An engineered bacterium with high production of ectoine, and a preparation method and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and discloses an engineered bacterium with high yield of ectoine as well as a preparation method and application thereof. The application uses Escherichia coli W3110 as a chassis cell, and uses synthetic biology to construct an ectoine metabolic pathway. By optimizing a constitutive promoter P glgs , preferably ectBAC encoding an ectoine synthesis gene as a reporter gene, a constitutive plasmid expressing an ectoine synthesis gene is constructed, the expression level of the gene is improved, and an engineered bacterium Escherichia coli ECT-W020 with high yield of ectoine is obtained, with a preservation number of GDMCC No: 65012. The use of the engineered bacterium of the application to produce ectoine eliminates the process of adding an inducer, and achieves the purpose of reducing production cost.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to an engineering bacterium with high ectoine production, a preparation method thereof and an 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 has used Ectoin in skin care products and launched Ectoine is a high-priced product. A "cell milking" fermentation process has been developed for the production of ectoine using a wild strain of Halomonas elongata, employing a "high-salt-induced synthesis followed by low-salt release." However, the high-salt culture medium used in this "bacterial milking" process not only corrodes equipment but also increases the difficulty of downstream processing. Furthermore, repeated exposure to high and low salt levels inhibits bacterial growth and reduces ectoine production.

[0004] The current main methods to solve this deficiency are: one is to screen new ectoine-producing strains, which can produce ectoine in low-salt environment, but the equipment requirement is high and the yield is low, which increases the difficulty of subsequent ectoine extraction and purification process. For example, in the Chinese invention patent document CN115651943A, a small molecule amino acid derivative ectoine fermentation preparation method and its application, 50g / L-100g / L of sodium chloride is used to stimulate halophilic bacteria to synthesize intracellular ectoine, which has long fermentation time, low ectoine yield and high requirement for production equipment, which is not conducive to industrial production. The other is to introduce the ectoine synthesis pathway in halophilic bacteria into non-halophilic microbial cell factories to realize the heterologous synthesis of ectoine. However, many recombinant strains need isopropyl-beta-D-thiogalactopyranoside (IPTG) and other inducers to synthesize and produce ectoine. IPTG is expensive, which increases the cost of fermentation production and complicates the fermentation process, making it difficult to purify ectoine later. For example, in the Chinese invention patent documents CN117866865A and CN118028204A, the engineering strains need to add IPTG during fermentation to increase the complexity of operation and prolong the fermentation period, which greatly increases the cost of ectoine industrial large-scale production and poses a significant challenge to economic benefits. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies of the prior art and provide an engineering strain for high-yield ectoine production and its preparation method and application.

[0006] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:

[0007] In a first aspect, the present application provides an engineering strain for high-yield ectoine production, which is Escherichia coli ECT-W020, and its preservation number is GDMCC No: 65012.

[0008] The strain ECT-W020 is named Escherichia coli ECT-W020, and its Latin name is Escherichia coli. It was preserved in Guangdong Microbial Culture Collection Center on August 15, 2024, with the preservation number GDMCC No: 65012 and the address of the preservation center being No. 59, Building 5, 100, Martyrs' Road, Guangzhou. The present application uses Escherichia coli W3110 as the chassis cell and applies synthetic biology to construct the ectoine metabolic pathway. By optimizing the constitutive promoter Pglgs The ectBAC gene encoding the ectoine synthesis gene is selected as a reporter gene, a constitutive plasmid expressing the ectoine synthesis gene is constructed, the expression level of the gene is improved, the process of adding an inducer is omitted, and the production cost is reduced.

[0009] In a second aspect, the application provides a method for preparing an engineered bacterium with high ectoine production, comprising the following steps:

[0010] (1) knocking out the EIIA Glc coenzyme gene crr, aspartate kinase / high serine dehydrogenase gene thrA, pyruvate formate lyase gene pflB, lactate dehydrogenase gene ldhA, and bifunctional aldehyde-alcohol dehydrogenase gene adhE;

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

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

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

[0014] The ectBAC gene cluster comprises, in order, the expression elements 2-aminobutyrate transaminase gene ectB, 2-aminobutyrate acetyltransferase gene ectA, and ectoine synthetase gene ectC.

