Genetically engineered corynebacterium glutamicum and application thereof in preparation of ectoine

Abstract

The invention discloses genetically engineered corynebacterium glutamicum and an application of the genetically engineered corynebacterium glutamicum in preparation of ectoine, and the genetically engineered corynebacterium glutamicum contains an L-diaminobutyric acid transaminase gene ectB, an L-diaminobutyric acid acetyltransferase gene ectA and an ectoine synthetase gene ectC, the invention relates to an aspartic acid transferase gene aspB, an aspartic acid kinase gene lysC and an aspartic acid semialdehyde dehydrogenase gene asd, wherein the nucleotide sequences of the gene aspB, the gene lysC and the gene asd are respectively as shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6. The invention further discloses a preparation method of the aspartic acid transferase gene aspB, the aspartic acid kinase gene lysC and the aspartic acid semialdehyde dehydrogenase gene asd. After the recombinant corynebacterium glutamicum constructed by the invention is fermented for 60 hours, the yield of ectoine can reach 50g / L. The genetically engineered corynebacterium glutamicum disclosed by the invention has important practical significance on industrial production and large-scale application of ectoine.

Description

Technical Field

[0001] This invention relates to a genetically engineered Corynebacterium glutamicum and its application in the preparation of tetrahydropyrimidine, belonging to the field of bioengineering technology. Background Technology

[0002] Many microorganisms accumulate compatible solutes to counteract external stresses such as high osmotic pressure, high temperature, dryness, and freezing. Among these compatible solutes, tetrahydropyrimidine (ectoine, also known as tetrahydromethylpyrimidine carboxylic acid, ectoine) stands out for its remarkable efficacy. It effectively protects halophilic cells from osmotic imbalance and maintains the stability of intracellular biomolecules. Recent studies have shown that tetrahydropyrimidine has the potential to whiten, moisturize, provide antioxidant and anti-aging effects, and protect against ultraviolet radiation when applied to human skin. It can also be used to treat inflammatory diseases such as allergic rhinitis, conjunctivitis, bronchitis, and lung inflammation. These high-value applications have driven its current price to approximately $1000 per kilogram, and have also spurred the development of production methods.

[0003] Currently, the production of tetrahydropyrimidine via microbial fermentation has progressed to a large scale, and various fermentation strategies have been developed to increase yield, reduce production costs, and simplify the production process. In recent decades, low-salt fermentation of tetrahydropyrimidine has been achieved in *Corynebacterium glutamicum*, *Corynebacterium glutamicum*, *Saccharomyces cerevisiae*, and *Bacillus pasteurellii*. *Corynebacterium glutamicum* (… Corynebacterium glutamicum Corynebacterium glutamicum is a type of Gram-positive bacterium with high CG content and is a major producer in the global amino acid fermentation industry. Because L-lysine and tetrahydropyrimidine share a common precursor, L-aspartic semialdehyde, Corynebacterium glutamicum lysine-producing strains are considered the optimal host for tetrahydropyrimidine production. Currently, research on heterologous synthesis of high-yield tetrahydropyrimidine using Corynebacterium glutamicum fermentation utilizes the tetrahydropyrimidine synthesis gene derived from *Pseudomonas schlegelii* (…). Stutzerimonas stutzeri ) and Halomonas elongata ( Halomonas elongata Furthermore, existing technologies also suffer from problems such as complex operation, long cycle time, high cost, and low output. Summary of the Invention

[0004] Objective of the Invention: To address the problems existing in the prior art, this invention provides a genetically engineered *Corynebacterium glutamicum* and its application in the preparation of tetrahydropyrimidine. The synthesis of tetrahydropyrimidine using this genetically engineered bacterium is economical, scientific, low-cost, simple, and has significant implications for large-scale production, providing the most reasonable method for the direct synthesis of tetrahydropyrimidine from microorganisms.

