Genetically engineered bacteria containing invasive orthogonal expression systems

Abstract

The present invention relates to genetically modified bacteria comprising an invasive orthogonal expression system and their 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.

Description

[Technical Field]

[0001] The present 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 present invention also relates to polynucleotides, vectors, and kits for expressing at least one protein of interest.

[0002] To address the challenges of climate change, alternatives to petroleum-based chemicals will likely need to be viable. Numerous biological production systems exist that use bacteria to synthesize molecules, and these consume far fewer resources than their petroleum-based counterparts.

[0003] However, current bioproduction systems can provide yields that are too low to be considered a comprehensive solution.

[0004] Therefore, there is a strong need for optimized biological production systems to improve bacterial production yields.

[0005] In this context, the present inventors have realized that current protein bioproduction systems share the transcriptional and translational capabilities of the host bacterium for the synthesis of all proteins within the cell.

[0006] The inventors then unexpectedly demonstrated that it is possible to shut down protein synthesis in host bacteria and redirect cellular resources to an orthogonal expression system. Such a system is said to be orthogonal in that it operates independently of the endogenous system of the bacterium. Furthermore, this system is invasive in that it disables the endogenous system. Therefore, the orthogonal expression system developed by the inventors makes it possible to maximize the allocation of resources dedicated to bioproduction by shutting down the endogenous expression system.

[0007] By exploiting the promoter sequence recognition function of the sigma factor, we cloned a heterologous sigma factor capable of autoinducing synthesis, as well as a repressor that controls the synthesis of the endogenous sigma factor. Interestingly, when synthesizing a test protein, the resulting bacteria were able to produce 35-fold more protein after 3 hours of induction than could be obtained using the best inducible promoter described in B. subtilis. Summary of the Invention

[0008] Thus, one aspect of the present invention is a genetically modified bacterium comprising: a first nucleotide sequence encoding an orthogonal sigma factor capable of interacting with an RNA polymerase of the genetically modified bacterium, wherein expression of the orthogonal sigma factor is inducible and under the control of a promoter recognized by the orthogonal sigma factor; a second nucleotide sequence whose expression product inhibits the action of an endogenous sigma factor of the genetically modified bacterium, wherein expression of the second nucleotide sequence is under the control of a promoter recognized by the orthogonal sigma factor; The present invention relates to a genetically modified bacterium comprising:

[0009] According to the present invention, the expression "sigma factor" (or σ) refers to a subunit of bacterial RNA polymerase that is required to initiate transcription. This protein plays a crucial role in the selection of the enzyme binding site.

[0010] According to the present invention, the expression "sigma factor" covers not only sigma factors but also phage-specific monomeric RNA polymerases such as T3 RNA polymerase or T7 RNA polymerase, which directly recognize specific promoter sequences.

[0011] According to the present invention, the term "endogenous" means that the designated element is naturally present in the genetically modified bacterial strain. According to the present invention, an endogenous sigma factor is naturally present in the genetically modified bacterial strain.

[0012] According to the present invention, the term "orthogonal" means that the designated element 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 present invention, an orthogonal sigma factor is not naturally present in the genetically modified bacterial strain, but when introduced and expressed therein, is able to initiate transcription therein, in parallel and independently of the sigma factor naturally expressed in this strain. In other words, an orthogonal sigma factor allows the introduction of a transcription system that is uncoupled from the transcription system naturally present in the genetically modified bacteria.

[0013] The orthogonal sigma factor does not naturally occur in the genetically modified bacterial strain, i.e., it may be derived from another bacterial species (i.e., a heterologous sigma factor), or it may be derived from another strain of the same species as the genetically modified bacterium, or from a bacteriophage that is not naturally occurring in the genetically modified strain. The orthogonal sigma factor may be naturally occurring or may have been genetically modified to recognize a different promoter sequence than the one it naturally recognizes in the organism from which it is derived.

[0014] In the present invention, expression of the orthogonal sigma factor is "inducible," i.e., not constitutive. Expression of the sigma factor is reversibly activated or inactivated in the presence of a given regulatory element. Without limitation, this regulatory element may be added directly to the culture medium or removed from the culture medium.

[0015] For example, in one embodiment, expression of the orthogonal sigma factor can be placed under the control of a xylose-inducible promoter, which can then be activated by adding xylose to the medium.

[0016] For example, in another embodiment, expression of the orthogonal sigma factor can be placed under the control of a promoter inducible by lactose (or allolactose or any functional analog), and this expression can then be shut off by the presence of the LacI regulatory protein and activated by the addition of IPTG, which binds to LacI and prevents it from binding to the "lactose" promoter.

[0017] For example, in other embodiments, expression of the orthogonal sigma factor is driven by a nisin-inducible promoter, a copper-inducible promoter, a mannose-inducible promoter (P manP ), surfactin-inducible promoter (P srfA ), sucrose-inducible promoter (P sacB ), mitomycin-inducible promoter (P dinR ), or can be under the control of a temperature-inducible promoter (P2).

[0018] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, further comprising a third nucleotide sequence encoding at least one protein of interest, wherein expression of said at least one protein of interest is under the control of a promoter recognized by an orthogonal sigma factor.

[0019] In one embodiment, the at least one protein of interest corresponds to a single protein of interest.

[0020] In one embodiment, the at least one protein of interest corresponds to two proteins of interest.

[0021] In one embodiment, the at least one protein of interest corresponds to two or more proteins of interest.

[0022] According to the present invention, the term "protein" refers to a polymer of amino acids, and also includes small peptides, for example, from 10 to 100 amino acids.

[0023] According to the present invention, the expression "protein of interest" generally refers to any protein of choice for carrying out the present invention. Such a protein is likely to be of commercial, therapeutic, industrial, technical or research interest. Indirectly, a protein of interest according to the present invention may also be an intermediate product or an enzyme that allows the conversion of a substrate into a particular protein of interest.

[0024] In one embodiment, the protein of interest is an enzyme selected from the group including, for example, amylase, xylanase, lichenase, lipase, cellulase, pectinase, hydrolase, pullulanase, nattokinase, agarase, galactosidase, protease, and peptidase.

[0025] In one embodiment, the protein of interest is a membrane protein.

[0026] In one embodiment, the protein of interest is an antigen.

