Novel escherichia coli host cell, and construction method therefor and use thereof

Abstract

Provided is a modified Escherichia coli host cell, the genome thereof comprising a T7 RNAP expression element, a mutated recA gene, a mutated fhuA gene, and a mutated ompT gene, wherein the T7 RNAP expression element comprises a promoter region and a T7 RNAP coding sequence, and optionally a selectable region between the promoter region and the T7 RNAP coding sequence. The strain modification strategy involved is simple and feasible, and the constructed Escherichia coli host cell has the following advantages: a clear genetic background, resistance to infection of an λDE3 phage, deletion of receptors required for infection of phages T1, T5, phi80, and UC-1, improved genetic stability, and better quality of an expressed recombinant protein.

Description

A novel Escherichia coli host cell, a construction method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of microbial biotechnology, and particularly relates to a novel Escherichia coli host cell, a construction method and application thereof. BACKGROUND

[0002] At present, BL21(DE3) and its related derivative strains integrated with T7 RNAP through lambda DE3 bacteriophage are widely used strains in the expression of recombinant proteins. However, the related strains are prone to lysis caused by bacteriophage pollution in the fermentation process, have poor genetic stability, and have low protein purity after purification.

[0003] The development of various gene editing technologies and the in-depth research on Escherichia coli make it possible to use the concept of synthetic biology to directionally modify strains. From the perspective of synthetic biology, in view of the problems of the above-mentioned BL21(DE3) and its related derivative strains, how to rationally design and integrate various excellent genetic shapes to modify more robust Escherichia coli host cells has important theoretical significance, and also has important industrial application value in the development of recombinant protein expression or other metabolic engineering fields. SUMMARY

[0004] In view of the above problems, the present application inserts T7 RNAP expression elements, modifies recombinase coding gene recA, bacteriophage receptor coding gene fhuA and outer membrane protease coding gene ompT at a specific position in the genome by RED recombination technology, and thus obtains a strain: clear genetic background, avoids the invasion of lambda DE3 bacteriophage genome, lacks the receptors required for the invasion of bacteriophages T1, T5, phi80 and UC-1, enhances the genetic stability of the strain, and the quality of the expressed recombinant protein is more excellent.

[0005] The first aspect of the present application provides a modified Escherichia coli host cell, the genome of which comprises a T7 RNAP expression element, a mutated recA gene, a mutated fhuA gene and a mutated ompT gene, wherein the T7 RNAP expression element comprises a promoter region and a T7 RNAP coding sequence, and optionally a selection region between the promoter region and the T7 RNAP coding sequence.

[0006] In some embodiments, the promoter region is a constitutive promoter or an inducible promoter.

[0007] In some preferred embodiments, the inducible promoter comprises PlacUV5, pTrc, P BAD , Pgap, Plac and Ptac.

[0008] In some embodiments, the T7 RNAP coding sequence is a DNA sequence capable of expressing a functional T7 RNAP or a variant thereof.

[0009] In some preferred embodiments, the T7 RNAP coding sequence is as shown in SEQ ID NO: 4.

[0010] In some embodiments, the addition, deletion, substitution or deletion of the optional region does not affect the function of the T7 RNAP expression element.

[0011] In some preferred embodiments, the sequence of the optional region is as shown in SEQ ID NO: 3.

[0012] In the present application, the T7 RNAP expression element inserted in the genome of the chassis strain by gene editing technology can be as shown in FIG. 1. For example, the sequence of the T7 RNAP expression element can be as shown in SEQ ID NO: 1. Among them, the sequence of the promoter region can be as shown in SEQ ID NO: 2, i.e. the Plac promoter; the sequence of the optional region can be as shown in SEQ ID NO: 3, which can be added, deleted, substituted or deleted without affecting the normal function of the T7 RNAP expression element; the T7 RNAP coding sequence can be as shown in SEQ ID NO: 4, which can also be equivalently replaced by any DNA sequence capable of expressing a functional T7 RNAP or a variant thereof.

[0013] In some embodiments, the T7 RNAP expression element is located in the recA gene region, the fhuA gene region, the ompT gene region, the non-essential region of the arabinose operon, the non-essential region of the lactose operon or the insertion repeat sequence region of the genome.

[0014] In the present application, the selection of the genomic integration site of the T7 RNAP expression element is a genomic location that does not affect or can improve the survival status or traits of E. coli.

[0015] In some embodiments, the mutation of the mutated recA gene includes insertion mutation, deletion mutation or substitution mutation of the coding region of the recA gene or the promoter region thereof.

