking at 42°C. The mutant strain W001 was obtained after knocking out thrA from the W3110 genome.

[0090] The same method was used above, with pTargetF-cadA as the template, using primers crr-CRISPR-F and crr-CRISPR-R, to amplify and construct the plasmid pTargetF-crr, and using primers crr-UP-F and crr-UP-R, crr-DOWN-F and crr-DOWN-R to amplify the crr replacement homology arm fragment, and knock out crr from the W001 / pCas genome, and identified with primers crr-UP-F and crr-check-R to obtain the knockout thrA and crr mutant strain W002. Continuously knock out ldhA and adhE on the W002 / pCas genome to obtain the knockout thrA, crr, ldhA, adhE mutant strain W003. Knock out pflB on the W003 / pCas genome to obtain the mutant strain W004 with knockout thrA, pflB, crr, ldhA and adhE.

[0091] Using the same method as above, using pTargetF-cadA as a template, crr, ldhA and adhE were continuously knocked out on the W3110 / pCas genome to obtain the mutant strain W005 with knockout of crr, ldhA and adhE. Primers pflB-CRISPR-F and pflB-CRISPR-R were used to amplify and construct the plasmid pTargetF-pflB, and primers pflB-UP-F and pflB-UP-R, pflB-DOWN-F and pflB-DOWN-R were used to amplify the pflB replacement homology arm fragment, and pflB was knocked out from the W005 / pCas genome. Primers pflB-UP-F and pflB-check-R were used to identify and obtain the mutant strain W006 with knockout of pflB, crr, ldhA and adhE.

[0092] The plasmid pTargetF-pykF was constructed by the same method as above, using pTargetF-cadA as a template and primers pykF-CRISPR-F and pykF-CRISPR-R. The upstream homology arm of pykF-UP was obtained by PCR amplification using pykF-UP-F and pykF-UP-R as primers and the W3110 genome as a template; the downstream homology arm of pykF-DOWN was obtained by using pykF-DOWN-F and pykF-DOWN-R primers. The trc-lysC-asd-aspC fragment was amplified using primers trc-lysC-asd-aspC-F and trc-lysC-asd-aspC-R using plasmid pACYC-glgs-etcBAC-trc-lysC-asd-aspC as a template. The pykF-UP upstream homology arm, trc-lysC-asd-aspC module and pykF-DOWN downstream homology arm were used The MultiSOne Step Cloning Kit was used to connect and assemble to form a complete knockout and integration fragment. The fragment △pykF-trc-lysC-asd-aspC was introduced into the mutant strain W004 / pCas by electroporation via the pTarget-pykF plasmid to obtain the mutant strain W007 with pykF knockout and trc-lysC-asd-aspC fragment integrated.

[0093] (2) Construction of recombinant strains

[0094] The plasmids pACYC-lpp-etcABC-trc-lysC-asd-aspC, pACYC-lpp-etcBAC-trc-lysC-asd-aspC, pAcYc-glgs-etcBAC-trc-lysc-asd-aspc and pAcYc-glgs-etcABC-trc-lysc-asd-aspc were introduced into the competent Escherichia coli cells W3110 by electroporation, 1 mL of LB medium was added and mixed by pipetting. The cells were cultured at 30°C and 200 rpm for 1 h, and then centrifuged at 8000 rpm for 1 min. The excess supernatant was discarded, and 100 μL of the supernatant was used to resuspend the bacteria. The culture was then spread on an LB plate containing chloramphenicol and cultured inverted overnight at 30°C. Colony PCR verification was performed using the corresponding primers for the strain, and a single colony of the positive clone was picked and transferred to LB liquid culture medium containing chloramphenicol, and cultured overnight at 37°C, 200rpm. The bacterial solution and 50% glycerol mixture were stored in a low-temperature environment of -80°C to obtain strains ECT-W001 (W3110, trc-lysc-asd-aspc, pAcYc-lpp-etcABC), ECT-W002 (W3110, trc-lysc-asd-aspc, pAcYc-lpp-etcBAC), ECT-W003 (W3110, trc-lysc-asd-aspc, pAcYc-glgs-etcBAC) and ECT-W004 (W3110, trc-lysc-asd-aspc, pAcYc-glgs-etcABC). Using the same method as above, the plasmid pAcYc-glgs-etcBAC-trc-lysc-asd-aspc was introduced into the mutant strain W001 to obtain strain ECT-W005 (W3110, ΔthrA, trc-lysc-asd-aspc, pAcYc-glgs-etcBAC).

