WO2016050680A1 - Yoqm-inactivation in bacillus - Google Patents

Yoqm-inactivation in bacillus Download PDF

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Publication number
WO2016050680A1
WO2016050680A1 PCT/EP2015/072230 EP2015072230W WO2016050680A1 WO 2016050680 A1 WO2016050680 A1 WO 2016050680A1 EP 2015072230 W EP2015072230 W EP 2015072230W WO 2016050680 A1 WO2016050680 A1 WO 2016050680A1
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Prior art keywords
bacillus
polypeptide
host cell
polynucleotide
yoqm
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PCT/EP2015/072230
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French (fr)
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Anne BREÜNER
Peter Rahbek Oestergaard
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Novozymes A/S
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Publication of WO2016050680A1 publication Critical patent/WO2016050680A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/32Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)

Definitions

  • Immobilized metal ion affinity chromatography is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, copper for the purification of histidine containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce a polyhistidine protein tag into the relevant gene in order to purify the encoded protein of interest by metal affinity chromatography.
  • Methods used to elute the tagged protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.
  • Polyhistidine-tags have been used for affinity purification of tagged recombinant proteins (Hengen, P (1995) Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences 20 (7): 285-6).
  • the instant invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt; as well as methods of producing a polyhistidine- tagged polypeptide of interest in such mutant host cells.
  • Ni-IDA iminodiacetic acid
  • Ni-NTA nitrilotriacetic acid
  • Co-CMA carboxyl-methylaspartate
  • Polyhistidine tagged polypeptides are known to bind effectively to affinity resins comprising bound bivalent nickel or cobalt ions, such as, sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl- methylaspartate (Co-CMA) for cobalt.
  • affinity resins are, therefore, routinely used in protein chemistry for the recovery or purification of poly-histidine tagged polypeptides of interest which are very often expressed in Bacillus hosts.
  • affinity-purifying a polyhistidine-tagged polypeptide produced in a Bacillus host cell is sometimes made difficult because a significant portion of the total affinity- bound polypeptides will consist of contaminants, i.e. not the intended polyhistidine tagged polypeptide of interest but other non-specifically bound contaminating polypeptides.
  • YoqM one native polypeptide, in Bacillus subtilis was responsible for a large part of the non-specifically bound contaminating polypeptides after affinity purificication.
  • the present invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having a least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; and (c) a polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 .
  • the invention in a second aspect, relates to methods of producing a polyhistidine-tagged polypeptide of interest, comprising the steps of: (a) cultivating a host cell of the first aspect under conditions conducive for production of the polypeptide; and, optionally, (b) recovering the polypeptide.
  • Figure 1 shows the Genetic map of the region of the Bacillus subtilis chromosome containing yoqM, the gene encoding YoqM, and flanking genes (black arrows). Chequered arrows: upstream and downstream homology regions used when replacing part of yoqM with spec. The region replaced by spec in AEB1779 is indicated by a horizontal line.
  • Figure 2 shows an SDS-PAGE analysis of supernatants from fermentations of various strains. Lanes marked M: Molecular weight marker. Sizes of relevant proteins are indicated. Lanes 1 , 5, and 9: A164 ⁇ 1 1. Lanes 2, 6, and 10: A164 ⁇ 1 1 producing the protein given above each gel. The position of the protein is indicated by an arrow, as is the position of YoqM. Lanes 3, 7, and 1 1 : AEB1779 producing the protein given above each gel. Lanes 4, 8, and 12: AEB1779.
  • Figure 3 shows an SDS-PAGE analysis of fractions from Ni-NTA column purifications of supernatants from strains expressing the same leucine aminopeptidase.
  • Lanes 1 +20 Molecular weight marker (97, 66, 45, 30, 20.1 , 14.4kDa).
  • Lanes 2-10 AYoqM strain AEB1779.
  • Lane 2 10 ⁇ 0.2 ⁇ filtrate
  • Lanes 3-10 1 ⁇ of fractions no 3-10 from elution of the Ni-NTA column.
  • Lanes 1 1 -19 YoqM wt strain ⁇ 164 ⁇ 1 1.
  • Lane 1 1 1 10 ⁇ 0.2 ⁇ filtrate
  • Lanes 12-19 1 ⁇ of fractions no 3-10 from elution of the Ni-NTA column.
  • the position of the aminopeptidase and of YoqM is indicated by arrows.
