WO2009022162A1

WO2009022162A1 - Bacillus with inactivated or downregulated htra and/or htrb

Info

Publication number: WO2009022162A1
Application number: PCT/GB2008/002796
Authority: WO
Inventors: Rocky Cranenburgh; Colin Harwood
Original assignee: Cobra Biologics Limited; Newcastle University
Priority date: 2007-08-15
Filing date: 2008-08-15
Publication date: 2009-02-19
Also published as: GB0715936D0

Abstract

A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated; and a method for producing a target protein using said strain.

Description

BACILLUS WITH INACTIVATED OR DOWNREGULATED HTRA AND/OR HTRB

All documents cited herein are incorporated by reference in their entirety. TECHNICAL FIELD

The present invention relates to Bacillus species, especially Bacillus subtilis (B. subtilis). In particular, it relates to a Bacillus strain that is useful for the recombinant expression of a range of proteins.

In addition, the invention relates to protein expression systems. In particular, it relates to the use of the Bacillus strain of the invention for producing a target protein, e.g. Protective Antigen (PA) of Bacillus anthracis.

BACKGROUND ART

Protein expression systems based on recombinant micro-organisms are well known in the art. A preferred form of protein expression system utilises the secretion of recombinant protein from bacterial cells cultured in a liquid growth medium. These processes often enable an increased yield, purity and solubility of the recombinant protein compared to processes wherein the protein remains in the cytoplasm and is released by cell lysis.

The most commonly used bacterium for recombinant protein production is the Gram-negative Escherichia coli. However, this bacterium does not have an active secretion system suitable for secreting a range of recombinant proteins. An alternative bacterium is the Gram-positive B. subtilis, which is capable of secreting several grams per litre of protein into its growth medium. B. subtilis is a promising strain for biopharmaceutical manufacture, but the proteins secreted at high yields tend to be native proteins, or proteins from closely related species. When heterologous proteins are expressed in B. subtilis, the proteins are degraded by extracytoplasmic proteases.

Eight extracytoplasmic proteases are known to be responsible for much of the degradation that takes place in B. subtilis expression systems. These are the seven extracellular proteases NprB, AprE, Epr, Bpr, NprE, Mpr and Vpr, and the cell wall-associated protease WprA. The inactivation of genes encoding these proteases has been proposed for improving yield in recombinant protein expression systems (see, for example, [1] and [2]).

The expression of heterologous proteins in B. subtilis places the bacterium under considerable secretion stress. Two further extracytoplasmic proteases, HtrA (formerly YkdA) and HtrB (formerly YvtA), are upregulated under conditions of secretion stress and are thought to facilitate the processing, maturation and secretion of extracellular proteins [3]. The ability of these enzymes to increase levels of extracellular protein may be due, in part, to a chaperone-like ability of HtrA that facilitates protein folding [4]. The importance of HtrA and HtrB to B. subtilis physiology is further demonstrated by the effect of inactivating the two genes: double mutant strains grow extremely slowly and give rise to small, round colonies with a mucoid appearance and sickly phenotype [3]. There is a need in the art to develop further Bacillus strains, particularly B. subtilis strains, for use in protein expression systems. Preferably, these strains would give rise to higher yields of recombinant protein than strains developed to date. In particular, there is a need for Bacillus strains, particularly B. subtilis strains, that are capable of expressing heterologous proteins at high yields.

The present invention provides one or more of the above needs.

DISCLOSURE OF THE INVENTION

Bacillus strains and their production

In one aspect, the present invention provides a Bacillus strain in which genes encoding HtrA and/or HtrB, and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In other words, the present invention provides a Bacillus strain in which i) gene(s) encoding a) HtrA, b) HtrB or c) HtrA and HtrB, and ii) gene(s) encoding at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

Surprisingly, it has been discovered that strains lacking HtrA or HtrB are capable of secreting heterologous proteins, and are therefore useful in protein expression systems. This is despite the known role of these enzymes in protein secretion. This result is particularly surprising when the loss of HtrA or HtrB is combined with the downregulation or inactivation of genes encoding at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA. Bacillus strains with such multiple mutations have unpredictable viability, and are typically less viable than wild type strains.