[0016] In some embodiments, the recombinase activity of the mutated recA gene is lost.

[0017] In some embodiments, the mutation of the mutated fhuA gene includes insertion mutation, deletion mutation or substitution mutation of the coding region of the fhuA gene or the promoter region thereof.

[0018] In some embodiments, the product encoded by the mutated fhuA gene loses activity.

[0019] In some embodiments, the mutation of the mutated ompT gene comprises an insertion mutation, a deletion mutation or a substitution mutation in the coding region of the ompT gene or the promoter region thereof.

[0020] In some embodiments, the mutation results in mutations in the appY gene and / or the envY gene located in the upstream and downstream regions of the ompT gene.

[0021] In the present application, the mutation of the ompT gene at least results in the mutation of the ompT gene, and can also include mutations in the related genes in the upstream and downstream regions of the ompT gene that do not affect the survival state of E. coli, such as appY, envY, etc. In some embodiments, the OmpT outer membrane protease encoded by the mutated ompT gene loses activity.

[0022] In some embodiments, the E. coli host cell is an E. coli cell of the B series or the K series.

[0023] In some preferred embodiments, the E. coli host cell comprises E. coli B wild type, WA834, W3110, MG1655 and BW25113.

[0024] The second aspect of the present application provides a method for constructing an engineered E. coli host cell, comprising:

[0025] (1) introducing a T7 RNAP expression element into the genome of the E. coli host cell by gene editing technology; and

[0026] (2) mutating the recA gene, the fhuA gene and the ompT gene on the genome of the E. coli host cell by gene editing technology,

[0027] wherein the T7 RNAP expression element comprises a promoter region and a T7 RNAP coding sequence, and optionally an optional region located between the promoter region and the T7 RNAP coding sequence.

[0028] In some embodiments, the promoter region is a constitutive promoter or an inducible promoter.

[0029] In some preferred embodiments, the inducible promoter comprises PlacUV5, pTrc, P BAD , Pgap, Plac and Ptac.

[0030] In some embodiments, the T7 RNAP coding sequence is a DNA sequence capable of expressing a functional T7 RNAP or a variant thereof.

[0031] In some preferred embodiments, the T7 RNAP coding sequence is as set forth in SEQ ID NO: 4.

[0032] In some embodiments, the addition, substitution, or deletion of the optional region does not affect the function of the T7 RNAP expression element.

[0033] In some preferred embodiments, the sequence of the optional region is as set forth in SEQ ID NO: 3.

[0034] In some embodiments, the T7 RNAP expression element is introduced into the recA gene region, the fhuA gene region, the ompT gene region, a non-essential region of the arabinose operon, a non-essential region of the lactose operon, or an insertion repeat region of the genome.

[0035] In some embodiments, the mutation comprises an insertion mutation, a deletion mutation, or a substitution mutation in the coding region or the promoter region of the recA gene, the fhuA gene, and the ompT gene.

[0036] In some preferred embodiments, the mutation of the ompT gene results in a mutation of the appY gene and / or the envY gene located in the upstream and downstream regions of the ompT gene.

[0037] In some embodiments, the mutation results in the loss of activity of the products encoded by the recA gene, the fhuA gene, and the ompT gene.

[0038] In the present application, the recA gene encodes a recombinase, the fhuA gene encodes a phage receptor, and the ompT gene encodes an outer membrane protease.

[0039] In some embodiments, the E. coli host cell is an E. coli cell of the B series or the K series.

[0040] In some preferred embodiments, the E. coli host cell comprises E. coli B wild type, WA834, W3110, MG1655, and BW25113.

[0041] In some embodiments, the gene editing technology is a gene editing technology that does not rely on phage integration.

[0042] In the present application, the gene editing technology can be any known or emerging gene editing technology that does not rely on phage integration.

[0043] In some preferred embodiments, the gene editing technique is a lambda-Red recombination technique or a CRISPR-Cas editing system.

[0044] The third aspect of the present application provides an engineered E. coli host cell obtained according to the method as described herein.

[0045] The fourth aspect of the present application provides use of the engineered E. coli host cell as described herein in recombinant protein expression or other metabolic engineering.

[0046] The strain engineering strategy involved in the present application is simple and easy to implement, and the E. coli host cell constructed has the following advantages: clear genetic background, avoidance of infection by lambda DE3 phage, deletion of receptors required for infection by phages T1, T5, phi80 and UC-1, improved genetic stability, and higher quality of expressed recombinant proteins. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a schematic diagram of a T7 RNAP expression element.

