WO1999014342A1 - Proteases from gram-positive organisms - Google Patents
Proteases from gram-positive organisms Download PDFInfo
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- WO1999014342A1 WO1999014342A1 PCT/US1998/018828 US9818828W WO9914342A1 WO 1999014342 A1 WO1999014342 A1 WO 1999014342A1 US 9818828 W US9818828 W US 9818828W WO 9914342 A1 WO9914342 A1 WO 9914342A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
- C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/189—Enzymes
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- D—TEXTILES; PAPER
- D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
- D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
- D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
- D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
Definitions
- the present invention relates to metallo-proteases derived from gram-positive microorganisms.
- the present invention provides nucleic acid and amino acid sequences of a metallo-protease identified in Bacillus subtilis.
- the present invention also provides methods for the production of the protease in host cells as well as the production of heterologous proteins in a host cell having a mutation or deletion of part or all of the proteases of the present invention.
- Gram-positive microorganisms such as members of the group Bacillus
- Gram-positive bacteria have been used for large-scale industrial fermentation due, in part, to their ability to secrete their fermentation products into the culture media.
- secreted proteins are exported across a cell membrane and a cell wall, and then are subsequently released into the external media usually maintaining their native conformation.
- proteases are produced in large quantities for industrial purposes.
- a negative aspect of the presence of proteases in gram- positive organisms is their contribution to the overall degradation of secreted heterologous or foreign proteins.
- proteases found in microorganisms are based on their catalytic mechanism which results in four groups: the serine proteases; metallo-proteases; cysteine proteases; and aspartic proteases. These categories, in general, can be distinguished by their sensitivity to various inhibitors. For example, the serine proteases are inhibited by phenylmethylsulfonylfluoride (PMSF) and diisopropylfluorophosphate (DIFP); the metallo- proteases by chelating agents; the cysteine enzymes by iodoacetamide and heavy metals and the aspartic proteases by pepstatin.
- PMSF phenylmethylsulfonylfluoride
- DIFP diisopropylfluorophosphate
- pepstatin the classification of proteases found in microorganisms.
- the serine proteases have alkaline pH optima, the metalloproteases are optimally active around neutrality, and the cysteine and aspa ⁇ ic enzymes have acidic pH optima (Biotechnology Handbooks. Bacillus, vol. 2. edited by Harwood, 1989 Plenum Press, New York).
- Metallo-proteases form the most diverse of the catalytic types of proteases.
- Family M23 contains bacterial enzymes such as the ⁇ -lytic endopeptidases of Lysobacter and Achromobacter and the Pseudomonas LasA protein and have specificity for Gly bonds, especially in Gly-Gly+Xaa-sequences (Methods in Enzymology, vol 248, Academic Press, Inc 1994)
- the enzymes of the M23 family contain zinc and a conserved His-Xaa-His motif
- the present invention relates to the discovery of a heretofore unknown metallo- protease (MP) found in gram positive microorganisms, uses of the MP in industrial applications, and advantageous strain improvements based on genetically engineering such microorganisms to delete, underexpress or overexpress that MP Due to the overall relatedness of MP with Pseudomonas lasA protein, including the presence of the motif His- Xaa-His, MP appears to be a member of the metallo-protease family M23
- Applicant's discovery in addition to providing a new and useful protease and methods of detecting DNA encoding such proteases in a gram positive microorganism, provides several advantages which may facilitate optimization and/or modification of strains of gram positive microorganisms, such as Bacillus, for expression of desired, e.g heterologous, proteins Such optimizations, as described below in detail, allow the construction of strains having decreased proteolytic degradation of desired expression products
- Applicant's invention is further based on the discovery of the presence of MP's in Gram-positive microorganisms
- the Gram-positive microorganism may be Bacillus and may also be selected from the group consisting of Bacillus subtilis, Bacillus stearothermoph ⁇ us, Bacillus lichemformis and Bacillus amyloliquifaciens
- the present invention further relies on the discovery that naturally occurring MP is encoded by nucleic acid found about 2248 kb from the point of origin of Bacillus subtilis 1-168 strain (Bacillus Genetic Stock Center, accession number 1 Al, Columbus, Ohio)
- the present invention relates to the MP encoded thereby, as well as the nucleic acid and amino acid molecules having the sequences disclosed in Figures 1A-1O
- the present invention thus provides methods for detecting gram positive microorganism homologs of B. subtilis MP that comprises hybridizing part or all of the nucleic acid encoding B. subtilis MP with nucleic acid derived from gram-positive organisms, either of genomic or cDNA origin Accordingly, the present invention provides a method for detecting a gram-positive microorganism MP, comprising the steps of hybridizing gram-positive microorganism nucleic acid under low stringency conditions to a probe, wherein the probe comprises part or all of the nucleic acid sequence shown in Figures 1A-1O; and isolating gram- positive nucleic acid which hybridizes to said probe.