[0015] The engineered bacterium with high ectoine production is constructed by the following aspects:

[0016] (1) constitutive promoter P glgs application: The application constructs a constitutive plasmid expressing the ectoine synthesis gene by selecting the constitutive promoter P glgs of the transcription factor-related gene. This promoter can improve the expression level of the gene, and the process of adding an inducer is omitted, which helps to reduce the production cost.

[0017] (2) selection of ectBAC reporter gene: the order of the ectBAC gene encoding the ectoine synthesis gene is selected.

[0018] (3) application of CRISPR / Cas9 technology: the EIIA GlcCoenzyme crr, reduces the consumption of PEP in the process of glucose transport. In addition, aspartokinase / homoserine dehydrogenase thrA, pyruvate formate lyase pflB, lactate dehydrogenase ldhA, bifunctional aldehyde-alcohol dehydrogenase adhE are also knocked out to select the best knockout combination to reduce the consumption of side chain by-products to the central carbon flow.

[0019] (4) Optimization of metabolic pathways: by knocking out pyruvate kinase pykF and integrating trc-lysC-asd-aspC module, the process of PEP conversion to pyruvate is inhibited, and the intracellular accumulation of oxaloacetate is increased. At the same time, under the control of strong promoter P trc , the expression level of aspartokinase (lysC), aspartate semialdehyde dehydrogenase (asd) and aspartate aminotransferase (aspC) is improved, the carbon flux from glucose to oxaloacetate is increased, and the biosynthesis and accumulation of ectoine is promoted.

[0020] As a preferred embodiment of the preparation method of the high-yield ectoine engineering bacteria of the present application, in step (1): the gene is knocked out by CRISPR / Cas9; the starting strain is Escherichia coli W3110.

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

[0022] As a preferred embodiment of the preparation method of the high-yield ectoine engineering bacteria of the present application, in step (2): the nucleotide sequence of the aspartokinase 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 preparation method of the high-yield ectoine engineering bacteria of the present application, in step (2): the trc-lysC-asd-aspC gene cluster is integrated; the trc-lysC-asd-aspC gene cluster sequentially includes the expression element promoter P trc , aspartokinase gene lysC, aspartate semialdehyde dehydrogenase gene asd and aspartate aminotransferase gene aspC; the nucleotide sequence of the promoter P trcthe 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.

[0024] As a preferred embodiment of the method for preparing the engineered bacteria with high yield of ectoine according to the present application, in step (3), the ectBAC gene cluster is expressed by a promoter P glgs transcription; the promoter P glgs the nucleotide sequence of which is shown as SEQ ID No. 14.

[0025] As a preferred embodiment of the method for preparing the engineered bacteria with high yield of ectoine according to the present application, in step (3), the nucleotide sequence of the 2-aminobutyrate transaminase gene ectB is shown as SEQ ID No. 3; the nucleotide sequence of the 2-aminobutyrate acetyltransferase gene ectA is shown as SEQ ID No. 1; and the nucleotide sequence of the ectoine synthetase gene ectC is shown as SEQ ID No. 5.

[0026] As a preferred embodiment of the method for preparing the engineered bacteria with high yield of ectoine according to the present application, in step (3), the ectBAC gene cluster is expressed by a promoter P

[0027] Preferably, the constitutive plasmid comprises a pACYC-Duet-1 plasmid, a pCDF, a pET, etc.

[0028] In a third aspect, the present application applies the engineered bacteria and the strains prepared by the method to the production of ectoine.

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

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

[0031] The engineered bacteria with high yield of ectoine according to the present application uses a constitutive promoter P glgsThe high-level expression of the ectoine synthesis gene can be continuously maintained without adding the inducer IPTG, thus simplifying the production process and reducing the additional cost in production. The preparation method of the high-ectoine-producing engineered bacteria of the application improves the carbon source utilization rate in the glucose transport process, reduces the generation of by-products in the metabolic process, and improves the accumulation of ectoine precursor substances, expands the metabolic flux, and improves the ectoine production efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The figure is a schematic diagram of the ectoine metabolic pathway optimization of the application.

