Microbial strains for antibiotic-free plasmid-based fermentation and methods for producing same

Abstract

The present invention relates to a means for recombinant production. In particular, the present invention relates to a recombinant bacterial cell comprising in its genome at least one essential gene, the endogenous promoter of said essential gene being replaced by a heterologous expression control sequence inducible by an inducer molecule, such that the expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule. Furthermore, methods of producing the recombinant bacterial cell of the present invention as well as methods of recombinant production of a compound of interest are contemplated. The present invention also provides the use of the recombinant bacterial cell of the present invention for the recombinant production of a compound of interest, and a kit for the production of a compound of interest.

Description

[Technical field]

[0001] The present invention relates to means for recombinant production. In particular, the present invention relates to a recombinant bacterial cell comprising in its genome at least one essential gene, the endogenous promoter of said essential gene being replaced by a heterologous expression control sequence inducible by an inducer molecule, such that expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule. Furthermore, methods of producing the recombinant bacterial cell of the present invention as well as methods of recombinant production of a compound of interest are contemplated. The present invention also provides the use of the recombinant bacterial cell of the present invention for the recombinant production of a compound of interest, and kits for the production of a compound of interest. [Background technology]

[0002] Plasmid-based expression of genes is an important tool for industrial-scale production of chemicals or pharmaceuticals. Typically, bacteria such as Escherichia coli (E. coli) are transformed with an expression plasmid encoding a gene of interest required for recombinant production of a desired chemical or pharmaceutical. Traditional culture methods require the use of a selection marker, such as a resistance gene to an antibiotic, in the expression plasmid and an antibiotic in the culture to select the transformed bacteria and subsequently stably maintain the expression plasmid in the transformed bacteria. However, the overuse of antibiotics is expensive and causes various environmental and health problems, promoting the generation of multidrug-resistant microorganisms, such as multidrug-resistant pathogens.

[0003] Several techniques have been developed for the recombinant production of desired products that do not rely on the use of antibiotics. For example, a toxin gene has been introduced into the bacterial chromosome and is expressed. Detoxification is achieved when a plasmid is introduced that expresses an antidote gene in addition to the gene of interest (Szpirer 2005).

[0004] In another approach, the conditionally essential gene dapD is downregulated by a repressor. The bacteria can grow only if an expression plasmid is introduced that contains the gene of interest and allows the removal of the repressor due to the presence of an operator that competes for binding with the repressor (hence the name repressor titration technique) (Cranenburgh 2001). This approach has the limitation that it requires the presence of an operator on the high copy number plasmid to titrate the repressor so that the bacteria can survive and for the necessary dapD gene expression. Furthermore, dapD is under the transcriptional control of the lac operator / promoter (lacO / P), which dramatically limits the use of this system. The widely used T7 expression system is not compatible with this system. Furthermore, Cranenbourg's approach requires a complex multi-step process to generate an expression strain.

[0005] RNA-based selectable markers have been developed. In such an approach, a marker on the chromosome is neutralized by an antisense RNA expressed from a successfully transformed expression plasmid containing the gene of interest (Mairhofer 2010).

[0006] Amino acid auxotrophy has been used in another approach: a genomic deletion of the proBA gene is complemented by proBA on an expression plasmid further carrying the gene of interest (Fiedler 2001).

[0007] There are certain drawbacks of the aforementioned methods, such as the inherent difficulties in the construction of the respective dapD, proBA or auxotrophic mutants and the need for a defined culture medium composition; even if the cultivation is carried out in a defined culture medium, there is still a risk of cross-feeding in favor of the growth of plasmid-deficient cells during fermentation as a result of secretion or cell lysis.

[0008] A similar approach of complementation has been reported for the essential infA gene, which is deleted in the genome and complemented by a successfully transformed expression plasmid such that the gene of interest becomes expressed in the transformed bacteria (Hagg et al, J. Biotechnol., (2004) and its corresponding PCT application WO2003 / 000881).

[0009] A further approach is based on bacteria stably transformed with a helper plasmid expressing the essential infA gene and containing a temperature-sensitive origin of replication (ori) and a resistance gene for a first selection marker. These bacteria are grown at a temperature that allows the ori to operate. After transformation with an expression plasmid carrying a normal ori and an additional selection marker, successfully transformed bacteria can be selected and grown at normal temperature and in the presence of a selection agent for the selection marker of the expression plasmid (WO2017 / 097383). However, bacteria treated by heat shock develop a stress response, which is poor for recombinant production.

[0010] In addition, the thermosensible approach also uses a leader sequence for infA. Endogenous infA expression is inhibited at lower temperatures by the leader sequence, which forms a hairpin structure at the 5' end of the transcript. The expression plasmid contains, in addition to the gene of interest, the infA gene, which can be properly transcribed and translated at lower temperatures. Thus, in such an approach, successfully transformed bacteria grow and are viable at lower temperatures, while non-transformed bacteria die because they cannot translate the endogenous infA transcript into the infA protein (WO2018 / 083116). Bacteria can only grow at low temperatures, which is poor for recombinant production.

[0011] The above mentioned methods for stabilizing plasmids in expression systems have several different drawbacks, such as: (i) the requirement to generate each expression strain in a laborious, time-consuming and expensive multi-step process that has to be repeated for every new product to be produced by said expression strain; (ii) the need to use several different plasmids containing selection markers such as antibiotics, toxin resistance genes or auxotrophic markers during the construction of the final expression strain; (iii) firstly, the need to delete the genomic copy of each essential gene after transformation of the plasmid containing the essential gene and the selection marker and secondly, if possible, to delete the selection marker from the plasmid; (iv) after construction of the production strain, there is a risk of cross-feeding during fermentation associated with loss of the expression plasmid and a combined reduction in production yield; (v) there is a risk of recombination of the essential gene from the plasmid into the genome; and / or (vi) a low success rate at each process step during the generation of the final expression strain, which leads to a low overall efficiency in strain construction. Summary of the Invention [Problem to be solved by the invention]

[0012] However, it would be highly desirable to develop production bacterial strains expressing desired genes of interest that do not rely on the complex and cumbersome cloning and previously mentioned mechanisms such as temperature shifting.

[0013] The technical problem underlying the present invention can be seen as the provision of means and methods for meeting the aforementioned needs. This technical problem is solved by the embodiments characterized in the claims and the following specification. [Means for solving the problem]

[0014] Thus, the present invention relates to a recombinant bacterial cell comprising in its genome at least one essential gene, the endogenous promoter of which has been replaced by a heterologous expression control sequence inducible by an inducer molecule, such that expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule. [Brief description of the drawings]

[0015] [Figure 1] Schematic diagram of the approach to generate a production strain using two selection genes and Red / ET recombination. A) Using Red / ET recombination, a first expression cassette containing, in 5' to 3' order, the rpsL promoter, the rpsL gene, the kmR gene and an I-SceI restriction site is recombined with the promoter region of the endogenous infA gene. B) An intermediate strain is obtained by selecting for kanamycin resistance, and C) using Red / ET recombination, the first expression cassette is replaced by a second expression cassette containing, in 5' to 3' order, the araC and pBAD promoters, and selection for suitable recombinant cells is performed by selecting for bacterial cells resistant to streptomycin (undesirable intermediate strains are sensitive to streptomycin), survival / growth in the presence of arabinose, and survival / growth in the presence of I-SceI. The platform strain is propagated in the presence of arabinose. [Diagram 2] Schematic diagram of the first approach to generate a production strain using Red / ET recombination and FLP recombination and one selection gene. A) Using Red / ET recombination, an expression cassette containing in 5' to 3' order an FRT site, an ampR gene, an FRT site, araC and the pBAD promoter is recombined with the promoter region of the endogenous infA gene. B) An intermediate strain is obtained by selecting for ampicillin resistance, which grows only in the presence of arabinose. C) Using FLP recombination, the ampR gene is removed. The platform strain grows in the presence of arabinose. [Diagram 3]Schematic diagram of the second approach to generate a production strain using Red / ET recombination and FLP recombination and one selection gene. A) Using Red / ET recombination, an expression cassette containing, in 5' to 3' order, araC, pBAD promoter, FRT site, rpsL promoter, kmR gene, and FRT site is recombined with the promoter region of the endogenous infA gene. B) An intermediate strain is obtained by selecting for kanamycin resistance, and C) FLP recombination is used to remove the kmR gene and the rpsL promoter, and the strain grows only in the presence of arabinose. The platform strain grows in the presence of arabinose. [Figure 4] Growth controls of the different strains on LB agar plates. A) Position of the analyzed strains on the plates. B) Growth controls on LB agar without arabinose or antibiotic addition (left plate), LB agar adjusted with 0.4% L-arabinose (middle plate) and LB agar adjusted with 15 μg / ml chloramphenicol (right plate). [Diagram 5] Restriction map of plasmid "pQE-T7 TNFα cmR-infA" (SEQ ID NO: 16). [Figure 6] Restriction map of plasmid "pQE-T7 TNFα infA" (sequence number 17). [Figure 7] Evaluation of plasmid maintenance of "pQE-T7 TNFα infA-cmR" by platform strain "HS996 araC-araBp-infA". Plasmid DNA was isolated from single colonies after transformation of the plasmid into the platform strain. No antibiotics were used to select for cells that had gained the plasmid during electroporation or to maintain the plasmid in overnight cultures. Plasmid DNA was analyzed by restriction digestion with NotI. All six clones showed the expected banding patterns (747 bp and 5289 bp). [Figure 8]Restriction digestion of the four clones obtained after transformation of the platform strain with the plasmid "pQE-TNFα infA". Plasmid DNA was digested with MluI+PacI. For all four clones analyzed, the expected restriction fragments of 2078 bp and 3211 bp were obtained. [Figure 9] Growth control of various platform strains on LB agar plates (left plate) and LB agar plates conditioned with 0.4% L-arabinose (right plate). The left half of each plate was streaked with a platform strain. On the right half of each plate, the corresponding strain harbors the plasmid pQE-T7 TNFα infA. A) Growth of platform strain E. coli BL21(DE3) araC-araBp-FRT-infA; B) Growth of E. coli TG10 araC-araBp-FRT-infA; C) Growth of E. coli HS996 araC-araBp-FRT-infA. [Figure 10] Growth control of the platform strain W3110 araC-araBp-FRT-infA on LB agar plates. The possibility of growing on LB agar was determined for the strain itself and after transformation of different plasmids with different copy numbers. A) Position of the analyzed strains on the plate. B) Growth control on LB agar without addition of arabinose (left plate) and LB agar adjusted with 0.4% L-arabinose (right plate). [Figure 11] Growth control of the platform strain HS996 pir+ araC-araBp-FRT-infA on LB agar. The possibility of growing on LB agar was determined for the strain itself and after transformation of different plasmids with different copy numbers and different origins of replication (GenH015-plasmid1=R6K origin, GenH015-plasmid2=p15A origin and GenH015-plasmid3=ColE1 origin). A) Position of the analyzed strains on the plate. B) Growth control on LB agar without the addition of arabinose. [Figure 12A-1]Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 12A-2] Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 12B-1] Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 12B-2]Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 12C-1] Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 12C-2] Restriction maps of various reporter plasmids. A) Plasmids based on the R6K origin of replication. On the left: "GenH015-plasmid1-cmR" (SEQ ID NO: 23). On the right: "GenH015-plasmid1" (SEQ ID NO: 26). B) Plasmids based on the p15A origin of replication. On the left: "GenH015-plasmid2-cmR" (SEQ ID NO: 24). On the right: "GenH015-plasmid2" (SEQ ID NO: 27). C) Plasmids based on the ColE1 origin of replication. On the left: "GenH015-plasmid3-cmR" (SEQ ID NO: 25). On the right: "GenH015-plasmid3" (SEQ ID NO: 28). [Figure 13]Schematic diagram of the approach to generate a production strain using Red / ET recombination and FLP recombination based on the frr gene. A) Using Red / ET recombination, an expression cassette containing, in 5' to 3' order, araC, pBAD promoter, FRT site, rpsL promoter, zeoR gene, and FRT site is recombined with the promoter region of the endogenous frr gene. B) An intermediate strain is obtained by selecting for zeocin resistance, and C) FLP recombination is used to remove the zeoR gene and the rpsL promoter, and the strain grows only in the presence of arabinose. The platform strain grows in the presence of arabinose. [Figure 14] Growth control of the platform strain "HS996 araC-araBp-FRT-frr" on LB agar (left plate) and LB agar adjusted with 0.4% L-arabinose (right plate). The left half of each plate was streaked with the platform strain. On the right half of each plate, the corresponding strain carries the plasmid "pQE-T7 TNFα frr." [Figure 15] Restriction map of plasmid "pQE-T7 TNFα zeoR-frr" (sequence number 35). [Figure 16] Restriction map of plasmid "pQE-T7 TNFα frr" (sequence number 36). [Figure 17]Growth control of clones propagated for over 120 generations without selection. Three different combinations of platform strains and plasmids were analyzed: "HS996 pir+ araC-araBp-FRT-infA" / "GenH015-plasmid1" (left plate), "T7E2 araC-araBp-FRT-infA" / "GenH015-plasmid2" (right plate) and "W3110 araC-araBp-FRT-infA" / "GenH015-plasmid3" (middle plate). A small aliquot from the last overnight culture was streaked onto an LB agar plate and incubated overnight at 37 °C to obtain single colonies. All colonies obtained showed the expected purple color, indicating the presence of the corresponding plasmid. Due to the low copy number of the p15A-based plasmid, the colonies obtained from the combination “T7E2 araC-araBp-FRT-infA” / “GenH015-plasmid2” (right plate) are less intense in color. [Figure 18] Growth control of clones (platform strain carrying plasmid “pQE-T7 TNFα zeoR-frr”) propagated over 120 generations without selection for cm resistance. 24 clones were streaked onto LB agar plates (left) and LB agar plates adjusted with 25 μg / ml Zeocin to confirm the presence of plasmid “pQE-T7 TNFα zeoR-frr” after 7 days of passaging without antibiotic addition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] It should be understood that, in this specification and claims, "a" or "an" can mean one or more, depending on the context in which it is used. Thus, for example, a reference to "an" item can mean that at least one item is available.

[0017] When used hereinafter, the terms "have", "comprise" or "include" are meant to have an open-ended or closed-ended meaning. Thus, when they have a closed-ended meaning, they may refer to a situation in which, in addition to the features introduced by these terms, no other features are present in the described embodiment, i.e., they have a closed-ended meaning in the sense of "consisting of" or "consisting essentially of". When they have a non-close-ended meaning, they refer to a situation in which, in addition to the features introduced by these terms, one or more other features are present in the described embodiment.

[0018] Furthermore, as used hereinafter, the terms "preferably", "more preferably", "most preferably", "particularly", "more particularly", "typically" and "more typically" or similar terms are used in conjunction with features to indicate that these features are preferred but not required features, i.e., these terms are intended to indicate that alternative features may also be envisaged in accordance with the present invention.

