Method for regulating and controlling synthesis of ectoine in escherichia coli

Abstract

The invention discloses a method for regulating and controlling synthesis of ectoine in escherichia coli, and relates to the technical field of biological medicines. According to the method for regulating and controlling the synthesis of the ectoine in the escherichia coli, disclosed by the invention, a gene segment of a tisB toxic protein is used for constructing a tisB toxic protein expression vector pET28a (+)-p2-22-tisB, and the tisB toxic protein expression vector is transferred into an escherichia coli engineering bacterium for synthesizing the ectoine, so that the escherichia coli engineering bacterium E.coli Ect pET28a (+)-p2-22-tisB for spontaneously inhibiting the synthesis of the ectoine is obtained, and meanwhile, the normal synthesis of the ectoine at an early stage can be ensured. The nucleotide sequence of the gene segment of the tisB toxic protein is as shown in SEQ ID NO. 1. In the first three rounds of fermentation, the yield of ectoine is close to that of the control engineering bacteria; after the seventh round, the yield of ectoine is sharply reduced to 2.575 g / L, and during the twentieth round of fermentation, the yield of ectoine is zero.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and more specifically, relates to a method for regulating the synthesis of ectoine in Escherichia coli. Background Art

[0002] With the widespread application of ectoine in the fields of cosmetics, biomedicine, etc., its market demand continues to grow. Constructing a high-yield ectoine cell factory through metabolic engineering and other means has become an important research direction. The maximum yield of ectoine of engineered Escherichia coli has reached a high level, providing strong support for industrial production. However, commercial espionage allows the above research to be plagiarized by other companies in a relatively cheap way, which is a huge loss for the above companies.

[0003] The tisB toxin protein plays an important physiological role in bacteria. It is a component of the toxin-antitoxin system. The toxin-antitoxin system is widely present in bacterial and fungal cells and is composed of a pair of small genetic control elements encoded by toxin genes and antitoxin genes. Among them, the toxin gene encodes a stable protein molecule, such as the tisB toxin protein, while the antitoxin gene encodes a protein with poor stability or an RNA with regulatory function. The specific function and mechanism of action of the tisB toxin protein are still under study. It is known that the toxin-antitoxin system is involved in a variety of physiological processes of bacteria, including cell growth, division, and retention state formation. The tisB toxin protein may exert its toxic effect by affecting the permeability of the cell membrane, interfering with the metabolic process in the cell, or interacting with other cell components. In addition, the study of the tisB toxin protein is of great significance for understanding the pathogenic mechanism of bacteria, developing new antibacterial drugs, and biotechnology applications. By in-depth research on the structure, function, and regulatory mechanism of the tisB toxin protein, new ideas and methods can be provided for the treatment and prevention of bacterial infections.

[0004] In the process of strain modification and metabolic optimization, one of the common strategies is to use promoters of different strengths to finely regulate the expression of target genes, coordinate and balance the synthesis and utilization of various intermediate metabolites, and improve the synthesis efficiency of metabolites. Many constitutive promoters of different strengths have been characterized in Escherichia coli. However, the regulatory elements of existing biological control gene circuits usually require additional inducers as signals, which makes the existing control circuits lack the ability to actively respond under inducer-free conditions and difficult to actively play a role after the engineered bacteria are leaked. The leaked engineered bacteria may survive and spread for a long time, increasing the risk of leakage of trade secrets. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for regulating the synthesis of ectoine in Escherichia coli, which is used to regulate the synthesis of ectoine in Escherichia coli at different stages.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for regulating ectoine synthesis in Escherichia coli comprises: transferring a gene fragment of tisB toxin protein into an ectoine synthesis strain to obtain an engineered bacterium that spontaneously inhibits ectoine synthesis;

[0008] The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1.

[0009] The method comprises:

[0010] 1) Construction of tisB protein expression vector;

[0011] 2) The constructed tisB toxin protein expression vector was transformed into the ectoine-synthesizing strain E. coli Ect to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

[0012] The tisB toxic protein expression vector is pET28a(+)-p2-22-tisB.

[0013] The construction method of the tisB toxic protein expression vector pET28a(+)-p2-22-tisB is as follows: constructing the gene fragment of the tisB toxic protein into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; constructing the constitutive promoter 2-22 fragment into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB;

[0014] The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1;

[0015] The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2.