In preferred embodiments of the strain of the present invention, the genes encoding HtrA and HtrB have been downregulated or inactivated. Accordingly, the present invention also provides a Bacillus strain in which genes encoding HtrA, HtrB and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

Surprisingly, it has been discovered that strains lacking both HtrA and HtrB can be produced that do not demonstrate the sickly phenotype observed in the prior art. This result is again particularly surprising when the loss of HtrA and HtrB is combined with the downregulation or inactivation of genes encoding at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA. Without wishing to be bound by theory, it is thought that the sickly phenotype can be avoided by carrying out the downregulation/inactivation of the genes encoding HtrA and HtrB after carrying out the downregulation/inactivation of the other genes.

As shown in the examples herein, strains of the present invention are capable of expressing heterologous proteins at surprisingly high yields. In specific embodiments of the strain of the present invention, the genes encoding HtrA and/or HtrB, and WprA have been downregulated or inactivated. In particular, the genes encoding HtrA and/or HtrB, WprA and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr and Vpr have been downregulated or inactivated.

In other specific embodiments of the strain of the present invention, the genes encoding HtrA and/or HtrB, and AprE have been downregulated or inactivated. In particular, the genes encoding HtrA and/or HtrB, AprE and at least one of NprB, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

In yet further specific embodiments of the strain of the present invention, the genes encoding HtrA and/or HtrB, and NprE have been downregulated or inactivated. In particular, the genes encoding HtrA and/or HtrB, NprE and at least one of NprB, AprE, Epr, Bpr, Mpr, Vpr and WprA have been downregulated or inactivated.

Alternatively, the genes encoding HtrA and/or HtrB, and NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and optionally WprA have been downregulated or inactivated.

Other combinations are also envisaged in the present invention, for example: a) A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least two of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any two of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE and NprE may have been downregulated or inactivated. Alternatively, AprE and WprA may have been downregulated or inactivated. Alternatively, NprE and WprA may have been downregulated or inactivated. These combinations are preferred because AprE, NprE and WprA are often particularly responsible for the degradation that takes place in expression systems. b) A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least three of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any three of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE, NprE and WprA may have been downregulated or inactivated. c) A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least four of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any four of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE, NprE, WprA and at least one of NprB, Epr, Bpr, Mpr and Vpr may have been downregulated or inactivated. d) A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least five of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any five of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE, NprE, WprA and at least two of NprB, Epr, Bpr, Mpr and Vpr may have been downregulated or inactivated. e) A Bacillus strain in which genes encoding HtrA and/or HtrB, and at {east six of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any six of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE, NprE, WprA and at least three of NprB, Epr, Bpr, Mpr and Vpr may have been downregulated or inactivated. f) A Bacillus strain in which genes encoding HtrA and/or HtrB and at least seven of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. In this embodiment, any seven of the genes encoding NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA may have been downregulated or inactivated. For example, AprE, NprE, WprA and at least four of NprB, Epr, Bpr, Mpr and Vpr may have been downregulated or inactivated.

In particularly preferred embodiments of the strain of the present invention, the genes encoding HtrA, HtrB, NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated. This strain has been found to be capable of expressing heterologous proteins at surprisingly high yields.

The sequences of the htrA, htrB, nprB, aprE, epr, bpr, nprE, mpr, vpr and wprA genes in B. subtilis (or their homologues in other Bacillus species) can be determined from the literature (see, in particular, [5] for B. subtilis; [6] and [7] for B. licheniformis; [8] and [9] for B. amyloliquefaciens; [10] and [11] for B. thuringiensis; [12] for B. cereus; [13] for B. clausii; [14] for B. coagulans; [15] and [16] for B. halodurans; and [17] for B. anthracis). Inactivation or downregulation of these genes may be carried out using any method known in the art. For example, the sequence of the gene may be partially or totally deleted, and additionally may be subject to allelic replacement, or may be subjected to mutation including insertional mutation in order to inactivate the encoded protein. Alternative methods of gene inactivation, such as the use of antisense RNA in order to prevent expression of the gene sequence or transposon mutagenesis in order to inactivate the gene, may also be employed. Typically, in the strain of the present invention, the genes have been downregulated or inactivated by being deleted (partially or totally).

In a particular method, the 5' and 3' regions flanking the target sequence to be deleted are amplified from Bacillus chromosomal DNA in two separate reactions. A first amplification reaction (for example a polymerase chain reaction (PCR) reaction) is carried out using 5XBA-5XHO and 3XHO-3XBA primer pairs. The two amplified flanks are then used to self-prime each other and generate the locus minus the target sequence. This truncate can then be amplified by external primers in a second amplification (PCR) reaction.