- the Bacillus is selected from the group consisting of B. lichemformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and B. thuringiensis.
- the production of desired heterologous proteins or polypeptides in gram-positive microorganisms may be hindered by the presence of one or more proteases, including MP, which degrade the produced heterologous protein or polypeptide.
- One advantage of the present invention is that it provides methods and expression systems which can be used to prevent that degradation, thereby enhancing yields of the desired heterologous protein or polypeptide.
- the present invention provides a gram-positive microorganism having a mutation or deletion of part or all of the gene encoding MP, which results in the inactivation of the MP proteolytic activity, either alone or in combination with mutations in other proteases, such as apr, npr, epr, mpr, bpf or isp for example, or other proteases known to those of skill in the art.
- the gram-positive organism is a member of the genus Bacillus.
- the Bacillus is selected from the group consisting of B. subtilis, B. lichemformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
- the Bacillus is Bacillus subtilis.
- the gram-positive host having one or more metallo-protease deletions or mutations is further genetically engineered to produce a desired protein.
- the desired protein is heterologous to the gram-positive host cell.
- the desired protein is homologous to the host cell.
- the present invention encompasses a gram-positive host cell having a deletion, mutation or interruption of the nucleic acid encoding the naturally occurring homologous protein, such as a protease, and having nucleic acid encoding the homologous protein re-introduced in a recombinant form.
- the host cell produces the homologous protein.
- the present invention also provides methods and expression systems for reducing degradation of heterologous proteins produced in gram-positive microorganisms.
- the gram- positive microorganism may be normally sporulating or non-sporulating.
- the gram positive host cell is a Bacillus.
- the Bacillus host cell is Bacillus.
- the Bacillus is selected from the group consisting of B. subtilis, B. lichemformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
- the metallo-protease MP may be used alone or in combination with other enzymes and/or mediators or enhancers.
- gram-positive MP is produced on an industrial fermentation scale in a microbial host expression system.
- the present invention provides a cleaning composition comprising a metalloprotease, MP, having the amino acid sequence shown in Figures 1 A- 10 or the amino acid encoded by the MP nucleic acid found at about 2248 kilobases from the point of origin of Bacillus subtilis.
- cleaning compositions comprising a metalloprotease having at least 80%, at least 90%, or at least 95% homology with the amino acid sequence shown in Figures 1 A- 10 or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1A- 10 under high stringency conditions.
- an animal feed comprising a metalloprotease, MP, having the amino acid sequence shown in Figures 1A-10. Also provided are animal feeds comprising a metalloprotease having at least 80%, at least 90%, and at least 95% homology with the amino acid sequence shown in Figures 1 A- 10 or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1A-10 under high stringency conditions.
- compositions for the treatment of a textile comprising a metalloprotease, MP, having the amino acid sequence shown in Figures 1 A-10.
- compositions for the treatment of a textile comprising a metalloprotease having at least 80%, at least 90%, or at least 95% homology with the amino acid sequence shown in Figures 1 A- 10 or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1A-1O under high stingency conditions.
- FIG. 1 A- 10 shows the DNA and amino acid sequence for Bacillus subtilis MP.
- Figure 2 show an amino acid alignment of Bacillus subtilis MP (designated as YOMI) and Pseudomonas LasA.
- the amino acid motif H-X-H is noted at amino acid 308 -310 in LasA.
- Bacillus includes all members known to those of skill in the art, including but not limited to B. subtilis, B. lichemformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautus and B. thuringiensis.
- the present invention relates to a newly characterized metallo-protease (MP) from gram positive organisms.
- the metallo-protease is obtainable from a gram-positive organism which is a Bacillus.
- the metalloprotease is obtainable from a Bacillus which is selected from the group consisting of B. subtilis, B. lichemformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautus and B. thuringiensis.
