Phosphatase expression system

Abstract

Provided herein, inter alia, are compositions and methods for expressing a recombinant phosphatase containing a plurality of disulfide bonds in a host cell.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 371,616, filed on August 16, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Incorporation of sequence listing by reference

[0004] This application is filed together with a sequence listing in electronic format. The sequence listing is provided as a file named NB42134 - WO - PCT.xml created on August 8, 2023, and having a size of 73,483 bytes. The information in the sequence listing in electronic format is incorporated herein by reference in its entirety. Technical field

[0005] Compositions and methods for expressing phosphatases are provided herein. Background art

[0006] The Gram - positive eubacterium Bacillus subtilis is well - known for its strong ability to secrete proteins both in its soil - based natural habitat and in biotechnological applications for the production of recombinant proteins (Darmo et al., Appl. Environ. Microbiol, 2006, 72(11):6876–85).

[0007] It is believed that most polypeptides secreted by Bacillus subtilis are exported from the cytoplasm in an unfolded form and must be efficiently folded after membrane translocation. However, the expression and secretion of polypeptides containing multiple disulfide bonds can induce a secretion stress response in Bacillus subtilis host cells, thereby reducing overall expression and secretion (Darmon et al., Applied and Environmental Microbiology, 2006, 72(11):6876–85). Other studies have found similar difficulties in expressing polypeptides containing disulfide bonds in Bacillus subtilis (Westers et al., Biochimica et Biophysica Acta, 2004, 1694:299–310).

[0008] In Bacillus and other Gram-positive bacteria, little is known about disulfide bond formation and isomerization. The existence of thiol-disulfide oxidoreductases in these organisms has long been questioned because disulfide bridges have not been found in the proteins they secrete, and the formation efficiency of disulfide bonds in heterologous proteins secreted is very low (Westers et al., Biochimica et Biophysica Acta, 2004, 1694:299–310; Bolhuis et al., Appl. Environ. Microbiol., 1999, 65:2934–41).

[0009] Accordingly, there is a need for a Bacillus subtilis host cell expression system that can produce polypeptides that are correctly folded and contain functional disulfide bonds in commercially relevant amounts.

[0010] The subject matter disclosed herein addresses these needs and provides additional benefits. SUMMARY OF THE INVENTION

[0011] The present invention particularly provides compositions and methods for expressing recombinant phosphatases (such as acid phosphatase and / or apyrase) containing multiple disulfide bonds in Gram-positive host cells (such as Bacillus subtilis host cells).

[0012] Accordingly, in some aspects, the present disclosure provides a recombinant Gram-positive host cell comprising a nucleic acid that is at least about 60% identical to SEQ ID NO:9 or SEQ ID NO:37 or a fragment thereof, wherein the host cell comprises a deletion of one or more endogenous genes encoding a protease. In some embodiments, the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:10 or SEQ ID NO:25 or a functional fragment thereof. In some embodiments, the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28 or a functional fragment thereof. In some embodiments of any of the embodiments disclosed herein, the host cell has one, two, three, four, five, six, seven, eight or nine endogenous genes encoding the deleted protease. In some embodiments, the host cell comprises nine endogenous genes encoding the deleted protease. In some embodiments of any of the embodiments disclosed herein, one or more endogenous genes encoding a protease comprise one or more of aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and / or nprB. In some embodiments of any of the embodiments disclosed herein, the polypeptide comprises one or more disulfide bonds. In some embodiments, the polypeptide comprises seven disulfide bonds. In some embodiments of any of the embodiments disclosed herein, the nucleic acid is expressed on an extrachromosomal vector. In some embodiments of any of the embodiments disclosed herein, the nucleic acid is integrated into the genome of the host cell. In some embodiments of any of the embodiments disclosed herein, the host cell is a Bacillus spp. In some embodiments, the Bacillus spp. is Bacillus subtilis. In some embodiments, the polypeptide is secreted from the host cell.

[0013] In a further aspect, the present disclosure provides a method for producing a recombinant protein, the method comprising culturing any of the host cells disclosed herein in a suitable medium. In some embodiments, the method further comprises purifying the recombinant protein.

[0014] Each of the aspects and embodiments described herein can be used together, unless explicitly or clearly excluded from the context of the embodiment or aspect.

[0015] Throughout the specification, various patents, patent applications and other types of publications are cited (e.g., journal articles, electronic database entries, etc.). For all purposes, the disclosures of all patents, patent applications and other publications cited herein are hereby incorporated by reference in their entireties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1It is a line graph depicting the production of apyrase from Glaciecola xiamenensis CRC22110 in Bacillus subtilis AP186 and the negative control strain AP128 during a time-course experiment.

[0017] Figure 2 It is a line graph depicting the comparison of ATPase activities in culture samples of Escherichia coli host strains AP391 (encoding the CRC22110 gene) and AP390 (lacking the gene of interest). Detailed implementation

[0018] The present invention is at least partially based on the inventors' unexpected discovery that expression of a phosphatase protein in a Gram-positive host cell, such as a Bacillus subtilis host cell, can result in useful functional enzyme yields. Despite the extensive disulfide bonding of the phosphatase protein, production of the functional protein still occurs. As discussed in the background section, the existing literature indicates that disulfide bond formation can induce a secretion stress response in Gram-positive expression hosts, such as Bacillus subtilis, thereby reducing overall expression and secretion.

[0019] I. Definitions

[0020] The term "acid phosphatase" (EC 3.1.3.2) (also known as acid phosphomonoesterase, phosphomonoesterase, glycerophosphatase, acid monophosphatase, acid phosphohydrolase, acid phosphomonoester hydrolase, uterotransferrin, acid nucleoside diphosphatase, acid phosphatase (class A), or orthophosphomonoester phosphohydrolase (acid optimum)) is used to mean an enzyme that has a pH optimum for mediating the hydrolysis of phosphoester bonds in a substrate at a pH of less than about pH 6.5, such as less than about pH 4.0. Acid phosphatases release attached phosphoryl groups from other molecules during digestion. It can be further classified as a phosphomonoesterase. Acid phosphatases are stored in lysosomes and function when lysosomes fuse with endosomes, which are acidified during their function; thus, acid phosphatases have an acidic pH optimum. The enzyme is present in many animal and plant species. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from Shigella. In some embodiments, the acid phosphatase used in the compositions and methods disclosed herein is not derived from mammals. In some embodiments, the enzyme expressed in any of the host cells disclosed herein (e.g., a Bacillus subtilis host cell) is an acid phosphatase.

[0021] As used herein, the “GDA1_CD39 superfamily” refers to enzymes consisting of nucleoside triphosphate diphosphohydrolases (NTPDases) that have a common motif in their protein sequences. The family is named after two proteins: yeast GDPase (GDA1) and lymphocyte activation antigen CD39. In some embodiments, these proteins are cell surface enzymes that hydrolyze a range of NTPs, including extracellular ATP. Non-limiting examples include ecto-ATPase, apyrase, CD39, and ecto-ATP diphosphohydrolase (ecto-ATP / Dase) (Knowles, 2011, Purinergic Signalling, Volume 7, pp. 21-45, which is incorporated herein by reference). In some embodiments, the enzymes expressed in any of the host cells disclosed herein (e.g., Bacillus subtilis host cells) are members of the GDA1_CD39 superfamily.

[0022] As used herein, the term "apyrase" refers to one or more of the calcium-activated enzymes (i.e., proteins belonging to the class EC.3.6.1.5) that have ATP-diphosphohydrolase activity and catalyze the hydrolysis of the γ-phosphate ester from ATP and the hydrolysis of the β-phosphate ester from ADP. Apyrases are found in all eukaryotes and some prokaryotic organisms, indicating that these enzymes retain their role in different species. They possess unique phosphohydrolase activity, nucleotide substrate specificity, divalent cation requirements, and sensitivity to inhibitors. (See, Plesner, Int. Rev. Cytol. [International Review of Cytology], 158:141 (1995); and Handa and Guidotti, Biochem. Biophys. Res. Commun. [Biochemical and Biophysical Research Communications], 218(3):916 (1996)). In mammals, apyrase is thought to function primarily as an extracellular hydrolase specific for ATP and ADP, a function that is important for the inactivation of synaptic ATP molecules following nerve stimulation. (See, Todorov et al., Nature [Nature], 387(6628):76 (1997)). Apyrase in mammals is also thought to be important for inhibiting ADP-induced platelet aggregation. (See, Marcus et al., J. Clin. Invest. [Journal of Clinical Investigation], 99(6):1351 (1997)). Recombinant apyrase is commercially available from New England Biolabs. In some embodiments, the apyrase used in the compositions and methods disclosed herein is not derived from potato. In other embodiments, the apyrase used in the compositions and methods disclosed herein is not derived from mammals. In some embodiments, the enzyme expressed in any host cell (e.g., a Bacillus subtilis host cell) disclosed herein is apyrase.

[0023] As used herein, "microorganism" or "microbe" refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0024] The terms "protein" and "polypeptide" refer to compounds containing amino acids linked via peptide bonds and can be used interchangeably. A "protein" or "polypeptide" contains a polymeric sequence of amino acid residues. Throughout this disclosure, the single-letter and 3-letter codes for amino acids as defined by the Joint Commission on Biochemical Nomenclature (JCBN) of the IUPAC-IUB are used. The single-letter X refers to any of the twenty amino acids. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence. The amino acid position in a given polypeptide sequence can be named by the single-letter code of the amino acid followed by the position number. For example, glycine (G) at position 87 is denoted as "G087" or "G87".

[0025] As used herein, when referring to an "amino acid sequence", it means the amino acid sequence of a protein or peptide molecule. An "amino acid sequence" can be deduced from the nucleic acid sequence encoding the protein. However, terms such as "polypeptide" or "protein" are not intended to limit the amino acid sequence to the deduced amino acid sequence, but can include post-translational modifications of the deduced amino acid sequence, such as amino acid deletions, additions, and modifications (such as glycosylation and addition of lipid moieties). Additionally, unless otherwise stated, the use of non-natural amino acids such as D-amino acids to improve stability or pharmacokinetic behavior falls within the scope of the term "amino acid sequence".

[0026] The term "mature" form of a protein, polypeptide, or peptide refers to the functional form of a protein, polypeptide, or enzyme without a signal peptide sequence and a propeptide sequence.

[0027] With respect to an amino acid sequence or a nucleic acid sequence, the term "wild-type" indicates that the amino acid sequence or nucleic acid sequence is a natural or naturally occurring sequence. As used herein, the term "naturally occurring" refers to any substance found in nature (e.g., a protein, amino acid, or nucleic acid sequence). In contrast, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant / engineered nucleic acid and protein sequences produced in the laboratory, or modifications of wild-type sequences).

[0028] As used herein, the terms "sequence identity" or "sequence similarity" mean that two polynucleotide sequences (a candidate sequence and a reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. When comparing the candidate sequence to the reference sequence, the candidate sequence may contain additions or deletions (i.e., gaps) as compared to the reference sequence used for the best alignment of the two sequences (which does not contain additions or deletions). The best sequence alignment for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (such as ALIGN or Megalign (DNASTAR, Inc.)), or by inspection.

[0029] As used herein, the term "percent (%) sequence identity" or "percent (%) sequence similarity" with respect to a reference sequence is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the residues in the reference polynucleotide sequence after the sequences have been optimally aligned and gaps, if necessary, are introduced to achieve the maximum percent sequence identity.

[0030] As used herein, with respect to an amino acid residue position, "corresponding to" or "corresponds to" or "correspond to" means the amino acid residue at the recited position in a protein or peptide, or an amino acid residue that is similar, homologous, or equivalent to the recited residue in a protein or peptide.

