Genetically modified bacteria comprising an invasive orthogonal expression system

Abstract

The invention relates to genetically modified bacteria comprising an invasive orthogonal expression system and to the use thereof for producing at least one protein of interest. The invention also relates to polynucleotides, vectors and kits for the expression of at least one protein of interest.

Description

[0001] GENETICALLY MODIFIED BACTERIA COMPRISING AN INVASIVE ORTHOGONAL EXPRESSION SYSTEM

[0002] The invention relates to genetically modified bacteria comprising an orthogonal transcription expression system and their use for the production of at least one protein of interest. The invention also relates to polynucleotides, vectors and kits for the expression of at least one protein of interest.

[0003] To address the challenge of climate change, it appears necessary to make alternatives to petroleum-based chemistry viable. There are many bioproduction systems that allow the synthesis of molecules using bacteria, which consume far fewer resources than their petroleum-based counterparts.

[0004] However, current bioproduction systems sometimes offer yields that are too unprofitable for them to be considered as a global solution.

[0005] There is therefore a real need for optimized bioproduction systems to improve production yield by bacteria.

[0006] In this context, the Inventors noted that current protein bioproduction systems share the transcription and translation capabilities of the host bacterium with the synthesis of all the cell's proteins.

[0007] Unexpectedly, the inventors then demonstrated that it is possible to stop the protein synthesis of the host bacterium and redirect cellular resources to an orthogonal expression system. Such a system is said to be orthogonal in that it is executed in a manner decoupled from the endogenous system of the bacterium. In addition, this system is invasive in that it outperforms the endogenous system. The orthogonal expression system developed by the inventors thus makes it possible to maximize the allocation of resources dedicated to bioproduction by blocking the endogenous expression system.

[0008] By taking advantage of the promoter sequence recognition function of sigma factors, the inventors cloned a heterologous sigma factor capable of self-inducing its synthesis and a repressor controlling the synthesis of the endogenous sigma factor. Interestingly, by synthesizing a test protein, the resulting bacterium allows, after a 3-hour induction, to produce a quantity of protein thirty-five times greater than the quantity that can be obtained using the best inducible promoter described in B. subtilis.

[0009] One aspect of the invention therefore relates to a genetically modified bacterium comprising: - a first nucleotide sequence encoding an orthogonal sigma factor capable of interacting with the RNA polymerase of the genetically modified bacterium, the expression of the orthogonal sigma factor being inducible and under the control of a promoter recognized by the orthogonal sigma factor, and

[0010] - a second nucleotide sequence whose expression product inhibits the action of an endogenous sigma factor of the genetically modified bacterium, the expression of the second nucleotide sequence being under the control of a promoter recognized by the orthogonal sigma factor.

[0011] According to the invention, the expression "sigma factor" (or o) designates the subunit of bacterial RNA polymerase necessary for the initiation of transcription. This protein plays a decisive role in the selection of the enzyme's binding sites.

[0012] According to the invention, the expression "sigma factor" covers not only sigma factors but also phage monomeric RNA polymerases, such as T3 or T7 RNA polymerases, which directly recognize specific promoter sequences.

[0013] According to the invention, the term "endogenous" means that the element it designates is naturally present in the genetically modified bacterial strain. Thus, according to the invention, the endogenous sigma factor is naturally present in the genetically modified bacterial strain.

[0014] According to the invention, the term "orthogonal" means that the element it designates is not naturally present in the genetically modified bacterial strain, but is capable of performing the same function as another element naturally present in this strain. Thus, according to the invention, an orthogonal sigma factor is not naturally present in the genetically modified bacterial strain, but when it is introduced and expressed in it, it is capable of initiating transcription there, in parallel and independently of the sigma factors naturally expressed in this strain. In other words, the orthogonal sigma factor makes it possible to introduce a transcription system decoupled from the transcription system naturally present in the genetically modified bacterium.

[0015] The orthogonal sigma factor is not naturally present in the genetically modified bacterial strain, which means that it can come not only from other bacterial species (i.e. a heterologous sigma factor), but also from other strains of the same species as the genetically modified bacterium or from a bacteriophage not naturally present in the genetically modified strain. This orthogonal sigma factor can be natural or genetically modified so as to recognize a promoter sequence different from the one it naturally recognizes in the organism from which it originates.

[0016] In the invention, the expression of the orthogonal sigma factor is "inducible", which means that it is not constitutive. The expression of the sigma factor is reversibly activated or deactivated in the presence of a given regulatory element. In a non-limiting manner, this regulatory element can be directly added to or removed from the culture medium.

[0017] For example, in one embodiment, expression of the orthogonal sigma factor may be placed under the control of a xylose-inducible promoter and may then be activated by the addition of xylose to the medium.

[0018] For example, in another embodiment, the expression of the orthogonal sigma factor can be placed under the control of a lactose (or allolactose or any functional analog) inducible promoter and can then be blocked by the presence of the regulatory protein Lac1 and activated by the addition of IPTG which will bind to Lac1 and prevent the binding of Lac1 to the "lactose" promoter.

[0019] For example, in other embodiments, expression of the orthogonal sigma factor may be placed under the control of a nisin-, copper-, mannose- (Pmanp), surfactin- (PsrfA), sucrose- (Psace), mitomycin- (Pdinp), or temperature- (P2)-inducible promoter.

[0020] In one embodiment, the invention relates to a genetically modified bacterium as defined above, further comprising:

[0021] - a third nucleotide sequence coding at least one protein of interest, the expression of said at least one protein of interest being under the control of a promoter recognized by the orthogonal sigma factor.

[0022] In one embodiment, said at least one protein of interest corresponds to a single protein of interest.

[0023] In one embodiment, said at least one protein of interest corresponds to two proteins of interest.

[0024] In one embodiment, said at least one protein of interest corresponds to two or more proteins of interest. According to the invention, the term "protein" designates a polymer of amino acids and also covers small peptides, for example from 10 to 100 amino acids.

[0025] According to the invention, the expression "protein of interest" generally designates any protein voluntarily selected for the implementation of the invention. In a non-limiting manner, such a protein is likely to be of commercial, therapeutic, industrial, technological or research interest. Indirectly, a protein of interest according to the invention may also be an intermediate product or an enzyme allowing the transformation of a substrate into a protein of particular interest.

[0026] In one embodiment, the protein of interest is an enzyme, for example selected from the group comprising an amylase, a xylanase, a lichenase, a lipase, a cellulase, a pectinase, a hydrolase, a pullulanase, a nattokinase, a pectinase, an agarase, a galactosidase, a protease and a peptidase.

[0027] In one embodiment, the protein of interest is a membrane protein.

[0028] In one embodiment, the protein of interest is an antigen.

[0029] In one embodiment, the protein of interest is a cytokine, in particular chosen from the group comprising interferon alpha, interferon beta and interferon gamma.