[0027] In one embodiment, the protein of interest is a cytokine, particularly selected from the group comprising alpha interferon, beta interferon, and gamma interferon.

[0028] In one embodiment, the protein of interest is a food additive.

[0029] In one embodiment, the protein of interest is a bacterial biofilm protein.

[0030] In one embodiment, the present invention provides a method for producing an orthogonal sigma factor comprising: a sigma factor derived from a bacterial species different from the bacterial species of the genetically modified bacterium; and sigma factors specific to extrachromosomal elements that do not naturally occur in genetically modified bacteria; The present invention relates to a genetically modified bacterium as defined above, which is selected from the group consisting of:

[0031] In one embodiment, the extrachromosomal element may be a plasmid or a bacteriophage such as T3 or T7. One advantage of choosing an extrachromosomal element is that it allows the use of sigma factors present in strains of the same species as the genetically modified bacterium, and therefore can function in that strain, while recognizing specific promoters that do not naturally occur in that strain.

[0032] In one embodiment, the invention relates to a genetically modified bacterium as defined above, wherein expression of the orthogonal sigma factor is controlled by two promoters in tandem, one promoter conferring inducible properties and another promoter recognized by the orthogonal sigma factor.

[0033] In one embodiment, the present invention relates to a method for producing a recombinant human ovarian cancer cell line comprising the steps of: transcriptional repressors, Interfering RNA, Proteases, and sigma factor inhibitors, The present invention relates to a genetically modified bacterium as defined above, which is selected from the group consisting of:

[0034] The expression product of the second sequence is designed to inhibit the action of the endogenous sigma factor, ie to switch off the transcription system naturally present in the genetically modified bacterium.

[0035] Without limitation, inhibition of the action of an endogenous sigma factor can be achieved by, for example, suppressing transcription of the gene encoding the endogenous sigma factor (e.g., via a transcriptional repressor), suppressing translation of the gene encoding the endogenous sigma factor (e.g., via an interfering RNA), degrading the endogenous sigma factor (e.g., via a protease that targets a cleavage site present in the endogenous sigma factor), or suppressing specific binding of the endogenous sigma factor (e.g., via a sigma factor inhibitor).

[0036] In one embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the native gene encoding an endogenous sigma factor has been inactivated and a copy of said gene has been placed under the control of a promoter that is recognized by a transcriptional repressor encoded by a second sequence.

[0037] In one embodiment, the invention relates to a genetically modified bacterium as defined above, wherein the native gene encoding an endogenous sigma factor is placed under the control of a promoter recognized by a transcriptional repressor encoded by a second sequence.

[0038] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein said endogenous sigma factor is a major sigma factor of the genetically modified bacterium.

[0039] According to the present invention, the expression "major sigma factors" refers to sigma factors involved in the transcription of housekeeping genes. Major sigma factors are generally considered to be essential for bacterial viability.

[0040] For example, in Bacillus subtilis, the major sigma factor is generally Sigma A (SigA or σ A ), and in Escherichia coli, the major sigma factor is generally Sigma 70 (Sig70 or σ 70 ) refers to the σ of B. subtilis. A and E. coli σ 70 The corresponding reference sequences are shown in Table 1.

[0041] [Table 1]

[0042] In one embodiment, the genetically modified bacterium is a strain of B. subtilis and the major sigma factor is a protein of the sequence set forth in SEQ ID NO:1.

[0043] In one embodiment, the genetically modified bacterium is a strain of E. coli and the major sigma factor is a protein of the sequence SEQ ID NO:2.

[0044] In one embodiment, in addition to inhibiting the principal sigma factor, other endogenous sigma factors can be inhibited to avoid competition for resources with orthogonal sigma factors. For example, in B. subtilis, in addition to inhibiting the principal sigma factor SigA, the alternative sigma factor SigD can also be inhibited.

[0045] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the orthogonal sigma factor is selected from the group comprising ZpdN (a sigma factor from Bacillus subtilis), Gp28 (a sigma factor from a phage), RpoS (a sigma factor from Escherichia coli), RpoN (a sigma factor from E. coli), SigB8 (a sigma factor from Prevotella bryantii), SigB (a sigma factor from B. subtilis), and t7p07 (a phage RNA polymerase), and is preferably ZpdN.

[0046] By way of example, reference sequences corresponding to orthogonal sigma factors suitable for use in B. subtilis are shown in Table 2.

[0047] [Table 2] TIFF2026508348000003.tif203170

[0048] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein said orthogonal sigma factor is 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.

[0049] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein said bacterium belongs to a species selected from Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus, and Lactococcus lactis, preferably B. subtilis.

[0050] In one embodiment, the genetically modified bacterium belongs to the species B. subtilis and the orthogonal sigma factor is ZpdN (SEQ ID NO: 3).

[0051] 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.

[0052] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein said bacterium is a GRAS (Generally Recognized As Safe) bacterium, i.e. a bacterium that is generally recognized as safe. Such bacteria are particularly suitable for the synthesis of proteins for food or therapeutic applications.

[0053] In one embodiment, the genetically modified bacterium is B. subtilis strain BSB1, which was deposited in accordance with the Budapest Treaty on February 1, 2023, at the Collection Nationale de Cultures de Microorganismes (CNCM) (Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France) under number CNCM I-5930.

[0054] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein at least the first and second nucleotide sequences, and optionally the third nucleotide sequence, are present on separate polynucleotides.

[0055] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein at least the first and second nucleotide sequences, and optionally the third nucleotide sequence, are present on the same polynucleotide.

[0056] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present on the same polynucleotide.

[0057] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the same promoter controls the expression of at least the orthogonal sigma factor encoded by the first nucleotide sequence and the expression product of the second nucleotide sequence, and optionally the protein of interest encoded by the third nucleotide sequence.

[0058] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the same promoter controls the expression of the orthogonal sigma factor encoded by the first nucleotide sequence, the expression product of the second nucleotide sequence, and the protein of interest encoded by the third nucleotide sequence.

[0059] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the protein of interest is a protein that is toxic to the genetically modified bacterium.