- the gram-positive organism is Bacillus subtilis and MP has the amino acid sequence encoded by the nucleic acid molecule having the sequence that occurs around 2248 kilobases from the point of origin of Bacillus subtilis 1-168.
- Bacillus subtilis has the nucleic acid and amino acid sequence as shown in Figures 1A-10.
- the present invention encompasses the use of amino acid variations of the amino acid sequences disclosed in Figures 1A-10 that have proteolytic activity.
- proteolytic amino acid variants can be used in the textile industry, animal feed and in cleaning compositions.
- the present invention also encompasses the use of B. subtilis amino acid variations or derivatives that are not proteolytically active. DNA encoding such variants can be used in methods designed to delete or mutate the naturally occurring host cell MP.
- nucleic acid refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded, whether representing the sense or antisense strand.
- amino acid refers to peptide or protein sequences or portions thereof.
- polynucleotide homolog refers to a gram-positive microorganism polynucleotide that has at least 80%, at least 90% and at least 95% identity to B.subtilis MP, or which is capable of hybridizing to B.subtilis MP under conditions of high stringency and which encodes an amino acid sequence having metallo-protease activity.
- isolated or purified refer to a nucleic acid or amino acid that is removed from at least one component with which it is naturally associated.
- heterologous protein refers to a protein or polypeptide that does not naturally occur in a gram-positive host cell.
- heterologous proteins include enzymes such as hydrolases including proteases, cellulases, amylases, carbohydrases, and Upases; isomerases such as racemases, epimerases, tautomerases, or mutases; transferases, kinases and phophatases.
- the heterologous gene may encode therapeutically significant proteins or peptides, such as growth factors, cytokines, ligands, receptors and inhibitors, as well as vaccines and antibodies.
- the gene may encode commercially important industrial proteins or peptides, such as proteases, carbohydrases such as amylases and glucoamylases, cellulases, oxidases and Upases.
- the gene of interest may be a naturally occurring gene, a mutated gene or a synthetic gene.
- homologous protein refers to a protein or polypeptide native or naturally occurring in a gram-positive host cell.
- the invention includes host cells producing the homologous protein via recombinant DNA technology.
- the present invention encompasses a gram-positive host cell having a deletion or interruption of the nucleic acid encoding the naturally occurring homologous protein, such as a protease, and having nucleic acid encoding the homologous protein re-introduced in a recombinant form.
- the host cell produces the homologous protein.
- the term "overexpressing" when referring to the production of a protein in a host cell means that the protein is produced in greater amounts than its production in its naturally occurring environment.
- proteolytic activity refers to a protein that is able to hydrolyze a peptide bond. Enzymes having proteolytic activity are described in Enzyme Nomenclature, 1992, edited Webb Academic Press, Inc.
- MP metallo-protease M23 family member
- the host cell is a gram-positive host cell that has a deletion or mutation in the naturally occurring nucleic acid encoding MP said mutation resulting in deletion or inactivation of the production by the host cell of the MP proteolytic gene product.
- the host cell may additionally be genetically engineered to produced a desired protein or polypeptide.
- host cells may also be desired to genetically engineer host cells of any type to produce a gram- 5 positive MP.
- Such host cells are used in large scale fermentation to produce large quantities of the protease which may be isolated or purified and used in cleaning products, such as detergents, in textile treatments and as animal feed additives.
- Gram-positive polynucleotide homologs of B.subtilis MP may be obtained by standard o procedures known in the art from, for example, cloned DNA (e.g. , a DNA "library”), genomic DNA libraries, by chemical synthesis once identified, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from a desired cell.
- cloned DNA e.g. , a DNA "library”
- genomic DNA libraries by chemical synthesis once identified, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from a desired cell.
- a preferred source is from genomic DNA.
- genomic DNA As will be understood by those of skill in the art, the polynucleotide sequence disclosed in Figures 1A-1O may reflect inadvertent errors inherent to nucleic acid sequencing technology. Moreover, the sequence of polynucleotides derived from related species, e.g., other Bacillus, will contain variations to the sequences specifically disclosed herein. Nonetheless, one of ordinary skill o in the art is fully capable of determining the correct sequences from the information provided herein regarding the invention. For example, as described below, it is possible to identify the MP of the invention by virtue of its location in the microorganism's genome. The present invention encompasses the naturally occurring nucleic acid molecule having the nucleic acid sequence obtained from the genomic sequence of Bacillus species.