Alternatively, the two flanking products from the first reaction may be cut with a suitable restriction endonuclease, e.g. Xhol, mixed and ligated. Suitably, the primers are designed such that the 5' flank amplicon is up to 700 base pairs in length whilst the 3' flank is 800 or more base pairs in length. This allows clear identification of the ligation, e.g. using gel chromatography. When the product of the ligation is run on a gel, three bands will be obtained. The target band will be of the order of 1.5 kbases in length.

This scheme is illustrated diagrammatically in Figure 1.

The ligation obtained in this way, or the product of the second PCR reaction forms the deleted locus which can then be used to form a suitable deletion plasmid. For example, the deleted locus may be cut with a restriction enzyme such as Xbal and cloned into pUC18. Suitable hosts such as E. coli DH5α can be used as the host for the initial cloning, although TG90 (which replicates high-copy number plasmids at low copy number) may be useful in some instances.

This is then subcloned into a suitable plasmid to form a deletion plasmid.

A particularly suitable plasmid is pORI240 (see Figure 2), which has been shown to work effectively in B. subtilis ([18]) and will allow unlabelled gene deletions or replacements to be made in bacterial chromosomes. It is derived from the lactococcal plasmid pWVOl, which lacks the repA gene (replication initiation protein), so will only replicate in strains expressing RepA in trans, such as E. coli EClOOO. It contains the tetracycline resistance gene (teή to enable selection of recombinants and the galactosidase gene (lacZ) to allow identification of revertants by blue/white selection. The lacZ is under the control of a p32 promoter (allowing expression in both Gram-positive and Gram- negative bacteria).

The deletion locus can then be subcloned into the pORI240 plasmid and transformants selected in a suitable host strain such as E. coli EClOOO by tetracycline resistance. Strains of Bacillus, particularly strains of B. subtilis such as 168, can then be transformed using this plasmid, and transformants selected on tetracycline. The plasmid is unable to replicate in this strain, so transformants will have undergone chromosomal integration.

Then a second recombination event (resolving the cointegrant) which will yield white colonies with X-gal present (integrants will remain blue) can be readily selected.

Figure 2 illustrates the strategy for gene deletion using pORI240, where the * symbol represents the deletion of the target gene (adapted from [18]). Using this method, either the wild type gene is restored, or the deleted copy is inserted in its place upon resolution of the integrated plasmid. Revertant clones can be screened out genotypically by PCR to confirm the deletion. Loss of protease activity may be confirmed phenotypically using Zymogram gel analysis (Novex) to confirm the loss of protease activity.

Other strategies for producing gene deletions can be employed. For example, the Xer-cise system ([19] and [20]) can be used. The Xer-cise system involves a double crossover homologous recombination event to direct gene deletion by antibiotic selection, followed by subsequent removal of the antibiotic resistance gene by site-specific recombination. Xer-cise uses a linear DNA cassette comprising an antibiotic resistance gene flanked by dif sites (recognition sequences of the native Xer site-specific recombinases; RipX and CodV in the case of B. subtilis), which is in turn flanked by sequence 5' and 3' of the target gene to be deleted (i.e. a deletion locus). Following transformation, the targeted gene is deleted by homologous recombination and the deletion mutants are selected by antibiotic resistance. Subsequent culture in the absence of antibiotic enables survival of cells that have undergone Xer-mediated site-specific recombination to remove the antibiotic resistance gene. The mechanism of gene deletion by Xer-cise is illustrated in Figure 3. A suitable method of deleting genes in B. subtilis using the Xer-cise system is given in Example 1.

In another aspect, the present invention therefore provides a method of producing a modified Bacillus strain comprising the step of downregulating or inactivating genes encoding HtrA and/or HtrB, and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA in a Bacillus strain. This method may be adapted to produce any of the strains of the invention referred to infra by downregulating or inactivating the mentioned genes in those strains. Preferably, the downregulation/inactivation of the gene(s) encoding HtrA and/or HtrB is carried out after the downregulation/inactivation of the other gene(s).

