MONOPHOSPHATE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of US Provisional Patent Application No. 61/553,385, filed 31 October 2011, the contents of which being hereby incorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention lies in the field of molecular biology and relates to polypeptides for the enzymatic synthesis of cyclic diadenosine monophosphate (c-di- AMP) and linear diadenosine monophosphate (5'-pA A) and nucleic acids encoding these polypeptides as well as cells comprising these nucleic acids. The present invention also relates to the recombinant production of the polypeptides of the invention and methods of synthesizing c-di-AMP and/or 5'-pApA using said polypeptides.

BACKGROUND OF THE INVENTION

[0003] Cyclic-di-AMP (c-di-AMP), or 3', 5'-cyclic diadenylate, is a bacterial messenger molecule that can be considered as a small cyclic RNA molecule containing two adenine bases linked by ribose and phosphate (Fig. 1). The existence of c-di-AMP in living cells was unknown until the recent serendipitous discovery of the cyclic dinucleotide bound by the DisA protein in Bacillus subtilis (Witte et al. (2008), Mol. Cell 30, 167-178). It was further found that c-di-AMP can be synthesized by the diadenylate cyclase (DAC) domain of DisA via condensation of two ATP molecules. C- di-AMP is likely to mediate DNA damage and repair because the DNA integrity scanning protein DisA scouts the chromosome for DNA double-stranded breaks (Witte et al., supra). Subsequent genomic mining revealed that the DAC domain proteins are widespread in bacteria and archaea, including some important human pathogens such as Staphococcus aureus (Romling (2008), Sci. Signal. 1, pe39). The wide occurrence of DAC domains hints that c-di-AMP may be another hidden nucleotide messenger mediating various cellular functions and phenotypes. Importantly, it was discovered in 2010 that c-di-AMP secreted by the intracellular pathogen Listeria monocytogenes c-di- AMP stimulates mammalian immune response by activating a host type I interferon response (Woodward et al., Science 328, 1703-1705). One of the great significances of this discovery is that c-di-AMP and its structural analogs could be exploited for the development of novel vaccine adjuvant.

[0004] The inventors recently discovered that a family of multidomain proteins co- occurs with a subset of the DAC domain proteins that include the homologs of YojJ and YbbP from B. subtilis. This group of proteins (COG3887), as represented by the B. subtilis protein ZfrYybT, contains two N-terminal transmembrane helices, a region that shares minimum sequence homology with some Per-Arnt-Sim (PAS) domains, a highly modified GGDEF domain, and a DHH domain. The inventors found that the DHH domain exhibited in vitro phosphodiesterase activity toward the c-di-AMP, indicating that YybT protein is the c-di-AMP specific phosphodiesterases responsible for the degradation of c-di-AMP to yield 5'-pApA in cells (Rao et al. (2010) Journal of Biological Chemistry 285, 473-482).

[0005] As one of the major metabolites of the c-di-AMP signaling network, 5'-pApA can also stimulate human innate immune response. Further investigation of the immune stimulatory property of c-di-AMP and 5'-pApA by in vitro and in vivo experiments requires large amount of the cyclic or linear dinucleotide (hundreds of grams). Currently, c-di-AMP and 5'-pApA are synthesized chemically in small quantity (Fig. 2) (Ching et al., Bioorganic & Medicinal Chemistry 18, 6657-6665). The labor-intensive and time-consuming chemical synthesis approaches only yield limited amount of c-di- AMP given the overall low yield of the multi-step synthesis.

[0006] Although the enzymatic activity of the diadenylate cyclase domain containing DisA proteins has been demonstrated using radio-isotope-labeled ATP (Witte et al., supra), the inventors found that the DisA proteins are not suitable for c-di-AMP production due to the relatively low enzymatic activity. Likewise, the homologs of YojJ and YbbP tested by the inventors only exhibit residual enzymatic activities for c-di- AMP synthesis. [0007] Currently, no enzymatic methods are avaible for synthesizing 5'-pApA. Hence, an efficient method for the preparation 5'-pApA is in demand for the synthesis of the linear dinucleotide.

[0008] Hence, there is need in the art for the more efficient synthesis of cyclic and linear diadenosine monophosphate.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the inventors' discovery and characterization of two thermophilic proteins (MJ1002 and G/YybT) from Methanocaldococcus jannaschii and Geobacillus thermodenitrificans that have diadenylate cyclase and c-di- AMP phosphodiesterase activities, respectively. The two enzymes exhibited much higher enzymatic activities and thermostability and thus are more suitable for the large- scale production of c-di-AMP and 5'-pApA (Figs. 7 and 8) as well as the synthesis of radio-isotope labeled dinucleotides. The inventors have further identified a number of homologs of the newly identified diadenylate cyclase termed MJ1002.

[00010] In a first aspect, the present invention is thus directed to an isolated polypeptide, wherein the isolated polypeptide

(i) comprises the amino acid sequence set forth in any one of SEQ ID Nos: 1 , 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a functional fragment or variant thereof;

(ii) is encoded by a nucleic acid molecule comprising the nucleotide

sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or a complement or fragment or codon-optimized variant thereof;

(iii) is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or a complement thereof;

(iv) has at least 50 % sequence identity to the polypeptide of (i) or (ii); and

(v) shares at least 65 % sequence homology with the polypeptide of (i) or (ii).

