System for the bacterial expression of heterologous proteins

Abstract

The present invention relates to an expression cassette for the bacterial expression of heterologous proteins, preferably Gram-positive bacteria; this cassette is characterised in that it contains the sequence coding for the ORF2 protein of Bacillus thuringiensis. The present invention also relates to recombinant expression vectors comprising such an expression cassette and to bacteria, preferably Gram-positive bacteria, transformed using this recombinant expression vector.

Description

[0001] DESCRIPTION TITLE: Bacterial expression system for heterologous proteins The present invention relates to a new system for the expression by bacteria of heterologous proteins in pseudo-crystalline form in a stable state, and protected from degradation; this system has a significant advantage in terms of purification of said proteins. The present invention relates more particularly to an expression cassette allowing the expression of heterologous proteins by bacteria, preferably Gram-positive bacteria; this cassette is characterized in that it contains the sequence coding for the ORF2 protein of Bacillus thuringiensis. The present invention also relates to recombinant expression vectors comprising such an expression cassette and to bacteria, preferably Gram-positive, transformed with this recombinant expression vector.The use of recombinant bacteria to produce heterologous proteins of interest, such as insulin and growth hormone, has been known for a long time and has been widely implemented, but it has also shown its limitations. Indeed, bacteria are unable to produce proteins with a complex structure such as antibodies or blood clotting factors. To be stable and active in vivo and therefore effective in humans, these proteins must undergo multiple post-translational modifications. In particular, when E. coli is used to produce proteins in large quantities, these proteins can end up misfolded or form insoluble aggregates. To overcome this problem, chaperone proteins can be co-expressed with the protein of interest, thus increasing the chances of obtaining a correctly folded and soluble protein. However, in general, these methods give limited results.Indeed, when the expression of insoluble aggregate-forming proteins was combined with the expression of E. coli chaperone proteins (DnaK, DnaJ, GrpE, GroES, GroEL), the result shows either a very small increase in soluble protein production (Malekian et al., 2019, Protein Expression and Purification 160:66-72), or the need to process insoluble aggregates in vitro by an ATP-dependent maturation system (Alfi et al., J Bioscience and. Bioengineering 128:290-295.). When this expression is combined with chaperone proteins of other origins, such as Spy (Spheroplast protein Y), the increase in soluble protein production is too small to be of industrial interest (Ruan et al., 2020, J. Biotech.307:130-138). Bacillus thuringiensis (Bt) is a spore-forming Gram-positive bacterium that produces large amounts of insecticidal proteins (Cry and Cyt proteins).These accumulate in the mother cell and form crystalline inclusions representing up to 25% of the dry weight of the bacterium. These inclusions are released, by cell lysis, at the end of the sporulation process (Agaisse and Lereclus, 1995). This massive production of proteins depends on several factors: i) the number of copies of the cry or cyt genes, ii) the strength of the promoter, iii) the stability of the transcripts, and iv) the formation of a stable crystal. The ability to produce some of these toxins (Cry or Cyt) in crystalline form is due either to a C-terminal domain of certain Cry proteins allowing the formation of crystalline inclusions, or to an additional "Helper" protein which could have a chaperone role intervening in the crystallization of Cyt proteins or certain Cry proteins, whose C-ter domain is absent (Adalat et al. 2017).By developing an original expression cassette allowing the co-expression of the Bt ORF2 protein and a heterologous protein of interest under the control of a strong promoter, the inventors have succeeded in producing significant quantities of heterologous proteins (other than Cry or Cyt proteins) in an asporogenous Bt strain, preferentially in stationary phase. In the absence of cell lysis, the heterologous protein thus produced accumulates in the bacterial cell in the form of a pseudo-crystalline inclusion; this provides a considerable advantage for the production of unstable or cytotoxic proteins that are difficult to express using current expression methods in microorganisms.Thus, the present invention relates to an expression cassette comprising: (i) a strong promoter active during the stationary phase; (ii) a sequence having at least 80%, preferably at least 90%, more preferably at least 95% and most preferably 100% identity, with the sequence SEQ ID No. 2 of the gene coding ORF2 of Bt of SEQ ID No. 