[0048] Figure 2 is an example of an artificial construction element for inserting a T7 RNAP expression element.

[0049] Figure 3 is a schematic diagram of a lambda-Red recombination system.

[0050] Figure 4 is PCR verification of related strains after insertion of a Plac-driven T7 RNAP expression element into the ompT gene locus.

[0051] Figure 5 is PCR verification of related strains after mutation of the fhuA gene using a lambda-Red recombination.

[0052] Figure 6 is PCR verification of related strains after mutation of the recA gene using a lambda-Red recombination.

[0053] Figure 7 is PCR verification of E. coli B strain containing a P BAD driven T7 RNAP expression element.

[0054] Figure 8 is a comparison of the quality of TNF-alpha expressed by the host of Example 5 and BL21(DE3). DETAILED DESCRIPTION

[0055] The following illustrates the embodiments of the present application by specific examples, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein.

[0056] Insertion of T7 RNAP expression element: the T7 RNAP expression element (SEQ ID NO: 1) as shown in Figure 1 is integrated into a specific region of the E. coli genome using a specific gene editing technique.

[0057] The specific sequence of the T7 RNAP expression element can be as shown in SEQ ID NO: 1; the promoter region in the T7 RNAP expression element can be selected from common constitutive promoters or inducible promoters, including but not limited to PlacUV5, pTrc, P BAD , Pgap, Plac, Ptac, etc., taking only the Plac promoter (SEQ ID NO: 2) as an example; the optional region in the T7 RNAP expression element can be increased, replaced or deleted without affecting the normal function of the expression element, and the sequence of the optional region can be as shown in SEQ ID NO: 3; the T7 RNAP coding gene in the T7 RNAP expression element can be replaced by any DNA sequence that can express a functional T7 RNAP or a variant thereof, and the sequence of the T7 RNAP coding gene can be as shown in SEQ ID NO: 4.

[0058] For the selection of the insertion position of the T7 RNAP expression element in the genome, it can be any genome position that does not affect or can improve the survival state or traits of E. coli, such as the ompT, recA, fhuA region, arabinose or lactose operon region, or the insertion repeat sequence region on the genome, etc.

[0059] Mutation of the gene ompT: mutation of the ompT gene in the genome by gene editing technology.

[0060] The mutation of the ompT gene will eventually result in the loss of activity of its encoded product OmpT protease; the mutant forms include but are not limited to insertion mutation, deletion mutation, substitution mutation, etc. of the coding region or the promoter region of the ompT gene.

[0061] Mutation of the gene recA: mutation of the recA gene in the genome by gene editing technology.

[0062] The mutation of the recA gene will eventually result in the loss of activity of its encoded product RecA recombinase; the mutant forms include but are not limited to insertion mutation, deletion mutation, substitution mutation, etc. of the coding region or the promoter region of the recA gene.

[0063] Mutation of the gene fhuA: mutation of the fhuA gene in the genome by gene editing technology.

[0064] The mutation of the fhuA gene will eventually result in the loss of activity of its encoded product; the mutant forms include but are not limited to insertion mutation, deletion mutation, substitution mutation, etc. of the coding region or the promoter region of the fhuA gene.

[0065] Embodiment

[0066] The technical solutions of the present application are described in detail below in combination with examples. The reagents and biological materials used below are commercial products unless otherwise specified.

[0067] Example 1 Synthesis of artificial construct element for insertion of Plac promoter-driven T7 RNAP expression element in genome

[0068] This example provides the synthesis of an artificial construct element containing Plac promoter (SEQ ID NO: 5): As shown in Figure 2, Suzhou Jinyu Biological Technology Co., Ltd. was commissioned to synthesize an element containing Plac promoter-driven T7 RNAP expression element (SEQ ID NO: 1), and at the same time, a resistance fragment sequence of FRT-Apramycin-FRT (SEQ ID NO: 21) was added at the 5' end of the element through gene synthesis for positive clone screening in the λ-Red recombination process. The above artificial element synthesized by gene synthesis was connected to the cloning vector pUC57 of Suzhou Jinyu Biological Technology Co., Ltd., and the template plasmid pWX-FRT-lac-T7RNAP-Apra for fragment amplification and modification in the λ-Red recombination process was obtained.