In preferred embodiments, the strain is asporogenic. The inability to form resistant spores avoids the possibility of environmental contamination when the strain is grown to large numbers, e.g. in a fermenter. Spore formation is regulated by a complex network of genes which function as a cascade [21]. Targeted downregulation or inactivation of sporulation genes, such as spoOA ([22]), spollAC ([1J), ftsA ([23]), spoIIIG ([24]) and/or spoIIIC ([24]) genes in B. subtilis (or their homologues in other Bacillus species), will result in a strain incapable of forming spores. Accordingly, in preferred embodiments of the strain of the present invention, at least one sporulation gene selected from the group consisting of spoOA, spollAC, ftsA, spoIIIG and spoIIIC has been downregulated or inactivated such that the strain is asporogenic. Preferablyy/X/4 or spollAC has been downregulated or inactivated such that the strain is asporogenic. Suitable methods for the downregulation or inactivation of these genes are described above. In particular, a suitable method for the downregulation or inactivation of spollAC is described in [I]. Typically, in the strain of the present invention, the sporulation gene(s) have been downregulated or inactivated by being deleted (partially or totally). The strain of the present invention may be a recombinant strain derived from any suitable Bacillus species. For example, the Bacillus species may be selected from the group consisting of B. subtilis, B. licheniformis, B. amyloliquefaciens, B. thuringiensis, B. cereus, B. clausii, B. coagulans, B. halodurans and B. anthracis.

The strain of the present invention is most preferably a B. subtilis strain. Preferably, the strain is derived from a strain of known provenance, such as B. subtilis 168 (the strain used in the complete genome sequencing project (see [5]), obtainable from the Institut Pasteur (CIP no. 106309)). Another preferred strain is B. subtilis natto.

The strain of the present invention may be transformed using conventional methods, so that it expresses a gene of choice, and in particular a heterologous gene. The strain can be used as a expression vehicle for a wide range of proteins, although it is particularly suited to the expression of antigens or proteins useful as vaccines such as PA of B. anthracis, or immunogenic fragments or domains or variants thereof. It may be particularly useful where more conventional expression hosts, such as E. coli, are unsuited to expression of a particular target gene, e.g. for reasons of toxicity of the target protein in that host.

Thus, in a preferred embodiment, the invention provides the strain as described above, wherein the strain is transformed with a gene for expression of a heterologous gene product. The gene may encode a wide range of desired proteins, for example pharmaceutical or agrochemical proteins or peptides, or other proteins which have commercial applications such as enzymes, for example cellulases and amylases, used in paper manufacture or detergent manufacture. It is specifically envisaged that the gene may encode a protein selected form the group consisting of antibodies, antigens, cytokines, hormones, enzymes, receptor proteins, receptor-binding proteins, structural proteins, membrane proteins, cell wall proteins, peptides, antibiotics, antivirals, DNA-binding proteins and heat shock proteins. Preferred pharmaceutical proteins that may be encoded by the gene include immunogenic proteins or peptides such as antigens, intended for use as a vaccine. Indeed, it is specifically contemplated that the heterologous gene encodes a protein for raising an immune response in a subject, e.g. a mammal, preferably a human. Other pharmaceutical proteins that may be encoded by the gene include hormones such as human growth hormone. Another pharmaceutical protein that may be encoded by the gene is an immunoglobulin, or a fragment thereof.

Typically, the strain will be transformed with a plasmid comprising the gene. The plasmid may be maintained within the strain in in vitro culture by the method of "operator repressor titration", as described in [25]. This method involves engineering a host cell, such that it contains a first chromosomal gene encoding a repressor and a second chromosomal gene essential for cell growth that has an operator sequence for the repressor in its control region. In the absence of a plasmid, expression of the second chromosomal gene is inhibited by binding of the repressor to the operator and the cell dies. The plasmids for maintenance in this host cell are engineered to contain the operator sequence such that in the presence of the plasmid, the repressor is titrated away from the operator for the gene essential for cell growth, the gene is expressed and the cell survives. This mechanism is also described in [26] and [27]. The plasmid may also be maintained within the strain by the method described in [28]. This method involves engineering a host cell, such that it contains a chromosomal gene that inhibits cell growth. The plasmids for maintenance in this host cell are engineered to encode an antisense sequence that inhibits the action of the chromosomal gene. Accordingly, the absence of a plasmid results in expression of the chromosomal gene and inhibited cell growth, while the presence of a plasmid results in cell growth. It is therefore preferred for the Bacillus strains of the present invention to additionally comprise the features of the host cells described in [25] and [28], particularly the features described in the claims thereof.

