F0RM2
THE PATENTS ACT, 1970
(39 of 1970)
COMPLETESPECIFICATION (See section 10 and rule 13)
High LeveJ Expression of Recombinant toxin protein CRM197 From Bacillus
We, Biological E Limited,
having office at Lakshmi Building, 3rd floor, Room Nos. 45 & 46, Sir P.M. Road, Fort, Mumbai 400 001, Maharashtra - India
The following specification described the invention and the manner in which it is to be performed :

Field Of The Invention
The present invention relates to the field of high level expression of recombinant microorganisms, more particularly recombinant toxin protein production in bacterial hosts, particularly are disclosed that have been engineered to express CRM197.
In particular, the present invention relates to Gram-positive microorganisms, such as Bacillus species having enhanced expression of CRM197, wherein one or more chromosomal genes of the Bacillus have been replaced with CRM197 gene.
Background Of The Invention
Microbial toxin proteins are used in medicine, as immunogens for vaccination against the toxin-producing microbe and as carrier proteins and adjuvants for other vaccines.
The naturally occurring, or wild-type. Diphtheria toxin may be obtained from toxin producing strains available from a variety of public sources including the American Type Culture Collection. A plasmid system for producing CRM197 protein in C. diphtheriae is described by, e.g., U.S. Pat. No. 5,614,382, "Plasmid for Production of CRM Protein and Diphtheria toxin,'' incorporated herein by reference in its entirety.
Therefore, The Microbial toxin proteins are used in medicine, as immunogens for vaccination against the toxin-producing microbe and as carrier proteins and adjuvants for polysaccharide based vaccines, and in scientific research as tools for studying molecular pathways.
Diphtheria toxin (DT) is a protein that is synthesized and secreted by toxigenic strains of Corynebacterium diphtheriae. Toxigenic strains contain a bacteriophage lysogen carrying the toxin gene. DT is synthesized as a 535-amino-acid polypeptide, which undergoes proteolysis to form the mature toxin. The mature toxin comprises two subunits, A and B, joined by a disulfide bridge. The B subunit, formed from the C-terminal portion of intact DT, enables binding and entry of DT through the cell

membrane and into the cytoplasm. Upon cell entry, the enzymatic A subunit, formed from the N terminal portion of intact DT, catalyzes ADP ribosylation of Elongation Factor 2 (EF-2). As a result, EF-2 is inactivated, protein synthesis stops, and the cell dies. Diphtheria toxin is highly cytotoxic; a single molecule can be lethal to a cell, and a dose of 10 ng/kg can kill animals and humans.
The CRM197 protein is a nontoxic, immunologically cross-reacting form of DT. It has been studied for its potential use as a DT booster or vaccine antigen. CRM197 is produced by C. diphtheriae that has been infected by the nontoxigenic phage β197tox—created by nitrosoguanidine mutagenesis of the toxigenic corynephage p. The CRM197 protein has the same molecular weight as DT but differs by a single base change (guanine to adenine) in the A subunit. This single base change results in an amino acid substitution (glutamic acid for glycine) and this point mutation results in a significant loss of toxicity.
The gene for CRM197 has a single base substitution, resulting in the incorporation of glutamic acid instead of glycine at residue 52. (Bishai, et al., 1987, "High-Level Expression of a Proteolytically Sensitive Diphtheria toxin Fragment in Escherichia coli," J. Bact. 169(11):5140-51, Giannini, et al., 1984, "The Amino-Acid Sequence of Two Non-Toxic Mutants of Diphtheria toxin: CRM45 and CRM197," Nucleic Acids Research 12(10): 4063-9, and GenBank Ace. No. 1007216A, all incorporated herein by reference.)
Conjugated polysaccharide vaccines that use CRM197 as a carrier protein have been approved for human use. These include: Menveo® (Novartis Vaccines and Diagnostics), a vaccine indicated for preventing invasive meningococcal disease caused by Neisseria meningitidis subgroups A, C, Y, and W-135; Menjugate (Novartis Vaccines), a meningococcal group C conjugate vaccine; and Prevnar® (Wyeth Pharmaceuticals, Inc.), a childhood pneumonia vaccine that targets seven serotypes of Streptococcus pneumoniae, and HibTITER® (Wyeth), a Haemophilus influenzae type b vaccine. In addition, CRM197 has potential use as a boosting antigen for C. diphtheria vaccination and is being investigated as a carrier protein for use in other vaccines.

Producing CRM197 in amounts sufficient to meet expanding needs has presented significant challenges. Various strategies were used to produce CRM197 by recombinant technology using E. coli, B. subtilis, Pseudomonos etc. The low yields of CRM197 in active form are due to degradation, improper folding, or both, depending on the specific characteristics, e.g., size and secondary structure, of the toxin. When expressed in higher levels, purification and in biologically active form poses challenge. Therefore, methods for producing large amounts of CRM 197, in soluble and active form and at low cost is highly needed.
Zhou et al., (J Tongji Med Univ. 1999;19(4):253-6) disclose cloning of 3 diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-E148S-K516A-F530A in B. Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS 118, but higher yield of 7.1 mg/L was observed in SMS300 compared with 2.1 mg/L in SMS 118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be degraded into two peptides of 37 ku and 21 ku.
US patent publication No. 2012/0128727 disclose production of proteins of pharmacological interest by means of artificial gene sequences, said sequences being inserted in expression vectors, the over-expression of the corresponding proteins in micro-organisms converted with said expression vectors, and a method for isolating the proteins expressed; in particular, it relates to the construction of an artificial gene encoding CRM197 as a whole and its derivatives, to the expression of CRM197 and its derivatives in Escherichia coli, and to a method for the isolation and purification of the protein CRM197.
US patent publication No. 2008/0193475 disclose a fermentation process for producing diphtheria toxin or a mutant thereof, in particular CRM197comprising a fermentation step of growing a strain of Corynebacterium diphtheria in medium in a fermenter under conditions of agitation sufficient to maintain a homogenous culture and limited aeration such that p02 within the culture falls to less than 4% for the majority of the fermentation step.

