Transgenic bacteria comprising invasive orthogonal expression system

Abstract

The present invention relates to a transgenic bacterium comprising an invasive orthogonal expression system and its use for the production of at least one protein of interest. The invention also relates to polynucleotides, vectors and kits for expressing at least one protein of interest.

Description

TECHNICAL FIELD

[0001] The present invention relates to a transgenic bacterium comprising an orthogonal transcriptional expression system and its use for the production of at least one protein of interest. The present invention also relates to polynucleotides, vectors and kits for expressing at least one protein of interest. BACKGROUND

[0002] To face the challenge of climate change, it seems necessary to make alternatives to petroleum-based chemicals viable. There are many biological production systems for synthesizing molecules using bacteria, which consume far fewer resources than their petroleum-based counterparts.

[0003] However, current biological production systems sometimes provide a yield that is too low to be considered as a comprehensive solution.

[0004] There is therefore a real need for optimized biological production systems to improve the production yield of bacteria. SUMMARY

[0005] In this context, the inventors have observed that current protein biological production systems share the transcriptional and translational capacity of the host bacteria to synthesize all proteins in the cell.

[0006] Unexpectedly, the inventors have then demonstrated that it is possible to stop the synthesis of proteins in the host bacteria and to repurpose the cellular resources to an orthogonal expression system. This system is considered orthogonal because it operates independently of the endogenous system of the bacteria. Moreover, this system is invasive because it overrides the endogenous system. The orthogonal expression system developed by the inventors thus makes it possible to maximize the allocation of resources dedicated to biological production by blocking the endogenous expression system.

[0007] By taking advantage of the promoter sequence recognition function of sigma factors, the inventors cloned a heterologous sigma factor able to auto-induce its synthesis and a repressor protein controlling the synthesis of the endogenous sigma factor. Interestingly, the resulting bacteria made it possible to produce, upon induction for 3 hours, the following amount of protein when synthesizing a test protein: 35 times the amount obtained with the best inducible promoter described in B. subtilis.

[0008] The present invention thus relates to a transgenic bacterium comprising:

[0009] - a first nucleotide sequence encoding an orthogonal sigma factor able to interact with the RNA polymerase of the transgenic bacterium,

[0010] the expression of said orthogonal sigma factor being inducible and controlled by a promoter recognized by said orthogonal sigma factor, and

[0011] - a second nucleotide sequence, the expression product of which inhibits the action of the endogenous sigma factor of the transgenic bacterium,

[0012] The expression of said second nucleotide sequence is controlled by a promoter recognized by said orthogonal sigma factor.

[0013] According to the application, the expression "sigma factor" (or sigma) denotes a subunit of the bacterial RNA polymerase required for initiating transcription. This protein plays a decisive role in selecting the enzyme binding site.

[0014] According to the application, the expression "sigma factor" encompasses not only sigma factors, but also phage-specific monomeric RNA polymerases such as T3 or T7 RNA polymerases, which directly recognize specific promoter sequences.

[0015] According to the application, the term "endogenous" means that the element in question naturally occurs in the transgenic bacterial strain. According to the application, the endogenous sigma factor naturally occurs in the transgenic bacterial strain.

[0016] According to the application, the term "orthogonal" means that the element in question does not naturally occur in the transgenic bacterial strain, but is capable of performing the same function as another element that naturally occurs in this strain. Thus, according to the application, the orthogonal sigma factor does not naturally occur in the transgenic bacterial strain, but when it is introduced into this and expressed, it is capable of initiating transcription in parallel and independently of the sigma factor that is naturally expressed in this strain. In other words, the orthogonal sigma factor makes it possible to introduce a transcription system that is separate from the transcription system that naturally occurs in the transgenic bacterium.

[0017] The orthogonal sigma factor does not naturally occur in the transgenic bacterial strain, which means that it can originate not only from other bacterial species (i.e. a heterologous sigma factor), but also from other strains of the same species as the transgenic bacterium or from a phage that does not naturally occur in the transgenic strain. This orthogonal sigma factor can be natural or genetically modified for recognizing a different promoter sequence than the promoter sequence that the orthogonal sigma factor naturally recognizes in the organism from which it originates.

[0018] In the present application, 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 inactivated in the presence of a given regulatory element. Without limitation, this regulatory element can be added directly to the culture medium or removed from the culture medium.

[0019] For example, in one embodiment, the expression of the orthogonal sigma factor can be under the control of a xylose-inducible promoter, which can thus be activated by adding xylose to the culture medium.

[0020] For example, in another embodiment, the expression of the orthogonal sigma factor can be under the control of a lactose (or allolactose or any functional analogue) inducible promoter, so this can be blocked by the presence of the LacI regulatory protein and activated by the addition of IPTG, which will bind to LacI and prevent LacI from binding to the "lactose" promoter.

[0021] For example, in other embodiments, the expression of the orthogonal sigma factor can be under the control of a streptolysin, copper, mannose (P manP ), surfactin (P srfA ), sucrose (P sacB ), mitomycin (P dinR ) or temperature (P2) inducible promoter.

[0022] In one embodiment, the application relates to a transgenic bacterium as defined above, further comprising:

[0023] - a third nucleotide sequence encoding at least one protein of interest,

[0024] The expression of the at least one protein of interest is under the control of a promoter recognized by the orthogonal sigma factor.

[0025] In one embodiment, the at least one protein of interest corresponds to a single protein of interest.

[0026] In one embodiment, the at least one protein of interest corresponds to two proteins of interest.

[0027] In one embodiment, the at least one protein of interest corresponds to two or more proteins of interest.

[0028] According to the application, the term "protein" denotes a polymer of amino acids and also encompasses small peptides, for example small peptides of 10 to 100 amino acids.

[0029] According to the application, the expression "protein of interest" generally denotes any protein which is actively chosen for the implementation of the application. Such a protein can have commercial, therapeutic, industrial, technical or research benefits. Indirectly, a protein of interest according to the application can also be an intermediate product or an enzyme capable of transforming a substrate into a protein of particular interest.

[0030] In one embodiment, the protein of interest is an enzyme, for example an enzyme chosen from amylases, xylanases, lichenases, lipases, cellulases, pectinases, hydrolases, pullulanases, nattokinases, pectinases, agarases, galactosidases, proteases and peptidases.

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

[0032] In one embodiment, the protein of interest is an antigen.

[0033] In one embodiment, the protein of interest is a cytokine, in particular a cytokine selected from the group consisting of alpha interferon, beta interferon and gamma interferon.

[0034] In one embodiment, the protein of interest is a food additive.

[0035] In one embodiment, the protein of interest is a bacterial biofilm protein.