  • Figure 4 shows an SDS-PAGE analysis of fractions from Ni-NTA column purifications of supernatants from strains expressing the same amylase; Lanes 1 -7: Fractions from purification of the amylase in the AYoqM host strain AEB1779, Lanes 9-15 Fractions from purification of the amylase in the YoqM wt host strain ⁇ 164 ⁇ 1 1 , Lane 8: MW marker (Molecular weights are given to the left in the figure). The position of the amylase and YoqM is indicated by arrows. Lane 1 : Fermentation broth from expression of the amylase in AEB1779. Lane 9: Fermentation broth from expression of the amylase in A164 ⁇ 1 1. Lanes 2 and 10: Flow through and wash from the Ni-NTA-columns, Lanes 3-10 and 1 1 -15: Fractions from Ni-NTA-columns.
  • polyhistidine tagged polypeptide means a polypeptide which has been modified to comprise at least six consecutive histidine residues, (H) 6 in its amino acid sequence or at least three alternating histidine residues within a region of six amino acid sequences, such as (HX) 3 , especially (HQ) 3 .
  • HX histidine residues
  • HQ alternating histidine residues within a region of six amino acid sequences
  • Coding sequence means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide.
  • the boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA.
  • the coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
  • control sequences means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention.
  • Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other.
  • control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
  • Expression includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • Expression vector means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
  • host cell means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Isolated means a substance in a form or environment that does not occur in nature.
  • isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
  • Mature polypeptide means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
  • one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
  • Mature polypeptide coding sequence means a polynucleotide that encodes a mature polypeptide of interest.
  • nucleic acid construct means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
  • Operably linked means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.
  • Sequence identity The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity”.
  • the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later.
  • the parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
  • the output of Needle labeled "longest identity" is used as the percent identity and is calculated as follows:
  • Very high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
  • High stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
  • Medium-high stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C.
  • Medium stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
  • Low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
  • Very low stringency conditions means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
  • the first aspect of the invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide selected from the group consisting of:
  • a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2;
  • a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having a least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; and
  • the mutant Bacillus host cell of the invention is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fastidiosus, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus megaterium, Bacillus methanolicus, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis host cell.
  • the mutant Bacillus host cell of the invention produces a polyhistidine-tagged polypeptide of interest; most preferably said polyhistidine-tagged polypeptide of interest is homologous or heterologous to the host cell; preferably it is an enzyme; more preferably the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase; and most preferably the enzyme is an alpha-glucosidase, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, glucocerebrosidase, alpha-glucosidase, beta-glucosi
  • the polyhistidine-tagged polypeptide of interest comprises at least six consecutive histidine residues, (H) 6 in its amino acid sequence or at least three alternating histidine residues in a region of six amino acid sequences, (HX) 3 , preferably (HQ) 3 ; still more preferably, the at least six consecutive histidine residues or the at least three alternating histidine residues in a region of six amino acid sequences are located in the N- terminal or C-terminal end of the polypeptide.
  • the YoqM polypeptide is capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt.
  • an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt.
  • a polypeptide of interest of the present invention may be obtained from microorganisms of any genus.
  • the term "obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted.
  • the polypeptide obtained from a given source is secreted extracellularly.
  • the polypeptide of interest is an enzyme, such as, a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably it is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta- glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucano-transferase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase
  • the polypeptide may be a bacterial polypeptide.
  • the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having [enzyme] activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
  • the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.
  • the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.
  • the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.
  • the polypeptide may be a fungal polypeptide.
  • the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora
  • the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.
  • the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysospohum keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusa
  • the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Center
  • the polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample.
  • the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).
  • the present invention also relates to polynucleotides encoding a YoqM polypeptide of interest, as described herein.
  • the techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof.
  • the cloning of the polynucleotides from genomic DNA can be effected, e.g. , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York.
  • Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • the polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding YoqM polypeptides from strains of different genera or species according to methods well known in the art.
  • probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein.
  • Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g. , at least 25, at least 35, or at least 70 nucleotides in length.
  • the nucleic acid probe is at least 100 nucleotides in length, e.g. , at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length.
  • Both DNA and RNA probes can be used.
  • the probes are typically labeled for detecting the corresponding gene (for example, with 32 P, 3 H, 35 S, biotin, or avidin). Such probes are encompassed by the present invention.
  • a genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a YoqM polypeptide.
  • Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques.
  • DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material.
  • the carrier material is used in a Southern blot.
  • hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to SEQ ID NO: 1 or a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
  • Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide.
  • the term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide.
  • These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like.
  • the variants may be constructed on the basis of a polynucleotide encoding a mature polypeptide, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence.
  • nucleotide substitutions see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.