[0005] Technical solution: To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered Corynebacterium glutamicum, wherein the genetically engineered Corynebacterium glutamicum contains an L-diaminobutyric acid transaminase gene ( ectB L-diaminobutyric acid acetyltransferase gene ( ectA ) and tetrahydropyrimidine synthase gene ( ectC ), and the exogenous gene aspartate aminotransferase gene ( aspB ), aspartate kinase gene ( lysC ) and aspartate semialdehyde dehydrogenase gene ( asd ); the gene aspB ,Gene lysC and genes asd The nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

[0006] As a specific implementation scheme, the L-diaminobutyric acid transaminase gene ( ectB L-diaminobutyric acid acetyltransferase gene ( ectA ) and tetrahydropyrimidine synthase gene ( ectC A co-expression vector was constructed by cloning into the pXMJ19 vector, and then expressed in Corynebacterium glutamicum.

[0007] As a specific implementation plan, the aspartate transaminase gene ( aspB ), aspartate kinase gene ( lysC ) and aspartate semialdehyde dehydrogenase gene ( asd An overexpression vector was constructed by cloning into the pECX99E vector, and then transformed into an expression vector. ectB, ectA and ectC The gene is expressed in Corynebacterium glutamicum.

[0008] As a specific implementation plan, the gene is described ectA ,Gene ectB and genes ectC It is derived from *Pseudomonas schrenckii*, and its nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

[0009] As a specific implementation plan, the gene is described aspB Derived from Bacillus tropicalis, its nucleotide sequence is shown in SEQ ID NO.4; the gene described lysC and genes asd It is derived from Escherichia coli, and its nucleotide sequence is shown in SEQ ID NO.5 and SEQ ID NO.6.

[0010] As a specific implementation plan, the chassis strain of the genetically engineered Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032.

[0011] Secondly, the present invention provides a method for preparing the above-mentioned genetically engineered Corynebacterium glutamicum, comprising the following steps: (1) Constructing a gene-containing ectA ,Gene ectB and genes ectC Recombinant plasmid 1, and containing gene aspB ,Gene lysC and genes asd Recombinant plasmid 2; (2) First, recombinant plasmid 1 is transformed into the chassis strain of Corynebacterium glutamicum, and then competent strains are prepared. Then, recombinant plasmid 2 is transformed into competent strains to obtain the genetically engineered Corynebacterium glutamicum.

[0012] Thirdly, the present invention provides the application of the above-mentioned genetically engineered Corynebacterium glutamicum in the preparation of tetrahydropyrimidine or products containing tetrahydropyrimidine; preferably, tetrahydropyrimidine or products containing tetrahydropyrimidine are synthesized using glucose as a carbon source.

[0013] Fourthly, the present invention provides a method for preparing tetrahydropyrimidine, comprising the following steps: The genetically engineered Corynebacterium glutamicum was cultured and induced in a glucose-containing medium.

[0014] As a specific implementation scheme, the culture medium contains yeast extract, peptone, sodium chloride, brain and heart extract and glucose; the culture is carried out at 28-37°C; the induction is performed using 0.1-1.0 mM IPTG.

[0015] More specifically, the method for preparing the tetrahydropyrimidine includes the following steps: (1) The genetically engineered Corynebacterium glutamicum is first cultured in a liquid culture medium containing chloramphenicol and glucose to obtain a first culture medium; at least a portion of the first culture medium is cultured in a chloramphenicol-free liquid culture medium containing glucose to obtain a second culture medium. (2) Isopropyl-β-D-thiogalactoside (IPTG) was added to the second culture medium and cultured for a while to obtain tetrahydropyrimidine.

[0016] In step (1), the liquid culture medium containing chloramphenicol can be liquid LBGB medium containing chloramphenicol; preferably, the liquid LBGB medium containing chloramphenicol contains 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 5 g glucose, and 18.5 g brain heart extract per 1 L of deionized water. The volume ratio of liquid culture medium to chloramphenicol in the liquid culture medium is 800–1200:1; preferably 900–1100:1. The liquid culture medium without chloramphenicol can be liquid LBGB medium; preferably, the liquid LBGB medium contains 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 5 g glucose, and 18.5 g brain heart extract per 1 L of deionized water. This facilitates the collection of successfully transformed *Corynebacterium glutamicum* plasmids and increases the yield of tetrahydropyrimidine.