[0060] The system of the present invention is particularly suitable for the production of proteins that are toxic to bacteria. In fact, in conventional expression systems, these proteins slow or even prevent bacterial growth, making it impossible to produce sufficient amounts of the protein. The system described in the present invention makes it possible to optimally cultivate bacteria and, once a sufficient bacterial population is reached, to switch the cellular machinery to the sole interest of producing the protein of interest.

[0061] In one embodiment, the present invention relates to a genetically modified bacterium as defined above, wherein the protein of interest is a protein secreted by the bacterium.

[0062] The system is particularly suitable for the production of secreted proteins, as it allows the production of large amounts of proteins in the culture medium, which can then be easily recovered, especially by keeping the bacteria alive.

[0063] In another aspect, the present invention relates to the use of a genetically modified bacterium as defined above for producing at least one protein of interest.

[0064] Another aspect of the present invention is a method for producing at least one protein of interest, comprising the steps of: i) culturing the genetically modified bacterium defined above; ii) inducing expression of the orthogonal sigma factor; The present invention relates to a method, including:

[0065] In one embodiment, step i) and / or step ii) is carried out in a bioreactor (or fermentor).

[0066] In one embodiment, the present invention relates to a method as defined above, further comprising a step of recovering the protein of interest. Methods for recovering the protein of interest are well known to those skilled in the art and may vary, in particular, depending on whether the protein of interest is intracellular or secreted by the bacteria into the culture medium.

[0067] In one embodiment, the present invention relates to a method as defined above, wherein the genetically modified bacterium is killed after the step of inducing expression of the orthogonal sigma factor is stopped.

[0068] Another aspect of the present 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 promoter conferring inducible properties and the other promoter recognized by the sigma factor.

[0069] In one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: transcriptional repressors, Interfering RNA, protease, or sigma factor inhibitors, The present invention relates to a polynucleotide as defined above, further comprising a second nucleotide sequence encoding

[0070] In one embodiment, the present invention 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.

[0071] In one embodiment, the present invention relates to a polynucleotide as defined above, wherein the expression of the first nucleotide sequence and the second nucleotide sequence is controlled by two promoters in tandem with each other.

[0072] In one embodiment, the present invention relates to a polynucleotide as defined above, further comprising a third nucleotide sequence encoding at least one protein of interest.

[0073] In one embodiment, the present invention 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.

[0074] In one embodiment, the present invention 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.

[0075] In one embodiment, the present 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 tandem promoter.

[0076] In one embodiment, the present invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the second nucleotide sequence are present in the form of an operon.

[0077] In one embodiment, the present invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence and the third nucleotide sequence are present in the form of an operon.

[0078] In one embodiment, the present invention relates to a polynucleotide as defined above, wherein the first nucleotide sequence, the second nucleotide sequence and the third nucleotide sequence are present in the form of an operon.

[0079] Another aspect of the present invention relates to an expression vector comprising at least one polynucleotide as defined above.

[0080] Another aspect of the present invention is a kit for recombinantly expressing at least one protein of interest, comprising at least: a genetically modified bacterium as defined above, a polynucleotide as defined above, and / or an expression vector as defined above, and Optionally, a molecule allowing the induction of the expression of said at least one protein of interest to be induced, The present invention relates to a kit comprising:

[0081] In one embodiment, the molecule allowing the induction of expression of said at least one protein of interest is selected from the group comprising xylose and IPTG.

[0082] The invention will now be explained in more detail with the aid of the following figures and examples, but the invention is in no way limited thereby. [Brief explanation of the drawings]

