Bacteriophage-resistant bacterial strains

Abstract

The present invention provides bacterial strains with modifications in the tolC and / or fhuA genes and methods of introducing those modifications in bacterial cells. The modifications result in increased resistance of the bacterial strains against bacteriophages, such as the TLS phage, the T1 phage and / or the T5 phage.

Description

[0001] Bacteriophage-resistant bacterial strains

[0002] DESCRIPTION

[0003] 1 Field of the present invention

[0004] The present invention relates to bacterial strains which are resistant to bacteriophages due to mutations in the tolC gene and / or the fhuA gene. Moreover, the present invention provides methods of generating such bacteriophage-resistant strains.

[0005] 2 Background

[0006] Bacteriophage infections are a major risk for industrial biotechnological processes which are based on bacteria, such as Escherichia coli. Several surface proteins which mediate bacteriophage infections have been identified (Hantke (2020)). For some specific bacteriophages, even the specific binding sites which are necessary for the infection are known.

[0007] Although the risk of a bacteriophage infection has been known for a long time, there are up to now no bacterial strains which show high bacteriophage resistance without impaired growth. For example, the commonly used E. coli strain NEB5-alpha has a mutated fhuA gen which confers resistance against the Tl- and T5 bacteriophages but shows reduced growth which limits its usefulness for industrial application.

[0008] The present invention addresses this need by providing bacterial strains, in particular E. coli strains, which have carefully engineered surface proteins in order to inhibit bacteriophage infection without impairing cell growth and viability.

[0009] 3 Summary of the present invention

[0010] The present invention provides bacterial strains with a modified tolC gene which confers resistance against phages such as the TLS phage without impairing cell growth or viability. The modified tolC gene comprises mutations in the region from position S279 to N304 of the TolC protein which comprises a loop which is essential for TLS phage infection. By modifying TolC in this region, it is possible to specifically abolish phage infection without interfering with protein function which is important for cell viability under industrially relevant conditions. Moreover, the bacterial strains of the present invention can additionally harbor a modified fhuA gene which confers resistance against phages asTl and T5 phages. The modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene. Surprisingly, it was found that keeping those last 641 base pairs is beneficial for cell viability and thus superior to the removal of the complete open reading frame of the / huA gene, especially in combination with the tolC modifications described above.

[0011] The present invention provides the following exemplary embodiments:

[0012] 1. A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less.

[0013] 2. The bacterial strain of embodiment 1, wherein said modified tolC gene comprises a) mutations encoding for two or more of the amino acid substitutions selected from S279P, Y283D, G302D, and Q303P; and / or b) mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with an unstructured linker, such as a linker comprising only amino acids selected from glycine, serine, alanine and proline. 3. The bacterial strain of embodiment 1 or 2, wherein said modified tolC gene comprises mutations encoding for the amino acid substitutions S279P, Y283D, and G302D.

[0014] 4. The bacterial strain of embodiment 1 or 2, wherein said modified tolC gene comprises mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with a. a linker comprising only glycine, serine, and alanine, b. a linker comprising only glycine and serine, c. the amino acid sequence GAGSASGSAGSGAAGSGAGASAGGAA (SEQ ID NO: 5), d. an amino acid sequence having at least 80% identity to SEQ ID NO: 5, or e. a linker comprising only alanine.

[0015] 5. The bacterial strain of embodiment 4, wherein said modified tolC gene comprises mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with the amino acid sequence GAGSASGSAGSGAAGSGAGASAGGAA.

[0016] 6. The bacterial strain of any of the previous embodiments, wherein the bacterial strain further comprises a modified fhuA gene, wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0017] 7. The bacterial strain of any of embodiment 6, wherein said modified fhuA gene comprises at least the last 692 base pairs of the open reading frame of the fhuA gene. The bacterial strain of any of embodiments 6 and 7, wherein said modified fhuA gene does not result in the translation of a FhuA protein. The bacterial strain of any of the previous embodiments, wherein the bacterial strain is a gram-negative bacterial strain. The bacterial strain of any of the previous embodiments, wherein the bacterial strain is an E. coli strain. The bacterial strain of any of the previous embodiments, wherein the bacterial strain is more resistant to infection with bacteriophages, compared to E. coli reference strains such as E. coli K12 MG1655 and E. coli K12 W3110. The bacterial strain of any of the previous embodiments, wherein the bacterial strain is more resistant to infection with a TLS phage, a T1 phage, and / or a T5 phage, compared to E. coli reference strains such as E. coli K12 MG1655 and E. coli K12 W3110. The bacterial strain of any of the previous embodiments, wherein the bacterial strain has no impaired growth, compared to E. coli reference strains such as E. coli K12 MG1655 and E. coli K12 W3110. A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less and optionally further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0018] 15. The method of embodiment 14, wherein said method includes using a CRISPR system to modify the tolC gene and / or the fhuA gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0019] 4 Brief description of the drawings

[0020] Figure 1: Growth curves of E. coli strains with mutations in the tolC and / or fhuA genes. Tested were the reference Strains 0 and 1 and Strains 2, 3, 4, and 5. Strain 2 carries a triple mutation in the tolC gene, Strain 3 carries the mutation to / C-loop (TolC loop substituted for an unstructured linker), Strain 4 carries the deletion hfhuA 5' (NT) (deletion of the / huA open reading frame except of the last 692 base pairs) and Strain 5 carries both the mutation to / C-loop and the deletion hfhuA 5' (NT). Shown is the OD600 as parameter for cell density (on the left) and the change of OD600 by time as parameter for the growth rate. There were no major differences in the growth rate between all tested E. coli strains.

[0021] Figure 2: Exemplary plates from a phage resistance test for E. coli strains with mutations in the tolC and / or fhuA genes. Shown are the results of the parent Strain 1 (Fig. 2A) and Strain

[0022] 5 (Fig. 2B) which carries both the mutation to / C-loop and the deletion hfhuA 5' (NT), tested was resistance to the TLS phage. 6 plates with 6 different phage titers (indicated as potency of 10, i.e. "-5" means 10-5) are shown. While Strain 1 displays abundant plague formation at phage titers of 10-5and 10-6and even plague formation at lower titers, there is no plague formation at all for Strain 5, even at very high phage titers like 101and 10-2. The plates are representative for an experiment performed in triplicates. Figure 3: Summary of the phage sensitivity, as determined in example 3, for the Strains 0- 5. Shown is the efficiency of plating (EOP) for each tested bacterial strain and each phage. A low efficiency of plating means high resistance.

[0023] Figure 4: Growth of the E. coli tolC double mutant Y283D G302D. The reference strain "Strain 1" was compared to the E. coli tolC double mutant Y283D G302D, which carries a modified tolC gene encoding for the amino acid substitutions Y283D and G302D.

