WO1999014341A2 - Proteases from gram-positive organisms - Google Patents
Proteases from gram-positive organisms Download PDFInfo
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- WO1999014341A2 WO1999014341A2 PCT/US1998/018677 US9818677W WO9914341A2 WO 1999014341 A2 WO1999014341 A2 WO 1999014341A2 US 9818677 W US9818677 W US 9818677W WO 9914341 A2 WO9914341 A2 WO 9914341A2
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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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- 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
Definitions
- the present invention relates to metallo-proteases derived from gram-positive microorganisms.
- the present invention provides nucleic acid and amino acid sequences of metallo-proteases identified in Bacillus.
- the present invention also provides methods for the production of the metallo-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 metallo- 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 can be distinguished by their sensitivity to various inhibitors.
- 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.
- the serine proteases have alkaline pH optima, the metalloproteases are optimally active around neutrality, and the cysteine and aspartic 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. About half of the families comprise enzymes containing the His-Glu-Xaa-Xaa-His (or HEXXH) motif which has been shown by X-ray crystallography to form part of the site for binding of the metal (normally zinc) atom. In one family of metalloproteases, a glutamic acid residue completes the metal-binding site, HEXXH + E. This family contains the most well characterized of the metallo-proteases, thermolysin. The three dimensional structure of thermolysin shows that, in the HEXXH motif, the His residues are zinc ligands and the Glu residue has a catalytic function. (Methods in Enzymology, vol. 248, Academic Press, Inc. 1994).
- HEXXH zinc metalloprotease motif
- the present invention relates to the discovery of a heretofore unknown metalloprotease (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.
- MP has overall amino acid relatedness to S. cerevisiae STE24 (Fujimura-Kamada et al., supra) and in part upon the unexpected discovery that nucleic acid encoding gram positive microorganism MP is found immediately downstream of nucleic acid encoding the major alkaline protease putative transcriptional terminator in gram-positive microorganisms.
- the present invention is also based, in part, upon Applicant's discovery that the characteristic metallo-protease amino acid motif HEXXH + E and putative transmembrane domains exist in Bacillus subtilis MP.
- the present invention is also based in part upon Applicant's discovery that Bacillus subtilis MP homologs are found in Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens.
- Applicant's discovery in addition to providing a new and useful group of proteases 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..
- MP may play a role in regulating and/or processing the major alkaline protease in Bacillus.
- MP can serve as a marker for identification of the major alkaline protease in Bacillus species.
- the metallo-protease is obtainable from a gram-positive microorganism which is a Bacillus.
- the metallo-protease is obtainable from a Bacillus which is preferably selected from the group consisting of Bacillus subtilis, Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens.
- the present invention encompasses the naturally occurring MP encoded by nucleic acid found immediately downstream from the transcriptional terminator of the major alkaline protease of a Bacillus species as well as the nucleic acid and amino acid molecules having the sequences disclosed in the Figures.
- the present invention encompasses the naturally occurring MP nucleic acid molecule having the sequence found in Bacillus subtilis 1-168 strain (Bacillus Genetic Stock Center, accession number 1 Al, Columbus, Ohio) in the region of about 1102 kb from the point of origin and immediately downstream of the putative transcriptional terminator of the aprE gene.
- Bacillus subtilis MP nucleic acid and amino acid molecules have the sequences as shown in Figures 1 A-1E.
- the present invention is also based in part upon the unexpected discovery of nucleic acid encoding portions of Bacillus subtilis MP homologs found in at least 3 non B. subtilis Bacillus species, including Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens.
- Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens MP is found downstream of the major alkaline protease of each Bacillus.
- the present invention encompasses the naturally occurring Bacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquifaciens MP.
- the MP is encoded by the nucleic acid molecules having the nucleic acid sequence that is immediately downstream of the putative transcriptional terminator of the major alkaline protease or subtilisn in the genome of Bacillus stearothermophilus, Bacillus licheniformis or Bacillus amyloliquifaciens.
- the Bacillus stearothermophilus MP comprises the amino acid sequence as shown in Figure 3.
- the Bacillus licheniformis MP comprises the amino acid sequence as shown in Figure 4.
- the Bacillus amyloliquifaciens MP comprises the amino acid sequence as shown in Figure 5.
- the present invention encompasses any nucleic acid molecule encoding Bacillus stearothermophilus, Bacillus licheniformis or Bacillus amyloliquifaciens MP.
- the present invention provides isolated polynucleotide and amino acid sequences for Bacillus subtilis MP in Figures 1A-1E. Due to the degeneracy of the genetic code, the present invention encompasses any nucleic acid sequence that encodes the Bacillus subtilis MP amino acid sequence.
- the present invention provides expression vectors and host cells comprising nucleic acid encoding a gram-positive MP.