Preferably, the gene for expression of a heterologous gene product encodes PA of B. anthracis or an immunogenic fragment or domain thereof, or a variant of any of these.

As used herein, the term "variant" refers to sequences of amino acids which differ from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for other amino acids. Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. "Non- conservative" substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide. Suitably variants will be at least 60% identical, preferably at least 75% identical, and more preferably at least 90% identical to the base sequence. In particular, the identity of a particular sequence to the sequence on which they are based may be assessed using the multiple alignment method described by Lipman and Pearson [29]. The "optimised" percentage score should be calculated with the following parameters for the Lipman-Pearson algorithm: ktup =1, gap penalty =4 and gap penalty length =12. The sequences for which similarity is to be assessed should be used as the "test sequence" which means that the base sequence for the comparison, such as the sequence of PA of B. anthracis should be entered first into the algorithm.

The term "fragment" as used herein refers to truncated regions which lack one of more amino acids as compared to the full length sequence. The fragment may comprise a domain. For example, particular protective domains of the PA of B. anthracis comprise domains 1 or 4 of the full length sequence, or protective regions of these domains. Domain 1 comprises amino acids 1-258 of full- length PA, while domain 4 comprises amino acids 596-736. These amino acids numbers refer to the sequence as shown in [30]. Domain 1 comprises two regions, designated Ia and Ib. Region Ia comprises amino acids 1-169 whereas region Ib is from amino acid 170-258. It appears that region Ia is important for the production of a good protective immune response. In a particularly preferred embodiment, a combination of domains 1 and 4, or protective regions thereof, are used as the immunogenic reagent which gives rise to an immune response protective against B. anthracis. This combination, for example as a fusion peptide, may be expressed using the expression system of the invention.

In some embodiments, the strain of the present invention does not comprise a chromosomally integrated antibiotic resistance gene.

Methods for producing a target protein

In a further aspect, the invention provides a method for producing a target protein, said method comprising transforming a strain of the invention with a nucleotide sequence which encodes said protein, culturing said transformed strain and recovering said target protein from the culture.

The invention also provides a method for producing a heterologous gene product, said method comprising culturing a strain of the invention transformed with a gene for expression of a heterologous gene product, and recovering said gene product from the culture.

Conventional methods, such as are generally used for the transformation and culture of microorganisms are suitably employed. In particular, the strains of the present invention can be grown in a range of media with suitable aeration, for example in a fed-batch fermentation.

General

The term "comprising" encompasses "including" as well as "consisting of e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The term "strain", as used herein, refers to a single cell or a culture of cells. The culture of cells comprises bacteria of a particular species having common characteristics. Typically, the culture of cells will be homogeneous, i.e. composed of the descendants of a single bacterium.

The terms "downregulated", "downregulating" and "downregulation", as used herein with reference to a gene in a Bacillus strain of the invention, refer to the reduction of expression of said gene relative to the level of expression of that gene in the corresponding, unmodified Bacillus strain under identical conditions. Similarly, the terms "inactivated", "inactivating" and "inactivation", as used herein with reference to a gene in a Bacillus strain of the invention, refer to the complete prevention of expression of said gene.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 illustrates the assembly of the gene deletion loci using PCR. The deletion loci for nprB, aprE, epr, bpr, nprE and mpr were assembled using splicing PCR or ligation as shown.

Figure 2 illustrates gene deletion using single crossover recombination (from [18]). This technique was used to delete nprB, aprE, epr and bpr from B. subtilis strain 168 to create strain 168RMC-04.

Figure 3 illustrates gene deletion using Xer-cise. This technique was used to delete mpr, nprE, vpr, wprA, htrA mά htrB from 168RMC-04 to create strain 168JH-10. Figure 4 provides a summary of the mutants described herein. Figure 5 illustrates the PA expression plasmid pHT28pagA.

Figure 6 comprises two western blots of the culture media of various B. subtilϊs strains, showing the level secretion of recombinant PA from these strains. A) lane 1 : 168; lane 2: 168AB-06; lane 3: 168AB-07; lane 4: 168JH-10; lane 5: recombinant PA protein reference; B) lane 1: 168AB-06; lane 2: 168AB-07; lane 3: 168JH-08; lane 4: 168JH-10; lane 5: recombinant PA protein reference. The western blots were probed with an anti-PA antibody.