[0033] Figure 2 The figure is a standard curve of the ectoine standard. DETAILED DESCRIPTION

[0034] The engineered bacteria of the application use E. coli W3110 as the chassis cell and apply synthetic biology to construct the ectoine metabolic pathway. Figure 1 As shown in the figure, the constitutive promoter P glgs is selected from the genes related to transcription factors in E. coli, the ectBAC encoding the ectoine synthesis gene is selected as the reporter gene, the constitutive plasmid expressing the ectoine synthesis gene is constructed, the expression level of the gene is improved, the process of adding the inducer is omitted, and the purpose of reducing the production cost is achieved.

[0035] The CRISPR / Cas9 technology is applied to knock out the EIIA Glc coenzyme crr in the phosphotransferase system (PTS) to reduce the consumption of PEP in the glucose transport process, the aspartate kinase / high serine dehydrogenase thrA, pyruvate formate lyase pflB, lactate dehydrogenase ldhA, and bifunctional aldehyde-alcohol dehydrogenase adhE are knocked out, the best knockout combination is selected to reduce the consumption of the side chain by-products such as ethanol, lactic acid, and threonine to the central carbon flow, improve the intracellular accumulation of precursors such as oxaloacetate and aspartate and acetyl coenzyme A, and expand the metabolic flux of ectoine.

[0036] Further metabolic pathway of ectoine: by knocking out pykF and integrating into the trc-lysC-asd-aspC module, the conversion of phosphoenolpyruvate (PEP) to pyruvate is inhibited, and the intracellular accumulation of oxaloacetate is increased. In addition, under the control of the strong promoter P trc , the expression levels of aspartate kinase (lysC), aspartate semialdehyde dehydrogenase (asd), and aspartate aminotransferase (aspC) are improved, the carbon flux from glucose to oxaloacetate is increased, the conversion of oxaloacetate to L-aspartate after L-aspartate is promoted, the conversion to L-aspartate-β-semialdehyde is accelerated, the accumulation of ectoine biosynthesis metabolism is accelerated, and the synthesis amount of ectoine is improved.

[0037] For better illustrating the purpose, technical scheme and advantages of the present application, the present application will be further described in combination with specific examples. Those skilled in the art should understand that the specific examples described herein are only used to explain the present application, and are not used to limit the present application. The test methods used in the examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available unless otherwise specified.

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

[0039] The chassis cell Escherichia coli W3110 DSM5911 was purchased from Guangzhou Qiyun Biotechnology Co., Ltd.; the pTargetF plasmid was purchased from Molecular Cloud Cat. No.: MC_0000012; the pACYC-Duet-1 plasmid was purchased from Novagen; the tryptone and the yeast powder were purchased from Oxoid Company; the DNA marker and the Primer Star Max were purchased from Takara Company; the 2xPhanta Max Master Mix, the 2xRapid Taq Master Mix and the Phanta HS Super-Fidelity DNA Polymerase were purchased from Thermo Fisher Scientific; the Ultra One Step Cloning Kit was purchased from Nanjing Nvwaizhan Biotechnology Co., Ltd.; the agarose gel DNA recovery kit and the plasmid extraction kit were purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.; the ectoine standard (chromatographically pure) was purchased from Tanmo Standard Material Center; the kanamycin and the spectinomycin were purchased from Shengong Biotech (Shanghai) Co., Ltd.; other medicines or reagents were imported or domestically produced analytical pure or chromatographically pure. The Ultra One Step Cloning Kit was purchased from Nanjing Nvwaizhan Biotechnology Co., Ltd.; the agarose gel DNA recovery kit and the plasmid extraction kit were purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd.; the ectoine standard (chromatographically pure) was purchased from Tanmo Standard Material Center; the kanamycin and the spectinomycin were purchased from Shengong Biotech (Shanghai) Co., Ltd.; other medicines or reagents were imported or domestically produced analytical pure or chromatographically pure.