[0031] As used herein, the terms "recombinant" or "non-natural" refer to an organism, microorganism, cell, nucleic acid molecule, vector, etc. that has at least one engineered genetic alteration or has been modified by the introduction of a heterologous nucleic acid molecule; or to a cell (e.g., a gram-positive cell) that has been altered such that the expression of a heterologous nucleic acid molecule or an endogenous nucleic acid molecule or gene can be controlled. Recombinant also refers to a cell that is derived from a non-natural cell or is the progeny of a non-natural cell that has one or more such modifications. Genetic alterations include, for example, modifications that introduce an expressible nucleic acid molecule encoding a protein, or other nucleic acid molecule additions, deletions, substitutions, or other functional alterations to the genetic material of a cell. For example, a recombinant cell may express the same or a homologous form of a gene or other nucleic acid molecule (e.g., a polynucleotide construct) that is not found in a natural (wild-type) cell, or may provide an altered pattern of endogenous gene expression, such as overexpression, underexpression, minimal expression, or no expression at all.

[0032] "Recombination", "recombining", or generating "recombined" nucleic acids generally refers to the assembly of two or more nucleic acid fragments, where the assembly results in a chimeric DNA sequence that would not otherwise be found in the genome.

[0033] The term "derived from" encompasses the terms "originated from", "obtained from", "obtainable from", and "produced from", and generally indicates that a specified material or composition finds its origin in another specified material or composition, or has characteristics that can be described with reference to another specified material or composition. For example, the recombinant Gram-positive bacterial cells of the present disclosure can be derived from / obtained from any known Gram-positive bacterial strain (e.g., Bacillus subtilis strain 168, etc.).

[0034] As used herein, an "endogenous gene" refers to a gene located in its natural position in the genome of an organism.

[0035] As used herein, a "heterologous" gene, "non-endogenous" gene, or "exogenous" gene refers to a gene (or gene coding sequence (CDS) / open reading frame (ORF)) that is not normally found in a host organism but is introduced into the host organism by gene transfer. The term "exogenous" gene includes a natural gene (or ORF) inserted into a non-natural organism and / or a chimeric gene inserted into a natural or non-natural organism.

[0036] As used herein, a "heterologous control sequence" refers to a gene expression control sequence (e.g., promoter, enhancer, terminator, etc.) that does not function inherently to regulate (control) the expression of a gene of interest. Typically, heterologous nucleic acids are not endogenous (natural) to the part of the cell or genome in which they are present and have been added to the cell by infection, transfection, transduction, transformation, microinjection, electroporation, etc. A "heterologous" nucleic acid construct can contain a combination of control sequences / DNA coding (ORF) sequences that is the same as or different from the combination found in a natural host cell.

[0037] As used herein, the terms "signal sequence" and "signal peptide" refer to a sequence of amino acid residues that can participate in the secretion or directed transport of a mature protein or a precursor form of a protein. Typically, the signal sequence is located at the N-terminus of the precursor or mature protein sequence. The signal sequence can be endogenous or exogenous. The signal sequence is generally not present in the mature protein. Typically, the signal sequence is cleaved from the protein by signal peptidase during translocation.

[0038] As used herein, the term "expression" refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid molecule of the present disclosure. Expression can also refer to the translation of mRNA into a polypeptide. Thus, the term "expression" includes any steps involved in polypeptide production, which include but are not limited to transcription, post-transcriptional modification, translation, post-translational modification, secretion, etc.

[0039] As used herein, "nucleic acid" refers to nucleotide or polynucleotide sequences and fragments or portions thereof, as well as DNA, cDNA, and RNA of genomic or synthetic origin, which may be double-stranded or single-stranded, whether representing the sense or antisense strand. It should be understood that due to the degeneracy of the genetic code, multiple nucleotide sequences can encode a given protein. It should be understood that the polynucleotides (or nucleic acid molecules) described herein include "genes", "vectors", and "plasmids". Accordingly, the term "gene" refers to a polynucleotide encoding a specific sequence of amino acids, which includes all or part of the protein-coding sequence and may include regulatory (non-transcribed) DNA sequences, such as promoter sequences, which determine, for example, the conditions under which the gene is expressed. The transcribed region of a gene may include untranslated regions (UTRs) (including introns, 5'-untranslated regions (UTRs), and 3'-UTRs) as well as the coding sequence.

[0040] As used herein, the term "coding sequence" refers to a nucleotide sequence that directly specifies the amino acid sequence of its (encoded) protein product. The boundaries of a coding sequence are typically determined by a reading frame (hereinafter, "ORF") that usually begins with an ATG start codon. Coding sequences typically include DNA, cDNA, and recombinant nucleotide sequences.

[0041] As used herein, the term "promoter" refers to a nucleic acid sequence capable of controlling the expression of a coding sequence or functional RNA. Typically, the coding sequence is located downstream (3′) of the promoter sequence. A promoter may be derived entirely from a native gene, or may be composed of different elements derived from different promoters found in nature, or may even contain synthetic nucleic acid segments.

[0042] As used herein, the term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment such that the function of one is affected by the other. For example, a promoter is effectively linked to a coding sequence (e.g., an ORF) when expression of the coding sequence can be achieved (i.e., the coding sequence is under the transcriptional control of the promoter). The coding sequence can be operably linked to the regulatory sequence in the sense or antisense direction. Thus, a nucleic acid is "effectively linked" to another nucleic acid sequence when placed in a functional relationship with the other nucleic acid sequence. For example, if DNA encoding a secretory leader sequence (i.e., a secretory signal sequence) is expressed as a preprotein involved in polypeptide secretion, the DNA encoding the secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader sequence, contiguous and in the reading phase. However, an enhancer need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide linkers or adaptors are used according to conventional practice.

[0043] As used herein, a "functional promoter sequence that controls the expression of a gene of interest (or its open reading frame) linked to the protein-coding sequence of the gene of interest" refers to a promoter sequence that controls the transcription and translation of the coding sequence in a desired Gram-positive host cell. For example, in certain embodiments, the present disclosure relates to a polynucleotide comprising an upstream (5') promoter (or 5' promoter region, or tandem 5' promoters, etc.) that is functional in a Gram-positive cell, wherein the promoter region is operably linked to a nucleic acid sequence (e.g., an ORF) encoding an acid phosphatase protein.

[0044] As used herein, a "suitable regulatory sequence" refers to a nucleotide sequence located upstream (5' non-coding sequence), within, or downstream (3' non-coding sequence) of a coding sequence and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, transcriptional leader sequences, RNA processing sites, effector binding sites, and stem-loop structures.

[0045] As used herein, the terms "modification" and "genetic modification" are used interchangeably and include, but are not limited to: (a) introducing, substituting, or removing one or more nucleotides in a gene (or its ORF), or introducing, substituting, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene or its ORF, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) specific mutagenesis of any one or more of the genes disclosed herein and / or (g) random mutagenesis.

[0046] As used herein, "disruption of a gene" or "gene disruption" are used interchangeably and broadly refer to any genetic modification that substantially prevents a host cell from producing a functional gene product (e.g., a protein). Thus, as used herein, gene disruption includes, but is not limited to, frameshift mutations, premature stop codons (i.e., such that a functional protein is not produced), substitutions that eliminate or reduce protein activity (such that a functional protein is not produced), internal deletions, insertions that disrupt the coding sequence, mutations that remove the operable connection between the native promoter and the open reading frame required for transcription, and the like.

[0047] As used herein, as used in phrases such as "introducing into a bacterial cell" or "introducing into a bacterial cell" at least one polynucleotide open reading frame (ORF), or its gene, or its vector, the term "introducing" includes methods known in the art for introducing polynucleotides into cells, including but not limited to protoplast fusion, natural or artificial transformation (e.g., calcium chloride, electroporation), transduction, transfection, conjugation, and the like.

[0048] As used herein, "transformed" or "transformation" means transforming a cell by using recombinant DNA technology. Transformation typically occurs by inserting one or more nucleotide sequences (e.g., a polynucleotide, ORF, or gene) into the cell. The inserted nucleotide sequence can be a heterologous nucleotide sequence (i.e., a sequence that is not naturally present in the cell to be transformed). Thus, transformation generally refers to introducing foreign DNA into a host cell such that the DNA remains as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0049] As used herein, "transforming DNA", "transforming sequence", and "DNA construct" refer to DNA used to introduce a sequence into a host cell or organism. Transforming DNA is DNA used to introduce a sequence into a host cell or organism. The DNA can be generated in vitro by PCR or any other suitable technique. In some embodiments, the transforming DNA contains an input sequence, while in other embodiments, it further contains an input sequence flanked by homology boxes. In still other embodiments, the transforming DNA contains other non-homologous sequences added to the ends (i.e., filler sequences or flanks). The ends can be closed such that the transforming DNA forms a closed loop, such as when inserted into a vector.

[0050] As used herein, an "input sequence" refers to a DNA sequence introduced into the chromosome of a Gram-positive host cell. In some embodiments, the input sequence is part of a DNA construct. In other embodiments, the input sequence encodes one or more proteins of interest. In some embodiments, the input sequence comprises a sequence that may or may not be present in the genome of the cell to be transformed (i.e., it may be a homologous or heterologous sequence). In some embodiments, the input sequence encodes one or more proteins of interest, genes, and / or mutated or modified genes. In alternative embodiments, the input sequence encodes a functional wild-type gene or operon, a functional mutated gene or operon, or a non-functional gene or operon. In some embodiments, a non-functional sequence can be inserted into a gene to disrupt its function. In another embodiment, the input sequence includes a selectable marker. In additional embodiments, the input sequence includes two homology boxes.

[0051] As used herein, a "homology box" or "homology arm" refers to a nucleic acid sequence that is homologous to a sequence in the chromosome of a Gram-positive bacterial cell. More particularly, according to the present invention, a homology box is an upstream or downstream region that has a sequence identity between about 80% and 100%, between about 90% and 100%, or between about 95% and 100% with the directly flanking coding regions of a gene or a portion of a gene to be deleted, disrupted, inactivated, etc. These sequences direct the integration position of the DNA construct in the chromosome of the Gram-positive bacterial cell and direct which part of the chromosome of the Gram-positive bacterial cell is replaced by the input sequence. While not intended to limit the disclosure, a homology box can include between about 1 base pair (bp) and 200 kilobases (kb). In some embodiments, the homology box includes between about 1 bp and 10.0 kb; between 1 bp and 5.0 kb; between 1 bp and 2.5 kb; between 1 bp and 1.0 kb; and between 0.25 kb and 2.5 kb. The homology box can also include about 10.0 kb, 5.0 kb, 2.5 kb, 2.0 kb, 1.5 kb, 1.0 kb, 0.5 kb, 0.25 kb, and 0.1 kb. In some embodiments, the 5' and 3' ends of the selectable marker are flanked by homology boxes, where the homology box contains a nucleic acid sequence that is closely flanked by the coding region of a gene.

[0052] As used herein, the term "nucleotide sequence encoding a selectable marker" refers to a nucleotide sequence that is capable of being expressed in a host cell and wherein the expression of the selectable marker confers upon the cell containing the expressed gene the ability to grow in the presence of the corresponding selective reagent or in the absence of an essential nutrient.

[0053] As used herein, the terms "selectable marker" and "selection marker" refer to nucleic acids (e.g., genes) that are capable of being expressed in a host cell and that permit easy selection of those hosts that contain the vector. Examples of such selectable markers include, but are not limited to, antimicrobial agents. Thus, the term "selectable marker" refers to a gene that provides an indication that the host cell has taken up the input DNA of interest or that some other reaction has occurred. Typically, a selectable marker is a gene that confers antimicrobial resistance or a metabolic advantage on the host cell to allow differentiation of cells containing foreign DNA from cells that have not received any foreign sequences during transformation.

[0054] A "residing selectable marker" is a marker that is located on the chromosome of the microorganism to be transformed. The residing selectable marker encodes a gene different from the selectable marker on the transformation DNA construct. Selection markers are well known to those of skill in the art. As noted above, markers can be antimicrobial resistance markers (e.g., ampR, phleoR, specR, kanR, eryR, tetR, cmpR, and neoR). Other markers useful according to the present invention include, but are not limited to, auxotrophic markers such as serine, lysine, tryptophan; and detection markers such as β-galactosidase.