- Nucleic acid encoding Bacillus subtilis MP starts around 2248 kilobases counting from the point of origin in the Bacillus subtilis strain 1-168 (Anagnostopala, 1961, J. Bacteriol. 81 :741-746 or Bacillus Genomic Stock Center, accession 1 Al, Columbus, Ohio). The Bacillus subtilis point of origin has been described in Ogasawara, N. (1995, Microbiology 141 :Pt.2 257-59). Bacillus subtilis MP has a length of 2285 amino acids. Based upon the location of the DNA encoding Bacillus subtilis MP, naturally occurring B.subtilis MP can be obtained by methods known to those of skill in the art including PCR technology.
- Oligonucleotide sequences or primers of about 10-30 nucleotides in length can be designed from the polynucleotide sequence disclosed in Figures 1A-10 and used in PCR technology to isolate the naturally occurring sequence from B.subtilis genomic sequences.
- Another general strategy for the "cloning" of B. subtilis genomic DNA pieces for sequencing uses inverse PCR.
- a known region is scanned for a set of appropriate restriction enzyme cleavage sites and inverse PCR is performed with a set of DNA primers determined from the outermost DNA sequence.
- the DNA fragments from the inverse PCR are directly used as template in the sequencing reaction.
- the newly derived sequences can be used to design new oligonucleotides. These new oligonucleotides are used to amplify DNA fragments with genomic DNA as template.
- the sequence determination on both strands of a DNA region is finished by applying a primer walking strategy on the genomic PCR fragments.
- Nucleic acid sequences derived from genomic DNA may contain regulatory regions in addition to coding regions. Whatever the source, the isolated MP gene should be molecularly cloned into a suitable vector for propagation of the gene.
- DNA fragments are generated, some of which will encode the desired gene.
- the DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
- the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. Once the DNA fragments are generated, identification of the specific DNA fragment containing the MP may be accomplished in a number of ways.
- a B.subtilis MP gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a gram- positive MP gene.
- a B.subtilis MP gene of the present invention or its specific RNA, or a fragment thereof, such as a probe or primer may be isolated and labeled and then used in hybridization assays to detect a gram- positive MP gene.
- Those DNA fragments sharing substantial sequence similarity to the probe will hybridize under stringent conditions.
- the present invention provides a method for the detection of gram- positive MP polynucleotide homologs which comprises hybridizing part or all of a nucleic acid sequence of B. subtilis MP with gram-positive microorganism nucleic acid of either genomic or cDNA origin. Also included within the scope of the present invention is the use of gram-positive microorganism polynucleotide sequences that are capable of hybridizing to the nucleotide sequence of B.subtilis MP under conditions of intermediate to maximal stringency. Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques, Methods in Enzvmology. Vol 152. Academic Press. San Diego CA) incorporated herein by reference, and confer a defined "stringency" as explained below.
- Tm melting temperature
- Maximum stringency typically occurs at about Tm-5°C (5°C below the Tm of the probe); “high stringency” at about 5°C to 10°C below Tm; “intermediate stringency” at about 10°C to 20°C below Tm; and “low stringency” at about 20°C to 25°C below Tm.
- a maximum stringency hybridization can be used to identify or detect identical polynucleotide sequences while an intermediate or low stringency hybridization can be used to identify or detect polynucleotide sequence homologs.
- hybridization shall include "the process by which a strand of nucleic acid joins with a complementary strand through base pairing" (Coombs J (1994) Dictionary of Biotechnology, Stockton Press, New York NY).
- PCR polymerase chain reaction
- B.subtilis MP amino acid sequences (shown in Figures 1 A- 1O) were identified via a BLAST search (Altschul, Stephen, Basic local alignment search tool, J. Mol. Biol. 215:403- 410) of Bacillus subtilis genomic nucleic acid sequences.
- B. subtilis MP (YOMI) was identified by its overall nucleic acid identity to the metallo-protease, Pseudomonas lasA, including the presence of the catalytic domain H-X-H as shown in Figure 2.
- the present invention provides host cells, expression methods and systems for the enhanced production and secretion of desired heterologous or homologous proteins in gram- positive microorganisms.
- a host cell is genetically engineered to have a deletion or mutation in the gene encoding a gram-positive MP such that the respective activity is deleted.