1; and (iii) the sequence of a gene coding a heterologous protein; said sequences of the genes coding ORF2 and the heterologous protein being in operon under the control of said strong promoter. Strong promoter A strong promoter is a promoter allowing a high level of transcription of the gene which is located downstream. Commonly, the expression of the promoters is evaluated with the activity of ß-galactosidase (AS, in units / mg of protein) according to the calculation described in Perchat et al. (“A cell-cell communication system regulates protease production during sporulation in bacteria of the Bacillus cereus group”, Molecular Biology, Vol.82, 3, p.619-633, 2011) : AS = (A. 420 x 1500000) / (T x V x C). A 420: absorbance at 420 nm T : reaction time in min V : volume of crude extract in µL C : protein concentration in µg / mL Using this formula, a promoter is considered highly expressed when the AS is greater than 500 U / mg prot. The strong promoter can originate from the host cell used for the expression of the heterologous protein; it can also be an exogenous promoter. Strong promoters are preferentially promoters activated at the beginning of stationary phase and / or which are functional during a large part of this physiological state. In particular, the strong promoter can be chosen from promoters that directly or indirectly require the Spo0A regulator such as those of the genes calY, inhA1, sipW, ces. According to this embodiment, the strong promoter is the promoter of the calY gene (accession number EU604076 in GenBank) and is designated PcalY of sequence SEQ ID No. 5, PinhA1 of SEQ ID No. 6 or Pces of SEQ ID No. 7.According to another embodiment of the invention, the strong promoter can be chosen from promoters which are repressed by the regulator CodY during the exponential growth phase, the strong promoter is then chosen from PoppA of SEQ ID No. 8 and PnppC of SEQ ID No. 9. The repression by CodY is lifted at the end of the exponential phase and these promoters are therefore specifically active during the stationary phase. According to yet another embodiment of the invention, the strong promoter is expressed during the stationary phase of Bacillus and more preferably the strong promoter is positively regulated by a regulator chosen from PlcR and NprR. The strong promoters are preferentially chosen from PpapR (SEQ ID No. 10), PplcB (SEQ ID No. 11), PnprA (SEQ ID No. 12), PnprR (SEQ ID No. 13). Thus, preferably, the highly active promoter in stationary phase is chosen from PcalY, PinhA1, Pces, PoppA, PnppC, PpapR, PplcB, PnprA and PnprR.Gene encoding ORF2 The Bt ORF2 protein of sequence SEQ ID No. 1 is co-transcribed with the gene encoding the Cry2A protein and has been described as a crystallization factor for this protein (Deng et al., Regulation of cry gene expression in Bacillus thuringiensis, Toxins, 2014; 6(7): 2194-2209); it is encoded by the nucleic sequence SEQ ID No. 2. Surprisingly, the Inventors have demonstrated that the co-expression of the gene for the heterologous protein with the gene encoding the ORF2 protein makes it possible to condense the heterologous proteins produced (see the experimental section below) in the host cells in the form of inclusions and thus to substantially increase the quantity of heterologous proteins produced. Gene encoding the heterologous protein Heterologous protein means a protein which is not naturally expressed by the host cell modified by an expression vector comprising the expression cassette according to the invention.Preferably, the heterologous protein is a protein of industrial or medical interest such as enzymes, such as proteases, lipases, amylases; hormones; antigens, for example, usable as immunogens, peptides or proteins for therapeutic use (bacteriocins, inhibitors or stimulators of the cellular response, etc.). It may also be cytolytic insecticidal toxins naturally expressed by Bt and used as a biopesticide; it may in particular be proteins of the Cry family (crystal proteins) or proteins of the Cyt family (cytolytic insecticidal toxins) or Vip3 proteins (toxins active against lepidopteran insects).The expression cassette according to the invention leads to the expression and condensation of heterologous proteins in the form of inclusion within bacteria, its use is particularly suitable for the production of heterologous proteins that are cytotoxic for the producing strain or unstable. Preferably, the ORF2 coding sequence is cloned in cis of the gene coding the heterologous protein of interest. It is preferentially cloned in operon with the gene coding the heterologous protein of interest.Other components of the expression cassette According to particular embodiments, the expression cassette according to the invention may further comprise: - an mRNA stabilizing sequence positioned downstream of the promoter and upstream of the sequence of the gene coding ORF2; preferably, it is STAB-SD of sequence SEQ ID No. 3; preferably, the