[0017] In this invention, the absorbance (OD600) of the second culture medium at a wavelength of 600 nm is 0.1–1; preferably 0.4–0.8. Isopropyl-β-D-thiogalactoside is a liquid with a concentration of 100 mmol / L; the volume ratio of the chloramphenicol-free liquid culture medium to the isopropyl-β-D-thiogalactoside liquid is 1700–2200:1; preferably 1900–2100:1. This is beneficial for the growth of genetically modified Corynebacterium glutamicum and the expression of its proteins.

[0018] Beneficial effects: This invention transforms the L-diaminobutyric acid transaminase gene, L-diaminobutyric acid acetyltransferase gene, and tetrahydropyrimidine synthase gene into conventional Corynebacterium glutamicum to form genetically engineered Corynebacterium glutamicum, and introduces exogenous genes. aspB ,Gene lysC and genes asd Through genetic modification, it can produce active L-diaminobutyric acid transaminase, L-diaminobutyric acid acetyltransferase, and tetrahydropyrimidine synthase. Furthermore, this invention utilizes this genetically engineered Corynebacterium glutamicum to produce tetrahydropyrimidine through fermentation. Even further, this method employs mild reaction conditions and yields a high amount of tetrahydropyrimidine. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the synthesis route of tetrahydropyrimidine according to the present invention.

[0020] Figure 2 This is a pXMJ19 plasmid map of the present invention.

[0021] Figure 3 This is the pECX99E plasmid map of the present invention. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] In this invention, nouns and terms include both singular and plural forms unless the context clearly indicates otherwise. For example, "sample" includes one or more samples and their equivalents known to those skilled in the art, etc. The terms "at least one" or "at least one" as used in this invention refer not only to cases containing "one" or "a kind," but more importantly, to cases containing "a plurality" or "multiple."

[0026] The specific methods, steps, reagents, and materials used in this invention, unless otherwise stated, are all commonly known in the art and readily available through publications or purchase. Specific publications can be found, for example, in Cold Spring Harbor's *Molecular Cloning: A Laboratory Manual*, Fourth Edition, etc.

[0027] The following describes the raw materials used in the embodiments: Liquid LBGB medium: Each 1L of deionized water contains 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 5 g glucose, and 18.5 g brain and heart extract.

[0028] LBGB plate medium: Add 1.5% (w / v) agar powder to liquid LBGB medium, then autoclave at 121℃ for 20 min; when the medium cools to about 50℃, pour the medium into sterile petri dishes to obtain antibiotic-free LBGB plate medium.

[0029] LBGB plate medium containing chloramphenicol: Chloramphenicol was added to the antibiotic-free LBGB plate medium to make the chloramphenicol concentration 40 μg / mL, thus obtaining LBGB plate medium with chloramphenicol resistance.

[0030] Liquid LBHIS medium: Each 1L of deionized water contains 5 g peptone, 2.5 g yeast extract, 5 g sodium chloride, 18.5 g brain heart extract, and 91 g sorbitol.

[0031] Liquid Epo medium: Each 1L of deionized water contains 10 g peptone, 5 g yeast extract, 10 g sodium chloride, 1 g Tween-80, and 25 g glycine. Example

[0032] Construction of genetically engineered Corynebacterium glutamicum recombinant plasmid EctBAC-pXMJ19 for Allatoin BAC The expression system was designed using strains that originally produced tetrahydropyrimidine. Stutzerimonas stutzeri Allatoin synthesis related genes ectA, ectB and ectC of Allatoin BAC gene fragment, (Base sequences are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3). First, it was synthesized by Genscript Biotech Co., Ltd. AspB-LysC-asd-pECX99E During synthesis, sequences homologous to both sides of the linearized vector were added. The pXMJ-19 plasmid was digested with restriction endonucleases to obtain the linearized vector. The target gene and the linearized vector were ligated using homologous recombination to obtain the tetrahydropyrimidine BAC operon overexpression plasmid EctBAC-pXMJ19.

[0033] 2. Constructing genetically engineered Corynebacterium glutamicum recombinant plasmids AspB-LysC-asd-pECX99E The specific construction method is the same as step 1.