[0030] In one embodiment, the protein of interest is a food additive.

[0031] In one embodiment, the protein of interest is a bacterial biofilm protein.

[0032] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the orthogonal sigma factor is chosen from:

[0033] - a sigma factor from a bacterial species different from that of the genetically modified bacterium, and

[0034] - a sigma factor specific to an extra-chromosomal element naturally absent from the genetically modified bacteria.

[0035] In one embodiment, the extrachromosomal element may be a plasmid or a bacteriophage, such as T3 or T7. One of the advantages of choosing an extrachromosomal element is that it allows the use of a sigma factor present in strains of the same species as the genetically modified bacterium, and therefore capable of functioning in the latter, while recognizing specific promoters not naturally present in the latter.

[0036] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the expression of the orthogonal sigma factor is controlled by two tandem promoters: one conferring the inducible character and the other being recognized by the orthogonal sigma factor.

[0037] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the expression product of the second sequence is chosen from:

[0038] - a transcriptional repressor,

[0039] - an interfering RNA,

[0040] - a protease, and

[0041] - a sigma factor inhibitor.

[0042] The expression product of the second sequence aims to inhibit the action of the endogenous sigma factor, that is, to turn off the transcription system naturally present in the genetically modified bacterium.

[0043] In a non-limiting manner, inhibition of the action of the endogenous sigma factor can be obtained for example by preventing the transcription of the gene encoding the endogenous sigma factor (eg via a transcriptional repressor), by preventing the translation of the gene encoding the endogenous sigma factor (eg via an interfering RNA), by degrading the endogenous sigma factor (eg via a protease targeting a cleavage site present on the endogenous sigma factor) or by preventing the specific binding of the endogenous sigma factor (eg via a sigma factor inhibitor).

[0044] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the native gene encoding the endogenous sigma factor has been inactivated and a copy of said gene has been placed under the control of a promoter recognized by a transcriptional repressor encoded by the second sequence.

[0045] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the native gene encoding the endogenous sigma factor is placed under the control of a promoter recognized by a transcriptional repressor encoded by the second sequence. In one embodiment, the invention relates to a genetically modified bacterium as defined above, said endogenous sigma factor being the primary sigma factor of the genetically modified bacterium.

[0046] According to the invention, the term "primary sigma factor" refers to the sigma factor that ensures the transcription of housekeeping genes. The primary sigma factor is generally considered essential for the survival of the bacterium.

[0047] For example, in Bacillus subtilis, the primary sigma factor is usually designated Sigma A (SigA or o A ); in Escherichia coli, the primary sigma factor is generally designated Sigma 70 (Sig70 or o70 ). For example, reference sequences corresponding to o A of B. subtilis and to o 70 of E. coli are presented in Table 1.

[0048] Table 1. Examples of primary sigma factors. In one embodiment, the genetically modified bacterium is a strain of B. subtilis and the primary sigma factor is a protein of sequence SEQ ID NO: 1.

[0049] In one embodiment, the genetically modified bacterium is a strain of E. coli and the primary sigma factor is a protein of sequence SEQ ID NO: 2. In one embodiment, other endogenous sigma factors may also be inhibited, in addition to inhibition of the primary sigma factor, to avoid resource competition with the orthogonal sigma factor. For example, in B. subtilis, the alternative sigma factor SigD may also be inhibited, in addition to inhibition of the primary sigma factor SigA.

[0050] In one embodiment, the invention relates to a genetically modified bacterium as defined above, said orthogonal sigma factor being selected from the group comprising ZpdN (a Bacillus subtilis sigma factor), Gp28 (a phage sigma factor), RpoS (an Escherichia coli sigma factor), RpoN (an E. coli sigma factor), SigB8 (a Prevotella bryantii sigma factor), SigB (a B. subtilis sigma factor) and t7p07 (a phage RNA polymerase), preferably ZpdN.

[0051] As an example, reference sequences corresponding to orthogonal sigma factors, suitable for use in B. subtilis, are given in Table 2.

[0052] Table 2. Examples of orthogonal sigma factors for B. subtilis.

[0053] In one embodiment, the invention relates to a genetically modified bacterium as defined above, said orthogonal sigma factor being selected from the group comprising SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, preferably SEQ ID NO: 3.

[0054] In one embodiment, the invention relates to a genetically modified bacterium as defined above, said bacterium belonging to a species selected from Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus and Lactococcus lactis, preferably B. subtilis.

[0055] In one embodiment, the genetically modified bacterium belongs to the species B. subtilis and the orthogonal sigma factor is ZpdN (SEQ ID NO: 3).

[0056] In one embodiment, the genetically modified bacterium belongs to the species B. subtilis, the orthogonal sigma factor is ZpdN (SEQ ID NO: 3) and the promoter recognized by the orthogonal sigma factor is selected from the group of sequences comprising SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0057] In one embodiment, the invention relates to a genetically modified bacterium as defined above, said bacterium being a GRAS (Generally Recognized As Safe) bacterium, i.e. generally recognized as safe. Such bacteria are particularly suitable for the synthesis of proteins intended for food or therapeutic applications.

[0058] In one embodiment, the genetically modified bacterium is the B. subtilis BSB1 strain. This isolated strain was deposited with the National Collection of Microorganism Cultures (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, 75724 PARIS, France) on 1 er February 2023 under number CNCM 1-5930, in accordance with the Budapest Treaty.

[0059] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which at least the first nucleotide sequence and the second nucleotide sequence, and optionally the third nucleotide sequence, are present on distinct polynucleotides.

[0060] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which at least the first nucleotide sequence and the second nucleotide sequence, and optionally the third nucleotide sequence, are present on the same polynucleotide.

[0061] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present on the same polynucleotide.

[0062] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the same promoter controls at least the expression of the orthogonal sigma factor encoded by the first nucleotide sequence and the expression product of the second nucleotide sequence, and optionally of the protein of interest encoded by the third nucleotide sequence.

[0063] In one embodiment, the invention relates to a genetically modified bacterium as defined above, in which the same promoter controls the expression of the orthogonal sigma factor encoded by the first nucleotide sequence, of the expression product of the second nucleotide sequence and of the protein of interest encoded by the third nucleotide sequence.

[0064] In one embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the protein of interest is a protein toxic to the genetically modified bacterium.

[0065] The system of the invention is particularly suitable for the production of proteins that are toxic to the bacteria. Indeed, in conventional expression systems, these proteins slow down, or even block, the growth of the bacteria, making it impossible to produce a sufficient quantity of this protein. The system of the invention makes it possible to optimally cultivate the bacteria, and then, when a sufficient bacterial population is reached, to switch the cellular machinery to the sole benefit of the production of the protein of interest.

[0066] In one embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the protein of interest is a protein secreted by the bacterium.

[0067] The system of the invention is particularly suitable for the production of secreted proteins because it thus makes it possible to produce in the culture medium a significant quantity of proteins which can be easily recovered, in particular by preserving the live bacteria.