[0016] The method described herein comprises the following specific steps:

[0017] 1) The gene fragment of tisB toxic protein was constructed into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; the constitutive promoter 2-22 fragment was constructed into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB;

[0018] The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1;

[0019] The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2;

[0020] 2) After incubating 50 ng of the recombinant expression vector pET28a(+)-p2-22-tisB with the competent strain of ectoine synthesis E. coli Ect on ice for 30 min, heat shock was performed at 42°C for 90 s to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

[0021] Application of tisB toxin protein expression vector pET28a(+)-p2-22-tisB in regulating ectoine synthesis in Escherichia coli.

[0022] The construction method of the tisB toxic protein expression vector pET28a(+)-p2-22-tisB is as follows: constructing the gene fragment of the tisB toxic protein into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; constructing the constitutive promoter 2-22 fragment into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB;

[0023] The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1;

[0024] The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2.

[0025] The application specifically comprises the following steps:

[0026] 1) The gene fragment of tisB toxic protein was constructed into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; the constitutive promoter 2-22 fragment was constructed into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB;

[0027] The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1;

[0028] The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2;

[0029] 2) After incubating 50 ng of the recombinant expression vector pET28a(+)-p2-22-tisB with the competent strain of ectoine synthesis E. coli Ect on ice for 30 min, heat shock was performed at 42°C for 90 s to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

[0030] The engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits the synthesis of ectoine stops synthesizing ectoine after multiple rounds of cyclic fermentation culture.

[0031] The multiple rounds of fermentation culture are ≥7 rounds of fermentation culture.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) This application starts with an engineered Escherichia coli that synthesizes ectoine, uses pET28a(+) as an expression vector, constructs an expression frame of the TisB toxin protein gene, and controls the expression by a constitutive promoter 2-22, thereby obtaining a recombinant engineered strain E. coli Ect pET28a(+)-p2-22-tisB that can spontaneously inhibit ectoine synthesis after multiple rounds of fermentation.

[0034] 2) After multiple rounds of fermentation culture, the results of the Escherichia coli engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB constructed in the present application that spontaneously inhibits the synthesis of ectoine showed that the production of ectoine in the first three rounds of fermentation was about 7.269 g / L, which was similar to that of the control engineered bacteria (7.191 g / L); after the seventh round, the production of ectoine dropped sharply to 2.575 g / L, and in the twentieth round of fermentation, the production of ectoine was zero.

[0035] 3) This application transforms tisB toxin protein into Escherichia coli, constructs an Escherichia coli engineered strain that can spontaneously inhibit the synthesis of ectoine after multiple rounds of culture and fermentation, and can ensure the normal synthesis of ectoine in the early stage. This method can be applied to the protection of industrial strains and biosafety protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1Schematic diagram of tisB toxic protein expression vector pET28a(+)-p2-22-tisB;

[0037] Figure 2 It is the liquid phase diagram of tetrahydropyrimidine;

[0038] Figure 3 This is a graph showing the production of ectoine from multiple rounds of culture and fermentation of recombinant engineered bacteria. DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0040] Example 1

[0041] 1. Construction of tisB protein expression vector

[0042] The primer sequences designed based on the tisB toxin protein gene sequence (NCBI Reference Sequence: NC_000913.3) are as follows:

[0043] tisB-F1:

[0044] 5'-aactttaagaaggagatataccatggATGAACCTGGTGGATATCGC-3' (SEQ ID NO.3),

[0045] tisB-R1:

[0046] 5'-caagcttgtcgacggagctcgaattcTTACTTCAGGTATTTCAGAACAGCAT-3' (SEQ ID NO. 4);

[0047] tisB-F3:

[0048] 5'-atctcgatcccgcgaaatgaactggttaaggtgatggaca-3' (SEQ ID NO.5),

[0049] tisB-R3:

[0050] 5'-ccttcttaaagttaaacaaaattatttctagagggcctttagtatagcatattaccacc-3' (SEQ ID NO. 6).

[0051] The tisB toxic protein gene fragment (SEQ ID NO.1) was amplified using high-fidelity PCR polymerase Prime Star and primers F1 / R1, and then assembled by Gibbson and integrated into pET28a(+) by homologous recombination to construct the recombinant expression vector pET28a(+)-tisB. On this basis, the constitutive promoter 2-22 fragment (SEQ ID NO.2) was amplified using primers F3 / R3 using Escherichia coli MG1655 as a template, and then assembled by Gibbson and integrated into pET28a(+)-tisB by homologous recombination to construct the recombinant expression vector pET28a(+)-p2-22-tisB ( Figure 1 ).