[0036] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the orthogonal sigma factor is selected from the group consisting of:

[0037] - a sigma factor from a bacterial species different from the transgenic bacterium, and

[0038] - a sigma factor specific for an extrachromosomal element naturally absent in the transgenic bacterium.

[0039] In one embodiment, the extrachromosomal element can be a plasmid or a bacteriophage, such as T3 or T7. One of the advantages of selecting an extrachromosomal element is that it makes it possible to use a sigma factor present in a strain belonging to the same species as the transgenic bacterium, which sigma factor is thus able to function in the transgenic bacterium while recognizing a specific promoter that is not naturally present in the transgenic bacterium.

[0040] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the expression of the orthogonal sigma factor is controlled by two promoters in series: one conferring an inducible character and the other being recognized by the orthogonal sigma factor.

[0041] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the expression product of the second sequence is selected from the group consisting of:

[0042] - a transcriptional repressor,

[0043] - an interfering RNA,

[0044] - a protease, and

[0045] - a sigma factor inhibitor.

[0046] The expression product of the second sequence is intended to inhibit the action of the endogenous sigma factor, i.e. to shut down the transcription system naturally present in the transgenic bacterium.

[0047] Without limitation, inhibiting the action of an endogenous sigma factor can be achieved, for example, by preventing transcription of a gene encoding an endogenous sigma factor (e.g., via a transcriptional repressor), preventing translation of a gene encoding an endogenous sigma factor (e.g., via an interfering RNA), degrading an endogenous sigma factor (e.g., via a protease targeting a cleavage site present on the endogenous sigma factor), or preventing specific binding of an endogenous sigma factor (e.g., via a sigma factor inhibitor).

[0048] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein 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, said transcriptional repressor being encoded by the second sequence.

[0049] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the native gene encoding the endogenous sigma factor is placed under the control of a promoter recognized by a transcriptional repressor, said transcriptional repressor being encoded by the second sequence.

[0050] In one embodiment, the application relates to a transgenic bacterium as defined above, said endogenous sigma factor being the major sigma factor of the transgenic bacterium.

[0051] According to the application, the expression "major sigma factor" means a sigma factor responsible for the transcription of housekeeping genes. Major sigma factors are generally considered to be essential for bacterial survival.

[0052] For example, in Bacillus subtilis, the major sigma factor is generally denoted as σΑ (SigA or σ A ); in Escherichia coli, the major sigma factor is generally denoted as σ

[0053] 70 (Sig70 or σ 70 ). For example, reference sequences corresponding to σ A of Bacillus subtilis and σ 70 of Escherichia coli are shown in Table 1.

[0054] Table 1. Examples of major sigma factors.

[0055]

[0056]

[0057] In one embodiment, the transgenic bacterium is a strain of Bacillus subtilis and the major sigma factor is the protein of sequence SEQ ID NO: 1.

[0058] In one embodiment, the transgenic bacteria is a strain of Escherichia coli and the primary sigma factor is the protein of sequence SEQ ID NO: 2.

[0059] In one embodiment, in addition to inhibiting the primary sigma factor, other endogenous sigma factors can be inhibited to avoid resource competition with the orthogonal sigma factor. For example, in Bacillus subtilis, in addition to inhibiting the primary sigma factor SigA, the alternative sigma factor SigD can be inhibited.

[0060] In one embodiment, the present application relates to a transgenic bacteria as defined above, said orthogonal sigma factor being selected from the group consisting of ZpdN (a sigma factor from Bacillus subtilis), Gp28 (a sigma factor from a bacteriophage), RpoS (a sigma factor from Escherichia coli), RpoN (a sigma factor from Escherichia coli), SigB8 (a sigma factor from Prevotella bryantii), SigB (a sigma factor from Bacillus subtilis) and t7p07 (a bacteriophage RNA polymerase), preferably ZpdN.

[0061] By way of example, reference sequences corresponding to orthogonal sigma factors suitable for use in Bacillus subtilis are given in Table 2.

[0062] Table 2. Examples of orthogonal sigma factors for Bacillus subtilis.

[0063]

[0064]

[0065]

[0066] In one embodiment, the present application relates to a transgenic bacteria as defined above, said orthogonal sigma factor being selected from the group consisting of 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.

[0067] In one embodiment, the present application relates to a transgenic bacteria as defined above, said bacteria belonging to a species selected from the group consisting of Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus and Lactococcus lactis, preferably Bacillus subtilis.

[0068] In one embodiment, the transgenic bacteria belongs to the species Bacillus subtilis and the orthogonal sigma factor is ZpdN (SEQ ID NO: 3).

[0069] In one embodiment, the transgenic bacterium belongs to the Bacillus subtilis species, the orthogonal sigma factor is ZpdN (SEQ ID NO: 3) and the promoter recognized by the orthogonal sigma factor is selected from the sequences comprising SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15.

[0070] In one embodiment, the application relates to a transgenic bacterium as defined above, which is a GRAS (Generally Recognized As Safe) bacterium, i.e. recognized as safe. Such a bacterium is particularly suitable for the synthesis of proteins for food or therapeutic applications.

[0071] In one embodiment, the transgenic bacterium is the Bacillus subtilis BSB1 strain. According to the Budapest Treaty, this isolated strain was deposited on February 1, 2023, under the number CNCM 1-5930 at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, 25 rue Dr Roux, 75724 Paris, France).

[0072] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the at least first and second nucleotide sequences and optionally the third nucleotide sequence are present in different polynucleotides.

[0073] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the at least first and second nucleotide sequences and optionally the third nucleotide sequence are present in the same polynucleotide.

[0074] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the first, second and third nucleotide sequences are present in the same polynucleotide.

[0075] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the same promoter controls the expression of the orthogonal sigma factor encoded by the first nucleotide sequence and the expression of the expression product of the second nucleotide sequence and optionally the expression of the protein of interest encoded by the third nucleotide sequence.

[0076] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the same promoter controls the expression of the orthogonal sigma factor encoded by the first nucleotide sequence, the expression of the expression product of the second nucleotide sequence and the expression of the protein of interest encoded by the third nucleotide sequence.

[0077] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the protein of interest is a protein that is toxic to the transgenic bacterium.

[0078] The system of the application is particularly suitable for the production of proteins that are toxic to bacteria. Indeed, in conventional expression systems, these proteins slow down or even block the growth of the bacteria, making it impossible to produce sufficient quantities of the protein. The system described in the application enables the bacteria to be optimally cultivated and then, once a sufficient number of bacteria has been reached, the cellular machinery can be switched to the sole benefit of the production of the protein of interest.

[0079] In one embodiment, the application relates to a transgenic bacterium as defined above, wherein the protein of interest is a protein that is secreted by the bacterium.

[0080] The system in question is particularly suitable for the production of secreted proteins, since it enables a large quantity of protein to be produced in the culture medium (which can then be easily recovered), in particular by preserving the viability of the bacteria.