US patent No. 8,530,171 describes a method for producing a recombinant toxin protein in a Pseudomonad host cell, said method comprising: ligating into an expression vector a nucleotide sequence encoding the toxin protein; transforming the Pseudomonad host cell with the expression vector; and culturing the transformed Pseudomonad host cell in a culture media suitable for the expression of the recombinant toxin protein; wherein the recombinant toxin protein is CRM 197, and wherein the recombinant protein is produced at a yield of soluble or active CRM197 protein of about 0.2 grams per liter to about 12 grams per liter.
WO 2011/042516 describes improved process for making a bacterial toxin by periplasmic expression comprising the steps of a) growing a culture of the bacterial host cell containing an expression vector in which particular signal sequences are linked to the sequence of a bacterial toxin and b) inducing expression of the polypeptide containing particular signal sequences linked to a bacterial toxin such that a bacterial toxin is expressed periplasmically.
However, above said methods, produce CRM 197 in an inactive form which needs further processing steps like solubulization, refolding in addition to the occurrence of un processed CRM 197 (CRM with leader peptide). Therefore, a method for economically producing CRM197 would greatly facilitate vaccine research and manufacture.
Objective of the invention
The main objective of the present intention is to CRM 197 as mentioned in pure form with high yield.
Summary Of The Invention
The present invention relates to a method for producing a recombinant toxin protein in a Bacillus host cell, said method comprising: ligating into a vector a nucleotide sequence encoding a toxin protein; transforming the Bacillus host cell; and culturing the transformed Bacillus host cell in a culture media suitable for the expression of the recombinant toxin protein CRM197.

Brief description of the drawings
Fig.l Shows the complete CRM197 Amino Acid and DNA Sequences.
A. Amino acid sequence (SEQ ID NO: 1). B. An optimized DNA sequence (SEQ ID NO:2) encoding the CRM197 protein, This optimized sequence is a non-limiting example of an optimized sequence useful in the methods of the present invention. Fig.2 Shows SDS PAGE expression and Immuno analysis of CRM CRM produced by genetically engineered B. subtilis by SDS-PAGE and Western Blot. After cell growth, the cells were pelleted down by centrifugation. The culture supernatant (20 ul) is analyzed by SDS-PAGE. The gel is stained by silver stain to visualize the bands. B. subtilis-expressed CRM197 migrated as a single band at "58 kDa on SDS-PAGE. After SDA-PAGE, the proteins were transferred to nitrocellulose membrane and the expression was detected using Polyclonal CRM specific antibodies. Lane 3 shows expression of CRM by Bacillus subtilis.
Fig.3 Shows Size exclusion HPLC profile of Purified CRM 197 obtained from Bacillus
The purified CRM197 subjected to size exclusion HPLC (SEC-HPLC) to determine
the size and presence of any aggregates. Fig 4 shows a clear single peak (monomer)
with RT of about 8.6 min (similar to CRM197 standard sample) without any
aggregates.
Fig.4a and 4b Shows RP HPLC profiles of Purified CRM 197 obtained from
Bacillus
The purified CRM197 subjected to RP HPLC (RP-HPLC) to determine the presence
of CRM as well as aggregate formation. Fig 5b shows a clear peak of Bacillus CRM
197 was matching with retention time of about 18.4 min of standard CRM197
(Sigma) without any aggregates (Fig 5a).
Fig.5 Shows DNA cleavage activity of CRM197
To determine that the extra cellularly produced CRM 197 by Bacillus is properly
folded, we determined the nuclease activity which is the characteristic feature of
CRM 197.
Fig 5 shows a clear hollow around the well of agar plate indicating the DNAse
activity (well no. 1 & 2). For comparison, standard CRM 197 supplied by Sigma
Aldrich was used as control (well 3).

Fig.6 Shows N terminal Amino acid analysis
To determine amino acid sequence similarity with standard CRM by N-terminus amino acid, automated sequencing was performed in Procise 492 system with "Modi_Pulsed liquid PVDF52B" method as per the standard operating procedure. Estimated sequence is matching with standard CRM 197 amino acid sequence.
Detailed Description Of The Invention
The present invention relates to a high level expression of recombinant toxin protein CRM 197 and method of producing from Bacillus thereof, for producing a recombinant toxin protein in a Bacillus host cell, said method comprising: ligating into a vector a-nucleotide sequence encoding a toxin protein; transforming the Bacillus host cell; followed by culturing the transformed Bacillus host cell in a culture media suitable for the expression of the recombinant toxin protein through which a recombinant toxin protein CRM197 is obatined.
In another embodiment, the present invention provides a high level expression of recombinant toxin protein CRM 197 in an altered Bacillus strain, comprising transforming a Bacillus strain with a DNA construct having a polynucleotide sequence flanked on each side by a fragment of an indigenous gene or a chromosomal region, or homologous sequence thereto, associated with amino acid biosynthetic and/or biodegradative pathways, without strain viability, by means of inactivating or replacing said indigenous gene or chromosomal region by homologous recombination between the DNA construct and the genome of said Bacillus strain, wherein, the indigenous gene or chromosomal region is chosen from the group consisting of regions namely abnA, amyE, aprE, aspB, bglS, bpr, dacB, dacF, dltD, epr, fliL, fliZ, lipB, lytC, lytD, lytF, mdf, mpr, nprB, pbp, pel B, penP, phoA, phoB,phrC, rpmG, sacB, sacC, spoIID, tyrA, vpr, wprA, xynA, ybbC, ybdN, ybfO, yckD, yddT, ydjM, yfhK, yfjS, yhaK, yhjA, yjdB, yjfA, ykol ykvv, ykwD, ylaE, yncM, yndA, yngK, yoaW, yobB, yocH, yojL, yolA, yomL, yoqM, ypbG, ypcP, ypmV, ypuA, yqfZ, yqgA, yqzC, yraJ, yurL, yvbX, yvcE, yvgO, ywaD, ywcL, yweA, ywmC, ywmD, ywqC, ywtD, ywtF, yxaK, yya B region and fragments thereof,