[0052] 2. Construction of Ectohydropyrimidine synthesis strain (E. coli Ect)

[0053] The genome of Halomonas elongatus (CGMCC:1.6329) was used as a template, and the primers ectABC-F / ectABC-R were used to amplify the ectABC gene fragment. The plasmid pSTOP1622 (MoBiTec) was used as a template, and the primers xylA-F / xylA-R were used to amplify the xylose repressor protein and the xylose-inducible promoters xylR and PxylA. The genome of Escherichia coli W3110 (ATCC 27325) was used as a template, and the upstream homology arm was amplified using the primers upstream yghX-F / upstream yghX-R, and the downstream homology arm was amplified using the primers downstream yghX-F / downstream yghX-R. Using Gibbson assembly, the upstream homology arm, downstream homology arm, ectABC, xylR, and PxylA were assembled into the expression cassette of "ectABC upstream homology arm-xylR-PxylA- ectABC-downstream homology arm"; pGRB was used as a template and primers pGRB-F / pGRB-R were used to construct the gRNA expression vector pGRB-yghX; the "upstream homology arm of the expression frame of ectABC-xylR-PxylA- ectABC-downstream homology arm" was incubated with the gRNA expression vector pGRB-yghX and E. coli W3110 competent cells on ice for 30 minutes, and then electroporated at 1800v and coated on LB plates containing 50 mg / L Amp resistance and 50 mg / L spectacular enzyme, and single colony PCR was picked using xylA-F / ectABC-R as primers to verify the positive clones; the positive clones were cultured overnight at 37°C, 200rpm to lose the plasmid pREDCas9 to obtain the ectoine synthesis strain E. coli Ect.

[0054] Table 1 Primer sequences

[0055]

[0056] 3. Construction of recombinant engineered bacteria

[0057] 50 ng of the recombinant expression vector pET28a(+)-p2-22-tisB was incubated with the competent Escherichia coli engineered bacteria (E. coli Ect) for synthesizing ectoine on ice for 30 minutes, and then heat-shocked at 42°C for 90 seconds to obtain the recombinant engineered bacterial expression strain E. coli Ect pET28a(+)-p2-22-tisB.

[0058] 4. Cultivation of recombinant engineered bacteria

[0059] A single colony of the recombinant E. coli expression strain E. coli Ect pET28a(+)-p2-22-tisB was picked and inoculated into 5 mL LB medium (with kanamycin added at a final concentration of 50 mg / L), and cultured overnight at 37°C and 200 rpm to obtain the seed culture solution of the recombinant engineered bacteria.

[0060] 5. Multiple rounds of cultivation of recombinant engineered bacteria

[0061] Glycerol was added to the seed culture of the recombinant engineered bacteria as the first round. A small amount of bacterial solution was dipped into the inoculation loop and streaked onto an LB plate containing kanamycin as the second round. After incubation at 37°C for 12 hours, a small amount of colonies was dipped into the inoculation loop and transferred to a fresh kanamycin LB plate with kanamycin, and then incubated for another 12 hours to obtain the third round, and this cycle was repeated.

[0062] The recombinant strains from different rounds were transferred at a ratio of 1% (v / v) to 50 mL of fermentation medium (2 g / L yeast powder, 3 g / L potassium dihydrogen phosphate, 25 g / L sodium hydrogen phosphate dodecahydrate, 16.0 g / L ammonium sulfate, 1 g / L magnesium sulfate heptahydrate, 0.01 g / L manganese sulfate heptahydrate, 20 g / L glucose, and kanamycin with a final concentration of 50 mg / L). The culture was carried out at 37°C until the OD600nm was 0.6-0.8. Xylose with a final concentration of 20 g / L was added. After induction at 37°C for 48 h, the culture was centrifuged at 8000 rpm at 4°C for 5 min. The supernatant was collected and the ectoine production was measured.

[0063] The chromatographic conditions of the preparative HPLC were as follows: chromatographic column: Shim-pack GIST C18; mobile phase: 2% acetonitrile / water (v / v); detection wavelength: 210 nm; flow rate: 1 mL / min; column temperature: 35 o C. The elution time was 10 min, and the peak time of ectoine was 2.9 min ( Figure 2 ).