STAB-SD sequence is downstream of the transcription +1 and at a position between approximately 100 and 500, preferably between 100 and 300 and even more preferably between 100 and 150 base pairs upstream of the ribosomal binding site (RBS); and / or - a terminator sequence of the cry1Ac gene, designated Tcry1Ac, for example a sequence having at least 90%, preferably at least 95% or at least 98%, identity with SEQ ID No. 4, preferably, it is SEQ ID No. 4; this sequence is cloned downstream of the gene coding the heterologous protein.The choice of these sequences should not, however, be considered as limiting because it is within the reach of those skilled in the art to substitute these sequences with sequences with equivalent functions. Thus, the expression cassette according to the invention may comprise: (i) a strong promoter active during the stationary phase; and the stabilizing sequence of the STAB-SD mRNA; (ii) the sequence of the gene coding ORF2; (iii) the sequence of a gene coding a heterologous protein; or (i) a strong promoter active during the stationary phase; (ii) the sequence of the gene coding ORF2; (iii) the sequence of a gene coding a heterologous protein; and the terminator sequence of the cry1Ac gene, Tcry1Ac; or (i) a strong promoter active during the stationary phase; and the stabilizing sequence of the STAB-SD mRNA; (ii) the sequence of the gene coding ORF2; (iii) the sequence of a gene encoding a heterologous protein; and the terminator sequence of the cry1Ac gene, Tcry1Ac.According to these embodiments, the strong promoter active during the stationary phase can be chosen from PcalY, PinhA1, Pces, PoppA, PnppC, PpapR, PplcB, PnprA and PnprR. recombinant plasmids The present invention also relates to a recombinant plasmid comprising the expression cassette according to the invention. The plasmids that can be used according to the invention conventionally comprise an origin of replication and at least one selection system consisting of one or more genes allowing the selection of transformed bacteria, for example antibiotic resistance genes. Any plasmid, preferably with a high copy number, adapted to the bacterial host used can be used. The plasmids suitable according to the invention are plasmids allowing expression in bacteria of the genus Bacillus.Preferably, the plasmids used are those exhibiting high segregational stability (e.g., the plasmid must remain present in the strain for at least 25 generations) and / or structural stability (i.e., it does not undergo molecular rearrangements during bacterial growth). The stability of plasmids derived from the pHT1030 replicon (e.g., plasmids (pHT304, pHT315 or pHT370) or the pBC16 replicon has been demonstrated (Lereclus, D., Arantes, O., 1992. spbA locus ensures the segregational stability of pHT1030, a novel type of Gram-positive replicon. Mol. Microbiol. 7: 35-46 doi: 10.1111 / j.1365-2958.1992.tb00835.x ; Arantes, O., Lereclus, D., 1991. Construction of cloning vectors for Bacillus thuringiensis. Gene 108: 115-119 doi: 10.1016 / 0378-1119(91)90495-w). The stability structural is defined as non-intramolecular recombination of the plasmid.The structural stability of plasmid pHT1030 and its derivatives is demonstrated by the fact that they can carry large exogenous DNA fragments (> 10 kb) without undergoing molecular rearrangements (Lereclus et al., 1989. Transformation and expression of a cloned delta-endotoxin gene in Bacillus thuringiensis. FEMS Microbiol. Lett. 60: 211-217). In the case of Bt, plasmids derived from pHT1030, pBC16, pE194, pC194 and any plasmid naturally present in this bacterium can be used, for example pHT8-1 present in Bt kurstaki strains HD73 and Bt 407, pHT73 present in Bt kurstaki strain HD73, or pBMB299 present in Bt kurstaki strain HD1. Preferably, these are high copy number plasmids, in particular derived from plasmid pHT1030, such as pHT3101, pHT304, pHT315 and pHT370; preferably pHT315.The construction of the expression cassette according to the invention and its incorporation into a plasmid are carried out by molecular biology techniques well known to those skilled in the art as illustrated in the experimental part. Transformed bacteria The present invention also relates to bacteria transformed with a plasmid according to the invention. Any bacterium in stationary phase of growth can be used. Preferably, these are Gram+ bacteria such as bacteria of the genus Bacillus, in particular Bacillus cereus, Bacillus megaterium, Bacillus sphaericus, Bacillus subtilis and Bacillus thuringiensis. Even more preferably, strains of the genus Bacillus, in particular Bacillus subtilis and Bt, asporogenous (natural or mutant), will be used.Asporogenous strains are advantageous in that they do not exhibit cell lysis, thus, the heterologous proteins produced are retained in the producing bacterium (mother cell) and protected from degradation by extracellular proteases. In order to suppress the sporulation