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

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

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

[0043] The working quality concentration of the antibiotic: chloramphenicol 34 mg / L, kanamycin 50 mg / L, spectinomycin 50 mg / L;

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

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

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

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

[0048] The synthesis of the gene sequences involved in the examples is as follows:

[0049] The PCR primers are shown in Table 1, synthesized by Suzhou Jinyu Zhi Biological Technology Co., Ltd.;

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

[0051]

[0052]

[0053]

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

[0055] Example 1: Cloning of target genes

[0056] The target genes ectB, ectA, ectC were amplified by PCR reaction program using the corresponding primer pairs in Table 2 as templates, and the plasmid backbone pMB1-trc was amplified by PCR reaction program using the pTargetF (Molecular Cloud Cat. No.: MC_0000012) plasmid as template, the backbone pACYC fragment was amplified by PCR reaction program using the pACYC-Duet-1 (Novagen) plasmid as template, and the other fragments lysC, asd and aspC as well as the promoters P lpp , P glgs and P trc were amplified by PCR reaction program using the corresponding primer pairs in Table 2 as templates.

[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] Reagent Amount (μL) Upstream primer 1 μL Downstream primer 1 μL Template DNA 0.5 μL ddH2O 17.5 μL 2x Phanta Max Master Mix (Dye Plus) 20 μL Total system 40 μL

[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°C; 2 min Step 5: cycle steps 2-4 Cycle 30 times in total Step 6: final extension 72°C; 5 min Step 7: preservation Preserve at 4°C

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

[0068] Example 2: Short fragment overlap extension

[0069] PCR products of Example 1 were used to connect the three gene clusters etcA, etcB and etcC in the ectoine biosynthesis pathway in order respectively as etcABC and etcBAC by overlap extension PCR method, and the gel was recovered to obtain ectoine synthesis gene cluster P etcABC (etcABC-F and etcABC-R) and P etcBAC (etcBAC-F and etcBAC-R). The two target genes were respectively connected with the promoter P lpp , P glgs Overlap extension PCR amplification verification, gel purification and recovery obtained 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, lysC, asd and aspC were connected in order by overlap extension PCR to obtain module P lysC -P asd -P aspC The template was connected with the promoter Ptrc to obtain product Ptrc-P lysC -P asd -P aspC Ptrc-P lysC -P asd -P aspC Gel electrophoresis verified that the PCR reaction was successful, and the DNA was purified and recovered at 4°C for cold storage.

[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 procedure 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 ligation and assembly. After digesting the plasmid template with DpnI, the product was transformed into E. coli Turbo cells by heat stimulation. The strain was screened on a chloramphenicol-resistant plate. After culturing for about 16 hours, a single colony was picked for colony PCR verification. The verified bacteria were cultured, and the plasmids were extracted and sent to Shanghai Meiji for sequencing. The recombinant plasmids with correct sequencing were named pACYC-lpp-etcABC-trc- lysC-asd-aspC pACYC-lpp-etcBAC-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-glgs-etcABC-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-etcBAC-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, MultiS One Step Cloning Kit connected assembly, operation as above. The correct recombinant plasmid is named trc- lysC-asd-aspC .

[0082] Example 4: Construction of recombinant strains

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

[0084] Heat shock method operation: take the competent W3110 cells from the-80℃ refrigerator and place on ice to thaw, transfer the above plasmids into the competent W3110 cells, blow and mix, ice bath for 30 min, 42℃ heat shock for 90 s, add 1 mL of antibiotic-free LB medium, incubate at 37℃ for 20 min, centrifuge at 8000 rpm for 1 min, discard the supernatant, take 300 μL of LB medium to resuspend and mix the bacterial solution, take 100 μL of bacterial solution to spread on chloramphenicol-resistant LB plates for screening, and plate in a 37℃ incubator for 16 h, pick multiple single colonies for gene identification by PCR, and streak on chloramphenicol-resistant plates for continuous passage to verify plasmid stability.

[0085] Example 5: Optimization of ectoine metabolic pathway

[0086] (1) Construction of E. coli knockout and interference gene plasmid

[0087] The CHOPCHOP website was used to design the knockout and interference N20 site of the gene, pTargetF-cadA was used as a template, and reverse PCR was performed using primers thrA-CRISPR-F and thrA-CRISPR-R to amplify the plasmid pTargetF-thrA sequence. After digestion of the plasmid template with DpnI, pTargetF-thrA was transformed into competent cells Turbo by heat shock method, and the strain was selected on a spectinomycin-resistant plate. After 16 h of culture, single colonies were picked for PCR identification, and the successfully identified strains were expanded for measurement, and the pTargetF-thrA plasmid was extracted.