[0081] In another aspect, the application relates to the use of a transgenic bacterium as defined above for the production of at least one protein of interest.

[0082] Another aspect of the application relates to a method for producing at least one protein of interest, comprising:

[0083] i) a step of cultivating a transgenic bacterium as defined above, and

[0084] ii) a step of inducing the expression of the orthogonal sigma factor.

[0085] In one embodiment, step i) and / or step ii) are carried out in a bioreactor (or fermenter).

[0086] In one embodiment, the application relates to a method as defined above, further comprising a step of recovering the protein of interest. The methods for recovering the protein of interest are well known to those skilled in the art and can vary, in particular depending on whether the protein of interest is intracellular or secreted into the culture medium by the bacteria.

[0087] In one embodiment, the application relates to a method as defined above, wherein the step of inducing the expression of the orthogonal sigma factor is stopped before the death of the transgenic bacteria.

[0088] Another aspect of the application relates to a polynucleotide comprising at least a first nucleotide sequence encoding a sigma factor under the control of two tandem promoters: one of the promoters confers an inducible property and the other is recognized by the sigma factor.

[0089] In one embodiment, the present application relates to a polynucleotide as defined above, further comprising a second nucleotide sequence, said second nucleotide sequence encoding:

[0090] - a transcriptional repressor,

[0091] - an interfering RNA,

[0092] - a protease, or

[0093] - a sigma factor inhibitor.

[0094] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the expression of the second nucleotide sequence is controlled by a promoter recognized by said sigma factor.

[0095] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the expression of the first nucleotide sequence and the second nucleotide sequence is controlled by the same two tandem promoters.

[0096] In one embodiment, the present application relates to a polynucleotide as defined above, further comprising a third nucleotide sequence encoding at least one protein of interest.

[0097] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the expression of the third nucleotide sequence is controlled by a promoter recognized by said sigma factor.

[0098] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the expression of the first nucleotide sequence and the third nucleotide sequence is controlled by the same two tandem promoters.

[0099] In one embodiment, the present application 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 tandem promoter.

[0100] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the second nucleotide sequence are in the form of an operon.

[0101] In one embodiment, the present application relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the third nucleotide sequence are in the form of an operon.

[0102] In one embodiment, the present application 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.

[0103] Another aspect of the present application relates to an expression vector comprising at least one polynucleotide as defined above.

[0104] Another aspect of the application relates to a kit for the recombinant expression of at least one protein of interest, comprising at least:

[0105] - a transgenic bacterium as defined above,

[0106] - a polynucleotide as defined above, and / or

[0107] - an expression vector as defined above, and

[0108] - optionally a molecule capable of inducing the expression of said at least one protein of interest.

[0109] In one embodiment, the molecule capable of inducing the expression of said at least one protein of interest is selected from the group consisting of xylose and IPTG.

[0110] The application will be described in more detail with the help of the following figures and examples, which in no way limit the application. BRIEF DESCRIPTION OF DRAWINGS

[0111] Figure 1 . Representation of the switching system between the two transcriptional programs.

[0112] To build the orthogonal transcriptional system, the gene encoding the orthogonal sigma factor (SigOrth) is under the control of the promoter P hy-span . This is repressed by the protein encoded by the lacI gene and induced by the addition of IPTG to the culture medium. The orthogonal sigma factor is able to recognize a specific promoter (P σorth ) and, in combination with the host's RNA polymerase, induces the transcription of the cloned gene of interest (goi). The promoter recognized by this orthogonal sigma factor is introduced upstream of the promoter P hy-spank to ensure the self-activation of the expression of the orthogonal sigma factor.

[0113] To build the system for the repression of the expression of sigma A, the gene encoding the sigma factor A is cloned in a single copy in the cell downstream of the promoter with the operator of the repressor protein TetR BD . The gene encoding this repressor protein is under the control of the promoter P hy-spank .

[0114] The switching between the two transcriptional programs is achieved by cloning the gene encoding the repressor protein TertR BD in the operon with the gene encoding the orthogonal sigma factor in the self-activation loop.

[0115] Figure 2 . Interaction between the orthogonal sigma factor and the host's RNA polymerase.

[0116] (A) Schematic of the strains tested. The gene encoding the sigma factor was cloned under the control of the IPTG inducible promoter P hy-spank . The construct enabling the induction of the expression of the gene encoding the sigma factor under the control of the promoter P hy-spank was placed in a B. subtilis strain producing a tagged RNA polymerase. This tag enables the purification of the protein complex, revealing the polypeptides interacting with the host RNA polymerase.

[0117] (B) Acrylamide gel analysis of the RNA polymerase protein complex. The purified RNA polymerase complex was analyzed on a gel. The subunits β, β' and α of the RNA polymerase as well as the housekeeping sigma factor SigA (indicated with a triangle) were detected in all purifications; the lowermost triangle indicates the expected size of the expressed sigma factor.

[0118] Figure 3 . Implementation of a specific reporter system for the orthogonal sigma factors Gp28 and ZpdN.

[0119] (A) Schematic of the strains tested. The genes encoding the sigma factors Gp28 and ZpdN or the T7 RNA polymerase were cloned under the control of the IPTG inducible promoter P hy-spank . The gene encoding GFP was under the control of a different promoter recognized by one of the two sigma factors.

[0120] (B) Histogram of the amount of fluorescence produced. The level of fluorescence (optical density) relative to the number of cells was measured in the different strains. For comparison purposes, a reference strain in which the gene encoding GFP is under the control of P hy-spank was used to normalize all these values. Each strain was tested three times in three technical replicates; the mean and standard deviation were calculated. The values obtained with the best promoter sequence tested are shown on a logarithmic scale.

[0121] Figure 4 . Influence of the auto-induction loop.

[0122] (A) Schematic of the strains tested. The promoter recognized by the sigma factor ZpdN (P hy-spank ) was cloned upstream of the construct P zpdG ::zpdN. The transcriptional activity associated with the production of the sigma factor ZpdN was demonstrated by the production of GFP: the sfgfp gene is under the control of the promoter P zpdG .

[0123] (B) Level of fluorescence produced by each of the two strains at different concentrations of IPTG. The GFP production per unit of DO hy-spank (i.e. optical density) was measured for the strains with and without the auto-activation loop ZpdN, relative to the reference strain, under different induction conditions of P 600 with increasing doses of IPTG.

[0124] Figure 5 . Inhibition of the housekeeping sigma factor of B. subtilis.

[0125] (A) Schematic of the strains tested. The gene encoding the transcriptional repressor TetR BD was cloned under the control of the IPTG inducible promoter P hy-spank . The gene encoding the housekeeping sigma factor was under the control of a promoter with an operator sequence recognized by the repressor TetR.