[0095] Using the same method as above, plasmid pAcYc-glgs-etcABC- trc-lysC-asd-aspC The mutant strain W003 was introduced to obtain strain ECT-W007 (W3110, ΔthrA, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcABC); plasmid pAcYc-glgs-etcBAC- trc-lysC-asd-aspC The mutant strain W003 was introduced to obtain strain ECT-W008 (W3110, ΔthrA, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcBAC); plasmid pAcYc-glgs-etcABC- trc-lysC-asd-aspCThe mutant strain W006 was introduced to obtain strain ECT-W009 (W3110, ΔpflB, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcABC); plasmid pAcYc-glgs-etcBAC- trc-lysC-asd-aspC The mutant strain W006 was introduced to obtain strain ECT-W010 (W3110, ΔpflB, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcBAC);

[0096] Using the same method as above, plasmid pAcYc-glgs-etcABC- trc-lysC-asd-aspC The mutant strain W004 was introduced to obtain strain ECT-W011 (W3110, ΔthrA, ΔpflB, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcABC); plasmid pAcYc-glgs-etcBAC- trc-lysC-asd-aspC The mutant strain W004 was introduced to obtain strain ECT-W012 (W3110, ΔthrA, ΔpflB, Δcrr, ΔldhA, ΔadhE, trc-lysc-asd-aspc, pAcYc-glgs-etcBAC).

[0097] Using the same method as above, the plasmid pAcYc-glgs-etcBAC-lysc-aspc was introduced into the mutant strain W007, and etcBAC was overexpressed to obtain the strain ECT-W020 (W3110, ΔpflB, ΔthrA, Δcrr, ΔldhA, ΔadhE, ΔpykF::trc-lysC-asd-aspC, pACYC-glgs-ectBAC); the plasmid pAcYc-glgs-etcABC-lysc-aspc was introduced into the mutant strain W007, and etcABC was overexpressed to obtain the strain ECT-W016 (W3110, ΔpflB, ΔthrA, Δcrr, ΔldhA, ΔadhE, ΔpykF::trc-lysC-asd-aspC, pACYC-glgs-etcABC); plasmid pACYC-lpp-etcBAC-trc-lysC-asd-aspC was introduced into mutant strain W007, and etcBAC was overexpressed to obtain strain ECT-W018 (W3110, ΔpflB, ΔthrA, Δcrr, ΔldhA, ΔadhE, ΔpykF::trc-lysC-asd-aspC, pACYC-lpp-etcBAC).

[0098] Example 6: Detection of Ecdoin Production of Recombinant Strains

[0099] Ultrapure water was used to prepare standard solutions of ectoine with mass concentrations of 10, 30, 50, 80, 100, 150, 200 and 300 mg / L, and the standard solutions of ectoine and samples were taken together for determination by HPLC. Chromatographic column: ZORBAXStableBond aq; mobile phase: 100% 0.02M potassium dihydrogen phosphate; flow rate: 0.5mL / min; detection wavelength: 210nm; column temperature: 25℃; sample volume: 10μL; a standard curve was drawn with the ectoine standard, as shown in Figure 2. Figure 2 shown.

[0100] The engineered strain of Ecdoin constructed in Example 5 was inoculated into LB plates containing chloramphenicol, cultured in a 37°C incubator for 16 hours, and a single clone was inoculated into a 5mL LB test tube. After overnight culture at 37°C shaker 200rpm, 10% of the bacterial solution was transferred to 100mL of basal medium containing chloramphenicol for culture, and fermented at 37°C, 200rpm for 43 hours. 800μL of the fermentation broth was added to 2.4mL of anhydrous ethanol, mixed and reacted for 30min, centrifuged at 8000r / min for 10min, the supernatant was taken, the solvent was removed by nitrogen blowing, 2.4mL of anhydrous ethanol was added for ultrasonic dissolution, the supernatant was taken by centrifugation, nitrogen blowing, 800μL of water was added for oscillation and dissolution, and HPLC was used for detection, and the yield of Ecdoin was calculated according to the standard curve.