[0055] As defined herein, the "genome" of a host cell, the "genome" of a Gram-positive bacterial (host) cell, the "genome" of a Bacillus species (host) cell, etc. includes chromosomal genes and extrachromosomal genes.

[0056] As used herein, the terms "plasmid", "vector", and "cassette" refer to extrachromosomal elements that typically carry genes that are not part of the central metabolism of the cell and that are usually in the form of circular double-stranded DNA molecules. Such elements can be linear or circular self-replicating sequences, genomic integration sequences, phages, or nucleotide sequences of single-stranded or double-stranded DNA or RNA derived from any source, wherein multiple nucleotide sequences have been ligated or recombined into a single construct that is capable of introducing a promoter fragment and a DNA sequence for a selected gene product, along with appropriate 3' untranslated sequences, into a cell.

[0057] As used herein, the term "plasmid" refers to a circular double-stranded (ds) DNA construct that is used as a cloning vector and forms an extrachromosomal self-replicating genetic element in many bacteria and some eukaryotes. In some embodiments, the plasmid is incorporated into the genome of the host cell. In some embodiments, the plasmid is present in the parental cell and is lost in the daughter cells.

[0058] As used herein, "transformation cassette" refers to a specific vector that contains a gene (or its ORF) and, in addition to the foreign gene, has elements that facilitate the transformation of a specific host cell.

[0059] As used herein, the term "vector" refers to any nucleic acid that can replicate (propagate) in a cell and can carry a new gene or DNA segment into a cell. Thus, the term refers to a nucleic acid construct designed to be transferred between different host cells. Vectors include viruses, bacteriophages, proviruses, plasmids, phagemids, transposons, and artificial chromosomes (such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), PLACs (plant artificial chromosomes)), etc., that act as "episomes" (i.e., they replicate autonomously) or can integrate into the chromosome of the host organism.

[0060] As used herein, the terms "expression cassette" and "expression vector" refer to nucleic acid constructs that are recombinantly or synthetically produced and have a series of designated nucleic acid elements (i.e., these are vectors or vector elements, as described above) that permit the transcription of a specific nucleic acid in a target cell. A recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes (among other sequences) the nucleic acid sequence to be transcribed and a promoter. In some embodiments, the DNA construct also includes a series of designated nucleic acid elements that permit the transcription of a specific nucleic acid in a target cell. In certain embodiments, the DNA constructs of the present disclosure contain selectable markers and inactivated chromosomes, or genes, or DNA segments as defined herein.

[0061] As used herein, a "targeting vector" is a vector that includes a polynucleotide sequence that is homologous to a region in the chromosome of the host cell into which the targeting vector is transformed and that can drive homologous recombination at that region. For example, a targeting vector can be used to introduce a mutation into the chromosome of a host cell by homologous recombination. In some embodiments, the targeting vector contains other non-homologous sequences, such as those added to the ends (i.e., filler sequences or flanking sequences). The ends can be closed such that the targeting vector forms a closed loop, such as in an insertion vector. For example, in certain embodiments, a cell (such as a Bacillus species (host) cell) is modified (e.g., transformed) by introducing one or more "targeting vectors" into the cell.

[0062] As used herein, "flanking sequence" refers to any sequence that is upstream or downstream of the sequence being discussed (e.g., for gene A-B-C, gene B is flanked by the A and C gene sequences). In certain embodiments, the input sequence is flanked on each side by a homeobox. In another embodiment, the input sequence and the homeobox are contained within a unit that is flanked on each side by a filler sequence. In some embodiments, the flanking sequence is present only on one side (3' or 5'), but in some embodiments, it is on each side of the sequence being flanked. The sequence of each homeobox is homologous to a sequence in the Bacillus chromosome. These sequences direct where in the Bacillus chromosome the new construct will be integrated and where part of the Bacillus chromosome will be replaced by the input sequence. In other embodiments, the 5' and 3' ends of the selectable marker are flanked by polynucleotide sequences that contain portions of inactivated chromosomal segments. In some embodiments, the flanking sequence is present only on one side (3' or 5'), while in other embodiments, it is present on each side of the sequence being flanked.

[0063] As used herein, "host cell" refers to a cell that has the ability to serve as a host or expression vehicle for newly introduced DNA sequences. In certain aspects, the host cells of the present disclosure are Gram-positive bacterial cells / strains. As will be understood by those skilled in the art, many Gram-positive host strains are generally regarded as safe (GRAS) according to US FDA guidelines, and thus, Gram-positive host cells are particularly useful protein production hosts as compared to Gram-negative hosts (e.g., E. coli expression systems) that require additional expensive processing steps to remove endotoxins (e.g., LPS).

[0064] As used herein, the Bacillus subtilis strain named "CBS12" was constructed to express and secrete apyrase (SEQ ID NO:10 or SEQ ID NO:25) derived from Gallaecimonas xiamenensis.

[0065] As used herein, the terms “purified,” “isolated,” or “enriched” mean that a biomolecule (e.g., a polypeptide or polynucleotide) has been altered from its native state by separating it from some or all of the naturally-occurring components with which it is associated in nature. Such separation or purification can be accomplished by separation techniques recognized in the art, such as ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation, to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition. Components that provide additional benefits, such as activators, anti-inhibitors, desired ions, pH-controlling compounds, or other enzymes or chemicals, can then be further added to the purified or isolated biomolecule composition.

[0066] As used herein, “protein preparation” is any material, typically a solution, usually aqueous, that contains one or more proteins.

[0067] As used herein, the terms “broth,” “culture broth,” “fermentation broth,” and / or “whole fermentation broth” are used interchangeably and refer to a preparation produced by cell fermentation that does not undergo a processing step after fermentation is complete. For example, whole fermentation broth is typically produced when a microbial culture is grown to saturation and incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of a protein by a host cell; and optionally secretion of the protein into the cell culture medium). Typically, whole fermentation broth is unfractionated and contains spent cell culture medium, metabolites, extracellular polypeptides, and microbial cells.

[0068] As used herein, the phrase “treated broth” refers to a broth that has been conditioned by altering its chemical composition and / or physical characteristics. Broth “conditioning” can include one or more treatments such as cell lysis, pH adjustment, heating, cooling, addition of chemicals (e.g., calcium, salts, flocculants, reducing agents, enzyme activators, enzyme inhibitors, and / or surfactants), mixing, and / or timed holding of the broth (e.g., 0.5 to 200 hours) without further processing.

[0069] As used herein, the “cell lysis” process includes any cell lysis technique known in the art, including but not limited to enzyme treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, sonication), simply holding the culture without feed, etc.

[0070] As used herein, the terms "recovery", "recovered", and "recovering" refer to the separation of a protein from at least a portion of one or more components of a microbial broth, and / or the separation of a protein from at least a portion of one or more solvents (e.g., water or ethanol) in the broth.

[0071] In some aspects, the broth in which host cells have been fermented to produce a phosphatase protein is clarified, with or without broth treatment. As used herein, "clarified" broth means broth that has been subjected to at least one clarification process to remove cell debris and / or other insoluble components. As understood in the art, clarification processes include, but are not limited to, centrifugation techniques, crossflow membrane filtration techniques, solid / liquid filtration techniques, and the like.

[0072] "Cell debris" refers to cell walls and other insoluble components released or formed after the rupture of cell membranes (e.g., after a cell lysis process).

[0073] In some aspects, as understood in the art, the separation of solvents includes, but is not limited to, ultrafiltration, evaporation, spray drying, and freeze drying. The resulting solution is referred to as "clarified broth concentrate", "UF concentrate", or "ultrafiltration concentrate".

[0074] Certain ranges are presented herein with the term "about" preceding a numerical value. The term "about" is used herein to provide literal support for the exact numerical value that follows as well as for numbers that are close to or approximate the number that follows. In determining whether a number is close to or approximates a particular recited number, the unrecited number that is close to or approximates the recited number can be a number that is a substantial equivalent of the recited number in the context in which it is presented. For example, with respect to numerical values, the term "about" means a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0075] Unless the context clearly indicates otherwise, as used herein, the singular terms "a / an" and "the" include plural referents.

[0076] It should be further noted that claims can be drafted to exclude any optional elements. Accordingly, this statement is intended to serve as a basis for the use of exclusive terms such as "solely", "only", etc. in relation to the recitation of claim elements or the use of "negative" limitations.

[0077] It should also be noted that, as used herein, the term "consisting essentially of" refers to a composition in which the components following the term are present in a total amount of less than 30% by weight of the total composition in the presence of other known components and do not affect or interfere with the function or activity of the components.

[0078] It is further noted that, as used herein, the term "comprising" means including but not limited to the components that follow the term "comprising". The components that follow the term "comprising" are necessary or mandatory, but the composition comprising the components may further include other non-mandatory or optional components.

[0079] It is also noted that, as used herein, the term "consisting of" means including and limited to the components that follow the term "consisting of". Thus, the components that follow the term "consisting of" are necessary or mandatory, and no other components are present in the composition.

[0080] Every maximum numerical limit given throughout this specification is intended to include every lower numerical limit, as if such lower numerical limits were expressly written herein. Every minimum numerical limit given throughout this specification will include every higher numerical limit, as if such higher numerical limits were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0081] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0082] Other definitions of terms may occur throughout this specification.

[0083] II. Compositions

[0084] A. Enzymes

[0085] The present invention provides an isolated protein having phosphatase activity. A phosphatase is an enzyme that dephosphorylates its substrate; that is, it hydrolyzes a phosphomonoester into a phosphate ion and a molecule having a free hydroxyl group. Phosphatases include ATP diphosphatase. This action is the opposite of that of phosphorylases and kinases, which attach a phosphate group to their substrates by using a high-energy molecule such as ATP.

[0086] The phosphatases for use in the compositions and methods disclosed herein are active at least at about pH 3 to pH 9 (such as any one of about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9) or at pH 3.5 to 5.5, or at pH 3.5 to 7, or at pH 5 to 7, or at pH 3.5 to 9, or at pH 5 to 9. In some embodiments, the enzyme comprises an amino acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) with the full-length amino acid sequence of SEQ ID NO:10 or SEQ ID NO:25 (CRC22110).In some embodiments, the enzyme comprises the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:25 (CRC22110).

[0087] In some embodiments, the enzyme comprises a nucleic acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with the full-length nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:37. In some embodiments, the enzyme comprises a nucleic acid sequence having at least 60% sequence identity with SEQ ID NO:9 or SEQ ID NO:37. In some embodiments, the enzyme comprises the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:37.

[0088] In some of any embodiments, the enzyme is a polypeptide having one or more modifications. In some embodiments, the modification comprises a deletion, an insertion or a substitution. In some embodiments, the modification comprises a truncation. In some embodiments, the truncation is an N-terminal truncation. In some embodiments, the truncation is a C-terminal truncation. In some embodiments, the modification comprises an insertion. In some embodiments, the insertion is located at the N-terminus. In some embodiments, the insertion is located at the C-terminus. In some embodiments, any polypeptide provided herein further contains a histidine (His) tag.

[0089] In some embodiments, the enzyme comprises an amino acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to the full-length amino acid sequence of SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28. In some embodiments, the enzyme comprises the amino acid sequence of SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28.

[0090] In additional embodiments, phosphatases for use in the compositions and methods disclosed herein: 1) at least about pH 3 to pH 7 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7) or at least about pH 3 to pH 9 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7, or pH 3.is active at a pH of 5 to 9 (or a pH of 5 to 9), 2) comprises an amino acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with the full-length amino acid sequence of one or more of SEQ ID NO: 10 or SEQ ID NO: 25; and 3) has amino acid residues S204, F205, L206, G207, L208 and G209 at positions corresponding to the numbering of SEQ ID NO: 10.