[0088] The CRISPR-Cas9 gene editing system was used to delete a gene fragment from the Escherichia coli genome. PCR amplification was performed using primers thrA-UP-F and thrA-UP-R and the W3110 genome as a template to obtain the upstream homology arm of thrA-UP. Similarly, primers thrA-DOWN-F and thrA-DOWN-R were used to obtain the downstream homology arm of thrA-DOWN. Subsequently, overlapping amplification was performed using primers thrA-UP-F and thrA-DOWN-R and the thrA-UP and thrA-DOWN fragments as templates to obtain the replacement homology arm 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 through 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. After knocking out thrA from the W3110 genome, the mutant strain W001 was obtained.

[0090] Using the same method as above, using pTargetF-cadA as a template and primers crr-CRISPR-F and crr-CRISPR-R, plasmid pTargetF-crr was amplified and constructed. Primers crr-UP-F and crr-UP-R, as well as crr-DOWN-F and crr-DOWN-R, were used to amplify the crr replacement homology arm fragment. The crr was knocked out from the W001 / pCas genome, and the knockout was confirmed using primers crr-UP-F and crr-check-R. The thrA and crr knockout mutant strain W002 was obtained. ldhA and adhE were sequentially knocked out in the W002 / pCas genome to obtain the thrA, crr, ldhA, and adhE knockout mutant strain W003. PflB was knocked out in the W003 / pCas genome to obtain the thrA, pflB, crr, ldhA, and adhE knockout mutant strain W004.

[0091] The same method was used to knockout crr, ldhA and adhE in W3110 / pCas genome with pTargetF-cadA as template to obtain mutant strain W005 with crr, ldhA and adhE knocked out. The plasmid pTargetF-pflB was constructed by amplifying pflB with primers pflB-CRISPR-F and pflB-CRISPR-R, and the pflB replacement homologous arm fragments were amplified with primers pflB-UP-F and pflB-UP-R, pflB-DOWN-F and pflB-DOWN-R. The mutant strain W006 with pflB, crr, ldhA and adhE knocked out was obtained by knocking out pflB from W005 / pCas genome and identified with primers pflB-UP-F and pflB-check-R.

[0092] The same method was used to knockout crr, ldhA and adhE in W3110 / pCas genome with pTargetF-cadA as template to obtain mutant strain W005 with crr, ldhA and adhE knocked out. The plasmid pTargetF-pflB was constructed by amplifying pflB with primers pflB-CRISPR-F and pflB-CRISPR-R, and the pflB replacement homologous arm fragments were amplified with primers pflB-UP-F and pflB-UP-R, pflB-DOWN-F and pflB-DOWN-R. The mutant strain W006 with pflB, crr, ldhA and adhE knocked out was obtained by knocking out pflB from W005 / pCas genome and identified with primers pflB-UP-F and pflB-check-R. The same method was used to knockout crr, ldhA and adhE in W3110 / pCas genome with pTargetF-cadA as template to obtain mutant strain W005 with crr, ldhA and adhE knocked out. The plasmid pTargetF-pflB was constructed by amplifying pflB with primers pflB-CRISPR-F and pflB-CRISPR-R, and the pflB replacement homologous arm fragments were amplified with primers pflB-UP-F and pflB-UP-R, pflB-DOWN-F and pflB-DOWN-R. The mutant strain W006 with pflB, crr, ldhA and adhE knocked out was obtained by knocking out pflB from W005 / pCas genome and identified with primers pflB-UP-F and pflB-check-R.