[0019] Furthermore, the term "at least one" as used herein is understood to mean that one or more of the items mentioned following the term may be used in accordance with the present invention. For example, if the term indicates that at least one item shall be used, this may be understood as one item, or two or more items, i.e., two, three, four, five, or any other number. Depending on the item to which the term refers, a person skilled in the art will understand what upper limit (if any) the term may refer to.

[0020] The term "recombinant" as used herein refers to a bacterial cell that has been genetically modified. Preferably, said genetic modification may be a permanent modification. Thus, according to the present invention, any modification of the genome of a bacterial cell is encompassed, including those achieved by recombinant technology and those achieved by gene editing technology. The modification of the genome may be carried out, for example, by using the CRISPR-Cas-9 system, the lambda Red recombination-based system (this technology is also called Red / ET recombination or Red / ET recombineering), site-specific recombination technology, such as the Cre-recombinase-based system, the FLP-FRT-based system, the transposon-based technology or the random integration technology.

[0021] The term "bacterial cell" as used herein refers to a gram-positive or gram-negative bacterial cell suitable for the recombinant production of a compound of interest.

[0022] Depending on the target compound to be produced and the amount of the target compound envisaged, the skilled artisan will be well aware which bacterial cells are suitable. Typically, bacterial cells are of the genera Escherichia, Vibrio, Bacillus, Lactobacillus, Acetobacter, Corynebacterium, Brevibacterium, Pseudomonas, Streptomyces, Gluconobacter, Clostridium, and the like. Clostridium, Streptococcus, Zooepidemicus, Basfia, Crytococcus, Rhodotorula, Nocardia, Erwinia, Xanthomonas, Leuconostoc and Klebsiella, e.g. Escherichia coli. coli, Vibrio natriegens, Bacillus species, preferably Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus stearothermophilus, Bacillus clausii, Bacillus alkalophilus, Bacillus coagulans, Bacillus circulans, Bacillus pumilus pumilus, Bacillus thuringiensisthuringiensis, Bacillus psuedofirmus, Bacillus marmarensis, Bacillus cellulolyticus, Bacillus hemicellulolyticus, Bacillus clarkia, Bacillus akibai, Bacillus gibsonii, Bacillus lautus, Bacillus megaterium or Bacillus halodurans, Lactobacillus species, preferably Lactobacillus plantarum, Lactobacillus brevis, brevis, Lactobacillus coryniformis, Lactobacillus sanfranciscensis, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus curvatus or Lactobacillus varians, and Acetobacter species, preferably Acetobacter aceti or Acetobacter pasteurianus, Corynebacterium species, preferably Corynebacterium glutamicum. glutamicum, Corynebacterium acetoglutamicum, Corynebacterium acetacidophyllumacetoacidophilum, Corynebacterium callunae, Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes, Corynebacterium melassecola, Corynebacterium effiziens, Corynebacterium efficiens, Corynebacterium deserti, Brevibacterium flavum, Brevibacterium lactofermentum lactofermentum, Brevibacterium divarecatum, Pseudomonas putida, Pseudomonas syringae, Streptomyces species, preferably Streptomyces coelicolor, Streptomyces lividans, Streptomyces albus, Streptomyces avermitilis, Gluconobacter oxydans, Gluconobacter morbifer, Gluconobacter thailandicus, thailandicus, Clostridium species, preferably Clostridium acetobutylicum, Clostridium saccharobutylicum, Clostridium beijerinckiibeijerinckii, Streptococcus species, preferably Streptococcus aureus, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, Streptococcus equi subsp., Zooepidemicus, Basfia succiniciproducens, Cryptococcus laurentii, Rhodotorula glutensis, Nocardia mediterranei, Erwinia herbicola, Preferably, the bacterial cell referred to herein is a Gram-negative bacterial cell. Preferably, said bacterial cell is an Escherichia cell, such as an E. coli cell. Furthermore, it is preferably envisaged that the bacterial genome is devoid of any gene capable of functionally complementing at least one essential gene in the absence of an inducer molecule.

[0023] The term "endogenous essential gene" as used herein refers to a gene that is naturally present in the genome of a bacterial cell and is required for the proper functioning of the bacterial cell. The absence of a functional endogenous essential gene, as referred to herein, typically results in the death and / or growth arrest of the bacterial cell. Preferably, the endogenous essential gene is required for proper cell growth and / or viability of said bacterial cell.

[0024] The term "essential gene" as used herein refers to a gene required for the survival of an organism, microorganism or cell, preferably a bacterial cell. For example, such essential genes are required for cell division (e.g., ftsL, ftsN, ftsZ or parC), or for maintaining cell structure (e.g., dapA, dapB, rodA or rodZ), for central metabolism, for gene translation into proteins (e.g., era, rpsB, grpE, mopA or mopB), or for cellular DNA replication (e.g., infA, infC, frr, dnaJ or dnaK), for RNA transcription (e.g., nusG or trmA), or for supporting transport processes within, into or out of the cell (e.g., secY or msbA). By definition, if an essential gene is inactivated, for example by deletion or knockout, the cell will die.

[0025] Essential genes can be classified as follows: (a) Conditionally essential genes, i.e. the gene is essential only under certain circumstances or growth conditions, i.e. the gene results in an auxotrophy or is inactivated only at certain temperatures. For example, an auxotrophy is the inability of a (micro)organism to synthesize a particular organic compound, i.e. a sugar, fatty acid or amino acid, required for its growth. (b) absolutely essential genes, i.e., they are absolutely required for the cell to grow, reproduce and survive even under optimal growth conditions. Said essential genes are essential for an organism, for example, for DNA replication, RNA, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration; (c) "redundant" essential genes, i.e., alternative metabolic pathways within a cell that lead to the same synthetic intermediate or end product often make an essential gene non-essential; for example, if there are two enzymes within a cell that synthesize the same product, the redundant enzyme can replace the other gene copy. If both genes are deleted and the product is not produced, the essentiality of the essential gene(s) becomes apparent; (d) Essential gene combinations, i.e., the gene alone is non-essential, but becomes essential when another gene is inactive and therefore another gene deficiency is present.

[0026] Preferably, the term "essential gene" as used herein refers to at least one gene that is an absolutely essential gene according to (b) or to at least one gene that is a combination of essential genes according to (d). Thus, in the bacterial cell of the invention, said at least one essential gene is (i) an absolutely essential gene that is absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or is essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration, or (ii) a combination of essential genes that, in combination, are absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or are essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration.

[0027] More preferably, the at least one endogenous essential gene is selected from the group consisting of infA, infC / IF-3, dnaJ, dnaK, era, frr, ftsL, ftsN, ftsZ, grpE, mopA, mopB, msbA, nusG, parC, rpsB, secY and trma. More preferably, such endogenous essential gene is selected from the group consisting of genes involved in supporting the DNA replication, RNA transcription or transport processes in, to or from the cell of the cell. In particular, it is selected from the group consisting of the following genes: infA, infC, frr, dnaJ, dnaK, nusG, trma, secY and msbA. Most preferably, said at least one essential gene is infA, secY or frr.

[0028] Nucleic acid sequences of the aforementioned essential genes are known in the art for various bacterial species, including those explicitly mentioned elsewhere herein. In addition to the nucleic acid sequences disclosed in the prior art, the aforementioned endogenous essential genes may also include variant sequences. Preferably, such variant sequences still encode gene products capable of exerting the biological function of the gene product of the essential gene. Typically, the variant nucleic acid sequences are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific sequences for the endogenous essential genes disclosed in the prior art. Furthermore, endogenous essential genes may also include those that contain a nucleic acid sequence that encodes a gene product, i.e., a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the specific sequences for gene products encoded by endogenous essential genes disclosed in the prior art. Nucleic acid sequence identity or amino acid sequence identity, as referred to herein, can be determined by aligning the sequences, preferably so that the highest order match is obtained, and determining the number of identical amino acids between two nucleic acid or amino acid sequences. It can be calculated using published techniques or methods, for example, encoded in computer programs such as BLASTP, BLASTN or FASTA. Alignment of sequences is preferably performed using the program "NEEDLE" [The European Molecular Biology Open Software Suite (EMBOSS)], which is based on the algorithm from Needleman and Wunsch [J. Mol. Biol. (1979) 48, p. 443-453], using default parameters (for example: gap open=10.0, gap extend=0.5 and matrix 0 EDNAFULL)].Percent sequence identity values ​​are typically calculated over the entire nucleic acid or amino acid sequence, or over a fragment of the nucleic acid or amino acid sequence that is at least 50% of the length of the entire sequence. The default settings of the aforementioned computer programs should typically be used to calculate the percentage sequence identity. Preferred nucleic acid sequences for the open reading frames of the aforementioned essential genes from E. coli infA, secY and frr are also shown in SEQ ID NO: 15 (infA), SEQ ID NO: 16 (secY) and SEQ ID NO: 37 (frr), respectively.

[0029] Furthermore, the promoter of the aforementioned essential gene is a region in the 5' upstream non-coding region of the gene, which contains at least one expression control sequence that naturally controls the expression of the essential gene. The promoter, as referred to herein, typically contains a binding site for a basic transcription factor, such as a sigma factor, which is required to recruit RNA polymerase and initiate transcription of RNA from the gene. In addition, the promoter also preferably contains an additional transcription factor binding site that is bound by a transcription factor that is naturally required for proper transcription of the essential gene. Typically, the promoter of the essential gene is located in a DNA segment upstream of the transcription start site. The DNA segment that begins with the transcription start may preferably have a length of up to 100bp, up to 200bp, up to 300bp, up to 400bp, up to 500bp, up to 750bp, or up to 1,000bp further upstream. The promoter regions for the essential genes referred to above are well known to those skilled in the art.

[0030] The endogenous promoter of at least one essential gene shall be replaced by a heterologous expression control sequence inducible by an inducer molecule. As a result of said replacement, the expression of the essential gene shall no longer be dependent on its endogenous promoter. Rather, as a result of the replacement, the expression of the at least one essential gene shall become dependent on the heterologous expression control sequence inducible by an inducer molecule, and thus dependent on the presence of said inducer molecule in said bacterial cell. It is understood that the promoter may be replaced, i.e. by exchanging a DNA segment having a promoter by a polynucleotide having a heterologous expression control sequence. Furthermore, the endogenous promoter may also be inactivated and a polynucleotide comprising a heterologous expression control sequence may be introduced into the genome of the bacterial cell such that the expression of the at least one essential gene becomes dependent on said heterologous expression control sequence. The inactivation of the endogenous promoter is typically the result of a genetic modification. In particular, it is envisaged that the inactivated endogenous promoter comprises at least one nucleotide deletion, substitution and / or addition compared to the wild-type version. The result of said genetic modification may be that the promoter is no longer able to initiate the transcription of the at least one essential gene. Preferably, the endogenous promoter in the genome of the bacterial system is inactivated by using the CRISPR-Cas-9 system, the lambda red recombination-based system, the site-specific recombination technique, such as the Cre-recombinase-based system, the FLP-FRT-based system, the transposon-based technique or the random integration technique. In addition, the endogenous promoter may also be inactivated by other techniques, such as random mutagenesis and selection for inactivated bacterial cells. In random mutagenesis, the bacterial cells are treated with mutagenic agents or radiation to randomly introduce DNA modifications, such as substitutions or deletions. More preferably, the replacement of the endogenous promoter may be carried out by any of the methods of the invention as described herein below or as described in the accompanying examples below.

[0031] It is understood that the elements of the at least one essential gene other than the promoter, i.e., the coding sequence of the gene (structural gene) and the 3' elements, shall preferably remain in their endogenous location. Thus, at least one essential gene in a bacterial cell of the invention having an endogenous promoter replaced as specified elsewhere herein will typically have, at least at the 3' end of the structural gene, the same flanking sequences as the at least one essential gene in its unmodified form.

[0032] The term "heterologous expression control sequence inducible by an inducer molecule" as referred to herein relates to a nucleic acid sequence capable of controlling the expression of said coding nucleic acid sequence of a gene when operably linked to said coding nucleic acid sequence of a gene. An expression control sequence is typically a promoter comprising transcriptional and translational regulatory elements. The expression control sequence shall be inducible by an inducer molecule, i.e. its function shall be dependent on the presence and / or amount of the inducer molecule. Such inducer-dependent expression control sequences are known in the art and preferably include the AraC / PBAD promoter, the RhaR-RhaS / rhaBAD promoter, the XylS / Pm promoter, the NitR / PnitA promoter, and the ChnR / Pb inducer / promoter (see, for example, Brautaset 2009, Microb Biotechnol 2(1): 15-30). Other inducer-dependent systems may be tetracycline-inducible promoters (tetON / OFF), lacI / IPTG promoters, ethanol-inducible promoters (AlcA / AlcR), steroid-inducible promoters (LexA / XVE) or vanillate-inducible promoters. Furthermore, the inducible expression control sequence referred to herein shall be heterologous with respect to the wild-type, i.e. naturally occurring, bacterial cell, i.e. it is an expression control sequence that is not naturally present in a recombinant bacterial cell or is not naturally associated with an endogenous essential gene in said bacterial cell. In other words, the term "heterologous" shall mean a gene or operon that is not naturally operably linked, e.g. in the genome of a wild-type microorganism, or that is naturally operably linked at a different place or location, e.g. in the genome of a wild-type microorganism, with respect to the inducible expression nucleic acid sequence that is operably linked or engineered to become operably linked to a second nucleic acid sequence, e.g. a promoter. By "not naturally associated" or "operably linked" is meant that the recombinant bacterial cell (= "host cell") does not naturally have this control sequence operably linked to the coding nucleic acid sequence of the essential gene (whose expression is controlled by the heterologous control sequence).The inducibly expressed nucleic acid sequences may be from the same microbial taxonomic species or from different species.

[0033] The term "inducer molecule", as referred to according to the present invention, relates to any molecule capable of acting as an inducer of the expression of a gene operably linked to a heterologous expression control sequence inducible by said inducer molecule. Typically, the inducer molecule may be a metabolite metabolized by the bacterium, such as a sugar, preferably arabinose, rhamnose, xylose or sucrose, a substituted benzene, a cyclohexanone related compound, ε-caprolactam, propionate, thiostrepton, an alkane, isopropyl β-D-1-thiogalactopyranoside, tetracycline, anhydrotetracycline or a peptide. Preferably, said inducer molecule is selected from the group consisting of arabinose, rhamnose, xylose, sucrose, tetracycline, anhydrotetracycline and IPTG.