MODES FOR CARRYING OUT THE INVENTION

Example 1

Generating the gene deletion mutant strains

To create the gene deletion loci for nprB, aprE, epr and bpr, the genes and flanking regions were amplified from chromosomal DNA using the proximal and distal primers of each set of four described in Table 1 (i.e. the ' 5XBA' and '3XBA' pairs). These PCR products were used as templates for amplification by PCR of the 5' and 3' flanks of the genes. 'Splicing PCR' was used to join these flanking PCR products for aprE, epr, bpr and mpr, whereby the homology between the 5' ends of the internal ('XHO') primers enabled the two resulting PCR products to self-prime in a second PCR (Figure 1). This method was unsuccessful for nprB, so an alternative method was used: cutting the two flanking PCR products at the introduced Xhol restriction site and ligating to assemble the deletion locus. Whether generated by splicing PCR or by ligation, each gene-deleted locus was cloned into plasmid pGEM-T Easy (Promega) as an Xbal restriction fragment.

The deletion loci for nprB, aprE, epr and bpr were then cloned into pORI240 (generating plasmids pORlΔnprB, pORIΔaprE, pORIΔepr and pORIΔbpr), which allows unlabelled gene deletions or replacements to be made in bacterial chromosomes ([18]). It is derived from the lactococcal plasmid pWVOl, which lacks the repA gene, (replication initiation protein), so will only replicate in strains expressing RepA in trans, such as E. coli EClOOO. The tet (tetracycline resistance) gene enables selection of recombinants, and lacZ (/3-galactosidase gene) allows identification of revertants by blue/white selection. lacZ is under the control of a p32 promoter, allowing expression in Gram- positive and negative bacteria. A second recombination event (resolving of the cointegrant) resulted in white colonies with X-gal present, with either i) the wild-type gene restored, or ii) the required gene deletion. Figure 2 shows gene deletion using pORI240, with the * symbol representing the deletion of the target gene (adapted from [18]). Using this method, either the wild type gene is restored, or the deleted copy inserted in its place upon resolution of the integrated plasmid. PCR was used to identify the deletion mutants. Table 1. Primers used in the construction of the gene deletion and expression plasmids

Gene deletions were achieved by making competent cells of B. subtilis 168 (following a protocol provided by F. Kunst, Institut Pasteur, Paris, France) and transforming with the covalently closed circular plasmids. B. sub tills 168 gene deletions were carried out in the order oϊ nprB, aprE, epr and bpr using plasmids based on pORI240, resulting in the quadruple protease deletion mutant 168RMC- 04. However, further attempts at deleting mpr and nprE using equivalent plasmids (construction not described) were unsuccessful using this technology, so an alternative method, Xer-cise, was applied.

The mpr and nprE deletion plasmids (pmpr-DifCAT and pnprE-DifCAT) were constructed as described in [19] and used to delete nprE and then mpr from 168RMC-04 as described above to create 168AB-06. To delete vpr, the primers SacTF and ywcHR were used to amplify vpr and its flanking regions from B. subtilis 168 gDNA, and this PCR product was cloned into plasmid pCR2.1- TOPO. The resulting plasmid, pTOPO-vpr, was cut with Ndel (blunted) and BspDI, and the dif- flanked chloramphenicol resistance gene (excised using HpaII and BfrBI) from pTOPO.bac-DifCAT ([19]) was 1 igated in to create the knockout plasmid pvpr-DIFCAT. This was linearised and used to transform 168AB-06 to delete vpr and generate 168AB-07, a strain with all the extracellular proteases deleted.

Deletion cassettes for wprA, htrA and htrB were synthesised and supplied in plasmid pPCR-Script (by Geneart, Regensburg, Germany) with two-200 bp flanking regions separated by 18 bp containing Agel and Nsil sites (Table X). The ^/-flanked chloramphenicol resistance gene from pTOPO.bac- DifCAT was then excised using Agel and Nsil and cloned into the Agel and Nsil sites of each deletion loci plasmid to create the Xer-cise deletion plasmids (pDwprA-DifCAT, pDhtrA-DifCAT, pDhtrB-DifCAT). The final mutant strain with ten genes deleted, 168JH-10, was generated from 168AB-07 by sequential deletion of wprA, htrA and htrB using these plasmids (Figure 4). Additionally, a strain with nine genes deleted, 168JH-AB, was created that retained functional wprA.