[0091] In additional embodiments, phosphatases for use in the compositions and methods disclosed herein: 1) at at least about pH 3 to pH 7 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7) or at at least about pH 3 to pH 9 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7, or pH 3.is active at a pH of 5 to 9 (or a pH of 5 to 9), 2) comprises an amino acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with the full-length amino acid sequence of one or more of SEQ ID NO:10 or SEQ ID NO:25; and 3) has amino acid residues S204, F205, L206, G207, L208 and G209 at positions corresponding to the numbering of SEQ ID NO:25.

[0092] In additional embodiments, phosphatases for use in the compositions and methods disclosed herein: 1) at least about pH 3 to pH 7 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7) or at least about pH 3 to pH 9 (such as about pH 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7, or pH 3.5 to 5.5, or pH 3.5 to 7, or pH 5 to 7, or pH 3.is active at a pH of 5 to 9, or a pH of 5 to 9), 2) comprises an amino acid sequence having at least 40% or at least 60% sequence identity (such as any one of about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) or at least 35% or at least 60% sequence identity (such as any one of about 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) with the full-length amino acid sequence of one or more of SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28; and 3) has amino acid residues S204, F205, L206, G207, L208 and G209 at the position.

[0093] B. host cell

[0094] As briefly set forth above and further described in the Examples section below, provided herein are exemplary Gram-positive bacterial cells that have been designed, constructed, and evaluated for the expression of heterologous proteins. For example, a polynucleotide (expression cassette) encoding a phosphatase protein (CRC22110; SEQ ID NO:10 or SEQ ID NO:25) or a truncated variant (SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28) can be introduced into a Bacillus cell and evaluated in a large-scale bioreactor. More particularly, an expression cassette encoding a phosphatase from Marinomonas xiamenensis (CRC22110; SEQ ID NO:10 or SEQ ID NO:25) or a truncated variant (SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28) is constructed for intracellular expression or for secreted expression of the phosphatase in a Bacillus cell. In some embodiments, an expression cassette encoding a phosphatase from Marinomonas xiamenensis (CRC22110; SEQ ID NO:10 or SEQ ID NO:25) or a truncated variant (SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28) is constructed for secreted expression of the phosphatase in a Bacillus cell. In some embodiments, the polypeptide is secreted from the host cell. In some embodiments, the polypeptide is secreted from the host cell into the culture medium. Accordingly, certain embodiments of the present disclosure provide recombinant Gram-positive bacterial cells that express one or more heterologous nucleic acids (polynucleotides) encoding a phosphatase protein.

[0095] In certain embodiments, the recombinant Gram-positive bacterial cells express a heterologous polynucleotide encoding a native phosphatase protein or a functional variant derived from a native phosphatase protein. In certain aspects, the heterologous polynucleotide encoding the phosphatase protein is an expression cassette introduced into the recombinant cell. In certain embodiments, at least one expression cassette is introduced into the Gram-positive bacterial cell. In other embodiments, at least two expression cassettes are introduced into the Gram-positive bacterial cell. Accordingly, in certain aspects, the Gram-positive host cell of the present disclosure comprises one or more phosphatase expression cassettes introduced therein, wherein the host cell expresses the phosphatase when cultured under suitable conditions.

[0096] In some aspects, Gram-positive bacterial cells include bacilli, clostridia, and mollicutes (e.g., including Lactobacillales having Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Oscillospiraceae, Streptococcaceae, and Bacillales having Alicyclobacellaceae, Bacillaceae, Caryophanaceae, Listeriaceae, Paenibacillaceae, Planococcaceae, Sporolactobacillaceae, Staphylococcaceae, Thermoactinomycetaceae, Turicibacteraceae).

[0097] In certain embodiments, species of the family Bacillaceae include Alkalibacillus, Amphibacillus, Anoxybacillus, Bacillus, Caldalkalibacillus, Cerasilbacillus, Exiguobacterium, Filobacillus, Geobacillus, Gracilibacillus, Halobacillus, Halolactibacillus, Jeotgalibacillus, Lentibacillus, Marinibacillus, Oceanobacillus, Ornithinibacillus, Paraliobacillus, Paucisalibacillus, Pontibacillus, Pontibacillus, Saccharococcus, Salibacillus, Salinibacillus, Tenuibacillus, Thalassobacillus, Ureibacillus, and Virgibacillus.

[0098] In other embodiments, the Bacillus species cells include, but are not limited to, Bacillus acidiceler, Bacillus acidicola, Bacillus acidocaldarius, Bacillus acidoterrestris, Bacillus aeolius, Bacillus aerius, Bacillus aerophilus, Bacillus agaradhaerens, Bacillus agri, Bacillus aidingensis, Bacillus akibai, Bacillus alcalophilus, Bacillus algicola, Bacillus alginolyticus, Bacillus alkalidiazo-trophicus, Bacillus alkalinitrilicus, Bacillus alkalitelluris, Bacillus altitudinis, Bacillus alveayuensis, Bacillus alvei, Bacillus amylolyticus, Bacillus aneurinilyticus, Bacillus aneurinilyticus, Bacillus anthracia, Bacillus aquimaris, Bacillus arenosi, Bacillus arseniciselenatis, Bacillus arsenicoselenatis, Bacillus arsenicus, Bacillus arvi, Bacillus asahii, Bacillus atrophaeus, Bacillus aurantiacus, Bacillus axarquiensis, Bacillus azotofixans, Bacillus azotoformans, Bacillus badius, Bacillus barbaricus, Bacillus bataviensis, Bacillus beijingensis, Bacillus benzoevorans, Bacillus bogoriensis, Bacillus boroniphilus, Bacillus potsdamensis, BacillusB. borstelenis, B. butanolivorans, B. carboniphilus, B. cecembensis, B. cellulosilyticus, B. centrosporus, B. chagannorensis, B. chitinolyticus, B. chondroitinus, B. choshinensis, B. cibi, B. circulans,.

[0099] Bacillus clarkii, Bacillus clausii, Bacillus coagulans, Bacillus coahuilensis, Bacillus cohnii, Bacillus curdianolyticus, Bacillus cycloheptanicus, Bacillus decisifrondis, Bacillus decolorationis, Bacillus dipsosauri, Bacillus drentensis, Bacillus edaphicus, Bacillus ehimensis, Bacillus endophyticus, Bacillus farraginis, Bacillus fastidiosus, Bacillus firmus, B. plexus, Bacillus foraminis, Bacillus fordii, Bacillus formosus, Bacillus fortis, Bacillus fumarioli, Bacillus funiculus, Bacillus fusiformis, Bacillus galactophilus, Bacillus galactosidilyticus, Bacillus gelatini, Bacillus gibsonii, Bacillus ginsengi, Bacillus ginsengihumi, Bacillus globisporus, Bacillus globisporus subsp. globisporus, Bacillus globisporus subsp. marinus, Bacillus glucanolyticus, Bacillus gordonae, Bacillus halmapalus, Bacillus haloalkaliphilus, Bacillus halodenitrificans, Bacillus halodurans, Bacillus halophilus, Bacillus hemicellulosilyticus, Bacillus herbersteinensis, Bacillus horikoshii, B.Horikoshii), Bacillus horti, Bacillus hemi, Bacillus hwajinpoensis, Bacillus idriensis, Bacillus indicus, Bacillus infantis, Bacillus infernus, Bacillus insolitus, Bacillus isabeliae, Bacillus jeotgali, Bacillus kaustophilus, Bacillus kobensis, Bacillus koreensis, Bacillus kribbensis, Bacillus krulwichiae, Bacillus laevolacticus, Bacillus larvae, Bacillus laterosporus, Bacillus lautus, Bacillus lehensis, Bacillus lentimorbus, Bacillus lentus, Bacillus litoralis, Bacillus luciferensis, B. macauensis, Bacillus macerans, Bacillus macquariensis, Bacillus macyae, Bacillus malacitensis, Bacillus mannanilyticus, Bacillus marinus subsp. marinus, Bacillus marisflavi, Bacillus marismortui, Bacillus massiliensis, Bacillus methanolicus, Bacillus migulanus, Bacillus mojavensis, Bacillus mucilaginosus, Bacillus muralis, Bacillus murimartini, Bacillus mycoides, Bacillus naganoensis, Bacillus nealsonii, Bacillus neidei, Bacillus niabensis, Bacillus niacini, Bacillus novalis, Bacillus odysseyi, B.B. odysseyi, B. okhensis, B. okuhidensis, B. oleronius, B. oshimensis, B. pabuli, B. pallidus, B. pallidus (illeg.), B. panaciterrae, B. pantothenticus, B. parabrevis, B. pasteurii, B. patagoniensis, B. peoriae, B. plakortidis, B. pocheonensis, B. polygoni, B. polymyxa, B. popilliae, B. pseudalcaliphilus, B. pseudofirmus, B. pseudomycoides, B. psychrodurans, B. psychrophilus, B. psychrosaccarolyticus, B. psychrotolerans, B. pulvifaciens, B. pycnus, B. qingdaonensis, B. reuszeri, B. runs, B. safensis, B. salarius, B. salexigens, B. saliphilus, B. schlegelii, B. selenatarsenatis, B. selenitrireducens, B. seohaeanensis, B. shackletonii, B. silvestris, B. simplex, B. siralis, B. smithii, B. soli, B. sonorensissonorensis), Bacillus sphaericus, Bacillus sporothermodurans, Bacillus stearothermophilus, Bacillus stratosphericus, Bacillus subterraneus, Bacillus subtilis subsp. spizizenii, Bacillus subtilis subsp. subtilis, Bacillus taeanensis, Bacillus tequilensis, Bacillus thermantarcticus, Bacillus thermoaerophilus, Bacillus thermoamylovorans, B. thermantarcticus, Bacillus thermocatenulatus, Bacillus thermocloacae, Bacillus thermodenitrificans, Bacillus thermoglucosidasius, Bacillus thermoleovorans, Bacillus thermoruber, Bacillus thermosphaericus, Bacillus thiaminolyticus, Bacillus thioparans, Bacillus thuringiensis, Bacillus tusciae, Bacillus validus, Bacillus vallismortis, Bacillus vedderi, Bacillus velezensis, Bacillus vietnamensis, Bacillus vireti, Bacillus vulcani, Bacillus wakoensis, and Bacillus weihenstephanensis.

[0100] In certain embodiments, the Bacillus species cells are selected from the group consisting of: Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus lautus, and Bacillus thuringiensis. As used herein, "Bacillus" includes Bacillus species that have been reclassified, including but not limited to Bacillus stearothermophilus, which is now named "Geobacillus stearothermophilus".

[0101] As generally set forth above and further described in the examples below, certain embodiments of the present disclosure relate to recombinant Gram-positive bacterial cells expressing heterologous phosphatase (including apyrase) proteins, recombinant polynucleotides (e.g., vectors, expression cassettes) encoding heterologous phosphatase proteins that are particularly suitable for introduction (e.g., transformation) into Gram-positive host cells (i.e., for expressing heterologous phosphatases), and the like. In other embodiments, the Gram-positive host cells of the present disclosure are rendered deficient in the production of one or more native (endogenous) proteins. In certain aspects, the recombinant host cells of the present disclosure comprise a deletion or disruption of one or more endogenous genes encoding one or more proteases that are native to the recombinant cell. For example, in certain embodiments, recombinant Gram-positive cells defective in the production of one or more native (endogenous) proteases can be used to mitigate phosphatase degradation (e.g., during fermentation and / or downstream processing of phosphatase preparations). Recombinant Gram-positive cells defective in the production of one or more native background proteases or other problematic (native) background proteins are also envisioned herein to facilitate downstream recovery and purification of phosphatases (e.g., by reducing unwanted host cell background (native) protein contaminants).