[0126] (B) Expression kinetics as a function of time of the fluorescence for different doses of the inducer. After induction of the expression of the gene tetR BD at t=0 with two concentrations of the inducer, the fluorescence level related to the number of cells (optical density) was measured as a function of time.

[0127] Figure 6 . Intrusive orthogonal transcription system.

[0128] (A) Schematic of the strains tested. The gene encoding the transcriptional repressor TetR BD and the gene encoding the sigma factor ZpdN were cloned under the control of the IPTG inducible promoter P hy-spank . The gene encoding the housekeeping sigma factor was under the control of a promoter with an operator sequence recognized by the repressor TetR.

[0129] (B) Fluorescence level of the expression as a function of the induction of DO 600nm . The fluorescence level related to the number of cells (optical density) was measured in a strain with a gene encoding GFP under the control of P veg (Left histogram) and in a strain with the whole system (right histogram). In the latter, the measurement was made without IPTG (full histogram) and after 3 hours of induction of P hy-spank expression (hatched histogram). The results are given as a function of the number of cells at the time of the induction (measured in DO 600nm equivalents).

[0130] Figure 7 . Cytometry analysis of the cells after induction of the transcriptional switch.

[0131] For each strain tested and for each sample, 100 000 cells were analyzed. The amount of fluorescence of each was measured. The graphs give the number of cells counted for each fluorescence intensity (on a logarithmic scale). Three reference strains were used: i) strain 168, which does not have a gene encoding sfGFP and makes it possible to measure the spontaneous fluorescence under the test conditions; ii) strain P veg- GFP, constitutively producing GFP; iii) a strain with GFP under the control of an IPTG inducible promoter.

[0132] Figure 8 . Cultivation of strains containing the complete production system in LB medium with and without IPTG.

[0133] Cells in the exponential phase were diluted at t=0 in LB without IPTG (empty symbols) or in the presence of 500 mM IPTG (solid symbols). Over time, the optical density of the cultures at 600 nm was measured (triangular symbols and dashed line). The cells were diluted and plated on LB boxes. After 24 hours, the number of colonies formed on the plates was determined (circular symbols and solid line).

[0134] Figure 9 . Analysis of bacterial survival by labelling with propidium iodide.

[0135] Live cells, dead cells and mixtures of both types of bacteria were treated with propidium iodide (left column). The cells were then analysed by cytometry to detect the red fluorescence of propidium iodide in the cytoplasm. The dead cells (alone and in mixture) fluoresced, indicating that the labelling clearly detected bacteria with altered membrane integrity. At the same time, cells cultivated for 3 hours without and with IPTG 500 nM were labelled and analysed in the same way (right column).

[0136] Figure 10 . Cultivation of strains containing the complete production system in LB medium with and without IPTG.

[0137] Cells in the exponential phase were diluted at t=0 in LB without IPTG (empty symbols) or in the presence of 20 mM IPTG (solid symbols). Over time, the optical density of the cultures at 600 nm was measured (triangular symbols and dashed line). The cells were diluted and plated on LB boxes. After 24 hours, the number of colonies formed on the plates was determined (circular symbols and solid line).

[0138] Figure 11 . Influence of the removal of the alternative sigma factor on the production of the protein of interest.

[0139] Cells containing the complete production system in a genetic background of the wild type (grey) and a genetic background in which the gene encoding the sigma factor SigD is deleted (black) were cultivated in microtiter plates. The optical density and the GFP-related fluorescence of three replicates were measured. The data of the GFP per unit of optical density in arbitrary units of the three biological replicates over time were plotted.

[0140] Figure 12 . Under the control of the promoter P zpdN or under the control of the promoter PzpcXYZ Comparison of strains with a gene encoding GFP under the control of the promoter P

[0141] At t=0, dilution of a strain with a sfgfp gene under the control of the promoter P zpdN (Gray) control or promoter P zpcXYZ (Black) control. For each culture, three replicates were performed, the optical density (circular symbols) and the GFP production (square symbols) of the culture were monitored. To avoid signal saturation, the sensitivity of the measuring device was reduced.

[0142] Figure 13 Growth of a strain expressing the nuclease of the phage Bil66.

[0143] At t=0, dilution of a strain with a gene encoding a protein complex with nuclease activity under the control of the promoter P zpdN (Black) in the presence of IPTG 500 nM or in the absence of IPTG. For each of the three replicate cultures, the optical density was monitored. Growth arrest and lysis were observed after induction of the nuclease. DETAILED DESCRIPTION

[0144] EMBODIMENT

[0145] MATERIALS AND METHODS

[0146] Culture of bacteria

[0147] Bacillus subtilis strains were grown at 37°C in LB medium and CH medium (chemically defined rich medium). CH medium contains (per liter): 9.4 g of hydrolyzed casein (Difco L-Tyrosine, without vitamins); 3.76 g of L-glutamic acid NaH2O; 1.22 g of L-alanine; 1.41 g of L- asparagine; 1.28 g of KH2PO4; 0.094 g of Na2SO4; 1.24 g of NH4CI; 0.094 g of NH4NO3; 0.036 g of CaCI2, 6H2O; 0.062 g of MgSO4 7H2O; 0.068 mg of MnSO4 H2O; 0.1 mg of FeCI3 6H2O; 0.02 mg of C6H8O7 H2O, supplemented with 0.5% of glucose. Solid medium was obtained by adding 1.5% of agar to the liquid medium. The competent cells of Bacillus subtilis cells were transformed according to the method described by Konkol et al. (Journal of Bacteriology 195: 18, 4085-4093, 2013). The final concentrations of 8, 15, 5, 0.5 and 100 μg.mL -1The antibiotics bleomycin, neomycin, chloramphenicol, erythromycin and spectinomycin.

[0148] E. coli strains were grown in LB medium at 37°C, as described by Hanahan et al. (Methods in Enzymology 204, 63-113, 1991) to prepare competent cells. Ampicillin at 100 pg.mL"1was used to select plasmid transformants.

[0149] Molecular biology procedures

[0150] DNA fragments were amplified using the Invitrogen Platinum SuperFi DNA Polymerase kit (Thermo Fisher Scientific). The Thermo Scientific DreamTaq DNA Polymerase kit (Thermo Fisher Scientific) was used for sample verification and colony PCR. These polymerases were used according to the manufacturer's recommendations. When the PCR template was modified DNA, for the genome of bacteriophage SPO1, a specific protocol as described by Stewart et al. (Journal of Molecular Biology 24, 388(1): 48-70, 2009) was used. The first 5 amplifications were performed at a hybridization temperature of 40°C, then the remaining 25 amplifications were performed at 50°C (standard hybridization temperature was used here).