[0069] Example 2 Synthesis of artificial construct element for insertion of P BAD promoter-driven T7 RNAP expression element in genome

[0070] This example provides the synthesis of an artificial construct element containing P BAD promoter (SEQ ID NO: 6): The Plac promoter region (SEQ ID NO: 2) of the artificial construct element (SEQ ID NO: 5) in Example 1 was replaced with the P BAD promoter (SEQ ID NO: 7) by gene synthesis, and the element was connected to the cloning vector pUC57 of Suzhou Jinyu Biological Technology Co., Ltd., and the template plasmid pWX-FRT-T7RNAP for fragment amplification and modification in the λ-Red recombination process was obtained.

[0071] Example 3 Insertion of Plac-driven T7 RNAP expression element and mutation of ompT gene in E. coli B and W3110

[0072] (1) According to the sequence of the ompT gene region, primers containing 50 bp homologous arms SEQ ID NO: 8 / SEQ ID NO: 9 were designed, and the fragment for modification containing the T7RNAP expression element (SEQ ID NO: 1) shown in Figure 2 was amplified using the plasmid pWX-FRT-lac-T7RNAP-Apra in Example 1 as the template.

[0073] (2) The above fragments were respectively electroporated into E. coli B or W3110 electroporation-competent cells containing plasmid pKD46 (purchased from HonorGene, which can be replaced by any equivalent plasmid containing the coding genes of Exo / Beta / Gams three RED recombination-related proteins), and coated on LB solid plates containing apramycin, and single colonies were obtained by inverted culture overnight.

[0074] (3) Strain PCR verification and PCR product sequencing were performed on the above clones using homology arm outside primers SEQ ID NO: 10 / SEQ ID NO: 11, and positive clones (strains after fragment recombination) were obtained.

[0075] (4) pCP20 (purchased from HonorGene, a plasmid for resistance removal, which can be replaced by any other equivalent plasmid containing the coding gene of FLP recombinase) was transformed into the above corresponding positive strains after fragment recombination, and single colonies were obtained by coating on LB solid plates containing chloramphenicol. The obtained single colonies were then coated on LB solid plates without antibiotics and incubated at 37°C / 42°C until single colonies grew. Strain PCR verification and PCR product sequencing were performed on the above clones using homology arm outside primers SEQ ID NO: 10 / SEQ ID NO: 11, and finally the positive strains with resistance fragments removed were obtained, i.e., strains with inserted T7 RNAP expression elements and ompT gene mutations (Figure 4).

[0076] Example 4: Mutating the fhuA gene of the E. coli B and W3110 derived strains obtained in Example 3

[0077] (1) Primers SEQ ID NO: 12 / SEQ ID NO: 13 containing 50 bp homology arms were designed according to the sequence of the fhuA gene region, and the resistance fragment FRT-apramycin-FRT without other elements for modification was amplified using the plasmid pWX-FRT-lac-T7RNAP-Apra in Example 1 as the template.

[0078] (2) The above resistance fragment was electroporated into the electroporation-competent cells prepared from the strains derived from Example 3 containing plasmid pKD46 (which can be replaced by any other equivalent plasmid), and coated on LB solid plates containing apramycin, and single colonies were obtained by inverted culture overnight.

[0079] (3) Strain PCR verification and PCR product sequencing were performed on the above clones using homology arm outside primers SEQ ID NO: 14 / SEQ ID NO: 15, and positive clones (strains after fragment recombination) were obtained.

[0080] (4) pCP20 (which can be replaced by any other equivalent plasmid) is transformed into the above strain, and the obtained single colony is then coated on an LB solid plate without antibiotics and incubated at 37°C / 42°C until a single colony grows. The above strain is verified by strain PCR using primers outside the homologous arms SEQ ID NO: 14 / SEQ ID NO: 15, and the PCR product is sequenced. Finally, a positive strain with the resistance fragment removed is obtained, which is a combined strain inserted with the T7 RNAP expression element, combined with the ompT gene mutation and the fhuA gene mutation (Figure 5).

[0081] Example 5 Mutating the recA gene of the E. coli B and W3110 derived strains obtained in Example 4

[0082] (1) The primers SEQ ID NO: 16 / SEQ ID NO: 17 containing 50 bp homologous arms are designed according to the sequence of the recA gene region, and the resistance fragment FRT- apramycin-FRT without other elements is amplified using the plasmid pWX-FRT-lac-T7RNAP-Apra in Example 1 as a template.

[0083] (2) The above resistance fragment is electroporated into the electrocompetent cells prepared from the strain derived from Example 4 containing the plasmid pKD46 (purchased from HonorGene, which can be replaced by any equivalent plasmid containing the three RED recombination related protein coding genes Exo / Beta / Gams), and coated on an LB solid plate containing apramycin, and single colonies are obtained by inverted culture overnight.

