Production-generation-controllable Escherichia coli engineering bacterium for hirudin and construction method and application of production-generation-controllable Escherichia coli engineering bacterium

Abstract

The invention discloses a hirudin escherichia coli engineering bacterium with controllable production generations as well as a construction method and application thereof, and relates to the technical field of biological medicines. The invention discloses a construction method of hirudin escherichia coli engineering bacteria with controllable production generations, which comprises the following steps: constructing a hokC protein gene as shown in the sequence of SEQ ID NO.1 and a constitutive promoter trxA as shown in the sequence of SEQ ID NO.2 into a recombinant expression vector pET28a (+)-ptrxA-hokC, and then transforming the recombinant expression vector into a hirudin synthetic strain E. coli Hirudin HV1-3 to obtain the hirudin escherichia coli engineering bacteria with controllable production generations. Through multiple rounds of culture, the hirudin yield of the engineering strain is reduced to 0 in the tenth round. The method is suitable for various hirudin types, and has the characteristics of reliable operation and high safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and more specifically, relates to an engineered strain of Escherichia coli with controllable generation of hirudin, its construction method and application. Background Technology

[0002] Sulfo-hirudin is the most potent thrombin inhibitor discovered to date and is widely used clinically for the treatment of cardiovascular diseases. Chinese invention patent ZL202111351835.8 describes the construction of a high-yield strain of *E. coli* that produces sulfo-hirudin, providing strong support for the industrial production of sulfo-hirudin.

[0003] The hokC toxin plays a crucial physiological role in bacteria, serving as a component of the toxin-antitoxin system. This system, widely present in bacterial and fungal cells, consists of a pair of small genetic control elements encoded by a toxin gene and an antitoxin gene. The toxin gene encodes a stable protein molecule, such as the hokC toxin, while the antitoxin gene encodes a less stable protein or a regulatory RNA. The specific function and mechanism of action of the hokC toxin are still under investigation. It is known that the toxin-antitoxin system participates in various bacterial physiological processes, including cell growth, division, and persistence. The hokC toxin may exert its toxic effects by influencing cell membrane permeability, interfering with intracellular metabolic processes, or interacting with other cellular components. Furthermore, research on the hokC toxin is significant for understanding bacterial pathogenic mechanisms, developing new antimicrobial drugs, and advancing biotechnological applications. In-depth research into the structure, function, and regulatory mechanisms of the hokC toxin can provide new insights and methods for the treatment and prevention of bacterial infections.

[0004] In the process of strain modification and metabolic optimization, using promoters of varying strengths to finely regulate the expression of target genes is a common strategy. However, inducible promoters require additional inducers as signals, which makes the control loop lack the ability to actively respond under inducer-free conditions, making it difficult for them to function actively after the engineered bacteria are leaked. Leaked engineered bacteria may survive and spread for a long time. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the technical problem to be solved by the present invention is to provide a method for constructing an engineered *Escherichia coli* strain capable of controlling the number of generations of hirudin production. Another technical problem to be solved by the present invention is to provide the engineered *Escherichia coli* strain capable of controlling the number of generations of hirudin production obtained by the aforementioned method. Finally, the technical problem to be solved by the present invention is to provide the application of the aforementioned engineered strain in hirudin synthesis.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for constructing an engineered strain of *Escherichia coli* capable of producing hirudin in a controlled manner includes: constructing a hokC toxin protein expression vector pET28a(+)-ptrxA-hokC, wherein the construction method of the hokC toxin protein expression vector pET28a(+)-ptrxA-hokC is as follows: constructing the hokC toxin protein gene fragment with the nucleotide sequence shown in SEQ ID NO.1 into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-hokC; constructing the constitutive promoter trxA fragment with the nucleotide sequence shown in SEQ ID NO.2 into the recombinant expression vector pET28a(+)-hokC to obtain the hokC toxin protein expression vector pET28a(+)-ptrxA-hokC; and transforming the constructed hokC toxin protein expression vector into a hirudin-producing strain to obtain an engineered strain of *Escherichia coli* capable of producing hirudin in a controlled manner.

[0008] In some embodiments, the hirudin-synthesizing strain is E. coli Hirudin HV1, E. coli Hirudin HV2, or E. coli Hirudin HV3.

[0009] In some embodiments, the method for constructing the hirudin-producing Escherichia coli engineered strain includes the following steps: incubating 50 ng of the recombinant expression vector pET28a(+)-ptrxA-hokC with competent cells of the hirudin-producing strain on ice for 30 min, followed by heat shock at 42°C for 90 s to obtain the hirudin-producing Escherichia coli engineered strain.

[0010] The hirudin-producing Escherichia coli engineered strain obtained by any of the construction methods described herein has a controllable production generation.

[0011] The application of the engineered Escherichia coli strain with controllable generation of hirudin in hirudin synthesis.

[0012] In some embodiments, the hirudin-producing Escherichia coli engineered strain, after 10 cycles of fermentation culture, reduces hirudin production to zero.