[0024] Figure 5: Graphical depiction of the sensitivity data from Table 8. The Figure shows resistance of the E. coli tolC double mutant Y283D G302D against the TLS phage.

[0025] 5 Detailed description of the present invention

[0026] 5.1 Definitions

[0027] Unless otherwise stated below, all terms used in this application, including the specification and claims, have the meaning usually given to them in the respective scientific field.

[0028] As used in the specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" include plural as well as singular referents unless the context clearly dictates otherwise.

[0029] "Wildtype" means a gene or protein sequence as found in naturally occurring reference strains, for E. coli e.g. in the strain K12 MG1655. For example, the sequence of the wildtype TolC protein is SEQ ID NO: 1 and the sequence of the wildtype FhuA protein is SEQ ID NO: 4. The sequence of the wildtype tolC gene open reading frame is SEQ ID NO: 31 and the sequence of the wildtype fhuA gene open reading frame is SEQ ID NO: 34.

[0030] A "modified" gene, as used herein, means a gene which comprises one or more mutations.

[0031] "Mutation", as used herein, means a difference in the gene compared to the respective wildtype gene (e.g. in the E. coli reference strain K12 MG1655). A mutation can be in the open reading frame or the promoter of a gene. Mutations can be substitutions, deletions, and / or insertions of one or more nucleotides. Mutations can lead to an altered amino acid sequence of the protein encoded by the gene comprising the mutations. Depending on the specific mutations, the encoded amino acid sequence can have amino acids substitutions, deletions and / or insertions compared to the amino acid sequence encoded by the wildtype gene. For example, mutations in the tolC gene can lead to amino acid amino acids substitutions, deletions and / or insertions compared to the wildtype TolC protein (SEQ ID NO: 1).

[0032] "Phage insensitive", "phage resistant" or "phage resistance", as used herein, means that a bacterial strain that is less sensitive, and preferably insensitive to infection and / or phage- mediated killing and / or phage-mediated growth inhibition. On the level of a single bacterial cell, "phage insensitive", "phage resistant" or "phage resistance" means that a bacterial cell is less likely to be infected, killed, inhibited in its growth, or to produce phage progeny. Assays for testing phage resistance, such as a double agar overlay assay, are well known in the art and described in the example section below. In some embodiments, increased phage insensitivity or increase phage resistance means that plaque formation in a double agar overlay assay is reduced.

[0033] "Bacteriophage", as used herein means a virus that infects and replicates within bacteria. The terms "bacteriophage" and "phage" are used interchangeably throughout this application.

[0034] "Unstructured Linker", as used herein, refers to an amino acid sequence that connects two specific amino acids within a protein without forming a particular secondary structure. In particular, a linker can connect amino acids 1278 and K305 of the TolC protein, thereby replacing amino acids 279 to 304 of the wild-type TolC protein. Typically, an unstructured linker comprises only amino acids selected from glycine, serine, alanine and proline.

[0035] A "GS-linker", as used herein, is a linker which comprises only glycine and serine. An alanine linker is a linker which comprises only alanine.

[0036] A "CRISPR system", as used herein, means a CRISPR protein, such as a Cas protein, and a compatible guide RNA (gRNA). The CRISPR protein and the gRNA of a CRISPR system can form a complex which is capable of modifying nucleic acids. There are multiple CRISPR systems known in the art. For example, a CRISPR system comprising the CRISPR protein MAD7 is described in Mund et al. (2023).

[0037] The expression that "the number of deletions is 5 or less" means that there are 5, 4, 3, 2, 1, or 0 deletions.

[0038] The term "wild type" refers to a known gene, protein, or organism as it occurs commonly in nature.

[0039] The term "substitution", as used herein in regard to protein sequences, refers to an exchange of an amino acid residue of the wild type sequence against another amino acid residue.

[0040] The term "N-terminal open reading frame of the fhuA gene", as used herein, means the open reading frame of the fhuA gene except of its last 600 base pairs, i.e. the last 600 base pairs of SEQ ID NO: 34.

[0041] The term "at least the last 641 base pairs of the open reading frame", as used herein, means that the last 641 base pairs and optionally further base pairs located 5' of said last 641 base pairs.

[0042] 5.2 Bacterial strains and methods of the present invention

[0043] 5.2.1 Bacterial strains

[0044] The present invention provides bacterial strains comprising a modified tolC gene. In some embodiments, the bacterial strain is a gram-negative bacterial strain. Examples of gramnegative bacteria include Salmonella, Escherichia, Shigella, Campylobacter, Fusobacterium, Bordetella, Pasteurella, Actinobacillus, Haemophilus and Histophilus. In some embodiments the bacterial strain is from the family of the Enterobacteriaceae, in particular a strain of the species Escherichia coli (E. coli).

[0045] Any suitable strain or genetic background of E. coli may be used in the present invention. Commonly used strains and genetic backgrounds of E. coli are well known in the art. Exemplary E. coli strains include, but are not limited to, laboratory strains such as K12 and its substrains (e.g., MG1655, Clifton wild type, W3110, DH5a, and the like), and E. coli B and its substrains (e.g., BL21, BL21(DE3), and the like).

[0046] 5.2.2 Bacteriophages

[0047] There are many different types of bacteriophages with varying mechanisms to attack bacteria. Bacteriophages can undergo lytic or lysogenic cycles within bacteria. During the lytic cycle, the bacteriophage genetic material is injected into a bacterium, where transcription, translation and replication take place, leading to the assembly and packaging of bacteriophage proteins and nucleic acids and eventually to lysis where many bacteriophages are released, ready to infect further bacteria. Some bacteriophages can also carry out a lysogenic cycle in which the bacteriophage genetic material is incorporated into a bacterial genome.

[0048] Bacteriophages which are particularly relevant for industrial production processes in E. coli include the Escherichia phage TolC and Lipopolysaccharide Specific (TLS) ("TLS phage", also known as Escherichia phage vB_EcoS_DanielBernoulli (Bas08)), the Escherichia phage T1 ("Tl phage", also known as Tunavirus Tl) and the Escherichia phage T5 ("T5 phage", also known as Tequintavirus T5). TLS phage infection is medicated by the host cell membrane protein TolC, while Tl and T5 phage infection is mediated by the host cell membrane protein FhuA.

[0049] The modifications of the tolC gene according to the present invention improve resistance against bacteriophages, in particular against the TLS phage. The modifications of the fhuA gene according to the present invention improve resistance against bacteriophages, in particular against the Tl phage, and / or the T5 phage.