[00011] The isolated polypeptide according to the first aspect may have diadenylate cyclase activity. [00012] In a second aspect, the present invention is directed to an isolated polypeptide, wherein the isolated polypeptide

(i) comprises the amino acid sequence set forth in SEQ ID NO:23 or a

functional fragment or variant thereof;

(ii) is encoded by a nucleic acid molecule comprising the nucleotide

sequence set forth in SEQ ID NO:24 or a complement or fragment or codon-optimized variant thereof;

(iii) is encoded by a nucleic acid molecule comprising a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:24 or a complement thereof;

(iv) has at least 80 % sequence identity to the polypeptide of (i) or (ii); and

(v) shares at least 90 % sequence homology with the polypeptide of (i) or (ii).

[00013] The isolated polypeptide of the second aspect may have c-di-AMP phosphodiesterase activity.

[00014] In another aspect, the present invention relates to an isolated nucleic acid molecule, wherein the isolated nucleic acid molecule encodes a polypeptide of the invention.

[00015] In still another aspect, the invention encompasses a recombinant cell comprising the nucleic acid molecule according to the invention. The recombinant cell may be a procaryotic cell, such as an E.coli cell.

[00016] In a still further aspect, the invention is directed to a method for the production of the isolated polypeptide according to the invention, the method comprising:

(i) incubating the recombinant cell according to the invention under conditions that allow expression of the polypeptide; and

(ii) isolating the expressed polypeptide.

[00017] In another aspect, the invention relates to an in vitro method for the synthesis of cyclic di-AMP (c-di-AMP) comprising incubating the isolated polypeptide according to the first aspect of the invention with ATP under conditions that allow production of c-di-AMP. [00018] In a still further aspect, the invention also covers an in vitro method for the synthesis of linear diadenosine monophosphate (5'pApA) comprising incubating the isolated polypeptide according to the second aspect of the invention with cyclic di-AMP under conditions that allow production of 5'pApA.

BRIEF DESCRIPTION OF THE DRAWINGS

[00017] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings.

[00018] Figure 1 shows the structure of cyclic and linear diadenosine monophosphate.

[00019] Figure 2 shows the synthetic route for the chemical sythesis of c-di- AMP and other cyclic dinucleotides, as reported by Ching et al. (Bioorg. Med. Chem. (2010) 18: 6657-65).

[00020] Figure 3 shows the metabolism of c-di-AMP and the enzymatic synthesis of c-di-AMP and 5'-pApA. Abbreviations used are: DACs: diadenylate cyclases; PDEs: c-di-AMP phosphodiesterases).

[00021] Figure 4 schematically shows the domain organization of MJ1002 and GtYybT as cloned from Methanocaldococcus jannaschii and Geobacillus denitrificans, respectively.

[00022] Figure 5 shows the enzymatic activity assays for MJ1002. The formation of c-di-AMP was monitored by HPLC with an RP-C 18 column by which separation of the product c-di-AMP from the starting material ATP can be achieved. A: HPLC analysis of the reaction mixture on an RP-C 18 column (reaction) ATP Std: ATP Standard; cyclic di-AMP Std: c-di-AMP standard; B: Concentration of c-di-AMP plotted against reaction time (seconds); C: Product turnover in % plotted against the enzyme concentration; D: Product yield plotted against the pH of the reaction buffer; E: MALDI mass spectrum of the product c-di-AMP.

[00023] Figure 6 shows the enzymatic activity assays for GtYybT. The formation of 5'-pApA was monitored by HPLC with an RP-C 18 column similar to c-di-AMP monitoring described above. A: Reaction scheme of the phosphoester linkage cleavage catalyzed by the PDE. B: HPLC analysis of the reaction mixture on an RP-C18 column (YybT + c-di-AMP: 3 min) and comparison with a 5'-pApA standard, a 3'-pApA standard and an AMP standard; C: Michaelis-Menten plot for GtYybT with c-di-AMP as a substrate.

DETAILED DESCRIPTION OF THE INVENTION

[00024] The terms used herein have, unless explicitly stated otherwise, the following meanings.

[00025] The present invention is based on the inventors' finding that a subgroup of proteins sharing sequence homology with the newly identified protein MJ1002, having the amino acid sequence set forth in SEQ ID NO: l, have high in vitro diadenylate cyclase activity and thus can be advantageously used to enzymatically synthesize c-di-AMP. Additionally, the inventors identified a phosphodiesterase, GtYybT, with the amino acid sequence set forth in SEQ ID NO:23, that is capable of cleaving c-di-AMP in vitro, thus, yielding linear diadenosine monophosphate (5'- pApA).

[00026] Based on this findings, the present invention relates to an isolated polypeptide, wherein the isolated polypeptide (i) includes or consists of the amino acid sequence set forth in any one of SEQ ID Nos: l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 or a functional fragment or variant thereof; or (ii) is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or a complement or fragment or codon-optimized variant thereof; or (iii) is encoded by a nucleic acid molecule comprising or consisting of a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or a complement thereof; or (iv) comprises or consists of an amino acid sequence that has at least 50 % sequence identity to the polypeptide of (i) or (ii); or (v) comprises or consists of an amino acid sequence that shares at least 65 % sequence homology with the polypeptide of (i) or (ii).