Table 2: Sequences synthesised as deleted loci (418 bp each). The region containing the Agel and Nsil sites is underlined.

Regardless of the method of gene deletion used, all deletion loci were identified by PCR and confirmed by DNA sequencing (Table 3). Table 3: Nucleotide sequences of the gene-deleted regions in the mutant strain chromosomes. Sequences are 100 bp flanking the 28 bp dif or 6 bp Xhol sites displayed in bold (the 'XhoF site in aprE was erroneously designed as GAGCTC). Introduced sequence is underlined.

Example 2

Construction of expression plasmid

The model protein chosen for expression studies was the recombinant protective antigen (PA) from

Bacillus anthracis ([3 I]). This is a component of the anthrax toxin (together with the lethal and oedema factors), but on its own it provides protective immunity against anthrax. An expression plasmid was created to produce secreted PA based on pHTOl (Mobitec, Germany) in which pagA expression was controlled from the P_grac promoter. Firstly, the 28 amino acid secretion signal sequence from the Bacillus licheniformis α-amylase was synthesised and cloned into the BamHI- Xbal sites of pHTOl, with a Pstl site incorporated into the 3' end between the BamHI and Xbal sites. The PA gene pagA was amplified in two separate PCRs using primer combinations S-pag28one/AS- pagone and S-pag28two/AS-pagtwo. The PCR products were then pooled, denatured briefly at 95 ⁰C, allowed to cool and therefore reanneal, and cloned into the /VKYZ>αI-cut plasmid to create pHT28pagA. The primers are displayed in Table 1, and Figure 4 shows the pHT28pagA plasmid with the sequence of the synthesised α-amylase signal sequence and pagA detailed. Example 3

Secretion of recombinant PA

To demonstrate the secretion of recombinant PA from B. subtilis, strains 168, 168AB-06, 168AB-07,

168JH-08 and 168 JH- 10 were transformed with pHT28pagA. Two separate growth studies were performed (A and B), with the inclusion of 168 as a control in A and 168JH-08 in B. Transformants were cultured overnight (10 ml culture in LB broth at 37 ⁰C and 200 rpm) and inoculated into 50 ml of medium in shake flasks at a dilution of 1/100. These were induced by the addition of 0.5 mM IPTG at inoculation. Culture samples were taken at hourly intervals thereafter, centrifuged to generate a cell pellet, and the cell-free supernatant frozen and stored at -80 ⁰C. These samples were thawed and 16 μl subjected to polyacrylamide gel electrophoresis (12 % acrylamide/bis-acrylamide in MOPS buffer). The gel was then western blotted: transferred to a nitrocellulose membrane, probed first with an anti-PA antibody, then a secondary alkaline phosphatase-conjugated rabbit anti-mouse antibody (AbCam), and developed using NBT/BCIP solution (Sigma) (Figure 6).

The western blot revealed that in the wild-type strain 168, there was significant degradation of the full-length PA. With the deletion of six (168AB-06) or all seven (168AB-07) of the extracellular proteases, degraded PA was still present. The subsequent deletion of wprA in 168JH-08 resulted in higher molecular mass degradation products of PA. By additionally deleting htrA and htrB to generate 168JH-10, virtually no degraded products are generated, thus increasing the yield and purity of the secreted recombinant protein.

It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.

REFERENCES (the contents of which are hereby incorporated by reference)

[I] WO 03/006649

[2] Wu et al Appl Environ Microbiol. 2002 68(7):326 L-9 [3] Noone et al J Bacterid. 2001 183(2):654-63 [4] Antelmann et al MoI Microbiol. 2003 49(1): 143-56 [5] Kunst et al Nature. 1997 390(6657):249-56 [6] Genbank accession number: NC_006270 [7] Veith et al J MoI Microbiol Biotechnol. 2004;7(4):204- l 1 [8] Genbank accession number: NC_009725 [9] Chen et al Nat. Biotechnol. (2007) In press [10] Genbank accession number: NC_005957 [1 1] Han et al J Bacteriol. 2006 188(9):3382-90 [12] Genbank accession number: NZ_ABDM01000005 [13] Genbank accession number: NC_006582 [14] Genbank accession number: NZ_AAWV01000084 [15] Genbank accession number: NC_002570 [16] Takami et al Nucleic Acids Res. 2000 28(21):43 17-31 [17] Genbank accession number: NZ_ABDN01000001 [18] Leenhouts et al MoI Gen Genet. 1996 253(l-2):217-24 [19] Bloor and Cranenburgh Appl Environ Microbiol. 2006 72(4):2520-5 [20] WO 2006/003412