[0034] Advantageously, according to the research underlying the present invention, it has been found that by replacing the endogenous promoters of endogenous essential genes in the genome of a bacterial cell by expression control sequences that can be controlled by an inducer molecule, it is possible to provide recombinant bacterial cells whose viability and / or growth are dependent on the presence or amount of the inducer molecule in the culture. Such recombinant bacterial cells can be used as a common platform for generating recombinant bacterial cells useful for the manufacture of various target compounds, i.e. protein-producing bacterial cells. By providing said recombinant bacterial cells as a common platform, it is no longer necessary to generate bacterial cells that produce the desired target compounds without the use of antibiotics in a cumbersome and inefficient manner. In particular, some prior art techniques require the individual generation of the producing bacterial cells from scratch, i.e. from unmodified bacterial cells, using a separate complementing plasmid in each case or, in the case of the knock-out approach, the application of a helper plasmid to remove the resistance gene from the final producing bacterial cell. According to the present invention, a common bacterial cell can be used as a platform. A given expression plasmid used to generate the producing bacterial cell can be easily modified, with less cloning effort, to comply with this system. There is no need for a helper plasmid and no need to remove the helper plasmid, for example by using a heat treatment step above the incubation temperature of 37°C.

[0035] It is understood that the production bacterial cells for the desired target compound to be used in the recombinant manufacturing process can be generated from the aforementioned recombinant bacterial cells by introducing an expression plasmid into said recombinant bacterial cells.

[0036] Thus, in a preferred embodiment of a recombinant bacterial cell of the invention, said bacterial cell comprises an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell.

[0037] In the aforementioned bacterial cells according to the invention, the plasmid is also maintained since the cell relies on a plasmid-borne copy of at least one essential gene, such as, inter alia, the infA gene as used in the following examples. However, the invention includes further important improvements: 1) Strain engineering allowing maintenance of essential gene-based plasmids is achieved using fewer steps and highly efficient lambda-Red-based recombineering, resulting in a faster and more robust protocol that can be performed in a variety of strain backgrounds, including difficult-to-engineer strains; 2) strain modifications that allow for maintenance of essential gene-based plasmids only need to be completed once per strain, rather than each time a new plasmid is used; and 3) The plasmid of interest can be transformed into the engineered strain without the need for an antibiotic resistance marker, making the system completely independent of antibiotic resistance genes.

[0038] An expression plasmid may have at least one origin of replication (= "ori") with a broad host range, i.e. the plasmid can replicate in many taxonomically distinct species (e.g. oriV, oriB or oriS), or at least one origin of replication with a narrow host range, i.e. the plasmid can replicate in few taxonomically distinct species or potentially only in one species (e.g. ColE1 ori). Broad host range plasmids are e.g. RK2, RP4, RP1, R68, pUC1, pSa, pSFA231, pBC1, pWVO1, pLF1311, pAP1, pBBR1, pLS1, pIP501, pJD4, RSF1010 / R1162 / R300B or pPS10, which are conditionally broad host range. Some of the plasmids have one ori that can initiate replication in one or more species, or several different oris, each capable of replicating in one or more species. Narrow host range plasmids are, for example, pET, pBR322, pUC18, pUC19 or pMB1.

[0039] Expression plasmids that can be used in the present invention include, for example, RK2, RP4, RP1, R68, pUC1, pSa, pSFA231, pPS10, pBC1, pEP2, pWVO01, pLF1311, pAP1, pBBR1, pLS1, pIP501, pJD4, pRSF1010 / R1162 / R300B, pPS10, pIP501, pRK290, pLAFR1, pLAFR5, pRS44, pJB6 53, pFAJ1700, pDSK509, pKT210, pAYC32, pAYC51 / 52, pJFF224-NX, pBHR1, pGEX, pSC101, p15A, pJF118EH, pKK223-3, pUC18, pUC19, pBR322, pACYC184, pASK-IBA3, pET, and any expression plasmids made therefrom or derived therefrom.

[0040] Depending on the at least one further nucleic acid of interest (see below for definition) in the plasmid, the skilled artisan knows which plasmid to choose for optimal expression of said nucleic acid of interest and for best production of said nucleic acid or a product derived therefrom. Some of the plasmids mentioned herein have a high copy number in the cell (e.g. pUC18, pMB1 or RSF1030), others have a lower copy number (e.g. pBR322, pSC101 or p15A) or potentially only one copy in the cell (e.g. F1 or P1).

[0041] Preferably, the expression plasmid is selected from the group consisting of pJF118EH, pKK223-3, pUC18, pBR322, pACYC184, pASK-IBA3, pET, and any expression plasmids related or derived therefrom. It is understood that some plasmids may contain selection markers, such as antibiotic resistance genes, but these genes are not required to use the recombinant bacterial cells according to the invention. Rather, expression of the plasmid copy of at least one essential gene shall be sufficient to select recombinant bacterial cells that have successfully incorporated the expression plasmid. More preferably, the expression plasmid may be a plasmid described in the accompanying examples below.

[0042] According to the present invention, it is envisaged that the expression of the plasmid copy of at least one essential gene is controlled by an expression control sequence that is biologically active under physiological conditions in the recombinant bacterial cell. Said expression control sequence may therefore be a constitutively active promoter that is permanently expressed in the cell. Alternatively, it may be a promoter that is constitutively expressed during the growth phase in the recombinant bacterial cell. Suitable expression control sequences are well known to those skilled in the art. They include, for example, the constitutively active promoters from E. coli described in Shimada et al. 2014, PLoS ONE 9(3): e90447. https: / / doi.org / 10.1371 / journal.pone.0090447. Similar constitutively active promoters can be found for other bacteria and have already been reported in the prior art. Preferably, said biologically active expression control sequence is an expression control sequence that is naturally associated with at least one endogenous essential gene. Thus, more preferably, the expression control sequence is a promoter selected from the group consisting of infA promoter, infAp2 promoter, infC / IF-3 promoter, dnaJ promoter, dnaK promoter, era promoter, frr promoter, ftsL promoter, ftsN promoter, ftsZ promoter, grpE promoter, mopA promoter, mopB promoter, msbA promoter, nusG promoter, parC promoter, rpsB promoter, secY promoter and trmA promoter. Most preferably, the at least one essential gene is the infA promoter.

[0043] Preferably, said expression plasmid comprises at least one additional nucleic acid of interest required for the recombinant production of a compound of interest in said recombinant bacterial cell.

[0044] The term "at least one further nucleic acid of interest" as used herein relates to a nucleic acid encoding a gene product, i.e. a protein, peptide or RNA, required to produce a compound of interest envisaged to be produced by the recombinant bacterial cell of the invention. It is understood that the compound of interest to be produced may also be the encoded gene product itself. Typically, however, the gene product is required for the recombinant production of the compound of interest. Preferably, the protein referred to herein may be an enzyme required for one or more catalytic transformations required during the recombinant production process. Alternatively, it may be a binding protein, such as a chaperone, which may bind to and protect the compound of interest, thus facilitating the purification of said compound. The peptide referred to herein may be useful for controlling the growth of bacterial cells in culture, or may be useful as an antimicrobial peptide, in cases where the peptide itself is envisaged to be produced as a compound of interest. The RNA referred to herein may be an inhibitory RNA, such as an siRNA or a microRNA, or an enzymatically active RNA. Both inhibitory RNAs or ribozymes are suitable agents for controlling the presence and / or amount of certain other RNAs, such as mRNAs encoding certain proteins or peptides. By controlling such mRNAs, inhibitory RNAs or ribozymes can control the presence or amount of biological activity of proteins present in bacterial cells, such as the presence or amount of enzymatic activity, and it is therefore understood that recombinant manufacturing processes can also be controlled by these agents.

[0045] It is understood that the nucleic acid of interest required for the recombinant production of the compound of interest shall be operably linked to an expression control sequence that is biologically active in the bacterial cell of the invention or that can be activated during cultivation. Thus, such expression control sequences may include constitutively active promoters as well as inducible promoters. Constitutively active promoters have already been described elsewhere herein. It is understood that the inducible promoter used to control the expression of the nucleic acid of interest shall not be controlled by the inducible molecule used to control the expression of the at least one introduced copy of said at least one essential gene according to the invention. Furthermore, it should be understood that when an inducible promoter is used to produce the compound of interest, said promoter must be induced, i.e. an inducing stimulus must be applied to the recombinant bacterial cell culture. A typical inducing stimulus applied in this context may be a change in physical conditions, for example the application of a heat stimulus (also called "heat shock") or the administration of an inducing molecule. Inducible promoters typically encompass not only heat-inducible promoters but also compound-inducible promoters such as those mentioned elsewhere herein. The skilled artisan will be well aware of which promoters can be used in this context.

[0046] It should be noted that an expression plasmid in principle allows the expression of both at least one plasmid copy of at least one essential gene referred to above and at least one further nucleic acid of interest as specified above. Therefore, both expression cassettes must be located in the expression plasmid in such a way that there is no interference of the expression of either expression cassette by the other expression cassette.

[0047] The term "compound of interest" as used according to the present invention refers to any compound that can be recombinantly produced in a recombinant bacterial cell of the present invention. The compound of interest is intended to be produced typically by expression and / or sequential reaction(s) of a gene product encoded by an additional nucleic acid of an expression plasmid in a producing bacterial cell as described hereinbefore. Preferably, the compound of interest belongs to a chemical class selected from the group consisting of lipids, amino acids, purines, such as nucleotides and nucleosides based thereon, pyrimidines, such as nucleotides and nucleosides based thereon, isoprenoids, proteins, peptides, nucleic acids, terpenes, carotenoids, sugars, polysaccharides, alkaloids, alcohols, antibiotics, vitamins, polyhydroxyalkanoates, polyamides, polylactic acids, enzymes, coenzymes, organic acids, etc. Furthermore, the compound of interest may also be the gene product itself encoded by an additional nucleic acid of interest contained in the expression plasmid. Typically, the compound of interest is in such a case a protein, peptide or RNA molecule. Furthermore, the expression plasmid itself or a part thereof, i.e. a nucleic acid sequence such as a DNA or RNA molecule, may be envisaged as the compound of interest produced by the producing recombinant bacterial cell of the present invention.

[0048] Preferably, the nucleic acid of interest, as referred to herein, may be a therapeutic nucleic acid, such as unmodified DNA or RNA, or a closely related compound used to treat a disease. Typically, plasmid DNA as a nucleic acid of interest according to the present invention may be used to prepare a pharmaceutical composition or a vaccine, or for use in gene therapy. For these applications, it is essential that antibiotic resistance marker genes are not present on the plasmid DNA. Since 1989, over 3,000 clinical trials have been reported, approximately 15% of which use plasmid DNA (pDNA) as a vector system (Alves et al. 2021). Compared to virus and RNA-based vectors, plasmid DNA has lower toxicity, higher stability, and is easier and cheaper to produce. However, pDNA vectors have some limitations when compared to viral vectors, namely lower transfection efficiency and sufficient expression over time. Transfection efficiency is improved by reducing the size of the pDNA vector (Oliveira and Mairhofer 2013). Loss of expression over time, or gene silencing, has recently been shown to occur when more than 1 kb of bacterial DNA is present in a pDNA vector (Lu et al. 2012). Furthermore, the presence of unmethylated CpG motifs, commonly found in prokaryotic DNA, can trigger an immune response (Alves et al. 2021), further highlighting the importance of minimizing the size of pDNA vectors. Thus, a small plasmid backbone is critical for therapeutic applications.

[0049] For example, the total length of the plasmid backbone consisting of the infA gene and the p15A origin of replication is about 1 kb. For example, the total length of the plasmid backbone consisting of the infA gene and the R6K origin of replication is about 800 bp in size. Thus, the plasmid maintenance systems described elsewhere herein are preferably well suited for the production of pDNA for gene therapy and other therapeutic applications.

[0050] Advantageously, according to the research underlying the present invention, it has further been found that the use of a recombinant bacterial cell comprising in its genome at least one essential gene, the endogenous promoter of said essential gene being replaced by a heterologous expression control sequence inducible by an inducer molecule, as a platform allows for easy generation of a variety of different production bacterial cells that can be applied for recombinant production of a desired target compound. The platform bacterial cell can be cultured in the presence of the inducer molecule. After transformation with the expression plasmid described herein, the transformed bacterial cell is no longer cultured in the presence of the inducer molecule, and only bacteria that have incorporated the expression plasmid allowing the expression of at least one essential gene can grow and / or survive. Once the inducer molecule is removed from the culture, antibiotic selection markers and culture under selection conditions are not required, since the selection of transformed bacteria is performed under physiological conditions. Thus, the recombinant bacterial cell of the present invention avoids the use of antibiotics required in conventional recombinant production processes for selection and culture. Neither the introduced expression plasmid nor the platform bacterial cell contains an antibiotic resistance marker gene. The expression plasmid in the producing bacterial cells is stably maintained in the cells even under high density fermentation conditions, and under optimal and suboptimal culture temperatures and conditions.

[0051] The present invention also provides (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a first selection gene that confers sensitivity to a first selection agent, such that the bacterial cell will not grow and / or die in the presence of said first selection agent; and (ii) a second selection gene that confers resistance to a second selection agent such that the bacterial cell can grow in the presence of said second selection agent. exchanging the at least one essential gene, the first and the second selection gene with a first polynucleotide comprising a selection cassette comprising a promoter capable of being active in a bacterial cell, operably linked to said first polynucleotide, such that expression of said at least one essential gene, the first and the second selection gene becomes dependent on said promoter; (b) using a second selection agent to select bacterial cells that have integrated the first polynucleotide into their genome; (c) replacing the first polynucleotide integrated in the genome with a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of at least one essential gene becomes dependent on the heterologous expression sequence; and (d) selecting bacterial cells that have integrated said second polynucleotide into their genome using the first selection molecule and an inducer molecule. The present invention also relates to a method for producing the recombinant bacterial cell of the present invention, comprising:

[0052] The method according to the invention may consist of the aforementioned steps (a) to (d) or may comprise further steps, such as a step of culturing and / or pre-treating the bacterial cells before step (a) and / or a step of culturing and / or treating the bacterial cells after step (d).

[0053] The term "promoter capable of being active in a bacterial cell" refers to an expression control sequence that controls the expression of a gene operably linked thereto in a bacterial cell. The promoter may be an endogenous promoter naturally present in the genome of the bacterial cell, or it may be a heterologous promoter, i.e. a promoter from another species known to operate in bacterial cells. Furthermore, the promoter may be preferably active in the bacterial cell as a constitutively active promoter or an inducible promoter. The skilled person is familiar with suitable promoters. More preferably, the promoter constitutively active in a bacterial cell is selected from the group consisting of rpsL promoter, infA promoter, infAp2 promoter, em7 promoter, and cat promoter. Most preferably, the promoter is the rpsL promoter. Also more preferably, the inducible promoter is selected from the group consisting of a heat-inducible promoter, a tetracycline-inducible promoter, a lacI / IPTG promoter, an ethanol-inducible promoter (AlcA / AlcR), a steroid-inducible promoter (LexA / XVE), and a vanillate-inducible promoter. It is understood that when an inducible promoter is used as the promoter that can be active in a bacterial cell according to the present invention, said promoter is preferably different from the heterologous expression control sequence that is inducible by an inducer molecule. In particular, the inducer for the inducible promoter and the inducer molecule that controls the expression of at least one essential gene shall be different.