[0083] [Figure 1] This diagram shows a system for switching between two transcriptional programs. To construct an orthogonal transcription system, a gene encoding an orthogonal sigma factor (SigOrth) is placed under the control of the Phy-spank promoter. This is repressed by a protein encoded by the lacI gene and induced by IPTG added to the culture medium. The orthogonal sigma factor recognizes a specific promoter (Pσorth) and, in cooperation with the host RNA polymerase, induces transcription of the cloned gene of interest (goi). The promoter recognized by this orthogonal sigma factor is introduced upstream of the Phy-spank promoter, ensuring self-activation of orthogonal sigma factor expression. To construct a system for suppressing sigma A expression, a single intracellular copy of the gene encoding sigma factor A is cloned downstream of a promoter carrying the operating sequence for the repressor TetRBD. The gene encoding this repressor is placed under the control of the Phy-spank promoter. Switching between the two transcriptional programs is achieved by cloning the gene encoding the repressor TertRBD into an operon together with a gene encoding an orthogonal sigma factor within an autoactivation loop. [Figure 2]Figure 1. Interaction between orthogonal sigma factors and host RNA polymerase. (A) Schematic of the strains tested. A construct allowing inducible expression of a gene encoding a sigma factor under the control of the promoter Phy-spank was placed into a B. subtilis strain producing a labeled RNA polymerase. This labeling allows purification of the protein complex, revealing the polypeptide that interacts with the host RNA polymerase. (B) Acrylamide gel of analysis of the RNA polymerase protein complex. The purified RNA polymerase complex is analyzed on a gel. The RNA polymerase subunits β, β', and α, as well as the housekeeping sigma factor SigA, are detected in all purifications (indicated by triangles). The expected sizes of the expressed sigma factors are indicated (bottom triangles). [Figure 3] Figure 1 shows the implementation of a specific reporting system for the orthogonal sigma factors Gp28 and ZpdN. (A) Schematic diagram of the strains tested. The genes encoding the sigma factors Gp28 and ZpdN or the gene 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 a different promoter recognized by one of these two sigma factors. (B) Histogram of the amount of fluorescence produced. Fluorescence level (optical density) was measured relative to cell number in various strains. For comparison purposes, all values ​​were normalized using a reference strain in which the gene encoding GFP was under the control of Phy-spank. Each strain was tested three times with three technical replicates, and the mean and standard deviation were calculated. The values ​​obtained using the best promoter sequence tested are shown on a logarithmic scale. [Figure 4]Figure 1. Effect of the autoinduction loop. (A) Schematic diagram of the tested strains. The promoter (PzpdG) recognized by the sigma factor ZpdN was cloned upstream of the construct Phy-spank::zpdN. Transcriptional activity associated with the production of the sigma factor ZpdN was demonstrated by the production of GFP, with the sfgfp gene under the control of the promoter PzpdG. (B) Levels of fluorescence produced in each of the two strains at various IPTG concentrations. The amount of GFP produced per unit DO600 (i.e., optical density), normalized to the reference strain, was measured under various induction conditions, Phy-spank, with increasing doses of IPTG for strains with and without the autoactivation loop ZpdN. [Figure 5] Figure 1. Repression of housekeeping sigma factor expression in B. subtilis. (A) Schematic diagram of the strains tested. The gene encoding the transcriptional repressor TetRBD was 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 with an operator sequence recognized by the repressor TetR. (B) Kinetics of fluorescence expression over time for various inducer doses. Expression of the tetRBD gene was induced at t=0 with two inducer concentrations, and then fluorescence levels (optical density) relative to cell number were measured over time. [Figure 6]Figure 1. Invasive orthogonal transcription system. (A) Schematic diagram of the tested strains. The genes encoding the transcriptional repressor TetRBD and the sigma factor ZpdN were cloned under the control of the IPTG-inducible promoter Phy-spank. The gene encoding a housekeeping sigma factor was placed under the control of a promoter with an operator sequence recognized by the repressor TetR. (B) Fluorescence expression levels versus DO600nm upon induction. Fluorescence levels (optical density) versus cell number were measured in strains carrying a GFP-encoding gene under the control of Pveg (left histogram) and in strains carrying the entire system (right histogram). In the latter case, measurements were performed without IPTG (solid histogram) and 3 hours after induction of Phy-spank expression (hatched histogram). Results are shown relative to cell number at induction (measured at DO600nm equivalent). [Figure 7] Figure 1 shows cytometric analysis of cells after induction of the transcription flip. For each strain and sample tested, 100,000 cells were analyzed. Fluorescence was measured for each. The graph shows the number of cells counted for each fluorescence intensity (on a logarithmic scale). Three reference strains were used: i) strain 168, which does not contain the gene encoding sfGFP and allows for measurable autofluorescence under the conditions tested; ii) strain Pveg-GFP, which constitutively produces GFP; and iii) a strain with GFP under the control of an ITPG-inducible promoter. [Figure 8] Figure 1 shows the cultivation of the strain comprising the entire production system in LB medium with and without IPTG. Cells in the exponential growth phase were diluted at t=0 into LB without IPTG (open symbols) or in the presence of 500 mM IPTG (filled symbols). Over time, the optical density of the cultures at 600 nm was measured (triangle symbols and dotted line). Cells were diluted and plated onto box LB. After 24 hours, the number of colonies formed on the plates was determined (circle symbols and solid line). [Figure 9]Analysis of bacterial viability by labeling with propidium iodide. Live cells, dead cells, and a mixture of both types of bacteria were treated with propidium iodide (left column). Cells were then analyzed by cytometry to detect the red fluorescence of propidium iodide in the cytoplasm. Dead cells (alone and in the mixture) fluoresce, demonstrating that labeling clearly detects bacteria with altered membrane integrity. In parallel, cells cultured without IPTG and with IPTG (500 nM) for 3 h were labeled and analyzed in the same way (right column). [Figure 10] Figure 1 shows the culture of the strain comprising the entire production system in LB medium with and without IPTG. Cells in the exponential growth phase were diluted at t=0 into LB without IPTG (open symbols) or in the presence of 20 mM IPTG (filled symbols). Over time, the optical density of the cultures at 600 nm was measured (triangle symbols and dotted line). Cells were diluted and plated onto box LB. After 24 hours, the number of colonies formed on the boxes was determined (circle symbols and solid line). [Figure 11] Figure 1 shows the effect of depletion of alternative sigma factors on the production of a protein of interest. Cells containing the complete production system in a wild-type genetic context (gray) and in a genetic context with a deletion of the gene encoding the sigma factor SigD (black) were cultured in microplates. Optical density and GFP-associated fluorescence were measured in triplicate. GFP data per unit optical density in arbitrary units are plotted over time for three biological replicates. [Figure 12] Figure 1 shows a comparison of strains carrying a gene encoding GFP under the control of the promoter PzpdN or the promoter PzpcXYZ. Strains carrying the sfgfp gene either under the control of the promoter PzpdN (gray) or under the control of the promoter PzpcXYZ (black) were diluted in the presence of IPTG (500 nM) at t=0. For each triplicate culture, the optical density (circle symbols) and GFP production (square symbols) of the cultures were monitored. The sensitivity of the measurement device was reduced to avoid signal saturation. [Figure 13] Growth of a strain expressing the phage Bil66 nuclease. A strain carrying a gene encoding a protein complex with nuclease activity under the control of the promoter PzpdN was diluted at t=0 in the presence (black) or absence of IPTG (500 nM). The optical density of each triplicate culture was monitored. Growth arrest and lysis are observed after nuclease induction. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0084] Materials and Methods Bacterial culture Bacillus subtilis strains were grown at 37°C in LB and CH media, which are rich media with chemically defined compositions. CH medium contained (per liter) 9.4 g hydrolyzed casein (Difco L-casamino acids, vitamin-free), 3.76 g L-glutamic acid NaH2O, 1.22 g L-alanine, 1.41 g L-asparagine, 1.28 g KH2PO4, 0.094 g Na2SO4, 1.24 g NH4Cl, 0.094 g NH4NO3, 0.036 g CaCl2·6H2O, 0.062 g MgSO4·7H2O, 0.068 mg MnSO4·H2O, 0.1 mg FeCl3·6H2O, and 0.02 mg C6H8O7·H2O, supplemented with 0.5% glucose. Solid medium was obtained by adding 1.5% agar to the liquid medium. Competent B. subtilis cells were transformed according to the method described by Konkol et al. (Journal of Bacteriology 195:18, 4085-4093, 2013). The antibiotics phleomycin, neomycin, chloramphenicol, erythromycin, and spectinomycin were added at 8 μg mL each. -1 , 15 μg mL -1 , 5 μg mL -1 , 0.5 μg mL -1 , and 100 μg mL -1 was added at a final concentration of

[0085] Escherichia coli strains were grown in LB medium at 37°C, and competent cells were prepared as described by Hanahan et al. (Methods in Enzymology 204, 63-113, 1991). -1 Plasmid transformants were selected using 100 ml of ampicillin.