[21] Hoch spoO genes, the phosphorelay, and the initiation of sporulation. In Bacillus and other Gram- positive bacteria. Edd. Sonenshein el al, chapter 51:747-755, American Society for Microbiology, Washington D. C

[22] Piggot and Hubert Curr Opin Microbiol. 2004 7(6):579-86 [23] Kemp et al J Bacteriol. 2002 184(14):3856-6 [24] Kim and Kim Biotechnol. Lett. 2001 23:999-1004 [25] WO 97/09435

[26] Williams et al Nucleic Acids Research 1999 26(9): 2120-2124 [27] Cranenburgh et al Nucleic Acid Research 2001 29(5): e26-e27 [28] WO 2005/052167

[29] Lipman and Pearson Science. 1985 227(4693): 1435-41 [30] Welkos et al Gene 1988 69(2):287-300 [31] Baillie et al J Appl Microbiol. 1998 May;84(5):741-6

Claims

1. A Bacillus strain in which genes encoding HtrA and/or HtrB, and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

2. The strain according to claim I, wherein said genes encoding HtrA, HtrB and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

3. The strain according to claim 1 , wherein said genes encoding HtrA and/or HtrB, and WprA have been downregulated or inactivated.

4. The strain according to claim 1 , wherein said genes encoding HtrA and/or HtrB, and AprE have been downregulated or inactivated.

5. The strain according to claim 1 , wherein said genes encoding HtrA and/or HtrB, and NprE have been downregulated or inactivated.

6. The strain according to claim 2, wherein said genes encoding HtrA, HtrB, WprA and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr and Vpr have been downregulated or inactivated.

7. The strain according to claim 2, wherein said genes encoding HtrA, HtrB, NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and optionally WprA have been downregulated or inactivated.

8. The strain according to claim 2, wherein said genes encoding HtrA, HtrB, NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA have been downregulated or inactivated.

9. The strain according to any one of the preceding claims wherein said genes have been deleted.

10. The strain according to any one of the preceding claims wherein the strain is asporogenic.

1 1. The strain according to claim 10, wherein at least one sporulation gene selected from the group consisting of spoOA, spoIIAC, ftsA, spoIIIG and spoIIIC has been downregulated or inactivated such that the strain is asporogenic.

12. The strain according to claim 1 1 , wherein said gene(s) have been deleted.

13. The strain according to any one of the preceding claims, wherein the strain is a Bacillus subtilis strain.

14. The strain according to claim 13, wherein the strain is derived from Bacillus subtilis strain 168.

15. The strain according to any one of the preceding claims, wherein the strain is transformed with a gene for expression of a heterologous gene product.

16. The strain according to claim 15 wherein said gene encodes a protein for raising an immune response in a subject.

17. The strain according to claim 16 wherein said gene encodes Protective Antigen of Bacillus anthracis or an immunogenic fragment or domain thereof, or a variant of any of these.

18. A method for producing a target protein, said method comprising transforming a strain according to any one of claims 1 to 14 with a nucleotide sequence which encodes said protein, culturing said transformed strain and recovering said target protein from the culture.

19. A method for producing a heterologous gene product, said method comprising culturing a strain according to any one of claims 15 to 17, and recovering said gene product from the culture.

20. A method of producing a modified Bacillus strain comprising the step of downregulating or inactivating genes encoding HtrA and/or HtrB, and at least one of NprB, AprE, Epr, Bpr, NprE, Mpr, Vpr and WprA in a Bacillus strain.

21. The method of claim 20, wherein the modified Bacillus strain is a Bacillus strain according to any of claims 1 to 17.

22. The method of claim 20 or claim 21, wherein the downregulation or inactivation of said gene(s) encoding HtrA and/or HtrB is carried out after the downregulation or inactivation of said other gene(s).