[0084] (3) The above strain is verified by strain PCR using primers outside the homologous arms SEQ ID NO: 18 / SEQ ID NO: 19, and the PCR product is sequenced to obtain a positive clone (strain after fragment recombination).

[0085] (4) pCP20 (which can be replaced by any other equivalent plasmid) is transformed into the above strain, and the obtained single colony is then coated on an LB solid plate without antibiotics and incubated at 37°C / 42°C until a single colony grows. The above strain is verified by strain PCR using primers outside the homologous arms SEQ ID NO: 14 / SEQ ID NO: 15, and the PCR product is sequenced. Finally, a positive strain with the resistance fragment removed is obtained, which is a combined strain inserted with the T7 RNAP expression element, combined with the ompT gene mutation, the fhuA gene mutation, and the recA gene mutation (Figure 6).

[0086] Example 6 P BADFinal strain construction with a promoter driving a T7 RNAP expression element

[0087] (1) Using the primers of SEQ ID NO: 8 / SEQ ID NO: 9 containing 50 bp homology arms in Example 3, the fragment for recombination containing the artificial construct element (SEQ ID NO: 6) was amplified from the plasmid pWX-FRT-T7RNAP in Example 2 as shown in Figure 2.

[0088] (2) Using the same procedure as in (2) to (4) in Example 3, the strain with the P BAD Strain with a promoter-driven T7 RNAP expression element and ompT gene mutation.

[0089] (3) Based on the strain in step (2), the same procedure in Example 4 was performed, i.e. the combined strain with the T7 RNAP expression element, combined ompT gene mutation and fhuA gene mutation was obtained.

[0090] (4) Based on the strain in step (3), the same procedure in Example 5 was continued, i.e. the final E. coli B strain with the P BAD T7 RNAP expression element driven by a promoter, combined ompT gene mutation, fhuA gene mutation, recA gene mutation (Figure 7).

[0091] Example 7 Quality comparison evaluation of TNF expression

[0092] (1) The coding sequence of TNF-a, SEQ ID NO: 20, was ligated into the pET28a expression vector after TTTAAGAAGGAGATATACC (SEQ ID NO: 22) by way of gene synthesis (performed by Suzhou Jinweizhi Biotechnology Co., Ltd.) to obtain the corresponding expression plasmid pET28aBRP-T7-TNF.

[0093] (2) The above plasmid was respectively transformed into BL21 (DE3) and the E. coli B and W3110 derived mutant strains obtained in Example 5, and positive clones were screened on LB solid plates containing kanamycin.

[0094] (3) The positive clones were respectively inoculated into 15 mL of LB liquid medium containing kanamycin, and cultured at 37°C, 220 rpm until the OD 600 When the OD reached 0.6-1.0, IPTG was added to a final concentration of 0.2 mM and expression was induced at 20°C for 16-20 h.

[0095] (4) For the sample after induction expression, a small sample was purified and subjected to CE-SDS detection. As can be seen from Figure 8, the quality of the recombinant protein expressed by the strain of the application is superior to that of BL21(DE3).

[0096] The above merely describes some preferred embodiments of the present application, and the present application is not limited to the contents of the embodiments. Any changes and modifications made by those skilled in the art within the concept of the technical solutions of the present application are within the protection scope of the present application.

Claims

1. A modified Escherichia coli host cell, characterized in that The genome of the E. coli host cell comprises a T7 RNAP expression element, a mutated recA gene, a mutated fhuA gene and a mutated ompT gene, The T7 RNAP expression element comprises a promoter region and a T7 RNAP coding sequence, and an optional region optionally located between the promoter region and the T7 RNAP coding sequence.

2. The modified Escherichia coli host cell according to claim 1, wherein The promoter region is a constitutive promoter or an inducible promoter.

3. The modified Escherichia coli host cell according to claim 2, wherein The inducible promoters include PlacUV5, pTrc, P BAD , Pgap, Plac and Ptac.

4. The modified Escherichia coli host cell according to claim 1, wherein The T7 RNAP coding sequence is a DNA sequence capable of expressing functional T7 RNAP or a variant thereof.

5. The modified Escherichia coli host cell according to claim 4, wherein The T7 RNAP coding sequence is shown in SEQ ID NO:

4.

6. The modified Escherichia coli host cell according to claim 1, wherein The addition, subtraction, replacement or deletion of the optional region does not affect the function of the T7 RNAP expression element.