In certain embodiments, the recombinant protein is produced in soluble and/or active toxin protein at an yield of about 0.1 grams per liter to about 12 grams per liter.
In specific embodiments, the yield of soluble and/or active toxin protein is about 0.05 g/L, about 0.1 g/L, about 0.2 g/L, about 0.3 g/L, about 0.4 g/L, about 0.5 g/L, about 0.6 g/L, about 0.7 g/L, about 0.8 g/L, about 0.9 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L. about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, about 11 g/L, about 12 g/L,
In another embodiment, the nucleotide sequence encoding the toxin protein is fused to a secretion signals coding sequence that when expressed directs transfer of the toxin protein into the medium. The host cell is defective in the expression of at least one protease or the host cell over expresses at least one folding modulator, or a combination thereof.
In another embodiment of the invention, autonomously replicating plasmids have been developed to express heterologous toxin protein but with cons of instability. Along with that, integrative plasmids have been developed which confers stability, integration of genes into the chromosome. Integration ensures that normally a single copy of the transgene is present, which can be maintained in the same copy number even in the absence of selection (Vazquez-Cruz et al., 1996).
In another embodiment, the recombinant toxin protein is CRM197 and the host cell is defective in the expression of nprE , aprE, epr, bpr, mpr, ble, nprB, bsr, vpr, wprA, hrc A, htr A and htr B, or a combination thereof.
In another embodiment, the host cell over expresses prsA and GroE proteins. In related embodiments, the recombinant toxin protein is fused to a secretion leader that is abnA, amyE, aprE, aspB, bglS, bpr, dacB, dacF, dltD, epr, fliL, fliZ, lipB, lytC, lytD, lytF, mdf, mpr, nprB, pbp, pel B, penP, phoA, phoB.phrC, rpmG, sacB, sacC, spoIID, tyrA. vpr, wprA, xynA, ybbC, ybdN, ybfO, yckD, yddT, ydjM, yfhK,

yfjS, yhaK, yhjA, yjdB, yjfA. ykoJ, ykw, ykwD, ylaE, yncM, yndA, yngK, yoaW, yobB, yocH, yojL, yolA, yomL, yoqM, ypbG, ypcP, ypmV, ypuA, yqfZ, yqgA, yqzC, yraJ, yurL. yvbX, yvcE, yvgO, ywaD, ywcL, yweA, ywmC, ywmD, ywqC, ywtD, ywtF, yxaK, yya B.
In another embodiment, the activity of the recombinant toxin protein is measured in an activity assay, wherein about 10% to about 100% of the active toxin protein produced is determined to be active. In related embodiments, the activity assay is an immunological assay, a receptor-binding assay, or an enzyme assay.
In yet another embodiment of the present invention, the expression vector comprises a native host derivative promoter operatively linked to the protein coding sequence, and wherein the culturing comprises growing the recombinant Bacillus species in synthetic, semi synthetic or media fortified with organic nutrients. The cells will be grown to a cell density of 5 to 200 OD (OD at 600 nm. The fermentation can be batch mode, fed batch mode or continuous mode. The pH of the media is from 5.0 to 10.0 and temperature used for cultivation is 10°C to 40°C.
In a preferred embodiment, the host cell is a Bacillus subtilis.
In yet another embodimentsof the invention, the nucleotide sequence has been optimized for expression in the Bacillus host cell. In related embodiments, the nucleotide sequence has been optimized for expression in the Bacillus host cell. In other related embodiments, the nucleotide sequence has been optimized for expression in the Bacillus subtilis host cell.
In further embodiments of the invention, the expression vector further comprises a tag sequence adjacent to the coding sequence for the secretion signal. In embodiments, the expression vector further comprises a tag sequence adjacent to the coding sequence for the toxin protein.
The present invention also provides a recombinant toxin protein produced according to the methods described herein.

The present invention describes production of high level expression of recombinant microorganisms, more particularly recombinant toxin protein production in bacterial hosts, particularly are disclosed that have been engineered to express as CRM 197 alone or in combination with recombinant genes encoding housekeeping chaperones.
Toxins
ADP-Ribosylating Toxins
ADP-ribosylating toxins (ADPRTs) facilitate scission of the N-glycosyl bond between nicotinamide and the N-ribose of NAD and transfer the ADP-ribose moiety to target proteins. ADPRTs are classified into four families based on their respective targets. In embodiments of the present invention, a recombinant toxin protein selected from a group including ADPRTs is produced. In embodiments, the group of Type II ADPRTs consists of DT CRM197.
CRM197
Cross-reacting material 197 (CRM 197) is a Diphtheria toxin (DT) variant produced from a DT gene having a missense mutation. DT is an ADP-ribosylating toxin; CRM197 lacks the ADP-ribosyltransferase (ADPRT) activity of DT5 and is thus nontoxic. The gene for CRM 197 has a single base substitution, resulting in the incorporation of glutamic acid instead of glycine at residue 52.
In embodiments of the present invention, CRM197 is produced using any of the host strains described herein in Example 1. in combination with any of the expression vectors (plasmids) described in Example 1. In embodiments, the nucleic acid sequence is optimized for expression in the Bacillus host cell. In embodiments, the expression vectors used contain constructs expressing any of the secretion leaders described in Table 1 fused to the recombinant CRM 197.
Host
The continuous discovery of new vaccines and therapeutics asks for the development of efficient systems for the production of pharmaceutical proteins. The choice of an appropriate host and suitable production conditions is crucial for the