- a gram-positive microorganism is genetically engineered to produce a metallo-protease of the present invention.
- the mutation is a non- reverting mutation.
- One method for mutating nucleic acid encoding a gram-positive metallo-protease is to clone the nucleic acid or part thereof, modify the nucleic acid by site directed mutagenesis and reintroduce the mutated nucleic acid into the cell on a plasmid.
- the mutated gene may be introduced into the chromosome.
- the parent host cell the result is that the naturally occurring nucleic acid and the mutated nucleic acid are located in tandem on the chromosome.
- the modified sequence is left in the chromosome having thereby effectively introduced the mutation into the chromosomal gene for progeny of the parent host cell.
- Another method for inactivating the metallo-protease proteolytic activity is through deleting the chromosomal gene copy.
- the entire gene is deleted, the deletion occurring in such as way as to make reversion impossible.
- a partial deletion is produced, provided that the nucleic acid sequence left in the chromosome is too short for homologous recombination with a plasmid encoded metalloprotease gene.
- nucleic acid encoding the catalytic amino acid residues are deleted.
- Deletion of the naturally occurring gram-positive microorganism metallo-protease can be carried out as follows.
- a metallo-protease gene including its 5' and 3' regions is isolated and inserted into a cloning vector.
- the coding region of the metallo-protease gene is deleted form the vector in vitro, leaving behind a sufficient amount of the 5' and 3' flanking sequences to provide for homologous recombination with the naturally occurring gene in the parent host cell.
- the vector is then transformed into the gram-positive host cell.
- the vector integrates into the chromosome via homologous recombination in the flanking regions. This method leads to a gram-positive strain in which the protease gene has been deleted.
- the vector used in an integration method is preferably a plasmid.
- a selectable marker may be included to allow for ease of identification of desired recombinant microorgansims.
- the vector is preferably one which can be selectively integrated into the chromosome. This can be achieved by introducing an inducible origin of replication, for example, a temperature sensitive origin into the plasmid. By growing the transformants at a temperature to which the origin of replication is sensitive, the replication function of the plasmid is inactivated, thereby providing a means for selection of chromosomal integrants. Integrants may be selected for growth at high temperatures in the presence of the selectable marker, such as an antibiotic. Integration mechanisms are described in WO 88/06623.
- Another method of inactivating the naturally occurring metallo-protease gene is to mutagenize the chromosomal gene copy by transforming a gram-positive microorganism with oligonucleotides which are mutagenic.
- the chromosomal metallo-protease gene can be replaced with a mutant gene by homologous recombination.
- the present invention encompasses host cells having additional protease deletions or mutations, such as deletions or mutations in apr, npr, epr, mpr and others known to those of skill in the art.
- One assay for the detection of mutants involves growing the Bacillus host cell on medium containing a protease substrate and measuring the appearance or lack thereof, of a zone of clearing or halo around the colonies. Host cells which have an inactive protease will exhibit little or no halo around the colonies.
- an expression vector comprising at least one copy of nucleic acid encoding a gram-positive microorganism MP, and preferably comprising multiple copies, is transformed into the host cell under conditions suitable for expression of the metallo-protease.
- polynucleotides which encode a gram-positive microorganism MP, or fragments thereof, or fusion proteins or polynucleotide homolog sequences that encode amino acid variants of B.subtilis MP may be used to generate recombinant DNA molecules that direct their expression in host cells.
- the gram-positive host cell belongs to the genus Bacillus.
- the gram positive host cell is B. subtilis.
- Codons preferred by a particular gram-positive host cell can be selected, for example, to increase the rate of expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
- Altered MP polynucleotide sequences which may be used in accordance with the invention include deletions, insertions or substitutions of different nucleotide residues resulting in a polynucleotide that encodes the same or a functionally equivalent MP homolog, respectively.
- a "deletion" is defined as a change in either nucleotide or amino acid sequence in which one or more nucleotides or amino acid residues, respectively, are absent.
- an "insertion” or “addition” is that change in a nucleotide or amino acid sequence which has resulted in the addition of one or more nucleotides or amino acid residues, respectively, as compared to the naturally occurring MP.
- substitution results from the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids, respectively.
- the encoded protein may also show deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent MP variant.
- Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and or the amphipathic nature of the residues as long as the variant retains the ability to modulate secretion.
- negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine, phenylalanine, and tyrosine.