[0151] To prepare DNA fragments, genomic DNA was extracted using the GenElute kit (Sigma-Aldrich). The DNA fragments of PCR were purified using the QIAquick PCR Purification kit according to the manufacturer's recommendations. For DNA assembly, the NEBuilder HiFi Assembly Master Mix kit (NEB), the enzyme BsaI (NEB) and the T4 DNA ligase (NEB) were used according to the manufacturer's recommendations.

[0152] Construction of strains and plasmids

[0153] Construction and correction of gp28

[0154] The genes encoding the orthogonal transcription system (fragments zpdN, gp28, sigB8, rpoS, rpoN, and T7p07) were amplified by PCR. The genes encoding the σ factor RpoS, RpoN, and RNA polymerase T7 were amplified using Escherichia coli ER2566 strain as a template; the genes encoding the transcription factors SigB and SigA (sigB fragment and sigA fragment) were amplified using genomic DNA of the BSB1 strain as a template; the gene encoding the Gp28 protein was obtained from the DNA of the bacteriophage SPO1; the gene encoding the SigB8 protein was obtained from the metagenomic fragment of the Prevotella brunneri strain; and the gene encoding sfGFP was amplified from the genomic DNA of the SG13 strain (Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016).

[0155] Plasmid pDR111 was used as a template to amplify the 3' end of the amyE gene and the lacI gene or the 5' end of the amyE gene, the spectinomycin resistance gene and the promoter P. hy-spank The two regions (amyB fragment and amyF fragment) were ligated by Gibson reaction on either side of the amplified fragment of the sequence of the gene sigB8, rpoS, sigA, gfp or sigB. The reaction products were integrated into the Bacillus subtilis chromosome. The obtained clones were checked by PCR and sequencing.

[0156] The RNA polymerases containing zpdN, gp28, rpoN or T7 RNA polymerase (rnaP T7 ) gene fragment is connected to the promoter P hy-spank The downstream linearized full-length plasmid pDR111 (fragment pDR111inv) was then added. The reaction product was contacted with competent E. coli TG1 cells. Transformants selected with ampicillin were analyzed. Plasmids verified by restriction and sequencing were used to transform Bacillus subtilis.

[0157] The sequencing results showed that the gp28 gene had two mutations (526-7 CA→TG ). Although these two mutations were found in several independent PCRs performed on the same phage preparation, it was decided to correct these two mutations to restore the published sequence. To this end, two complementary oligonucleotides containing the modified regions were synthesized. The plasmid pDR11+gp28 was used to 526-7CA-TG These two oligonucleotides, serving as templates, were used to amplify the complete plasmid by correcting the mutation. This plasmid was recircularized by the Gibson reaction. The reaction product was introduced into E. coli, and clones containing the corrected pDR11+gp28 plasmid were selected and introduced into Bacillus subtilis.

[0158] Construction of reporter systems and various promoters

[0159] 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), a gene encoding tetracycline resistance (fragment tetL) and a gene encoding sfGFP with an upstream terminator (fragment term). This plasmid allows the replacement of the chromosomal region denoted "prophage 1" by the sfGFP gene and the tetracycline resistance gene by homologous recombination.

[0160] This plasmid pJSM3T was amplified by inverse PCR with primers having at their extremity 5' the promoter sequence P zpdG . The PCR fragment was looped and then integrated into the B. subtilis chromosome. The obtained strain has a single copy of the sfGFP gene under the control of the promoter P zpdG . For the reporter constructs of the sigma factor Gp28 and the RNA polymerase of the phage T7, the various fragments carrying the promoters were obtained by PCR and cloned into the plasmid pJSM3T also amplified by PCR. In these constructs, the RBS R0 was added to the primers used to amplify the plasmid.

[0161] Construction of the auto-activation loop

[0162] The plasmid pDR111-zpdN was amplified with primers located upstream of the promoter P hy-spank . The fragment containing P zpdG was generated independently. The overhanging tails of the primers were constructed so that after assembly, a region of 80 pb separates the two promoter sequences. The two amplified sequences were ligated using the Gibson method and the sequences were purified from the template using the enzyme DpnI. The fragment to be integrated into the chromosome was then amplified at the end of the homology regions carried by pDR111 (Amy5' and Amy3') with primers and then this PCR product was directly transformed into a B. subtilis strain containing the construct P zpdG sfgfp.

[0163] SigA reporter module

[0164] The glyA and upp / atpl regions, the erythromycin resistance gene (erm), the constitutive promoter (P ref, Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016) and the mKate2 gene. Different PCR fragments were assembled by Gibson reaction. The complete construct was amplified with primers at the end of the fragments homologous to the B. subtilis chromosome and the PCR product was transformed into the strain (BSB1).

[0165] Repressor of GFP expression

[0166] The gene encoding the repressor TetR (BD) (Schmitter et al., Molecular Microbiology, 105:413-425, 2017) was amplified by PCR and assembled by Gibson method with the promoter P hy-spank Downstream linearized pDR111 assembly. The fragments to be integrated were amplified by PCR and transformed into B. subtilis.

[0167] The gene sfgfp was amplified using primers with two boxes tetO located between the box -35 and -10 of the promoter spoVG and downstream of the box -10, respectively. The resistance gene to bleomycin (ble) and two DNA fragments carrying the genes nirC and sacP, respectively, were amplified by PCR and assembled using the Golden gate method. The fragment to be inserted into the B. subtilis chromosome was amplified and transformed into the strain carrying the construct P hy-spank tetR DB .

[0168] Inhibitory expression of SigA and deletion of endogenous SigA

[0169] The gene sigA under the control of P tetO was amplified using the same construct as for the construct of the gene sfgfp.

[0170] The two regions flanking the gene sigA and the chloramphenicol resistance gene were assembled using the Gibson method and cloned into the plasmid pUC19 in the strain Escherichia coli TG1. The plasmid pUC19_sigA was linearized by digestion with the restriction enzyme Ndel (sites located outside the region of interest). The linearized plasmid was transformed into the strain SMS185 (P hy-spank sigA) in the presence of IPTG.

[0171] Complete system

[0172] The plasmid pDR111_P zpdG _P hy-spank- zpdN was linearized and the plasmid was assembled with a PCR fragment carrying tetR BD Gene assembly by PCR fragments. The fragments to be integrated were then amplified and transformed into B. subtilis strains.

[0173] The constructs implemented are shown in Table 3.

[0174] Table 3. Constructs formed for the development of the invasive orthogonal expression system.