downstream processing of a pharmaceutical grade product. Escherichia coli and members of the species Bacillus are the most frequently used prokaryotes for the industrial production of recombinant proteins. These organisms are above all favored due to the fact that the cultivation of these bacteria in large-scale production systems at high cell densities is easy and usually inexpensive. Bacillus species have been major workhorse industrial microorganisms with roles in applied microbiology.
For the economical production of recombinant proteins, the existence of stable expression systems is a necessity. At present, about 60% of the commercially available enzymes are produced by Bacillus species, mostly being homologous proteins that are naturally secreted in the growth medium, such as alkaline proteases as washing agent or amylases for the starch industry. These roles have continually expanded and evolved over the past century. The development of strains and production strategies has recently been influenced or facilitated by the application of molecular biology techniques to strain development. Bacillus species are attractive industrial organisms for a variety of reasons, including their high growth rates leading to short fermentation cycle times, their capacity to secrete proteins into the extracellular medium, and the GRAS (generally regarded as safe) status with the Food and Drug Administration for species. For that reason, the use of B. subtilis for the production of food products is highly favored over the use of E. coli. The outer cell membrane of most Gram-negative bacteria, e.g. E. coli, contains lipopolysaccharides (LPS), generally referred to as endotoxins, which are pyrogenic in humans and other mammals. These endotoxins complicate product purification, because the end-product should be completely endotoxin- free.
Bacillus subtilis is a Gram-positive, nonpathogenic organism which is widely used as a host for enzyme production, due to its ability to secrete large amounts of proteins into the growth medium (Simonen et al., 1993; Westers et al., 2004). The secretion of a target protein leads to the natural separation of the product from cell components, which simplifies downstream processing of the protein. Accordingly, there has been a great deal of research performed regarding protein production in B. subtilis (Simonen et al., 1993; Westers et al., 2004). In B. subtilis, one long-standing major problem is the presence of high levels of extracellular protease for the

production of heterologous proteins. In recent years, many proteases have been identified via the completed genome sequence of B. subtilis (Kunst et al., 1997; Westers et al., 2004), thereby allowing the construction of many protease-depleted strains for the production of heterologous proteins In addition, considerable efforts have been targeted at developing B. subtilis as a host for the production of heterologous proteins (Westers et al., 2004; Kodama et al, 2012).
Host Secretory System
Protein export from the cytoplasm to destinations outside the cell is a phenomenon that takes place in all domains of life. Most bacterial proteins destined to leave the cytoplasm are exported via the highly conserved SecA-YEG (Sec) pathway. In addition, more specialized bacterial export pathways are used for the export of specific subsets of extracellular proteins. Most exported proteins are synthesized as precursors with an N-terminal signal peptide. These pre proteins are first recognized by soluble targeting factors for their transport to the translocation machinery in the cell membrane. Next, the polypeptide chain is transported through a proteinacious channel in the membrane, a process driven by a translocation motor that binds and hydrolyzes nucleotide triphosphates. Finally, the signal peptide is removed, resulting in the release of the mature protein from the membrane. The mature protein folds into its native conformation shortly after the release from the translocase, unless it is translocated in a folded state.
Although the soil bacterium B. subtilis has a relatively simple cell structure, proteins can at least be delivered to, or retained at, five (sub) cellular locations: the cytoplasm, the cytoplasmic membrane, the membrane/cell wall interface, the cell wall, and the growth medium. The final destination of a protein is governed by the presence or absence of signal peptides and/or retention signals. Nearly all proteins of B. subtilis lacking transport signals are retained in the cytoplasm and fold, with or without the aid of chaperones, into their native conformation. Other proteins contain membrane-spanning domains that are required for their insertion into the cytoplasmic membrane (Harwood, CR, Cranenburgh R (2008).
The Bacillus secretory pathway can be divided into three functional stages: (1) early stages involving the synthesis of secretory pre-proteins, their interaction (if any)

with chaperones and binding to the translocase; (2) translocation across the cell membrane via the Sec (protein secretion of unfolded proteins) or Tat translocase (twin-arginine translocation of folded proteins); (3) late stages, including removal of the signal peptide, release from the translocase, folding on the trans side of the cell membrane and passage through the cell wall (Simonen et al, 1993]. Thus, several different factors are involved before the produced protein reaches its final destination and conformation and therefore each of these factors can be a bottleneck for high-level production. For production of heterologous proteins in the medium of B. subtilis, it is necessary to use a signal peptide that directs the protein very efficiently to the translocase and that is cleaved efficiently by the signal peptidases. Therefore, modulation of a signal peptide to obtain an efficient signal peptidase cleavage site is sometimes a necessity. Furthermore, an increased expression of signal peptidases could enhance the capacity of the secretion machinery, e.g. for AmyQ as shown by Tjalsma et al.
Signal peptides
For the production of a heterologous protein in the culture medium of B. subtilis, it is necessary to use a signal peptide that directs the protein very efficiently to the translocase. However, heterologous protein secretion often results in inefficient and un satisfyingly low improvement of heterologous yields protein Secretion by Bacillus subtilis s. The relationship between signal peptides and target proteins remains unknown. Accordingly, previous studies have indicated the need for individually optimal signal peptides for every heterologous secretion target. Recently, Brockmeier et al. (2006) established a new strategy for the optimization of heterologous protein secretion in B. subtilis, by screening a library of all natural signal peptides of the strain. Accordingly, the best signal peptide for the secretion of one target protein is not automatically the best, or even sufficient, for the secretion of a different target protein (Brockmeier et al.. 2006).
In embodiments, a sequence encoding a secretion signal peptides is fused to the sequence encoding the toxin protein. In embodiments, the secretion leader is an extra cellular secretion leader. In embodiments, the secretion leader is the native secretion leader.