[0050] The bacterial strains of the present invention can be completely resistant against one or more bacteriophages. Resistance can be determined by measuring the efficiency of plating, as known in the field and described in example 3 below. A strain is regarded as completely resistant if the ratio between its efficiency of plating and that of a reference strain which is suitable to phage infection is less than 0.00001 (IE-5). In one embodiment, the bacterial strain is completely resistant to the TLS phage. In one embodiment the bacterial strain is completely resistant to the TLS phage and the T1 phage. In one embodiment the bacterial strain is completely resistant to the TLS phage and the T5 phage. In one embodiment the bacterial strain is completely resistant to the TLS phage, the T1 phage, and the T5 phage.

[0051] The bacterial strains of the present invention can be more resistant against one or more bacteriophages compared to a reference strain, such as E. coli K12 MG1655 and E. coli K12 W3110. This increased resistance can be quantified by measuring the efficiency of plating, as known in the field and described in example 3 below. In some embodiments, the ratio between the efficiency of plating of a strain of the present invention is 0.1 or less, 0.01 or less, 0.001 or less, or 0.0001. That means that the bacterial strain of the present invention is at least 10-fold, at least 100-fold, at least 1000-fold, or at least 10,000-fold more resistant to the bacteriophage. In one embodiment, the bacterial strain is more resistant to the TLS phage. In one embodiment the bacterial strain is more resistant to the TLS phage and the T1 phage. In one embodiment the bacterial strain is more resistant to the TLS phage and the T5 phage. In one embodiment the bacterial strain is more resistant to the TLS phage, the T1 phage, and the T5 phage.

[0052] 5.2.3 TolC

[0053] TolC is a multifunctional outer membrane protein found in E. coli and other Gram-negative bacteria. Encoded by the tolC gene, this protein plays a crucial role in various cellular processes, primarily related to efflux systems and molecular transport. TolC forms a trimeric structure with a p-barrel in the outer membrane and a long a-helical barrel that extends into the periplasm. TolC homologs are found in many Gram-negative bacteria, indicating its evolutionary importance, with the structure and function of TolC being highly conserved across different bacterial species.

[0054] One of TolC's primary functions is its role as an essential component of several export ABC transporters, including AcrAD-TolC, MdtEF-TolC, EmrAB-TolC, and MacAB-TolC. These systems are responsible for exporting antibiotics, detergents, dyes, and other toxic compounds from the cell, contributing significantly to bacterial survival and multidrug resistance. TolC facilitates the export of various substrates, including antibiotics, heavy metals, and organic solvents. It was shown that loss or inactivation of TolC has a major impact on the physiology of enterobacteria and that enterobacteria require TolC for adaptation to their environment, such as acidic pH, metabolite concentrations, and stress conditions (Zgurskaya et al. (2011)). Thus, under industrially relevant conditions, it is important to that bacterial strains harbor a functional TolC protein.

[0055] In addition to its transport functions, TolC serves as the entry receptor for the TLS phage, as reported by German and Misra (2001). To interfere with this function as TLS phage entry receptor without losing the physiologically important functions of TolC, it is crucial to perform careful engineering of TolC instead of generating a complete TolC knock-out. The present invention provides such modified versions of TolC which still fulfill the protein functions which are crucial for the viability of the bacteria, but do not provide a binding site for the TLS phage and thus make the bacterial strains phage resistant. The important region for the interaction between TolC and the TLS phage is a loop between position S279 to position N304 of the TolC protein. It is known that single amino acid exchanges or deletion of a larger portion of the loop can impact TLS binding (see WO2021074182). However, the known single mutations do not provide complete resistance to the TLS phage, while a larger deletion impairs the function of the TolC protein and thus cell viability. The present invention overcomes those problems by introducing several amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 while keeping the overall protein structure intact because the number of deletions is limited to 5. In some embodiments, the number of deletions is 5 or less. In some embodiments, the number of deletions is 4 or less. In some embodiments, the number of deletions is 3 or less. In some embodiments, the number of deletions is 2 or less. In some embodiments, the number of deletions is 1 or less. In some embodiments, there is no deletion.

[0056] The sequence of the wild-type TolC protein of E. coli is represented by SEQ ID NO: 1. SEQ ID NO: 1 is encoded by the nucleotide sequence of SEQ ID NO: 31. In some embodiments, TolC is an E. coli TolC and the bacterial strain is an E. coli stain. The present invention provides bacterial strains with modified versions of the TolC protein, resulting from mutations in the tolC gene, such as SEQ ID NO: 2 and SEQ ID NO: 3. SEQ ID NO: 2 can be encoded e.g. by the nucleotide sequence of SEQ ID NO: 32. SEQ ID NO: 3 can be encoded e.g. by the nucleotide sequence of SEQ ID NO: 33. "to / C-loop" is the mutation in the tolC gene in which the amino acids between position 279 and position 304 of the wildtype TolC protein are substituted with the amino acid sequence GAGSASGSAGSGAAGSGAGASAGGAA (SEQ ID NO: 5).

[0057] In an embodiment, the modified tolC gene may comprise mutations encoding at least two amino acid substitutions selected from the group consisting of S279P, Y283D and G302D. For example, the modified tolC gene may comprise or consist of mutations encoding for the amino acid substitutions S279P, Y283D and G302D. Alternatively, the modified tolC gene may comprise or consist of mutations encoding the amino acid substitutions Y283D and G302D.

[0058] Unstructured linkers in the TolC loop

[0059] In some embodiments, the region from position S279 to position N304 is substituted with an unstructured linker. This abolishes TLS phage binding while keeping the spatial orientation in the protein intact and thus ensuring cell viability. The number of deletions of the linker compared to the wt TolC loop is 5 or less. As the wt TolC loop has 26 amino acids, this means that the linker has 21 or more amino acids.

[0060] In some embodiments, the linker has 21 amino acids. In some embodiments, the linker has 22 amino acids. In some embodiments, the linker has 23 amino acids. In some embodiments, the linker has 24 amino acids. In some embodiments, the linker has 25 amino acids. In some embodiments, the linker has 26 amino acids. In some embodiments, the linker has 27 amino acids. In some embodiments, the linker has 28 amino acids. In some embodiments, the linker has 29 amino acids. In some embodiments, the linker has 30 amino acids. In some embodiments, the linker has more than 30 amino acids.

[0061] In some embodiments, the linker has 21-30 amino acids. In some embodiments, the linker has 22-30 amino acids. In some embodiments, the linker has 23-30 amino acids. In some embodiments, the linker has 24-30 amino acids. In some embodiments, the linker has 25- 30 amino acids. In some embodiments, the linker has 26-30 amino acids. In some embodiments, the linker has 27-30 amino acids. In some embodiments, the linker has 24- 28 amino acids. In some embodiments, the linker has 25-27 amino acids.

[0062] In some embodiments, such linkers comprise only amino acids selected from glycine, serine, alanine, and proline. An example of an unstructured linker is a GSA-linker which comprises only glycine, serine, and alanine. Another example is a GS-linker which comprise only glycine and serine.

[0063] Table 1 indicates some possible unstructured linkers according to the present invention.