[0175]

[0176]

[0177]

[0178] Tandem affinity purification

[0179] Strains were grown in LB medium supplemented with 1 mM IPTG until DO 600= 1. 100 ml of culture were harvested and centrifuged, the pellet was washed in buffer A (10 mM Tris-HCl pH 7.5, 150 mM NaCl) and then directly frozen in liquid nitrogen. The pellet was resuspended in 600 μL of buffer B (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA and 0.1 % Triton X-100) with 6 μL of lysozyme (Sigma-Aldrich) and 1.5 μL of Benzonase (25 U / μL, Novagen). The suspension was then incubated at 37°C for 15 min and then on ice for 30 min; centrifuged at 20000 g for 30 min at 4°C. The supernatant was collected and transferred to a column (BioSpin-Biorad) containing 200 μL of anti-FLAG M2 resin (Sigma) previously equilibrated in buffer B. The column was agitated overnight at 4°C. The next day, the resin was resuspended in 200 μL of buffer TEV (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1 % Triton X-100 and 1 mM DTT) with 50 units of TEV protease (Invitrogen). The column was agitated overnight at 4°C and then eluted in a second column containing calmodulin-agarose 4B resin (Amersham Bioscience) previously equilibrated in buffer CBB (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 0.1 % Triton X-100, 1 mM DTT and 2 mM CaCl2). The column was washed twice with 500 μL of buffer CBB and then left overnight at 4°C. The column was washed twice with 500 μL of buffer CBB and then 200 μL of buffer CWB (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1 % Triton X-100, 1 mM DTT and 0.1 mM CaCl2). The protein complex was eluted twice with 500 μL of buffer CEB (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT and 3 mM EGTA). The protein was precipitated with acetone and then analysed in a SDS-PAGE gel or by liquid chromatography-tandem mass spectrometry (LC-MS / MS) in a resuspension solution (1 M urea and 100 mM Tris-HCl pH 8.5).

[0180] Viability test on solid medium

[0181] In a 96-well plate ( Bacillus subtilis strains were precultured in LB medium for about 20 hours in Greiner Bio-One Cellstar® 96-well plates. In the morning, they were diluted 10-fold in the test medium. When these precultures reached a DO 600 ≈0.3-0.4, they were diluted again 20-fold in the medium of interest. At each stage, the cultures were inoculated into microplates and incubated at 37°C under constant stirring (360 rpm) in a multimode plate reader Synergy 2 TM 2 Systematic tests were performed with the reference strains P hy-spank -gfp and BSB1. Each strain was grown in at least three replicates and measured in three independent experiments. DO 600 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 every 10 minutes.

[0182] To compare the promoters with other non-inducing strains, the fluorescence was normalized to the reference strain. For constitutive promoters, the relative expression units were calculated:

[0183] REU(t i ) = [(Fluo(t i ) / DO 600 (t i )] 菌株 / [(Fluo(t i ) / DO 600 (t i )] ref

[0184] In the case of inducible promoters, the fluorescence value obtained in DO without inducer was subtracted from the fluorescence value obtained under induction before reporting it relative to the reference strain:

[0185] REU(t i ) = {[(Fluo(t 600 ) / DO i (t 菌株 ) + ind-[(Fluo(t i ) / DO 600 (t i )] 菌株-ind} / [(Fluo(t i ) / DO 600 (t réf

[0186] The average and standard deviation of all experiments were then calculated.

[0187] Propidium iodide viability test

[0188] Propidium iodide is a colored molecule that cannot accumulate in bacteria that have lost membrane integrity, i.e. if the cell is dead. To test the viability of the cells, propidium iodide was added to the culture samples at a final concentration of 167 μΜ. After 10 minutes of incubation, the cells were analyzed by flow cytometry to detect the coloration of the internalized propidium iodide inside the cells. To validate this coloration, samples of cells BSB1 in the exponential phase were directly treated. Another culture sample was centrifuged and then resuspended in isopropanol for 60 minutes to kill the cells, then centrifuged. The cells were treated with 10 mM Tris + 150 mM NaCl and colored in the same way. The test was validated with these two samples.

[0189] Inhibition of alternative sigma factors

[0190] The resistance gene to erythromycin was amplified from the erythromycin resistant strain. The upstream and downstream regions of each of the genes encoding the alternative sigma factors SigB, D and H of B. subtilis were amplified by PCR. The primers were designed to introduce a site Bsal at the end of all these fragments. For each construct, the upstream and downstream fragments and the resistance cassette were cloned using a "Golden gate" reaction. The linear fragments obtained were integrated into the genome of the strain BSB1 and then transferred to the chromosome of the strain of the complete production system containing GFP. The constructed strains are summarized in Table 4 and the protein sequences of the alternative sigma factors are shown in Table 5.

[0191] Table 4 Strains in which the alternative sigma factors have been inhibited and constructs

[0192]

[0193] Table 5 Sequences of the alternative sigma factors SigB, SigB and SigH.

[0194]

[0195]

[0196] Test of the promoter recognized by the sigma factor ZpdN

[0197] Using genomic DNA from a strain with a complete production system, the promoter P zpdGThe oligonucleotides were designed such that the two promoters to be tested were introduced as well as a 30 base pair overlap region. The two DNA fragments were ligated by Gibson reaction and then introduced into the strain overproducing the sigma factor ZpdN and the strain containing all parts of the production system except for GFP. The strains constructed are summarized in Table 6 and the sequences of the promoters tested are shown in Table 7.

[0198] Table 6 Strains and constructs formed for testing other promoters

[0199]

[0200] Table 7 Sequences of the promoter regions.

[0201]

[0202] Cloning of the proteins of interest LacZ, AmyS, YqaH and NucA

[0203] To test if other proteins than GFP are 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 start codon ATG and downstream of the stop codon was amplified by PCR by introducing the sites Bsal at both ends. In the same way, the different reading frames were amplified by creating sites Bsal compatible with the previous ones. The different fragments were ligated by 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.

[0204] Table 8 Strains and constructs formed for testing the proteins LacZ, AmyS, NucA and YqaH

[0205]

[0206] Table 9 Sequences of the proteins LacZ, AmyS, YqaH and NucA.

[0207]

[0208]

[0209] Results

[0210] To construct the orthogonal invasive transcription system, several modules were constructed. Figure 1

[0211] ​The first module consists in putting the orthogonal system under the control of an inducible promoter and the transcription of the gene of interest (e.g. a gene encoding GFP) under the control of the orthogonal transcription system.

[0212] The second module consists in establishing an auto-activation loop for the transcription of the orthogonal transcription system to amplify the production of the orthogonal transcription system.

[0213] The third module consists in establishing an inhibition of the expression of the B. subtilis housekeeping sigma factor SigA.

[0214] Finally, the auto-activation loop of the sigma factor and the inhibition of the expression of the sigma A are coupled.

[0215] Example 1 : Construction of the first module

[0216] Several orthogonal transcription systems were cloned in single copy into the B. subtilis chromosome, in the amyE locus, under the control of the IPTG inducible promoter P hy-spank Figure 2 (A).