[0102] Accordingly, certain embodiments particularly relate to nucleic acids, polynucleotides (e.g., plasmids, vectors, expression cassettes), regulatory elements, etc., suitable for use in constructing recombinant Gram-positive host cells. Thus, as shown in the examples and generally described herein, the recombinant cells of the present disclosure can be constructed by those skilled in the art using standard and conventional recombinant DNA and molecular cloning techniques well known in the art. Methods for genetically modifying cells include, but are not limited to, (a) introducing, substituting, or removing one or more nucleotides in a gene, or introducing, substituting, or removing one or more nucleotides in a regulatory element required for transcription or translation of a gene, (b) gene disruption, (c) gene conversion, (d) gene deletion, (e) gene downregulation, (f) site-directed mutagenesis, and / or (g) random mutagenesis.

[0103] In certain embodiments, the recombinant (modified) cells of the present disclosure can be constructed by reducing or eliminating the expression of a gene using methods well known in the art (e.g., insertion, disruption, substitution, or deletion). The portion of the gene to be modified or inactivated can be, for example, the coding region or a regulatory element required for the expression of the coding region.

[0104] Examples of such regulatory or control sequences can be a promoter sequence or a functional portion thereof (i.e., a portion sufficient to affect the expression of a nucleic acid sequence). Other control sequences for modification include, but are not limited to, leader sequences, propeptide sequences, signal sequences, transcription terminators, transcription activators, etc.

[0105] In certain other embodiments, modified cells are constructed by gene deletion to eliminate or reduce the expression of a gene. Gene deletion techniques enable the partial or complete removal of one or more genes, thereby eliminating their expression or expressing non-functional (or reduced-activity) protein products.

[0106] In such a method, the deletion of a gene can be accomplished by homologous recombination using a plasmid that has been constructed to continuously contain the 5' and 3' regions flanking the gene. By way of example, the contiguous 5′ and 3′ regions can be introduced into Bacillus cells (e.g., on a temperature-sensitive plasmid such as pE194) associated with a second selectable marker at a permissive temperature to allow the plasmid to establish in the cells. The cells are then transferred to a non-permissive temperature to select for cells in which the plasmid has integrated into one of the chromosomal homologous flanking regions. Selection for plasmid integration is achieved by selecting the second selectable marker. After integration, the recombination event at the second homologous flanking region is stimulated by transferring the cells to the permissive temperature for several generations without selection. The cells are plated to obtain single colonies, and the colonies are examined for loss of both selectable markers.

[0107] Thus, those skilled in the art can readily identify nucleotide regions (suitable for complete or partial deletion) in the coding sequence and / or the non-coding sequence of a gene.

[0108] In other embodiments, modified cells are constructed by introducing, substituting or removing one or more nucleotides in a gene or in regulatory elements required for its transcription or translation.

[0109] For example, nucleotides can be inserted or removed so as to result in the introduction of a stop codon, the removal of a start codon or a frameshift of the reading frame. Such modifications can be accomplished by site-directed mutagenesis or mutagenesis generated by PCR according to methods known in the art. Thus, in certain embodiments, the gene of the present disclosure is inactivated by complete or partial deletion.

[0110] In another embodiment, modified cells are constructed by a gene conversion process. For example, in a gene conversion method, a nucleic acid sequence corresponding to a gene is mutagenized in vitro to produce a defective nucleic acid sequence, and then the defective nucleic acid sequence is transformed into a parental Bacillus cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous gene. It may be desirable that the defective gene or gene fragment also encodes a marker that can be used to select transformants containing the defective gene. For example, the defective gene can be associated with a selectable marker and introduced on a non-replicating or temperature-sensitive plasmid. Selection of plasmid integration is achieved by selecting for the marker under conditions that do not permit plasmid replication. Selection of the second recombination event leading to gene replacement is achieved by examining whether the colony has lost the selectable marker and has acquired the mutated gene. Alternatively, the defective nucleic acid sequence can contain an insertion, substitution or deletion of one or more nucleotides of the gene, as described below.

[0111] In other embodiments, modified cells are constructed by established antisense techniques using a nucleotide sequence complementary to the nucleic acid sequence of a gene. More particularly, the expression of a gene in a host cell can be reduced (down-regulated) or eliminated by introducing a nucleotide sequence complementary to the nucleic acid sequence of the gene, which can be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions that permit the complementary antisense nucleotide sequence to hybridize to the mRNA, the amount of the translated protein is thus reduced or eliminated. Such antisense methods include, but are not limited to, RNA interference (RNAi), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides, etc., all of which are well known to those skilled in the art.

[0112] In still other embodiments, modified cells are constructed by random or specific mutagenesis using methods well known in the art, including but not limited to chemical mutagenesis and transposition. Gene modification can be carried out by subjecting parental cells to mutagenesis and screening for mutant cells in which gene expression has been reduced or eliminated. Mutagenesis, which can be specific or random, can be carried out, for example, by using suitable physical or chemical mutagens, by using suitable oligonucleotides, or by subjecting a DNA sequence to mutagenesis generated by PCR. In addition, mutagenesis can be carried out by using any combination of these mutagenesis methods. Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, ethyl methane sulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When using such reagents, mutagenesis is typically carried out by incubating the parental cells to be mutagenized in the presence of the selected mutagen under suitable conditions and selecting mutant cells that exhibit reduced or no expression of the gene.

[0113] PCT Publication No. WO 2003 / 083125 (incorporated herein by reference) discloses methods for modifying Bacillus cells, such as using PCR fusion to generate Bacillus deletion strains and DNA constructs to bypass Escherichia coli. PCT Publication No. WO 2002 / 14490 (incorporated herein by reference) discloses methods for modifying Bacillus cells, including (1) constructing and transforming an integration plasmid (pComK), (2) randomly mutagenizing coding sequences, signal sequences, and propeptide sequences, (3) homologous recombination, (4) increasing transformation efficiency by adding non-homologous flanks to the transforming DNA, (5) optimizing double crossover integration, (6) site-directed mutagenesis, and (7) markerless deletion.

[0114] Suitable methods for introducing polynucleotide sequences into bacterial cells (e.g., Escherichia coli, Bacillus species) are well known to those skilled in the art. In fact, methods such as transformation, including protoplast transformation and plate aggregation, transduction, and protoplast fusion, are known and suitable for the present disclosure. The transformation method is particularly suitable for introducing the DNA constructs of the present disclosure into host cells.

[0115] In addition to the common methods, in some embodiments, the host cell is directly transformed (i.e., without using an intermediate cell to amplify the DNA construct or otherwise process the DNA construct before introducing it into the host cell). Introducing the DNA construct into the host cell includes those physical and chemical methods known in the art for introducing DNA into a host cell without inserting it into a plasmid or vector. Such methods include, but are not limited to, calcium chloride precipitation, electroporation, naked DNA, liposomes, etc. In additional embodiments, the DNA construct is co-transformed with a plasmid without inserting into the plasmid. In additional embodiments, a selectable marker is deleted or substantially excised from a modified Gram-positive bacterial strain by methods known in the art. In some embodiments, the vector is excised from the host chromosome, leaving the flanking regions on the chromosome while removing the native chromosomal region.

[0116] Promoters and promoter sequence regions for expressing a gene, its open reading frame (ORF), and / or its variant sequences in Gram-positive cells are generally known to those skilled in the art. The promoter sequences of the present disclosure are typically selected such that they function in Gram-positive host cells (e.g., Bacillus cells such as Bacillus licheniformis cells, Bacillus subtilis cells, Bacillus amyloliquefaciens, etc.). For example, promoters that can be used to drive gene expression in Bacillus cells include, but are not limited to, the Bacillus subtilis alkaline protease (aprE) promoter, the α-amylase promoter (amyE) of Bacillus subtilis, the α-amylase promoter (amyL) of Bacillus licheniformis, the α-amylase promoter of Bacillus amyloliquefaciens, the neutral protease (nprE) promoter from Bacillus subtilis, the mutant aprE promoter, or any other promoter from Bacillus licheniformis or other related Bacillus. Methods for screening and generating a library of promoters with a range of activities (promoter strength) in Bacillus cells are described in PCT Publication No. WO 2002 / 14490 (incorporated herein by reference).

[0117] C. enzyme composition

[0118] Any of the enzymes used in the methods disclosed herein can be formulated into a composition (e.g., a pharmaceutical or nutritional composition). In some embodiments, the proteins having phosphatase activity at the acidic pH range described herein according to the present invention can be used for treating and / or preventing diseases associated with digestive tract inflammation. In one embodiment, the present invention provides a composition, preferably a pharmaceutical composition, comprising the protein according to the present invention. The pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0119] The composition can be presented in any form, such as as tablets, as injectable fluids or as infusion fluids, etc. In addition, the composition, protein, nucleotide and / or carrier according to the present invention can be administered via different routes, such as intravenously, rectally, bronchially or orally. Another suitable route of administration is the use of a duodenal drip.

[0120] In one embodiment, the route of administration used is the intravenous route. It will be clear to the person skilled in the art that preferably an effective amount of the protein according to the present invention is delivered. As a starting point, 1 - 50,000 U / kg / day can be used. Another suitable route (e.g., for HPP) is the subcutaneous route. If the intravenous route of administration is used, the protein according to the present invention can be administered via continuous infusion (for at least a certain amount of time).

[0121] The composition according to the present invention may optionally contain pharmaceutically acceptable excipients, stabilizers, activators, carriers, permeants, propellants, disinfectants, diluents and preservatives. Suitable excipients are well known in the field of pharmaceutical formulations and can be readily found and applied by the person skilled in the art, with references such as Remmington's Pharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pennsylvania, 17th edition 1985.

[0122] For oral administration, the protein can be administered, for example, in solid dosage forms such as capsules, tablets (e.g., with enteric coatings) and powders, or in liquid dosage forms such as elixirs, syrups and suspensions. Acid phosphatase and / or apyrase can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talc, magnesium carbonate, etc. Examples of additional inactive ingredients that can be added to provide the desired color, taste, stability, buffering capacity, dispersibility or other known desired characteristics are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink, etc. Similar diluents can be used to prepare compressed tablets. Both tablets and capsules can be manufactured as sustained release products for use in continuously releasing the drug over a number of hours. Compressed tablets can be sugar-coated or film-coated to mask any unpleasant taste and protect the tablets from the atmosphere, or can be enteric-coated to selectively disintegrate in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring agents to enhance patient acceptance.

[0123] Enteric coatings prevent the release of active compounds from orally ingestible dosage forms. Depending on the composition and / or thickness, enteric coatings tolerate gastric acid for a desired period of time before they start to disintegrate and allow for the slow release of acid phosphatase and / or adenosine triphosphate diphosphatase in the lower stomach, small intestine, or large intestine. Some examples of enteric coatings are disclosed in U.S. Patent No. 5,225,202 (incorporated by reference). Examples of enteric coatings include beeswax and glyceryl monostearate; beeswax, shellac, and cellulose, optionally containing a neutral copolymer with polymethacrylate; a copolymer of methacrylic acid and methyl methacrylate or a neutral copolymer of polymethacrylate containing a metal stearate (for references on enteric coatings, see: U.S. Patent Nos. 4,728,512, 4,794,001, 3,835,221, 2,809,918, 5,225,202, 5,026,560, 4,524,060, 5,536,507). Most enteric coating polymers become soluble at pH 5.5 and higher, with maximum solubility at pH values above 6.5. Enteric coatings can also include subcoating and overcoating steps, such as for pharmaceutical compositions intended for specific delivery in the lower GI tract, i.e., in the colon (pH 6.4 to 7.0, ileum pH 6.6), as opposed to the pH in the upper intestine, where the pH range in the duodenum of the small intestine is 7.7 - 8 (after addition of pancreatic juice and bile). The pH difference in the intestine can be utilized to target enteric-coated acid phosphatase and / or adenosine triphosphate diphosphatase compositions to specific regions in the digestive tract. It also allows for the selection of specific acid phosphatases and / or adenosine triphosphate diphosphatases that are most active at a specific pH in the intestine.

[0124] In addition to the fact that the proteins according to the invention can be bound to pharmaceutical compositions, such acid phosphatases and / or adenosine triphosphate diphosphatases can also be part of a nutritional composition or nutritional product.