[00027] In various embodiments, the polypeptide has diadenylate cyclase activity meaning that it can enzymatically catalyze the synthesis of c-di-AMP from ATP. [00028] "Isolated", as used herein, relates to a polypeptide or nucleic acid that has been at least partially separated from other cellular components it naturally associates or occurs with. Accordingly, "isolated" encompasses purified polypeptides or nucleic acid molecules that are essentially free of other components, such as other proteins, nucleic acids or cellular debris in general. In various embodiments, an "isolated polypeptide" or "isolated nucleic acid" is at least 80 %, preferably at least 90 %, more preferably at least 95 % pure, i.e. comprises no more than 20, 10, and 5 % impurities, respectively.

[00029] The terms "protein" and "polypeptide" are used interchangeably throughout the specification to designate a linear polymer of amino acid residues connected to each other by peptide bonds. A protein or polypeptide according to the present invention preferably comprises 50 or more, more preferably 100 or more amino acid residues.

[00030] The term "fragment", as used in connection with a polypeptide sequence herein, relates to a peptide or protein sequence which is in comparison to a reference peptide or protein sequence C-terminally, N-terminally or on both ends shortened such that a contiguous strand starting from the N-terminus, C-terminus or comprising a middle portion of the reference protein remains. In some embodiments, such fragments may have a length of at least 50 amino acids, preferably at least 100 amino acids. "Functional fragment", as used herein, refers to a fragment that retains, at least partially, the activity of the reference polypeptide, e.g. diadenylate cyclase activity or phosphodiesterase activity. "At least partially" means in this context that it retains at least 25, preferably at least 50, more preferably at least 75, most preferably at least 95 % of the enzymatic activity of the full length protein.

[00031] "Nucleic acid", "nucleic acid molecule" and "nucleotide sequence", as used interchangeably herein, relate to linear polymers of nucleotides, preferably the natural nucleotides represented by the mono-, di- or triphosphates of guanosine (G), adenosine (A), cytosine (C), thymidine (T)/uridine (U). The nucleic acids may be DNA, RNA, DNA/RNA hybrids or peptide nucleic acids, but are preferably DNA or RNA.

[00032] The term "fragment", as used herein in connection with a nucleic acid, relates to a nucleic acid sequence which is shorter than its reference nucleic acid sequence. The shortening occurs starting from the 5' end such that a contiguous strand starting from the 3' end remains (3' fragment). Alternatively, the fragment may be a 5' fragment, i.e. the shortening occurs starting from the 3' end such that a contiguous strand starting from the 5' end remains. In a further alternative, the fragment may be 3'- and 5 '-truncated. The fragments preferably have a length of at least 100 nucleotides, more preferably at least 300 nucleotides and most preferably at least 500 nucleotides.

[00033] "Complement", as used herein in relation to nucleic acids, relates to a nucleic acid molecule that is complementary to a reference nucleic acid sequence in that it comprises or consists of a sequence that can form Watson-Crick base pairing over parts or the whole length of the reference sequence or parts or the whole length of the complement molecule. The complement can be a perfect complement in that each of its nucleotides corresponds to the Watson-Crick base pairing partner in the reference sequence. In various embodiments, either each nucleotide of the reference nucleic acid is Watson-Crick base paired with the complement sequence or each nucleotide of the complement is Watson-Crick base paired with the reference sequence.

[00034] "Sequence identity", as used herein in relation to amino acid and nucleotide sequences means that a residue of a given molecule is identical to that at the corresponding position of a reference molecule.

[00035] "Sequence homology" or "sequence similarity", as interchangeably used herein in relation to amino acid sequences, means that a residue of a given molecule is identical or homologous or similar to that at the corresponding position" of a reference molecule. Similarity or homology means that the residue has similar characteristics as the one in the corresponding position of the reference molecule but is not identical. One example for such similarity is a "conservative amino acid replacement", where one amino acid is replaced by another with similar properties. Examples of conservative substitutions are the replacements among the members of the following groups: 1) alanine, serine, and threonine; 2) aspartic acid and glutamic acid; 3) asparagine and glutamine; 4) arginine and lysine; 5) isoleucine, leucine, methionine, and valine; and 6) phenylalanine, tyrosine, and tryptophan.

[00036] The term "variant" as used in the present invention in relation to polypeptides, relates to derivatives of a protein or peptide that comprise modifications of the amino acid sequence, for example by substitution, deletion, insertion or chemical modification. Preferably, such modifications do not reduce or change the functionality of the protein or peptide. Such variants include proteins, wherein one or more amino acids have been replaced by their respective D-stereoisomers or by amino acids other than the naturally occurring 20 amino acids, such as, for example, ornithine, hydroxyproline, citrulline, homoserine, hydroxylysine, norvaline.

[00037] "Codon-optimized variant", as used herein in relation to nucleic acid sequences, means that the nucleotide sequence of a given nucleic acid molecule has been altered such that, due to the redundancy of the genetic code, it still encodes the same amino acid sequence but is more efficiently expressed in a given host organism. It is routinely used in the art to facilitate or increase gene expression in heterologous expression systems. In fact, numerous service providers exist that offer custom codon optimization of a gene sequence for any given host organism (for example GenScript, USA, or GeneArt® (Life Technologies Ltd, UK)). In brief, codon optimization is carried out by replacing each codon of a given nucleotide sequence encoding a specific amino acid by that codon preferably or predominantly used by the host organism for the same amino acid. In particular, codon-optimization for allowing expression of heterologous gene sequences in E.coli is standard practice in the art. A codon-optimized variant of a given nucleic acid is thus a synthetic nucleotide sequence that yields the same translated amino acid sequence but has different codons. Such a codon-optimized sequence may, depending on the degree of substitutions necessary, only share a very low degree of sequence identity with the reference sequence. Each codori-optimized sequence is routinely checked for undesired sequences that may be generated by codon replacement, such as poly-A sequences, undesired restriction enzyme recognition sites, intron-splice recognition sites, etc.