  • the present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
  • the polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
  • the control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the polypeptide.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.
  • E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene ⁇ dagA), and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25).
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription.
  • the terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease ⁇ aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
  • control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
  • mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471 ).
  • the control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway.
  • the 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide.
  • the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence.
  • a foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence.
  • a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide.
  • any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
  • Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases ⁇ nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
  • the control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide.
  • the resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases).
  • a propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease ⁇ aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
  • the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
  • regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell.
  • regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems.
  • Other examples of regulatory sequences are those that allow for gene amplification.
  • the present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals.
  • the various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites.
  • the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vector may be a linear or closed circular plasmid.
  • the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome.
  • the vector may contain any means for assuring self-replication.
  • the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.
  • the vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells.
  • a selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
  • the vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
  • the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination.
  • the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s).
  • the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination.
  • the integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
  • the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question.
  • the origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell.
  • the term "origin of replication" or "plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.
  • bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ⁇ permitting replication in Bacillus.
  • More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide.
  • An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
  • the present invention also relates to recombinant host cells, comprising a polynucleotide operably linked to one or more control sequences that direct the production of a polypeptide of interest.
  • a construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector as described earlier.
  • host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
  • the host cell may be any Bacillus cell useful in the recombinant production of a polypeptide of the present invention, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cell.
  • Bacillus alkalophilus e.g., Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacill
  • the introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 -1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271 -5278).
  • any method known in the art for introducing DNA into a host cell can be used.
  • the present invention also relates to methods of producing a polypeptide, comprising
  • the cell is a Bacillus cell. In another aspect, the cell is a Bacillus subtilis cell.
  • the host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art.
  • the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated.
  • the cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
  • the YoqM polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
  • the polypeptide may be recovered using methods known in the art.
  • the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
  • a fermentation broth comprising the polypeptide is recovered.
  • the polypeptide may be purified or detected by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
  • chromatography e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion
  • electrophoretic procedures e.g., preparative isoelectric focusing
  • differential solubility e.g., ammonium sulfate precipitation
  • SDS-PAGE or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York,
  • the present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting or deleting a YoqM-encoding polynucleotide, for example as shown in SEQ ID NO:1 , or a portion thereof, encoding a YoqM polypeptide of the present invention, which results in the mutant cell producing less of the YoqM polypeptide than the parent cell when cultivated under the same conditions.
  • the polynucleotide inactivation is a disruption or a partial or full deletion of the YoqM-encoding polynucleotide.
  • the mutant cell may be constructed by reducing or eliminating expression of the polynucleotide using methods well known in the art, for example, insertions, disruptions, replacements, or deletions.
  • the polynucleotide is inactivated.
  • the polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for expression of the coding region.
  • An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the polynucleotide.
  • Other control sequences for possible modification include, but are not limited to, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.
  • Modification or inactivation of the polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the polynucleotide has been reduced or eliminated.
  • the mutagenesis which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
  • Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
  • UV ultraviolet
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • EMS ethyl methane sulphonate
  • sodium bisulphite formic acid
  • nucleotide analogues examples include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide ana
  • the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.
  • Modification or inactivation of the polynucleotide may be accomplished by insertion, substitution, or deletion of one or more nucleotides in the gene or a regulatory element required for transcription or translation thereof.
  • nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame.
  • modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art.
  • the modification may be performed in vivo, i.e., directly on the cell expressing the polynucleotide to be modified, it is preferred that the modification be performed in vitro as exemplified below.
  • An example of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption.
  • a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene.
  • the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed.
  • the polynucleotide is disrupted with a selectable marker such as those described herein.
  • the present invention also relates to methods of inhibiting the expression of the YoqM polypeptide in a Bacillus subtilis cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention.
  • the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
  • the dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA).
  • siRNA small interfering RNA
  • miRNA micro RNA
  • the dsRNA is small interfering RNA for inhibiting transcription.
  • the dsRNA is micro RNA for inhibiting translation.
  • the present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the polypeptide coding sequence of SEQ ID NO:1 for inhibiting expression of the polypeptide in a cell.
  • dsRNA double-stranded RNA
  • the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs.
  • ssRNA single-stranded RNA
  • RNAi RNA interference
  • the dsRNAs of the present invention can be used in gene-silencing.
  • the invention provides methods to selectively degrade RNA using a dsRNAi of the present invention.
  • the process may be practiced in vitro, ex vivo or in vivo.
  • the dsRNA molecules can be used to generate a loss-of-function mutation in a cell, an organ or an animal.
  • Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559; 6,51 1 ,824; and 6,515,109.
  • the present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a polynucleotide encoding the polypeptide or a control sequence thereof or a silenced gene encoding the polypeptide, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell.
  • the polypeptide-deficient mutant cells are particularly useful as host cells for expression of native and heterologous polypeptides. Therefore, the present invention further relates to methods of producing a native or heterologous polypeptide, comprising (a) cultivating the mutant cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
  • heterologous polypeptides means polypeptides that are not native to the host cell, e.g., a variant of a native protein.
  • the host cell may comprise more than one copy of a polynucleotide encoding the native or heterologous polypeptide.
  • the methods used for cultivation and purification of the product of interest may be performed by methods known in the art.
  • the present invention relates to a protein product essentially free from YoqM polypeptide that is produced by a method of the present invention.
  • the present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
  • Bacillus subtilis 168 B. subtilis type strain (BGSC 1A1 , Bacillus Genetic Stock Center, Columbus, OH)
  • Bacillus subtilis 168 ⁇ 4 B subtilis 168 with deletions in the spollAC, aprE, nprE, and amyE genes. The deletion of these four genes was performed essentially as described for Bacillus subtilis ⁇ 164 ⁇ 5 in U.S. Patent No. 5,891 ,701.
  • A164 Bacillus subtilis wild type strain ATCC 6051 a
  • ⁇ 164 ⁇ 10 Bacillus subtilis A164 with deletions in the genes sigF, nprE, aprE, amyE, srfAC, wprA, bpr, vpr, mpr, and epr, rendering them all inactive.
  • Connelly MB et al (2004) "Extracellular Proteolytic activity Plays a Central Role in Swarming Motility in Bacillus subtilis" J. Bacteriol. 186(13) 4159-4167.
  • ⁇ 164 ⁇ 1 1 Bacillus subtilis ⁇ 164 ⁇ 10 with a disruption in the ispA gene, rendering it inactive as disclosed in WO 2012/127002.
  • AEB1766 Bacillus subtilis 186 ⁇ 4 with the yoqM gene inactivated by insertion of spec the gene as disclosed herein.
  • the Bacillus subtilis yoqM DNA sequence is provided in SEQ ID NO:1 and the encoded YoqM amino acid sequence is provided in SEQ ID NO:2.
  • AEB1779 Bacillus subtilis ⁇ 164 ⁇ 1 1 with the yoqM gene inactivated by insertion of spec as disclosed herein.
  • Enzymes for DNA manipulation were used essentially as recommended by the supplier.
  • Competent cells and transformation of B. subtilis was obtained as described in Yasbin et al. (1975): Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121 , 296-304.
  • Example 1 Obtaining a SOE-PCR fragment for replacing yoqM with spec
  • the chromosomal region upstream of the yoqM gene in B. subtilis A164, the spectinomycin resistance marker gene, spec, and the region downstream from the yoqM gene were joined by SOE-PCR.
  • the following templates and primers were used for obtaining the three fragments:
  • Upstream region Template, chromosomal DNA from B. subtilis A164; primers, pab785 and pab786.
  • spec gene Template, chromosomal DNA from a B. subtilis strain in which the spec gene and flanking regions from Tn554 from Staphylococcus aureus have been inserted.
  • the flanking regions should include at least 142 bp upstream of the spec gene and 100 bp downstream for the primers to be able to anneal. Primers, pab784 and pab787.
  • Downstream region Template, chromosomal DNA from B. subtilis A164; primers, pab788 and pab790.
  • FIG. 1 A map of the yoqM region of B. subtilis A164 is shown in Figure 1 , with the upstream and downstream amplified regions indicated, as well as the region replaced by spec.
  • the yoqM gene in B. subtilis ⁇ 164 ⁇ 1 1 was inactivated by insertion of spec by preparing chromosomal DNA from AEB1766, obtained in example 2, and transforming it into B. subtilis ⁇ 164 ⁇ 1 1. By selecting for resistance towards spectinomycin, strains in which the spec gene was inserted in the chromosome could be isolated. Correct insertion of spec in yoqM was verified by PCR on chromosomal DNA from spectinomycin resistant transformants with appropriate primers. One correct strain was named AEB1779.
  • Expression constructs were prepared of four bacterial genes, representing four different enzyme classes: a bacterial amylase (glycoside hydrolase family 13), a peptidase (family M28 leucine aminopeptidase), a phytase (histidine acid phosphatase) and a kappa-carrageenase (glycoside hydrolase family 16). All the constructs were designed so that His-tags were fused to the expressed enzymes.