activity of a Bt strain, it is possible to inactivate any gene essential for sporulation such as the genes involved in the expression of the sporulation sigma factors Spo0A, SigE, SigH and SigK; preferably, a Bt ΔsigE or ∆spo0A strain, preferentially Bt 407-ΔsigE (Bravo et al., 1996) or Bt HD73 ∆spo0A, is used. Inactivation of these genes can be achieved by interruption or modification of the coding sequence, or by deletion of all or part of the gene.The deletion is obtained by double crossing-over between the adjacent regions located upstream and downstream of the gene, using plasmids whose replication is thermosensitive, for example the plasmids pRN5101 (Lereclus et al., 1992) or pMAD (Arnaud et al., 2004), and using the protocols described in these articles. Preferably, when the inactivated sporulation gene is Spo0A, the strong promoter used is chosen from PpapR, PoppA, PnppC, PnprA, PnprR and PplcB, and when the inactivated sporulation gene is sigE, the strong promoter is PcalY, PinhA1 or Pces. The plasmid according to the invention can be introduced into the host bacterium according to techniques known to those skilled in the art; In particular, transformation of the host bacterium can be achieved by electroporation (Lereclus et al., 1989) or by heterogram conjugation (Tieu-Cuot et al., 1987).The expression cassette containing the gene of interest can also be introduced onto the bacterial chromosome or onto a resident plasmid by homologous recombination (Lereclus et al., 1992). Method for producing a heterologous protein The present invention also relates to a method for producing a heterologous protein comprising the steps of: - preparing a transformed bacterium according to the invention; - culturing said transformed bacterium in stationary phase; and - optionally, purifying said heterologous protein. The culture of the transformed bacterium is carried out on a culture medium containing at least one source of nitrogen and glucose in appropriate concentrations at a temperature preferably between 25 and 35°C, preferably the temperature is of the order of 30°C; for example, the culture medium is LB medium.The purification of the heterologous protein can be carried out by centrifugation and solubilization of the crystals; in addition, the methods of exclusion chromatography, ion exchange chromatography or even affinity chromatography can be implemented. The present invention also relates to the use of a sequence having at least 80%, preferably at least 90%, more preferably at least 95% and most preferably 100% identity, with the sequence coding ORF2 of Bt in an expression cassette also comprising a strong promoter active in stationary phase and the sequence coding a heterologous protein, for the production of said heterologous protein. FIGURES [Fig 1] Schematic representation of the plasmid pHT-Cal'orf2-gfp-Tcry1Ac. [Fig 2] Effect of the ORF2 protein on the production and condensation of proteins during the stationary phase of Bt.Fluorescence microscope observation of Bt407-ΔsigE cells transformed with the plasmid pHTCal'gfp-Tcry1Ac (a) and with the plasmid pHT-Cal'orf2-gfp-Tcry1Ac (b), then with the addition of PEG6000 in the culture medium (c). [Fig 3] Purification of GFP produced in operon with ORF2. Photo of migration on a 10% SDSPAGE gel of the soluble (FS) and insoluble (FI) fractions resulting from Bt 407ΔsigE - pHTCal'ORF2-gfp-TCry cultures after 24 h at 30°C in LB medium. [Fig 4 ] Effect of the Helper protein ORF2 on GFP production. Western blot: the gene coding for GFP is cloned in the presence (ORF2-GFP) or absence (GFP) of orf2 in the construct shown in Figure 1, except that the promoter used is PoppA instead of the PcalY promoter. Cytoplasmic extracts are made from the same cell density and loaded onto the gel. GFP 1.5 µg is a purified GFP standard and Seeblue ladder is a size marker.The GFP protein is detected using an anti-His-tag antibody from GeneScript. EXAMPLES 1. Materials and Methods 1.1. Construction of the plasmid according to the invention pHT-Cal'orf2-gfp-Tcry1Ac and the control plasmid pHTCal'-gfp-Tcry1Ac Plasmid pHT-. The promoter region of the calY gene (PcalY) was amplified with oligos PcalY1 and PcalY2 from the chromosomal DNA of Bt kurstaki strain HD73. The resulting DNA fragment was digested with enzymes Xba1 and Asc1 and then purified as an XbaI / AscI fragment. The Tcry1aC terminator region was amplified with oligos Tcry1 and Tcry2 from plasmid DNA of strain HD73. The resulting DNA fragment was digested with enzymes Kpn1 and EcoR1 and then purified as a KpnI / EcoRI fragment. These two DNA fragments were inserted between the XbaI / AscI and KpnI / EcoRI sites, respectively, into a plasmid pHT315 already containing the STAB-SD region between the AscI and BamHI sites. The orf2 gene was amplified with oligos ORF2(1) and ORF2(2) from plasmid DNA of Bt kurstaki strain HD1. The gfp gene was amplified with oligos GFP1 and GFP2 from plasmid pHT304-gfpbte (laboratory stock). This gfp gene encodes a His-tagged GFP protein.These two DNA fragments were assembled by “splicing by overlap extension PCR” and inserted into the plasmid (previously linearized by PCR with oligos LinP1 and LinP2).Oligonucleotides : SEQ ID N°14 - PcalY1 5'-GCTCTAGAAAGCAAGACTAGTAATATTTACGAG-3' site XbaI underlined SEQ DI N°15 - PcalY2 5'-GGCGCGCCCACAATCAATTCCCCCTAGCTTT-3' site AscI underlined SEQ - ID N°116 5'-GGGGTACCAACTCAGGTTTAAATATCGTTTTC-3' site KpnI underlined SEQ ID N°17 - Tcry2 5'-GGAATTCCCAAATAAAAAGAAAAAGCAGAG-3' site EcoRI underlined SEQ ID N°18 - ORF2(1) 5'-CATTTTATGGACCTAACTGACTAT SEQ ID N°17 - Tcry2 N°19 - ORF2(2) 5'-CATTCATGGTGTCACCTCCTAAAAATAAAGTTTTATATTAACTAAACTCTTTTGATA-3' SEQ ID N°20 - GFP1 5'-TATCAAAGAGTTTAGTTTTAATAAAACTTTATTTTTAAGGAGGTGACACCATG-3'Q N°21 - GFP 5'-ACCTGAGTTGGTACCTTAATGATGATGATGATGAGA-3' SEQ ID N°22 - LinP1 5'-GGTACCAACTCAGGTTTAAATATC-3' SEQ ID N°23 - LinP2 5'-GGATCCATAAAATGATTTTTCAT-3' The plasmid pHT-Cal-Calc shown schematically in Figure 1. pHT-Cal'gfp-Tcry1 plasmid The promoter region of the calY gene (P calY) was amplified with oligos PcalY1 and PcalY2 from the chromosomal DNA of Bt kurstaki strain HD73. The resulting DNA fragment was digested with enzymes Xba1 and Asc1 and then purified as an XbaI / AscI fragment. The STAB-SD region was amplified with oligos StabSD1 and StabSD2 from a plasmid containing the entire promoter region of the cry3A gene. The resulting DNA fragment was digested with enzymes AscI and BamHI and then purified as an AscI / BamHI fragment. The Tcry1aC terminator region was amplified with oligos Tcry1 and Tcry2 from the plasmid DNA of strain HD73. The resulting DNA fragment was digested with enzymes Kpn1 and EcoR1 and then purified as a KpnI / EcoRI fragment. These three DNA fragments were inserted between the XbaI / AscI, AscI / BamHI and KpnI / EcoRI sites, respectively, in a plasmid pHT315.The gfp gene was amplified with GFP1' (GFP1 bis) and GFP2' oligos from pHT304-gfpbte plasmid (laboratory stock) and inserted into the plasmid (previously linearized by PCR with LinP1 and LinP2 oligos). SEQ ID N°14 - PcalY1 5'-GCTCTAGAAAGCAAGACTAGTAATATTTACGAG-3' site XbaI underlined SEQ DI N°15 - PcalY2 5'-GGCGCGCCAACAATCAATTCCCCCTAGCTTT-3' site AscI underlined SEQ ID N°26 - StabSD1 5'-GGCGCGCCTCTTGAAAGGAGGGATGCCT-3' site AscI underlined SEQ ID N°27 - StabSD2 5'-CGGGATCCATAAAATGATTTTTCATAAATC-3' site BamHI underlined SEQ ID N°16 - Tcry1 5'-GGGGTACCAACTCAG GTTTCAGTTCGT site-3'underlined SEQ ID N°27 - StabSD2 ID N°17 - Tcry2 5'-GGAATTCCCAAATAAAAAAAGAAAAAGCAGAG-3' site EcoRI underlined SEQ ID N°28 - GFP1 bis 5'- TCATTTTATGGATCCGAAATAAAATGCATCTGTATTTGAA-3' SEQ ID N°21 - GFP2 5'-ACCTGAGTTGGTACCTTAATGATGATGATGATGAGA-3' SEQ ID N°22 - LinP1 5'-GGTACCAACTCAGGTTTAAATATC-3' SEQ ID N°23 - LinP2 5'-GGATCCATAAAATGATTTTTCAT-3' 1.2.Preparation and transformation of the asporogenous strain Bt407-ΔsigE The mutant Bt 407-ΔsigE is prepared according to Bravo et al., 1996. The bacteria are grown in LB medium at 37°C until OD. 600nm= 1 and transformed by electroporation as described in Lereclus et al., 1989. 1.3. GFP production The asporogenous strain Bt407-ΔsigE was transformed with each of the two constructed plasmids, pHT304-18-Cal'orf2-gfp-Tcry1Ac and pHTCal'-gfp-Tcry1Ac (control plasmid), and then cultured in LB medium at 30°C. The culture was stopped 24 h after the start of culture and the cells fixed with formaldehyde, then observed under a fluorescence microscope (Figure 2). 2. Results Observation under a fluorescence microscope of the cells containing the control plasmid reveals a diffuse expression of GFP in the cells after 24 h of culture (Figure 2 a). Fluorescence microscopic observation of the Bt407-ΔsigE pHTCal'orf2-gfp-Tcry1Ac strain reveals the appearance of the beginning of the formation of GFP crystals or pseudo-crystals (Figure 2 b). The presence of ORF2 therefore makes it possible to produce GFP in a crystalline or pseudo-crystalline form.The co-transcription of the ORF2 protein and GFP genes into an operon is sufficient for the formation of these structures. Interestingly, when Bt407-ΔsigE-pHT-Cal'orf2-gfp-Tcry1Ac cells are centrifuged to stop their growth after 24 hours and a known crystallographic precipitant, PEG6000, is added at 20%, an increase in the size of crystals or pseudocrystals in the cells is observed (Figure 2 c). After 24 hours of culture in LB medium at 30°C, Bt407-ΔsigE-pHT-Cal'orf2-gfp-Tcry1Ac cells and control cells are broken using a French Press at 2 kbar pressure, and then the culture medium containing the broken cells is centrifuged to separate the soluble fraction from the insoluble fraction. 2 μg of these fractions are deposited on a 10% SDS-page gel (figure 3).GFP is found this time in the soluble fraction, meaning that the inclusions become soluble when the cells are broken in a buffer of 200 mM NaCl, 20 mM Tris pH 7.5. The same results are observed when the breaking medium is acidified to pH 5.5.