[0093] (2) Construction of recombinant strain

[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 Escherichia coli competent cells W3110 by electroporation, 1 mL of LB medium was added and pipetted to mix, and the cells were cultured at 30°C and 200 rpm for 1 hour. The cells were centrifuged at 8000 rpm for 1 minute, and the excess supernatant was discarded. 100 μL of the supernatant was used to resuspend the bacteria, which were then spread on LB plates containing chloramphenicol and cultured inverted at 30°C overnight. Colony PCR verification was performed using the corresponding primers for the strain, and single colonies of positive clones were picked and transferred to LB liquid culture medium containing chloramphenicol. The culture was cultured overnight at 37°C and 200 rpm. The mixture of the bacterial solution and 50% glycerol was 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-aspCStrain ECT-W009 (W3110, ApflB, Acrr, AldhA, AdhE, trc-lysc-asd-aspC, pAcYc-glgs-etcABC) was obtained by introducing mutant strain W006 with plasmid pAcYc-glgs-etcABC- trc- lysC-asd-aspC Strain ECT-W010 (W3110, ApflB, Acrr, AldhA, AdhE, trc-lysc-asd-aspC, pAcYc-glgs-etcBAC) was obtained by introducing mutant strain W006 with plasmid pAcYc-glgs-etcBAC-

[0096] Strain ECT-W009 (W3110, ApflB, Acrr, AldhA, AdhE, trc-lysc-asd-aspC, pAcYc-glgs-etcABC) was obtained by introducing mutant strain W006 with plasmid pAcYc-glgs-etcABC- trc- lysC-asd-aspC Strain ECT-W012 (W3110, AthrA, ApflB, Acrr, AldhA, AdhE, trc-lysc-asd-aspC, pAcYc-glgs-etcBAC) was obtained by introducing mutant strain W004 with plasmid pAcYc-glgs-etcBAC. Figure 2 Strain ECT-W012 (W3110, AthrA, ApflB, Acrr, AldhA, AdhE, trc-lysc-asd-aspC, pAcYc-glgs-etcBAC) was obtained by introducing mutant strain W004 with plasmid pAcYc-glgs-etcBAC.

[0097] Strain ECT-W020 (W3110, ApflB, AthrA, Acrr, AldhA, AdhE, ApykF::trc-lysC-asd-aspC, pACYC-glgs-etcBAC) was obtained by introducing mutant strain W007 with plasmid pAcYc-glgs-etcBAC-lysc-aspc to overexpress etcBAC; Strain ECT-W016 (W3110, ApflB, AthrA, Acrr, AldhA, AdhE, ApykF::trc-lysC-asd-aspC, pACYC-glgs-etcABC) was obtained by introducing mutant strain W007 with plasmid pAcYc-glgs-etcABC-lysc-aspc to overexpress etcABC; Strain ECT-W018 (W3110, ApflB, AthrA, Acrr, AldhA, AdhE, ApykF::trc-lysC-asd-aspC, pACYC-lpp-etcBAC) was obtained by introducing mutant strain W007 with plasmid pACYC-lpp-etcBAC-trc-lysC-asd-aspC to overexpress etcBAC.

[0098] Example 6: Detection of ectoine production of recombinant strains

[0099] Ectoine standard solutions with mass concentrations of 10, 30, 50, 80, 100, 150, 200 and 300 mg / L were prepared with ultrapure water, and the ectoine standard solutions and samples were determined by HPLC. The chromatographic column was ZORBAX Stable Bond aq, the mobile phase was 100% 0.02 M potassium dihydrogen phosphate, the flow rate was 0.5 mL / min, the detection wavelength was 210 nm, the column temperature was 25°C, the sample volume was 10 μL, and the standard curve was plotted with ectoine standard as shown in Recombinant strain .

[0100] The ectoine engineering strains constructed in Example 5 were inoculated into LB plates containing chloramphenicol, and cultured in a 37°C incubator for 16 h. Single colonies were inoculated into 5 mL of LB test tube and cultured overnight at 37°C with 200 rpm shaking. Then 10% of the bacterial solution was transferred into 100 mL of basic medium containing chloramphenicol and cultured at 37°C with 200 rpm shaking for 43 h. 800 μL of the fermentation broth was added to 2.4 mL of anhydrous ethanol, mixed and reacted for 30 min, centrifuged at 8000 r / min for 10 min, and the supernatant was collected, the solvent was removed by nitrogen blowing, 2.4 mL of anhydrous ethanol was added and ultrasonically dissolved, centrifuged to collect the supernatant, nitrogen blown, 800 μL of water was added and shaken to dissolve, and HPLC was used for detection. The yield of ectoine was calculated according to the standard curve.