  • Gene expression cassettes were inserted by homologous recombination in the pectate lyase locus of the two Bacillus subtilis hosts ⁇ 164 ⁇ 1 1 and AEB1779 under the control of a triple promoter system as described in W099/43835. The gene coding for chloramphenicol acetyltransferase was used as a marker (as described in Diderichsen et al., 1993, Plasmid 30:312-315).
  • the yoqM wildtype and yoqM deleted strains both expressing a His-tagged leucine aminopeptidase as described in Example 4 were fermented in CAL 18-2 medium and culture supernatants were purified by filtering through a Nalgene 0.2 ⁇ filtration unit in order to remove the rest of the Bacillus host cells.
  • the pH of the 0.2 ⁇ filtrate was adjusted to pH 7.5 with 3M Tris-base and applied to a Ni-NTA column (Ni-NTA Superflow from Qiagen) equilibrated in 50mM HEPES, 5mM Imidazole, 500mM NaCI, pH 7.5.
  • Example 4 were fermented in CAL 18-2 medium and culture supernatants were purified essentially as described in Example 5, and fractions were analysed by SDS-PAGE ( Figure 4). Expression of the amylase was similar in both strains (Fig. 4, lanes 1 and 9) as was also found in Example 4, but the yield of amylase in the Ni-NTA-column eluent was clearly higher from the yoqM deleted host.

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Abstract

The instant invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl- methylaspartate (Co-CMA) for cobalt; as well as methods of producing a polyhistidine-tagged polypeptide of interest in such mutant host cells.

Description

YoqM-inactivation in Bacillus
Reference to a Sequence Listing
This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.
Background of the Invention
Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, copper for the purification of histidine containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce a polyhistidine protein tag into the relevant gene in order to purify the encoded protein of interest by metal affinity chromatography. Methods used to elute the tagged protein of interest include changing the pH, or adding a competitive molecule, such as imidazole. Polyhistidine-tags have been used for affinity purification of tagged recombinant proteins (Hengen, P (1995) Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences 20 (7): 285-6).
Field of the Invention
The instant invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt; as well as methods of producing a polyhistidine- tagged polypeptide of interest in such mutant host cells.
Summary of the Invention
Polyhistidine tagged polypeptides are known to bind effectively to affinity resins comprising bound bivalent nickel or cobalt ions, such as, sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl- methylaspartate (Co-CMA) for cobalt. Such affinity resins are, therefore, routinely used in protein chemistry for the recovery or purification of poly-histidine tagged polypeptides of interest which are very often expressed in Bacillus hosts.
However, the process of affinity-purifying a polyhistidine-tagged polypeptide produced in a Bacillus host cell is sometimes made difficult because a significant portion of the total affinity- bound polypeptides will consist of contaminants, i.e. not the intended polyhistidine tagged polypeptide of interest but other non-specifically bound contaminating polypeptides.
The inventors of the present application found to their surprise that especially one native polypeptide, YoqM, in Bacillus subtilis was responsible for a large part of the non-specifically bound contaminating polypeptides after affinity purificication. The inactivation of the YoqM- encoding gene led to much improved yields as well as improved purities of several different affinity-purified polyhistidine tagged polypeptide of interest produced in B. subtilis.
Accordingly, in a first aspect the present invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; (b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having a least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; and (c) a polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 .
In a second aspect, the invention relates to methods of producing a polyhistidine-tagged polypeptide of interest, comprising the steps of: (a) cultivating a host cell of the first aspect under conditions conducive for production of the polypeptide; and, optionally, (b) recovering the polypeptide.
Brief Description of the Figures
Figure 1 shows the Genetic map of the region of the Bacillus subtilis chromosome containing yoqM, the gene encoding YoqM, and flanking genes (black arrows). Chequered arrows: upstream and downstream homology regions used when replacing part of yoqM with spec. The region replaced by spec in AEB1779 is indicated by a horizontal line.
Figure 2 shows an SDS-PAGE analysis of supernatants from fermentations of various strains. Lanes marked M: Molecular weight marker. Sizes of relevant proteins are indicated. Lanes 1 , 5, and 9: A164 Δ1 1. Lanes 2, 6, and 10: A164Δ1 1 producing the protein given above each gel. The position of the protein is indicated by an arrow, as is the position of YoqM. Lanes 3, 7, and 1 1 : AEB1779 producing the protein given above each gel. Lanes 4, 8, and 12: AEB1779.