[0054] The term "operably linked" as used herein means that two genetic elements referred to herein are functionally linked to each other. For example, an expression control sequence can be functionally linked to the coding sequence of a gene of interest if it is capable of controlling the expression of the gene of interest. Typically, the expression sequence can be located physically close to the coding sequence of the gene, i.e., at its 5' end. However, it is also possible to place the expression control sequence at a certain distance from the coding sequence, as long as the expression control sequence is still capable of controlling the expression of the coding sequence. Those skilled in the art are well aware of how such operative linkage can be achieved depending on the given expression control sequence.

[0055] The term "first selection gene" refers to a gene encoding a gene product whose expression in a bacterial cell confers sensitivity to a first selection agent to the bacterial cell. A first selection gene may typically encode a protein that, when expressed in a bacterial cell, inhibits the growth of said bacterial cell and / or is detrimental to the viability of said bacterial cell, potentially lethal, in the presence of a first selection agent. Suitable proteins may be enzymes, ribosomal proteins and other proteins required for translation, transcription factors, cell cycle proteins, etc. Preferably, the first selection gene, as referred to according to the present invention, is selected from the group consisting of ribosomal S12 protein gene (rpsL), levansucrase (sacB), tRNA synthetase for phenylalanine (pheS), tetR, galactokinase (galK), thymidylate synthetase (thyA), and tolC. More preferably, the first selection gene is rpsL.

[0056] The term "first selection agent" as used herein refers to a compound that is detrimental to the viability and / or growth of bacterial cells expressing a first selection gene that confers sensitivity to the first selection agent. Preferably, the first selection agent is selected from the group consisting of streptomycin, sucrose, chloro-phenylalanine, chlortetracycline and fusaric acid, galactose, trimethoprim and its derivatives, and e.g. bacteriocins, e.g. colicins, e.g. pore-forming colicins, e.g. colicin A, B, E1, Ia, Ib, N, etc.; colicin D, which is a translation factor inhibitor; colicin M, which blocks peptidoglycan synthesis, or colicin E3 or E3 with nuclease activity, preferably colicin E1. More preferably, the first selection agent is streptomycin.

[0057] It is well known which first selection genes confer sensitivity to which of the above-mentioned first selection agents, however the following table lists preferred pairs.

[0058] [Table 1]

[0059] The term "second selection gene" refers to a gene encoding a gene product whose expression in a bacterial cell confers resistance to a second selection agent to the bacterial cell. A second selection gene may typically encode a protein that, when expressed in a bacterial cell, allows said bacterial cell to survive and grow in the presence of a second selection agent. In the absence of the protein encoded by the second selection gene, the bacterial cell dies or is inhibited from growing in the presence of said second selection agent. Suitable proteins may be enzymes, transporter proteins, etc. Preferably, the second selection gene, as referred to according to the present invention, is selected from the group consisting of kmR, zeoR, ampR, tetR, cmR, hygR, specR, and BSD. More preferably, the second selection gene is kmR or zeoR. However, the skilled artisan is well aware that other resistance genes, preferably antibiotic resistance genes, can be used in a similar manner.

[0060] The term "second selection agent" as used herein refers to a compound that is detrimental to the viability and / or growth of bacterial cells that do not express a second selection gene that confers resistance to the second selection agent. Preferably, the second selection agent is selected from the group consisting of kanamycin, zeocin, ampicillin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin. More preferably, the second selection agent is kanamycin or zeocin. However, the skilled artisan is well aware that other resistance genes, preferably antibiotic resistance genes, can be used in a similar manner. Moreover, the skilled artisan is well aware that in the described process, the first and second selection agents must be different from each other.

[0061] It is well known which first selection genes confer sensitivity to which of the above-mentioned first selection agents, however the following table lists preferred pairs.

[0062] [Table 2]

[0063] The term "exchanging" as used herein refers to any technique that results in replacing the endogenous promoter of at least one essential gene by a first polynucleotide comprising a selection cassette comprising a promoter that may be active in a bacterial cell, operably linked to (i) a first selection gene that confers sensitivity to a first selection agent, such that the bacterial cell does not grow and / or dies in the presence of said first selection agent, and (ii) a second selection gene that confers resistance to said second selection agent, such that the bacterial cell grows in the presence of a second selection agent, such that the expression of the at least one essential gene, the first and the second selection genes becomes dependent on said promoters. Preferably, such exchange can be performed by using site-specific or random recombination. It is therefore understood that the first polynucleotide may comprise sequences of the bacterial genome that naturally occur upstream and downstream of the endogenous promoter of at least one essential gene, upstream of the promoter that may be active in a bacterial cell, and downstream of the second selection gene that confers resistance to the second selection agent. Said first polynucleotide is typically integrated into the genome of the bacterial cell by using the CRISPR-Cas-9 system, lambda Red recombination, site-specific or random recombination techniques, in particular lambda Red recombination (also called Red / ET recombination or Red / ET recombineering), which are described in more detail elsewhere herein. How to carry out such recombination techniques is well known to those skilled in the art.

[0064] It will be appreciated that viability and / or growth of the recombinant bacterial cell after carrying out the aforementioned step (a) of the method of the invention will become dependent on the expression of a second selection gene conferring resistance to a second selection agent such that the bacterial cell is capable of growing and / or surviving in the presence of said second selection agent.

[0065] Bacterial cells that have properly integrated the first polynucleotide into their genome can be selected by culturing the recombinant bacterial cells in the presence of a second selection agent in step (b). The selection is typically carried out by culturing the bacterial cells in the presence of a selection agent for a time and under conditions that allow selection. Exemplary conditions and times are described in the accompanying examples and can be determined by the skilled artisan without further ado.

[0066] In step (c), the first polynucleotide integrated into the genome is replaced by a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of at least one essential gene becomes dependent on said heterologous expression sequence.

[0067] Bacterial cells that have properly integrated said second polynucleotide into their genome can be selected by culturing the recombinant bacterial cells in the presence of an inducer molecule and a first selection agent, which in this context interferes with the viability and / or growth of bacterial cells in which the first polynucleotide has not been replaced by the second polynucleotide and which therefore still express the first selection gene that confers sensitivity to the first selection agent.

[0068] Preferably, said selection cassette further comprises a restriction enzyme cleavage site at the 3' end of the second selection gene. Preferably, the selection of bacterial cells that have properly integrated the second polynucleotide can be further improved by treating the cells with a restriction enzyme that cleaves the first polynucleotide at the cleavage site at the 3' end of the second selection gene. Thus, the method of the present invention further comprises, after step (c), treating the bacterial cells with a restriction enzyme such that the genome of the bacterial cells still comprising the first polynucleotide comprising the selection cassette is cleaved between the second selection gene and at least one essential gene, while the genome of the bacterial cells in which the first polynucleotide comprising the selection cassette integrated in the genome has been replaced by a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that the expression of the at least one essential gene becomes dependent on said heterologous expression sequence remains unaffected by the restriction enzyme. More preferably, said restriction enzyme is selected from the group consisting of I-SceI, I-CeuI, I-PpoI, PI-SceI and PI-PspI. More preferably, the restriction enzyme is I-SceI.

[0069] Preferably, the bacterial cells are cultured in the presence of inducer molecules in and after step (d). By culturing recombinant bacterial cells in the presence of inducer molecules, a cell culture of recombinant bacterial cells can be generated that can be used as a common platform for the production of various target compounds. Depending on the desired target compound, the recombinant bacterial cells of said culture may be individually transformed with an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence biologically active in said bacterial cells, said expression plasmid comprising a target nucleic acid required for the production of the target compound. Successful transformants grow in the absence of inducer molecules, whereas untransformed bacterial cells require the presence of inducer molecules for growth and / or survival.

[0070] Particular preferred details for carrying out the above-described methods can be found in the accompanying Examples below.

[0071] The present invention also provides (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a selection gene that confers resistance to a selective agent, flanked by recognition sites for site-specific recombination in bacterial cells; and (ii) a heterologous expression control sequence inducible by an inducer molecule; such that expression of at least one essential gene becomes dependent on said heterologous expression control sequence and such that the bacterial cell grows in the presence of said inducer molecule and in the presence of said selection agent, (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent and an inducer molecule; (c) removing the selection gene by using a recombinase capable of performing site-specific recombination using recognition sites flanking the selection gene such that expression of at least one essential gene remains dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide that lacks a selection gene. The present invention also encompasses a method for producing a recombinant bacterial cell of the present invention, comprising:

[0072] The term "selection gene" refers to a gene encoding a gene product whose expression in a bacterial cell confers resistance to a selection agent to the bacterial cell. A selection gene may typically encode a protein that, when expressed in a bacterial cell, allows said bacterial cell to survive and grow in the presence of a second selection agent. In the absence of the protein encoded by the second selection gene, the bacterial cell dies or is inhibited from growing in the presence of said selection agent. Suitable proteins may be enzymes, transporter proteins, etc. Preferably, said selection gene in the aforementioned method of the invention is selected from the group consisting of ampR, kmR, zeoR, tetR, cmR, hygR, specR, and BSD. More preferably, the selection gene in the aforementioned method is ampR. The skilled artisan will know that other selection genes for antibiotics, toxins or other killing agents can be used in a similar manner.

[0073] The term "selection agent" as used herein refers to a compound that is detrimental to the viability and / or growth of bacterial cells that do not express a selection gene that confers resistance to the selection agent. Preferably, said selection agent in the aforementioned method of the present invention is selected from the group consisting of ampicillin, kanamycin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin. More preferably, the selection agent in the aforementioned method is ampicillin.

[0074] The term "recognition site for site-specific recombination" as used herein refers to a DNA sequence that is specifically recognized by an enzyme capable of mediating site-specific recombination. Such an enzyme is typically a recombinase, such as a Cre-recombinase-based system (Cre / loxP), a Dre-recombinase-based system (Dre / rox), a FLP-FRT-based system or a transposon-based technique. Typically, such recognition sites are located in DNA segments that flank the DNA segments that will be affected by the recombination event, thus marking the DNA region affected by recombination. Preferably, said recognition site for site-specific recombination in bacterial cells is a FRT sequence. Also preferably, said recombinase is a flippase (FLP) recombinase. Alternatively, the recognition site for site-specific recombination in bacterial cells is preferably a loxP sequence and the recombinase is preferably a Cre recombinase. The skilled person is well aware how to use recognition sites for site-specific recombination with a suitable recombinase in the method of the present invention.

[0075] In step (a) of the aforementioned method of the present invention, the endogenous promoter of at least one essential gene in the genome of the bacterial cell is replaced by a polynucleotide comprising (i) a selection gene conferring resistance to a selection agent, flanked by recognition sites for site-specific recombination in the bacterial cell, and (ii) a heterologous expression control sequence inducible by an inducer molecule. Preferably, such replacement can be performed by using Lambda Red recombination (also called Red / ET recombination or Red / ET recombineering) or the CRISPR-Cas-9 system. It is understood that if site-specific recombination is used for the replacement in step (a), preferably a different recognition site than that present in the polynucleotide referred to in step (a) and a different recombinase than that used in step (c) shall be used. How to perform such recombination techniques is well known to the skilled person. As a result of the successful replacement performed in step (a), the expression of the at least one essential gene shall become dependent on the heterologous expression control sequence and the bacterial cell shall grow in the presence of the inducer molecule and in the presence of the selection agent.

[0076] Thus, in step (b) of the method, bacterial cells that have integrated said polynucleotide into their genome are selected using a selection agent and an inducer molecule. Said selection is typically carried out by culturing bacterial cells in the presence of a selection agent and an inducer molecule for a time and under conditions that allow selection. Exemplary conditions and times are described in the accompanying examples and can be determined by the skilled artisan without further ado. As a result of removal of the selection gene, expression of at least one essential gene shall remain dependent on said heterologous expression control sequence.

[0077] After selection of bacterial cells that have properly integrated the polynucleotide described above, the segment of the polynucleotide containing the selection gene is removed in step (c) of the method by using a recombinase capable of performing site-specific recombination using recognition sites flanking the selection gene. As a result of removal of the selection gene, expression of at least one essential gene shall remain dependent on the heterologous expression control sequence.

[0078] Since the heterologous expression control sequence is inducible by an inducer molecule, bacterial cells that have integrated into their genome the polynucleotide lacking the selection gene can be selected in step (d) using the inducer molecule. Said selection is typically carried out by culturing the bacterial cells in the presence of the inducer molecule for a time and under conditions that allow selection. Typical conditions and times are described in the accompanying examples and can be determined by the skilled artisan without further ado.

[0079] Particular preferred details for carrying out the above-described methods can be found in the accompanying Examples below.

[0080] Furthermore, the present invention relates to a method for producing a (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a heterologous expression control sequence inducible by an inducer molecule; (ii) a promoter that can be active in a bacterial cell, and (iii) a selection gene that confers resistance to a selection agent; (ii) a promoter that can be active in a bacterial cell and (iii) a selection gene, wherein the segment of the polynucleotide comprising the selection gene is flanked by recognition sites for site-specific recombination in the bacterial cell, while the segment of the polynucleotide comprising the heterologous expression control sequence is not flanked by said recognition sites; (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent; (c) removing a segment of the polynucleotide comprising a promoter and a selection gene that may be active in the bacterial cell by using a recombinase capable of performing site-specific recombination using recognition sites flanking a segment of the polynucleotide such that expression of at least one essential gene becomes dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide lacking the segment flanked by the recognition sites. The present invention also relates to a method for producing the recombinant bacterial cell of the present invention, comprising:

[0081] Preferably, the selection gene in the aforementioned method of the present invention is selected from the group consisting of kmR, ampR, zeoR, tetR, cmR, hygR, specR and BSD. More preferably, the selection gene in the aforementioned method is kmR.

[0082] Preferably, the selection agent in the aforementioned method of the present invention is selected from the group consisting of kanamycin, ampicillin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin. More preferably, the selection agent in the aforementioned method is kanamycin.