[00038] In various other aspects, the invention relates to an isolated polypeptide, wherein the isolated polypeptide (i) includes or consists of the amino acid sequence set - forth in SEQ ID NO:23 or a functional fragment or variant thereof; or (ii) is encoded by a nucleic acid molecule comprising or consisting of the nucleotide sequence set forth in SEQ ID NO:24 or a complement or fragment or codon-optimized variant thereof; or (iii) is encoded by a nucleic acid molecule comprising or consisting of a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO:24 or a complement thereof; or (iv) comprises or consists of an amino acid sequence that has at least 80 % sequence identity to the polypeptide of (i) or (ii); or (v) comprises or consists of an amino acid sequence that shares at least 90 % sequence homology with the polypeptide of (i) or (ii).

[00039] The polypeptide of the amino acid of SEQ ID NO:23 or its functional fragment or variant has, in various embodiments, phosphodiesterase activity for c-di- AMP.

[00040] The fragment of the protein comprising or consisting of the amino acid sequence set forth in SEQ ID NO:23 may comprise or consist of amino acid residues 55-658. It was found that this fragment (SEQ ID NO:25) comprising the cytoplasmic domain of GtYybT is surprisingly stable and has a high enzymatic activity. The nucleotide sequence encoding said fragment is set forth in SEQ ID NO:26 and is one preferred embodiment of the nucleic acid molecules of the invention. Specifically, also codon-optimized variants and fragments and complements thereof are contemplated in the present invention. Similarly also encompassed are nucleic acid molecules that share a certain degree of sequence similarity with this sequence as defined below.

[00041] The present invention further relates to isolated nucleic acid molecules that encode the polypeptides defined above. Accordingly, in various embodiments, the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in any one of SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or the complement thereof; comprises or consists of a nucleotide sequence that has at least 50 % sequence identity to the nucleotide sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or the complement thereof; or comprises or consists of a nucleotide sequence that is obtainable by codon optimization of the nucleotide sequence set forth in SEQ ID Nos:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or the complement thereof for expression in a given species.

[00042] In various embodiments, the sequence identity may be at least 60, at least 70, at least 80 or at least 90 %.

[00043] The nucleic acid molecules of the invention may further comprises additional nucleotide sequence besides the encoding sequence. Such nucleotide sequences include sequence elements which contain information regarding to transcriptional and/or translational regulation, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage in this context is a linkage in which the regulatory sequence elements and the sequence to be expressed are connected in a way that enables gene expression. The precise nature of the regulatory regions necessary for gene expression may vary among species, but in general these regions comprise a promoter which, in prokaryotes, contains both the promoter per se, i.e. DNA elements directing the initiation of transcription, as well as DNA elements which, when transcribed into RNA, will signal the initiation of translation. Such promoter regions normally include 5' non-coding sequences involved in initiation of transcription and translation, such as the -35/- 10 boxes and the Shine- Dalgarno element in prokaryotes or the TATA box, CAAT sequences, and 5'-capping elements in eukaryotes. These regions can also include enhancer or repressor elements as well as translated signal and leader sequences for targeting the native polypeptide to a specific compartment of a host cell. In addition, the 3' non-coding sequences may contain regulatory elements involved in transcriptional termination, polyadenylation or the like. If, however, these termination sequences are not satisfactory functional in a particular host cell, then they may be substituted with signals functional in that cell.

[00044] In various other embodiments, such additional nucleic acid sequences encode for another protein or peptide, for example a tag that facilitates purification and isolation of the expressed polypeptides and can, optionally, be cleavable by a protease. Said protease recognition site may also be encoded by said additional nucleic acid sequence.

[00045] In various embodiments, the nucleic acid molecules of the invention are comprised in a vector or any other kind of cloning vehicle, including, but not limited to a plasmid, a phagemid, a phage, a baculovirus, a cosmid, or an artificial chromosome.

[00046] Such cloning vehicles can include, aside from the regulatory sequences described above and a nucleic acid sequence of the present invention, replication and control sequences derived from a species compatible with the host cell that is used for expression as well as selection markers conferring a selectable phenotype on transformed or transfected cells. Large numbers of suitable cloning vectors are known in the art, and are commercially available.

[00047] In certain embodiments the nucleic acid molecules disclosed herein are comprised in a cloning vector. In some embodiments the nucleic acid molecules disclosed herein are comprised in an expression vector. The vectors may comprise regulatory elements for replication and selection markers. In certain embodiments, the selection marker may be selected from the group consisting of genes conferring resistance to an antibiotic, including but not limited to ampicilin, kanamycin, chloramphenicol, tetracycline, blasticidin, spectinomycin, gentamicin, hygromycin, and/or zeocin.

[00048] An above-described nucleic acid molecule of the present invention, comprising a nucleic acid sequence encoding for a protein of interest, if integrated in a vector, must be integrated such that the peptide or protein of interest can be expressed. Therefore, a vector of the present invention can comprise any of the above-mentioned regulatory elements.