[0064] Table 1: Examples of unstructured linkers. In some embodiments, the unstructured linker has an amino acid sequence having at least 80% identity to one of the sequences in Table 1. In particular, the unstructured linker can have an amino acid sequence having at least 80% identity to SEQ ID NO: 5.

[0065] 5.2.4 fhuA

[0066] The ferric hydroxamate uptake (FhuA) protein, also known as ferrichrome outer membrane transporter / phage receptor, ferrichrome-iron receptor, or ferric hydroxamate receptor, is a protein encoded by the fhuA gene in E. coli and other Gram-negative bacteria. FhuA is a 714-amino acid protein that forms a p-barrel structure in the outer membrane. Its primary function is to serve as a receptor for ferrichrome, a hydroxamate siderophore, facilitating iron uptake in iron-limited environments.

[0067] FhuA is also involved in phage infection. It serves as the primary attachment receptor for T1 phage, T5 phage, 4>8O phage, and UC-1 phage.

[0068] It was found that a complete knock-out of fhuA confers resistance to T1 and T5 phage infection (citation). However, it also impairs bacterial growth and viability. In the present invention it was discovered that this effect is not due to the FhuA protein itself, which is dispensable at least under common industrial conditions, but due to an important effect of the 3' portion of the fhuA open reading frame for the neighboring genes fhuCDB. It was found that the negative consequences of the fhuA knock-out are prevented if at least the last (3') 600 base pairs, in particular even at least the last 641 base pairs, of the open reading frame of the / huA gene are still present. Thus, the present invention provides a knock-out of the fhuA gene which completely abolishes the production of the FhuA protein but preserves at least the last 641 base pairs of the open reading frame of the fhuA gene. This can e.g. be achieved by removing the complete fhuA open reading frame except of at least the last 641 bases, or by removing parts of the 5' region of the open reading frame except for at least the last 641 bases, or by removing at least parts of the promoter of the fhuA gene. Such a modified fhuA gene does not lead to the translation of a FhuA protein. In some embodiments, at least parts of the promoter of the fhuA gene are removed. There have also been approaches to introduce mutations in fhuA to (e.g. Endriss et al., (2003)) which are intended to abolish phage binding while preserving the FhuA protein function. The approach of the present invention has the advantage that the FhuA protein, which is not required under common industrial conditions, is not produced which saves resources of the bacterial cells. The present approach is also superior to an inactivating point mutation because it cannot spontaneously revert.

[0069] In some embodiments, the present invention provides a modified / huA gene, wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0070] In some embodiments, said modified fhuA gene comprises at least the last 600 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 610 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 620 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 630 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 640 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 650 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 660 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 670 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 680 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 690 base pairs of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 692 base pairs (SEQ ID NO: 35) of the open reading frame of the fhuA gene. In some embodiments, said modified fhuA gene comprises at least the last 700 base pairs of the open reading frame of the fhuA gene. The sequence of the wild-type FhuA protein of E. coli is represented by SEQ ID NO: 4. SEQ ID NO: 4 is encoded by SEQ ID NO: 34. In some embodiments, FhuA is an E. coli FhuA and the bacterial strain is an E. coli stain.

[0071] 5.2.5 Uses of the bacterial strains of the invention

[0072] The bacterial strains of the present invention, for example E. coli, Serratia, or Salmonella strains, in particular E. coli strains, can be used to produce a variety of molecules. Examples are proteins (such as recombinant proteins, enzymes, antibodies, antibody fragments, DARPINs, bacterial proteins), peptides (such as proinsulin or insulin), nucleic acids (such as plasmid DNA, RNA, mRNA, tRNA, rRNA, siRNA, gRNA), metabolites (such as amino acids, sugars, organic acids, vitamins, lipids, phospholipids, fatty acids, or alcohols), antibiotics, pigments, or synthetic polymers.

[0073] Examples for recombinant proteins which can be produced in bacterial strains comprise antibodies and fragments thereof, such as single-domain antibodies, scFvs, or Fab fragments; cytokines, such as interferons, interleukins; interleukin receptors, interleukin receptor antagonists; growth factors, such as G-CSF, GM-CSF, M-CSF, stem cell growth factors, tumor necrosis factors, growth hormones, insulin-like growth factors, fibroblast growth factors, platelet-derived growth factors, transforming growth factors, hepatocyte growth factors, bone morphogenetic factors, nerve growth factors, brain-derived neurotrophic factors (BDNF), glial cell line-derived neurotrophic factors; protein kinases, protein hormones, angiogenesis inhibitors, tissue plasminogen activators, blood coagulation factors, trypsin inhibitors, elastase inhibitors, complement constituents, hypoxia-induced stress proteins, proto-oncogenic products, transcription factors, virus- constitutive proteins, parathyroid hormone, prourokinase, erythropoietin, thrombopoietin, neurotrophin, glucocerebrosidase, superoxide dismutase, renin, lysozyme P450, prochymosin, lipocortin, reptin, serum albumin, streptokinase, tenecteplase, and cyclodextrin glycosyltransferases. Techniques for the production of such molecules, e.g. suitable plasmids and culture conditions, are known in the art. For example, for E. coli growth the preferred temperature ranges from about 20°C to about 39°C, in particular from about 25°C to about 37°C, in particular at about 30°C. The pH of the medium may be any suitable pH. For E. coli, a particularly suitable pH range is from about 5 to about 9, in particular from about 6.8 to about 7.4.

[0074] The present invention also provides the use of any of the bacterial strains as described below for the production of molecules as described below, for example for the production of proteins and nucleic acids.

[0075] 5.3 Methods of the invention

[0076] In one aspect, the present invention provides a method for generating a phage-resistant bacterial strain. The bacterial strain can be any of the strains described above in 5.2.1. The method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein. The modification in the tolC gene can result in any of the modified tolC genes and / or TolC proteins described above in 5.2.3.

[0077] The method optionally further comprises modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene. The modification in the / huA gene can result in any of the modified fhuA genes described above in 5.2.4.

[0078] The modification of the tolC and / or fhuA gene according to the present invention can be done by using any targeted genome editing technique, such as a CRISPR system, zinc finger nucleases, or TALENs. In particular, the modification of the tolC and / or fhuA gene can be done by using a CRISPR system. As defined above, such a CRIPSR system comprises a CRISPR protein and a compatible guide RNA (gRNA). Any CRISPR protein which is active in a bacterial cells is suitable for performing the modifications according to the present invention. Exemplary CRISPR proteins include Cas9, MAD7, Casl2a (Cpfl), Casl3, Cas3, Casl4, and Casl2b. In some embodiments, the CRISPR protein is Cas9 or MAD7. In some embodiments, the CRISPR protein is MAD7. Any gRNA which is capable of associating which suitable CRISPR protein and mediate a DNA modification by said CRIPSR protein is suitable for performing the modifications according to the present invention. The requirements for gRNAs of the respective CRISPR enzymes are known in the art. For example, a CRISPR system comprising the CRISPR protein MAD7 which is capable of modifying genes in E. coli is described in Mund et al. (2023).