[0101] The results are as follows:

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

[0103] The results are shown in Table 7. The strain containing the constitutive promoter glgs has stronger ectoine synthesis ability than the strain containing the lpp promoter, and the plasmid constructed in the order of EctB, EctA and EctC is beneficial to the synthesis of ectoine.

[0104] Without the need to add an inducer during fermentation, the process flow and cost of producing ectoine are avoided, which is beneficial to the industrialized production of ectoine in the future.

[0105] Table 7: Ectoine production of ECT-W001 to ECT-W004

[0106] Exodin yield ECT-W001 16.92 ± 0.52 mg / L ECT-W002 42.61 ± 0.72 mg / L ECT-W003 113.78 ± 12.23 mg / L ECT-W004 29.58 ± 0.58 mg / L Recombinant strain

[0107] 2. Effect of knocking out thrA, pflB, adhE, IdhA and crr on ectoine production

[0108] (1) The effect of thrA gene deletion on exedin production

[0109] In the ECT-W003 genome, the thrA gene, which encodes aspartokinase / homoserine dehydrogenase, was knocked out to improve the exedin production of the recombinant strain ECT-W005 to 150.64±0.58 mg / L, which is 5 times of the initial strain ECT-W003. The thrA gene was knocked out to reduce the synthesis of threonine and increase the carbon flow to exedin. The thrA gene deletion inhibits the aspartokinase activity, and the lysC gene, which encodes aspartokinase feedback resistance in C. glutamicum, is introduced to compensate for the enzyme activity deficiency and supplement aspartokinase.

[0110] Table 8 The effect of thrA gene knockout on exedin production

[0111] Exodin yield ECT-W003 115.93 ± 6.92 mg / L ECT-W005 550.64 ± 15.8 mg / L Recombinant strain

[0112] (2) The effect of recombinant strains ECT-W005 to ECT-W012 on exedin synthesis in shake flask fermentation

[0113] ECT-W007 and ECT-W008 both knock out crr, ldhA and adhE successively on the basis of thrA knockout to promote the synthesis of exedin. Similarly, ECT-W009 and ECT-W010 knock out pflB successively on the basis of crr, ldhA and adhE knockout, which is also conducive to improving the yield of exedin; strains ECT-W011 and ECT-W012 knock out thrA and pfIB genes successively on the basis of crr, ldhA and adhE knockout, which significantly improves the yield of exedin, and the yield of recombinant exedin with ectBAC gene cluster as the promoter is significantly higher than that with ectABC gene cluster as the promoter. glgs lpp The yield of recombinant exedin with ectBAC gene cluster as the promoter is significantly higher than that with ectABC gene cluster as the promoter. The yield of exedin of recombinant strain ECT-W012 in the basic medium is 1542.74±32.40 mg / L, while the yield of exedin of ECT-W011 is only 522.15±7.15 mg / L. The gene pflB encodes pyruvate formate lyase, ldhA encodes lactate dehydrogenase, adhE gene encodes bifunctional aldehyde-alcohol dehydrogenase, and crr encodes EIIA Glc coenzyme in the phosphotransferase system (PTS) system, which converts phosphoenolpyruvate (PEP) to pyruvate. This indicates that knocking out thrA, pflB, ldhA and adhE reduces the consumption of by-products such as threonine, ethanol, pyruvate and lactic acid to the central carbon flow, and increases the accumulation of precursor oxaloacetate, acetyl coenzyme A and L-aspartate-β-semialdehyde in the cell.

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

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

[0116] 3. Further optimization of the ectoine metabolic pathway

[0117] The recombinant strains ECT-W016, ECT-W018 and ECT-W020 further knock out the gene pykF on the basis of knocking out thrA, pflB, adhE, IdhA, crr and integrate into the trc-lysC-asd-aspC module, the yield of ectoine is increased by several times, the conversion of phosphoenolpyruvate (PEP) to pyruvic acid is inhibited, the intracellular accumulation of oxaloacetate is increased, and lysC, asd and aspC are supplemented to promote the conversion of oxaloacetate to aspartate and accelerate the accumulation of ectoine biosynthesis metabolism. ECT-W020, after optimization of the ectoine metabolic pathway, the yield of ectoine was 25.51±0.24g / L after 43 hours of fermentation in a shake flask, and was 6 times the yield of ectoine of strains ECT-W016 and ECT-W018.