[0013] In some embodiments, the culture medium used in each round of fermentation contains kanamycin at a final concentration of 50 mg / L.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] This invention involves transferring the hokC protein gene into *E. coli*, constructing a recombinant expression vector containing the hokC toxin protein gene, and transforming it into a specific hirudin-synthesizing strain. After multiple rounds of culture, the hirudin production in *E. coli* can be reduced to zero by the 10th round of continuous culture, effectively controlling the hirudin synthesis process and adjusting its yield as needed. The constitutive promoter trxA fragment used eliminates the need for additional inducers, allowing for active response even in the absence of inducers, enhancing the convenience and autonomy of regulation, and making it more suitable for actual industrial production environments. This method is applicable to various hirudin-synthesizing strains (*E. coli* Hirudin HV1, HV2, HV3), possessing a certain degree of versatility and meeting the regulatory needs of different hirudin types, providing strong support for the production and management of hirudin in the field of biomedicine. Attached Figure Description

[0016] Figure 1 A schematic diagram of the hokC toxin expression vector pET28a(+)-ptrxA-hokC;

[0017] Figure 2 A diagram illustrating the construction process of the hirudin-synthesizing strain E. coli Hirudin HV1;

[0018] Figure 3 Production yield of hirudin-synthetic strain E. coli Hirudin HV1 after multiple fermentation cycles;

[0019] Figure 4 A diagram illustrating the construction process of the hirudin-synthesizing strain E. coli Hirudin HV2;

[0020] Figure 5 Figure showing the yield of hirudin synthesizing strain E. coli Hirudin HV2 after multiple fermentation cycles;

[0021] Figure 6 A diagram illustrating the construction process of the hirudin-synthesizing strain E. coli Hirudin HV3;

[0022] Figure 7 Figure showing the yield of hirudin synthesizing strain E. coli Hirudin HV3 after multiple fermentations. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0024] Example 1

[0025] 1. Constructing the hokC toxin protein expression vector

[0026] Primer sequences were designed based on the hokC toxin gene sequence (NCBI Reference Sequence: NC_000913.3) as follows:

[0027] hokC-F1:

[0028] 5'-GTTTAACTTTAAGAAGGAGATATACCATGAAGCAGCATAAGGCGATG-3' (SEQ ID NO.4),

[0029] hokC-R1:

[0030] 5'-GCTTTGTTAGCAGCCGGATCTTACTCGGATTCGTAAGCCGTG-3' (SEQ ID NO.5);

[0031] hokC-28a-F2:

[0032] 5'-GGTATATCTCCTTCTTAAAGTTAAAC-3' (SEQ ID NO.6),

[0033] hokC-28a-R2:

[0034] 5'-GATCCGGCTGCTAACAAAGC-3' (SEQ ID NO.7);

[0035] trxA-F3:

[0036] 5'-CGAGATCTCGATCCCGCGAAATGCGAAGTCGGAAAACTTCTGTTC-3' (SEQ ID NO.8),

[0037] trxA-R3:

[0038] 5'-CTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGATATAACTCCACAGGAATAAGCCTGGC-3' (SEQ ID NO. 9).

[0039] trxA-hokC-28a-F4:

[0040] 5'-ATTTCGCGGGATCGAGATCTCG-3' (SEQ ID NO. 10),

[0041] trxA-hokC-28a-R4:

[0042] 5'-CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG-3' (SEQ ID NO. 11).

[0043] The hokC toxin gene fragment (SEQ ID NO.1) was amplified using the high-fidelity PCR polymerase Prime Star with primers hokC-F1 / hokC-R1, and the pET28a(+) linear fragment was amplified using primers hokC-28a-F2 / hokC-28a-R2. After Gibbson assembly, the hokC and pET28a(+) fragments were homologously recombined to construct the recombinant expression vector pET28a(+)-hokC. Based on this, using *E. coli* MG1655 as a template, the constitutive promoter trxA fragment (SEQ ID NO.2) was amplified using primers trxA-F3 / trxA-R3, and the linear fragment pET28a(+)-hokC was amplified using primers trxA-hokC-28a-F4 / trxA-hokC-28a-R4. Following Gibbson assembly, homologous recombination integrated the trxA and pET28a(+)-hokC fragments to construct the recombinant expression vector pET28a(+)-ptrxA-hokC(SEQ ID NO.2). Figure 1 ).

[0044] 2. Constructing a hirudin-synthesizing strain (E. coli Hirudin HV1) through integrated expression.

[0045] Using the hirudin HV1 disclosed in the previous patent (Chinese Patent: ZL202111351835.8) as a template, the HV1 gene fragment was amplified using primers HV1-F / HV1-R; using the Escherichia coli genome W3110 (ATCC 27325) as a template, the strong promoter P1-16 (SEQ ID NO.3) of E. coli was amplified using primers P1-16-F1 / P1-16-R1; using the commercial vector pET28a(+) as a template, the T7 terminator T7ter was amplified using primers T7ter-F1 / T7ter-R1; using the E. coli genome W3110 (ATCC 27325) as a template, the upstream homologous arm was amplified using primers upstream yghX-F / upstream yghX-R; and using primers downstream yghX-F / downstream... The downstream homologous arm was amplified using yghX-R; using Gibbson assembly, the upstream homologous arm, downstream homologous arm, HV1, P1-16, and T7ter were assembled into the HV1 expression cassette “upstream homologous arm-P1-16-HV1-T7ter-downstream homologous arm”; using pGRB as a template, the expression vector pGRB-gRNA with guide sequence N20 was constructed using primers pGRB-F / pGRB-R; the HV1 expression cassette “upstream homologous arm-P1-16-HV1-T7ter-downstream homologous arm”, along with the gRNA expression vector pGRB-gRNA and E. coli W3110 pREDCas9 competent cells, were co-incubated on ice for 30 min, electrolyzed at 1800V, and then plated on LB agar plates containing 50 mg / L ampicillin and 50 mg / L spectinomycin. Single colony PCR was performed using primers P1-16 F1 / T7terR1 to verify positive clones. Successfully validated positive clones were inoculated into LB solid medium containing 0.1 mM IPTG (while maintaining 50 mg / L ampicillin and 50 mg / L spectinomycin) and cultured at 30°C for 12-20 hours to induce high-level expression of the homologous recombinase Red. Simultaneously, constitutively expressed Cas9 protein, guided by gRNA, recognizes the target site and cleaves it at the target site on the chromosome; the inducibly expressed homologous recombinase Red promotes homologous recombination repair at the target site, allowing the HV1 expression cassette "upstream homologous arm-P1-16-HV1-T7ter-downstream homologous arm" to integrate into the genome through the homologous arm, completing genome editing. Subsequently, the obtained positive transformants were picked and inoculated into liquid medium containing LB containing 50 mg / L ampicillin and cultured at 30°C for 1-2 hours until OD. 600=0.6~0.8, arabinose was added to a final concentration of 2 g / L to induce the expression of sgRNA that cleaves the pGRB-gRNA plasmid in the pREDCas9 plasmid, thus eliminating the pGRB-gRNA plasmid. The recombinant bacteria after eliminating the pGRB-gRNA plasmid were then inoculated into LB liquid medium and cultured overnight at 37°C without antibiotics to cause the loss of the temperature-sensitive helper plasmid pREDCas9. Finally, an engineered strain with a stable integration of hirudin expression units in its genome and without any exogenous helper plasmids was obtained, named E. coli HirudinHV1 (…). Figure 2 The primer sequences are shown below:

[0046] HV1-F:

[0047] 5'-ATGGTTGTTTACGACACCTGTACCGAG-3' (SEQ ID NO. 12),

[0048] HV1-R:

[0049] 5'-TTATTGCAAGTATTCTTCTGGAATTTCTTCGAAATC-3' (SEQ ID NO. 13);

[0050] P1-16-F1:

[0051] 5'-TTATGATAAGTTCACTGTTGGCTAAAGGG-3' (SEQ ID NO. 14),

[0052] P1-16-R1:

[0053] 5'-CTCGGTACAGGTGTCGTAAACAACCATGGTATATCTCCTTCTAAGTTCATTATGACTTACACTCCCTTTAGC-3' (SEQ ID NO. 15);

[0054] T7ter-F1:

[0055] 5'-GATTTCGAAGAAATTCCAGAAGAATACTTGCAATAACTAGCATAACCCCTTGGGGC-3' (SEQ IDNO.16),

[0056] T7ter-R1:

[0057] 5'-CAAAAAACCCCTCAAGACCCGTTTAG-3' (SEQ ID NO. 17);

[0058] upstream yghX-F:

[0059] 5‘-gctcaccggcaaccatgc-3’(SEQ ID NO.18),

[0060] upstream yghX-R:

[0061] 5‘-CCCTTTAGCCAACAGTGAACTTATCATAAtaggtttatctcttacgggattacgtcttaaac-3’(SEQ ID NO.19);

[0062] downstream yghX-F:

[0063] 5‘-CTAAACGGGTCTTGAGGGGTTTTTTGTAGTAATCCAGCAACTCTTGTGGG-3’(SEQ IDNO.20),

[0064] downstream yghX-R:

[0065] 5‘-ggctttggtcgaggctgg-3’(SEQ ID NO.21);

[0066] pGRB-F1:

[0067] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTgcggcagatttgtcataacggggTTTTTCGGTGATGACGGTGAAAACC-3’(SEQ ID NO.22),

[0068] pGRB-R1:

[0069] 5‘-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3’(SEQID NO.23);

[0070] pGRB-F2:

[0071] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTaggccagccctcgttgattcgggTTTTTCGGTGATGACGGTGAAAACC-3’(SEQ ID NO.24),

[0072] pGRB-R2:

[0073] 5'-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3' (SEQ ID NO. 25);

[0074] pGRB-F3:

[0075] 5'-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTatcttcgccacatcggcagtgggTTTTTCGGTGATGACGGTGAAAACC-3' (SEQ ID NO. 26),

[0076] pGRB-R3:

[0077] 5'-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3' (SEQ ID NO. 27).

[0078] 3. Constructing recombinant engineered bacteria

[0079] 50 ng of the recombinant expression vector pET28a(+)-ptrxA-hokC was co-incubated with competent E. coli Hirudin HV1 cells that synthesize hirudin on ice for 30 min, followed by heat shock at 42℃ for 90 s to obtain the recombinant engineered bacterial expression strain E. coli Hirudin HV1 pET28a(+)-ptrxA-hokC.

[0080] 4. Cultivation of recombinant engineered bacteria

[0081] A single colony of the recombinant Escherichia coli expression strain E. coli Hirudin HV1 pET28a(+)-ptrxA-hokC was picked and inoculated into 5 mL of LB medium (with kanamycin added to a final concentration of 50 mg / L). The culture was incubated overnight at 37°C and 200 rpm to obtain the seed culture of the recombinant engineered bacteria.

[0082] 5. Multiple rounds of culture of recombinant engineered bacteria

[0083] Glycerol was added to the seed culture medium of the recombinant engineered bacteria as the first round. A small amount of bacterial culture was streaked onto an LB plate containing kanamycin using an inoculation loop as the second round. After incubation at 37°C for 12 hours, a small amount of colonies was collected using an inoculation loop and transferred to a fresh kanamycin-containing LB plate, and incubated for another 12 hours to obtain the third round. This cycle was then repeated.