[0064] The results are as follows Figure 3As shown, in the first three rounds of fermentation, the production of ectoine was about 7.269 g / L, which was close to that of the control engineered bacteria (7.191 g / L). After the seventh round, the production of ectoine dropped sharply to 2.575 g / L. In the twentieth round of fermentation, the production of ectoine was zero.

[0065] In summary, the tisB toxin protein was transformed into Escherichia coli to construct an Escherichia coli engineered strain that can spontaneously inhibit the synthesis of ectoine after multiple rounds of culture and fermentation, while ensuring the normal synthesis of ectoine in the early stage. This method can be applied to the protection of industrial strains and biosafety protection.

[0066] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for regulating the synthesis of ectoine in Escherichia coli, characterized in that: The gene fragment of tisB toxin protein is transferred into the ectoine synthesis strain to obtain the engineered bacteria that spontaneously inhibits the synthesis of ectoine; The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.

1.

2. The method for regulating the synthesis of ectoine in Escherichia coli according to claim 1, characterized in that: include: 1) Construction of tisB protein expression vector; 2) The constructed tisB toxin protein expression vector was transformed into the ectoine-synthesizing strain E. coli Ect to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

3. The method for regulating the synthesis of ectoine in Escherichia coli according to claim 2, characterized in that: The tisB toxin protein expression vector is pET28a(+)-p2-22-tisB.

4. The method for regulating the synthesis of ectoine in Escherichia coli according to claim 3, characterized in that: The construction method of the tisB toxic protein expression vector pET28a(+)-p2-22-tisB is as follows: constructing the gene fragment of the tisB toxic protein into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; constructing the constitutive promoter 2-22 fragment into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB; The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1; The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.

2.

5. The method for regulating the synthesis of ectoine in Escherichia coli according to claim 2, characterized in that: The specific steps include: 1) The gene fragment of tisB toxic protein was constructed into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; the constitutive promoter 2-22 fragment was constructed into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB; The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1; The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2; 2) After incubating 50 ng of the recombinant expression vector pET28a(+)-p2-22-tisB with the competent strain of ectoine synthesis E. coli Ect on ice for 30 min, heat shock was performed at 42°C for 90 s to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

6. Use of the tisB toxin protein expression vector pET28a(+)-p2-22-tisB obtained in claim 5 in regulating the synthesis of ectoine in Escherichia coli.

7. The use of the tisB toxin protein expression vector pET28a(+)-p2-22-tisB according to claim 6 in regulating the synthesis of ectoine in Escherichia coli, characterized in that: The construction method of the tisB toxic protein expression vector pET28a(+)-p2-22-tisB is as follows: constructing the gene fragment of the tisB toxic protein into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; constructing the constitutive promoter 2-22 fragment into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB; The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1; The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.

2.

8. Use of the tisB toxin protein expression vector pET28a(+)-p2-22-tisB according to claim 6 in regulating the synthesis of ectoine in Escherichia coli, characterized in that: The specific steps include: 1) The gene fragment of tisB toxic protein was constructed into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-tisB; the constitutive promoter 2-22 fragment was constructed into the recombinant expression vector pET28a(+)-tisB to obtain the tisB toxic protein expression vector pET28a(+)-p2-22-tisB; The nucleotide sequence of the gene fragment of the tisB toxin protein is shown in SEQ ID NO.1; The nucleotide sequence of the constitutive promoter 2-22 fragment is shown in SEQ ID NO.2; 2) After incubating 50 ng of the recombinant expression vector pET28a(+)-p2-22-tisB with the competent strain of ectoine synthesis E. coli Ect on ice for 30 min, heat shock was performed at 42°C for 90 s to obtain the engineered bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits ectoine synthesis.

9. The use of the tisB toxin protein expression vector pET28a(+)-p2-22-tisB according to claim 8 in regulating the synthesis of ectoine in Escherichia coli, characterized in that: The engineered E. coli bacteria E. coli Ect pET28a(+)-p2-22-tisB that spontaneously inhibits the synthesis of ectoine stops synthesizing ectoine after multiple rounds of cyclic fermentation culture.

10. Use of the tisB toxin protein expression vector pET28a(+)-p2-22-tisB according to claim 9 in regulating the synthesis of ectoine in Escherichia coli, characterized in that: The multiple rounds of fermentation culture are ≥7 rounds of fermentation culture.

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