[0125] The proteins according to the invention can be added to a nutrient (such as milk), but can also be produced within the nutrient (e.g., by molecular engineering). In addition, tablets and / or capsules can be prepared and subsequently added to the nutrient or taken directly by a human.

[0126] In yet another aspect, the present invention features beverage and food products comprising acid phosphatase and / or apyrase effective for treating or preventing gastrointestinal inflammation in a subject in need thereof. The beverage product may contain from 1 unit / ml to 10,000 units / ml, such as from 1 unit / ml to 200 units / ml, from 200 units / ml to 500 units / ml, from 500 units / ml to 1,000 units / ml, from 1,000 units / ml to 5,000 units / ml, or from 5,000 units / ml to 10,000 units / ml. The food product may contain from 1 unit / g to 10,000 units / g, such as from 1 unit / g to 200 units / g, from 200 units / g to 500 units / g, from 500 units / g to 1,000 units / g, from 1,000 units / g to 5,000 units / g, or from 5,000 units / g to 10,000 units / g.

[0127] III. Methods

[0128] A. cell culture method

[0129] In certain embodiments, the present disclosure provides recombinant cells capable of producing a protein of interest (such as a phosphatase). More particularly, certain embodiments are genetically modified (recombinant) Gram-positive bacterial cells that express a heterologous phosphatase. Accordingly, certain embodiments relate to culturing (fermenting) Gram-positive cells for the production of a phosphatase protein. Generally, Gram-positive cells are fermented using fermentation methods well known in the art.

[0130] In some embodiments, the cells are grown under batch or continuous fermentation conditions. Classical batch fermentation is a closed system where the composition of the medium is set at the start of fermentation and the composition does not change during fermentation. At the start of fermentation, the medium is inoculated with the desired organism. In this method, fermentation occurs without adding any components to the system. Typically, batch fermentation qualifies as "batch" with respect to the addition of a carbon source, and attempts are often made to control factors such as pH and oxygen concentration. The metabolite and biomass composition of the batch system changes continuously until fermentation ceases. In batch culture, cells progress through a stationary lag phase to a high-growth logarithmic phase and finally into a stationary phase where the growth rate decreases or stops. If untreated, cells in the stationary phase eventually die. Generally, cells in the logarithmic phase are responsible for the bulk production of the product.

[0131] A suitable variant of the standard batch system is the "fed-batch fermentation" system. In this variant of the typical batch system, substrate is added incrementally as the fermentation progresses. The fed-batch system is useful when catabolite repression might inhibit the metabolism of the cells and when a limited amount of substrate is desired in the culture medium. Measurement of the actual substrate concentration in a fed-batch system is difficult and thus it is estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases (such as CO2). Batch and fed-batch fermentations are commonly used and well-known in the art.

[0132] Continuous fermentation is an open system where a defined fermentation medium is continuously added to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the culture at a constant high density where the cells are mainly in the logarithmic growth phase. Continuous fermentation allows for the regulation of one or more factors that affect cell growth and / or product concentration. For example, in one embodiment, a limiting nutrient (such as a carbon source or a nitrogen source) is maintained at a fixed rate and all other parameters are allowed to be adjusted. In other systems, many factors that affect growth can be continuously changed while the cell concentration measured by the turbidity of the medium remains constant. The continuous system strives to maintain steady-state growth conditions. Thus, the cell loss due to the withdrawal of the medium should be balanced with the cell growth rate in the fermentation. Methods for regulating nutrients and growth factors for continuous fermentation processes and techniques for maximizing the rate of product formation are well-known in the field of industrial microbiology.

[0133] Cultivation / fermentation is typically done in a growth medium that contains an aqueous mineral salt medium, organic growth factors, a carbon source and an energy source material, molecular oxygen, and of course, an initial inoculum of the microbial host to be used.

[0134] In addition to the carbon source and energy source, oxygen, assimilable nitrogen, and the inoculum of the microorganism, it is also necessary to supply an appropriate amount of mineral nutrients in the proper proportions to ensure proper microbial growth, maximize the assimilation of the carbon source and energy source by the cells during the microbial conversion process, and obtain the maximum cell yield and maximum cell density in the fermentation medium.

[0135] The composition of the aqueous mineral medium can vary within a wide range, which depends in part on the microorganism and substrate used, as is known in the art. In addition to nitrogen, these mineral media should also include an appropriate amount of phosphorus, magnesium, calcium, potassium, sulfur, and sodium in suitable soluble assimilable ionic and combined forms, and preferably certain trace elements such as copper, manganese, molybdenum, zinc, iron, boron, and iodine and others should also be present in suitable soluble assimilable forms, all as is known in the art.

[0136] The fermentation reaction is an aerobic process in which the required molecular oxygen is supplied by a molecular oxygen-containing gas such as air, oxygen-enriched air or even substantially pure molecular oxygen, provided that the contents of the fermentation vessel are maintained at a suitable oxygen partial pressure that can be effectively used to help the microbial species grow vigorously.

[0137] The fermentation temperature can vary slightly, but for most Gram-positive cells, the temperature will generally be in the range of about 20 °C to 40 °C.

[0138] The microorganisms also require an assimilable nitrogen source. The assimilable nitrogen source can be any nitrogen-containing compound or capable of releasing nitrogen in a form suitable for metabolic utilization by the microorganisms. Although various organic nitrogen source compounds such as protein hydrolysates can be used, generally inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea and various ammonium salts (such as ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, ammonium chloride or various other ammonia compounds) can be utilized. Ammonia gas itself is convenient for large-scale operation and can be used in a suitable amount by bubbling through the aqueous fermentation broth (fermentation medium). At the same time, such ammonia can also be used to help with pH control.

[0139] The pH range in the aqueous microbial fermentation broth (fermentation mixture) should be in the exemplary range of about 2.0 to 8.0. The preference for the microbial pH range depends to some extent on the medium used and the specific microorganism and thus changes slightly with the change of the medium, as can be easily determined by those skilled in the art.

[0140] In some aspects, the fermentation is carried out in such a way that the carbon-containing substrate can be controlled as a limiting factor, so as to provide good conversion of the carbon-containing substrate for the cells and avoid contamination of these cells by a substantial amount of unconverted substrate. The latter situation is not a problem for water-soluble substrates because any remaining trace substances can be easily washed away. However, in the case of non-water-soluble substrates, this may be a problem and additional product processing steps such as suitable washing steps are required.

[0141] As described above, the time to reach this level is not critical and can vary with the specific microorganism and the fermentation process carried out. However, it is well known in the art how to determine the carbon source concentration in the fermentation medium and whether the desired carbon source level has been reached.

[0142] If desired, before feeding the aqueous mineral medium into the fermenter, part or all of the carbon source and energy material and / or part of the assimilable nitrogen source (such as ammonia) can be added to the aqueous mineral medium.

[0143] Preferably, each feed stream introduced into the reactor is controlled at a predetermined rate or in response to a demand determined by monitoring, such as the concentration of carbon and energy substrates, pH, dissolved oxygen, oxygen or carbon dioxide in the off-gas from the fermenter, cell density measurable by dry cell weight, light transmittance, etc. The feed rates of the various materials can be varied to obtain the fastest possible cell growth rate consistent with the efficient utilization of the carbon and energy sources, in order to obtain the highest possible microbial cell yield relative to substrate variations.

[0144] In batch operation or preferably fed-batch operation, all equipment, reactors or fermentors, vessels or containers, pipes, attached circulation or cooling equipment, etc. are sterilized at the beginning, usually by applying steam, such as at about 121 °C for at least about 15 minutes. Then, in the presence of all the required nutrients, including oxygen and carbon-containing substrates, the sterilized reactor is inoculated with a culture of the selected microorganism. The type of fermenter used is not important.

[0145] B. protein recovery

[0146] As further detailed below and presented in the examples, the present disclosure further describes and illustrates particularly suitable processes (methods) for harvesting, clarifying, recovering, purifying, etc. a fermentation broth in which one or more phosphatase proteins have been produced. Thus, certain embodiments particularly relate to collecting the broth at the end of fermentation, harvesting the collected broth, and recovering one or more phosphatases from the harvested broth (e.g., clarifying the harvested broth, concentrating the clarified broth, purifying the clarified broth concentrate, etc.). In certain aspects, the purified protein preparation is derived from the fermentation broth collected and harvested as described herein.

[0147] Certain other aspects of the present disclosure particularly provide novel methods for recovering and optionally purifying recombinantly produced proteins (e.g., phosphatases) obtained from recombinant cells (e.g., recombinant Gram-negative cells, recombinant Gram-positive cells, recombinant plant (e.g., tobacco) cells, etc.) that express the recombinantly produced proteins. Certain other aspects of the present disclosure particularly provide novel methods for recovering and optionally purifying phosphatases obtained from naturally occurring sources.

[0148] Accordingly, in some aspects, phosphatase protein preparations are recovered according to the compositions and methods of the present disclosure. In other aspects, phosphatase preparations are recovered and purified according to the methods of the present disclosure. As used herein, the terms "purified", "isolated", or "enriched" with respect to a protein mean the conversion of a phosphatase from a less pure state by separating it from some or all of the contaminants with which it is associated. Contaminants include, but are not limited to, microbial cells, metabolites, solvents, chemicals, colors, inactive forms of the target phosphatase, aggregates, processing aids, inhibitors, fermentation media, cell debris, nucleic acids, proteins other than the target phosphatase protein, host cell proteins, cross-contaminants from production equipment, and the like.

[0149] Accordingly, in the context of "purifying a phosphatase" as used herein, purification can be accomplished by separation techniques well known in the art, including but not limited to ion exchange chromatography, affinity chromatography, hydrophobic separation, dialysis, protease treatment, heat treatment, ammonium sulfate precipitation or other protein salt precipitation, crystallization, centrifugation, size exclusion chromatography, filtration, microfiltration, gel electrophoresis, or gradient separation to remove unwanted whole cells, cell debris, impurities, foreign proteins, or enzymes from the final composition.

[0150] Then, ingredients that provide additional benefits, such as activators, anti-inhibitors, desired ions, pH-controlling compounds, or other enzymes or chemicals, can be further added to the purified or isolated phosphatase composition.

[0151] As used herein, phosphatase "purity" is a relative term and is not intended to be limiting when used in phrases such as "the recovered phosphatase has a higher purity, the same purity, or a lower purity than before the recovery process". For example, the relative "purity" of a protein before and after a recovery process can be determined using methods known in the art, including but not limited to general quantitative methods (e.g., Bradford assay, UV-Vis, activity assay), electrophoretic analysis (SDS-PAGE), analytical HPLC, mass spectrometry, hydrophobic interaction chromatography, and the like.

[0152] Accordingly, in some aspects, a phosphatase protein preparation is recovered from a fermentation broth, wherein the recovered phosphatase preparation has a higher purity after undergoing one or more of the recovery processes described herein. For example, the fermentation broth (e.g., the whole broth at the end of fermentation) can be subjected to one or more protein recovery processes, including but not limited to broth conditioning processes, broth clarification processes, protein enrichment and / or protein purification processes (e.g., protein concentration, filtration, precipitation, crystallization, crystal separation, crystal sludge dissolution processes, etc.), buffer exchange processes, sterile filtration processes, and the like. In some aspects, the fermentation broth is subjected to a broth treatment (broth conditioning) process to improve subsequent broth treatment characteristics.

[0153] In certain embodiments, such as when a Gram-positive host cell has been engineered for intracellular phosphatase expression, the fermentation broth is subjected to a cell lysis process. For example, the cell lysis process includes, but is not limited to, enzymatic treatment (e.g., lysozyme, proteinase K treatment), chemical means (e.g., ionic liquids), physical means (e.g., French press, sonication), simply maintaining the culture without feed, etc. Thus, in certain preferred embodiments, the broth lysis process releases the intracellular phosphatase into the (lysed) cell broth.