[0084] Recombinant strains from different rounds were transferred at a ratio of 1% (v / v) to 50 mL of fermentation medium (each liter of medium contained 23.6 g yeast extract, 11.8 g peptone, 9.4 g dipotassium hydrogen phosphate, 2.2 g potassium dihydrogen phosphate, and 40 g glucose, with kanamycin added to a final concentration of 50 mg / L). The culture was then incubated at 37°C. o C, cultured to OD 600 nm The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. (37) o C, after induction for 5 hours, 8000 rpm, 4 o Centrifuge at 400°C for 5 min to collect bacterial cells. Wash and resuspend the cells in pre-cooled Tris-HCl (pH 7.0) to 50 mL, then sonicate on ice under the following conditions: 400 W, 2 s on, 3 s off, 200 cycles until the bacterial solution is clear, 14000 rpm, 4 o Centrifuge at C for 20 min, collect the supernatant, which is the crude hirudin.

[0085] The fermentation medium formula is as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 1-8 mL glycerol.

[0086] The fermentation medium can also be formulated as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 20-100 g glucose.

[0087] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method for hirudin analysis were as follows: column: Inertsil ODS-3 4.6 × 250 mm; mobile phase: 0.065% aqueous solution of trifluoroacetic acid (A), 0.05% trifluoroacetic acid acetonitrile solution (B); detection wavelength: 220 nm; flow rate: 1 mL / min. The elution program was as follows: time (min) 0.01: pump A 95% + pump B 5%; time (min) 25.00: pump A 35% + pump B 65%; time (min) 25.01: pump A 35% + pump B 65%; time (min) 27.00: pump A 5% + pump B 95%; time (min) 27.01: pump A 95% + pump B 5%; time (min) 32.00: pump A 95% + pump B 5%.

[0088] The results are as follows Figure 3As shown, in the first three rounds of fermentation, the yield of hirudin in the recombinant strain E. coli Hirudin HV1 pET28a(+)-ptrxA-hokC was approximately 1.563-1.587 g / L, which was similar to that of the control engineered strain E. coli Hirudin HV1 (1.538-1.608 g / L). After the fifth round, the yield of hirudin in the recombinant strain dropped sharply to 0.324 g / L, and in the tenth round of fermentation, the yield of hirudin in the recombinant strain was zero.

[0089] Example 2

[0090] 1. Constructing the hokC toxin protein expression vector

[0091] Primer sequences were designed based on the hokC toxin gene sequence (NCBI Reference Sequence: NC_000913.3) as follows:

[0092] hokC-F1:

[0093] 5'-GTTTAACTTTAAGAAGGAGATATACCATGAAGCAGCATAAGGCGATG-3',

[0094] hokC-R1:

[0095] 5'-GCTTTGTTAGCAGCCGGATCTTACTCGGATTCGTAAGCCGTG-3';

[0096] hokC-28a-F2:

[0097] 5'-GGTATATCTCCTTCTTAAAGTTAAAC-3',

[0098] hokC-28a-R2:

[0099] 5'-GATCCGGCTGCTAACAAAGC-3';

[0100] trxA-F3:

[0101] 5'-CGAGATCTCGATCCCGCGAAATGCGAAGTCGGAAAACTTCTGTTC-3',

[0102] trxA-R3:

[0103] 5'-CTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGATATAACTCCACAGGAATAAGCCTGGC-3'.

[0104] trxA-hokC-28a-F4:

[0105] 5'-ATTTCGCGGGATCGAGATCTCG-3',

[0106] trxA-hokC-28a-R4:

[0107] 5'-CCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAG-3'.

[0108] The hokC toxin gene fragment (SEQ ID NO.1) was amplified using the high-fidelity PCR polymerase Prime Star with primers hokC-F1 / hokC-R1, and the pET28a(+) linear fragment was amplified using primers hokC-28a-F2 / hokC-28a-R2. After Gibbson assembly, the hokC and pET28a(+) fragments were homologously recombined to construct the recombinant expression vector pET28a(+)-hokC. Based on this, using *E. coli* MG1655 as a template, the constitutive promoter trxA fragment (SEQ ID NO.2) was amplified using primers trxA-F3 / trxA-R3, and the linear fragment pET28a(+)-hokC was amplified using primers trxA-hokC-28a-F4 / trxA-hokC-28a-R4. Following Gibbson assembly, homologous recombination integrated the trxA and pET28a(+)-hokC fragments to construct the recombinant expression vector pET28a(+)-ptrxA-hokC(SEQ ID NO.2). Figure 1 ).

[0109] 2. Construct a hirudin-synthesizing strain (E. coli Hirudin HV2) through integrated expression.