- the MP polynucleotides of the present invention may be engineered in order to modify the cloning, processing and/or expression of the gene product.
- mutations may be introduced using techniques which are well known in the art, eg, site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns or to change codon preference, for example.
- a gram-positive microorganism MP polynucleotide may be ligated to a heterologous sequence to encode a fusion protein.
- a fusion protein may also be engineered to contain a cleavage site located between the metallo-protease nucleotide sequence and the heterologous protein sequence, so that the metallo-protease may be cleaved and purified away from the heterologous moiety.
- Expression vectors used in expressing the metallo-proteases of the present invention in gram-positive microorganisms comprise at least one promoter associated with a metalloprotease selected from the group consisting of MP, which promoter is functional in the host cell.
- the promoter is the wild-type promoter for the selected metallo-protease and in another embodiment of the present invention, the promoter is heterologous to the metallo-protease, but still functional in the host cell.
- nucleic acid encoding the metallo-protease is stably integrated into the microorganism genome.
- the expression vector contains a multiple cloning site cassette which preferably comprises at least one restriction endonuclease site unique to the vector, to facilitate ease of nucleic acid manipulation.
- the vector also comprises one or more selectable markers.
- selectable marker refers to a gene capable of expression in the gram-positive host which allows for ease of selection of those hosts containing the vector. Examples of such selectable markers include but are not limited to antibiotics, such as, erythromycin, actinomycin, chloramphenicol and tetracycline.
- a variety of host cells can be used for the production Bacillus subtilis MP or MP homologs including bacterial, fungal, mammalian and insects cells.
- General transformation procedures are taught in Current Protocols In Molecular Biology (vol. 1, edited by Ausubel et al., John Wiley & Sons, Inc. 1987, Chapter 9) and include calcium phosphate methods, transformation using DEAE-Dextran and electroporation. Plant transformation methods are taught in Rodriquez (WO 95/14099, published 26 May 1995).
- the host cell is a gram-positive microorganism and in another preferred embodiment, the host cell is Bacillus.
- nucleic acid encoding one or more MP(s) of the present invention is introduced into a host cell via an expression vector capable of replicating within the Bacillus host cell.
- Suitable replicating plasmids for Bacillus are described in Molecular Biological Methods for Bacillus, Ed. Harwood and Cutting, John Wiley & Sons, 1990, hereby expressly incorporated by reference; see chapter 3 on plasmids.
- Suitable replicating plasmids for B. subtilis are listed on page 92.
- nucleic acid encoding MP is stably integrated into the microorganism genome.
- Preferred host cells are gram-positive host cells. Another preferred host is Bacillus. Another preferred host is Bacillus subtilis.
- Plasmid marker rescue transformation involves the uptake of a donor plasmid by competent cells carrying a partially homologous resident plasmid (Contente et al., Plasmid 2:555-571 (1979); Haima et al., Mol. Gen. Genet. 223: 185-191 (1990); Weinrauch et al., J. Bacteriol.
- a host cell has been transformed with a mutated or a naturally occurring gene encoding a gram-positive MP
- detection of the presence/absence of marker gene expression can suggest whether the gene of interest is present
- its expression should be s confirmed.
- the nucleic acid encoding an MP of the present invention is inserted within a marker gene sequence
- recombinant cells containing the insert can be identified by the absence of marker gene function.
- a marker gene can be placed in tandem with nucleic acid encoding the MP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the MP as well.
- host cells which contain the coding sequence for a metallo-protease and express the protein may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridization and protein bioassay or immunoassay techniques which include membrane-based, solution- based, or chip-based technologies for the detection and/or quantification of the nucleic acid or 5 protein.
- the presence of the metallo-protease polynucleotide sequence can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of B.subtilis MP.
- Means for determining the levels of secretion of a heterologous or homologous protein in a gram-positive host cell and detecting secreted proteins include, using either polyclonal or monoclonal antibodies specific for the protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul MN) and Maddox DE et al (1983, J Exp Med 158: 1211).
- ELISA enzyme-linked immunosorbent assay
- RIA radioimmunoassay
- FACS fluorescent activated cell sorting
- Means for producing labeled hybridization or PCR probes for detecting specific polynucleotide sequences include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
- the nucleotide sequence, or any portion of it may be cloned into a vector for the production of an mRNA probe.
- Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3 or SP6 and labeled nucleotides.
- reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like.