[0079] In general, a method for modifying a gene in E. coli using a CRISPR system can comprise the following steps:

[0080] • Identifying one or more target sequences in the bacterial genome

[0081] • Ensuring that the target sequences are adjacent to a PAM (Protospacer Adjacent Motif) recognized by the CRISPR enzyme to be used, e.g. MAD7

[0082] • Designing one or more gRNAs complementary to the target sequence, typically around 20 nucleotides long

[0083] • Designing a suitable donor DNA sequence containing the desired genetic modification for gene insertion or replacement in the tolC and / or fhuA gene.

[0084] • Preparing one or more plasmids encoding the MAD7 gene and the gRNA(s)

[0085] • Preparing the donor DNA or a plasmid encoding the donor DNA

[0086] • Providing competent bacterial cells (e.g. E. coli)

[0087] • Introducing the plasmid(s) encoding the CRISPR enzyme and the gRNA(s) as well as the donor DNA into the cells using an appropriate method (e.g., electroporation, heat shock).

[0088] • Allowing the transformed bacteria to recover and express the CRISPR components.

[0089] • Allowing the CRISPR enzyme (e.g. MAD7 nuclease), guided by the gRNA(s), to create a double-strand break at the target site(s) • Allowing the homology-directed repair (HDR) pathway to incorporate the donor DNA at the break site.

[0090] • Optionally selecting modified bacteria, for example antibiotic selection if a resistance marker was included

[0091] • Screening colonies for the desired modification, e.g. using PCR, DNA sequencing, or phenotypic assays to identify successfully modified bacteria.

[0092] • Optionally, verifying the absence of off-target modifications in other parts of the genome and removal of plasmids encoding the components of the CRISPR system components, e.g. by curing the bacteria from the plasmids

[0093] In some embodiments, the method includes using a CRISPR system, wherein the method comprises one or more of the above steps. In some embodiments, the method includes using a CRISPR system, wherein the method comprises all of the above steps.

[0094] In some embodiments, the method includes using a CRISPR system, wherein the CRISPR system comprises a MAD7 nuclease, and wherein the method comprises one or more of the above steps. In some embodiments, the method includes using a CRISPR system, wherein the CRISPR system comprises a MAD7 nuclease, and wherein the method comprises all of the above steps.

[0095] Instead of introducing plasmids into the bacterial cells, it is also possible to prepare the MAD7 nuclease and the gRNA in a cell-free system and to introduce them in the bacterial cells.

[0096] In some embodiments, the bacterial strain of the methods of the present invention is a gram-negative bacterial strain. In some embodiments, the bacterial strain of the methods of the present invention is an E. coli strain.

[0097] 5.4 Embodiments of the invention

[0098] The following exemplary embodiments of the present invention are given for illustrative purposes. E. coli

[0099] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein the bacterial strain is E. coli.

[0100] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein the bacterial strain is E. coli K12. tolC modifications and resistance

[0101] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein the bacterial strain is E. coli.

[0102] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein the bacterial strain is E. coli.

[0103] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for four amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein the bacterial strain is E. coli.

[0104] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein the bacterial strain is E. coli. A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein the bacterial strain is E. coli.

[0105] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein the bacterial strain is E. coli.

[0106] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0107] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0108] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110. A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0109] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0110] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0111] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 1000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or

[0112] E. coli K12 W3110.

[0113] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 1000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0114] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 1000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0115] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 1000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0116] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 1000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0117] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 10,000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110. A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 10,000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0118] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 10,000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0119] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 10,000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0120] A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is E. coli, and wherein the strain exhibits at least 10,000-fold increased resistance against TLS phage compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0121] Combination with fhuA

[0122] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0123] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0124] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0125] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

[0126] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene. A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

[0127] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

[0128] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

[0129] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

[0130] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

[0131] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0132] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0133] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli

[0134] K12 W3110.

[0135] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 641 base pairs of the open reading frame of the / huA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0136] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0137] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0138] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the / huA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0139] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0140] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110. A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0141] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0142] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110. A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 692 base pairs of the open reading frame of the / huA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0143] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0144] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage and T1 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12

[0145] W3110. A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified to / Cgene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0146] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for at least three amino acid substitutions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, and wherein said modified / huA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0147] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110. A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 692 base pairs of the open reading frame of the / huA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0148] A bacterial strain comprising a modified tolC gene and a modified fhuA gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is E. coli, and wherein the strain exhibits complete resistance against TLS phage, T1 phage, and T5 phage with an efficiency of plating ratio < 0.00001 compared to the reference strains E. coli K12 MG1655 and / or E. coli K12 W3110.

[0149] Methods

[0150] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is an E. coli strain.

[0151] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0152] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at three two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is an E. coli strain.

[0153] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least three amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0154] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain.

[0155] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 24-30 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0156] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain.

[0157] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 25-27 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0158] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain.

[0159] A method for generating a phage-resistant bacterial strain, wherein the method comprises modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease. A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain.

[0160] A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 692 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain.

[0161] A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease. A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least three amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain.

[0162] A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least three amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0163] A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain. A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for an unstructured linker of 26 amino acids replacing the region from position S279 to position N304 of the TolC wildtype protein, and further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene, wherein the bacterial strain is an E. coli strain, and wherein said method includes using a CRISPR system to modify the tolC gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

[0164] 5.5 Further aspects of the invention

[0165] In another aspect, the invention provides a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the tolC gene for CRISPR system-mediated genome editing. The invention also provides a combination of a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the to / C gene for CRISPR system-mediated genome editing and a nucleic acid vector encoding a CRISPR enzyme, such as MAD7. The invention also provides a combination of a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the to / C gene for CRISPR system-mediated genome editing, a nucleic acid vector encoding a CRISPR enzyme, such as MAD7, and a recombination template.

[0166] In another aspect, the invention provides a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the fhuA gene for CRISPR system-mediated genome editing. The invention also provides a combination of a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the fhuA gene for CRISPR system-mediated genome editing and a nucleic acid vector encoding a CRISPR enzyme, such as MAD7. The invention also provides a combination of a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the fhuA gene for CRISPR system-mediated genome editing, a nucleic acid vector encoding a CRISPR enzyme, such as MAD7, and a recombination template.

[0167] In another aspect, the present invention provides a recombinant vector comprising a nucleic acid molecule encoding a modified TolC protein, wherein said nucleic acid molecule comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region corresponding to position S279 to position N304 of the TolC protein, compared to the wildtype tolC gene, wherein the number of deletions is 5 or less.