Figure 3 shows an SDS-PAGE analysis of fractions from Ni-NTA column purifications of supernatants from strains expressing the same leucine aminopeptidase. Lanes 1 +20: Molecular weight marker (97, 66, 45, 30, 20.1 , 14.4kDa). Lanes 2-10: AYoqM strain AEB1779. Lane 2: 10μΙ 0.2μηι filtrate, Lanes 3-10: 1 μΙ of fractions no 3-10 from elution of the Ni-NTA column. Lanes 1 1 -19: YoqM wt strain Α164Δ1 1. Lane 1 1 : 10μΙ 0.2μηι filtrate, Lanes 12-19: 1 μΙ of fractions no 3-10 from elution of the Ni-NTA column. The position of the aminopeptidase and of YoqM is indicated by arrows.
Figure 4 shows an SDS-PAGE analysis of fractions from Ni-NTA column purifications of supernatants from strains expressing the same amylase; Lanes 1 -7: Fractions from purification of the amylase in the AYoqM host strain AEB1779, Lanes 9-15 Fractions from purification of the amylase in the YoqM wt host strain Α164Δ1 1 , Lane 8: MW marker (Molecular weights are given to the left in the figure). The position of the amylase and YoqM is indicated by arrows. Lane 1 : Fermentation broth from expression of the amylase in AEB1779. Lane 9: Fermentation broth from expression of the amylase in A164Δ1 1. Lanes 2 and 10: Flow through and wash from the Ni-NTA-columns, Lanes 3-10 and 1 1 -15: Fractions from Ni-NTA-columns.
Definitions
Polyhistidine-tagged: The term "polyhistidine tagged polypeptide" means a polypeptide which has been modified to comprise at least six consecutive histidine residues, (H)6 in its amino acid sequence or at least three alternating histidine residues within a region of six amino acid sequences, such as (HX)3, especially (HQ)3. Usually, but not always, the at least six consecutive histidine residues or the at least three alternating histidine residues within a region of six amino acid sequences are located in the N-terminal or C-terminal end of the polypeptide.
Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.
Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.
Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1 ) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).
Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide. It is also known in the art that different host cells process polypeptides differently, and thus, one host cell expressing a polynucleotide may produce a different mature polypeptide (e.g., having a different C-terminal and/or N-terminal amino acid) as compared to another host cell expressing the same polynucleotide.
Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide of interest.
Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.
Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence. Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".
For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment)
Very high stringency conditions: The term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 70°C.
High stringency conditions: The term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 65°C.
Medium-high stringency conditions: The term "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 60°C. Medium stringency conditions: The term "medium stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 55°C.
Low stringency conditions: The term "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 50°C.
Very low stringency conditions: The term "very low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2X SSC, 0.2% SDS at 45°C.
Detailed Description of the Invention
The first aspect of the invention relates to mutant Bacillus host cells which comprise an inactivation of a polynucleotide encoding a YoqM polypeptide selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2; preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having a least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; and
(c) a polypeptide encoded by a polynucleotide that hybridizes under very low stringency conditions with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 ; preferably under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions or very high stringency conditions with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1.
In a preferred embodiment, the mutant Bacillus host cell of the invention is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fastidiosus, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus megaterium, Bacillus methanolicus, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis host cell.
Preferably, the mutant Bacillus host cell of the invention produces a polyhistidine-tagged polypeptide of interest; most preferably said polyhistidine-tagged polypeptide of interest is homologous or heterologous to the host cell; preferably it is an enzyme; more preferably the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase; and most preferably the enzyme is an alpha-glucosidase, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, urokinase, or xylanase.
It is also preferable that the polyhistidine-tagged polypeptide of interest comprises at least six consecutive histidine residues, (H)6 in its amino acid sequence or at least three alternating histidine residues in a region of six amino acid sequences, (HX)3, preferably (HQ)3; still more preferably, the at least six consecutive histidine residues or the at least three alternating histidine residues in a region of six amino acid sequences are located in the N- terminal or C-terminal end of the polypeptide.
In a preferred embodiment of the invention, the YoqM polypeptide is capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt.
Sources of Polypeptides of interest
A polypeptide of interest of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.
Preferably, the polypeptide of interest is an enzyme, such as, a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase; more preferably it is an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, asparaginase, beta-galactosidase, beta- glucosidase, beta-xylosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, green fluorescent protein, glucano-transferase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or a xylanase.
The polypeptide may be a bacterial polypeptide. For example, the polypeptide may be a Gram-positive bacterial polypeptide such as a Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces polypeptide having [enzyme] activity, or a Gram-negative bacterial polypeptide such as a Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma polypeptide.
In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide.
In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide.
In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide.