[0002] SEQUENCES SEQ ID N°1 – ORF2 de Bacillus thuringiensis MLKYHFPNVCEDELINIYSYGDFKGQGKYICLFKIENQSFLFWRNDKGNKIYTNLESISVEIINTNNTYN QSQNVCPQDLVDTYNQSQNVCPQDLVDTYNQSQNVCPQDLVDTYNQSQNVCPQDLVDTYNQSQ NVCPQDLVDTYNQSQNVCPQDLVDTYNQSQNVYTQDLIDTYNQSQNVCPQDLVDTYNQSQNVCP QDLVDTYNQSQNVCPQDLVDTYNQSQNVCPQDLNVYTQDLIDTYNQSQNCDCGCK SEQ ID N°2 ATGCTAAAATATCATTTTCCTAATGTATGTGAAGATGAATTAATTAATATATATTCTTATGGGGATT TTAAAGGACAAGGAAAATATATATGCCTCTTTAAAATTGAAAACCAATCATTCTTATTTTGGAGAA ACGATAAAGGAAATAAAATATATACAAATTTAGAATCTATCAGTGTAGAAATAATAAACACGAAT AATACGTATAATCAAAGTCAGAATGTATGCCCACAAGATTTAGTTGATACGTATAATCAAAGTCA GAATGTATGCCCACAAGATTTAGTTGATACGTATAATCAAAGTCAGAATGTATGCCCACAAGATTT AGTTGATACGTATAACCAAAGTCAGAATGTATGCCCACAAGATTTAGTTGATACGTATAATCAAA GTCAGAATGTATGCCCACAAGATTTAGTTGATACGTATAACCAAAGTCAGAATGTATGCCCACAA GATTTAGTTGATACGTATAACCAAAGTCAGAATGTATACACACAAGATTTAATTGATACGTATAAC CAAAGTCAGAATGTATGCCCACAAGATTTAGTTGATACGTATAACCAAAGTCAGAATGTATGCCC ACAAGATTTAGTTGATACGTATAACCAAAGTCAGAATGTATGCCCACAAGATTTAGTTGATACGTATAACCAAAGTCAGAATGTATGCCCACAAGATTTGAATGTATACACACAAGATTTAATTGATACGT ATAATCAAAGTCAGAATTGTGATTGTGGTTGTAAG SEQ ID N°3 - STAB-SD Bacillus thuringiensis gaaaggaggg atgcc SEQ ID N°4 - Tcry1Ac - Bacillus thuringi act ttaaatatcg ttttcaaatc aattgtccaa gagcagcatt acaaatagat aagtaatttg ttgtaatgaa aaacggacat cacctccatt gaaacggagt gatgtccgtt ttactatgtt attttctagt aatacatatg tatagagcaa cttaatcaag cagagatatt ttcacctatac gat tgactta SEQTTTTTTTTTTTTTT N°5 - PcalY Promoter region of the calY gene of Bacillus thuringiensis. 5'- AAGCAAGACTATATATTTACGAGTGTGAGGAACGTTAGCCCTCACCTTTGTTTTTCTTTT TTCTTATAGTTAATGATTTGAGTGAAAAAAGTTATATTATTTGTTTTTCTGAAAAG TCTAAATGATATTGAAATAAAATAACTGAAATTTAGTTTAGTTTAGTTTT GTTGTTCCGTTATAATGAACATAAGGTTTTTAAAAGAACACATATTAGCTAAGCTAATATAGTTT TCTTTACATCTCTTATTGAAAAATAAGTGATAAAAATAAAAAAAAGCTAGGGGGAATTTGATT-3' Underlined by a dash: region 10 (TATAAT) of the promoter. Highlighted by two dashes: the -35 (TTGTGT) region of the promoter. Hebold: the +1 of transcription determined by RACE PCR. SEQ ID N°6 - Promoter region of the inhA1 gene of Bacillus thuringiensis - PinhA1 AATTGTGATATATTCGTATGCTAACTATGAAATTTTTCAAAATATTAAATATTACATGATATG ACTAAATATTGAAAAAATATTGAATTTTTAATAATTCAATTTGTAATTTGATAGATTAGATTAGATGATG GAGGAAATATGGGATG Pces de SEQ ID N°7 AAGTAATCGTTGCCGTACCCGTATGAAATTGAAATAAAATTAATTTTAATTCTAATAATTCGT ATTGCGTGAATATTAATAATGTTTATAATGAAATTGTATAATTTGGTTAATTAACTTGTGCGAA