[0110] Using the hirudin HV2 disclosed in the previous patent (Chinese Patent: ZL202111351835.8) as a template, the HV2 gene fragment was amplified using primers HV2-F / HV2-R; using the Escherichia coli genome W3110 (ATCC 27325) as a template, the strong promoter P1-16 (SEQ ID NO.3) of E. coli was amplified using primers P1-16 F1 / P1-16 R2; using the commercial vector pET28a(+) as a template, the T7 terminator T7ter was amplified using primers T7ter F2 / T7ter R1; using the E. coli genome W3110 (ATCC 27325) as a template, the upstream homologous arm was amplified using primers upstream yghX-F / upstream yghX-R; and using primers downstream yghX-F / downstream... The downstream homologous arm was amplified using yghX-R; using Gibbson assembly, the upstream homologous arm, downstream homologous arm, HV2, P1-16, and T7ter were assembled into the HV2 expression cassette “upstream homologous arm-P1-16-HV2-T7ter-downstream homologous arm”; using pGRB as a template, the expression vector pGRB-gRNA with the guide sequence N20 was constructed using primers pGRB-F / pGRB-R; the HV2 expression cassette “upstream homologous arm-P1-16-HV2-T7ter-downstream homologous arm” was co-incubated with the gRNA expression vector pGRB-gRNA and E. coli W3110 pREDCas9 competent cells on ice for 30 min, followed by electroporation at 1800V and plating onto LB agar plates containing 50 mg / L ampicillin and 50 mg / L spectinomycin, and then plated with P1-16… Using F1 / T7terR1 as primers, single colonies were selected for PCR verification of positive clones. Successfully verified positive clones were inoculated into LB solid medium containing 0.1 mM IPTG (while maintaining 50 mg / L ampicillin and 50 mg / L spectinomycin) and cultured at 30°C for 12-20 hours to induce high-level expression of the homologous recombinase Red. Simultaneously, constitutively expressed Cas9 protein, guided by gRNA, recognizes the target site and cleaves it at the target site on the chromosome; the inducible expression of homologous recombinase Red promotes homologous recombination repair at the target site, allowing the HV2 expression cassette "upstream homologous arm-P1-16-HV2-T7ter-downstream homologous arm" to integrate into the genome through the homologous arm, completing genome editing. Subsequently, the obtained positive transformants were inoculated into liquid medium containing LB containing 50 mg / L ampicillin and cultured at 30°C for 1-2 hours until OD. 600=0.6~0.8, arabinose was added to a final concentration of 2 g / L to induce the expression of sgRNA that cleaves the pGRB-gRNA plasmid in the pREDCas9 plasmid, thus eliminating the pGRB-gRNA plasmid. The recombinant bacteria after eliminating the pGRB-gRNA plasmid were then inoculated into LB liquid medium and cultured overnight at 37°C without antibiotics to cause the loss of the temperature-sensitive helper plasmid pREDCas9. Finally, an engineered strain with a stable integration of hirudin expression units in its genome and without any exogenous helper plasmids was obtained, named E. coli HirudinHV2 ( Figure 4 The primer sequences are shown below:

[0111] HV2-F:

[0112] 5'-ATGATTACTTACACTGATTGTACTGAATCTGGTC-3' (SEQ ID NO. 28),

[0113] HV2-R:

[0114] 5'-TTATTGCAAGTATTCTTCTGGAATTTCTTCAAAATCTC-3' (SEQ ID NO. 29);

[0115] P1-16-F1:

[0116] 5'-TTATGATAAGTTCACTGTTGGCTAAAGGG-3',

[0117] P1-16-R2:

[0118] 5'-GACCAGATTCAGTACAATCAGTGTAAGTAATCATGGTATATCTCCTTCTAAGTTCATTATGACTTACACTCCCTTTAGC-3' (SEQ ID NO. 30);

[0119] T7ter-F2:

[0120] 5'-GAGATTTTGAAGAAATTCCAGAAGAATACTTGCAATAACTAGCATAACCCCTTGGGGC-3' (SEQ ID NO.31),

[0121] T7ter-R1:

[0122] 5'-CAAAAAACCCCTCAAGACCCGTTTAG-3';

[0123] upstream yghX-F:

[0124] 5‘-gctcaccggcaaccatgc-3’,

[0125] upstream yghX-R:

[0126] 5‘-CCCTTTAGCCAACAGTGAACTTATCATAAtaggtttatctcttacgggattacgtcttaaac-3’;

[0127] downstream yghX-F:

[0128] 5‘-CTAAACGGGTCTTGAGGGGTTTTTTGTAGTAATCCAGCAACTCTTGTGGG-3’,

[0129] downstream yghX-R:

[0130] 5‘-ggctttggtcgaggctgg-3’;

[0131] pGRB-F1:

[0132] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTgcggcagatttgtcataacggggTTTTTCGGTGATGACGGTGAAAACC-3’,

[0133] pGRB-R1:

[0134] 5‘-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3’;

[0135] pGRB-F2:

[0136] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTaggccagccctcgttgattcgggTTTTTCGGTGATGACGGTGAAAACC-3’,

[0137] pGRB-R2:

[0138] 5‘-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3’;

[0139] pGRB-F3:

[0140] 5'-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTatcttcgccacatcggcagtgggTTTTTCGGTGATGACGGTGAAAACC-3',

[0141] pGRB-R3:

[0142] 5'-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3'.

[0143] 3. Constructing recombinant engineered bacteria

[0144] 50 ng of the recombinant expression vector pET28a(+)-ptrxA-hokC was co-incubated with competent cells of E. coli Hirudin HV2, a strain that synthesizes hirudin, on ice for 30 min, followed by heat shock at 42℃ for 90 s to obtain the recombinant engineered bacterial expression strain E. coli Hirudin HV2 pET28a(+)-ptrxA-hokC.

[0145] 4. Cultivation of recombinant engineered bacteria

[0146] Single colonies of the recombinant Escherichia coli expression strain E. coli Hirudin HV2 pET28a(+)-ptrxA-hokC were picked and inoculated into 5 mL of LB medium (with kanamycin added to a final concentration of 50 mg / L). The culture was incubated overnight at 37°C and 200 rpm to obtain the seed culture of the recombinant engineered bacteria.