[0086] molecular biology manipulation DNA fragments were amplified using the Invitrogen Platinum SuperFi DNA Polymerase Kit (ThermoFisher Scientific). Sample verification and colony PCR were performed using the Thermo Scientific DreamTaq DNA Polymerase Kit (ThermoFisher Scientific). These polymerases were used according to their manufacturer's recommendations. When the PCR template was modified DNA, a specific protocol was used as described in Stewart et al. (Journal of Molecular Biology 24, 388(1):48-70, 2009), for the genome of phage SPO1. The first five amplifications were performed at a hybridization temperature of 40°C, followed by the remaining 25 amplifications at 50°C (the standard hybridization temperature used here).

[0087] To prepare DNA fragments, genomic DNA was extracted using the GenElute kit (Sigma-Aldrich). PCR DNA fragments were purified using a QIAquick PCR purification kit according to the manufacturer's recommendations. DNA assembly was performed using the NEBuilder HiFi Assembly Master Mix kit (NEB), the enzymes BsaI (NEB), and T4 DNA ligase (NEB), according to the manufacturer's recommendations.

[0088] Strain and plasmid construction Construction and modification of gp28 Genes encoding the orthogonal transcription system were amplified by PCR (fragment zpdN, fragment gp28, fragment sigB8, fragment rpoS, fragment rpoN, and fragment T7p07). Genes encoding the sigma factors RpoS, RpoN, and RNA polymerase T7 were amplified using the E. coli ER2566 strain as a template. Genes encoding the transcription factors SigB and 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 SPO1. The gene encoding the SigB8 protein was obtained from a metagenomic fragment of a Prevotella briantii strain. 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).

[0089] Using the plasmid pDR111 as a template, either 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 Two regions (amyB fragment and amyF fragment) containing the sigB8, rpoS, sigA, gfp, or sigB sequences were amplified. These two fragments were ligated by Gibson reaction on either side of the amplified fragment. The reaction products were integrated into the B. subtilis chromosome. The resulting clones were examined by PCR and sequencing.

[0090] The zpdN gene, the gp28 gene, the rpoN gene, or the T7 RNA polymerase (rnaP T7 ) gene fragment was inserted into the promoter P hy-spank The downstream fragment was ligated to the linearized full-length plasmid pDR111 by Gibson reaction (fragment pDR111inv). The reaction product was contacted with competent E. coli TG1 cells. Transformants selected with ampicillin were analyzed. Plasmids verified by restriction digestion and sequencing were used to transform B. subtilis.

[0091] The sequencing results showed that the gp28 gene had two mutations (526-7) compared with the sequences stored in the database. CA→TG These two mutations were found in several independent PCRs performed on the same phage preparation, but we decided to correct these two mutations to restore the published sequence. To this end, we synthesized two complementary oligonucleotides containing the modified regions. Plasmid pDR11+gp28 526-7CA-TG Using these two oligonucleotides as a template, the complete plasmid was amplified by correcting the mutation. This plasmid was then recircularized by the Gibson reaction. The reaction product was transformed into E. coli, and clones containing the corrected pDR11+gp28 plasmid were selected and transformed into B. subtilis.

[0092] Construction of reporting systems and various promoters A pUC19-derived plasmid was constructed by Gibson reaction to clone two regions of the B. subtilis chromosome (fragment glmS and fragment ybdG, positions 201,048 bp to 202,483 bp and 220,083 bp to 221,532 bp in the reference strain B. subtilis 168-GenBank AL009126.3), a gene encoding tetracycline resistance (fragment tetL), and a gene encoding sfGFP (fragment sfgfp) with an upstream terminator (fragment term). This plasmid replaced the chromosomal region designated "prophage 1" with the sfgfp gene and tetracycline resistance gene by homologous recombination.

[0093] This plasmid pJSM3T was cloned with the promoter sequence P zpdG The PCR fragment was amplified by reverse PCR using primers containing the promoter P. The PCR fragment was circularized and then integrated into the B. subtilis chromosome. The resulting strain was engineered to contain the promoter P. zpdGThe reporter constructs for the sigma factor Gp28 and phage T7 RNA polymerase contained a single copy of the sfGFP gene under the control of . For the reporter constructs for the sigma factor Gp28 and phage T7 RNA polymerase, various promoter fragments were obtained by PCR and cloned into the plasmid pJSM3T, which was also amplified by PCR. For these constructs, the RBS R0 was added to the primers used to amplify the plasmids.

[0094] Building a self-activating loop Plasmid pDR111-zpdN was cloned with promoter P hy-spank The primers used for amplification were located upstream of P. zpdG The fragments containing the promoter sequences were independently generated. The floating tails of the primers were constructed so that after assembly, the two promoter sequences would be separated by an 80 bp region. The two amplified sequences were joined using the Gibson method and purified from the template using the enzyme DpnI. The fragment to be integrated into the chromosome was then amplified using primers at the ends of the homologous section (Amy5' and Amy3') contained in pDR111, and this PCR product was used as construct P. zpdG It was directly transformed into a B. subtilis strain containing sfgfp.

[0095] SigA reporter module glyA region and upp / atpl region, erythromycin resistance gene (erm), constitutive promoter (P ref The mKate2 gene was amplified by PCR. The various PCR fragments were assembled by Gibson reaction. The complete construct was amplified using primers at the ends of the fragment homologous to the B. subtilis chromosome, and the PCR product was transformed into a strain (BSB1).

[0096] Repressor of GFP expression The gene encoding the repressor TetR (BD) (Schmitter et al., Molecular Microbiology, 105:413-425, 2017) was amplified by PCR and transfected with the promoter P by the Gibson method. hy-spank The fragment to be integrated was amplified by PCR and transformed into B. subtilis.

[0097] The gene sfgfp was amplified using primers containing two boxes, tetO, located between box-35 and box-10 of the promoter spoVG and downstream of box-10, respectively. Two DNA fragments containing the phleomycin resistance gene (ble) and 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 named construct P. hy-spank tetR DB The vector was transformed into a strain carrying the vector.