[0068] In another aspect, the invention relates to the use of a genetically modified bacterium as defined above for the production of at least one protein of interest.

[0069] Another aspect of the invention relates to a method for producing at least one protein of interest comprising: i) a step of culturing a genetically modified bacterium as defined above, and ii) a step of inducing the expression of the orthogonal sigma factor.

[0070] In one embodiment, step i) and / or step ii) is carried out in a bioreactor (or fermenter).

[0071] In one embodiment, the invention relates to a method as defined above further comprising a step of recovering the protein of interest. The methods of recovering the proteins of interest are well known to those skilled in the art and may vary, in particular depending on whether the proteins of interest are intracellular or secreted into the culture medium by the bacteria.

[0072] In one embodiment, the invention relates to a method as defined above in which the step of inducing the expression of the orthogonal sigma factor is stopped before the death of the genetically modified bacterium.

[0073] Another aspect of the invention relates to a polynucleotide comprising at least a first nucleotide sequence encoding a sigma factor under the control of two promoters in tandem: one conferring an inducible character and the other being recognized by said sigma factor.

[0074] In one embodiment, the invention relates to a polynucleotide as defined above, further comprising a second nucleotide sequence encoding: - a transcriptional repressor,

[0075] - an interfering RNA,

[0076] - a protease, or

[0077] - a sigma factor inhibitor.

[0078] In one embodiment, the invention relates to a polynucleotide as defined above, in which the expression of the second nucleotide sequence is controlled by the promoter recognized by said sigma factor.

[0079] In one embodiment, the invention relates to a polynucleotide as defined above, in which the expression of the first nucleotide sequence and the second nucleotide sequence is controlled by the same two promoters in tandem.

[0080] In one embodiment, the invention relates to a polynucleotide as defined above, further comprising a third nucleotide sequence encoding at least one protein of interest.

[0081] In one embodiment, the invention relates to a polynucleotide as defined above, in which the expression of the third nucleotide sequence is controlled by the promoter recognized by said sigma factor.

[0082] In one embodiment, the invention relates to a polynucleotide as defined above, in which the expression of the first nucleotide sequence and the third nucleotide sequence is controlled by the same two promoters in tandem.

[0083] In one embodiment, the invention relates to a polynucleotide as defined above, wherein the expression of the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence is controlled by the same promoters in tandem.

[0084] In one embodiment, the invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the second nucleotide sequence are in the form of an operon.

[0085] In one embodiment, the invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the third nucleotide sequence are in the form of an operon. In one embodiment, the invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are in the form of an operon.

[0086] Another aspect of the invention relates to an expression vector comprising at least one polynucleotide as defined above.

[0087] Another aspect of the invention relates to a kit for the recombinant expression of at least one protein of interest comprising at least:

[0088] - a genetically modified bacterium as defined above,

[0089] - a polynucleotide as defined above, and / or

[0090] - an expression vector as defined above, and

[0091] - optionally, a molecule making it possible to induce the expression of said at least one protein of interest.

[0092] In one embodiment, the molecule making it possible to induce the expression of said at least one protein of interest is chosen from the group comprising xylose and IPTG.

[0093] The present invention will be described in more detail with the aid of the following figures and examples which do not limit the invention in any way.

[0094] FIGURES

[0095] Figure 1. Representation of the system for switching between two transcription programs.

[0096] To construct the orthogonal transcription system, the gene encoding the orthogonal sigma factor (SigOrth) is placed under the control of the Phy spank promoter. This is repressed by the protein encoded by the lad gene and induced by IPTG added to the medium. The orthogonal sigma factor allows the recognition of specific promoters (P o0 rth) and induces, in association with the host RNA polymerase, the transcription of the cloned genes of interest (goi). A promoter recognized by this orthogonal sigma factor is introduced upstream of the Phy-spank promoter to ensure auto-activation of the expression of the orthogonal sigma factor. To construct the system for repressing the expression of sigma A; the single copy in the cell of the gene encoding the sigma factor A is cloned downstream of a promoter possessing the operator sequences of the TetR repressor B D- The gene coding for this repressor is placed under the control of the Phy spank- promoter

[0097] The switch between the two transcription programs is achieved by cloning the gene encoding the TertR repressor B D in operon with the gene encoding the orthogonal sigma factor in the auto-activation loop.

[0098] Figure 2. Interaction between orthogonal sigma factors and host RNA polymerase.

[0099] (A) Schematic representation of the strain tested. Constructs allowing inducible expression of genes encoding sigma factors under the control of the Phy spank promoter were placed in a B. subtilis strain producing a tagged RNA polymerase. This tag allows the purification of protein complexes, thus revealing the polypeptides that interact with the host RNA polymerase.

[0100] (B) Acrylamide gel of RNA polymerase protein complex analysis. Purified RNA polymerase complexes are analyzed on the gel. RNA polymerase subunits: P, P', and α, as well as the housekeeping sigma factor SigA are detected in all purifications (indicated by triangles); the expected size for expressed sigma factors is indicated (by the lowest triangles).

[0101] Figure 3. Establishment of a reporter system specific to the orthogonal sigma factors Gp28 and ZpdN.

[0102] (A) Schematic representation of the strain tested. The genes encoding the sigma factors Gp28 and ZpdN or encoding T7 RNA polymerase were cloned under the control of the IPTG-inducible promoter Phy spank- The gene encoding GFP was placed under the control of different promoters recognized by one of these two sigma factors.

[0103] (B) Histogram of the amount of fluorescence produced. The level of fluorescence relative to the number of cells (optical density) was measured in the different strains. In order to be compared with each other, these values ​​are all normalized using the reference strain in which the gene encoding GFP is under the control of Phy spank- Each strain is tested three times in three technical replicates; the mean and standard deviation are calculated. The values ​​obtained with the best promoter sequence tested are represented on a logarithmic scale.

[0104] Figure 4. Effect of the autoinduction loop. (A) Schematic representation of the tested strain. The promoter recognized by the sigma factor ZpdN (PZPÜG) was cloned upstream of the Ph construct y-S pank::zpdN. Transcriptional activity linked to the production of the sigma factor ZpdN is highlighted by the production of GFP: the sfgfp gene being under the control of the P promoterzp dG- (B) Fluorescence level produced for each of the two strains for different concentrations of IPTG. GFP production per unit of DOgoo (Le. optical density) normalized to the reference strain was measured under different Phy spank induction conditions with increasing doses of IPTG for strains possessing or lacking the ZpdN auto-activation loop. Figure 5. Repression of B. subtilis housekeeping sigma factor expression.