[0083] In step (a) of the aforementioned method of the present invention, the endogenous promoter of at least one essential gene is transduced by a polynucleotide comprising (i) a heterologous expression control sequence inducible by an inducer molecule, (ii) a promoter that can be active in a bacterial cell, and (iii) a selection gene that confers resistance to a selection agent. The segment of the polynucleotide comprising (ii) the promoter that can be active in a bacterial cell and (iii) the selection gene is flanked by recognition sites for site-specific recombination in a bacterial cell, while the segment of the polynucleotide comprising the heterologous expression control sequence is not flanked by said recognition sites. Preferably, the exchange in step (a) of the method can be carried out by using Lambda Red recombination (also called Red / ET recombination or Red / ET recombineering) or the CRISPR-Cas-9 system. It is understood that if site-specific recombination is used for the exchange in step (a), preferably a recognition site different from that present in the polynucleotide referred to in step (a) and a recombinase different from the recombinase used in step (c) shall be used. How to carry out such recombination techniques is well known to those skilled in the art. As a result of the successful exchange carried out in step (a), expression of at least one essential gene in the bacterial cell becomes dependent on a promoter that may be active in the bacterial cell.

[0084] Thus, in step (b) of the method, bacterial cells which have integrated said polynucleotide into their genome are selected using a selection agent. Said selection is typically carried out by culturing the bacterial cells in the presence of the selection agent for a time and under conditions which allow selection. Exemplary conditions and times are described in the accompanying examples and can be determined by the skilled artisan without further ado.

[0085] After selection of bacterial cells that have properly integrated the polynucleotide described above, the segment of the polynucleotide comprising the selection gene and the promoter that can be active in the bacterial cell is removed in step (c) of the method by using a recombinase capable of performing site-specific recombination using recognition sites flanking said segment. As a result of the removal of the selection gene and the promoter that can be active in the bacterial cell (i.e. the segment of the polynucleotide flanked by the recognition sites), the expression of at least one essential gene shall become dependent on the heterologous expression control sequence.

[0086] Since the heterologous expression control sequence is inducible by an inducer molecule, bacterial cells that have integrated into their genome the polynucleotide lacking the segment flanked by the recognition sites can be selected in step (d) using the inducer molecule. Said selection is typically carried out by culturing the bacterial cells in the presence of the inducer molecule for a time and under conditions that allow selection. Typical conditions and times are described in the accompanying examples and can be determined by the skilled artisan without further ado.

[0087] Particular preferred details for carrying out the above-described methods can be found in the accompanying Examples below.

[0088] The present invention also provides (a) introducing into a recombinant bacterial cell of the invention an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell; and (b) culturing the recombinant bacterial cells obtained in step a) in the absence of inducer molecules. The present invention relates to a method for recombinantly producing a compound of interest, comprising the steps of:

[0089] The term "expression control sequence biologically active in said bacterial cell" refers to any promoter naturally occurring in a bacterial cell or any heterologous or artificial promoter biologically active in said cell. Preferably, said expression control sequence biologically active in a bacterial cell may be a promoter biologically active in a bacterial cell as defined elsewhere herein. Alternatively, said expression control sequence may be a promoter of an essential gene mentioned herein, such as the infA, secY or frr promoter, or any other promoter of an essential gene disclosed herein.

[0090] Preferably, said biologically active expression control sequence is an expression control sequence naturally associated with at least one endogenous essential gene, details of which can be found elsewhere herein.

[0091] Preferably, said expression plasmid comprises at least one additional nucleic acid of interest required for the recombinant production of a compound of interest in said recombinant bacterial cell.

[0092] In step (a) of the above method, an expression plasmid containing at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence biologically active in said bacterial cell, is introduced into said cell. Typically, the expression plasmid can be introduced by any conventional transformation method, such as electroporation, TSS transformation technique, CaCl transformation technique or RbCl transformation technique, or virus-mediated transduction. Those skilled in the art are well aware of how such transformation techniques can be carried out.

[0093] Once the expression plasmid has been introduced, for example by any of the aforementioned transformation techniques, the recombinant bacterial cells are cultured in step (b) of the aforementioned method of the invention in a suitable culture medium free of antibiotics and under suitable culture conditions. In particular, the culture medium shall be free of inducer molecules, in order to achieve selection of recombinant bacterial cells that have successfully taken up the expression plasmid. Furthermore, the culture conditions shall typically allow recombinant production of the compound of interest by the recombinant bacterial cells being cultured. As described elsewhere herein, this may require application of an expression stimulus to said cultured cells. However, the compound of interest may also be constitutively produced in the recombinant bacterial cells. Preferably, the culture is carried out until a desired level of the compound of interest is produced by the recombinant bacterial cells in the culture and by said cells, after which the cells, and / or the culture medium used to culture the cells, are harvested to obtain the compound of interest. Particularly preferred techniques are described in the examples below.

[0094] The cultivation of recombinant bacterial cells may also include several cultivation steps: for example, to avoid a lag phase of the cells in the bioreactor culture or at least to shorten said lag phase in the bioreactor, the cultivation may include culturing the recombinant bacterial cells in a preculture until the cells grow exponentially, and then inoculating the culture in a production vial, such as a bioreactor, with said preculture.

[0095] Such a culturing step may include, but is not limited to, culturing the bacterial cells on a solid growth medium or growing in a liquid medium. Such a medium and / or culture may be (i) a minimal medium containing the bare minimum required for the growth of a wild-type organism (e.g., containing only the nutrients essential for the growth of bacterial cells, such as water, N and / or C sources, vitamins, and various salts containing the essential elements required for protein and nucleic acid synthesis), (ii) a defined medium, which is fully understood in its composition but generally does not contain a complex nutrient composition, such as yeast extract, or (iii) a complex medium, in which the types and amounts of nutrients are not defined, such as yeast extract, peptone from plants or animals, tryptone, corn steep liquor, or similar protein sources. Any medium may be used for the expression of genes according to the present invention.

[0096] A culture medium, as referred to herein, is preferably a solution containing compounds required for proper growth and viability of the recombinant bacterial cells of the present invention. Typically, a culture medium, as referred to herein, contains compounds that act as nitrogen and / or carbon sources for the bacterial cells. Said compounds may be chemically defined compounds mixed together or may be obtained as non-chemically defined mixtures from biological sources. Chemically defined compounds useful in culture media may be carbohydrates, organic acids, hydrocarbons, alcohols or mixtures thereof. Preferred carbohydrates are selected from the group consisting of glucose, fructose, sucrose, galactose, xylose, arabinose, sucrose, maltose, maltotriose, lactose, dextrins, maltodextrins, starch and inulin, and mixtures thereof. Preferred alcohols are selected from the group consisting of glycerol, methanol and ethanol, inositol, mannitol and sorbitol, and mixtures thereof. Preferred organic acids are selected from the group consisting of acetic acid, propionic acid, lactic acid, formic acid, malic acid, citric acid, fumaric acid and higher alkanoic acids and mixtures thereof. The chemically undefined mixture of compounds may be an extract from or produced from microbial, animal or plant materials or cells, such as vegetable protein preparations, soybean meal, corn meal, pea meal, corn gluten, corn steep liquor, cotton meal, peanut meal, potato meal, meat, casein, gelatin, whey, fish meal, yeast protein, yeast extract, tryptone, peptone, bacto-tryptone, bacto-peptone, waste from the processing of microbial cells, plants, meat or animal bodies, and combinations thereof.Depending on the recombinant bacterial cell to be used, i.e. a platform recombinant bacterial cell, whose growth and / or viability depends on the presence or a certain amount of an inducer molecule in the culture medium, or a production bacterial cell obtained after transformation with an expression plasmid as specified herein, whose growth and / or viability in the absence of an inducer molecule in the culture medium depends on the correct uptake of the expression plasmid, the culture medium to be used according to the present invention may or may not comprise further components such as an inducer molecule.

[0097] A bioreactor, as referred to herein, is a vessel in which large-scale cell cultivation and large-scale production of a desired product takes place. After completion of the cultivation in the production bioreactor, the fermentation broth is harvested and the desired product is recovered. The bioreactor may contain inlets and outlets, for example for the medium, and various sensors, for example for measuring pH and temperature during the fermentation process. The fermentation medium in the production bioreactor may be the same or different from the fermentation medium used in the seed fermenter or the last seed fermenter in the seed train. The production bioreactor may have a volume of 500L, 1,000L, 5,000L, 10,000L, 20,000L, 50,000L, or 100,000L or more.

[0098] The previously mentioned culture may be carried out in a batch mode, in which the cells are cultured in the culture medium that is initially present, without changing the medium composition or the volume of the medium. Thus, in the batch mode, a substantial or significant amount of fresh liquid culture medium is not added to the cell culture, and a substantial or significant amount of liquid culture medium is not removed from the cell culture during the culture. Alternatively, the culture may be carried out in a fed-batch mode, in which the cells are cultured in the culture medium that is initially present, and a feed solution is added periodically or continuously, without substantially or significantly removing the liquid culture medium during the culture. The fed-batch culture may include various feeding regimes and times, such as daily feeding, feeding more than once per day, or feeding less than once per day. Furthermore, alternatively, the culture may be carried out in a continuous fermentation mode, in which the cells are cultured in the culture medium that is initially present, and fresh culture medium is continuously fed to the bioreactor, and the fermented culture medium is removed from the bioreactor at the same rate, so that the volume in the bioreactor is constant. The skilled person knows that such batch, semi-batch or continuous fermentation processes can be carried out without nutrient limitation during the fermentation process, but typically such processes are carried out by limiting at least one nutrient during or during a certain period of the fermentation, typically such limitation is carried out, for example, by limiting the C, N and / or P source or by limiting oxygen.

[0099] Thus, in a preferred embodiment of the aforementioned method of the present invention, said method comprises the steps of: (c) obtaining the compound of interest produced by the recombinant bacterial cell. Further includes:

[0100] After the aforementioned method of the invention has been carried out, the compound of interest may be obtained by purification from the culture medium used for the cultivation or from the cultivated recombinant bacterial cell(s). Typically, the fermentation broth and the bacterial cells are separated from each other, for example by centrifugation, sedimentation or filtration techniques.

[0101] If the compound of interest is present in the culture medium, further purification steps may depend on the chemical nature and properties of the compound of interest. Such purification steps may include affinity chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic chromatography, chromatofocusing chromatography, and size exclusion chromatography), extraction, electrophoresis (e.g., preparative isoelectric focusing), filtration, evaporation, spray drying, precipitation (e.g., using ammonium sulfate) or crystallization, or a combination thereof.

[0102] When the compound of interest is contained in a recombinant bacterium, release of the compound of interest from said cells may be required. Release from the cells can be achieved, for example, by cell lysis using techniques well known to those skilled in the art, such as lysozyme treatment, sonication, French press or a combination thereof. The compound of interest released from the recombinant bacterial cells can then be further purified by the purification steps mentioned above. Preferably, for the purification of pDNA vectors, several types of chromatographic methods (e.g., hydrophobic interaction, anion exchange, multimodal, affinity) can be applied after primary isolation. Details of said methods are well known in the art (see, for example, Alves et al. 2021 and references therein).

[0103] The method of the present invention may also include further steps to formulate the desired compound in a desired form, which may include the production of granules, tablets, powders, solutions, gels, gaseous formulations, any 3D items, nanoparticles, coatings, etc. from the compound of interest.

[0104] Depending on the nature of the target compound, the formulation of the target compound may be carried out under certain standard conditions, such as GMP.

[0105] The present invention also relates generally to the use of the recombinant bacterial cells of the invention for the recombinant production of compounds of interest.

[0106] Furthermore, the present invention relates to a method for producing a (i) a recombinant bacterial cell of the invention, and (ii) an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell;

[0023] A kit for recombinant production of a compound of interest is provided, comprising:

[0107] The term "kit" as used herein refers to a collection of components required to carry out the method of the present invention for recombinant production of a compound of interest. The kit shall include a recombinant bacterial cell of the present invention and an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell. Preferably, said biologically active expression control sequence is an expression control sequence that is naturally associated with at least one endogenous essential gene. Also preferably, said expression plasmid comprises at least one additional nucleic acid of interest required for recombinant production of a compound of interest in said recombinant bacterial cell.

[0108] Typically, the components of the kit are provided in separate containers or in a single container. The container also typically contains instructions for carrying out the method of the invention for recombinant production of the compound of interest. Furthermore, the kit may preferably contain further components necessary for carrying out the method of the invention, such as culture media, washing solutions, solvents, and / or reagents or means required for purification of the compound of interest.

[0109] The following embodiments are particularly preferred embodiments envisaged according to the present invention: All definitions and explanations of terms made above apply mutatis mutandis.

[0110] Embodiment 1. A recombinant bacterial cell comprising in its genome at least one essential gene, wherein the endogenous promoter of said essential gene has been replaced by a heterologous expression control sequence inducible by an inducer molecule, such that expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule.

[0111] Embodiment 2: The recombinant bacterial cell according to embodiment 1, wherein said at least one essential gene is (i) an absolutely essential gene that is absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or is essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration, or (ii) a combination of essential genes that, in combination, are absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or are essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration.

[0112] Embodiment 3. The bacterial cell is selected from the group consisting of Escherichia, Vibrio, Bacillus, Lactobacillus, Acetobacter, Corynebacterium, Brevibacterium, Pseudomonas, Streptomyces, Gluconobacter, Clostridium, and the like. 3. The recombinant bacterial cell according to embodiment 1 or 2, which is from a genus selected from the group consisting of: Acinetobacter, Bacillus subtilis, Bacillus anguineus, Bacillus subtilis, Bacillus moniliforme, Bacillus subtilis ...

[0113] Embodiment 4. A recombinant bacterial cell according to any one of embodiments 1 to 3, wherein the at least one essential gene is a gene required for cell growth and / or viability, preferably selected from the group consisting of infA, infC / IF-3, dnaJ, dnaK, era, frr, ftsL, ftsN, ftsZ, grpE, mopA, mopB, msbA, nusG, parC, rpsB, secY and trma.

[0114] Embodiment 5. The recombinant bacterial cell of embodiment 4, wherein the at least one essential gene is infA, secY, or frr.

[0115] Embodiment 6. A recombinant bacterial cell according to any one of embodiments 1 to 5, wherein the bacterial genome lacks any gene capable of functionally complementing at least one essential gene in the absence of an inducer molecule.

[0116] Embodiment 7. A recombinant bacterial cell according to any one of embodiments 1 to 6, wherein the bacterial cell comprises an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence biologically active in the bacterial cell.

[0117] Embodiment 8. The recombinant bacterial cell of embodiment 7, wherein said biologically active expression control sequence is an expression control sequence naturally associated with at least one endogenous essential gene.

[0118] Embodiment 9. The recombinant bacterial cell of embodiment 7 or 8, wherein the expression plasmid comprises at least one additional nucleic acid of interest required for recombinant production of a compound of interest in the recombinant bacterial cell.

[0119] Embodiment 10. The recombinant bacterial cell of any one of embodiments 1-9, wherein the inducer molecule is selected from the group consisting of arabinose, rhamnose, xylose, sucrose, tetracycline, anhydrotetracycline, and IPTG.