[0098] Repressible expression of SigA and deletion of endogenous SigA Using the same construct used for gene sfgfp, P tetO The gene sigA was amplified under the control of

[0099] The gene sigA and the two regions on either side of the chloramphenicol resistance gene were assembled using the Gibson method and cloned into the plasmid pUC19 in the E. coli strain TG1. The plasmid pUC19_sigA was linearized by digestion with the restriction enzyme NdeI (a site located outside the region of interest). The linearized plasmid was transformed into the strain SMS185 (P hy-spank sigA) was transformed into

[0100] Complete system Plasmid pDR111_P zpdG _P hy-spank-zpdN was linearized by PCR using primers located downstream of the zpdN gene and tetR was cloned using the Gibson method. BD The PCR fragment containing the gene was then assembled. The fragment to be integrated was then amplified and transformed into a strain of B. subtilis.

[0101] The construction carried out is shown in Table 3.

[0102] [Table 3] TIFF2026508348000005.tif187170

[0103] Tandem Affinity Purification The strains were grown in LB medium supplemented with 1 mM IPTG. 600The cells were grown until the RI was 1. A 100 ml culture was harvested and centrifuged, and the pellet was washed in buffer A (10 mM Tris-HCl (pH 7.5), 150 mM NaCl) and then frozen directly 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) containing 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, then on ice for 30 min, and centrifuged at 20,000 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) pre-equilibrated in buffer B. The column was left overnight at 4°C with agitation. 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) containing 50 units of TEV protease (Invitrogen). The column was left overnight at 4°C with agitation and then eluted over a second column containing Calmodulin-Sepharose 4B resin (Amersham Bioscience) pre-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 CaCl). 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, followed by 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 CaCl). 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).Proteins were precipitated with acetone and then analyzed on SDS-PAGE gels or in resuspension solution (1 M urea and 100 mM Tris-HCl, pH 8.5) and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS / MS).

[0104] Viability test on solid medium B. subtilis strains were pre-cultured in LB medium in 96-well plates (CELLSTAR™, Greiner Bio-One) for approximately 20 hours. They were diluted 10-fold in test medium in the morning. These pre-cultures were DO 600 Once the P value reached ≈0.3-0.4, they were diluted 20-fold again in the medium of interest. At each stage, the cultures were seeded into microplates and incubated at 37°C under constant agitation (360 rpm) in a multimode plate reader, Synergy™ 2 (Biotek™). hy-spank -gfp and BSB1 were systematically tested. Each strain was grown in at least triplicate and measured in three independent experiments. Every 10 minutes, 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.

[0105] To compare promoters to other uninduced strains, fluorescence was normalized to the reference strain. For constitutive promoters, relative expression units are calculated: REU(t i )=[(fluorescence(t i ) / DO 600 (t i )] 菌株 / [(fluorescence(t i ) / DO 600 (t i )] 参照

[0106] For inducible promoters, the fluorescence value with DO obtained without inducer is subtracted from the fluorescence value obtained with induction, and this is reported to the reference strain: REU(t i )={[(fluorescence(t i ) / DO 600 (t i )] 誘導ありの菌株 -[(fluorescence(t i ) / DO 600 (t i )] 誘導なしの菌株} / [(fluorescence(t i ) / DO 600 (ti)] 参照

[0107] For all experiments, the mean and standard deviation are then calculated.

[0108] Propidium iodide viability test Propidium iodide is a color molecule that cannot accumulate in bacteria that have lost membrane integrity, i.e., dead cells. To test cell viability, propidium iodide was added to a culture sample at a final concentration of 167 μM. After 10 minutes of incubation, the cells were analyzed by flow cytometry to detect color development due to intracellularly incorporated propidium iodide. To verify this color development, a sample of exponentially growing BSB1 cells was directly processed. Another culture sample was centrifuged and resuspended in isopropanol for 60 minutes to kill the cells, followed by centrifugation. The cells were treated with 10 mM Tris + 150 mM NaCl and similarly colored. These two samples were used to verify the test.

[0109] Inhibition of alternative sigma factors The erythromycin resistance gene was amplified from an erythromycin-resistant strain. The upstream and downstream regions of the genes encoding the alternative sigma factors Sig B, Sig D, and Sig H of B. subtilis were amplified by PCR. Primers were designed to introduce Bsa1 sites at the ends of all of these fragments. For each construct, the upstream and downstream fragments and the resistance cassette were cloned using the "Golden Gate" reaction. The resulting linear fragments were integrated into the genome of strain BSB1 and then transferred to the chromosome of a strain containing the entire GFP production system. The constructed strains are summarized in Table 4, and the protein sequences of the alternative sigma factors are shown in Table 5.

[0110] [Table 4]

[0111] [Table 5]

[0112] Examination of promoters recognized by the sigma factor ZpdN Genomic DNA from a strain with a complete production system was used to construct the promoter P containing the integration region (glmS gene). zpdG The upstream region of this promoter and the downstream region of this promoter (sfgfp, tetL, and YbdG genes) were amplified. Oligonucleotides were designed to introduce a 30-base pair overlap region along with the two promoters being tested. The two DNA fragments were ligated by Gibson reaction and then introduced into a strain overproducing the sigma factor ZpdN and a strain containing all parts of the production system except for GFP. The constructed strains are summarized in Table 6, and the sequences of the promoters tested are listed in Table 7.

[0113] [Table 6]

[0114] [Table 7]

[0115] Cloning of the target LacZ, AmyS, YqaH, and NucA proteins To test whether proteins other than GFP could be expressed, the open reading phase of GFP was replaced with that of a gene encoding LacZ, NucA, YqaH, or AmyS. The region upstream of the ATG start codon and downstream of the stop codon were amplified by PCR by introducing Bsa1 sites at both ends. Similarly, various reading phases were amplified by creating Bsa1 sites compatible with the previous ones. The various fragments were joined by Golden Gate reaction and then transferred into the final strain. The constructed strains are summarized in Table 8, and the protein sequences are shown in Table 9.

[0116] [Table 8]

[0117] [Table 9]

[0118] result To construct the orthogonal invasive transcription system, several modules were constructed (Fig. 1).

[0119] The first module consists of placing an orthogonal system under the control of an inducible promoter and placing transcription of a gene of interest (eg, a gene encoding GFP) under the control of the orthogonal transcription system.

[0120] The second module consists of setting up a self-activating loop for transcription of the orthogonal transcription system to amplify the production of the orthogonal transcription system.