[0101] The results are as follows:

[0102] 1. Effects of the constitutive promoters glgs and lpp and the coding order of the ectoine synthesis gene cluster on ectoine production

[0103] The results are shown in Table 7. The strain containing the constitutive promoter glgs has a stronger ability to synthesize ectoine than the strain containing the lpp promoter, and the construction of the plasmid with the coding sequence of the three synthetic genes related to ectoine in the order of EctB, EctA, and EctC is conducive to the synthesis of ectoine.

[0104] There is no need to add inducers during the fermentation process, which avoids increasing the process flow and cost of producing ectoine, and is conducive to the future industrial production of ectoine.

[0105] Table 7 Output of ECT-W001 to ECT-W004 Ecdoin

[0106] Recombinant strains Ecdoin production ECT-W001 16.92±0.52mg / L ECT-W002 42.61±0.72mg / L ECT-W003 113.78±12.23mg / L ECT-W004 29.58±0.58mg / L

[0107] 2. Effects of knocking out thrA, pflB, adhE, IdhA, and crr on the yield of ectoine

[0108] (1) Effect of thrA gene deletion alone on ectoine production

[0109] In the ECT-W003 genome, the thrA gene, which can encode aspartate kinase / homoserine dehydrogenase at the same time, was knocked out, and the ectoine production of the recombinant bacterial group ECT-W005 was increased to 150.64±0.58mg / L, which is 5 times that of the initial strain ECT-W003. Analysis of knocking out the thrA gene reduced threonine synthesis and increased carbon flow to ectoine. The loss of the thrA gene inhibits the activity of aspartate kinase. The feedback resistance lysC of Corynebacterium glutamicum, which encodes aspartate kinase, was introduced to compensate for the enzyme activity deficiency and supplement aspartate kinase.

[0110] Table 8 Effect of knocking out thrA gene on edodene yield

[0111] Recombinant strains Ecdoin production ECT-W003 115.93±6.92mg / L ECT-W005 550.64±15.8mg / L

[0112] (2) Shake flask fermentation to study the effect of ectoine synthesis in recombinant strains ECT-W005 to ECT-W012

[0113] ECT-W007 and ECT-W008 both knocked out crr, ldhA and adhE in succession on the basis of knocking out thrA, which promoted the synthesis of ectoine. Similarly, ECT-W009 and ECT-W010 knocked out crr, ldhA and adhE in succession and knocked out pflB, which was also beneficial to increase the yield of ectoine. Strain ECT-W011 and ECT-W012 knocked out thrA and pfIB genes at the same time on the basis of knocking out crr, ldhA and adhE, which significantly increased the yield of ectoine, and the yield of P glgs The yield of recombinant ectoine produced by the promoter and ectBAC gene cluster was significantly higher than that produced by the promoter and ectoine synthesis gene cluster. lpp The recombinant strain ECT-W012 produced 1542.74±32.40 mg / L of ectoine in the basal medium, while the ectoine production of ECT-W011 was only 522.15±7.15 mg / L. The gene pflB encodes pyruvate formate lyase, ldhA encodes lactate dehydrogenase, adhE encodes bifunctional aldol dehydrogenase, and crr encodes EIIA in the phosphotransferase system (PTS) Glc The results showed that knocking out thrA, pflB, ldhA and adhE reduced the consumption of central carbon flow by byproducts such as threonine, ethanol, pyruvate and lactate, and increased the accumulation of precursor substances oxaloacetate, acetyl-CoA and L-aspartate-β-semialdehyde in cells.