- Patents teaching the use of such labels include US Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.
- recombinant immunoglobulins may be produced as shown in US Patent No. 4,816,567 and incorporated herein by reference.
- Gram positive host cells transformed with polynucleotide sequences encoding heterologous or homologous protein may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
- the protein produced by a recombinant gram-positive host cell comprising a mutation or deletion of the metallo-protease activity will be secreted into the culture media.
- Other recombinant constructions may join the heterologous or homologous polynucleotide sequences to nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins (Kroll DJ et al (1993) DNA Cell Biol 12:441-53).
- Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3:263-281), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
- metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals (Porath J (1992) Protein Expr Purif 3:263-281), protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle WA).
- a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego CA) between the purification domain and the heterologous protein can be used to
- the present invention provides genetically engineered host cells comprising mutations, preferably non-revertable mutations, or deletions in the naturally occurring gene encoding MP such that the proteolytic activity is diminished or deleted altogether.
- the host cell may contain additional protease deletions, such as deletions of the mature subtilisn protease and/or mature neutral protease disclosed in United States Patent No. 5,264,366.
- the host cell is further genetically engineered to produce a desired protein or polypeptide.
- the host cell is a Bacillus.
- the host cell is a Bacillus subtilis.
- a host cell is genetically engineered to produce a gram- positive MP.
- the host cell is grown under large scale fermentation conditions.
- the MP is isolated and/or purified and used in the textile industry, the feed industry and in cleaning compositions such as detergents.
- MP can be useful in formulating various cleaning compositions.
- a number of known compounds are suitable surfactants useful in compositions comprising the MP of the invention. These include nonionic, anionic, cationic, anionic or zwitterionic detergents, as disclosed in US 4,404,128 and US 4,261,868.
- a suitable detergent formulation is that described in Example 7 of US Patent 5,204,015. The art is familiar with the different formulations which can be used as cleaning compositions.
- MP can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning solutions or products, peptide hydrolysis, waste treatment, textile applications, as fusion- cleavage enzymes in protein production, etc.
- MP may comprise enhanced performance in a detergent composition (as compared to another detergent protease).
- enhanced performance in a detergent is defined as increasing cleaning of certain enzyme sensitive stains such as grass or blood, as determined by usual evaluation after a standard wash cycle.
- MP can be formulated into known powdered and liquid detergents having pH between 6.5 and 12.0 at levels of about .01 to about 5% (preferably .1% to .5%) by weight.
- These detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, Upases or endoglycosidases, as well as builders and stabilizers.
- MP can be used in a cleaning composition without detergents, again either alone or in combination with builders and stabilizers.
- Proteases can be included in animal feed such as part of animal feed additives as described in, for example, US 5,612,055; US 5,314,692; and US 5,147,642.
- One aspect of the invention is a composition for the treatment of a textile that includes
- composition can be used to treat for example silk or wool as described in publications such as RD 216,034; EP 134,267; US 4,533,359; and EP 344,259.
- a B.subtlis MP polynucleotide, or any part thereof, provides the basis for detecting the presence of gram-positive microorganism MP polynucleotide homologs through hybridization techniques and PCR technology.
- one aspect of the present invention is to provide for nucleic acid hybridization and PCR probes which can be used to detect polynucleotide sequences, including genomic and cDNA sequences, encoding gram-positive MP or portions thereof.
- an MP polynucleotide can be used in hybridization technology to detect the major protease of a gram-positive microorganism due to the proximity of the MP with the major protease.
- Genomic DNA from Bacillus cells is prepared as taught in Current Protocols In o Molecular Biology vol. 1, edited by Ausubel et al., John Wiley & Sons, Inc. 1987, chapter 2. 4.1.
- Bacillus cells from a saturated liquid culture are lysed and the proteins removed by digestion with proteinase K.
- Cell wall debris, polysaccharides, and remaining proteins are removed by selective precipitation with CTAB, and high molecular weight genomic DNA is recovered from the resulting supernatant by isopropanol precipitation. If s exceptionally clean genomic DNA is desired, an additional step of purifying the Bacillus genomic DNA on a cesium chloride gradient is added.
- the DNA is subjected to Sau3A digestion.
- Sau3A recognizes the 4 base pair site GATC and generates fragments compatible with several convenient phage lambda and cosmid vectors.
- the DNA is subjected to partial digestion to o increase the chance of obtaining random fragments.