[0121] The third module is responsible for repressing the expression of the Bacillus subtilis household sigma factor SigA.

[0122] Finally, the autoactivation loop of sigma factors is linked to the repression of sigma factor A expression.

[0123] Example 1: Construction of the first module Several orthogonal transcription systems were developed using the IPTG-inducible promoter P hy-spank It was cloned in single copy into the B. subtilis chromosome at the amyE locus under the control of (Fig. 2, A).

[0124] In these strains, we introduced a transcriptional fusion between subunit β of RNA polymerase and tagged SPA. The RNA polymerase complexes were purified, and the protein partners were analyzed on an acrylamide gel (Fig. 2B).

[0125] Gel analysis revealed that the 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 3A). The promoter P hy-spank Similar constructions were carried out in strains producing phage T7 RNA polymerase under the control of .

[0126] This construct was integrated in single copy into the B. subtilis chromosome between the ybbU and ybdG genes in the region proximal to the chromosomal origin of replication.

[0127] For each constructed strain, the amount of GFP produced was measured over time in LB medium without IPTG or in LB medium containing 500 μM IPTG (fluorescence / DO 600nm ) (Figure 3, B).

[0128] The relative fluorescence of induction of orthogonal sigma factors was calculated ((fluorescence / DO 600nm) IPTG500μM -(Fluorescence / DO 600nm ) IPTGなし To compare strains, the relative fluorescence was calculated using the promoter P hy-spank The GFP activity was normalized to that of a reference strain carrying a gene encoding GFP under the control of sigma factor Gp28 (GFP / OD normalization). The results show that no GFP activity was detected in constructs containing the promoter recognized by sigma factor Gp28 (Table 10). For sigma factor ZpdN, the tested promoters showed high transcriptional activity similar to that observed with polymerase T7 RNA.

[0129] [Table 10]

[0130] We continued to build the system using ZpdN and verified the first module.

[0131] Example 2: Construction of a second module In the second module, the active promoter (P) recognized by the sigma factor ZpdN used in module 1 was inserted. zpdG ) as promoter P hy-spank It was cloned upstream of (Fig. 4, A).

[0132] This autoinduction loop amplifies the induction response of ZpdN expression and allows ZpdN to be expressed independently of housekeeping sigma factors in B. subtilis.

[0133] The normalized specific activity was measured at various IPTG concentrations for strains with and without the autoinduction loop (Figure 4B). The results show that at low doses of IPTG (20 μM or less), the autoactivation loop allows for the production of more GFP. At higher concentrations (above 100 μM), the strain with the autoactivation loop produced slightly less GFP; it should be noted that in this strain, the ZpdN promoter is present in two copies on the chromosome. A second module was constructed and is active.

[0134] Example 3: Construction of the third module In the third module, the repressor TetR DB The gene encoding hy-spank (Fig. 5, A).

[0135] To test the repression efficiency of this protein, TetR BD A promoter containing an operator region recognized by a repressor (P tetO ) was cloned in front of the GFP gene.

[0136] Cultures with and without IPTG (promoter P tetO Measurement of the fluorescence produced in the cultures (with and without IPTG) reveals a very large difference (1.7±0.1 arbitrary units without IPTG and 0.11±0.08 arbitrary units with IPTG).

[0137] Under the control of this promoter, a greater than 15-fold repression of the GFP gene transcription / translation rate was measured.

[0138] TetR BD After verifying the repression, the gene sigA was transformed into promoter P tetO The natural copy of the sigA locus was then replaced with a gene encoding chloramphenicol resistance.

[0139] Next, we verified that the growth of this strain is sensitive to ITPG. After adding IPTG to the culture, the growth of the strain is significantly affected.

[0140] After 30 min, the slope of the optical density curve becomes steeper (Figure 5B), the generation time increases, and growth ceases after 3 h. A very large decrease in viability (100,000-fold) was observed on the inducer-free LB agar boxes after 3 h of incubation.

[0141] In the latter strain, fluorescent protein production is dependent on the sigma A promoter. ref By using the mkate2 reporter (Guiziou et al., Nucleic Acids Research, 44(15):7495-508, 2016), the repressor TetR BD The induction of SigA resulted in a decrease in the amount of fluorescence, suggesting that competition between SigA and ZpdN may occur.

[0142] Thus, the third module, which allows for the repression of expression of housekeeping sigma factors in B. subtilis, is functional.

[0143] Example 4: Tying of an Autoactivating Loop The final step consisted of linking the autoactivation loop to the repression of the expression of housekeeping sigma factors.

[0144] To achieve this, the repressor TetR BD The gene encoding the sigma factor ZpdN is driven by the promoter P hy-spank The genes were cloned into a single operon under the control of (Fig. 6, A).

[0145] In this strain, the housekeeping sigma factor SigA is expressed by the promoter P tetO is under the control of

[0146] In the absence of an inducer, the sigA gene is expressed and the transcriptional program for B. subtilis proteins becomes active.

[0147] Addition of IPTG to this strain induces i) the expression of the sigma factor ZpdN, which further induces the expression of GFP and its own expression, and ii) the expression of the repressor TetR, which prevents the expression of the sigA gene.

[0148] The housekeeping transcriptional program that produces proteins that enable B. subtilis growth is shut down, and the transcriptional program that produces the protein of interest is activated, allowing all of the cell's resources to be directed toward producing the protein of interest.

[0149] In this strain, the GFP production level was significantly higher than that of DO during IPTG induction. 600 The maximum level of GFP produced at 3 hours of induction was dependent on the promoter P veg The level of GFP produced by P hy-spank This is much higher than the level of GFP produced after induction of

[0150] Cytometric analysis of cells after induction of the transcriptional program switch (FIG. 7) was performed.

[0151] After 1 hour of induction, some cells switched to the GFP-producing program, and after 2 hours, all cells were producing GFP very uniformly (the peak was very narrow). No cells that did not produce GFP were detected, but the construct P veg In the case of the strain carrying gfp, approximately 1% of the cells did not produce GFP. After 3 hours of induction, GFP production increased, where a high percentage of cells produced significant amounts of GFP (2 × 10 6 The GFP production level was significantly higher than that of strain P. hy-spank The levels are much higher than those obtained with GFP and the population is much more homogeneous.