The polypeptide may be a fungal polypeptide. For example, the polypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.
In another aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.
In another aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium inops, Chrysospohum keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia setosa, Thielavia spededonium, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.
It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above- mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al, 1989, supra).
Polynucleotides
The present invention also relates to polynucleotides encoding a YoqM polypeptide of interest, as described herein. The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from genomic DNA can be effected, e.g. , by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used.
The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding YoqM polypeptides from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g. , at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g. , at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a YoqM polypeptide. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with SEQ ID NO: 1 or a subsequence thereof, the carrier material is used in a Southern blot.
For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to SEQ ID NO: 1 or a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film or any other detection means known in the art.
Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of a polynucleotide encoding a mature polypeptide, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107. Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.
The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis crylllA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301 -315), Streptomyces coelicolor agarase gene {dagA), and prokaryotic beta- lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731 ), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21 -25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.
The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease {aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).
The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.
Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis crylllA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471 ).
The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used.
Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 1 1837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, Bacillus stearothermophilus neutral proteases {nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.
The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease {aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.
Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that cause expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory sequences in prokaryotic systems include the lac, tac, and trp operator systems. Other examples of regulatory sequences are those that allow for gene amplification.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used. The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance.
The vector preferably contains an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.
Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB1 10, pE194, pTA1060, and ρΑΜβΙ permitting replication in Bacillus.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et ai, 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising a polynucleotide operably linked to one or more control sequences that direct the production of a polypeptide of interest. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or vector is maintained as a chromosomal integrant or as a self- replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any Bacillus cell useful in the recombinant production of a polypeptide of the present invention, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis cell.
The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 1 1 1 -1 15), competent cell transformation (see, e.g., Young and Spizizen, 1961 , J. Bacteriol. 81 : 823-829, or Dubnau and Davidoff-Abelson, 1971 , J. Mol. Biol. 56: 209-221 ), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751 ), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271 -5278). However, any method known in the art for introducing DNA into a host cell can be used.
Methods of Production
The present invention also relates to methods of producing a polypeptide, comprising
(a) cultivating a cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the polypeptide. In one aspect, the cell is a Bacillus cell. In another aspect, the cell is a Bacillus subtilis cell.
The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cells may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed- batch, or solid state fermentations) in laboratory or industrial fermentors in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.
The YoqM polypeptide may be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide.
The polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a fermentation broth comprising the polypeptide is recovered.
The polypeptide may be purified or detected by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
Removal or Reduction of a polypeptide by gene inactivation
The present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting or deleting a YoqM-encoding polynucleotide, for example as shown in SEQ ID NO:1 , or a portion thereof, encoding a YoqM polypeptide of the present invention, which results in the mutant cell producing less of the YoqM polypeptide than the parent cell when cultivated under the same conditions.
In a preferred embodiment, the polynucleotide inactivation is a disruption or a partial or full deletion of the YoqM-encoding polynucleotide.
The mutant cell may be constructed by reducing or eliminating expression of the polynucleotide using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. In a preferred aspect, the polynucleotide is inactivated. The polynucleotide to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the polynucleotide. Other control sequences for possible modification include, but are not limited to, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.
Modification or inactivation of the polynucleotide may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the polynucleotide has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.
Modification or inactivation of the polynucleotide may be accomplished by insertion, substitution, or deletion of one or more nucleotides in the gene or a regulatory element required for transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the polynucleotide to be modified, it is preferred that the modification be performed in vitro as exemplified below.
An example of a convenient way to eliminate or reduce expression of a polynucleotide is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous polynucleotide is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous polynucleotide. It may be desirable that the defective polynucleotide also encodes a marker that may be used for selection of transformants in which the polynucleotide has been modified or destroyed. In an aspect, the polynucleotide is disrupted with a selectable marker such as those described herein. The present invention also relates to methods of inhibiting the expression of the YoqM polypeptide in a Bacillus subtilis cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more duplex nucleotides in length.
The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA for inhibiting translation.
The present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the polypeptide coding sequence of SEQ ID NO:1 for inhibiting expression of the polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).
The dsRNAs of the present invention can be used in gene-silencing. In one aspect, the invention provides methods to selectively degrade RNA using a dsRNAi of the present invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can be used to generate a loss-of-function mutation in a cell, an organ or an animal. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559; 6,51 1 ,824; and 6,515,109.
The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a polynucleotide encoding the polypeptide or a control sequence thereof or a silenced gene encoding the polypeptide, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell.