[0105] (A) Schematic representation of the strain tested. The gene encoding the TetR transcription repressor BD was cloned under the control of the IPTG-inducible promoter; the Phy spank- The gene encoding the housekeeping sigma factor was placed under the control of a promoter possessing the operator sequences recognized by the TetR repressor. (B) Kinetics of fluorescence expression as a function of time for different doses of inducer. The level of fluorescence relative to the number of cells (optical density) was measured over time after induction at t=0 of tetR gene expression. B o at two concentrations of inducer.

[0106] Figure 6. Invasive orthogonal transcription system.

[0107] (A) Schematic representation of the strain tested. The gene encoding the TetR transcription repressor BD and the gene encoding the sigma factor ZpdN were cloned under the control of the IPTG-inducible promoter Phy spank- The gene encoding the housekeeping sigma factor was placed under the control of a promoter possessing the operator sequences recognized by the repressor TetR.

[0108] (B) Fluorescence expression level as a function of induction DOgoonm. The fluorescence level relative to the number of cells (optical density) was measured in the strain possessing the gene coding for GFP under the control of P ve g (left histogram) and in the strain possessing the entire system (right histograms). In the latter, the measurements were made without IPTG (full histogram) and after 3 hours of induction of Ph expression y.S p O nk(hatched histogram).

[0109] The results are given as a function of the quantity of cells (measured in DOgoonm equivalent) at the time of induction. Figure 7. Cytometric analysis of cells after induction of the transcription switch. For each strain tested and for each sample, 100,000 cells were analyzed. For each, the quantity of fluorescence was measured. The graphs give for each fluorescence intensity (on a logarithmic scale) the number of cells counted. Three reference strains were used: i) strain 168 which does not have the gene coding for sfGFP and which allows the measurement of autofluorescence under the conditions tested, ii) strain P veg -GFP which constitutively produces GFP, and iii) the strain which has GFP under the control of an ITPG-inducible promoter.

[0110] Figure 8. Culture of the strain containing the entire production system in LB medium with and without IPTG.

[0111] Exponential phase cells were diluted at t=0 in LB without IPTG (open symbols) or in the presence of 500 mM IPTG (solid symbols). Over time, the optical density at 600 nm of the culture was measured (triangle symbols and dotted line). The cells were diluted and plated on LB plates. After 24 h, the number of colonies formed on plates was determined (round symbols and solid lines).

[0112] Figure 9. Analysis of bacterial survival by propidium iodide labeling.

[0113] Live cells, dead cells, and a mixture of both types of bacteria were treated with propidium iodide (left column). The cells were then analyzed by cytometry to detect the red fluorescence of propidium iodide in the cytoplasm. Dead cells (alone and in a mixture) are fluorescent, indicating that the labeling clearly allows the detection of bacteria with altered membrane integrity. In parallel, cells cultured for 3 hours without and with 500nM IPTG were labeled and analyzed in the same way (right column).

[0114] Figure 10. Culture of the strain containing the entire production system in LB medium with and without IPTG.

[0115] Exponential phase cells were diluted at t=0 in LB without IPTG (open symbols) or in the presence of 20 mM IPTG (solid symbols). Over time, the optical density at 600 nm of the culture was measured (triangle and dotted line symbols). The cells were diluted and plated on LB plates. After 24 h, the number of colonies formed on plates was determined (round symbols and solid lines).

[0116] Figure 11. Effect of deletion of alternative sigma factors on the production of the protein of interest. Cells containing the complete production system in the wild-type genetic background (gray) and in the genetic background of the deletion of the gene encoding the sigma factor SigD (black) were cultured in microplates. For three replicates, optical density and GFP-related fluorescence were measured. GFP data per optical density unit in arbitrary units are plotted over time for the three biological replicates.

[0117] Figure 12. Comparison of strains possessing the gene encoding GFP under the control of the PzpdN promoter or under the control of the P promoter ZPC XYZ-

[0118] Strains possessing the sfgfp gene are either under the control of the P promoter zp dN (in gray), either under the control of the P promoter ZPCXYZ (in black) were diluted to t=0 in the presence of 500nM IPTG. For each triplicate culture, the culture optical density (round symbols) and GFP production (square symbols) were monitored. To avoid signal saturation, the sensitivity of the measuring device was reduced.

[0119] Figure 13. Growth of the strain expressing the phage Bil66 nuclease.

[0120] The strain possessing the genes coding for the protein complex having nuclease activity under the control of the P promoter zp dN was diluted to t=0 in the presence of 500nM IPTG (in black) or in the absence of IPTG. For each of the triplicate cultures, the optical density was monitored. Growth arrest and lysis were observed after nuclease induction.

[0121] EXAMPLES

[0122] Materials and methods

[0123] Culture of bacteria

[0124] Bacillus subtilis strains were grown at 37°C in LB medium and in CH medium: a rich medium of chemically defined composition. CH medium contains (per L.): hydrolyzed casein (Difco Casamino L-acids, vitamin-free) 9.4 g.; L-glutamic acid NaH2O 3.76 g.; L-alanine 1.22 g.; L-asparagine 1.41 g.; KH2PO41.28 g.; Na2SO4, 0.094 g.; NH4CI, 1.24 g.; NH4NO3, 0.094 g.; CaCI2,6H2O 0.036 g.; MgSO47H2O 0.062 g.; MnSO4H2O 0.068 mg.; FeCI36H2O 0.1 mg. C6H8O7H2O 0.02 mg supplemented with 0.5% glucose. Solid media were obtained by adding 1.5% agar to liquid media. Competent B. subtilis cells were transformed according to the method described in Konkol et al. (Journal of Bacteriology 195:18, 4085-4093, 2013). The antibiotics phleomycin, neomycin, chloramphenicol, erythromycin, and spectinomycin were added at final concentrations of 8, 15, 5, 0.5, and 100 pg.mL, respectively.Escherichia coli strains were grown at 37°C in LB medium, competent cells were prepared as described in Hanahan et al. (Methods in Enzymology 204, 63-113, 1991). Ampicillin at 100 pg.mL-1 was used to select plasmid transformants.

[0125] Molecular biology manipulations

[0126] The Invitrogen Platinum SuperFi DNA Polymerase Kit (ThermoFisher Scientific) was used for DNA fragment amplification. The Thermo Scientific DreamTaq DNA Polymerase Kit (ThermoFisher Scientific) was used for sample verification and colony PCR. These polymerases were used according to their manufacturer's recommendations. When the PCR template is modified DNA, such as for the SP01 phage genome, a specific protocol was used as described in Stewart et al. (Journal of Molecular Biology 24, 388(l):48-70, 2009). The first 5 amplifications were performed with an annealing temperature of 40°C, then the other 25 amplifications were performed at 50°C (the standard annealing temperature used here).

[0127] For DNA fragment preparation, genomic DNA was extracted using the GenElute kit (Sigma-Aldrich). PCR DNA fragments were purified using the QIAquick PCR purification kit from QIAGEN according to the manufacturer's recommendations. For DNA assembly, the NEBuilder HiFi Assembly Master Mix kit (NEB), the enzyme Bsal (NEB), and T4 DNA Ligase (NEB) as ligase were used according to the manufacturer's recommendations.