[0168] In another aspect, the invention provides a kit for improving bacteriophage resistance in bacterial strains, such as E. coli, comprising: a) a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the tolC gene for CRISPR system-mediated genome editing; b) a nucleic acid vector encoding a CRISPR enzyme, such as MAD7; c) optionally a recombination template; and d) instructions for use.

[0169] In another aspect, the invention provides the use of the nucleic acid vectors or kit as described above for improving bacteriophage resistance in a bacterial strain.

[0170] In another aspect, the invention provides a kit for improving bacteriophage resistance in bacterial strains, such as E. coli, comprising: a) a nucleic acid vector comprising a nucleic acid sequence encoding a guide RNA targeting the / huA gene for CRISPR system-mediated genome editing; b) a nucleic acid vector encoding a CRISPR enzyme, such as MAD7; c) optionally a recombination template; and d) instructions for use.

[0171] In another aspect, the invention provides the use of the nucleic acid vectors or kit as described above for improving bacteriophage resistance in a bacterial strain.

[0172] 5.6 Industrial applicability

[0173] The bacterial strains and methods of the present invention may be for example used in the commercial production of a variety of molecules, such as therapeutic proteins, for example insulin or antibodies, or in the production of nucleic acids, such as DNA or RNA. 6 Examples

[0174] 6.1 Example 1: Generation of E. coli strains with tolC and fhuA mutations

[0175] To improve the resistance of E. coli strains against the TLS, Tl, and T5 bacteriophages, their tolC and / or fhuA genes were modified as described below.

[0176] 6.1.1 Methods

[0177] Genome editing of E. coli was performed using the CRISPR-MAD7 toolkit for E. coli previously developed and published (Mund et al., (2023)). In brief, electrocompetent cells of the strain were transformed with the pRED-MAD7 plasmid by electroporation (2 mm cuvette, 2.5 kV, 25 pF, 200 Q) and a single clone was selected from LB-Agar containing 100 pg / mL ampicillin. The clone was cultured overnight in 5 mL SOB medium (0.5 % yeast extract, 2 % Phytone, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCl2, 10 mM MgSO4) containing 100 pg / mL ampicillin (SOB-Amp) at 30 °C and 220 rpm in a 14 mL round bottom tube. lOOmL SOB-Amp were inoculated to an OD600 of 0.05 with the overnight culture and incubated at 30°C and 220rpm. When the culture reached an OD600 of 0.3-0.4, X-RED expression was induced by addition of 6.5 mL l M arabinose solution for 45 min. The culture was cooled for 10 min on ice. Cells were pelleted by centrifugation at 3000xg ref and 4°C for 10 min and washed three times with sterile 4°C cold H2O. Cells were then washed in 2 mL cold 10% glycerol and resuspended in 0.8 mL 10% glycerol, of which 50 pL aliquots were stored at -80°C.

[0178] For each genome edit, the respective the gene-specific gRNA expression plasmid (pGRNA) and donor DNA were designed and generated as previously described (Mund et al., (2023)). In brief for each desired edit, a single guide RNA (gRNA) target sequence was identified close to the desired edit (typically <20 nt) or within the deleted region and 3' of a 5'-YTTN- 3' PAM. All target sequences were designed based on the genome sequence of E. coli MG1655 as reference (Blattner et al., (1997)). gRNA and donor DNA were chosen and designed such that MAD7 no longer cleaves the genomic DNA at the edited locus. This was ensured by a design such that the PAM was mutated and / or the protospacer was modified at more than 7 positions using synonymous mutations, with a special focus on the seed region.

[0179] For cloning of the pGRNAs, the target sequence was then synthesized as oligonucleotides. The respective forward and reverse oligonucleotides (10 pmol each) were hybridized in 50 pL annealing buffer (50mM NaCI, lOmM Tris, ImM EDTA) by heating to 95 °C for 5 min followed by 0.1°C / s cooling to 4°C in a thermocycler. 1 pL of doublestrand-oligonucleotide solution was ligated into 50 ng Bsal-digested pGRNA- sacB-ccdB (Mund et al., (2023)) using T4 DNA Quick Ligase (New England Biolabs). Plasmid sequences were confirmed by Sanger sequencing.

[0180] Donor DNA for deletions was synthesized as 100 bp-long single stranded oligonucleotide (50 bp upstream and downstream of deleted region, respectively). Donor DNA for the introduction of single, multiple or stretches of point mutations, was obtained using benchtop DNA assembly (BioXP, Telesis Bio) of 1000 kb double-stranded DNA (500 bp upstream and downstream of deleted region, respectively).

[0181] For CRISPR-MAD7 genome editing, 250 ng pGRNA and donor DNA were co-transformed by electroporation (2 mm cuvette, 2.5 kV, 25 pF, 200 Q), and cells were recovered in 1 mL SOC medium (New England Biolabs, #B9020) at 30 °C and for 90 min. Cells were pelleted again, resuspended in 2 mL LB containing 100 pg / mL ampicillin and 50 pg / mL kanamycin and incubated at 30 °C 220 rpm in a 14 mL round bottom tube for 2 h. 20 pL and 200 pL of the culture were plated on LB-Agar containing 100 pg / mL ampicillin and 50 pg / mL kanamycin and plates were incubated for >24 h at 30 °C.

[0182] Successful editing was confirmed by amplification of the edited locus by colony PCR and subsequent Sanger sequencing.

[0183] To cure cells from pGRNA, cells were singled out on LB-Agar containing lOOg / L sucrose and

[0184] 100 pg / mL ampicillin and incubated at 30 °C for >24 h. Single colonies were sequentially replicated on LB-agar plates containing either ampicillin (lOOpg / mL), kanamycin (50pg / mL) or no antibiotic (as control). Plates were incubated overnight at 30°C, and plasmid presence was scored by growth on the respective antibiotic. To cure pRED-MAD7, cells were singled out on LB-Agar and grown overnight at 37 °C. Single colonies were sequentially replicated on LB-agar plates containing either ampicillin (100 pg / mL) or no antibiotic. Plates were incubated overnight at 37 °C, and presence of pRED-MAD7 was scored by growth on LB- ampicillin.

[0185] The final, plasmid-free genome-edited strain was serially streaked twice to ensure clonality. A single colony was then used to inoculate a 20 mL culture in 100 mL Eppendorf shake flasks in LB media (vegetal, product number) and cultivated to an OD600 of 0.8 - 1.5. The culture was then added to an equal volume of 50 % glycerol and frozen as cryostocks for further use.

[0186] Table 2 provides an overview of the used gRNA and donor sequences.

[0187] Table 2: gRNA and donor sequences

[0188] 6.1.2 Results

[0189] Table 3 provides an overview of the E. coli strains generated in this study. Strains 2 to 5 were generated starting from strain 1 using the methods described above.