GAAAAGTTAATAATGTTAAGGAAAATGAATGTTTTTGTATTTTACTTTTTTAAAAGAGGG SEQ ID N°8 Promoter region of the gene oppA of Bacillus thuringiensis - PoppA CTAGACCGTCAAAATATTACTAGAATTATTATACTATAAAAGCTATAATAAGTACTAGGATTAATT TTTTGAAAATTACGCAATTAAAGGTCTGATTTTAAGATGGTAGCAATGTTAATGTATCCCT TTACGAAAATTTTAAAATAATATTTTTAAAATTTTAACAAAAACAAAACTATATTCA CAATTGTTATATTATGTATAATGAAAATTTGAAACTTGGAGATTTTATTCAATTCTTTTCA TTAACAAGTGAGGGTACAGGAAGTGCAATTAGGGGAGG SEQ ID N°9 - Promoter region of Bacillus thuringiensis nppC gene - PnppCATTCCACTTTTATATAAAGATTTTTGAATATTTTCAACTTACCCTTGTCAAACATTGTCAATTT ATATTAAAATAACAACATACAAAATATTTAGTCTTTTCAGAAAAGATGGAGGGATAGCCATG SEQ ID N°10 - Promoter region of the papR gene of Bacillus thuringiRepensis - PparingiRepensis CTAGAACCATGAATTAAAAGAATCACTTATAAAAAATGAAGAAATAAAAAAGACATAAAGAA CAAATATGCATAATTGCATAAAGTCTGGATAATTTTCATGATATTTAAAGAAAAAATGCGG SEQ ID N°11 - Promoter region of the plcB gene of Bacillus thuringiensis - PplcB AGCATGTGTATGCTTTACTCTTTTTCATTTAGTTAGACAGCCTTAATAATAGTTTTAAAAT GAAAGTGTATTCATTCATTATTCACTGTGTATAAAGTTATAATGATATGAACATTTGCATATTTT AATTTTAGTGATAGAAATTTCGTGAAAGGTGGGATATTCTAGTCATAGGACCGGACCTAACCA TAGGATCCTAACAAAATGTTTACAATAATTCAATTATAAAATGGAGG SEQ ID N°12 - promoter region of the nprA gene of Bacillus thuringiensis PnprA TTAATAGTGGAAACATAATGCTAATATGAAACTACTCTTTTTCAAAAAAATTTTTATTAGGGGGAA GGTTCATG SEQ ID N°13 - promoter region of the inhA1 gene of Bacillus thuringiensis PnprRGAAGT ANDATIONS TO THE N° N° N°14 - PcalY14 - PcalY1 5'-GCTATAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG-3' - PcalY2 5'-GGCGCGCCCACAATCAATTCCCCCTAGCTTT-3' SEQ ID N°16 - Tcry1 5'-GGGGTACCAACTCAGGTTTAAATATCGTTTTC-3' SEQ ID N°17 - Tcry2 5'-GGAATTCCCAAATAAAAAAAGAAAGCAGRF SEQ ID N°18 - OQ18 5'-CATTTTATGGATCCCCCAAAGGATTTAACTATGCACGA-3' SEQ ID N°19 - ORF2(2) 5'-CATTCATGGTGTCACCTCCTTAAAATAAAGTTTTATATTAACTAAACTCTTTTGATA-3' SEQ ID N°20 - GFP1 5'-TATCAAAAGGTTTAGTTTTAATAAAACTTTATTTTTAAGGAGGTGACACCATGAATG-3' SEQ ID N°21 - GFP2 5'-ACCTGAGTTGGTACCTTAATGATGATGATGAGA-3' SEQ ID N°22 - LinP1 5'-GGTACACAATA SEQ ID-3'QTC-3' LinP2 5'-GGATCCATAAAATGATTTTTCAT-3' SEQ ID N°24 - PcalY1 oligo 5'-GCTCTAGAAAGCAAGACTAGTAATATTTACGAG-3' SEQ ID N°25 – PcalY2 oligo 5'-GGCGCCCACAATCAATTCCCCCTTT-3'QST SEQ ID N°16 5'-GGCGCGCCTCTTGAAAGGAGGGATGCCT-3' SEQ ID N°27 -StabSD2 5’-CGGGATCCATAAAATGATTTTTCATAAATC-3’ SEQ ID N°28 - GFP1 bis 5’- TCATTTTATGGATCCGAAATAAAATGCATCTGTATTTGAA-3’