Table 1: leader sequence list

1 abnA MKKKKTWKRFLHFSSAALAAGLIFTSAAPAEA
2 amyE MFAKRFKTSLLPLFAGFLLLFHLVLAGPAAASA
3 aprE MRSKKLWISLLFALTLIFTMAFSNMSVQA
4 aspB MKLAKRVSALTPSTTLAITAKA
5 bglC MKRSISIFITCLLITLLTMGGMIASPASA
6 bglS MPYLKRVLLLLVTGLFMSLFAVTATASA
7 bpr MRKKTKNRLISSVLSTVVISSLLFPGAAGA
8 dacB MRIFKKAVFVIMISFLIATVNVNTAHA
9 dacF MKRLLSTLLIGIMLLTFAPSAFA
10 dltD MKKRFFGPI1LAFILFAGAIA
11 epr MKNMSCKLVVSVTLFFSFLTIGPLAHA
12 fliL MKKKLMIILLIILIVIGALGAAA
13 fliZ MKKSQYFIVFICFFVLFSVHPIAAAAA
14 lipB MKKVLMAFIICLSLILSVLAAPPSGAKA
15 lytC MRSYIKVLTMCFLGLILFVPTALA
16 lytD MKKRLIAPMLLSAASLAFFAMSGSAQA
17 lytF MKKKLAAGLTASAIVGTTLVVTPAEA
18 mdr MDTTTAKQASTKFVVLGLLLGILMSAiMDNTIVATA
19 mpr MKLVPRFRKQWFAYLTVLCLALAAAVSFGVPAKA
20 nprB MRNLTKTSLLLAGLCTAAQMVFVTHASA
21 pbp MKKSIKLYVAVLLLFVVASVPYMHQAALA
22 pel MKKVMLATALFLGLTPAGANA
23 penP MKLKTKASIKFG1CVGLLCLSITGFTPFFNSTHAEA
24 phoA MKKMSLFQNMKSKLLPIAAVSVLTAGIFAGA
25 phoB MKKFPKKLLPIAVLSSIAFSSLASGSVPEASA
26 phrC MKLKSKLFVICLAAAAIFTAAGVSANA
27 rpmG MRKKITLACKTCGNRNYTTMKSSASA
28 sacB MNIKKFAKQATVLTFTTALLAGGATQAFA
29 sacC MKKRLIQVMIMFTLLLTMAFSADA
30 spoIID MKQFAITLSVLCALILLVPTLLVIPFQHNKEAGA
31 tyrA MNQMKDTILLAGLGLIGGSIALA

32
vpr MKKGIIRFLLVSFVLFFALSTGITGVQAAPA
33 wprA MKRRKFSSVVAAVLIFALIFSLFSPGTKAAA
34 xynA MFKFKKNFLVGLSAALMSISLFSATASA

35 ybbC MRKTIFAFLTGLMMFGTITAASA
36 ybdN MVKKWLIQFAVMLSVLSTFTYSASA
37 ybfO MKRM1VRMTLPLL1VCLAFSSFSASARA
38 yckD MKRITINIITMFIAAAVISLTGTAEA
39 yddT MRKKRVITCVMAASLTLGSLLPAGYASA
40 ydjM MLKKVILAAFILVGSTLGAFSFSSDASA
41 yfhK MKKKQVMLALTAAAGLGLTALHSAPAAKA
42 yfjs MKWMCSICCAAVLLAGGAAQA
43 yhaK MRTWKRIPKTTMLISLVSPFLLITPVLFYAALA
44 yhjA MKKAAAVLLSLGLVFGFSYGAGHVAEA
45 yjdB MNFKKTVVSALSISALALSVSGVASA
46 yjfA MKRLFMKASLVLFAVVFVFAVKGAPAKA
47 ykoJ MLKKKWMVGLLAGCLAAGGFSYNAFA
48 ykvV MLTKRLLTIYIMLLGLIAWFPGAAQA
49 ykwD MKKAFILSAAAAVGLFTFGGVQQASA
50 ylaE MKKTFVKKAMLTTAAMTSAALLTFGPDAASA
51 yncM MAKPLSKGGILVKKVLIAGAVGTAVLFGTLSSGIPGLPAADA
52 yndA MRFTKVVGFLSVLGLAAVFPLTAQA
53 yngK MKVCQKSIVRFLVSLIIGTFVISVPFMANA
54 yoaW MKKMLMLAFTFLLALTIHVGEASA
55 yobB MKIRKILLSSALSFGMLISAVPALA
56 yocH MKKT1MSFVAVAALSTTAFGAHA
57 yojL MKKKIVAGLAVSAVVGSSMAAAPAEA
58 yolA MKKRITYSLLALLAVVAFAFTDSSKAKA
59 yomL MRKKRVITCVMAASLTLGSLLPAGYATA
60 yoqM MKLRKVLTGSVLSLGLLVSASPAFA
61 ypbG MKLSVKIAGVLTVAAAAMTAKMYATA
62 ypcP MNNNKLLLVDGMALLFRAFFATA
63 ypmB MRKKALIFTVIFGIIFLAVLLVSASIYKSAMA

64
ypuA MKKIWIGMLAAAVLLLMVPKVSLADA
65 yqfZ MKRLTLVCSIVFILFILFYDLKIGTIPIQDLPVYEASA
66 yqgA MKQGKFSVFLILLLMLTLVVAPKGKAEA
67 yqxl MFKKLLLATSALTFSLSLVLPLDGHAKA
68 yqzC MTKRGIQAFAGGIILATAVLAAVFYLTDEDQAAA
69 yqzG MMIKQCVICLSLLVFGTTAAHA
70 yraJ MTLTKLKMLSMLTVMIASLFIFSSQALA
71 yurl MTKKAWFLPLVCVLLISGWLAPAASASA
72 yvbX MKKWLIIAVSLAIAIVLFMYTKGEAKA
73 yvcE MRKSLITLGLASVIGTSSFLIPFTSKTASA
74 yvgO MKRIRIPMTLALGAALTIAPLSFASA
75 ywaD MKKLLTVMTMAVLTAGTLLLPAQSVTPAAHA
76 ywcl MKRLLVSLRVWMVFLMNWVTPDRKTARA
77 yweA MLKRTSFVSSLFISSAVLLSILLPSGQAHA
78 ywmC MKKRFSLIMMTGLLFGLTSPAFA
79 ywmD MKKLLAAGIIGLLTVSIASPSFA
80 ywqC MGESTSLKEILSTLTKRILLIMIVTAAATA
81 ywtD MNTLANWKKFLLVAVIICFLVPIMTKAE1AEA
82 ywtF MEERSQRRKKKRKLKKWVKVVAGLMAFLVIAAGSVGAYA
83 yxaK MVKSFRMKALIAGAAVAAAVSAGAVSDVPAAKVLQPTAAYA
84 yyaB MVYQTKRDVPVTLMIVFLILLIQADA
Codon Optimization
In heterologous expression systems, optimization steps may improve the ability of the host to produce the foreign protein. Optimization can thus address any of a number of sequence features of the heterologous gene. As a specific example, a rare codon-induced translational pause can result in reduced heterologous protein expression. One method of improving optimal translation in the host organism includes performing codon optimization which can result in rare host codons being removed from the synthetic polynucleotide sequence.