[0190] Table 3: List of E. coli strains

[0191] 6.2 Example 2: Growth rate determination

[0192] To assess cell viability, the growth rate of the E. coli Strains 3, 4, and 5 was compared with the parent Strain 1 (which comprises a wild type tolC andfhuA genes).

[0193] 6.2.1 Methods

[0194] For cell growth analysis, 50 mL media in 300 mL Eppendorf shake flasks were inoculated with the respective E. coli strain. Growth was quantified in complex LB media and minimal media (M9 minimal media). Cell growth was monitored by online backscatter measurements (CellGrowthQuantifier, ScientificBioprocessing), followed by normalization of the data to offline OD600 measurements at the end of cultivation. Strain growth was regarded unimpaired if the maximum growth speed (biomass change in OD600 per hour) during exponential growth as well as final OD600 deviated less than 10 % from the unedited reference Strains 0 and 1.

[0195] 6.2.2 Results

[0196] The results of the growth rate determination are shown in Fig. 1. It was found that Strains 2, 3, 4, and 5 did not show impaired growth speed, indicating that the introduced mutations do not reduce cell growth and viability.

[0197] 6.3 Example 3: Bacteriophage resistance test

[0198] The effect of the modifications in the tolC and fhuA genes was assessed by testing the resistance of the strains against the bacteriophages TLS, T1 and T5. 6.3.1 Methods

[0199] To test the effect of the above mutations on phage resistance, the E. coli strains harboring the above mutations were test for resistance against the following bacteriophages:

[0200] - Escherichia phage vB_EcoS_DanielBernoulli (Bas08) (DSM 112886) („TLS phage")

[0201] - Escherichia phage T1 (Tunavirus Tl) (DSM 5801) („T1 phage")

[0202] - Escherichia phage T5 (Tequintavirus T5) (DSM 16353). ("T5 phage")

[0203] Phages were purchased from DSMZ.

[0204] For phage resistance-testing, the E. coli strains were analyzed using the Double Agar Overlay Plaque Assay (described e.g. in Daubie et al. (2022)). In brief, the E. coli strains to be tested were cultivated for 6 h in LB and mixed with phage at a specific dilution. 10 pL of this solution was added to 2.5 mL of 45 °C LB containing 0.5% agar, and the mixture was added to the surface of LB 1.5% agar plates to form soft agar overlay plates. Due to the high dilution of phage, instead of lysing the whole bacteria population, phage activity results only in specific localized zones without bacteria growth, so-called "plaques". After 24 h incubation (37 °C for Tl and T5, 30 °C for TLS phage, 60% humidity), plaques were counted, yielding the number of plaque-forming units ("pfu"). The concentrations (CFU / mL) were calculated as follows: Number of pfu x dilution factor x 10 (because only 100 pL of phage were used for each plate).

[0205] The activities of the TLS, T1 and T5 phages were determined in triplicate against each tested E. coli strain. For each phage, 6-10 serial dilutions were tested, depending on the phage stock concentration and phage sensitivity of the strain, ensuring that the highest dilution did not result in plaque lysis.

[0206] The activity of the phages in the tested strains was compared to their activity against the reference host strain E. coli K12 MG1655 (Strain 0). This activity is expressed as "Efficiency of Plating" and is determined with the following formula (Daubie et al., (2022)):

[0207] (PFU / mF) tested strain

[0208] Efficiency of Plating (EOP) =

[0209] (PFU / mL) ref erence host strain Strains were determined to be resistant to the respective phages if the efficiency of plating was < 0.00001.

[0210] 6.3.2 Results

[0211] Exemplary plates of the Double Agar Overlay Plaque Assay are shown in Fig. 2. Shows are plates of the resistance test against the TLS phage for Strain 1 (Fig. 2A) and Strain 5 (Fig.

[0212] 2B). Fig. 2A exemplifies a strain which is not resistant and shows therefore abundant plaque formation. In contrast, Fig. 2B exemplifies a resistant strain so that no plaque formation can be observed.

[0213] Tables 4-6 indicate the sensitivity of all tested strains for the TLS, Tl, and T5 phage. Table 4: Sensitivity for TLS phage. The dilution of the phage stock is expressed as potency of 10 (for instance, -1 means 101= 1:10).

[0214] While the strain with / hu4 deletion, but wt tolC (Strain 4) showed a similar sensitivity for the TLS phage compared to the reference strains (Strains 0 and 1), the strains with tolC mutation (Strains 2, 3, and 5) showed complete resistance to TLS infection. Not even an 1:10 dilution of the TLS phage stock could induce plaque formation in those strains.

[0215] Table 5: Sensitivity for T1 phage. The dilution of the phage stock is expressed as potency of 10 (for instance, -1 means 101= 1:10).

[0216] While the strains with mutations in tolC but wt fhuA (Strains 2 and 3) showed a similar sensitivity for the T1 bacteriophage than the reference strains (Strain 0 and 1), the strains with / hu4 deletion (Strains 4 and 5) showed complete resistance to T1 infection. Not even a 1:10 dilution of the T1 phage stock could induce place formation in those strains.

[0217] Table 6: Sensitivity for T5 phage. The dilution of the phage stock is expressed as potency of 10 (for instance, -1 means 101= 1:10).

[0218] While the strains with mutations in tolC but wtfhuA (Strains 2 and 3) showed only a slight reduction in sensitivity for the T5 bacteriophage compared to the reference strains (Strains 0 and 1), the strains with fhuA deletion (Strains 4 and 5) showed strongly increased resistance to T5 infection. The combination of a tolC mutation and a fhuA deletion (Strain

[0219] 5) resulted in complete T5 resistance where not even a 1:10 dilution of the T5 phage stock could induce plaque formation in that strain.

[0220] The above results of the efficiency of plating (EOP) are graphically depicted in Fig. 3. Table 7 provides an overview of the tested E. coli strains and bacteriophages. A strain is defined as resistant if the efficiency of plating was < 0.00001.

[0221] Table 7: Summary of resistance to bacteriophages The strains with / huA deletion (Strains 4 and 5) were resistant against the T1 phage while the strains with the tolC mutations (Strains 2, 3, 5) were resistant against the TLS phage. Regarding the T5 phage, only Strains 4 and 5 showed resistance, with Strain 5 showing an even higher degree of resistance than Strain 4.