[0217] In these strains, a transcriptional fusion between the subunit beta of the RNA polymerase and the marker SPA was introduced. The complex of the RNA polymerase was purified and the chaperone molecules were analyzed on an acrylamide gel Figure 2 (B).

[0218] Gel analysis showed that the sigma factors Gp28 and ZpdN interact with the RNA polymerase on the gel. For both candidates, a different promoter recognized by one or the other of the two sigma factors was cloned in front of a gene encoding GFP Figure 3 (A). Similar constructions were performed in strains producing the RNA polymerase of the bacteriophage T7 under the control of the promoter P hy-spank

[0219] The constructs were integrated in single copy into the B. subtilis chromosome, between the ybbU and ybdG genes in a region close to the replication origin of the chromosome.

[0220] For each strain constructed, the amount of GFP production (fluorescence / DO 600nm ) was measured over time in LB medium without or with 500 mM IPTG Figure 3 (B).

[0221] The specific fluorescence induced by the orthogonal sigma factor ((fluorescence / DO 600nm ) IPTG500μM (fluorescence / DO 600nm ) -IPTG ) was calculated. To compare the strains, the specific fluorescence was normalized relative to the strain with the promoter P​​hy-spank Normalization was performed using a reference strain containing a gene encoding GFP under the control of the GFP gene (GFP / OD norm). The results showed that no GFP activity was detected for constructs containing a promoter recognized by the sigma factor Gp28 (Table 10). With regard to the sigma factor ZpdN, the promoters tested showed high transcriptional activity similar to that observed for polymerase T7 RNA.

[0222] Table 10 GFP activity of the different strains tested.

[0223]

[0224]

[0225] The system construction was continued using ZpdN and the first module was verified.

[0226] Example 2: Construction of the second module

[0227] For the second module, the active promoter (P zpdG ) cloned into promoter P hy-spank Upstream ( Figure 4 , A).

[0228] This autoinduction loop enhances the induction response of ZpdN expression and makes ZpdN expression independent of the Bacillus subtilis housekeeping sigma factor.

[0229] The normalized specific activity of the strains with and without the autoinduction loop was measured at different IPTG concentrations ( Figure 4 , B). The results show that at low doses of IPTG (less than or equal to 20 μM), the self-activation loop leads to increased GFP production. At higher concentrations (above 100 μM), the strain with the self-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.

[0230] Example 3: Construction of the third module

[0231] For the third module, the repressor protein TetR DB The gene was cloned at the amyE locus on the chromosome of Bacillus subtilis as a promoter under the inducible promoter P hy-spank Control ( Figure 5 , A).

[0232] To test the inhibitory efficacy of this protein, a TetR-expressing protein was cloned in front of the GFP gene. BD The promoter of the operator region recognized by the repressor protein (P tetO ).

[0233] The measurement of the fluorescence produced in cultures with and without IPTG (with and without inhibition of the promoter P tetO reveals a very significant difference (1.7 + / - 0.1 arbitrary units without IPTG and 0.11 + / - 0.08 arbitrary units with IPTG).

[0234] Under the control of this promoter, an inhibition of more than 15 times the transcription / translation rate of the GFP gene was measured.

[0235] Once the inhibitory TetR BD protein was validated, the gene sigA was cloned under the control of the promoter P tetO .Then, the natural copy of the sigA locus was replaced by a gene encoding chloramphenicol resistance.

[0236] The growth of this strain was then demonstrated to be sensitive to ITPG. After the addition of IPTG in the culture, the growth of the strain was strongly affected.

[0237] From 30 minutes, the slope of the optical density curve becomes steeper Figure 5 , B): the generation time increases. Then, after 3 hours, the growth stops. In the box of LB agar medium without inducer, a very strong decrease of the viability (100000 times) was observed in the 3 hours culture.

[0238] The use of the P ref mkate2 reporter gene (Guiziou et al., Nucleic Acids Research, 44(15): 7495-508, 2016) whose production of fluorescent protein depends on the sigma A promoter, in the latter strain shows that the induction of the repressor TetR BD decreases the amount of fluorescence, suggesting a possible competition between SigA and ZpdN.

[0239] Thus, the third module capable of inhibiting the expression of the housekeeping sigma factor of Bacillus subtilis is functional.

[0240] Example 4: Coupling of the auto-activation loop

[0241] The last step consists in coupling the auto-activation loop with the inhibition of the expression of the housekeeping sigma factor.

[0242] To achieve this coupling, the gene encoding the repressor TetR BD and the gene encoding the sigma factor ZpdN are cloned in an operon under the control of the promoter P hy-spank (A). Figure 6

[0243] In this strain, the housekeeping sigma factor SigA is under the control of the promoter P tetO .

[0244] In the absence of the inducer, the sigA gene is expressed: the transcriptional program of the B. subtilis proteins is active.

[0245] The addition of IPTG to this strain induces: i) the expression of the sigma factor ZpdN, which in turn induces the expression of GFP and its own expression; and ii) the expression of the repressor TetR, which blocks the expression of the sigA gene.

[0246] The housekeeping transcriptional program for the production of proteins for the growth of B. subtilis is stopped, while the transcriptional program for the production of the protein of interest is activated: this switch of transcriptional programs makes all the cellular resources available for the production of the protein of interest.

[0247] In this strain, the level of GFP production depends on the DO 600 ( Figure 6 , B) at the time of induction by IPTG. The maximum level of GFP produced after 3 hours of induction is equivalent to that obtained by the promoter P veg after 7 hours of culture and is much higher than the level of GFP produced after induction of the promoter P hy-spank .

[0248] The cells after induction of the transcriptional program switch are subjected to cytometry analysis Figure 7 .

[0249] After one hour of induction, some cells switch to the GFP production program; after two hours, all the cells produce GFP in a very homogeneous manner (very narrow peak). No cells are detected that do not produce GFP, while in the case of the strain carrying the construct P veg gfp, about 1% of the cells do not produce GFP. After 3 hours of induction, the production of GFP increases and the proportion of cells producing a large amount of GFP is very high (more than 2 x 10 6 arbitrary units). The level of production of GFP is much higher than that obtained with the strain P hy-spank gfp, and the population is more homogeneous.

[0250] The various embodiments mentioned in no way limit the scope of the invention claimed; they are given by way of illustration in order to better understand the invention.

[0251] Example 5: Characterization of the viability of the cells after the induction production phase

[0252] After induction of GFP production, the state of the cells was characterized. The cells were diluted and grown in the presence of IPTG. The optical density stopped increasing after two hours, indicating that the growth had stopped. No decrease in optical density was observed, so the population did not lyse.

[0253] During the kinetic process, samples were taken, diluted and plated on rich medium without inducer. After 24 hours of incubation at 37°C, the colonies that had formed were counted. From one hour of induction, the cells were no longer cultivable, even after the induction had stopped (almost no colonies formed on the plates) Figure 8 ).