- the partially digested Bacillus genomic DNA is subjected to size fractionation on a 1% agarose gel prior to cloning into a vector.
- size fractionation on a sucrose gradient can be used.
- the genomic DNA obtained from the size fractionation step is purified away from the agarose and ligated into a cloning vector appropriate for use in a host cell and 5 transformed into the host cell.
- gram-positive microorganism MP 0 DNA derived from a gram-positive microorganism is prepared according to the methods disclosed in Current Protocols in Molecular Biology, Chap. 2 or 3. The nucleic acid is subjected to hybridization and/or PCR amplification with a probe or primer derived from MP. The nucleic acid probe is labeled by combining 50 pmol of the nucleic acid and 250 mCi of [gamma - ⁇ P] adenosine triphosphate (Amersham, Chicago LL) and T4 polynucleotide kinase (DuPont NEN®, Boston MA). The labeled probe is purified with Sephadex G-25 super fine resin column (Pharmacia). A portion containing 10 ' counts per minute of each is used in a typical membrane based hybridization analysis of nucleic acid sample of either genomic or cDNA origin.
- the DNA sample which has been subjected to restriction endonuclease digestion is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham NH). Hybridization is carried out for 16 hours at 40 degrees C. To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. The blots are exposed to film for several hours, the film developed and hybridization patterns are compared visually to detect polynucleotide homologs of B.subtilis MP. The homologs are subjected to confirmatory nucleic acid sequencing.
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Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
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US09/308,375 US6300117B1 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
EP98946010A EP0958368A1 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
CA002272048A CA2272048A1 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
AU93123/98A AU9312398A (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
US10/927,590 US7220716B2 (en) | 1997-09-15 | 2004-08-25 | Proteases from gram-positive organisms |
US10/927,615 US7241575B2 (en) | 1997-09-15 | 2004-08-25 | Proteases from gram-positive organisms |
US10/926,729 US7189555B2 (en) | 1997-09-15 | 2004-08-25 | Proteases from gram-positive organisms |
Applications Claiming Priority (2)
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GBGB9719636.4A GB9719636D0 (en) | 1997-09-15 | 1997-09-15 | Proteases from gram-positive organisms |
GB9719636.4 | 1998-10-13 |
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US09/308,375 A-371-Of-International US6300117B1 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
US09/932,183 Continuation US6833265B2 (en) | 1997-09-15 | 2001-08-17 | Proteases from gram-positive organisms |
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WO1999014342A1 true WO1999014342A1 (en) | 1999-03-25 |
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AU (1) | AU9312398A (en) |
CA (1) | CA2272048A1 (en) |
GB (1) | GB9719636D0 (en) |
WO (1) | WO1999014342A1 (en) |
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Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0369817A2 (en) * | 1988-11-18 | 1990-05-23 | Omnigene Bioproducts, Inc. | Bacillus strains |
-
1997
- 1997-09-15 GB GBGB9719636.4A patent/GB9719636D0/en active Pending
-
1998
- 1998-09-08 CA CA002272048A patent/CA2272048A1/en not_active Abandoned
- 1998-09-08 AU AU93123/98A patent/AU9312398A/en not_active Abandoned
- 1998-09-08 EP EP98946010A patent/EP0958368A1/en not_active Withdrawn
- 1998-09-08 WO PCT/US1998/018828 patent/WO1999014342A1/en active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0369817A2 (en) * | 1988-11-18 | 1990-05-23 | Omnigene Bioproducts, Inc. | Bacillus strains |
Non-Patent Citations (3)
Title |
---|
EMBL/GENBANK DATABASES Accession no AF020713 Sequence reference AF020713 8 April 1998 LAZAREVIC V ET AL: "The complete nucleotide sequence of the Bacillus subtilis SPbeta2 prophage" * |
EMBL/GENBANK DATABASES Accession no O31976, Sequence reference O31976 01 January 1998 KUNST F ET AL:"The complete genome sequence of the Gram-positive bacterium Bacillus subtilis" * |
NATURE, vol. 390, 20 November 1997 (1997-11-20), pages 249 - 256, XP000208013 * |
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CA2272048A1 (en) | 1999-03-25 |
GB9719636D0 (en) | 1997-11-19 |
EP0958368A1 (en) | 1999-11-24 |
AU9312398A (en) | 1999-04-05 |
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