[0147] 5. Multiple rounds of culture of recombinant engineered bacteria

[0148] Glycerol was added to the seed culture medium of the recombinant engineered bacteria as the first round. A small amount of bacterial culture was streaked onto an LB plate containing kanamycin using an inoculation loop as the second round. After incubation at 37°C for 12 hours, a small amount of colonies was collected using an inoculation loop and transferred to a fresh kanamycin-containing LB plate, and incubated for another 12 hours to obtain the third round. This cycle was then repeated.

[0149] Recombinant strains from different rounds were transferred at a ratio of 1% (v / v) to 50 mL of fermentation medium (each liter of medium contained 23.6 g yeast extract, 11.8 g peptone, 9.4 g dipotassium hydrogen phosphate, 2.2 g potassium dihydrogen phosphate, and 60 g glucose, with kanamycin added to a final concentration of 50 mg / L). The culture was then incubated at 37°C. o C, cultured to OD 600 nm The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. (37)o C, after induction for 5 hours, 8000 rpm, 4 o Centrifuge at 400°C for 5 min to collect bacterial cells. Wash and resuspend the cells in pre-cooled Tris-HCl (pH 7.0) to 50 mL, then sonicate on ice under the following conditions: 400 W, 2 s on, 3 s off, 200 cycles until the bacterial solution is clear, 14000 rpm, 4 o Centrifuge at C for 20 min, collect the supernatant, which is the crude hirudin.

[0150] The fermentation medium formula is as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 1-8 mL glycerol.

[0151] The fermentation medium can also be formulated as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 20-100 g glucose.

[0152] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method for hirudin analysis were as follows: column: Inertsil ODS-3 4.6 × 250 mm; mobile phase: 0.065% aqueous solution of trifluoroacetic acid (A), 0.05% trifluoroacetic acid acetonitrile solution (B); detection wavelength: 220 nm; flow rate: 1 mL / min. The elution program was as follows: time (min) 0.01: pump A 95% + pump B 5%; time (min) 25.00: pump A 35% + pump B 65%; time (min) 25.01: pump A 35% + pump B 65%; time (min) 27.00: pump A 5% + pump B 95%; time (min) 27.01: pump A 95% + pump B 5%; time (min) 32.00: pump A 95% + pump B 5%.

[0153] The results are as follows Figure 5 As shown, in the first three rounds of fermentation, the yield of hirudin in the recombinant strain E. coli Hirudin HV2 pET28a(+)-ptrxA-hokC was approximately 1.785-1.804 g / L, which was similar to the hirudin yield in the control engineered strain E. coli Hirudin HV2 (1.765-1.836 g / L). After the fifth round, the yield of hirudin in the recombinant strain dropped sharply to 0.252 g / L, and in the tenth round of fermentation, the yield of hirudin in the recombinant strain was zero.

[0154] Example 3

[0155] 1. Constructing the hokC toxin protein expression vector

[0156] NCBI Reference Sequence: NCBI Reference Sequence: NC_000913.3

[0157] hokC-F1:

[0158] 5'-GTTTAACTTTAAGAAGGAGATACCATGAAGCAGCATAAGGCGATG-3',

[0159] hokC-R1:

[0160] 5'-GCTTTGTTAGCAGCCGGATCTTACTCGGATTCGTAAGCCGTG-3';

[0161] hokC-28a-F2:

[0162] 5'-GGTATATCTCCTTCTTAAAGTTAAAC-3',

[0163] hokC-28a-R2:

[0164] 5'-GATCCGGCTGCTAACAAAGC-3';

[0165] trxA-F3:

[0166] 5'-CGAGATCTCGATCCCGCGAAATGCGAAGTCGGAAAACTTCTGTTC-3',

[0167] trxA-R3:

[0168] 5'-CTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGATATAACTCCACAGGAATAAGCCTGGC-3'。

[0169] trxA-hokC-28a-F4:

[0170] 5'-ATTTCGCGGGATCGAGATCTCG-3',

[0171] trxA-hokC-28a-R4:

[0172] 5'-CCTCTAGAAATATTTTGTTTAACTTTAAGAAGGAG-3'。

[0173] The hokC toxin gene fragment (SEQ ID NO.1) was amplified using the high-fidelity PCR polymerase Prime Star with primers hokC-F1 / hokC-R1, and the pET28a(+) linear fragment was amplified using primers hokC-28a-F2 / hokC-28a-R2. After Gibbson assembly, the hokC and pET28a(+) fragments were homologously recombined to construct the recombinant expression vector pET28a(+)-hokC. Based on this, using *E. coli* MG1655 as a template, the constitutive promoter trxA fragment (SEQ ID NO.2) was amplified using primers trxA-F3 / trxA-R3, and the linear fragment pET28a(+)-hokC was amplified using primers trxA-hokC-28a-F4 / trxA-hokC-28a-R4. Following Gibbson assembly, homologous recombination integrated the trxA and pET28a(+)-hokC fragments to construct the recombinant expression vector pET28a(+)-ptrxA-hokC(SEQ ID NO.2). Figure 1 ).

[0174] 2. Constructing a hirudin-synthesizing strain (E. coli Hirudin HV3) through integrated expression.