[0254] 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 cells are "viable but non-culturable" cells. Thus, the integrity of the bacterial membrane is maintained. This is advantageous because it allows the integrity of the cells to be maintained, so that purification steps can be performed by centrifugation.

[0255] Example 6: Reversibility of the induction

[0256] To determine whether it is possible to maintain viable cells, induction was performed with a lower concentration of IPTG (20 mM).

[0257] After three hours, the cells were still able to grow on medium without IPTG Figure 10 ). Thus, the cells are 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 colonies formed per unit of optical density, it appears that more than half of the bacteria are still viable. They retain all the selection markers, so they have not lost any part of the system. Under this concentration of inducer, the induction is reversible, without selecting the subpopulation that the production system would lose.

[0258] Example 7: Effect of the absence of the secondary sigma factor of Bacillus subtilis

[0259] Certain other alternative sigma factors present in the cell can compete with ZpdN for contact with the RNA polymerase. Thus, fewer RNA polymerase + ZpdN complexes will be formed in the cell, limiting the transcription of the gene encoding GFP. To verify this hypothesis, the alternative sigma factors produced during the exponential phase were inhibited in a strain with the complete system. After deletion of the genes encoding the sigma factors B, D and H, respectively, the level of GFP production was tested.

[0260] Only the inhibition of the sigma factor encoded by the sigD gene showed an impact on this productionFigure 11 ). Previous results (Fehler et al., Microbial Cell Factories, 21 : 131, 2022) have shown that inactivation of genes encoding proteins of the flagellum induces an increase in amylase production. These genes are controlled by the sigma factor D. Thus, the observed results do not seem to be due to competition between sigma factors, but rather to the release of resources, since the transcriptional program induced by the production of the Sig factor D is not active. This result shows that a modification of the strain that is favorable to protein production can produce a cumulative effect in the constructed system.

[0261] Example 8: Testing of other promoters recognized by the sigma factor ZpdN

[0262] 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.

[0263] Two different promoters were cloned in front of the ribosome binding site R0 and the gene encoding sfGFP. In these strains, induction of the production of the sigma factor ZpdN allowed the level of GFP production to be multiplied by about two compared to the level produced by the previous construct (Table 11).

[0264] Table 11: GFP production as a function of the tested promoter.

[0265] Genotype Promoter RBS GFP / OD norm P hs ::zpdN]] P zpdG ]]> R0 14.6+ / -0.6 P zpcJK ]]> RO 38.7+ / -1.8 P zpcXYZ ]]> R0 39.9+ / -1.7

[0266] The best of the two promoters was integrated into a strain with the complete production system. A comparison was made between this new construct and the previous one. The results show that the growth curves of the two strains are similar, indicating that the growth arrest due to the loss of the sigma factor A is identical in the two strains Figure 12 ). However, it is clear that the new construct allows more GFP to be produced in the cell, between 4 and 5 times.

[0267] Example 9: Extension to the production of other proteins

[0268] The first cloned protein was beta-galactosidase, a very high molecular weight protein: 116 kDa. After 3 hours of induction, beta-galactosidase activity was detected in the cell, forming about 8500 nmol of ortho-nitrophenyl-beta-galactoside (ONP) per minute per mg of protein. By comparison, previous results have shown that for a single copy of the inducible promoter P spac , the level of activity measured after complete induction of the subpromoter was about 1000 nmol of ONP per minute per mg of protein.

[0269] The second cloned protein was a nuclease encoded by two polypeptides in phage Bil66. The generation times (calculated in 6 replicates) of the strain without induction and with and without the nuclease gene were similar to strains with genes for sfGFP (41 + / - 4 minutes) or beta-galactosidase (42 + / - 1 minutes) and to the strain with the nuclease (40 + / - 1 minutes) without induction. This result indicates that expression is turned off without induction. Induction of the system in the strain with the nuclease gene led to rapid lysis of the cells, indicating that the nuclease was indeed produced Figure 13

[0270] The third cloned protein was the YqaH protein, which inhibits replication of the chromosomal DNA of B. subtilis and thus blocks cell growth. The gene encoding this protein was cloned in the complete production system. In this example, the protein was successfully cloned and induced to produce after which a rapid stop of growth was induced.

[0271] The fourth cloned protein was the amylase AmyS of B. stearothermophilus. This protein has a signal peptide that can be recognized by the Sec-dependent secretion system of B. subtilis. Preliminary results indicate that protein secretion is still possible after induction of protein production in the constructed strain.​

Claims

1. A genetically modified bacterium comprising: - a first nucleotide sequence encoding an orthogonal sigma factor capable of interacting with the RNA polymerase of the transgenic bacterium, expression of the orthogonal sigma factor is inducible and is controlled by a promoter recognized by the orthogonal sigma factor, and - a second nucleotide sequence, the expression product of which inhibits the action of the endogenous sigma factor of the transgenic bacteria, The expression of the second nucleotide sequence is controlled by a promoter recognized by the orthogonal sigma factor.

2. The transgenic bacterium according to claim 1, further comprising: - a third nucleotide sequence encoding at least one protein of interest, The expression of the at least one protein of interest is controlled by a promoter recognized by an orthogonal sigma factor.

3. The genetically modified bacterium according to claim 1 or 2, wherein The orthogonal σ factor is selected from: - a sigma factor from a bacterial species different from that of the transgenic bacterium, and - Sigma factors specific for extrachromosomal elements naturally deleted in transgenic bacteria.

4. The transgenic bacterium according to any one of claims 1 to 3, which belongs to a species selected from the group consisting of Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus and Lactococcus lactis, preferably Bacillus subtilis.

5. A method for producing at least one protein of interest, comprising: - a step of cultivating a transgenic bacterium as defined in any one of claims 1 to 4, and - A step of inducing the expression of an orthogonal sigma factor.

6. A polynucleotide comprising at least a first nucleotide sequence encoding a sigma factor under the control of two tandem promoters: one promoter conferring induction properties and the other promoter being recognized by the sigma factor.

7. The polynucleotide according to claim 6, further comprising a second nucleotide sequence encoding: - transcriptional repressor proteins, -interfering RNA, - proteases, or -Sigma factor inhibitor.

8. The polynucleotide of claim 7, further comprising a third nucleotide sequence encoding at least one protein of interest.

9. An expression vector comprising at least one polynucleotide as defined in any one of claims 6 to 8.

10. A kit for recombinant expression of at least one protein of interest, comprising at least: - A transgenic bacterium as defined in any one of claims 1 to 4, - a polynucleotide as defined in any one of claims 6 to 8, and / or - an expression vector as defined in claim 9, and - optionally a molecule capable of inducing the expression of said at least one protein of interest.

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