[0114] Table 9 ECT-W005-ECT-W012 shake flask fermentation results

[0115] Recombinant strains Ecdoin production ECT-W005 562.14±33.37mg / L ECT-W007 501.96±42.60mg / L ECT-W008 2675.39±57.79mg / L ECT-W009 375.40±24.48mg / L ECT-W010 1374.96±49.60mg / L ECT-W011 822.15±57.15mg / L ECT-W012 6263.24±62.32mg / L

[0116] 3. Further optimize the metabolic pathway of Ecdoin

[0117] The recombinant strains ECT-W016, ECT-W018 and ECT-W020 further knocked out the gene pykF and integrated it into the trc-lysC-asd-aspC module based on the knockout of thrA, pflB, adhE, IdhA, and crr. The production of ectoine was increased several times, inhibiting the conversion of phosphoenolpyruvate (PEP) to pyruvate, increasing the intracellular accumulation of oxaloacetate, and supplementing lysC, asd and aspC to promote the conversion of oxaloacetate to aspartate, accelerating the accumulation of ectoine biosynthesis metabolism. After the optimization of the ectoine metabolic pathway, the ectoine production of ECT-W020 was 25.51±0.24g / L after 43 hours of fermentation in a shake flask, which was 6 times that of strains ECT-W016 and ECT-W018.

[0118] Table 10 Further optimization of ectoine metabolic pathway

[0119] Recombinant strains Ecdoin production ECT-W012 7.13±0.13g / L ECT-W016 4.78±0.19g / L ECT-W018 4.2±0.18g / L ECT-W020 25.51±0.24g / L

[0120] The strain ECT-W020 was named Escherichia coli ECT-W020, and its Latin name was named Escherichia coli. It was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 15, 2024, with the deposit number GDMCC No: 65012, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0121] Example 7: Production of ectoine by recombinant strain ECT-W020 and initial strain ECT-W003 in a 50L fermenter

[0122] (1) Bacterial activation: The recombinant strain ECT-W020 and the initial strain ECT-W003 were streaked on LB plates for activation, cultured at 37°C for 24 h, and single colonies were inoculated into 10 mL of LB medium and cultured at 37°C, 200 rpm for 12 h.

[0123] (2) Seed culture: transfer 10% of the first-level seed liquid to 1L of LB medium, culture at 37°C, 200rpm for 16h, then transfer all to a 50L fermenter filled with 20L sterilized basic fermentation medium, culture at 37°C, initial speed of 200rpm, and initial air volume of 100L / min. During the fermentation process, the system automatically adds ammonia water to control the pH at 7.2, maintains dissolved oxygen at not less than 20% by stirring and changing the ventilation volume, and the associated speed is 200rpm-800rpm. Ferment for 52h, and detect OD by sampling every hour 600 , wet weight of bacteria, residual sugar content, when the dissolved oxygen is lower than 70%, 40% concentration of glucose is added, and the control feedback is used to adjust the feeding rate to control the residual sugar in the fermentation liquid to 2g / L.

[0124] After the recombinant engineered strain ECT-W020 and the initial strain ECT-W003 were fermented in a 50L fermenter for 52 hours, the ectoine production of ECT-W020 in the basic fermentation medium reached 78.25 g / L, while the production of the initial strain ECT-W003 was only 378.83 mg / L, indicating that the patented method for optimizing the ectoine metabolic pathway is effective.

[0125] Table 11 Yield of ECT-W020 and ECT-W003 in basal medium

[0126]

[0127] Example 8: Production of ectoine by recombinant strain ECT-W020 and initial strain ECT-W003 in a 50L fermenter

[0128] (1) Bacterial activation: The recombinant strain ECT-W020 and the initial strain ECT-W003 were streaked on LB plates for activation, cultured at 37°C for 24 h, and single colonies were inoculated into 10 mL of LB medium and cultured at 37°C, 200 rpm for 12 h.

[0129] (2) Seed culture: transfer 10% of the first-level seed liquid to 1L of LB medium, culture at 37°C, 200rpm for 16h, then transfer all to a 50L fermenter filled with 20L of sterilized optimized fermentation medium, culture at 37°C, initial speed of 200rpm, and initial air volume of 100L / min. During the fermentation process, the system automatically adds ammonia water to control the pH at 7.2, maintains dissolved oxygen at not less than 20% by stirring and changing the ventilation volume, and the associated speed is 200rpm-800rpm. Fermentation is 52h, and OD is detected by sampling every hour. 600, wet weight of bacteria, residual sugar content, when the dissolved oxygen was lower than 70%, 40% concentration of glucose was started to be fed, and the control feedback was used to adjust the feeding rate to control the residual sugar in the fermentation liquid to 2g / L and start to feed 20% peptone at a constant rate of 160mL / h.