Interfering secondary structures also can result in reduced heterologous protein expression. Stem loop structures can also be involved in transcriptional pausing and attenuation. Another feature that can effect heterologous protein expression is the presence of restriction sites. By removing restriction sites that could interfere with subsequent sub-cloning of transcription units into host expression vectors a polynucleotide sequence can be optimized.
In another embodiment of the invention, the general codon usage in a host organism, such as B. subtilis, can be utilized to optimize the expression of the heterologous polynucleotide sequence. The percentage and distribution of codons that rarely would be considered as preferred for a particular amino acid in the host expression system can be evaluated. Values of 5% and 10% usage can be used as cut off values for the determination of rare codons.
The present invention contemplates the use of any coding sequence for the toxins produced, including any sequence that has been optimized for expression in the Bacillus host cell being used. Sequences contemplated for use can be optimized to any degree as desired, including, but not limited to, optimization to eliminate: codons occurring at less than 5% in the Bacillus host cell, codons occurring at less than 10% in the Bacillus host cell, a rare codon-induced translational pause, a putative internal RBS sequence, an extended repeat of G or C nucleotides, an interfering secondary structure, a restriction site, or combinations thereof.
Furthermore, the amino acid sequence of any secretion leader useful in practicing the methods of the present invention can be encoded by any appropriate nucleic acid sequence.
Promoters
A promoter having the nucleotide sequence of a promoter native to the selected bacterial host cell also may be used to control expression of the transgene encoding the target polypeptide. The promoters used in accordance with the present invention may be constitutive promoters or regulated promoters.

Table 2: Promoters

S.No Promoter Type
1 PGrac Constitutive/ Inducible
2. P43 Constitutive/ Inducible
3. PVeg Constitutive/ Inducible
4. AprE Constitutive/ Inducible
5. Est P Constitutive/ Inducible
6. YjfA Constitutive/ Inducible
7. YncM Constitutive/ Inducible
8 Amy E Constitutive/ Inducible
9 SacB Constitutive/ Inducible
Regulated promoters utilize promoter regulatory proteins in order to control transcription of the gene of which the promoter is a part. Where a regulated promoter is used herein, a corresponding promoter regulatory protein will also be part of an expression system according to the present invention. Examples of promoter regulatory proteins include: activator proteins, e.g., Protease proteins, e.g., YncM, YjfA and AprE; and starch utilizing proteins e.g Amy E protein. Many regulated-promoter/promoter-regulatory-protein pairs are known in the art.
Promoter regulatory proteins interact with an effector compound, i.e., a compound that reversibly or irreversibly associates with the regulatory protein so as to enable the protein to either release or bind to at least one DNA transcription regulatory region of the gene that is under the control of the promoter, thereby permitting or blocking the action of a transcriptase enzyme in initiating transcription of the gene. Effectors compounds are classified as either inducers or co-repressors, and these compounds include native effector compounds and gratuitous inducer compounds. Many regulated-promoter/promoter-regulatory-protein/effector-compound trios are known in the art. Although an effector compound can be used throughout the cell culture or fermentation, in a preferred embodiment in which a regulated promoter is used, after growth of a desired quantity or density of host cell biomass, an appropriate effector compound is added to the culture to directly or indirectly result in expression of the desired gene(s) encoding the protein or polypeptide of interest.

Host Strains
Bacterial hosts, including Bacillus s, and closely related bacterial organisms are contemplated for use in practicing the methods of the invention. In certain embodiments, the Bacillus host cell is Bacillus sublilis.
Bacterial Growth Conditions
Growth conditions useful in the methods of the provided invention can comprise a temperature of about 4° C. to about 42° C. and a pH of about 5.7 to about 8.8. When an expression construct with promoters (listed in Table 1) used, expression can be induced by adding sucrose to a culture at a final concentration of about 0.05 mM to about 1.0 mM.
The pH of the culture can be maintained using pH buffers and methods known to
those of skill in the art. Control of pH during culturing also can be achieved using
aqueous sodium hydroxide. In embodiments, the pH of the culture is about 5.7 to
about 8.8
In embodiments, the growth temperature is maintained at about 4° C. to about 42°
C.
In other embodiments, the temperature is changed during culturing. In one
embodiment, the temperature is maintained at about 37° C. before an agent to
induce expression from the construct. After adding the induction agent, the
temperature is reduced to about 25° C.
Induction
As described elsewhere herein, inducible promoters as well as constitutive promoters can be used in the expression construct to control expression of the recombinant toxin protein, e.g., Amy and Sac promoter.
In embodiments, a sac promoter derivative is used, and recombinant protein expression is induced by the addition of sucrose to a final concentration of about 0.05 mM to about 1.0 mM, when the cell density has reached a level identified by an OD575 of about 20 to about 120.

In another embodiment, wherein a non-sac type promoter is used, as described herein and in the literature, other inducers or effectors can be used. In one embodiment, the promoter is a constitutive promoter.
After adding inducing agent, cultures can be grown for a period of time, for example about 24 hours, during which time the recombinant protein is expressed. After adding an inducing agent, a culture can be grown for about 1 to 24 hrs. Cell cultures can be concentrated by centrifugation, and the culture supernatant can be applicable for the subsequent purification procedure.
Evaluation of Product
Numerous assay methods are known in the art for characterizing proteins. Use of any appropriate method for characterizing the yield or quality of the recombinant toxin protein is contemplated herein.
Protein Yield
Protein yield in any purification fraction as described herein can be determined by methods known, for example, by HPLC, and Western blot analysis. Activity assays, as described herein and known in the art, also can provide information regarding protein yield. In embodiments, these or any other methods known in the art are used to evaluate proper processing of a protein, e.g., proper secretion leader cleavage.
Useful measures of protein yield include, e.g., the amount of recombinant protein per culture volume (e.g., grams or milligrams of protein/litre of culture), percent or fraction of recombinant protein measured in the supernatant), percent or fraction of active protein (e.g.. amount of active protein/amount protein used in the assay).
In embodiments, the methods of the present invention can be used to obtain active and/or properly processed (e.g., having the secretion leader cleaved properly) recombinant toxin protein or subunit protein yield of about 0.05 grams per litre to about 12 grams per litre.