[0175] Using the hirudin HV3 disclosed in the previous patent (Chinese Patent: ZL202111351835.8) as a template, the HV3 gene fragment was amplified using primers HV3-F / HV3-R; using the E. coli genome W3110 (ATCC 27325) as a template, the strong promoter P1-16 of E. coli was amplified using primers P1-16 F1 / P1-16 R3; using the commercial vector pET28a(+) as a template, the T7 terminator T7ter was amplified using primers T7ter F3 / T7ter R1; using the E. coli genome W3110 (ATCC 27325) as a template, the upstream homologous arm was amplified using primers upstream yghX-F / upstream yghX-R; and using primers downstream yghX-F / downstream... The downstream homologous arm was amplified using yghX-R; using Gibbson assembly, the upstream homologous arm, downstream homologous arm, HV3, P1-16, and T7ter were assembled into the HV3 expression cassette “upstream homologous arm-P1-16-HV3-T7ter-downstream homologous arm”; using pGRB as a template, the expression vector pGRB-gRNA with guide sequence N20 was constructed using primers pGRB-F / pGRB-R; the HV3 expression cassette “upstream homologous arm-P1-16-HV3-T7ter-downstream homologous arm”, along with the gRNA expression vector pGRB-gRNA and E. coli W3110 pREDCas9 competent cells, were co-incubated on ice for 30 min, electrolyzed at 1800V, and then plated on LB agar plates containing 50 mg / L ampicillin and 50 mg / L spectinomycin. Single colony PCR was performed using primers P1-16 F1 / T7ter R1 to verify positive clones. Successfully validated positive clones were inoculated into LB solid medium containing 0.1 mM IPTG (while maintaining 50 mg / L ampicillin and 50 mg / L spectinomycin) and cultured at 30°C for 12-20 hours to induce high-level expression of the homologous recombinase Red. Simultaneously, constitutively expressed Cas9 protein, guided by gRNA, recognizes the target site and cleaves it at the target site on the chromosome; the inducibly expressed homologous recombinase Red promotes homologous recombination repair at the target site, allowing the HV3 expression cassette "upstream homologous arm-P1-16-HV3-T7ter-downstream homologous arm" to integrate into the genome through the homologous arm, completing genome editing. Subsequently, the obtained positive transformants were picked and inoculated into liquid medium containing LB containing 50 mg / L ampicillin and cultured at 30°C for 1-2 hours until OD. 600=0.6~0.8, arabinose was added to a final concentration of 2 g / L to induce the expression of sgRNA that cleaves the pGRB-gRNA plasmid in the pREDCas9 plasmid, thus eliminating the pGRB-gRNA plasmid. The recombinant bacteria after eliminating the pGRB-gRNA plasmid were then inoculated into LB liquid medium and cultured overnight at 37°C without antibiotics to cause the loss of the temperature-sensitive helper plasmid pREDCas9. Finally, an engineered strain with a stable integration of hirudin expression units in its genome and without any exogenous helper plasmids was obtained, named E. coli Hirudin HV3. Figure 6 The primer sequences are shown below:

[0176] HV3-F:

[0177] 5'-ATGATTACCTACACTGACTGTACTGAGTCC-3' (SEQ ID NO. 32),

[0178] HV3-R:

[0179] 5'-TTAGTCGTAAGCGTCCTCTGG-3' (SEQ ID NO. 33);

[0180] P1-16-F1:

[0181] 5'-TTATGATAAGTTCACTGTTGGCTAAAGGG-3',

[0182] P1-16-R3:

[0183] 5'-GGACTCAGTACAGTCAGTGTAGGTAATCATGGTATATCTCCTTCTAAGTTCATTATGACTTACACTCCCTTTAGC-3' (SEQ ID NO. 34);

[0184] T7ter-F3:

[0185] 5'-CCAGAGGACGCTTACGACTAACTAGCATAACCCCTTGGGGC-3' (SEQ ID NO.35),

[0186] T7ter-R1:

[0187] 5'-CAAAAAACCCCTCAAGACCCGTTTAG-3';

[0188] upstream yghX-F:

[0189] 5‘-gctcaccggcaaccatgc-3’,

[0190] upstream yghX-R:

[0191] 5‘-CCCTTTAGCCAACAGTGAACTTATCATAAtaggtttatctcttacgggattacgtcttaaac-3’;

[0192] downstream yghX-F:

[0193] 5‘-CTAAACGGGTCTTGAGGGGTTTTTTGTAGTAATCCAGCAACTCTTGTGGG-3’,

[0194] downstream yghX-R:

[0195] 5‘-ggctttggtcgaggctgg-3’;

[0196] pGRB-F1:

[0197] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTgcggcagatttgtcataacggggTTTTTCGGTGATGACGGTGAAAACC-3’,

[0198] pGRB-R1:

[0199] 5‘-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3’;

[0200] pGRB-F2:

[0201] 5‘-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTaggccagccctcgttgattcgggTTTTTCGGTGATGACGGTGAAAACC-3’,

[0202] pGRB-R2:

[0203] 5‘-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3’;

[0204] pGRB-F3:

[0205] 5'-GACAGCTAGCTCAGTCCTAGGTATAATACTAGTatcttcgccacatcggcagtgggTTTTTCGGTGATGACGGTGAAAACC-3',

[0206] pGRB-R3:

[0207] 5'-GGTTTTCACCGTCATCACCGAAAAAACTAGTATTATACCTAGGACTGAGCTAGCTGTC-3'.

[0208] 3. Constructing recombinant engineered bacteria

[0209] 50 ng of the recombinant expression vector pET28a(+)-ptrxA-hokC was co-incubated with competent E. coli Hirudin HV3 strains that synthesize hirudin on ice for 30 min, followed by heat shock at 42℃ for 90 s to obtain the recombinant engineered bacterial expression strain E. coli Hirudin HV3 pET28a(+)-ptrxA-hokC.