Claims

CLAIMS 1. An expression cassette comprising: (i) a strong stationary-phase active promoter; (ii) a sequence having at least 80% identity with the SEQ ID No. 2 sequence of the Bt ORF2-encoding gene; and (iii) the sequence of a gene encoding a heterologous protein; said ORF2-encoding gene sequences and the heterologous protein sequence being in operon under the control of said strong promoter.

2. An expression cassette according to claim 1, characterized in that said strong stationary-phase active promoter is selected from PcalY, PinhA1, Pces, PoppA, PnppC, PpapR, PplcB, PnprA, and PnprR.

3. An expression cassette according to claim 1 or claim 2, characterized in that it further comprises an mRNA stabilizing sequence and / or a cry1Ac gene terminator sequence.

4. Expression cassette according to claim 3, characterized in that the mRNA stabilizing sequence is the STAB-SD sequence. 5.Recombinant plasmid comprising the expression cassette according to any one of claims 1 to 4.

6. Recombinant plasmid according to claim 5, characterized in that said plasmid is a plasmid with high segregational and / or structural stability allowing expression in bacteria of the genus Bacillus.

7. Recombinant plasmid according to claim 5 or claim 6, characterized in that said plasmid is selected from pHT3101, pHT304, pHT315, pHT370, pBC16, pE194, pC194, pHT8-1, pHT73 and pBMB299 or from a derived plasmid.

8. A transformed bacterium comprising a plasmid according to any one of claims 5 to 7.

9. A transformed bacterium according to claim 8, characterized in that it is an asporogenous bacterium of the genus Bacillus.

10. A transformed bacterium of the genus Bacillus according to claim 9, characterized in that the inactivated sporulation gene is Spo0A and said highly active stationary-phase promoter is selected from PpapR, PoppA, PnppC, PnprA, PnprR, and PplcB.

11. A transformed bacterium of the genus Bacillus according to claim 9, characterized in that the inactivated sporulation gene is sigE and said highly active stationary-phase promoter is selected from PcalY, PinhA1, and Pces.

12. A method for producing a heterologous protein comprising the steps of: - preparing a transformed bacterium according to any one of claims 8 to 11; - culture of said transformed bacterium in stationary phase; and - optionally, purification of said heterologous protein. 13.Use of a sequence having at least 80% identity with the SEQ ID No. 2 sequence encoding ORF2 of Bt in an expression cassette also comprising a strong stationary-phase active promoter and the sequence encoding a heterologous protein, for the production of said heterologous protein.

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