[0128] Construction of strains and plasmids

[0129] Building and fixing gp28

[0130] The genes encoding orthogonal transcription systems were amplified by PCR (fragment zpdN, gp28, sigB8, rpoS, rpoN and T7p07). The genes encoding the sigma factors RpoS, RpoN and T7 RNA polymerase were amplified using the E. coll ER2566 strain as a template; the genes encoding the transcription factors SigB, SigA (fragments sigB and sigA) were amplified using the genomic DNA of the BSB1 strain as a template; the gene encoding the Gp28 protein was obtained from the DNA of the phage SP01; the gene encoding the SigB8 protein was obtained from a metagenomic fragment of a Prevotella bryantii strain; The gene encoding sfGFP was amplified from genomic DNA of strain SG13 (Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016).

[0131] Using plasmid pDR111 as a template, the two regions containing either the 3' end of the amyE gene and the lad gene, or the 5' end of the amyE gene, the spectinomycin resistance gene, and the Phy-spank promoter were amplified (amyB and amyF fragments). These two fragments were joined by the Gibson reaction on either side of the amplification fragments of the sigB8, rpoS, sigA, gfp, or sigB gene sequences. The reaction products were integrated into the B. subtilis chromosome. The resulting clones were verified by PCR and sequencing.

[0132] Fragments containing the zpdN, gp28, rpoN, or T7 RNA polymerase (rnaPr) genes were ligated, by Gibson reaction, to the complete plasmid pDRlll linearized downstream of the Phy-spank promoter (fragment pDRlllinv). The reaction products were contacted with competent E. coli TGI cells. Transformants selected on ampicillin were analyzed. Plasmids validated by restriction and sequencing were used to transform B. subtilis.

[0133] The sequencing results showed that the gp28 gene had two mutations (526-7 CA^TG) compared to the sequence deposited in the databases. Although these two mutations were found on several independent PCRs carried out on the same phage preparation, it was decided to correct these two mutations to restore the published sequence. For this purpose, two complementary oligonucleotides containing the modified regions were synthesized. These two oligonucleotides using the plasmid pDRll+gp28526-7CA-re as template were used to amplify the complete plasmid by correcting the mutations. This plasmid was recircularized by Gibson reaction. The reaction products were introduced into E. coli and a clone containing the corrected plasmid pDRll+gp28 was selected and introduced into B. subtilis.

[0134] Construction of the reporter system and the different promoters

[0135] A plasmid derived from pUC19 was constructed by Gibson reaction to clone two regions of the B. subtilis chromosome (fragments glmS and ybdG, positions 201048 to 202483 bp and 220083 to 221532 bp on the reference strain B. subtilis 168 - GenBank AL009126.3), the gene encoding tetracycline resistance (fragment tetL) and the gene encoding sfGFP (fragment sfgfp) with an upstream terminator (fragment term). This plasmid replaced by homologous recombination the region of the chromosome denoted "prophage 1" by the sfgfp genes and the tetracycline resistance gene.

[0136] This plasmid pJSM3T was amplified by inverse PCR with primers having at their 5' end the promoter sequence P zp dG- The PCR fragment was circularized and then integrated into the chromosome of B. subtilis. The resulting strain possesses the sfGFP gene under the control of the P promoter zpdG single copy. For the reporter constructs of the sigma factor Gp28 and the phage T7 RNA polymerase, the different fragments carrying the promoters were obtained by PCR and cloned into the plasmid pJSM3T also amplified by PCR. In these constructs, the RBS RO was added to the primers used to amplify the plasmid.

[0137] Construction of the self-activation loop

[0138] Plasmid pDR11-zpd / V was amplified with primers located upstream of the Phy-spank promoter. A fragment containing the P zpdG was generated independently. The floating tails of the primers were constructed such that after assembly, an 80 bp region separated the two promoter sequences. The two amplified sequences were joined using the Gibson method and purified from the template using the enzyme DpnL. Then, the fragment to be integrated into the chromosome was amplified with primers at the ends of the homology zones (Amy5' and Amy3') carried by pDR111. This PCR product was then directly transformed into the B. subtilis strain containing the P construct. Z CEO fgfp.

[0139] SigA reporter module

[0140] The glyA and upp / atpl regions, the erythromycin resistance gene (erm); the constitutive promoter (Pref, Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016) and the mKate2 gene were amplified by PCR. The different PCR fragments were assembled by Gibson reaction. The complete construct was amplified with primers at the ends of the fragments homologous to the B. subtilis chromosome, the PCR product was transformed into the strain (BSB1).

[0141] GFP expression repressor

[0142] The gene encoding the TetR(BD) repressor (Schmitter et al., Molecular Microbiology, 105:413-425, 2017) was amplified by PCR and assembled by the Gibson method with linearized pDR111 downstream of the Phy-spank promoter. The fragment to be integrated was amplified by PCR and transformed into B. subtilis.

[0143] A primer with two tetO boxes placed between the -35 and -10 boxes and downstream of the -10 box of the spoVG promoter respectively; was used to amplify the sfgfp gene. The phleomycin resistance gene (ble) and the two DNA fragments carrying the nirC and sacP genes respectively were amplified by PCR and assembled by the Golden Gate method. The fragment to be inserted into the B. subtilis chromosome was amplified and transformed into the strain carrying the Ph construct y - spanktetRoB-

[0144] Repressible expression of SigA and deletion of endogenous SigA The same construct as that with the sfgfp gene was made to amplify the sigA gene under the control of P te to-

[0145] The two regions located on either side of the sigA gene and the chloramphenicol resistance gene were assembled by the Gibson method and cloned into the plasmid pUC19 in the E. coli TGI strain. The plasmid pUC19_sigA was linearized by digestion with the restriction enzyme NdeI (site located outside the region of interest). The linearized plasmid was transformed into the strain SMS185 (Ph y . spankSigA) in the presence of IPTG.

[0146] Complete system

[0147] The plasmid pDR.lll_P Z pdG_Phy-spank-zpdN is linearized by PCR using primers located downstream of the zpdN gene and assembled with PCR fragments carrying the tetR gene B o by the Gibson method. The fragment to be integrated is then amplified and transformed into the B. subtilis strain.

[0148] The constructions carried out are presented in Table 3.