[0154] Thus, as described herein, the methods / processes of the present disclosure are not intended to be limiting, as one of ordinary skill in the art can readily adapt or modify one or more of the compositions and / or methods disclosed herein for the recovery of specific phosphatase proteins and / or combinations thereof.

[0155] The invention can be further understood by reference to the following examples, which are provided for illustrative purposes only and not for limitation.

[0156] Exemplary Embodiments

[0157] 1. A recombinant Gram-positive host cell comprising a nucleic acid that is at least about 60% identical to SEQ ID NO:9 or SEQ ID NO:37 or a fragment thereof, wherein the host cell comprises a deletion of one or more endogenous genes encoding proteases.

[0158] 2. The host cell according to embodiment 1, wherein the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:10 or SEQ ID NO:25 or a functional fragment thereof.

[0159] 3. The host cell according to embodiment 1 or embodiment 2, wherein the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 or a functional fragment thereof.

[0160] 4. The host cell according to any one of embodiments 1-3, wherein the host cell has one, two, three, four, five, six, seven, eight, or nine endogenous genes encoding the deleted protease.

[0161] 5. The host cell according to any one of embodiments 1-4, wherein the host cell comprises nine endogenous genes encoding the deleted protease.

[0162] 6. The host cell according to any one of embodiments 1-5, wherein one or more endogenous genes encoding the protease comprise one or more of aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA, and / or nprB.

[0163] 7. The host cell according to any one of embodiments 2-6, wherein the polypeptide comprises one or more disulfide bonds.

[0164] 8. The host cell according to embodiment 7, wherein the polypeptide comprises seven disulfide bonds.

[0165] 9. The host cell according to any one of embodiments 1-8, wherein the nucleic acid is expressed on an extrachromosomal vector.

[0166] 10. The host cell according to any one of embodiments 1-9, wherein the nucleic acid is integrated into the genome of the host cell.

[0167] 11. The host cell according to any one of embodiments 1-10, wherein the host cell is a Bacillus species.

[0168] 12. The host cell according to embodiment 11, wherein the Bacillus species is Bacillus subtilis.

[0169] 13. The host cell according to any one of embodiments 2-12, wherein the polypeptide is secreted from the host cell.

[0170] 14. A method for producing a recombinant protein, the method comprising culturing the host cell according to any one of embodiments 1-13 in a suitable medium.

[0171] 15. The method according to embodiment 14, the method further comprising purifying the recombinant protein.

[0172] Examples

[0173] Example 1: Expression of Xiamen galliciae ATP diphosphatase in Bacillus subtilis

[0174] The first DNA fragment (5’ skfA gene FR, SEQ ID NO:7) containing the flanking region of the skfA gene (5’) is operably linked to a polynucleotide construct (such as an expression cassette), which polynucleotide construct contains the DNA sequence of the Bacillus subtilis rrnI-p2 promoter region (SEQ ID NO:24) (5’), which DNA sequence is operably linked to the nucleotide sequence of the 5’ untranslated region (5’UTR) of Bacillus subtilis aprE (SEQ ID NO:1), which nucleotide sequence is operably linked to the nucleotide sequence encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO:2) (which nucleotide sequence encodes the aprE signal sequence SEQ ID NO:3), which nucleotide sequence is operably linked to the nucleotide sequence of the codon-optimized gene for Bacillus spp. encoding the mature Xiamen galiensis ATP diphosphatase (CRC22110) including the “AGK” peptide at the N-terminus (SEQ ID NO:9), which nucleotide sequence is operably linked to the Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:4), which terminator is operably linked to the flanking region of the skfA gene (3’ skfA gene FR) (SEQ ID NO:8).

[0175] The second DNA fragment (5’aprE gene FR) (SEQ ID NO:5) containing the flanking region of the aprE gene (5’) is operably linked to a polynucleotide construct (such as an expression cassette), which construct contains the nucleotide sequence of the Bacillus subtilis rrnI-p2 (SEQ ID NO:24) promoter region, which nucleotide sequence is operably linked to the nucleotide sequence of the 5’ untranslated region (5’UTR) of Bacillus subtilis aprE (SEQ ID NO:1), which nucleotide sequence is operably linked to the nucleotide sequence encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO:2), which nucleotide sequence is operably linked to the nucleotide sequence of the gene encoding the mature Xiamen galiensis ATP diphosphatase (SEQ ID NO:9), which nucleotide sequence is operably linked to the Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:4), which terminator is operably linked to the flanking region of the aprE gene (3’aprE gene FR) (SEQ ID NO:6).

[0176] More particularly, these DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes.

[0177] Nine protease Bacillus subtilis strains were used to transform two apyrase cassettes from the sources described above. The nine proteases deleted in this strain are aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA, and nprB.

[0178] The Bacillus subtilis strain AP186 (a Δnine protease strain) containing the apyrase expression sequence was constructed by integrating the first and second DNA fragments described above into the genome (where the first and second fragments contain the same expression cassette). The secreted apyrase protein was measured using the malachite green method and ATP substrate described above and normalized at several time points relative to a negative control (the Bacillus subtilis host strain AP128 that does not express apyrase). The Bacillus subtilis AP186 expressing the apyrase gene and the negative control strain AP128 (not expressing the apyrase gene) were grown in a medium (a semi-synthetic medium enriched with MOP buffer, urea as the main nitrogen source, maltodextrin as the main carbon source, supplemented with 3% soy peptone for robust cell growth) in a 24-well MTP in an orbital shaker at 37 °C, 250 rpm, and 70% humidity for three (3) days. The clarified and desalted culture supernatant was used to measure (assay) the reported apyrase activity to determine the productivity level, where samples were collected at 16.8, 24.8, 40.8, 48, and 65-hour time points.

[0179] Figure 1 Measurements of the ATPase activity of CRC22110 during a 65-hour time course of culturing strains AP186 and AP128 are depicted. Time-dependent ATPase activity was detected in the culture of the Bacillus subtilis host strain AP186 compared to the negative control Bacillus subtilis strain AP128 that does not express apyrase.

[0180] The clarified culture supernatant from the Bacillus subtilis CBS12 strain expressing CRC22110 was used for enzyme purification. HiPrep TM ButylFF column was used as the first purification step. Fractions containing the target protein were identified by SDS-PAGE and malachite green assay (using ATP substrate). The active enzyme fractions were combined and buffer-exchanged into 20 mM Tris pH 7.5 and finally loaded onto HiPrep TMOn the QFF column for subsequent separation steps. A NaCl 0 - 1.0 M gradient was used for the elution fractions and the active fractions identified as described above. The combined fractions were then run on a Superdex 75 column with 20 mM Tris pH 7.5, 150 mM NaCl, 5% propylene glycol. The active enzyme fractions were identified, combined, adjusted to 40% glycerol, and stored at -20 °C. Enzyme purity was evaluated by SDS-PAGE and determined to be >90%.

[0181] The purified protein sample was analyzed by size-exclusion liquid chromatography - electrospray ionization mass spectrometry (SEC-LC-ESI / MS) as described herein. An Orbitrap Eclipse Tribrid mass spectrometer coupled with a Vanquish UPLC (Thermo) with an Acquity BEH-SEC, 200A HPLC column was used for separation and analysis. The MS parameters were ESI(+) ionization, HESI probe, and 4000 V, set to intact protein / low pressure mode. Data analysis was performed using the Freestyle (Thermo) program and the MSRefiner (Genedata) tool. The observed deconvoluted mass spectra were consistent with the presence of the full-length mature CRC22110 polypeptide with 7 Cys-Cys intramolecular bonds. SEQ ID NO:10 corresponds to the full-length mature CRC22110 adenosine triphosphate diphosphatase polypeptide with an N-terminal AGK peptide. Polypeptides consistent with C-terminal truncations of 2 to 20 amino acids were also observed. The masses of all these truncated polypeptides matched the expected protein with 7 intramolecular disulfide bonds. Sample heterogeneity consistent with the presence of full-length and truncated peptides was also observed using hydrophobic interaction chromatography (HIC), where all protein fractions separated by HIC had enzyme activity in the ATP substrate phosphate release assay.

[0182] Example 2: Expression of Xiamen galliciae ATP diphosphatase in Escherichia coli

[0183] Synthesize the first DNA fragment (SEQ ID NO:14) encoding the mature Xiamen galiensis apyrase with codon optimization for Escherichia coli. Insert this fragment into the Escherichia coli plasmid pINT (SEQ ID NO:16), which contains an origin of replication, an antibiotic marker, and a constitutive lacI promoter (SEQ ID NO:20) driving the lacI gene (SEQ ID NO:19), such that the Xiamen galiensis apyrase sequence is operably linked to a T7 RNA polymerase promoter (SEQ ID NO:22), which is operably linked to a lacI operator sequence (SEQ ID NO:21). A T7 terminator (SEQ ID NO:23) is operably linked to the 3'-end of the apyrase. Assemble these DNA fragments using standard molecular biology techniques and use them as a template to construct the circular DNA plasmid pINT-CRC2210 containing the apyrase expression cassette.

[0184] Synthesize the second DNA fragment (SEQ ID NO:13) encoding the mature Xiamen galiensis apyrase with codon optimization for Escherichia coli. Insert this second fragment into a suitable Escherichia coli plasmid pSCR (SEQ ID NO:17). This second plasmid consists of an origin of replication, an antibiotic marker, a constitutive lacI promoter (SEQ ID NO:20) driving the lacI gene (SEQ ID NO:19), an Escherichia coli pelB signal nucleotide sequence (SEQ ID NO:11) encoding the Escherichia coli pelB signal peptide SEQ ID NO:12, and a T7 RNA polymerase promoter. The T7 RNA polymerase promoter (SEQ ID NO:22) is operably linked to a lacI operator sequence (SEQ ID NO:21), which is operably linked to the Escherichia coli pelB signal sequence. Insert this second DNA fragment into the pSCR plasmid such that the Escherichia coli pelB signal sequence is operably linked to the Xiamen galiensis apyrase, which is used to direct the mature protein to the periplasm. The 3'-side of the apyrase sequence is operably linked to a T7 terminator (SEQ ID NO:23). Assemble these DNA fragments using standard molecular biology techniques and use them as a template to construct the circular DNA plasmid pSCR-CRC22110 containing the apyrase expression cassette.

[0185] The Escherichia coli strain AP390 was generated by transforming the Escherichia coli plasmid pSCR, which does not contain the apyrase fragment, into the Escherichia coli strain BL21(DE3). The Escherichia coli strain AP391 was generated by transforming the Escherichia coli plasmid pSCR-CRC22110 into the Escherichia coli strain BL21(DE3).

[0186] The Escherichia coli strain AP392 was generated by transforming the Escherichia coli plasmid pINT, which does not contain the apyrase fragment, into the Escherichia coli strain BL21(DE3)pLysS (carrying pLysS, a compatible plasmid that produces T7 lysozyme), thereby reducing the basal expression of the target gene and providing even higher stringency. The Escherichia coli strain AP393 was generated by transforming the Escherichia coli plasmid pINT-CRC22110 into the Escherichia coli strain BL21(DE3)pLysS.