Agar Well Diffusion DNAse Activity assay:
The "activity" of a protein, though related qualities, are generally determined by different means. Protein activity, which can be evaluated using DNAse test agar assay, is another indicator of proper protein conformation. Activity levels can be described, e.g., in absolute terms or in relative terms, as when compared with the activity of a standard or control sample, or any sample used as a reference (Fig 5).
Immunoassays
Activity assays for evaluating toxins are known in the art and described in the literature. Activity assays include immunological or antibody binding assays, e.g., Western Blot analysis and ELISA, e.g., CRM197 can be evaluated by Diphtheria toxin. Antibodies usedl in these assays are 3B6 antibody were commercially available (Fig 2).
RP HPLC Analysis
A specific, precise, accurate, rapid and reliable RP-HPLC method has been developed and validated. It has short runtime 25 min and retention time 19.4 min to allow analysis of large number of samples in a short period of time. The supernatants were centrifuged at 20,800x g for 10 minutes (4°C) and the debris was removed using manual or automated liquid handling. The supernatants were subjected to Reverse Phase HPLC column (PLRP-S, 300 A0, 5uM, 150X4.6 MM) (Agilent technologies).
Representative graphs showing the results of the RP HPLC analysis of the sample is shown in Fig4a and 4b.
Size Exclusion HPLC Analysis
A specific, precise, accurate, rapid and reliable GPC-HPLC method has been developed and validated. It has short runtime 20 min and retention time 8.6 min to allow analysis of large number of samples for aggregate formation in a short period of time. The supernatants were centrifuged at 20,800x g for 10 minutes (4°C) and the debris was removed using manual or automated liquid handling. The supernatants were subjected to GPC HPLC column (Shodex KW-802.5, E 909092) (Agilent technologies).

Representative graphs showing the results of the GPC HPLC analysis of the sample is shown in Figure 3 in comparison with Sigma CRM 197 standard.
N terminal amino acid analysis
N-Terminal Sequencing uses a chemical process based on the technique developed by Pehr Edman in the 1950's where by: The N-terminal amino acid reacts with phenylisothiocyanate (PITC). The derivatizing process results in a phenylthiohydantoin (PTH) - amino acid. This amino acid is then sequentially removed while the rest of the peptide chain remains intact. Each derivatization process is a cycle. Each cycle removes a new amino acid. The amino acids are sequentially analyzed to give the sequence of the protein or peptide. Routinely used for the analysis of membrane bound gel electrophoresis separated proteins, HPLC separated tryptic digest fragments (Fig 6).
Sample Information and Preparation Required
Protein can be submitted dry, in solution, as an SDS or native polyacrylamide gel piece, or blotted to PVDF membrane. Nitrocellulose is not compatible with the Edman chemistry. Blotting is done as for a Western, but without blockers or antibodies and must be thoroughly washed. Membrane was stained with ponceaue red. Destain blots enough to leave target bands visible, wash with distilled and deionised water and dry. They can then be stored @4 °C for several weeks.
EXAMPLE 1
High Throughput Expression of a Recombinant CRM197 Protein
CRM197 expression strains were constructed and the amount of CRM197 protein produced in the strains was analyzed using RP HPLC. Based on the resulting data, certain strains were selected for use in large-scale expression.
Construction and Growth of CRM 197 Expression Strains
The CRM 197 coding sequence was constructed using B. subtilis preferred codons to encode the CRM197 amino acid sequence. SEQ ID NO: 1 shows the amino acid

sequence encoded by the expressed synthetic optimized CRM197 gene SEQ ID NO: 2 shows the DNA sequence of the expressed synthetic optimized CRM197 gene.
Basically, two different vector systems have been developed, autonomously replicating plasmid vectors, integrative vectors. Plasmids carrying the optimized CRM197 sequence, fused to ten B. subtilis secretion leaders as shown in Table 1, were constructed. The CRM197 coding sequence was fused in frame with that of B. subtilis secretion leaders to target the protein to the supernatant for recovery in the active form.
Tablel. Secretion leaders used for CRM197 expression screen
Constructs containing the ten secretion leaders fused to the recombinant CRM197 coding sequence were tested in B. subtilis host. Host cells were electroporated with the indicated plasmids, resuspended in growth medium with trace minerals. The cultures were incubated at 30°C with shaking for 48 hours. Ten micro liters of each of the seed cultures were transferred into triplicate test tubes, each tube containing 5 ml of growth medium supplemented with trace elements, and incubated as before for 24 hours.
PD = Protease Deletion (listed proteases are deleted); FMO = Folding Modulator Over expressor (listed folding modulators are over expressed. Sucrose was added to each well to a final concentration of 0.05 raM to induce the expression of target proteins. Sucrose was added to each well to a final concentration of 1% to induce the expression of folding modulators in folding modulator over-expressing strains, and the temperature was reduced to 25°C. Twenty four hours after induction, supernatant samples were frozen for later processing.
Product Analysis
The supernatants were centrifuged at 20,800x g for 10 minutes (4°C) and the debris was removed using manual or automated liquid handling. The supernatants were subjected to Reverse Phase HPLC, GPC HPLC, DNAse activity assay, western analysis and N terminal sequencing.