- the present invention also provides methods of making the gram positive MP.
- the present invention encompasses novel amino acid variations of gram positive MP amino acid sequences disclosed herein that have proteolytic activity. Naturally occurring gram positive MP as well as proteolytically active amino acid variations or derivatives thereof, have application in the textile industry, in cleaning compositions and in animal feed.
- the present invention 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 1 A- IE; and isolating gram-positive nucleic acid which hybridizes to said probe.
- the present invention provides a means of detecting the major alkaline protease of gram-positive microorganisms species based upon nucleic acid hybridization to B. subtilis MP.
- the gram-positive microorganism is a Bacillus.
- the Bacillus is selected from the group consisting of B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
- the production of desired heterologous proteins or polypeptides in gram-positive microorganisms may be hindered by the presence of one or more proteases 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. licheniformis, 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 present invention also encompasses amino acid variations or derivatives of gram positive MP that do not have the characteristic proteolytic activity as long as the nucleic acid sequences encoding such variations or derivatives would have sufficient 5' and 3' coding regions to be capable of being integrated into a gram-positive organism genome.
- Such variants would have applications in gram-positive expression systems where it is desirable to delete, mutate, alter or otherwise incapacitate the naturally occurring metallo-protease in order to diminish or delete its proteolytic activity.
- Such an expression system would have the advantage of allowing for greater yields of recombinant heterologous proteins or polypeptides.
- 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. licheniformis, 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.
- the present invention provides a cleaning composition comprising a metalloprotease of the present invention having the amino acid sequence shown in Figures 1 A- IE or the amino acid encoded by the MP nucleic acid found at about 1102 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- IE or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1 A- IE under high stringency conditions.
- an animal feed comprising a metalloprotease, MP, having the amino acid sequence shown in Figures 1A-1E.
- 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- IE or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1 A- IE under high stringency conditions.
- a composition for the treatment of a textile comprising a metalloprotease, MP, having the amino acid sequence shown in Figures 1 A- IE.
- 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- IE or comprising a metalloprotease encoded by a gene that hybridizes with the nucleic acid shown in Figures 1 A- IE under high stingency conditions.
- a Gram-positive MP is produced on an industrial fermentation scale in a microbial host expression system.
- Figures 1 A- IE shows the DNA, SEQ ID NO: 1, and amino acid sequence, SEQ ID NO:2, for Bacillus subtilis MP.
- Figures 2A-2B show an amino acid alignment of S. cerevisiae STE24, SEQ ID NO:3; 5 Bacillus subtilis MP, designated as YHFN.PRO, SEQ ID NO:2; AFC1_S-1 PRO from
- Figure 4 shows an amino acid alignment of MP with Bacillus licheniformis alkaline protease.
- Figure 5 shows and amino acid alignment of MP with Bacillus amyloliquifaciens i5 alkaline protease gene.
- Figure 6 shows the amino acid alignment of MP with Bacillus subtilis subtilisn.
- Bacillus includes all members known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. ciculans, B. lautus andB. thuringiensis.
- the present invention relates to a newly characterized metallo-protease (MP) from gram 25 positive organisms.
- MP metallo-protease
- the gram-positive organisms is a Bacillus.
- Bacillus is selected from the group consisting of B. subtilis,
- the gram-positive organism is Bacillus subtilis and 30 MP has the amino acid sequence encoded by the nucleic acid molecule having the sequence that occurs around 1102 kilobases from the point of origin of Bacillus subtilis 1-168.
- nucleic acid encoding the B. subtilis MP is immediately downstream from the aprE putative transcriptional terminator.
- Bacillus subtilis has the nucleic acid and amino acid sequence as shown in Figures 1A-1E.
- the present invention encompasses the use of amino acid variations of the amino acid sequences disclosed in Figures 1A-1E that have proteolytic activity. Such 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” as used herein 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 given gram-positive host cell.
- heterologous proteins include enzymes such as hydrolases including proteases, cellulases, amylases, carbohydrases, and lipases; 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 lipases.
- 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 given 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.
- the unexpected discovery of the metallo-protease MP found in translated uncharacterised B.subtilis genomic sequences provides a basis for producing host cells, expression methods and systems which can be used to prevent the degradation of recombinantly produced heterologous proteins.
- 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.
- MP may also be involved in regulation/processing of the major alkaline protease and can serve as a marker for the detection of the major alkaline protease of Bacillus species.
- host cells may also be desired to genetically engineer host cells of any type to produce a gram- positive metallo-protease.
- host cells are used in large scale fermentation to produce large quantities of the metallo-protease which may be isolated or purified and used in cleaning products, such as detergents.
- Gram positive polynucleotides having the nucleic acid sequence immediately downstream of the gram positive microorganism's major alkaline protease or subtilisn transcriptional terminator encode the gram positive MP.