[0222] 6.4 Example 4: Growth rate determination and bacteriophage resistance test for the tolC Y238D G302D double mutant

[0223] Growth rate determination as carried out in Example 2 and the bacteriophage resistance test as carried out in Example 3 was repeated for the tolC Y238D G302D double mutant. The tolC Y238D G302D double mutant was generated from strain 1 using the CRISPR-MAD7 toolkit as described for strains 2 to 5 in section 6.1.1 above. 6.4.1 Methods growth rate determination tolC Y238D G302D double mutant

[0224] For cell growth analysis, 50 mL media in 300 mL Eppendorf shake flasks were inoculated with the respective E. coli strain. Growth was quantified in minimal media (M9 minimal media). Cell growth was monitored by online backscatter measurements (CellGrowthQuantifier, ScientificBioprocessing), followed by normalization of the data to offline OD600 measurements at the end of cultivation. Strain growth was regarded unimpaired if the maximum growth speed (biomass change in OD600 per hour) during exponential growth as well as final OD600 deviated less than 10 % from the unedited reference Strains 0 and 1.

[0225] 6.4.2 Methods bacteriophage resistance test tolC Y238D G302D double mutant

[0226] To test bacteriophage resistance of the tolC Y238D G302D double mutant, the mutant strain was challenged with the following phage:

[0227] - Escherichia phage vB_EcoS_DanielBernoulli (Bas08) (DSM 112886) („TLS phage")

[0228] The phage was purchased from DSMZ.

[0229] The phage resistance testing was carried out as described in section 6.3.1 above.

[0230] 6.4.3 Results

[0231] The results of the growth rate determination is depicted in Fig. 4. Fig. 4 shows that the tolC Y238D G302D double mutant does not show impaired growth speed as the growth rate was comparable to the genetic background. This indicates that the Y238D G302D double mutations in the tolC protein do not reduce cell growth and cell viability in the mutant strain. The results of the bacteriophage resistance testing is provided in Table 8 below. The values of Table 8 are graphically depicted in Fig. 5. Both Table 8 and Fig. 5 show that the genetic background strain is highly susceptible to the TLS phage whereas the tolC Y238D G302D double mutant strain has a much lower susceptibility. The tolC Y238D G302D double mutant strain shows a high resistance to the TLS phage without being as resistant as strains

[0232] 2, 3 and 5 while being far more resistant than strain 4 (see Table 4).

[0233] Table 8: Sensitivity for TLS phage of Strain 0 versus tolC Y238D G302D double mutant. The dilution of the phage stock is expressed as potency of 10 (for instance, -1 means 101= 1:10). "TC" means that the plaque was too much to count. "ND" means "not detected".

[0234] References

[0235] Blattner et al. (1997). The complete genome sequence of Escherichia coli K-12E coli genome reference. Science. 1997 Sep 5;277(5331):1453-62. Daubie, V. et al. (2022). Determination of phage susceptibility as a clinical diagnostic tool: A routine perspective. Front. Cell. Infect. Microbiol. 12, 1000721.

[0236] Endriss et al. (2003). Mutant Analysis of the Escherichia coli FhuA Protein Reveals Sites of FhuA Activity. J Bacteriol. 2003 Aug;185(16):4683-4692. German and Misra (2001). The TolC protein of Escherichia coli serves as a cell-surface receptor for the newly characterized TLS bacteriophage. J Mol Biol. 2001 May ll;308(4):579-85

[0237] Hantke (2020). FEMS Microbiol Lett. 2020 Jan l;367(2):fnaa013

[0238] Mund et al. (2023). A MAD7- based genome editing system for Escherichia coli. Microb Biotechnol. 2023 May;16(5):1000-1010

[0239] Zgurskaya et al. (2011), Mechanism and function of the outer membrane channel TolC in multidrug resistance and physiology of enterobacteria. Front Microbiol. 2011 Sep 16:2:189 WO2021074182

Claims

55CLAIMS1. A bacterial strain comprising a modified tolC gene, wherein said modified tolC gene comprises mutations encoding for at least two amino acid substitutions, deletions, or / and insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less.

2. The bacterial strain of claim 1, wherein said modified tolC gene comprises a) mutations encoding for two or more of the amino acid substitutions selected from S279P, Y283D, G302D, and Q303P; and / or b) mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with an unstructured linker, such as a linker comprising only amino acids selected from glycine, serine, alanine and proline.

3. The bacterial strain of claim 1 or 2, wherein said modified tolC gene comprises mutations encoding for the amino acid substitutions S279P, Y283D, and G302D.

4. The bacterial strain of claim 1 or 2, wherein said modified tolC gene comprises mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with a. a linker comprising only glycine, serine, and alanine, b. a linker comprising only glycine and serine, c. the amino acid sequence GAGSASGSAGSGAAGSGAGASAGGAA (SEQ ID NO: 5), d. an amino acid sequence having at least 80% identity to SEQ ID NO: 5, or e. a linker comprising only alanine.

565. The bacterial strain of claim 4, wherein said modified tolC gene comprises mutations encoding for a replacement of the region from position S279 to position N304 of the TolC protein with the amino acid sequence GAGSASGSAGSGAAGSGAGASAGGAA.

6. The bacterial strain of any of the previous claims, wherein the bacterial strain further comprises a modified fhuA gene, wherein said modified fhuA gene lacks at least part of the N-terminal open reading frame of the / huA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

7. The bacterial strain of any of claim 6, wherein said modified fhuA gene comprises at least the last 692 base pairs of the open reading frame of the fhuA gene.

8. The bacterial strain of any of claims 6 and 7, wherein said modified fhuA gene does not result in the translation of a FhuA protein.

9. The bacterial strain of any of the previous claims, wherein the bacterial strain is a gram-negative bacterial strain.

10. The bacterial strain of any of the previous claims, wherein the bacterial strain is an E. coli strain.

11. The bacterial strain of any of the previous claims, wherein the bacterial strain is more resistant to infection with bacteriophages, compared to E. coli reference strains such as E. coli K12 MG1655 and E. coli K12 W3110.

12. The bacterial strain of any of the previous claims, wherein the bacterial strain is more resistant to infection with a TLS phage, a T1 phage, and / or a T5 phage, compared to E. coli reference strains such as E. coli K12 MG1655 and E. coli K12W3110.5713. The bacterial strain of any of the previous claims, wherein the bacterial strain has no impaired growth, compared to E. coli reference strains such as E. coli K12MG1655 and E. coli K12 W3110.

14. A method for generating a phage-resistant bacterial strain, wherein the method comprises a) modifying the tolC gene of said bacterial strain, thereby generating a modified tolC gene which comprises mutations encoding for at least two amino acid substitutions, deletions or insertions in the region from position S279 to position N304 of the TolC protein, compared to the TolC wildtype protein, wherein the number of deletions is 5 or less and optionally further comprises b) modifying the fhuA gene in said bacterial strain, thereby generating a modified fhuA gene which lacks at least part of the N-terminal open reading frame of the fhuA gene but comprises at least the last 641 base pairs of the open reading frame of the fhuA gene.

15. The method of claim 14, wherein said method includes using a CRISPR system to modify the tolC gene and / or the / huA gene, optionally wherein the CRISPR system comprises a MAD7 nuclease.

Images