[0120] Embodiment 11. (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a first selection gene that confers sensitivity to a first selection agent, such that the bacterial cell will not grow and / or die in the presence of said first selection agent; and (ii) a second selection gene that confers resistance to a second selection agent such that the bacterial cell can grow in the presence of said second selection agent. exchanging the at least one essential gene, the first and the second selection gene with a first polynucleotide comprising a selection cassette comprising a promoter capable of being active in a bacterial cell, operably linked to said first polynucleotide, such that expression of said at least one essential gene, the first and the second selection gene becomes dependent on said promoter; (b) using a second selection agent to select bacterial cells that have integrated the first polynucleotide into their genome; (c) replacing the first polynucleotide integrated in the genome with a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of at least one essential gene becomes dependent on the heterologous expression sequence; and (d) selecting bacterial cells that have integrated said second polynucleotide into their genome using the first selection molecule and an inducer molecule. 11. A method for producing a recombinant bacterial cell according to any one of embodiments 1 to 10, comprising:

[0121] Embodiment 12. The method of embodiment 11, wherein the first selection gene is selected from the group consisting of rpsL, sacB, pheS, tetR, galK, thyA, tolC and ccdB.

[0122] Embodiment 13. The method of any one of embodiments 11 or 12, wherein the first selective agent is selected from the group consisting of streptomycin, sucrose, chloro-phenylalanine, chlortetracycline and fusaric acid, galactose, trimethoprim and its derivatives, and a bacteriocin, preferably a colicin, preferably colicin A, B, E1, Ia, Ib, N, D, M or E3, more preferably colicin E1.

[0123] Embodiment 14. The method of any one of embodiments 11 to 13, wherein the second selection gene is selected from the group consisting of kmR, ampR, zeoR, tetR, cmR, hygR, specR, and BSD.

[0124] Embodiment 15. The method of any one of embodiments 11 to 14, wherein the second selective agent is selected from the group consisting of kanamycin, ampicillin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

[0125] Embodiment 16 The method of any one of embodiments 11 to 15, wherein the bacterial cells are cultured in the presence of an inducer molecule after step (d).

[0126] Embodiment 17. The method of any one of embodiments 11 to 16, wherein the selection cassette further comprises a restriction enzyme cleavage site at the 3' end of the second selection gene.

[0127] Embodiment 18. The method of embodiment 17, wherein the method further comprises, after step (c), treating the bacterial cell with a restriction enzyme such that the genome of the bacterial cell still comprising the first polynucleotide comprising the selection cassette is cut between the second selection gene and the at least one essential gene, while the genome of the bacterial cell in which the first polynucleotide comprising the selection cassette integrated in the genome has been replaced by a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of the at least one essential gene becomes dependent on said heterologous expression sequence remains unaffected by the restriction enzyme.

[0128] Embodiment 19. The method of embodiment 17 or 18, wherein the restriction enzyme is selected from the group consisting of I-SceI, I-CeuI, I-PpoI, PI-SceI, and PI-PspI.

[0129] EMBODIMENT 20. (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a selection gene that confers resistance to a selective agent, flanked by recognition sites for site-specific recombination in bacterial cells; and (ii) a heterologous expression control sequence inducible by an inducer molecule; such that expression of at least one essential gene becomes dependent on said heterologous expression control sequence and such that the bacterial cell grows in the presence of said inducer molecule and in the presence of said selection agent, (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent and an inducer molecule; (c) removing the selection gene by using a recombinase capable of performing site-specific recombination using recognition sites flanking the selection gene such that expression of at least one essential gene remains dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide that lacks a selection gene. 11. A method for producing a recombinant bacterial cell according to any one of embodiments 1 to 10, comprising:

[0130] Embodiment 21. The method of embodiment 20, wherein the selection gene is selected from the group consisting of ampR, kmR, zeoR, tetR, cmR, hygR, specR, and BSD.

[0131] Embodiment 22. The method of embodiment 20 or 21, wherein the selection agent is selected from the group consisting of ampicillin, kanamycin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

[0132] EMBODIMENT 23. (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a heterologous expression control sequence inducible by an inducer molecule; (ii) a promoter that can be active in a bacterial cell, and (iii) a selection gene that confers resistance to a selection agent; (ii) a promoter that can be active in a bacterial cell and (iii) a selection gene, wherein the segment of the polynucleotide comprising the selection gene is flanked by recognition sites for site-specific recombination in the bacterial cell, while the segment of the polynucleotide comprising the heterologous expression control sequence is not flanked by said recognition sites; (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent; (c) removing a segment of the polynucleotide comprising a promoter and a selection gene that may be active in the bacterial cell by using a recombinase capable of performing site-specific recombination using recognition sites flanking a segment of the polynucleotide such that expression of at least one essential gene becomes dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide lacking the segment flanked by the recognition sites. 11. A method for producing a recombinant bacterial cell according to any one of embodiments 1 to 10, comprising:

[0133] Embodiment 24 The method of embodiment 23, wherein the selection gene is selected from the group consisting of kmR, ampR, zeoR, tetR, cmR, hygR, specR, and BSD.

[0134] Embodiment 25. The method of embodiment 23 or 24, wherein the selection agent is selected from the group consisting of kanamycin, ampicillin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

[0135] Embodiment 26. The method of any one of embodiments 20 to 25, wherein the recognition site for site-specific recombination in a bacterial cell is an FRT or loxP sequence.

[0136] Embodiment 27. The method of any one of embodiments 20 to 26, wherein the recombinase is FLP recombinase or Cre recombinase.

[0137] Embodiment 28. The method of any one of embodiments 11 to 27, wherein the promoter that can be active in a bacterial cell is a constitutively active promoter or an inducible promoter in a bacterial cell.

[0138] Embodiment 29. The method of embodiment 28, wherein the promoter constitutively active in the bacterial cell is selected from the group consisting of the rpsL promoter, the infA promoter, the infAp2 promoter, the em7 promoter, and the cat promoter.

[0139] Embodiment 30. The method of embodiment 28, wherein the inducible promoter is selected from the group consisting of a heat-inducible promoter, a tetracycline-inducible promoter, a lacI / IPTG promoter, an ethanol-inducible promoter (AlcA / AlcR), a steroid-inducible promoter (LexA / XVE), and a vanillate-inducible promoter.

[0140] Embodiment 31. The recombinant bacterial cell of any one of embodiments 1 to 10 or the method of any one of embodiments 11 to 30, wherein the heterologous expression control sequence inducible by an inducer molecule is selected from the group consisting of AraC / PBAD promoter, RhaR-RhaS / rhaBAD promoter, XylS / Pm promoter, NitR / PnitA promoter, ChnR / Pb inducer / promoter, tetracycline-inducible promoter (tetON / OFF), lacI / IPTG promoter, ethanol-inducible promoter (AlcA / AlcR), steroid-inducible promoter (LexA / XVE), and vanillate-inducible promoter.

[0141] Embodiment 32. The method of any one of embodiments 11 to 31, wherein the inducer molecule is selected from the group consisting of arabinose, rhamnose, xylose, sucrose, tetracycline, anhydrotetracycline, and IPTG.

[0142] EMBODIMENT 33. (a) introducing into a recombinant bacterial cell according to any one of the preceding claims an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell; and (b) culturing the recombinant bacterial cells obtained in step a) in the absence of inducer molecules. A method for recombinantly producing a compound of interest, comprising:

[0143] Embodiment 34 The method of embodiment 33, wherein the expression plasmid comprises at least one additional nucleic acid of interest required for recombinant production of a compound of interest in the recombinant bacterial cell.

[0144] Embodiment 35 The method further comprises: (c) obtaining the compound of interest produced by the recombinant bacterial cell. 35. The method of embodiment 33 or 34, further comprising:

[0145] Embodiment 36 The method of any one of embodiments 33 to 35, wherein the biologically active expression control sequence is an expression control sequence naturally associated with at least one endogenous essential gene.

[0146] Embodiment 37. Use of a recombinant bacterial cell according to any one of embodiments 7 to 10 for the recombinant production of a compound of interest.

[0147] 38. (i) a recombinant bacterial cell according to any one of embodiments 1 to 6, and (ii) an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in said bacterial cell; A kit for recombinant production of a compound of interest comprising:

[0148] Embodiment 39. The kit of embodiment 38, wherein the biologically active expression control sequence is an expression control sequence naturally associated with at least one endogenous essential gene.

[0149] Embodiment 40. The kit of embodiment 38 or 39, wherein the expression plasmid comprises at least one additional nucleic acid of interest required for the recombinant production of a compound of interest in the recombinant bacterial cell.

[0150] All references cited herein are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned herein. EXAMPLES

[0151] The following examples are intended merely to illustrate the invention and are not to be construed in any way as limiting its scope.

[0152] The sequence numbers referred to below indicate: SEQ ID NO:1: "rpsL-zeoR" cassette: 219 bp SEQ ID NO: 2: PCR primer "CS-primer up": 86 bp SEQ ID NO: 3: PCR primer "CS-primer down": 77bp SEQ ID NO: 4: "araC-araBp-infA" knock-in cassette: 1442 bp SEQ ID NO: 5: PCR primer "CS2-primer up": 70bp SEQ ID NO: 6: PCR primer "CS2-primer down": 17bp SEQ ID NO: 7: Genomic infA locus after modification (aat-infA segment): 2064 bp SEQ ID NO: 8: "FRT-ampR-FRT-araC-araBp" knock-in cassette: 2682 bp SEQ ID NO: 9: PCR primer "FRT-primer up": 70 bp SEQ ID NO: 10: PCR primer "FRT-primer down": 22bp SEQ ID NO: 11: Modified genomic infA locus (aat-infA segment): 2184 bp SEQ ID NO: 12: "araC-araBp-FRT-kmR-FRT" knock-in cassette: 2647 bp SEQ ID NO: 13: PCR primer "FRT2-primer up": 22 bp SEQ ID NO: 14: Modified genomic infA locus (aat-infA segment): 2114 bp SEQ ID NO: 15: E. coli infA gene: 219 bp SEQ ID NO: 16: E. coli secY gene: 1332 bp SEQ ID NO: 17: Plasmid "pQE-T7 TNF alpha": 5746bp SEQ ID NO: 18: PCR primer "infA-primer up": 68 bp SEQ ID NO: 19: PCR primer "infA-primer down": 77 bp SEQ ID NO: 20: "infA-cmR" expression cassette: 1327 bp SEQ ID NO: 21: Plasmid "pQE-T7 TNFα cmR-infA": 6036 bp SEQ ID NO: 22: Plasmid "pQE-T7 TNFα infA": 5289 bp SEQ ID NO: 23: Plasmid "GenH015-plasmid1-cmR": 3184 bp SEQ ID NO: 24: Plasmid "GenH015-plasmid2-cmR": 3417bp SEQ ID NO: 25: Plasmid "GenH015-plasmid3-cmR": 3596 bp SEQ ID NO: 26: Plasmid "GenH015-plasmid1": 2437bp SEQ ID NO: 27: Plasmid "GenH015-plasmid2": 2670bp SEQ ID NO: 28: Plasmid "GenH015-plasmid3": 2849 bp SEQ ID NO: 29: araC-araBp-FRT-zeoR-FRT Knock-in cassette for the frr locus: 2423 bp SEQ ID NO: 30: PCR primer "FRT-frr-primer up": 19 bp SEQ ID NO: 31: PCR primer "FRT-frr-primer down": 23bp SEQ ID NO: 32: Genomic frr locus after modification (pyrH-araC-araBp-FRT-zeoR-FRT-frr segment): 3432 bp SEQ ID NO: 33: Genomic frr locus after FLP recombination (pyrH-araC-araBp-FRT-frr segment): 2888 bp SEQ ID NO: 34: "zeoR-frr" expression cassette: 1484 bp SEQ ID NO: 35: Plasmid "pQE-T7 TNFα zeoR-frr": 6176 bp SEQ ID NO: 36: Plasmid "pQE-T7 TNFα frr": 5604 bp SEQ ID NO: 37: E. coli frr gene: 558 bp

[0153] Example 1: Preparation of a platform strain with an arabinose-inducible infA gene a) Preparation of platform strains by direct replacement of the endogenous infA promoter with an arabinose-inducible promoter using the rpsL-zeo counterselection system An rpsL-based counterselection system was applied to replace the endogenous infA promoter with an arabinose-inducible promoter. This selection and counterselection system was developed by Gene Bridges ("Counter-Selection BAC Modification Kit" Gene Bridges, Cat. No. K002). It takes advantage of the fact that the S12 ribosomal protein is the target of the antibiotic streptomycin. Mutations in the rpsL gene that encodes this protein cause resistance to high concentrations of streptomycin (Chumpolkulwong, N. et al. 2004). Resistance is recessive in merodiploid strains. When both wild-type and mutant alleles of rpsL are expressed in the same strain, the strain is sensitive to streptomycin. Most of the commonly used E. coli strains (e.g., DH10B, HS996, TOP10) carry mutations in the rpsL gene that result in streptomycin resistance.

[0154] Other strains can be readily converted to a streptomycin-resistant phenotype by introducing the required point mutation (A / G transition at position 42; K42R) in the rpsL gene by Red / ET recombination (Zhang, Y. et al. 2000; Murphy, KC, Campellone, KG and Poteete, AR 2000).

[0155] aa) Replacement of the endogenous infA promoter with the rpsL-zeo counterselection cassette A 1026 bp DNA fragment (SEQ ID NO: 1) consisting of the rpsL gene and its promoter linked to the Zeocin resistance marker gene and flanked by 50 bp long homology arms to the infA genomic locus was amplified using primers CS-primer up (SEQ ID NO: 2) and CS-primer down (SEQ ID NO: 3).

[0156] Using Red / ET recombination (Zhang, Y. et al. 2000; Murphy, KC, Campellone, KG and Poteete, AR 2000), the resulting PCR product was inserted upstream of the infA start codon of E. coli strain HS996, replacing the endogenous promoter. The recombineering reaction was carried out as described in the manual for the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Cat. No. K006). Selection for cells with chromosomal integration of the cassette was carried out on LB agar plates containing 25 μg / ml Zeocin. Correct knock-in was confirmed by PCR across the modified infA locus and subsequent sequencing of the amplicon. Furthermore, cells were analyzed for their ability to grow on LB agar plates containing 200 μg / ml streptomycin. Only colonies sensitive to streptomycin were selected for the subsequent counterselection step.

[0157] ab) Replacement of the rpsL-zeo counterselection cassette with the araC-araBp cassette to obtain an arabinose-inducible infA gene. A 1442 bp sized DNA fragment consisting of the araC gene together with the araB promoter and 50 bp / 119 bp long homology arms to the infA genomic locus at the ends was amplified using primers CS2-primer up (SEQ ID NO: 5) and CS2-primer down (SEQ ID NO: 6). The resulting PCR product (SEQ ID NO: 4) was inserted between the aat and infA genes by Red / ET recombination. The sequence of the genomic locus after insertion of the arabinose-inducible promoter is given in SEQ ID NO: 7.