7. The modified Escherichia coli host cell according to claim 6, wherein The sequence of the optional region is shown in SEQ ID NO:

3.

8. The modified Escherichia coli host cell according to claim 1, wherein The T7 RNAP expression element is located in the recA gene region, the fhuA gene region, the ompT gene region, the non-essential region of the arabinose operon, the non-essential region of the lactose operon or the inserted repeat sequence region of the genome.

9. The modified Escherichia coli host cell according to claim 1, wherein The mutation of the mutated recA gene includes an insertion mutation, a deletion mutation or a substitution mutation in the coding region or the promoter region of the recA gene.

10. The modified Escherichia coli host cell according to claim 9, wherein The recombinase activity encoded by the mutated recA gene is lost.

11. The modified Escherichia coli host cell according to claim 1, wherein The mutation of the mutated fhuA gene includes an insertion mutation, a deletion mutation or a substitution mutation in the coding region or the promoter region of the fhuA gene.

12. The modified Escherichia coli host cell according to claim 11, wherein The product encoded by the mutated fhuA gene loses activity.

13. The modified Escherichia coli host cell according to claim 1, wherein The mutation of the mutated ompT gene includes an insertion mutation, a deletion mutation or a substitution mutation in the coding region or the promoter region of the ompT gene.

14. The modified Escherichia coli host cell according to claim 13, wherein The mutation causes the appY gene and / or envY gene located in the upstream and downstream regions of the ompT gene to mutate.

15. The modified Escherichia coli host cell according to claim 13, wherein The OmpT outer membrane protease activity encoded by the mutated ompT gene is lost.

16. The modified Escherichia coli host cell according to any one of claims 1 to 15, wherein The Escherichia coli host cells are B series or K series Escherichia coli cells.

17. The modified Escherichia coli host cell according to claim 16, wherein The E. coli host cells include E. coliB wild type, WA834, W3110, MG1655 and BW25113.

18. A method for constructing a modified Escherichia coli host cell, characterized in that: The method comprises: (1) introducing a T7 RNAP expression element into the genome of the Escherichia coli host cell by gene editing technology; and (2) mutating the recA gene, fhuA gene, and ompT gene on the genome of the Escherichia coli host cell by gene editing technology, The T7 RNAP expression element comprises a promoter region and a T7 RNAP coding sequence, and an optional region optionally located between the promoter region and the T7 RNAP coding sequence.

19. The method according to claim 18, wherein The promoter region is a constitutive promoter or an inducible promoter.

20. The method according to claim 19, wherein The inducible promoters include PlacUV5, pTrc, P BAD , Pgap, Plac and Ptac.

21. The method of claim 18, wherein: The T7 RNAP coding sequence is a DNA sequence capable of expressing functional T7 RNAP or a variant thereof.

22. The method according to claim 21, wherein The T7 RNAP coding sequence is shown in SEQ ID NO:

4.

23. The method of claim 18, wherein: The addition, subtraction, replacement or deletion of the optional region does not affect the function of the T7 RNAP expression element.

24. The method according to claim 23, wherein The sequence of the optional region is shown in SEQ ID NO:

3.

25. The method of claim 18, wherein: The T7 RNAP expression element is introduced into the recA gene region, the fhuA gene region, the ompT gene region, the non-essential region of the arabinose operon, the non-essential region of the lactose operon or the insertion repeat sequence region of the genome.

26. The method of claim 18, wherein: The mutation includes an insertion mutation, a deletion mutation or a substitution mutation in the coding region or the promoter region of the recA gene, the fhuA gene and the ompT gene.

27. The method according to claim 26, wherein The mutation of the ompT gene may cause the appY gene and / or envY gene located in the upstream and downstream regions of the ompT gene to mutate.

28. The method of claim 18, wherein: The mutation causes the activities of the products encoded by the recA gene, the fhuA gene and the ompT gene to be lost.

29. The method of claim 18, wherein The Escherichia coli host cells are B series or K series Escherichia coli cells.

30. The method of claim 29, wherein: The E. coli host cells include E. coliB wild type, WA834, W3110, MG1655 and BW25113.

31. The method of claim 18, wherein The gene editing technology is a gene editing technology that does not rely on phage integration.

32. The method of claim 31, wherein The gene editing technology is λ-Red recombination technology or CRISPR-Cas editing system.

33. A modified E. coli host cell obtained according to the method of any one of claims 18-32.

34. Use of the modified Escherichia coli host cell according to any one of claims 1 to 17 or 33 in recombinant protein expression or other metabolic engineering.

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