[0034] 3. Preparation of Corynebacterium glutamicum electrocompetent cells Corynebacterium glutamicum was inoculated into LBGB medium and cultured overnight at 30°C and 200 rpm. It was then transferred to 30 mL of Epo medium at a specific ratio to control the initial OD. 610 The concentration is 0.2-0.3. Incubate at 200 rpm and 30°C for 3-5 h until OD reaches 0.2-0.3. 610The concentration should reach approximately 0.9. Incubate the bacterial culture on ice for 30 min, then centrifuge at 4000 rpm and 4°C for 10 min. Take 30 mL of pre-chilled 10% glycerol, thoroughly resuspend the cells, and centrifuge at 4000 rpm and 4°C for 10 min. Repeat the washing process twice with pre-chilled 10% glycerol. Resuspend the cells in 400 μL of pre-chilled 10% glycerol, aliquot into 1.5 mL centrifuge tubes (80 μL per tube), and freeze at -80°C for storage or use directly for electroporation.

[0035] 4. Plasmid electroconversion Competent cells were placed on ice, and plasmids were added and mixed thoroughly. The mixture was incubated on ice for 10 min. After incubation, the cells were transferred to pre-chilled electroporation cuvettes with a 0.1 cm spacing and electroporated at 1.8 kV for 5 ms. Then, 1 mL of preheated LBHIS medium (46°C) was added, mixed, and transferred to a 1.5 mL centrifuge tube. The cells were heat-shocked at 46°C for 6 min. The cells were incubated at 30°C and 120 rpm for 1.5 h, followed by centrifugation at 10,000 rpm for 1 min. The supernatant was aspirated to approximately 100 μL, mixed thoroughly, and spread onto LBHIS plates containing the appropriate antibiotic. Transformants were cultured at 30°C for approximately 36 h. Transformant colonies were picked for PCR verification.

[0036] Following the electroporation and competent cell preparation methods described above, the recombinant plasmid EctBAC-pXMJ19 was first transformed into Corynebacterium glutamicum chassis strains, then competent strains were prepared, and subsequently, the recombinant plasmid was... ​ The genetically engineered Corynebacterium glutamicum was obtained by transforming the strain into competent cells. The validated strain was then placed in a glycerol tube to obtain the genetically engineered Corynebacterium glutamicum, which was stored at -80°C for later use.

[0037] 5. Cultivation process for the fermentation production of tetrahydropyrimidine by engineered bacteria Genetically engineered Corynebacterium glutamicum was inoculated into 5 mL of liquid LBGB medium, followed by the addition of 5 μL of chloramphenicol. The culture was incubated for 12 h in a shaker (30°C, 220 rpm) to obtain the first culture medium. 1 vol% of this first culture medium was then inoculated into 50 mL of liquid LBGB medium and incubated in a shaker (30°C, 220 rpm) until OD (digesterone) was reached. 600 When the concentration was 0.6, isopropyl-β-thiogalactopyranoside (IPTG, concentration 100 mmol / L) was added to a final concentration of 0.5 mM, and the mixture was cultured in a shaker (temperature 30℃, speed 220 rpm) for 48 h to obtain a bacterial culture containing tetrahydropyrimidine.

[0038] 6. Determination of tetrahydropyrimidine concentration in fermentation broth To analyze the products synthesized by Corynebacterium glutamicum in tetrahydropyrimidine, the main methods employed were high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).

[0039] Fermentation broth pretreatment: Take 1 mL of fermentation broth into a 2 mL EP tube, and incubate at 10000 r·min -1 Centrifuge for 5 min. Collect the supernatant, dilute with deionized water to an appropriate ratio, and filter through a 0.22 μm filter membrane for later use.

[0040] HPLC analysis method: The sample was loaded onto an amino column (Platisil, 5 μm, 250 mm × 4.6 mm) with 70% acetonitrile solution as the mobile phase and a flow rate of 1.0 mL / min. -1 The UV absorbance was measured at 210 nm, the column temperature was 30℃, and the retention time was 20 min.