Representative pictures of above mentioned test were depicted in art (Fig 2, 3, 4a. 4b. 5 and 6). Both secretion leader and host strain along with autonomous replication and integration factors showed a significant impact on CRM197 expression.
Expression ranged from no detectable yield to more than 0.05 g/L at the 5mL scale were observed for clone BE 6633. Both high and low yields were observed in the same host strain depending on the leader used and both high and low yields were observed using the same leader in different host strains.
EXAMPLE 2
Large-scale Expression of a Recombinant CRM197 Protein
Recombinant CRM197 protein was produced in Bacillus subtilis BE-6633 I n 35 liter fermentor. Cultures were grown in 35 liter fermentor containing semi synthetic medium as described herein.
Culture conditions were maintained at 37 °C and pH 7.0 through the addition of sodium hydroxide. Dissolved oxygen was maintained in excess through increases in agitation and flow of sparged air and oxygen into the fermentor. Glucose was delivered to the culture throughout the fermentation to maintain excess levels. These conditions were maintained until a target culture cell density (optical density at 575nm (A 575) for induction is reached, at which time sucrose is added to initiate CRM 197 production. Cell density at induction could be varied from A 575 of 40 to 200 absorbance units (AU). Sucrose concentrations could be varied in the range from 0.02 to 0.4 mM. pH from 6 to 7.5 and temperature 20 to 35 °C. After 16-24 hours, the culture from each bioreactor was harvested by centrifugation and the culture supernatant frozen at -80 °C. Samples were analyzed by RP HPLC and Western analysis for product formation.
Multiple fermentation conditions were evaluated resulting in top CRM 197 expression as determined by RP HPLC. The identities of the induced proteins were confirmed by Western blot analysis using a diphtheria toxin specific antibody (Figure 2).

Product Analysis
The supernatants were centrifuged at 20,800x g for 10 minutes (4°C) and the debris was removed using manual or automated liquid handling. The supernatants were subjected to Reverse Phase HPLC, GPC HPLC, DNAse activity assay, western
analysis.
Representative pictures of above mentioned test were not discussed in the art. Expression ranged from no detectable yield to more than 0.05 g/L at the 5mL scale were observed for clone BE 6633. Both high and low yields were observed in the same host strain depending on the leader used and both high and low yields were observed using the same leader in different host strains.

We claim :
1. A high level expression of recombinant toxin protein CRM 197 and method for producing in a Bacillus host cell, said method comprising: ligating into a vector a nucleotide sequence encoding a toxin protein; transforming the Bacillus host cell; followed by culturing the transformed Bacillus host cell in a culture media suitable for the expression of the recombinant toxin protein CRM197.
2. The method as claimed in claim 1, wherein the Bacillus host cell is Bacillus subtilis.
3. The method as claimed in claim 1, wherein the culturing comprises of growing the recombinant Bacillus strain in synthetic, semi synthetic or media fortified with organics nutrients to a cell density of 20 to 200 OD.
4. A high level expression of recombinant toxin protein CRM 197 in an altered Bacillus strain, comprising transforming a Bacillus strain with a DNA construct having a polynucleotide sequence flanked on each side by a fragment of an indigenous gene or a chromosomal region, or homologous sequence thereto, associated with amino acid biosynthetic and/or biodegradative pathways, without strain viability, by means of inactivating or replacing said indigenous gene or chromosomal region by homologous recombination between the DNA construct and the genome of said Bacillus strain.
5. The method as claimed in claim 1 and 4. wherein the indigenous gene or chromosomal region is chosen from the group consisting of regions namely abnA, amyE, aprE, aspB, bglS, bpr, dacB, dacF, dltD. epr, fliL, fliZ. lipB, lytC, lytD, lytF. mdf, mpr, nprB, pbp, pel B. penP, phoA, phoB,phrC, rpmG, sacB, sacC, spoIID, tyrA, vpr, wprA, xynA, ybbC, ybdN, ybfO, yckD, yddT, ydjM, yfhK, yfjS, yhaK, yhjA, yjdB, yjfA, ykoJ, ykvv, ykwD. ylaE, yncM, yndA, yngK, yoaW, yobB, yocH, yojL, yolA, yomL, yoqM, ypbG, ypcP, ypmV, ypuA, yqfZ, yqgA, yqzC, yraJ, yurL, yvbX, yvcE. yvgO, ywaD. ywcL. yweA, ywmC. ywmD, ywqC, ywtD, ywtF, yxaK. yya B region and fragments thereof.

6. The method as claimed in claim 4. wherein the nucleotide sequence has been optimized for expression in the Bacillus and encoding the toxin protein is fused to a secretion signals coding sequence that when expressed directs transfer of the toxin protein extra-eellularly to the medium.
7. The method as claimed in any of the claims 4 to 6, wherein the recombinant toxin protein is CRM197 and the host cell is defective in the expression of nprE , aprE, epr, bpr, mpr, ble, nprB, bsr, vpr, wprA, hrc A, htr A and htr B. or a combination thereof, or wherein the host cell over expresses prsA, Gro E.
8. The method as claimed in claim 4, wherein a DNA construct comprising an incoming sequence, wherein said incoming sequence comprises a nucleic acid sequence optimized for expression in the Bacillus and encoding a protein of interest, and a selective marker flanked on each side with a homology box, wherein said homology box includes nucleic acid sequences having 80 to 100% sequence identity to the sequence immediately flanking the coding regions of at least one gene selected from the group consisting abnA, amyE, aprE, aspB, bglS, bpr, dacB. dacF, dltD, epr, fliL, fliZ, lipB, lytC: lytD, lytF, mdf, mpr, nprB, pbp, pel B, penP, phoA, phoB.phrC, rpmG, sacB, sacC, spoIID, tyrA, vpr, wprA, xynA, ybbC, ybdN, ybfO. yckD, yddT, ydjM, yfhK, yfjS, yhaK, yhjA, yjdB, yjfA, ykoJ. ykvv, ykwD, ylaE, yncM, yndA, yngK, yoaW, yobB, yocH, yojL. yolA, yomL, yoqM, ypbG. ypcP, ypmV, ypuA, yqfZ, yqgA, yqzC. yraJ, yurL, yvbX, yvcE, yvgO, ywaD, ywcL, yweA, ywmC, ywmD, ywqC, ywtD, ywtF, yxaK. yya B.
9. The method as claimed in claims 4 to 8, wherein the vector comprises a native host derivative promoter operatively linked to the protein coding sequence which integrates precisely in to the host chromosome an propagates as an extra chromosomal element or combination thereof.

10. The method as claimed in claim 4, further comprising measuring the activity of the recombinant toxin protein in an activity assay, wherein the soluble toxin protein produced is determined to be active.