[00049] In another preferred embodiment, a vector comprising a nucleic acid molecule of the present invention comprises a promoter sequence and a transcriptional termination sequence, if not already present in the gene sequence. Suitable promoters for prokaryotic expression are, for example, the araBAD promoter, the tet-promoter, the lacUV5 promoter, the CMV promotor, the EF1 alpha promotor, the AOX1 promotor, the tac promotor, the T7 promoter, or the lac promotor. Examples of promoters useful for expression in eukaryotic cells are the SV40 promoter or the CMV promoter. Furthermore, a nucleic acid molecule of the invention can comprise transcriptional regulatory elements, e.g., repressor elements, which allow regulated transcription and translation of coding sequences comprised in the nucleic acid molecule. Repressor element may be selected from the group consisting of the Lac-, AraC-, or MalR- repressor.

[00050] The vector may be effective for prokaryotic or eukaryotic protein expression. In particular, the nucleic acid molecules of the present invention may be comprised in a vector for prokaryotic protein expression. Suitable systems are known to those skilled in the art.

[00051] By transfecting or transforming a host cell with the nucleic acid molecule according to the invention, recombinant cells harboring the nucleic acid molecules of the invention can be generated. The present invention thus, in one aspect, encompasses cells that comprise one or more nucleic acid molecules of the invention.

[00052] The above described vectors of the present invention may be used for the transformation or transfection of a host cell in order to achieve expression of a peptide or protein which is encoded by an above described nucleic acid molecule and comprised in the vector DNA. Thus, in a further aspect, the present invention also relates to a host cell comprising a vector as disclosed herein.

[00053] Also contemplated herein are host cells, which comprise a nucleic acid molecule as described herein integrated into their genomes. The skilled person is aware of suitable methods for achieving the nucleic acid molecule integration. For example, the molecule may be delivered into the host cells by means of liposome transfer or viral infection and afterwards the nucleic acid molecule may be integrated into the host genome by means of homologous recombination. In certain embodiments, the nucleic acid molecule is integrated at a site in the host genome, which mediates transcription of the peptide or protein of the invention encoded by the nucleic acid molecule. In various embodiments, the nucleic acid molecule further comprises elements which mediate transcription of the nucleic acid molecule once the molecule is integrated into the host genome and/or which serve as selection markers.

[00054] In certain embodiments, the nucleic acid molecule of the present invention is transcribed by a polymerase natively encoded in the host genome. In various embodiments, the nucleic acid molecule is transcribed by a R A-polymerase which is non-native to the host genome. In such embodiments, the nucleic acid molecule of the present invention may further comprise a sequence encoding for a polymerase and/or the host genome may be engineered or the host cell may be infected to comprise a nucleic acid sequence encoding for an exogenous polymerase.

[00055] The host cell may be specifically chosen as a host cell capable of expressing the gene. In addition or otherwise, in order to produce a peptide or protein, a fragment of the peptide or protein or a fusion protein of the peptide or protein with another polypeptide, the nucleic acid coding for the peptide or protein can be genetically engineered for expression in a suitable system. Transformation can be performed using standard techniques (Sambrook, J. et al. (2001), supra).

[00056] Prokaryotic or eukaryotic host organisms comprising such a vector for recombinant expression of a peptide or protein of interest as described herein form also part of the present invention. Suitable host cells can be prokaryotic cell. In certain embodiments the host cells are selected from the group consisting of gram positive and gram negative bacteria. In some embodiments, the host cell is a gram negative bacteria, such as E.coli. In certain embodiments, the host cell is E. coli, in particular E. coli BL21 (DE3) or other E. coli K12 derivatives. In further embodiments, the host cell is selected from the group consisting of Escherichia coli (E. coli), Pseudomonas, Serratia marcescens, Salmonella, Shigella (and other enterobacteriaceae), Neisseria, Hemophilus, Klebsiella, Proteus, Enterobacter, Helicobacter, Acinetobacter, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, Legionella, acetic acid bacteria, Bacillus, Bacilli, Carynebacterium, Clostridium, Listeria, Streptococcus, Staphylococcus, and Archaea cells. Suitable eukaryotic host cells are among others CHO cells, insect cells, fungi, yeast cells, e.g., Saccharomyces cerevisiae, S. pombe, Pichia pastoris.

[00057] The transformed host cells are cultured under conditions suitable for expression of the nucleotide sequence encoding a peptide or protein of the invention.

[00058] For producing the recombinant peptide or protein of interest, a vector of the invention is introduced into a suitable prokaryotic or eukaryotic host organism by means of recombinant DNA technology (as already outlined above). For this purpose, the host cell is first transformed with a vector comprising a nucleic acid molecule according to the present invention using established standard methods (Sambrook, J. et al. (2001), supra). The host cell is then cultured under conditions, which allow growth of the host cell and/or expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide. Subsequently, the polypeptide is recovered either from the cell or from the cultivation medium.

[00059] In various embodiments, the present invention thus relates to methods for the production of the polypeptide according to the invention, where the method comprises incubating the recombinant host cells according to the invention under conditions that allow expression of the polypeptide; and isolating the expressed polypeptide.

[00060] For expression of the peptides and proteins of the present invention several suitable protocols are known to the skilled person.