[0187] All Escherichia coli strains were cultured in a suitable growth medium using standard molecular biology techniques. The expression of the apyrase protein was induced by adding 1 mM isopropyl β-D-1-thiogalactopyranoside to the cell culture. The production of the apyrase protein was measured using the malachite green method and an adenosine triphosphate (ATP) substrate as described above and normalized at two time points relative to an appropriate negative control (the Escherichia coli strain AP390 that does not express apyrase). Figure 2 A measure of the ATPase activity of AP391 containing the pSCR-CRC22110 plasmid during a 20-hour time course of culturing the strain is depicted compared to the appropriate negative control AP390 strain containing the control plasmid pSCR. Negligible levels of ATPase activity were detected from the analysis of the AP392 and AP393 samples (and negative control AP390). The lack of intracellular apyrase expression in the AP391 strain is consistent with the need to use a signal sequence, such as pelB, to direct the apyrase polypeptide to the periplasm, enabling the proper formation of disulfide bonds and the expression of active apyrase in a specialized environment. Example 3. Expression of a truncated form of the target enzyme

[0188] This example describes the generation of various C-terminal truncations of the CRC22110 apyrase described in Example 1. The truncated variants were designed to remove C-terminal residues up to the final cysteine, which is involved in one of the 7 disulfide bonds in the mature enzyme: CRC22110-V1 (SED ID NO:26), CRC22110-V2 (SED ID NO:27), and CRC22110-V3 (SED ID NO:28). The following describes the method of expressing the truncated forms of CRC22110. In some cases, the predicted N-terminus of the mature polypeptide sequence includes an additional 3 residues (AGK) (CRC22110-V1 (SED ID NO:34), CRC22110-V2 (SED ID NO:35), and CRC22110-V3 (SED ID NO:36) correspond to the apyrase proteins expressed with N-terminal AGK). A first DNA fragment containing the (5’) flanking region of the skfA gene (5’ skfA gene FR, SEQ ID NO:7) is operably linked to a polynucleotide construct (e.g., an expression cassette) that contains the upstream (5’) DNA sequence of the Bacillus subtilis rrnI-p2 promoter region (SEQ ID NO:24), which is operably linked to the DNA sequence of the Bacillus subtilis aprE 5’ untranslated region (5’ UTR) (SEQ ID NO:5), which is operably linked to the DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO:2), which is operably linked to the DNA sequence encoding the desired C-terminal truncated Xiamen galiciana apyrase with the nucleotide sequence encoding the N-terminal tripeptide AGK (SEQ ID NO:31, 32, or 33) (polypeptide sequences SEQ ID NO:34, 35, and 36), which is operably linked to the Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:4), which is operably linked to the (3’) flanking region of the skfA gene (3’ skfA gene FR) (SEQ ID NO:8).A second DNA fragment (5’ amyE gene FR) (SEQ ID NO:29) containing the flanking region of the (5’) amyE gene is operably linked to a polynucleotide construct (e.g., an expression cassette) that contains an upstream (5’) Bacillus subtilis rrnI-p2 (SEQ ID NO:24) promoter region DNA sequence which is operably linked to a DNA sequence of the Bacillus subtilis aprE 5’ untranslated region (5’UTR) (SEQ ID NO:5), which is operably linked to a DNA encoding the Bacillus subtilis aprE signal sequence (SEQ ID NO:2), which is operably linked to a DNA sequence encoding the desired C-terminal truncated Xiamen galiensis apyrase with the addition of a nucleotide sequence encoding the N-terminal tripeptide AGK (polypeptide sequences SEQ ID NO:34, 35 and 36), and the DNA sequence is operably linked to a Bacillus amyloliquefaciens BPN terminator (SEQ ID NO:4), which is operably linked to a (3’) amyE gene flanking region (3’ amyE gene FR) (SEQ ID NO:30). More particularly, these DNA fragments are assembled using standard molecular biology techniques and used as templates to develop linear DNA expression cassettes for generating two-copy strains. Using standard molecular biology techniques, a Bacillus subtilis strain containing nine protease deletions as described in Example 1 is used to integrate the above first and second linear DNA expression cassette fragments into the genome.

[0189] Example 4. Biochemical evaluation of truncated forms of CRC22110 apyrase

[0190] As described below, the relative enzyme activity of the truncated forms of CRC22110 apyrase towards the ATP substrate was determined. Purified samples of CRC22110 (SEQ ID NO:10), CRC22110-V1 (SEQ ID NO:34), CRC22110-V2 (SEQ ID NO:35) and CRC22110-V3 (SEQ ID NO:36) were prepared and quantified as described below.

[0191] Enzyme isolation: The culture supernatant from Bacillus subtilis fermentation was obtained by filtration and ammonium sulfate and 1M Tris pH8 were added to a final concentration of 1M ammonium sulfate and 20mM Tris pH8. The sample was centrifuged and filtered. The filtrate was loaded onto a 300ml phenyl sepharose column equilibrated in 20mM Tris and 1M ammonium sulfate pH8. A linear gradient of 0% to 100% 20mM Tris pH8 was run at 10ml / min over 300 minutes. The elution fractions were collected and the ATPase activity was monitored using an ATPase activity assay. The active fractions were pooled, concentrated and buffer exchanged into 20mM Tris pH8. This pool was loaded onto a 25ml Q sepharose column equilibrated in 20mM Tris pH8. It was then eluted with step gradients of 50mM NaCl in 20mM Tris, followed by 100mM NaCl, 200mM NaCl, 400mM and 500mM NaCl in 20mM Tris (pH8). The active fractions and pure fractions (checked by SDS-PAGE >95%) were combined as the purified protein and then quantified.

[0192] Protein quantification by UPLC: Protein quantification method: Protein concentration was determined by UPLC (Ultra Performance Liquid Chromatography) and OD280 densitometry. For UPLC determination, the purified enzyme was diluted in 20mM Tris pH8 and the protein components were separated by using a Zorbax 300SB-C3 column (Agilent), and a linear gradient of 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B) was run, and detection was carried out at 220nm on the UHPLC to determine the concentration. 10ul of the sample was loaded onto the column and the peak area of the diluted sample was determined. The enzyme concentration of the sample was calculated using a standard curve of a purified reference enzyme (e.g., full-length CRC22110). The protein concentration was also determined by OD280 measurement. The purified enzyme was diluted in 20mM Tris pH8 and its OD280 was measured in a quartz cuvette. The protein concentration was calculated based on their respective extinction coefficients. The final concentration was calculated based on the average of the UPLC method and OD280 determination.

[0193] ATPase activity comparison: ATPase activity assays were performed using Bacillus subtilis strains expressing the mature full-length CRC22110 apyrase and the truncated variants CRC22110-V1, CRC22110-V2, and CRC22110-V3. These strains were cultured in a standard soy peptone MOPS-based medium for up to 48.75 hours together with the Bacillus subtilis host strain CBS12-1 (negative control) that does not express apyrase. As previously described, an apyrase assay that measures apyrase activity on an ATP substrate was used to determine the production of apyrase protein in the clarified culture supernatant. A 10 ul sample of the diluted enzyme was added to 0.25 mM ATP in the assay buffer to initiate the reaction. The reaction mixture was incubated at 25 °C for 10 minutes, and then 50 ul of the reaction mixture was added to 100 ul of QuantiChrom TM malachite green reagent (VWR catalog number 75878), and then the reaction mixture was incubated at 25 °C for 20 minutes. The activity values were normalized relative to the culture biomass measured by OD600 absorbance, and the results are shown in Table 1 as fold change relative to the negative control. The 3 truncated variants showed similar expression to the full-length CRC22110 parental molecule.

[0194]

[0195] Example 5. Disulfide Bond Mapping

[0196] This example describes the disulfide bond mapping of CRC22110-V1.

[0197] Briefly, a CRC22110-V1 protein sample diluted in an equal volume of freshly prepared 2 M urea solution in 0.1 M phosphate buffer (pH = 6.9) was incubated at room temperature for 30 minutes in the presence of 1 mM N-ethylmaleimide (final concentration) to derivatize reduced (free) cysteine residues. Then, a trypsin / LysC mixture (Promega, Madison) was added to the N-ethylmaleimide-treated sample at an enzyme-protein ratio of 1:25 (w / w), and the mixture was incubated at 25 °C overnight.

[0198] Chromatographic separation of the digested peptides was performed using a Vanquish UPLC coupled to an Eclipse Orbitrap Tribrid mass spectrometer (Thermo Fisher Scientific). The peptides were separated on a Kinetex XB-C18 (150x1 mm, 2.6 uM, 100A) column with a gradient elution of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) at a solvent flow rate of 100 uL / min at 50 °C. The gradient consisted of 5% B for 2 minutes, increasing to 50% B from 2 to 40 minutes, increasing to 100% B from 40 to 45 minutes, holding at 100% B from 45 to 47 minutes, and then returning to the initial condition of 5% B.

[0199] For mass spectrometry (MS) analysis, the eluate from the column was introduced into the mass spectrometer using a heated electrospray ionization (HESI) probe operating in positive mode, and MS / MS spectra were collected after HCD or ETD fragmentation of the peptides using standard applications for peptide mapping. The data collected were processed by MS Refiner (GenData Corporation) software.

[0200] Peptides containing N-ethylmaleimide-modified cysteine residues were not detected, indicating that all cysteine residues in the protein formed disulfide bonds.

[0201] Identification of the Cys9-Cys93, Cys181-Cys184, Cys226-Cys242, and Cys345-Cys350 cysteine pairs involved in disulfide bond formation was evaluated by MS and MS / MS data; the corresponding peptide pairs and their HCD fragments supported those assignments with high confidence. No interfering disulfide bonds were detected. Since all four cysteine residues were contained within the same tryptic peptide covering amino acid residues 280 to 314 [280-314], the exact interactions of Cys298, Cys299, Cys305, and Cys312 could not be determined. However, the match between the predicted mass of this peptide with two disulfide bonds and the experimentally determined mass (within less than 2.8 ppm for +4 and +3 ions) and the MS / MS fragment pattern indicated that all four cysteine residues were involved in disulfide bond formation within the same peptide.

[0202] Identification of the disulfide bond pairs involving residues Cys345-Cys350 and Cys358-Cys365 was based on the MS / MS spectra of aggregates of three disulfide-linked peptides ([315-346], [347-359], and [362 / 363-367]), in which several y ions corresponding to the masses of the disulfide-linked peptides [353-(C358)-359]=[362 / 363-(C365)-367] and [356-(C358)-359]=[362 / 363-(C365)-367], as well as ETD fragments corresponding to the masses of the peptide pairs [315-346]=[347-359] and [347-359]=[63-367] (in this notation "=" means disulfide bond), were detected. No MS peaks that could be attributed to disulfide bond arrangements different from those described were found.

Claims

1. A recombinant Gram - positive host cell comprising a nucleic acid that is at least about 60% identical to SEQ ID NO:9 or SEQ ID NO:37 or a fragment thereof, wherein the host cell comprises a deletion of one or more endogenous genes encoding a protease.

2. The host cell according to claim 1, wherein the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:10 or SEQ ID NO:25 or a functional fragment thereof.

3. The host cell according to claim 1 or claim 2, wherein the nucleic acid encodes a polypeptide that is at least about 80% identical to SEQ ID NO:26, SEQ ID NO:27 or SEQ ID NO:28 or a functional fragment thereof.

4. The host cell according to any one of claims 1 - 3, wherein the host cell has one, two, three, four, five, six, seven, eight or nine endogenous genes encoding a deleted protease.

5. The host cell according to any one of claims 1 - 4, wherein the host cell comprises nine endogenous genes encoding a deleted protease.

6. The host cell according to any one of claims 1 - 5, wherein the one or more endogenous genes encoding a protease comprise one or more of aprE, nprE, epr, ispA, bpr, mpr, vpr, wprA and / or nprB.

7. The host cell according to any one of claims 2 - 6, wherein the polypeptide comprises one or more disulfide bonds.

8. The host cell according to claim 7, wherein the polypeptide comprises seven disulfide bonds.

9. The host cell according to any one of claims 1 - 8, wherein the nucleic acid is expressed on an extrachromosomal vector.

10. The host cell according to any one of claims 1 - 9, wherein the nucleic acid is integrated into the genome of the host cell.

11. The host cell according to any one of claims 1 - 10, wherein the host cell is a Bacillus spp.

12. The host cell according to claim 11, wherein the Bacillus spp. is Bacillus subtilis.

13. The host cell according to any one of claims 2 - 12, wherein the polypeptide is secreted from the host cell.

14. A method for producing a recombinant protein, the method comprising culturing a host cell according to any one of claims 1 - 13 in a suitable medium.

15. The method according to claim 14, the method further comprising purifying the recombinant protein.

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