[0130] After the initial strain ECT-W003 and the recombinant engineered strain ECT-W020 were fermented in 50L fermentation medium for 52h, the growth performance of strain ECT-W003 was limited and the yield of ectoine did not increase with the growth of the strain, and the synthetic yield was only 1.32g / L. After ECT-W020 was fermented for 52h, the yield of ectoine was 188.59g / L. This shows that the recombinant ectoine strain ECT-W020 obtained by optimizing the metabolic pathway of this patent is effective and greatly improves the yield of ectoine. This strain can be used as an engineered strain for industrial production of ectoine.

[0131] Table 12 Yield of ECT-W020 and ECT-W003 in optimized fermentation medium

[0132]

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An engineered bacterium with high yield of ectoine, characterized in that: The engineered bacteria is Escherichia coli ECT-W020, and its deposit number is GDMCC No: 65012.

2. A method for preparing an engineered bacterium with high yield of ectoine, characterized in that: The following steps are involved: (1) Knockout of EIIA in the starting strain Glc the coenzyme gene crr, the aspartate kinase / homoserine dehydrogenase gene thrA, the pyruvate formate lyase gene pflB, the lactate dehydrogenase gene ldhA, and the bifunctional aldol dehydrogenase gene adhE; (2) knocking out the pyruvate kinase gene pykF of the starting strain and integrating the lysC-asd-aspC gene cluster; The lysC-asd-aspC gene cluster sequentially includes the expression elements aspartate kinase gene lysC, aspartate semialdehyde dehydrogenase gene asd and aspartate aminotransferase gene aspC; (3) Exogenous introduction of ectBAC gene cluster; The ectBAC gene cluster sequentially includes the expression elements 2-aminobutyrate aminotransferase gene ectB, 2-aminobutyrate acetyltransferase gene ectA, and ectoine synthase gene ectC.

3. The method for preparing the high-yield edodene engineering bacteria according to claim 2, characterized in that: In step (1): CRISPR / Cas9 is used to knock out the gene; the starting strain is Escherichia coli W3110.

4. The method for preparing the high-yield edodene engineering bacteria according to claim 2, characterized in that: In step (2): the lysC-asd-aspC gene cluster is driven by the promoter P trc Start transcription; the promoter P trc The nucleotide sequence is shown as SEQ ID No.

13.

5. The method for preparing the high-yield edodene engineering bacteria according to claim 2, characterized in that: In step (2): the nucleotide sequence of the aspartate kinase gene lysC is shown as SEQ ID No.7; the nucleotide sequence of the aspartate semialdehyde dehydrogenase gene asd is shown as SEQ ID No.9; and the nucleotide sequence of the aspartate aminotransferase gene aspC is shown as SEQ ID No.

11.

6. The method for preparing the high-yield edoin engineering bacteria according to claim 2, characterized in that: In step (3): the ectBAC gene cluster is driven by the promoter P glgs Start transcription; the promoter P glgs The nucleotide sequence is shown in SEQ ID No.

14.

7. The method for preparing the high-yield edoin engineering bacteria according to claim 2, characterized in that: In step (3): the nucleotide sequence of the 2-aminobutyrate aminotransferase gene ectB is shown in SEQ ID No.3; the nucleotide sequence of the 2-aminobutyrate acetyltransferase gene ectA is shown in SEQ ID No.1; the nucleotide sequence of the ectoine synthase gene ectC is shown in SEQ ID No.

5.

8. The method for preparing the high-yield edoin engineering bacteria according to claim 2, characterized in that: In step (3): the exogenous introduction uses a constitutive plasmid to express the ectBAC gene cluster.

9. Use of the engineered bacteria described in claim 1 and the strains obtained by the preparation methods described in claims 2-8 in the production of ectoine.

10. Use of the engineered bacteria according to claim 1 or the strains obtained by the preparation methods according to claims 2-8 in the production of food, medicine, health products and / or cosmetics containing ectoine.

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