[00061] Very generally, the expression of a recombinant peptide or protein of the present invention may be achieved by the following method comprising: (a) transforming a suitable host cell with a vector as disclosed herein, wherein the vector encodes the recombinant peptide or protein; (b) cultivating the host cell in a culture medium under conditions that allow growth of the host cell; (c) cultivating the host cell in a culture medium under conditions that allow expression of the recombinant peptide or protein; and optionally (d) purifying the protein. Thus, the present invention also relates to such methods.

[00062] In some embodiments, the transformation may be achieved using electroporation or heat shock treatment of the host cell.

[00063] Generally, any known culture medium suitable for growth of the selected host may be employed in this method. In various embodiments, the medium is a rich medium or a minimal medium. Also contemplated herein is a method, wherein step (b) and step (c) comprise the use of different media. For example, step (b) may be performed using a rich medium which is replaced by a minimal medium in step (c). In certain cases, the medium is selected from the group consisting of LB medium, TB medium, 2YT medium, and minimal medium.

[00064] In some embodiments, glycerol is added to the culture medium in concentrations of 0.1 v/v% to up to 50 v/v%.

[00065] The inventors surprisingly found that the addition of detergents to the growth medium positively influences the amount and activity of the expressed diadenylate cyclases. It is hypothesized that this effect is due to the solubilization and stabilization of the expressed DAC proteins. Suitable detergents are known in the art and include, for example, Triton X-100 (Ci ₄ H ₂₂ 0 ^' (C ₂ H ₄ 0)„), a nonionic surfactant which has a hydrophilic polyethylene oxide chain (on average it has 9.5 ethylene oxide units) and an aromatic hydrocarbon lipophilic/hydrophobic group. The hydrocarbon group is a 4-(l,l,3,3-tetramethylbutyl)-phenyl group. Other nonionic surfactants suitable for this purpose generally include alkoxylated fatty acid alcohols, block copolymers of polyalkenylglycols, such as and polyethyleneglycol and polyproypylenglycol and the like. The detergent may be present in the medium in a concentration of between about 0.1 to about 5 % by weight, preferably between about 0.5 and 2 % by weight, more preferably at about 1 % by weight.

[00066] In certain embodiments, the host cell is a prokaryotic cell, such as E.coli, in particular E.coli BL21 (DE3).

[00067] The obtained polypeptides may be separated from the cells or the medium and subjected to a variety of purification steps. For example, the recombinant peptide may be purified using a method selected from affinity chromatography, ion exchange chromatography, reverse phase chromatography, size exclusion chromatography, and combinations thereof.

[00068] The polypeptides of the invention having diadenylate cyclase activity may be used for the synthesis of cyclic di-AMP (c-di-AMP). In various embodiments, such a synthesis is an in vitro method comprising incubating the isolated polypeptide having DAC activity according to the invention with ATP under conditions that allow production of c-di-AMP.

[00069] The reaction may be carried out in a suitable reaction medium, such as an aqueous solution that can contain various excipients and auxiliaries, including, but not limited to pH buffering agents, salts, stabilizers, and surfactants. In various embodiments, the reaction medium contains magnesium ions.

[00070] In various embodiments of the present invention, the reaction is carried out at a pH greater than 7, preferably greater than 7.5, more preferably in the range between 8 and 10, most preferably about 9. For pH adjustment, various buffer substances known in the art may be used, including, but not limited to Tris (tris(hydroxymethyl)aminomethane), HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid), phosphates, bicarbonates and the like.

[00071] The reaction may be carried out at elevated temperature, for example in the range of between about 25 and 80 °C, preferably between about 35 and 75°C, more preferably between about 50 and 70 °C.

[00072] The present invention further covers the use of the phosphodiesterases of the invention for the synthesis of linear diadenosine monophosphate. An exemplary in vitro method for the synthesis of linear diadenosine monophosphate (5'pApA) comprises incubating the isolated polypeptide having phosphodiesterase activity according to the invention with cyclic di-AMP under conditions that allow production of 5'pApA. The reaction may be carried out under the conditions described above in connection with the synthesis of c-di-AMP. In other embodiments, the reaction temperature may preferably be in the range of between 20 and 50 °C, more preferably between 25 and 40 °C.

[00073] In one embodiment, the two methods can be combined so that in a first step c-di-AMP is produced which is then, in a second step, cleaved to yield 5'-pApA. This combined production method may be done in a one pot synthesis. [00074] The present invention thus also covers the uses of the invented polypeptides for the synthesis of c-di-AMP and 5 'pApA, respectively.

[00075] The above methods and uses can also be employed to facilitate the production of radioactively labelled c-di-AMP and 5'pApA. To achieve this, for example radioactively labelled ATP may be used as the educt, for example ³² P-labelled ATP. Other suitable radiolabels include ¹⁴ C and ³ H.

[00076] The enzymes used in the above-described synthesis methods may be recycled in that after termination of the reaction they are recovered and, if necessary, purified before being used in another round of synthesis.

[00077] After synthesis of c-di-AMP and linear 5'-pApA, these may be separated from other components of the reaction mixture by any suitable technique, including but not limited to High Performance Liquid Chromatography (HPLC). If HPLC is used, the column used may be a reverse phase (RP) CI 8 column and the solvent system may comprise Triethyl ammonium bicarbonate (pH 7.0, adjusted with acetic acid) and 9% methanol.