[0149] Table 3. Constructs constructed for the development of the invasive orthogonal expression system. Tandem affinity purification

[0150] Strains were grown in LB medium supplemented with 1 mM IPTG until DOgoo = 1. 100 ml of culture were harvested and centrifuged, the pellets were washed in buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl), then directly frozen in liquid nitrogen. The pellets were resuspended in 600 μL of buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA and 0.1% TritonX-100) with 6 μL of lysozyme (Sigma-Aldrich) and 1.5 μL of benzonase (25 U / μL, Novagen). The suspension was then incubated for 15 min at 37°C then 30 min on ice; centrifuged for 30 min at 20,000 g at 4°C. The supernatant is collected and transferred to a column (BioSpin - Biorad) containing 200 pL of anti-FLAG M2 resin (Sigma) previously equilibrated in buffer B. The columns are left overnight at 4°C with shaking.The next day, the resin was resuspended in 200 μL of TEV buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1% TritonX-100 and 1 mM DTT) with 50 units of TEV protease (Invitrogen). The columns were left overnight at 4°C with shaking, then eluted in a second column containing Calmodulin-Sepharose 4B resin (Amersham Bioscience) previously equilibrated in CBB buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, TritonX-1000, 1%, 1 mM DTT and 2 mM CaCl). The columns were washed twice with 500 μL of CBB buffer, then left overnight at 4°C. The columns were washed twice with 500 μL of CBB buffer, followed by 200 μL of CWB buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% TritonX-100, 1 mM DTT and 0.1 mM CaCl). The protein complexes were eluted twice with 500 μL of CEB buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT and 3 mM EGTA).Proteins are precipitated with acetone and then analyzed on SDS-PAGE gel or in a resuspension solution (Urea.

[0151] 1 M and 100 mM Tris-HCl pH 8.5) for analysis by liquid chromatography coupled with tandem mass spectrometry, LC-MS / MS.

[0152] Viability tests on solid media

[0153] B. subtilis strains were pre-cultured in LB medium for approximately 20 hours in 96-well plates (CELLSTAR®, Greiner Bio-One). In the morning, they were diluted 10 times in the medium of interest. When these precultures reached an ODgoo ~ 0.3-0.4, they were then diluted again 20 times in the medium of interest. At each step, the cultures were seeded into microplates and incubated at 37°C with constant shaking (360 rpm) in a Synergy™ multi-mode plate reader.

[0154] 2 (Biotek®). The reference strains, Phy-spank-gfp and BSB1, were systematically tested. Each strain was grown in triplicate at least, and measured in three independent experiments. Every 10 minutes, the ODœ0 and fluorescence (GFP: excitation spectrum 485 ± 20 nm, emission spectrum 528 ± 20 nm; mKate2: excitation spectrum 590 ± 20 nm, emission spectrum 635 ± 20 nm) were measured. In order to compare the promoter with other uninduced strains, the fluorescence was normalized to a reference strain. For a constitutive promoter, the Relative Expression Unit was calculated:

[0155] REU(ti )=[(Fluo(ti) / D06oo(ti)]strain / [(Fluo(ti) / D06oo(ti)]ref In the case of an inducible promoter, the fluorescence value per OD obtained without inducer is subtracted from that obtained with induction before reporting it to the reference strain:

[0156] REU(ti) ={[(Fluo(ti) / DO600(ti)]strain+ind“[(Fluo(ti) / DO600(ti)]strain-ind} / [(Fluo(ti) / OD600(ti)]ref

[0157] For all experiments, the mean values ​​and standard deviation are then calculated.

[0158] Propidium Iodide Viability Test Propidium iodide is a colored molecule that cannot be accumulated in bacteria that have lost membrane integrity, i.e., if the cells are dead. To test cell viability, propidium iodide at a final concentration of 167 pM was added to a sample of the cultures. After 10 minutes of incubation, the cells were analyzed by flow cytometry to detect staining by propidium iodide internalized in the cells. To validate this staining, a sample of BSB1 cells in exponential phase was treated directly. Another sample of the culture was centrifuged, then resuspended in isopropanol for 60 minutes to kill the cells, then centrifuged. The cells were taken up in 10 mM Tris + 150 mM NaCl, then stained in the same way. The test was validated with these two samples.

[0159] Deletion of alternative sigma factors The erythromycin resistance gene was amplified from an erythromycin-resistant strain. The downstream and upstream regions of each of the genes encoding the B. subtilis alternative sigma factors Sig B, D, and H were amplified by PCR. Primers were designed to introduce BsaI sites at the ends of all these fragments. For each construct, the upstream and downstream fragments, and the resistance cassette, were cloned by a Golden Gate reaction. The resulting linear fragments were integrated into the genome of strain BSB1 and then transferred to the chromosome of the strain containing the entire GFP production system. The strains constructed are summarized in Table 4, and the protein sequences of the alternative sigma factors are shown in Table 5.

[0160] Table 4. Strains and constructs in which alternative sigma factors were deleted.

[0161] Table 5. Sequences of the alternative sigma factors SigB, SigB and SigH.

[0162] Promoter assays recognized by the sigma factor ZpdN

[0163] Using genomic DNA from the strain possessing the complete production system, the region upstream of the P promoter zpdG containing the integration region (glmS gene) and the region downstream of this promoter (sfgfp, tetL, and YbdG genes) were amplified. The oligonucleotides were designed to introduce the two promoters to be tested, as well as a 30-base pair overlapping region. The two DNA fragments were joined by a Gibson reaction and then introduced into the strain overproducing the sigma factor ZpdN and into the strain containing all parts of the production system except GFP. The strains constructed are summarized in Table 6, and the sequences of the tested promoters are shown in Table 7.

[0164] Table 6. Strains and constructs made to test the other promoters.

[0165] Table 7. Sequences of promoter regions.

[0166] Cloning of the proteins of interest LacZ, AmyS, YqaH and NucA

[0167] To test whether other proteins other than GFP could be expressed, the open reading frame of GFP was replaced by the open reading frame of the genes encoding LacZ, NucA, YqaH, or AmyS. The region upstream of the ATG start codon and the region downstream of the stop codon were amplified by PCR by introducing the BsaI site at the ends. In the same way, the different reading frames were amplified by creating BsaI sites compatible with the previous ones. The different fragments were joined by a Golden Gate reaction and then transferred into the final strain. The strains constructed are summarized in Table 8, and the protein sequences are shown in Table 9.

[0168] Table 8. Strains and constructs made to test LacZ, AmyS, NucA and YqaH proteins.

[0169] Table 9. Sequences of LacZ, AmyS, YqaH and NucA proteins.

[0170] Results

[0171] In order to construct the invasive orthogonal transcription system, several modules were constructed (Figure 1). The first module consists of placing the orthogonal system under the control of an inducible promoter and the transcription of the gene of interest (e.g., the gene encoding GFP) under the control of the orthogonal transcription system.

[0172] The second module involves setting up a self-activation loop of the orthogonal transcription system transcription in order to amplify the production of the orthogonal transcription system.

[0173] The third module consists of implementing a repression of the expression of the housekeeping sigma factor SigA of Bacillus subtilis.

[0174] Finally, the self-activation loop of sigma factor and the repression of sigma factor A expression are coupled.