[0041] LC-MS analysis method: Detector: Waters Acquity PDA det tetrahydropyrimidine (200-400 nm); Column: Waters Accquity UPLC BEH C18 column (2.1 mm × 50 mm). Mobile phase A: 100% methanol; Mobile phase B: 0.1% formic acid; Column temperature: 45℃; Flow rate: 0.3 mL·min⁻¹; Gradient elution. Mass spectrometry detection conditions: Electrospray ionization positive ion source (ESI+), isocratic elution; Cone voltage: 30 V; Capillary voltage: 3.5 kV; Desolvent gas temperature: 400℃; Ion source temperature: 100℃; Cone gas flow rate: 50 L·h⁻¹; Desolvent gas flow rate: 700 L·h⁻¹; Ionization energy: 6 eV; Detector voltage: 1800 V; Mass range: 50-2000 m·z⁻¹.

[0042] The tetrahydropyrimidine in the bacterial culture obtained in step 5 was detected, and its final yield was 50 g / L.

[0043] This invention is not limited to the above-described embodiments. Any modifications, improvements, or substitutions that can be conceived by those skilled in the art without departing from the essential content of this invention fall within the scope of this invention.

Claims

1. A genetically engineered Corynebacterium glutamicum, characterized in that, The genetically engineered Corynebacterium glutamicum contains the L-diaminobutyric acid transaminase gene. ectB L-diaminobutyrate acetyltransferase gene ectA and tetrahydropyrimidine synthase gene ectC And the exogenous gene aspartate transaminase gene aspB Aspartate kinase gene lysC and aspartate semialdehyde dehydrogenase gene asd The gene aspB ,Gene lysC and genes asd The nucleotide sequences are shown in SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, respectively.

2. The genetically engineered Corynebacterium glutamicum according to claim 1, characterized in that, The L-diaminobutyric acid transaminase gene ectB L-diaminobutyrate acetyltransferase gene ectA and tetrahydropyrimidine synthase gene ectC The co-expression vector was constructed by cloning into the pXMJ19 vector, and then transformed into Corynebacterium glutamicum for expression.

3. The genetically engineered Corynebacterium glutamicum according to claim 1, characterized in that, The aspartate transaminase gene aspB Aspartate kinase gene lysC and aspartate semialdehyde dehydrogenase gene asd The overexpression vector was constructed by cloning into the pECX99E vector, and then transformed into an expression vector. ectB, ectA and ectC The gene is expressed in Corynebacterium glutamicum.

4. The genetically engineered Corynebacterium glutamicum according to claim 1, characterized in that, The aforementioned gene ectA ,Gene ectB and genes ectC It is derived from *Pseudomonas schrenckii*, and its nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3, respectively.

5. The genetically engineered Corynebacterium glutamicum according to claim 1, characterized in that, The aforementioned gene aspB Derived from tropical Bacillus; the gene described lysC and genes asd It originates from Escherichia coli.

6. The genetically engineered Corynebacterium glutamicum according to claim 1, characterized in that, The chassis strain of the genetically engineered Corynebacterium glutamicum is Corynebacterium glutamicum ( Corynebacterium glutamicum ATCC 13032.

7. The method for preparing genetically engineered Corynebacterium glutamicum according to any one of claims 1 to 6, characterized in that, Includes the following steps: (1) Constructing a gene-containing ectA ,Gene ectB and genes ectC Recombinant plasmid 1, and containing gene aspB ,Gene lysC and genes asd Recombinant plasmid 2; (2) First, recombinant plasmid 1 is transformed into the chassis strain of Corynebacterium glutamicum, then competent strains are prepared, and then recombinant plasmid 2 is transformed into competent strains to obtain the genetically engineered Corynebacterium glutamicum.

8. The use of the genetically engineered Corynebacterium glutamicum according to any one of claims 1 to 6 in the preparation of tetrahydropyrimidine or products containing tetrahydropyrimidine.

9. A method for preparing tetrahydropyrimidine, characterized in that, This includes culturing and inducing the genetically engineered Corynebacterium glutamicum according to any one of claims 1 to 6 in a culture medium containing glucose.

10. The method according to claim 9, characterized in that, The culture medium contains yeast extract, peptone, sodium chloride, brain and heart extract, and glucose; the culture is carried out at 28–37°C; and the induction is performed using 0.1–1.0 mM IPTG.

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