[00078] The present invention is further illustrated by the following examples. However, it should be understood, that the invention is not limited to the exemplified embodiments.

EXAMPLES

Example 1: Screening of genes

[00079] Screening of genes that encode thermophilic DAC proteins. The genomes of thermophilic bacteria such as Thermotoga maritime, Thermoanaerobacter ethanolicus X514, Methanocaldococcus jannaschii, Geobacillus thermodenitrificans, Thermoanaerobacter tengcongensis, Fervidobacterium nodosum RT17-B1, Thermosipho melanesiensis BI429, Moorella thermoacetica were screened for non- membrane DAC domains proteins that potentially contain enzymatically competent DAC domain. Bioinformatic tools were used to predict the cellular location and solubility of the proteins. Among several proteins selected for protein expression test based on the analysis results, MJ1002 was found to be the most stable and enzymatically active candidate. The protein and DNA sequences of MJ1002 are set forth in SEQ ID Nos. 1 and 2, respectively. Protein and DNA sequences of homologs of MJ1002 that originate from other thermophilic bacteria such as Methanococcus maripaludis, Methanococcus vannielii, Methanococcus aeolicus, Methanocaldococcus fervens, Methanocaldococcus vulcanius, Methanocaldococcus sp. strain DSM 12094, and Methanothermococcus okinawensis were subsequently identified and are set forth in SEQ ID Nos. 3-22.

[00080] Screening of genes that encode thermophilic YybT proteins. The genomes of thermophilic bacteria such as Thermotoga maritime, Thermoanaerobacter ethanolicus X514, Thermoanaerobacter tengcongensis, Fervidobacterium nodosum RT17-B1, Methanocaldococcus jannaschii, Geobacillus thermodenitrificans, Thermosipho melanesiensis BI429, Moorella thermoacetica were screened for YybT domains proteins that potentially contain enzymatically competent PDE domain. Bioinformatic tools were used to predict the solubility of the proteins. Among several proteins selected for protein expression test based on the analysis results, the truncated (j/YybT protein with the amino acid sequence set forth in SEQ ID NO:25 was found to be the most stable and enzymatically active one. The protein and DNA sequences of GtYybT full length are set forth in SEQ ID Nos. 23 and 24, and the respective sequences of the truncated version are set forth in SEQ ID Nos. 25 and 26. Example 2: Cloning, expression and purification of the enzymes

[00081] A DNA construct encoding for the protein MJJ002 gene (SEQ ID NO:2) was codon-optimized and synthesized and the DNA construct was digested from the plasmid and cloned into the expression vector pET28b (+) (Novagen) between the Ndel and Xhol restriction sites. The plasmid harboring the DNA construct and the (His) ₆ tag- encoding sequence was transformed into Escherichia coli strain BL21 (DE3). Cell stocks were stored in 20% glycerol at -80°C.

[00082] For protein expression, 2 ml of inoculum from the cell stock was added to 1 L of Luria-Bertani (LB) medium. Bacterial culture was grown at 37 °C up to OD 0.8 before being induced with 0.8 mM isopropyl-b-D-thiogalactoside (IPTG) at 16°C for overnight. The cells were harvested by centrifugation and the pellets were lysed by sonication in 20 ml of lysis buffer containing 20 mM Tris, 300mM NaCl, 5% Glycerol, pH 8 with 0.5% triton 0.1% 3-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride (PMSF). After centrifugation at 25,000 rpm for 30 min, the supernatant was filtered and then incubated with 2 ml of Ni -nitrilotriacetic acid (NTA) resin (Qiagen) for 30 min at 4 °C. The resin was washed with 50 ml of Wl buffer (lysis buffer with 20 mM imidazole) and 30 ml of W2 buffer (lysis buffer with 50 mM imidazole). The proteins were eluted using a stepped gradient method, with the elution buffer containing 20 mM Tris (pH 8.0), 200 mM NaCl, 5% glycerol, and 200, 300, and 500 mM imidazole. After sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) analysis, fractions with purity higher than 95% were pooled together and further purified using size exclusion chromatography as described below.

[00083] The gene encoding GtYybT from Geobacillus thermodenitrificans (C?tYybT _55-658 ; SEQ ID NO:26) was codon-optimized and cloned into pET28(a+) (Novagen) between the Ndel and Xhol restriction sites. Escherichia coli strain BL21 (DE3) was transformed with the plasmid for the expression of the N-terminal (His) ₆ - tagged recombinant construct GtYybT ₅₅ . ₆₅₈ . One liter of inoculated bacterial culture (LB medium) was grown up to O.D. of 0.8 before inducing with 0.8 mM IPTG. The culture was kept shaking at 16 °C for ~12 hours before it was pelleted by centrifugation. The cells were lysed in 20 ml lysis buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 5% Glycerol, 0.1 % /3-mercaptoethanol, and 1 mM PMSF). After the centrifugation at 25,000 rpm for 30 min, the supernatant was filtered and then incubated with 2 ml of Ni- NTA resin (Qiagen) for 1 hour at 4 °C. The resin was washed with 50 ml of Wl buffer (lysis buffer with 20 mM imidazole) and 20 ml of W2 buffer (lysis buffer with 50 mM imidazole). The proteins were eluted using a step gradient method with the elution buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5% glycerol and 200mM, 300 mM, or 500 mM imidazole. After analyzed by SDS-PAGE, fractions with purity higher than 95% were pooled together. Size-exclusion chromatography was carried out at 4 °C using the AKTA FPLC system equipped with a Superdex 200 HR 16/60 column (Amersham Biosciences). The buffer used for gel filtration was comprised of 50 mM Tris-HCl (pH 8.0), 150 mM NaCl and 5% Glycerol. The proteins were stored in -80 °C freezer after flash freezing and concentration measured by Bradford assay method.