[0175] Example 1: Construction of the first module

[0176] Several orthogonal transcription systems were cloned into the B. subtilis chromosome as a single copy at the amyE locus under the control of the IPTG-inducible promoter: Phy-spank (Figure 2, A).

[0177] In these strains, a transcriptional fusion between the P subunit of RNA polymerase and the SPA tag was introduced. The RNA polymerase complexes were purified and the protein partners were analyzed on acrylamide gel (Figure 2, B).

[0178] Gel analysis revealed that the gel-staining sigma factors Gp28 and ZpdN interact with RNA polymerase. For these two candidates, different promoters recognized by either of these two sigma factors were cloned in front of the gene encoding GFP (Figure 3, A). Similar constructs were made in the RNA polymerase-producing strain of phage T7 under the control of the Phy-spank- promoter.

[0179] The constructs were integrated as a single copy into the B. subtilis chromosome between the ybbU and ybdG genes in a region close to the chromosome origin of replication.

[0180] For each strain constructed, the amount of GFP produced was measured over time (fluorescence / DOgoonm) in LB medium without or with 500 pM IPTG (Figure 3, B).

[0181] The specific fluorescence of orthogonal sigma factor induction was calculated ((fluorescence / D06oonm)iPTG5oo liM-(fluorescence / DOgoonml-iPTc). In order to compare the strains with each other, the specific fluorescences were normalized with respect to a reference strain possessing the gene encoding GFP under the control of the Phy-spank promoter (GFP / OD norm). The results show that no GFP activity was detected for constructs containing promoters recognized by the sigma factor Gp28 (Table 10). Regarding the sigma factor ZpdN, the tested promoter exhibits strong transcriptional activity similar to that observed for T7 RNA polymerase. Table 10. GFP activity of the different strains tested.

[0182] The construction of the system was continued with ZpdN and the first module was validated. e 2: construction of the second module

[0183] For the second module, the active promoter recognized by the sigma factor ZpdN used in module 1 (Pzpds) was cloned upstream of the hy-spank promoter (Figure 4, A).

[0184] This auto-induction loop allows for amplification of the induction response of ZpdN expression and allows for ZpdN expression independent of the B. subtilis housekeeping sigma factor.

[0185] The normalized specific activities were measured for strains with and without the auto-induction loop at different IPTG concentrations (Figure 4, B). The results show that at a low dose of IPTG (less than or equal to 20 pM), the auto-activation loop allows for a greater production of GFP. At higher concentrations (more than 100 pM), the strain with the auto-activation loop produces slightly less GFP; it should be noted that, in this strain, the ZpdN promoter is present in two copies on the chromosome. The second module is constructed and active. e 3: construction of the third module

[0186] For the third module, the gene encoding the TetR repressor DB was cloned under the control of the inducible Phy-spank promoter at the amyE locus in the B. subtilis chromosome (Figure 5, A).

[0187] To test the repression efficiency of this protein, a promoter containing the operator regions recognized by the TetR repressor B D (Pteto) was cloned in front of the GFP gene.

[0188] Measurement of fluorescence produced in cultures without and with IPTG (without and with repression of the Pteto promoter) shows a very significant difference (1.7+ / -0.1 arbitrary units without IPTG and 0.11+ / -0.08 arbitrary units with IPTG).

[0189] A more than 15-fold repression of the GFP gene transcription / translation rate was measured under the control of this promoter.

[0190] Once the repression by TetR B D validated, the sigA gene was cloned under the control of the P promoter tet0. Then, the natural copy at the sigA gene locus was replaced by a gene encoding chloramphenicol resistance.

[0191] It was then verified that the growth of this strain is sensitive to IPTG. After adding IPTG to the culture, the growth of the strain is strongly affected. From 30 minutes, the slope of the optical density monitoring curve is inflected (Figure 5, B): the generation time increases. Then after 3 hours, the growth stops. In the 3-hour cultures, a very strong reduction (100,000 times) of the viability on LB Agar medium plates without inducer is observed.

[0192] The use in this latter strain of a P reporter re fmkate2 (Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016), for which the production of the fluorescent protein is dependent on a sigma A promoter, showed that the induction of TetR repressor BD reduces the amount of fluorescence indicating possible competition between SigA and ZpdN.

[0193] The third module allowing the repression of the expression of the housekeeping sigma factor of B. subtilis is therefore functional.

[0194] Example 4: Coupling the self-activation loop

[0195] The final step was to couple the self-activation loop and the repression of the expression of the housekeeping sigma factor.

[0196] For this, the gene encoding the TetR repressor BD and the gene encoding the sigma factor ZpdN were cloned into an operon under the control of the Phy-spank promoter (Figure 6, A).

[0197] In this strain, the housekeeping sigma factor SigA is under the control of the P promoter tet0 .

[0198] Without inducer, the sigA gene is expressed: the B. subtilis protein transcription program is active.

[0199] The addition of IPTG in this strain induces i) the expression of the sigma factor ZpdN which, in turn, will induce the expression of GFP and its own expression and ii) the expression of the repressor TetR which will block the expression of the sigA gene.

[0200] The housekeeping transcription program for the production of proteins enabling the growth of B. subtilis is stopped and the transcription program for the production of proteins of interest is activated: this switching of transcription programs then makes it possible to redirect all the cell's resources towards the production of proteins of interest.

[0201] In this strain, the level of GFP production is dependent on DOgoo at the time of IPTG induction (Figure 6, B). The maximum level of GFP produced within 3 hours of induction is comparable to the level of GFP produced by the P promoter vegin 7 hours of culture and significantly higher than the level of GFP produced after induction of Phy-spank- Cytometry analysis of cells after induction of the switch in transcription programs (Figure 7) was carried out.

[0202] After one hour of induction, some of the cells have switched to the GFP production program; after 2 hours, all the cells produce GFP and this in a very homogeneous manner between the cells (the peak is very slender). No non-GFP-producing cells are detected, whereas, in the case of the strain carrying the P construct V e g gfp, approximately 1% of cells do not produce GFP. After 3 hours of induction, GFP production increased with a high proportion of cells producing a significant amount of GFP (greater than 2x10 6arbitrary units). The level of GFP production is significantly higher than that obtained with the Phy-spankgfp strain and the population is much more homogeneous.

[0203] The various embodiments mentioned do not in any way limit the scope of the invention as claimed; they are given for the purpose of clarification in order to better understand the invention.

[0204] Example 5: Characterization of cell viability after induction of the production phase

[0205] The state of the cells was characterized after induction of GFP production. The cells were diluted and cultured in the presence of IPTG. The optical density stopped increasing after two hours, indicating growth arrest. No decrease in optical density was observed, therefore there was no lysis in the population.

[0206] During the kinetics, samples were taken, diluted, and then spread on plates of rich medium without inducer. After 24 hours of incubation at 37°C, the colonies formed were counted. From 1 hour of induction, the cells were no longer cultivable (virtually no colonies formed on the plates), even after stopping the induction (Figure 8).