[0210] 4. Cultivation of recombinant engineered bacteria

[0211] A single colony of the recombinant Escherichia coli expression strain E. coli Hirudin HV3 pET28a(+)-ptrxA-hokC was picked and inoculated into 5 mL of LB medium (with kanamycin added to a final concentration of 50 mg / L). The culture was incubated overnight at 37°C and 200 rpm to obtain the seed culture of the recombinant engineered bacteria.

[0212] 5. Multiple rounds of culture of recombinant engineered bacteria

[0213] Glycerol was added to the seed culture medium of the recombinant engineered bacteria as the first round. A small amount of bacterial culture was streaked onto an LB plate containing kanamycin using an inoculation loop as the second round. After incubation at 37°C for 12 hours, a small amount of colonies was collected using an inoculation loop and transferred to a fresh kanamycin-containing LB plate, and incubated for another 12 hours to obtain the third round. This cycle was then repeated.

[0214] Recombinant strains from different rounds were transferred at a rate of 1% (v / v) to 50 mL of fermentation medium (each liter of medium contained 23.6 g yeast extract, 11.8 g peptone, 9.4 g dipotassium hydrogen phosphate, 2.2 g potassium dihydrogen phosphate, and 4 mL glycerol, with a final concentration of 50 mg / L kanamycin added). The culture was incubated at 37°C. o C, cultured to OD 600 nm The concentration was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. 37 oC, after induction for 5 hours, 8000 rpm, 4 o Centrifuge at 400°C for 5 min to collect bacterial cells. Wash with pre-cooled Tris-HCl (pH 7.0), resuspend the cells in 50 mL, and sonicate on ice under the following conditions: 400 W, 2 s on, 3 s off, 200 cycles until the bacterial solution is clear, 14000 rpm, 4... o Centrifuge at C for 20 min, collect the supernatant, which is the crude hirudin.

[0215] The fermentation medium formula is as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 1-8 mL glycerol.

[0216] The fermentation medium can also be formulated as follows: each liter of medium contains 5-40 g yeast powder, 5-20 g peptone, 2-15 g dipotassium hydrogen phosphate, 0.5-6 g potassium dihydrogen phosphate and 20-100 g glucose.

[0217] The chromatographic conditions for the high-performance liquid chromatography (HPLC) method for hirudin analysis were as follows: column: Inertsil ODS-3 4.6 × 250 mm; mobile phase: 0.065% aqueous solution of trifluoroacetic acid (A), 0.05% trifluoroacetic acid acetonitrile solution (B); detection wavelength: 220 nm; flow rate: 1 mL / min. The elution program was as follows: time (min) 0.01: pump A 95% + pump B 5%; time (min) 25.00: pump A 35% + pump B 65%; time (min) 25.01: pump A 35% + pump B 65%; time (min) 27.00: pump A 5% + pump B 95%; time (min) 27.01: pump A 95% + pump B 5%; time (min) 32.00: pump A 95% + pump B 5%.

[0218] The results are as follows Figure 7 As shown, in the first three rounds of fermentation, the yield of hirudin HV3 in the recombinant engineered strain E. coli Hirudin HV3 pET28a(+)-ptrxA-hokC was approximately 1.437-1.572 g / L, which was similar to the yield of hirudin in the control engineered strain E. coli Hirudin HV3 (1.407-1.578 g / L). After the fifth round, the yield of hirudin in the recombinant engineered strain dropped sharply to 0.226 g / L, and in the tenth round of fermentation, the yield of hirudin in the recombinant engineered strain was zero.

[0219] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A method for constructing engineered Escherichia coli strains capable of producing hirudin with controllable generation rates, characterized in that, include: The hokC toxin protein expression vector pET28a(+)-ptrxA-hokC was constructed as follows: the hokC toxin protein gene fragment with the nucleotide sequence shown in SEQ ID NO.1 was constructed into the pET28a(+) vector to obtain the recombinant expression vector pET28a(+)-hokC; the constitutive promoter trxA fragment with the nucleotide sequence shown in SEQ ID NO.2 was constructed into the recombinant expression vector pET28a(+)-hokC to obtain the hokC toxin protein expression vector pET28a(+)-ptrxA-hokC; the constructed hokC toxin protein expression vector was transformed into a hirudin-producing strain to obtain an engineered Escherichia coli strain with controllable generation of hirudin production.

2. The method for constructing engineered Escherichia coli with controllable generation of hirudin production according to claim 1, characterized in that, The hirudin-producing strains are E. coli Hirudin HV1, E. coli Hirudin HV2, or E. coli Hirudin HV3.

3. The method for constructing engineered Escherichia coli with controllable generation of hirudin production according to claim 1, characterized in that, The specific steps include: incubating 50 ng of the recombinant expression vector pET28a(+)-ptrxA-hokC with competent cells of the hirudin-producing strain on ice for 30 min, followed by heat shock at 42℃ for 90 s to obtain engineered Escherichia coli strains with controllable generation of hirudin production.

4. The engineered Escherichia coli with controllable production generations of hirudin obtained by the construction method according to any one of claims 1-3.

5. The application of the engineered Escherichia coli strain with controllable generation of hirudin as described in claim 4 in the synthesis of hirudin.

6. The application according to claim 5, characterized in that, After 10 cycles of fermentation culture, the hirudin-producing Escherichia coli engineered strain with controllable production generations reduced hirudin production to zero.

7. The application according to claim 6, characterized in that, Each round of fermentation culture uses a medium containing kanamycin at a final concentration of 50 mg / L.

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