[0158] The Red / ET recombination step was carried out according to the manufacturer's instructions, except that 0.4% L-arabinose was added to the medium immediately after transformation of the cells to induce expression of infA by the araB promoter. Cells were grown for 2 h at 37° C. in 1 mL of LB medium supplemented with 0.4% L-arabinose but without the addition of antibiotics.

[0159] Selection for cells with chromosomal integration of the cassette was performed on LB agar plates containing 200 μg / ml streptomycin and 0.4% L-arabinose. The plates were incubated overnight at 37° C. The next day, single colonies were patched onto two different LB agar plates, one with 0.4% L-arabinose and 25 μg / ml Zeocin, and the other with 0.4% L-arabinose only. Colonies sensitive to Zeocin were further analyzed. Correct knock-in of the araC-araBp cassette was confirmed by PCR and subsequent sequencing of the amplicon.

[0160] ac) Growth control of platform strains Cells were streaked onto LB agar plates with and without the addition of 0.4% L-arabinose to confirm that the resulting platform strain grows only in the presence of L-arabinose (Figure 4). The E. coli strain "HS996 araC-araBp-infA" is based on the E. coli K12 strain HS996. The platform strain does not carry an antibiotic resistance marker gene in the genome. Resistance to streptomycin used in the above protocol does not come from an antibiotic resistance gene, but rather is the result of a K43T point mutation in the endogenous ribosomal S12.

[0161] b) Preparation of platform strains with the aid of a selection marker flanked by recognition sites for site-specific recombinases Replacement of the endogenous infA promoter with an arabinose-inducible promoter can be performed using a DNA fragment consisting of araC-araBp linked to a selectable marker flanked by FRT sites (FLP recombination target sites). An FRT site is a 34-bp DNA sequence containing two 13-bp palindromes separated by an asymmetric 8-bp core. A site-specific recombinase ("FLP") originally isolated from Saccharomyces cerevisiae mediates recombination between the two FRT sites such that the intervening DNA fragment (e.g., the selectable marker) is deleted, leaving behind a single 34-bp FRT site (Kilby et al. 1993). To replace the endogenous infA promoter with an arabinose-inducible promoter, an FRT-flanked selectable marker was ligated either upstream or downstream of the arabinose-inducible promoter sequence, and the DNA fragment was inserted into the genome by Red / ET recombination at the infA promoter locus. After replacement of the endogenous infA promoter, an FLP recombination step was performed to remove the antibiotic resistance marker gene, leaving one FRT site in the genome either upstream of the inducible promoter or downstream between the promoter and the start codon of infA.

[0162] b) Replacement of the endogenous infA promoter with the FRT-ampR-FRT-araC-araBp cassette A DNA fragment 2682 bp in size (SEQ ID NO: 8) consisting of the FRT-flanked ampR resistance marker gene linked to araC-araBp flanked by homology arms to the infA genomic locus was amplified using primers FRT-primer up (SEQ ID NO: 9) and FRT-primer down (SEQ ID NO: 10).

[0163] Using Red / ET recombination (Zhang, Y. et al. 2000; Murphy, KC, Campellone, KG and Poteete, AR 2000), the resulting PCR product was inserted upstream of the infA start codon of E. coli strain HS996, replacing the endogenous promoter. The recombineering reaction was carried out as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Cat. No. K006), except that 0.4% L-arabinose was added to the medium immediately after transformation of the cells with the DNA fragment (SEQ ID NO: 8) to induce expression of infA. The cells were grown in 1 mL of LB medium supplemented with 0.4% L-arabinose at 37°C for 2 hours. Selection for cells with chromosomal integration of the cassette was carried out on LB agar plates containing 0.4% L-arabinose and 50 μg / ml ampicillin. Correct knock-in was confirmed by PCR across the modified infA locus and subsequent sequencing of the amplicon.

[0164] The ampicillin resistance marker was subsequently removed by FLP recombination as described in section bc. The sequence of the genomic locus after insertion of the arabinose-inducible promoter and FLP recombination is given in SEQ ID NO:11.

[0165] bb) Replacement of the endogenous infA promoter with the araC-araBp-FRT-kmR-FRT cassette A 2647 bp sized DNA fragment (SEQ ID NO: 12) consisting of araC-araBp linked to the FRT-flanked kmR resistance marker gene and flanked by homology arms to the infA genomic locus was amplified using primers FRT2-primer up (SEQ ID NO: 13) and FRT-primer down (SEQ ID NO: 10).

[0166] Using Red / ET recombination (Zhang, Y. et al. 2000; Murphy, KC, Campellone, KG and Poteete, AR 2000), the resulting PCR product was inserted upstream of the infA start codon of E. coli strain T7E2, replacing the endogenous promoter. The recombineering reaction was carried out as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Cat. No. K006), except that 0.4% L-arabinose was added to the medium immediately after transformation of the cells with the DNA fragment (SEQ ID NO: 12) to induce expression of infA. The cells were grown in 1 mL of LB medium supplemented with 0.4% L-arabinose at 37°C for 2 hours. Selection for cells with chromosomal integration of the cassette was carried out on LB agar plates containing 0.4% L-arabinose and 15 μg / ml kanamycin. Correct knock-in was confirmed by PCR across the modified infA locus and subsequent sequencing of the amplicon.

[0167] The same protocol was applied to prepare further platform strains based on E. coli BL21(DE3), W3110, TG10 and HS996 pir+.

[0168] The kanamycin resistance marker was subsequently removed by FLP recombination as described in section bc. The sequence of the genomic locus after insertion of the arabinose-inducible promoter and FLP recombination is given in SEQ ID NO: 14.

[0169] bc) Removal of resistance marker genes by FLP recombination The FLP recombination steps were carried out as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Catalog No. K006), except that 0.4% L-arabinose was added to the medium during all steps. The FLP expression plasmid "708-FLPe cmR" (Gene Bridges, Catalog No. A105) was selected for removal of the resistance marker.

[0170] Cells were transformed with the FLP expression plasmid and grown for 2 h at 30° C. in 1 mL of LB medium supplemented with 0.4% L-arabinose but without the addition of antibiotics. Selection for cells carrying the FLP expression plasmid was performed on LB agar plates containing 15 μg / ml chloramphenicol and 0.4% L-arabinose. The plates were incubated overnight at 30° C. The next day, a single colony was inoculated into 1 mL of LB medium supplemented with 0.4% L-arabinose and incubated at 30° C. for 3 h. The incubation temperature was then increased to 37° C. and incubation continued for another 3 h. An aliquot of the culture was streaked onto LB agar plates conditioned with 0.4% L-arabinose and the plates were incubated overnight at 37° C. The resulting colonies were analyzed for FLP-mediated marker removal by assessing their sensitivity to the corresponding antibiotic. Correct removal of the antibiotic resistance marker gene was confirmed by PCR and subsequent sequencing of the amplicon.

[0171] bd) Growth control of platform strains Growth control of the platform strains was performed as described in section ac. Cells were streaked onto LB agar plates with and without the addition of 0.4% L-arabinose to confirm that the resulting platform strains grew only in the presence of L-arabinose (Figure 4). Two platform strains based on infA were prepared as described above using two E. coli strain backgrounds. E. coli strain "T7E2 araC-araBp-FRT-infA" is based on the T7E2 strain, a derivative of BL21(DE3) that lacks approximately 89% of the DE3 prophage. E. coli strain "HS996 FRT-araC-araBp-infA" is based on the E. coli K12 strain HS996. The platform strains do not carry antibiotic resistance marker genes in the genome.

[0172] Four additional platform strains based on infA were prepared as described above using the following E. coli strain backgrounds: BL21(DE3), W3110, TG10, and HS996 pir+, all of which grow only in the presence of L-arabinose (Figures 9-11).

[0173] Example 2: Preparation of expression plasmids a) Preparation of expression plasmid “pQE-T7 TNFα cmR-infA” Plasmid "pQE-T7 TNFα" (SEQ ID NO: 17) was selected to ensure that the plasmid encoding the infA gene was maintained in the platform strain without antibiotic selection. A DNA fragment consisting of a chloramphenicol resistance marker gene flanked by NotI sites was ligated to the genomic infA fragment and subsequently amplified using primers infA-primer up (SEQ ID NO: 18) and infA-primer down (SEQ ID NO: 19). The resulting PCR product (SEQ ID NO: 20) was recombined with plasmid "pQE-T7 TNFα" using the recombineering proficiency E. coli strain GB08-red (Fu et al. 2010). Plasmid modification was performed as described in the publication by Fu et al. A 50 base pair long homology arm was selected for recombination with plasmid "pQE-T7 TNFα" to replace the kmR marker in the original plasmid by the "cmR-infA" cassette. The resulting plasmid "pQE-T7 TNFα cmR-infA" (Figure 5, SEQ ID NO:21) was used as the first test plasmid for subsequent plasmid maintenance experiments.

[0174] An aliquot of 100 ng of plasmid DNA from "pQE-T7 TNFα cmR-infA" was digested with the restriction enzyme NotI and then religated to obtain a test plasmid lacking the antibiotic resistance marker gene. This second test plasmid was named "pQE-T7 TNFα infA" (Figure 6, SEQ ID NO:22).

[0175] b) Preparation of reporter plasmids with various origins of replication Three additional plasmids, "GenH015-plasmid1-cmR", "GenH015-plasmid2-cmR" and "GenH015-plasmid3-cmR" (Figure 12, SEQ ID NOs:23-25), were assembled, consisting of a chloramphenicol resistance marker gene flanked by NotI sites, the genomic infA fragment already used in "pQE-T7 TNFα infA", a fluorescent marker cassette, and either the R6K, p15A or ColE1 origin of replication. Purple coloration of the clones indicates the presence of the reporter plasmid, while E. coli cells without the plasmid remain white.

[0176] Aliquots of 100 ng of DNA from each plasmid were digested with NotI and subsequently religated to obtain the corresponding reporter plasmids without the antibiotic resistance marker gene. The resulting three plasmids were named "GenH015-plasmid1", "GenH015-plasmid2" and "GenH015-plasmid3" (Figure 12, SEQ ID NOs:26-28).

[0177] Example 3: Confirmation of plasmid maintenance based on "pQE-T7 TNFα infA" Electrocompetent cells were prepared from the platform strain "T7E2 araC-araBp-FRT-infA" as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Cat. No. K006). Approximately 10 ng of the plasmid "pQE-T7 TNFα infA-cmR" was electroporated into the cells of the platform strain. The cells were resuspended in 1 ml of LB medium (without antibiotics or L-arabinose) after electroporation and incubated at 37°C for 2 hours with shaking at 800 rpm. After this recovery step, the cells were streaked onto LB agar plates (without antibiotics or L-arabinose) and incubated overnight at 37°C. The next day, more than 100 colonies were visible on the plate.

[0178] Six colonies were inoculated into LB medium without antibiotic supplementation and grown overnight at 37° C. at 800 rpm. Plasmid DNA was isolated from these clones and analyzed with restriction enzymes to confirm the presence of the plasmid in the cells. All analyzed colonies show the expected restriction pattern (FIG. 7).

[0179] The experiment was repeated with the plasmid "pQE-T7 TNFα infA". More than 50 colonies were obtained. Four colonies were inoculated into LB medium without antibiotic addition and grown overnight at 37°C at 800 rpm. Plasmid DNA was isolated from these clones and analyzed with restriction enzymes to confirm the presence of the plasmid in the cells. All analyzed colonies show the expected restriction pattern (Figure 8).

[0180] No antibiotic selection was used to insert or maintain the plasmid in the cells. The cells are viable only in the presence of a plasmid carrying a copy of the infA gene driven by an appropriate promoter. The plasmid does not carry an antibiotic resistance marker gene.

[0181] Example 4: Confirmation of plasmid maintenance based on various reporter plasmids "GenH015-plasmid1", "GenH015-plasmid2" and "GenH015-plasmid3" Electrocompetent cells were prepared from platform strain W3110 araC-araBp-FRT-infA as described in Example 3. Approximately 10 ng of plasmids "pQE-T7 TNFα infA", "GenH015-plasmid2" and "GenH015-plasmid3" were electroporated into cells of the platform strain. Cells were resuspended in 1 ml of LB medium (without antibiotics or L-arabinose) after electroporation and incubated at 37°C for 2 hours with shaking at 800 rpm. After this recovery step, cells were streaked onto LB agar plates (without antibiotics or L-arabinose) and incubated overnight at 37°C. The next day, more than 100 colonies were visible on the plates. All colonies obtained after electroporation with "GenH015-plasmid2" and "GenH015-plasmid3" showed the expected purple color, indicating the presence of the corresponding plasmids. All three different plasmids tested were able to complement the platform strain and allow normal growth on LB medium lacking L-arabinose (Figure 10).

[0182] The gamma origin of replication of the broad-host-range plasmid R6K (oriR6Kγ) has the advantage of being very small in overall size. Replication of these plasmids requires the π protein encoded by the pir gene. This protein is generally expressed in trans from the pir gene integrated in the chromosome. To test whether plasmid maintenance can be obtained with plasmids using oriR6Kγ, the platform strain HS996 pir+ araC-araBp-FRT-infA was transformed with the three reporter plasmids "GenH015-plasmid1", "GenH015-plasmid2" and "GenH015-plasmid3". The next day, more than 100 colonies were found on all plates. All the colonies obtained showed the expected purple color, which indicated the presence of the corresponding plasmid. All three different plasmids tested were able to complement the platform strain and allow normal growth on LB medium without L-arabinose (Figure 11).

[0183] To verify long-term plasmid maintenance in the absence of antibiotics, three different combinations of platform strains and plasmids were further analyzed: “HS996 pir+ araC-araBp-FRT-infA” / “GenH015-plasmid1”, “T7E2 araC-araBp-FRT-infA” / “GenH015-plasmid2” and “W3110 araC-araBp-FRT-infA” / “GenH015-plasmid3”.

[0184] A single colony of the platform strain carrying the corresponding plasmid was used to inoculate 1.5 ml of LB medium. The culture was incubated overnight at 37° C. with shaking at 800 rpm. The next day, 10 μl of the overnight culture was taken to inoculate another 1.5 ml of LB medium. The culture was again incubated at 37° C. with shaking at 800 rpm until the next morning. This procedure was repeated for 5 days. A small aliquot from the last overnight culture was streaked onto an LB agar plate and incubated overnight at 37° C. to obtain single colonies. All the colonies obtained showed the expected purple color, indicating the presence of the corresponding plasmid (FIG. 17). Due to the low copy number of the p15A-based plasmid, the color of the colonies obtained from the “T7E2 araC-araBp-FRT-infA” / “GenH015-plasmid2” combination is less intense.