[00084] The domain structures of the proteins MJ1002 (SEQ ID NO: l) and GtYybT full length (SEQ ID NO:23) and the 55-658 fragment (SEQ ID NO:25) are shown in Figure 4.

Example 3: Enzyme assays

[00085] The enzymatic synthesis of c-di-AMP and 5'pApA is schematically illustrated in Figure 3.

[00086] An enzymatic assay was conducted to determine the diadenylate cyclase activity of MJ1002 (SEQ ID NO: l) with the buffer containing 50 mM Tris-HCl (pH 8.0), 250 mM NaCl, 10 mM MgCl ₂ , 100 μΜ ATP, 0.1 to 1 μΜ enzyme. Enzyme assays were conducted to determine the optimal pH for the enzymatic reaction, with the buffer containing 50 mM Tris-HCl (pH 5.0 - 9.0), 250 mM NaCl, 10 mM MgCl ₂ , 100 μΜ ATP, and 1 μΜ enzyme. For enzymatic reaction, the reaction mixtures were incubated in a water bath at 60° for 15 min. The reaction was stopped by the addition of 100 mM EDTA and centrifuging at 13,000 rpm for 5 min to remove protein precipitate, the supernatant was loaded onto an Eclipse XDB-C18 column (4.6 X 150 mm) for quantifying the product c-di-AMP using an Agilent LCI 200 system (mobile phase: 20 mM triethylammoniumbicarbonate [pH 7.0, pH adjusted using acetic acid] and 9% methanol; flow rate: 1 ml/min).

[00087] The assay for GtYybT protein (SEQ ID NO:25) was carried out by monitoring the formation of 5'-pApA using HPLC. Assay buffer and HPLC conditions are the same as described above. Initial velocity at a certain substrate concentration was obtained from a series of reactions that were stopped at various time points.

Example 4: Production of c-di-AMP and 5'-pApA

[00088] c-di-AMP was synthesized by incubating MJ1002 (SEQ ID NO: l) and ATP 60 or 70 °C in a reaction vessel that that contains 30-50 ml of 50 mM Tris buffer (pH 7.5), 250 mM NaCl, 20 mM MgCl ₂ , 1 mM ATP, 1 mM DTT and 5-10 μΜ enzyme. After more than 95% of ATP was converted to c-di-GMP as estimated by HPLC analysis (usually within 10 min), fresh ATP was supplemented in batches. After the reaction, the protein was precipitated and removed by heat treatment and the supernatant was concentrated using a rotary evaporator (Eyela) with the waterbath temperature set at 45 °C. The concentrated supernatant was loaded onto a semi- preparative Eclipse XDB-C18 (9.4x250 mm) column for purification by using the LC1200 system (Agilent). Sample injection and collection of c-di-AMP fraction were automated with an autosampler (Loading volume: 1 ml) and a fraction collector with 6 ml collecting vials. C-di-AMP was eluted using the same mobile phase described above with a flow rate of 3 ml/min. The fractions that contain c-di-AMP were pooled, concentrated by evaporation, and finally lyophilized to yield the white powder that was dissolved into 5 mM Tris buffer (pH 7.0) for storage at -20°C. The identity of the product was confirmed by MALDI-Mass spectrometry and comparison with standard from commercial source (Biolog, Germany).

[00089] Production of 5'-pApA was performed by incubating c-di-AMP with GtYybT (SEQ ID NO: 25) in a similar fashion as described above under the reaction conditions of 100 mM Tris (pH8.3), 20 mM KC1, 0.5 mM MnCl _2, 30 °C. Complete turnover of 10 mg of c-di-AMP to 5'-pApA was observed with 10 μΜ enzyme within 15 min.

Example 5: Synthesis of ³² P-labeIled c-di-AMP and 5'-pApA

[00090] ³² P-labelled c-di-AMP was synthesized by incubating MJ1002 (SEQ ID NO: 1) and ³² P-labelled ATP 60 °C in 250 μΐ buffer that contains 50 mM Tris buffer (pH 7.5), 250 mM NaCl, 20 mM MgCl ₂ , 1 mM DTT and 1 μΜ enzyme for 10 min. After the reaction, the protein was precipitated and removed by heat treatment and filtration. [00091] Production of ³² -P labeled 5'-pApA was performed by incubating the in situ prepared P-c-di-AMP with GtYybT in a similar fashion as described above under the reaction conditions of 100 raM Tris (pH8.3), 20 mM KC1, 0.5 mM MnCl _2, 30 °C. Complete turnover of can be obtained within 15 min.

[00092] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[00093] One skilled in the art would readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. Further, it will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods^ procedures, treatments, molecules and specific compounds described herein are presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art which are encompassed within the spirit of the invention are defined by the scope of the claims. The listing or discussion of a previously published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[00094] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. The word "comprise" or variations such as "comprises" or "comprising" will accordingly be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or group of integers. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by exemplary embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[00095] The content of all documents and patent documents cited herein is incorporated by reference in their entirety.