- nucleic acid sequence immediately downstream of the putative transcriptional terminator of aprE was subjected to nucleic acid sequencing and has the nucleic acid sequence and deduced amino acid sequence shown in Figures 1 A- IE.
- a variety of polynucleotides can encode the Bacillus subtilis MP.
- the present invention encompasses all such polynucleotides.
- the present invention encompasses the use of MP polynucleotide homologs encoding gram-positive microorganism metallo-proteases MP which have at least 80%, or at least 90% or at least 95% identity to B.subtilis MP as long as the homolog encodes a protein that has proteolytic activity.
- a preferred MP polynucleotide homolog has 96% homology to B. subtilis MP.
- Gram-positive polynucleotide homologs of B.subtilis MP may be obtained by standard 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.
- Figures 1A-1E and amino acid sequences disclosed in Figures 3, 4 and 5 may reflect inadvertent errors inherent to nucleic acid sequencing technology. Nonetheless, one of ordinary skill in the art is fully capable of determining the correct sequences from the information provided herein regarding the invention.
- the present invention encompasses the naturally occurring nucleic acid molecule having the nucleic acid sequence obtained from the genomic sequence of Bacillus species.
- Bacillus subtilis MP starts around 1102 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 1A1, Columbus, Ohio).
- the Bacillus subtilis point of origin has been described in Ogasawara, N. (1995, Microbiology 141, Pt.2 257-59) and
- Bacillus subtilis MP has a length of 426 amino acids. Based upon the location of the DNA encoding Bacillus subtilis MP and its proximity and relatedness to aprE, 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 1 A- IE 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 gram-positive MP gene For example, 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.
- 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.
- 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 Enzymology, Vol 152. Academic Press. San Diego CA incorporated herein by reference, and confer a defined "stringency” as explained below.
- 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). The process of amplification as carried out in polymerase chain reaction (PCR) technologies is described in Dieffenbach CW and GS Dveksler (1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview NY).
- PCR polymerase chain reaction
- a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides from B. subtilis MP preferably about 12 to 30 nucleotides, and more preferably about 20-25 nucleotides can be used as a probe or PCR primer.
- B.subtilis MP amino acid sequences (shown in Figures 1 A- IE) were identified via a BLAST search (Altschul, Stephen, Basic local alignment search tool, J. Mol. Biol. 215:403- 410) o ⁇ Bacillus subtilis genomic nucleic acid sequences.
- B. subtilis MP (YhfN) was identified by its overall nucleic acid identity to the metallo-protease, STE24 from Saccharomyces cerevisiae, including the presence of the catalytic domain HEXXH +E as shown in Figures 2A- 2B.
- 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 metallo- protease 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.
- 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 metallo-protease(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 a metallo-protease(s) of the present invention 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 confirmed.
- the nucleic acid encoding a metallo-protease 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 metallo-protease under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the metallo- protease 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 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. VII Assay of Protease Activity
- 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
- MP can be used, for example, in bar or liquid soap applications, dishcare formulations, contact lens cleaning ⁇ o 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.
- i5 MP can be formulated into known powdered and liquid detergents having pH between
- detergent cleaning compositions can also include other enzymes such as known proteases, amylases, cellulases, lipases or endoglycosidases, as well as builders and stabilizers.
- any temperature and pH suitable for the detergent is also suitable for the present compositions as long as the pH is within the above range, and the temperature is below the described MP's denaturing temperature.
- 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 MP.
- the 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 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 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 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 Sau3 A digestion.
- Sau3 A 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 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 transformed into the host cell.
- 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 32 P] adenosine triphosphate (Amersham, Chicago IL) and T4 polynucleotide kinase
- 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 (6)
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US09/486,192 US6521440B1 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
AU93069/98A AU9306998A (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
EP98945934A EP1015606A2 (en) | 1997-09-15 | 1998-09-08 | Proteases from gram-positive organisms |
US10/328,459 US6905868B2 (en) | 1997-09-15 | 2002-12-23 | Proteases from gram-positive organisms |
US11/008,331 US7326531B2 (en) | 1997-09-15 | 2004-12-08 | Proteases from gram-positive organisms |
US11/008,352 US8101563B2 (en) | 1997-09-15 | 2004-12-08 | Proteases from gram-positive organisms |
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GBGB9719637.2A GB9719637D0 (en) | 1997-09-15 | 1997-09-15 | Proteases from gram-positive organisms |
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US10/328,459 Division US6905868B2 (en) | 1997-09-15 | 2002-12-23 | Proteases from gram-positive organisms |
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Publication number | Publication date |
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GB9719637D0 (en) | 1997-11-19 |
AU9306998A (en) | 1999-04-05 |
WO1999014341A3 (en) | 1999-05-06 |
EP1015606A2 (en) | 2000-07-05 |
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