[0185] The results demonstrate effective maintenance of the plasmid during long-term passaging without the use of antibiotics.

[0186] Example 5: Preparation of platform strains based on the gene frr To replace the endogenous frr promoter with an arabinose-inducible promoter, an FRT-flanked Zeocin selection marker was linked downstream of the arabinose-inducible promoter sequence and the corresponding DNA fragment was inserted into the genome by Red / ET recombination at the frr promoter locus. After replacement of the endogenous frr promoter, an FLP recombination step was performed to remove the antibiotic resistance marker gene. One FRT site remained in the genome either upstream of the inducible promoter or downstream between the promoter and the start codon of frr (Figure 13).

[0187] a) Replacement of the endogenous frr promoter with the araC-araBp-FRT-zeoR-FRT cassette A 2423 bp sized DNA fragment (SEQ ID NO: 29) consisting of araC-araBp linked to the FRT-flanked zeoR resistance marker gene and flanked by homology arms to the frr genomic locus was amplified using primers FRT-frr-primer up (SEQ ID NO: 30) and FRT-frr-primer down (SEQ ID NO: 31).

[0188] Using Red / ET recombination (Zhang, Y. et al. 2000; Murphy, KC, Campellone, KG and Poteete, AR 2000), the resulting PCR product was inserted upstream of the frr start codon of E. coli strain HS996, replacing the endogenous promoter. The recombineering reaction was carried out as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Cat. No. K006), except that 0.4% L-arabinose was added to the medium immediately after transformation of the cells with the DNA fragment (SEQ ID NO: 29) to induce the expression of frr. The cells were grown in 1 mL of LB medium supplemented with 0.4% L-arabinose at 37°C for 2 hours. Selection for cells with chromosomal integration of the cassette was carried out on LB agar plates containing 0.4% L-arabinose and 25 μg / ml Zeocin. Correct knock-in was confirmed by PCR across the modified frr locus and subsequent sequencing of the amplicon.

[0189] The Zeocin resistance marker was subsequently removed by FLP recombination as described in Example 1, section bc. The sequence of the genomic locus after insertion of the arabinose-inducible promoter and FLP recombination is given in SEQ ID NO:32.

[0190] b) Removal of resistance marker genes by FLP recombination The FLP recombination steps were carried out as described in the manual of the "Quick & Easy E. coli Gene Deletion Kit" (Gene Bridges, Catalog No. K006), except that 0.4% L-arabinose was added to the medium during all steps. The FLP expression plasmid "708-FLPe cmR" (Gene Bridges, Catalog No. A105) was selected for removal of the resistance marker.

[0191] Cells were transformed with the FLP expression plasmid and grown for 2 h at 30° C. in 1 mL of LB medium supplemented with 0.4% L-arabinose but without the addition of antibiotics. Selection for cells carrying the FLP expression plasmid was performed on LB agar plates containing 15 μg / ml chloramphenicol and 0.4% L-arabinose. The plates were incubated overnight at 30° C. The next day, a single colony was inoculated into 1 mL of LB medium supplemented with 0.4% L-arabinose and incubated at 30° C. for 3 h. The incubation temperature was then increased to 37° C. and incubation continued for another 3 h. An aliquot of the culture was streaked onto LB agar plates conditioned with 0.4% L-arabinose and the plates were incubated overnight at 37° C. The resulting colonies were analyzed for FLP-mediated marker removal by assessing their sensitivity to the corresponding antibiotic. Correct removal of the antibiotic resistance marker gene was confirmed by PCR and subsequent sequencing of the amplicon. The sequence of the genomic locus after removal of the Zeocin resistance marker gene is given in SEQ ID NO:33.

[0192] c) Growth control of platform strains Growth control of the platform strains was performed as described in Example 1, section ac. Cells were streaked onto LB agar plates with and without the addition of 0.4% L-arabinose to confirm that the resulting platform strains grow only in the presence of L-arabinose (FIG. 14). The platform strains do not carry antibiotic resistance marker genes in their genomes.

[0193] d) Preparation of expression plasmid “pQE-T7 TNFα frr” Plasmid "pQE-T7 TNFα" (SEQ ID NO: 17) was selected to ensure that the plasmid encoding the frr gene was maintained in the platform strain without antibiotic selection. A synthetic DNA fragment (SEQ ID NO: 34) consisting of a Zeocin resistance marker gene flanked by NotI sites and linked to the genomic frr fragment was selected to replace the "cmR-infA" cassette in plasmid "pQE-T7 TNFα cmR-infA" (Figure 5, SEQ ID NO: 21) using the recombineering proficiency E. coli strain GB08-red (Fu et al. 2010).

[0194] The resulting plasmid "pQE-T7 TNFα zeoR-frr" (Figure 15, SEQ ID NO:35) was used as the first test plasmid for subsequent plasmid maintenance experiments.

[0195] An aliquot of 100 ng of plasmid DNA from "pQE-T7 TNFα zeoR-frr" was digested with the restriction enzyme NotI and then religated to obtain a test plasmid lacking the antibiotic resistance marker gene. This second test plasmid was named "pQE-T7 TNFα frr" (Figure 16, SEQ ID NO:36).

[0196] e) Verification of long-term plasmid maintenance in the absence of frr-based antibiotics To verify that the frr-based plasmids were maintained in the platform strains as desired, a single colony of the platform strain carrying the plasmid "pQE-T7 TNFα zeoR-frr" was used to inoculate 1.5 ml of LB medium. The culture was incubated overnight at 37°C with shaking at 800 rpm. The next day, 10 μl of the overnight culture was taken to inoculate another 1.5 ml of LB medium. The culture was again incubated at 37°C with shaking at 800 rpm until the next morning. This procedure was repeated for 5 days. A small aliquot from the last overnight culture was streaked onto an LB agar plate and incubated overnight at 37°C to obtain single colonies. 24 single colonies were streaked onto two LB agar plates, one conditioned with 25 μg / ml zeocin and the other without the addition of antibiotic. The plates were incubated overnight at 37°C. All clones grew on both plates, confirming that the plasmid "pQE-T7 TNFα zeoR-frr" was maintained in all clones even though the cells were grown for 7 days without the addition of Zeocin (Figure 18). This result demonstrates the effective maintenance of the plasmid during long-term passaging without the use of antibiotics.

[0197] References TIFF2025507940000003.tif212167TIFF2025507940000004.tif232168

Claims

1. A recombinant bacterial cell comprising in its genome at least one essential gene, wherein the endogenous promoter of said essential gene has been replaced by a heterologous expression control sequence inducible by an inducer molecule, such that expression of said essential gene in the recombinant bacterial cell is dependent on the presence of said inducer molecule.

2. The recombinant bacterial cell of claim 1, wherein the at least one essential gene is (i) an absolutely essential gene that is absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or is essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration, or (ii) a combination of essential genes that, in combination, are absolutely required for proper cell growth, reproduction and / or survival even under optimal growth conditions or are essential for DNA replication, RNA synthesis, protein synthesis, central metabolism, cell wall or membrane synthesis or regeneration.

3. The bacterial cell is selected from the group consisting of Escherichia, Vibrio, Bacillus, Lactobacillus, Acetobacter, Corynebacterium, Brevibacterium, Pseudomonas, Streptomyces, Gluconobacter, Clostridium, and the like. dium, Streptococcus, Zooepidemicus, Basfia, Crytococcus, Rhodotorula, Nocardia, Erwinia, Xanthomonas, Leuconostoc and Klebsiella, preferably from a genus selected from the group consisting of E. coli.

3. The recombinant bacterial cell of claim 1 or 2, wherein the recombinant bacterial cell is a (E. coli) cell, and the at least one essential gene is a gene required for cell growth and / or viability, preferably selected from the group consisting of infA, infC / IF-3, dnaJ, dnaK, era, frr, ftsL, ftsN, ftsZ, grpE, mopA, mopB, msbA, nusG, parC, rpsB, secY and trma.

4. 4. The recombinant bacterial cell according to claim 1, wherein the bacterial cell comprises an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence that is biologically active in the bacterial cell.

5. 5. The recombinant bacterial cell of claim 4, wherein the expression plasmid comprises at least one additional target nucleic acid required for recombinant production of a target compound in the recombinant bacterial cell.

6. A method for producing a recombinant bacterial cell according to any one of claims 1 to 5, comprising the steps of: (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a first selection gene that confers sensitivity to a first selection agent, such that the bacterial cell will not grow and / or die in the presence of said first selection agent; and (ii) a second selection gene that confers resistance to a second selection agent such that the bacterial cell can grow in the presence of said second selection agent. exchanging the at least one essential gene, the first and the second selection gene with a first polynucleotide comprising a selection cassette comprising a promoter capable of being active in a bacterial cell, operably linked to said first polynucleotide, such that expression of said at least one essential gene, the first and the second selection gene becomes dependent on said promoter; (b) using a second selection agent to select bacterial cells that have integrated the first polynucleotide into their genome; (c) replacing the first polynucleotide integrated in the genome with a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of at least one essential gene becomes dependent on the heterologous expression sequence; and (d) selecting bacterial cells that have integrated said second polynucleotide into their genome using the first selection molecule and an inducer molecule. The method comprising:

7. 7. The method of claim 6, wherein the first selection gene is selected from the group consisting of rpsL, sacB, pheS, tetR, galK, thyA, tolC and ccdB, and the first selection agent is selected from the group consisting of streptomycin, sucrose, chloro-phenylalanine, chlortetracycline and fusaric acid, galactose, trimethoprim and its derivatives, and a bacteriocin, preferably a colicin, preferably colicin A, B, E1, Ia, Ib, N, D, M or E3, more preferably colicin E1.

8. 8. The method of claim 6 or 7, wherein the second selection gene is selected from the group consisting of kmR, ampR, zeoR, tetR, cmR, hygR, specR, and BSD, and the second selection agent is selected from the group consisting of kanamycin, ampicillin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

9. 9. The method according to any one of claims 6 to 8, wherein the selection cassette further comprises a restriction enzyme cleavage site at the 3' end of the second selection gene, and the method further comprises, after step (c), treating the bacterial cell with a restriction enzyme such that the genome of the bacterial cell still comprising the first polynucleotide comprising the selection cassette is cleaved between the second selection gene and the at least one essential gene, while the genome of the bacterial cell in which the first polynucleotide comprising the selection cassette integrated in its genome has been replaced by a second polynucleotide comprising a heterologous expression control sequence inducible by an inducer molecule such that expression of the at least one essential gene becomes dependent on said heterologous expression sequence remains unaffected by the restriction enzyme.

10. A method for producing a recombinant bacterial cell according to any one of claims 1 to 5, comprising the steps of: (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a selection gene that confers resistance to a selection agent, flanked by recognition sites for site-specific recombination in bacterial cells; and (ii) a heterologous expression control sequence inducible by an inducer molecule; such that expression of at least one essential gene becomes dependent on said heterologous expression control sequence and such that the bacterial cell grows in the presence of said inducer molecule and in the presence of said selection agent, (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent and an inducer molecule; (c) removing the selection gene by using a recombinase capable of performing site-specific recombination using recognition sites flanking the selection gene such that expression of at least one essential gene remains dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide that lacks a selection gene. The method comprising:

11. 11. The method of claim 10, wherein the selection gene is selected from the group consisting of ampR, kmR, zeoR, tetR, cmR, hygR, specR, and BSD, and the selection agent is selected from the group consisting of ampicillin, kanamycin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

12. A method for producing a recombinant bacterial cell according to any one of claims 1 to 5, comprising the steps of: (a) identifying in the genome of a bacterial cell an endogenous promoter of at least one essential gene; (i) a heterologous expression control sequence inducible by an inducer molecule; (ii) a promoter that can be active in a bacterial cell, and (iii) a selection gene that confers resistance to a selection agent; (ii) a promoter that can be active in a bacterial cell and (iii) a selection gene, wherein the segment of the polynucleotide comprising the selection gene is flanked by recognition sites for site-specific recombination in the bacterial cell, while the segment of the polynucleotide comprising the heterologous expression control sequence is not flanked by said recognition sites; (b) selecting bacterial cells that have integrated the polynucleotide into their genome using a selection agent; (c) removing a segment of the polynucleotide comprising a promoter and a selection gene that may be active in the bacterial cell by using a recombinase capable of performing site-specific recombination using recognition sites flanking a segment of the polynucleotide such that expression of at least one essential gene becomes dependent on said heterologous expression control sequences; and (d) using an inducer molecule to select bacterial cells that have integrated into their genome the polynucleotide lacking the segment flanked by the recognition sites. The method comprising:

13. 13. The method of claim 12, wherein the selection gene is selected from the group consisting of kmR, ampR, zeoR, tetR, cmR, hygR, specR, and BSD, and the selection agent is selected from the group consisting of kanamycin, ampicillin, zeocin, tetracycline, chloramphenicol, hygromycin, spectinomycin, and blasticidin.

14. 14. The method of claim 12 or 13, wherein the recognition sites for site-specific recombination in bacterial cells are FRT or loxP sequences and the recombinase is FLP or Cre recombinase.

15. The method according to any one of claims 12 to 14, wherein the promoter capable of being active in a bacterial cell is a constitutively active promoter selected from the group consisting of rpsL promoter, infA promoter, infAp2 promoter, em7 promoter, and cat promoter.

16. 16. The recombinant bacterial cell of any one of claims 1 to 5 or the method of any one of claims 6 to 15, wherein the heterologous expression control sequence inducible by an inducer molecule is selected from the group consisting of AraC / PBAD promoter, RhaR-RhaS / rhaBAD promoter, XylS / Pm promoter, NitR / PnitA promoter, ChnR / Pb inducer / promoter, tetracycline inducible promoter (tetON / OFF), lacI / IPTG promoter, ethanol inducible promoter (AlcA / AlcR), steroid inducible promoter (LexA / XVE), and vanillate inducible promoter.

17. 17. The recombinant bacterial cell of any one of claims 1 to 5, the method of any one of claims 6 to 15, or the recombinant bacterial cell or method of claim 16, wherein the inducer molecule is selected from the group consisting of arabinose, rhamnose, xylose, sucrose, tetracycline, anhydrotetracycline and IPTG.

18. 1. A method for recombinantly producing a compound of interest, comprising: (a) introducing into a recombinant bacterial cell according to any one of claims 1 to 5 an expression plasmid comprising at least one plasmid copy of at least one essential gene, said plasmid copy being under the control of an expression control sequence which is biologically active in said bacterial cell; and (b) culturing the recombinant bacterial cells obtained in step a) in the absence of inducer molecules. Including, A recombinant production method, wherein said expression plasmid comprises at least one additional nucleic acid of interest required for the recombinant production of a compound of interest in said recombinant bacterial cell.

Images