[0207] To characterize their viability, the cells were incubated with propidium iodide, which only labels dead cells. After three hours of induction, no cells were stained with propidium iodide, indicating that there were no dead cells in the population (Figure 9). These are so-called "viable but non-culturable" cells. The integrity of the bacterial membrane is therefore preserved. This situation is advantageous because it maintains the integrity of the cells and therefore allows for a purification step by centrifugation.

[0208] Example 6: Reversibility of induction

[0209] To determine whether it is possible to preserve viable cells, induction at a lower concentration of IPTG (20 mM) was performed. After three hours, the cells are still able to grow on medium lacking IPTG (Figure 10). The cells are therefore still viable after three hours under these induction conditions of the system. It is possible to reverse the induction and recover viable cells on solid medium without inducer. By calculating the number of colony-forming particles per unit of optical density, it appears that more than half of the bacteria are still alive. They retain all the selection markers and therefore have not lost any part of the system. At this concentration of inducer, the induction is reversible and no subpopulation in which the production system would be lost is selected.

[0210] Example 7: Effect of deletion of secondary sigma factors in B. subtilis

[0211] Some other alternative sigma factors present in the cell could compete with ZpdN for access to RNA polymerase. As a result, less RNA polymerase + ZpdN complex would be formed in the cell, thus limiting the transcription of the gene encoding GFP. To test this hypothesis, the alternative sigma factors produced during the exponential phase were deleted in the strain possessing the entire system. The level of GFP production was tested after deletion of the genes encoding sigma factors B, D and H respectively.

[0212] Only the deletion of the sigma factor encoded by the sigD gene showed an effect on this production (Figure 11). Previous results (Fehler et al., Microbial Cell Factories, 21:131, 2022) have shown that inactivation of genes encoding flagellum proteins induces an increase in amylase production. These genes are under the control of the sigmaD factor. It therefore appears that what is observed is not due to competition between sigma factors, but rather to a release of resources due to the fact that the transcription program induced by the production of the SigD factor is not active. This result indicates that strain modifications favorable to protein production can have a cumulative effect in the constructed system.

[0213] Example 8: Tests of other promoters recognized by the sigma factor ZpdN

[0214] In order to produce several proteins simultaneously in the same cell, it would be interesting to have several promoters recognized by the sigma factor ZpdN.

[0215] Two different promoters were cloned in front of the RO ribosome binding site and the gene encoding sfGFP. In these strains, induction of sigma factor ZpdN production induced a level of GFP production approximately twice that produced by the previous construct (Table 11).

[0216] Table 11. GFP production as a function of the promoter tested.

[0217] The best of the two promoters was integrated into the strain with the full production system. A comparison between this new construct and the previous one was made. The results show that the growth curves of the two strains are similar, indicating that the growth arrest due to the loss of sigma A is the same in both strains (Figure 12). However, it is clear that the new construct allows for the production of between 4 and 5 times more GFP in the cells.

[0218] Example 9: Generalization to the production of other proteins

[0219] The first cloned protein is beta-galactosidase, a protein of very high molecular weight: 116 kDa. After 3 hours of induction, a beta-galactosidase activity of approximately 8500 nmol of ortho-nitrophenyl-p-galactoside (ONP) formed per minute per milligram of protein is detected in the cells. For comparison, previous results show that, for the inducible promoter P S pac in a single copy in the chromosome, the level of activity measured after complete induction of the subpromoter is approximately 1000 nmol of ONP formed per minute per milligram of protein.

[0220] The second cloned protein is a nuclease encoded by two polypeptides of the phage Bil66. In the absence of induction, the generation times (calculated on 6 replicates) of the strain containing or not the nuclease genes are similar for the strain with the sfGFP gene (41+ / -4 minutes) or beta-galactosidase gene (42+ / -1 minutes) and for the strain with the nuclease (40+ / -1 minutes). This result indicates that in the absence of induction, the expression is indeed closed. In the strain with the nuclease gene, induction of the system induces rapid lysis of the cells, which indicates that the nuclease is indeed produced (Figure 13).

[0221] The third cloned protein is the YqaH protein, which inhibits chromosomal DNA replication in B. subtilis, thus blocking cell growth. The gene encoding this protein is cloned into the complete production system. In this example, the protein was successfully cloned and, after induction of production, induced rapid growth arrest. The fourth cloned protein is the AmyS amylase from Bacillus stearothermophilus. This protein has a signal peptide that is recognized by the Sec secretion system in B. subtilis. Preliminary results showed that protein secretion was still possible after induction of protein production in the constructed strain.

Claims

Claims 1. Genetically modified bacteria comprising: - a first nucleotide sequence encoding an orthogonal sigma factor capable of interacting with the RNA polymerase of the genetically modified bacterium, the expression of the orthogonal sigma factor being inducible and under the control of a promoter recognized by the orthogonal sigma factor, and - a second nucleotide sequence whose expression product inhibits the action of an endogenous sigma factor of the genetically modified bacterium, the expression of the second nucleotide sequence being under the control of a promoter recognized by the orthogonal sigma factor.

2. Genetically modified bacteria according to claim 1, further comprising: - a third nucleotide sequence coding at least one protein of interest, the expression of said at least one protein of interest being under the control of a promoter recognized by the orthogonal sigma factor.

3. Genetically modified bacteria according to claims 1 or 2, in which the orthogonal sigma factor is chosen from: - a sigma factor from a bacterial species different from that of the genetically modified bacterium, and - a sigma factor specific to an extra-chromosomal element naturally absent from the genetically modified bacteria.

4. Genetically modified bacteria according to any one of claims 1 to 3, said bacteria belonging to a species chosen from Bacillus subtilis, Escherichia coll, Geobacillus stearothermophilus and Lactococcus lactis, preferably B. subtilis.

5. Method for producing at least one protein of interest comprising: - a step of culturing a genetically modified bacterium as defined according to any one of claims 1 to 4, and - a step of induction of the expression of the orthogonal sigma factor.

6. Polynucleotide comprising at least a first nucleotide sequence coding a sigma factor under the control of two tandem promoters: one conferring an inducible character and the other being recognized by said sigma factor.

7. Polynucleotide according to claim 6, further comprising a second nucleotide sequence encoding: - a transcriptional repressor, - an interfering RNA, - a protease, or - a sigma factor inhibitor.

8. Polynucleotide according to claim 7, further comprising a third nucleotide sequence encoding at least one protein of interest.

9. Expression vector comprising at least one polynucleotide defined according to any one of claims 6 to 8.

10. Kit for the recombinant expression of at least one protein of interest comprising at least: - a genetically modified bacterium defined according to any one of claims 1 to 4, - a polynucleotide defined according to any one of claims 6 to 8, and / or - an expression vector defined according to claim 9, and - optionally, a molecule making it possible to induce the expression of said at least one protein of interest.

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