[0152] The various embodiments described in no way limit the scope of the invention as set forth in the claims, but are provided as examples for a better understanding of the invention.

[0153] Example 5: Characterization of cell viability after induction of the production phase The state of the cells was characterized after induction of GFP production. The cells were diluted and cultured in the presence of IPTG. After 2 hours, the optical density stopped increasing, indicating that growth had ceased. No decrease in optical density was observed, indicating no lysis in this population.

[0154] For kinetic studies, samples were collected, diluted, and then plated onto inducer-free rich medium boxes. After 24 hours of incubation at 37°C, the colonies formed were counted. After 1 hour of induction, the cells were no longer cultivable (virtually no colonies formed on the boxes), even after induction was stopped (Figure 8).

[0155] To characterize cell viability, cells were incubated with propidium iodide, which only labels dead cells. Three hours after induction, the cells were not stained by propidium iodide, indicating that no dead cells were present in the population (Figure 9). These are "viable but non-culturable" cells. Therefore, the integrity of the bacterial membrane is maintained. This situation is advantageous because it allows the cell integrity to be maintained, and therefore, a purification step can be performed by centrifugation.

[0156] Example 6: Reversibility of induction To determine whether viable cells could be maintained, induction with a lower concentration of IPTG (20 mM) was performed.

[0157] After 3 hours, the cells were still able to grow on medium without IPTG (Figure 10). Thus, under the inducing conditions of the system, the cells were still viable after 3 hours. 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 viable. Since they all retain the selection marker, no part of the system has been lost. At this inducer concentration, induction is reversible and does not select for a subpopulation that would otherwise have lost production.

[0158] Example 7: Effects of secondary sigma factor deletion in B. subtilis Certain other alternative sigma factors present in cells may compete with ZpdN for access to RNA polymerase. As a result, fewer RNA polymerase + ZpdN complexes are formed in the cells, limiting transcription of the gene encoding GFP. To test this hypothesis, alternative sigma factors produced during exponential growth were suppressed in a strain containing the entire system. GFP production levels were examined after deletion of the genes encoding sigma factor B, sigma factor D, and sigma factor H, respectively.

[0159] Only the suppression of the sigma factor encoded by the sigD gene had 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 flagellar proteins induces increased amylase production. These genes are under the control of sigma factor D. Therefore, the observed results are not due to competition between sigma factors, but rather to the release of resources due to the inactivity of the transcriptional program induced by sigma factor D production. This result indicates that strain modifications favoring protein production can have a cumulative effect in the constructed system.

[0160] Example 8: Testing other promoters recognized by the ZpdN sigma factor To produce several proteins simultaneously in the same cell, it would be interesting to have several promoters recognized by the sigma factor ZpdN.

[0161] Two different promoters were cloned in front of the gene encoding the ribosome binding site R0 and sfGFP. In these strains, induction of production of the sigma factor ZpdN resulted in GFP production at levels nearly twice those produced by the previous construct (Table 11).

[0162] [Table 11]

[0163] The best two promoters were integrated into a strain containing the entire production system. A comparison was made between this new construct and the previous construct. The results show that the growth curves of the two strains are similar, indicating that growth arrest due to loss of sigma factor A is the same in both strains (Figure 12). However, it is clear that the new construct appears to be capable of producing between four and five times more GFP intracellularly.

[0164] Example 9: Extension to the production of other proteins The first protein cloned was β-galactosidase, a protein with a very high molecular weight of 116 kDa. Three hours after induction, β-galactosidase activity was detected in the cells, with approximately 8500 nmol of ortho-nitrophenyl-β-galactoside (ONP) formed per minute per milligram of protein. In comparison, previous results showed that expression of a single copy of the inducible promoter P in the chromosome spac In the case of , activity levels measured after full induction of the subpromoter indicate the formation of nearly 1000 nmol of ONP per minute per milligram of protein.

[0165] The second cloned protein is a nuclease encoded by two polypeptides in phage Bil66. In the absence of induction, the generation times (calculated over six replicates) for strains with and without the nuclease gene were similar for strains with the sfGFP gene (41 ± 4 min) or β-galactosidase gene (42 ± 1 min), and for strains with the nuclease gene (40 ± 1 min). This result indicates that expression is shut off in the absence of induction. In the strain with the nuclease gene, induction of the system caused rapid cell lysis, indicating that the nuclease is indeed produced (Figure 13).

[0166] The third protein cloned was YqaH, a protein that inhibits cell growth by inhibiting chromosomal DNA replication in B. subtilis. The gene encoding this protein was then cloned into a complete production system. In this example, the protein was successfully cloned, causing a rapid cessation of growth after induction of production.

[0167] The fourth cloned protein is the amylase AmyS from Bacillus stearothermophilus. This protein has a signal peptide recognized by the Sec-dependent B. subtilis secretion system. Preliminary results show that protein secretion is still possible after induction of protein production in the constructed strain.

Claims

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

2. 2. The genetically modified bacterium of claim 1, further comprising a third nucleotide sequence encoding at least one protein of interest, wherein expression of the at least one protein of interest is under the control of a promoter recognized by the orthogonal sigma factor.

3. The orthogonal sigma factors are A sigma factor derived from a bacterial species different from the bacterial species of the genetically modified bacterium; and a sigma factor specific to an extrachromosomal element that does not naturally occur in said genetically modified bacterium; The genetically modified bacterium according to claim 1 or 2, wherein the bacterium is selected from the group consisting of:

4. 4. The genetically modified bacterium of any one of claims 1 to 3, wherein the bacterium belongs to a species selected from Bacillus subtilis, Escherichia coli, Geobacillus stearothermophilus, and Lactococcus lactis, and is preferably B. subtilis.

5. 1. A method for producing at least one protein of interest, comprising: Culturing the genetically modified bacterium defined in any one of claims 1 to 4; Inducing expression of an orthogonal sigma factor; A method comprising:

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

7. transcriptional repressors, Interfering RNA, protease, or sigma factor inhibitors, 7. The polynucleotide of claim 6, further comprising a second nucleotide sequence encoding:

8. 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 recombinantly expressing at least one protein of interest, comprising at least: A genetically modified bacterium according to 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 allowing the induction of the expression of said at least one protein of interest to be induced, Includes a kit.

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