[0118] Table 10 Further optimization of the ectoine metabolic pathway

[0119] Exodin yield ECT-W012 7.13 ± 0.13 g / L ECT-W016 4.78 ± 0.19 g / L ECT-W018 4.2 ± 0.18 g / L ECT-W020 25.51 ± 0.24 g / L ​

[0120] The strain ECT-W020 is named Escherichia coli ECT-W020, and its Latin name is classified as Escherichia coli, which was deposited in the Guangdong Microbial Culture Collection Center on August 15, 2024, with the accession number GDMCC No: 65012, and the address of the deposit is No. 59, Building 5, 100, Martyrs Road, Guangzhou.

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

[0122] (1) Activation of bacterial cells: ectoine recombinant strains ECT-W020 and initial strains ECT-W003 were streaked on LB plates and incubated at 37°C for 24h, and single colonies were inoculated into 10mL of LB medium and incubated at 37°C, 200rpm for 12h.

[0123] (2) Seed culture: 10% of the first seed liquid was transferred to 1 L of LB medium, and cultured at 37°C, 200 rpm for 16 h, and then all were transferred to a 50 L volume fermenter containing 20 L of sterilized basic fermentation medium, and cultured at 37°C, with an initial rotation speed of 200 rpm and an initial air flow of 100 L / min. The pH was controlled at 7.2 by automatic ammonia flow addition throughout the fermentation process, and the dissolved oxygen was maintained at no less than 20% by stirring and changing the aeration rate, with a correlation rotation speed of 200 rpm-800 rpm. The fermentation was carried out for 52 h, and OD 600 , the wet weight of the bacterial cells, and the residual sugar content were detected every hour. When the dissolved oxygen was less than 70%, 40% concentration glucose was added, and the residual sugar content in the fermentation broth was controlled at 2 g / L by feedback control of the feeding speed throughout the process.

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

[0125] Table 11: Ectoine yield of ECT-W020 and ECT-W003 in the basic medium

[0126]

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

[0128] (1) Bacterial activation: The ectoine recombinant strain ECT-W020 and the initial strain ECT-W003 were streaked on LB plates and 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: 10% of the first seed liquid was transferred to 1 L of LB medium, and cultured at 37°C, 200 rpm for 16 h, and then all were transferred to a 50 L volume fermenter containing 20 L of sterilized basic fermentation medium, and cultured at 37°C, with an initial rotation speed of 200 rpm and an initial air flow of 100 L / min. The pH was controlled at 7.2 by automatic ammonia flow addition throughout the fermentation process, and the dissolved oxygen was maintained at no less than 20% by stirring and changing the aeration rate, with a correlation rotation speed of 200 rpm-800 rpm. The fermentation was carried out for 52 h, and OD 600, the wet weight of the bacteria, the residual sugar amount, when the dissolved oxygen is less than 70%, the 40% concentration of glucose is started to be added, the feedback control is used throughout the process to adjust the feeding speed to control the residual sugar in the fermentation broth to 2g / L, and the constant speed of 20% protein peptone 160mL / h is started to be added.

[0130] After the initial strain ECT-W003 and the recombinant engineering strain ECT-W020 are fermented in the 50L fermentation medium for 52h, the growth performance of the strain ECT-W003 is limited and the ectoine yield is not synthesized with the proliferation of the strain, and the yield is only 1.32g / L. The ectoine yield of ECT-W020 is 188.59g / L after 52h of fermentation. It is proved that the ectoine recombinant strain ECT-W020 obtained by optimizing the metabolic pathway is effective, and the ectoine yield is greatly improved. The strain can be used as an engineering strain for industrial production of ectoine.

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

[0132]

[0133] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not a limitation on the protection scope of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. An engineered bacterium for high production of ectoine, characterized in that, The engineered bacteria is Escherichia coli (E. coli) Escherichia coli ) ECT-W020, the preservation number of which is GDMCC No: 65012.

2. Use of the engineered bacteria of claim 1 in the production of an ectoine.

3. Use of the engineered bacteria of claim 1 in the production of food, pharmaceutical and / or cosmetic products containing an ectoine.

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