The polypeptide-deficient mutant cells are particularly useful as host cells for expression of native and heterologous polypeptides. Therefore, the present invention further relates to methods of producing a native or heterologous polypeptide, comprising (a) cultivating the mutant cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptides" means polypeptides that are not native to the host cell, e.g., a variant of a native protein. The host cell may comprise more than one copy of a polynucleotide encoding the native or heterologous polypeptide.
The methods used for cultivation and purification of the product of interest may be performed by methods known in the art.
In a further aspect, the present invention relates to a protein product essentially free from YoqM polypeptide that is produced by a method of the present invention. The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Strains
Bacillus subtilis 168: B. subtilis type strain (BGSC 1A1 , Bacillus Genetic Stock Center, Columbus, OH)
Bacillus subtilis 168Δ4: B subtilis 168 with deletions in the spollAC, aprE, nprE, and amyE genes. The deletion of these four genes was performed essentially as described for Bacillus subtilis Α164Δ5 in U.S. Patent No. 5,891 ,701.
A164: Bacillus subtilis wild type strain ATCC 6051 a
Α164Δ10: Bacillus subtilis A164 with deletions in the genes sigF, nprE, aprE, amyE, srfAC, wprA, bpr, vpr, mpr, and epr, rendering them all inactive. Connelly MB et al (2004) "Extracellular Proteolytic activity Plays a Central Role in Swarming Motility in Bacillus subtilis" J. Bacteriol. 186(13) 4159-4167.
Α164Δ1 1 : Bacillus subtilis Α164Δ10 with a disruption in the ispA gene, rendering it inactive as disclosed in WO 2012/127002.
AEB1766: Bacillus subtilis 186Δ4 with the yoqM gene inactivated by insertion of spec the gene as disclosed herein. The Bacillus subtilis yoqM DNA sequence is provided in SEQ ID NO:1 and the encoded YoqM amino acid sequence is provided in SEQ ID NO:2.
AEB1779: Bacillus subtilis Α164Δ1 1 with the yoqM gene inactivated by insertion of spec as disclosed herein.
Table 1. Oligonucleotide primers
Figure imgf000020_0001
Molecular biological methods
DNA manipulations and transformations were performed by standard molecular biology methods as described in:
• Sambrook et al. (1989): Molecular cloning: A laboratory manual. Cold Spring Harbor laboratory, Cold Spring Harbor, NY.
• Ausubel et al. (eds) (1995): Current protocols in Molecular Biology. John Wiley and Sons.
• Harwood and Cutting (eds) (1990): Molecular Biological Methods for Bacillus. John Wiley and Sons.
Enzymes for DNA manipulation were used essentially as recommended by the supplier.
Competent cells and transformation of B. subtilis was obtained as described in Yasbin et al. (1975): Transformation and transfection in lysogenic strains of Bacillus subtilis: evidence for selective induction of prophage in competent cells. J. Bacteriol. 121 , 296-304.
Example 1. Obtaining a SOE-PCR fragment for replacing yoqM with spec
The chromosomal region upstream of the yoqM gene in B. subtilis A164, the spectinomycin resistance marker gene, spec, and the region downstream from the yoqM gene were joined by SOE-PCR. The following templates and primers were used for obtaining the three fragments:
Upstream region: Template, chromosomal DNA from B. subtilis A164; primers, pab785 and pab786.
spec gene: Template, chromosomal DNA from a B. subtilis strain in which the spec gene and flanking regions from Tn554 from Staphylococcus aureus have been inserted. The flanking regions should include at least 142 bp upstream of the spec gene and 100 bp downstream for the primers to be able to anneal. Primers, pab784 and pab787.
Downstream region: Template, chromosomal DNA from B. subtilis A164; primers, pab788 and pab790.
To join the three fragments by a final SOE-PCR reaction was performed with the three fragments as templates and primers pab564 and pab789.
A map of the yoqM region of B. subtilis A164 is shown in Figure 1 , with the upstream and downstream amplified regions indicated, as well as the region replaced by spec.
Example 2. Inactivating yoqM in Bacillus subtilis 168Δ4 by insertion of spec
The SOE-PCR fragment with upstream yogM-spec-downstream yoqM obtained in example 1 was transformed into B. subtilis 168Δ4, selecting for resistance against spectinomycin, since the spec gene on the SOE-PCR fragment renders the strain resistant to this antibiotic. Correct insertion of the fragment was tested in spectinomycin resistant transformants by PCR on chromosomal DNA and sequencing of the resulting PCR fragment using appropriate primers. One correct strain was named AEB1766. Example 3. Inactivating yoqM in Bacillus subtilis Α164Δ11 by insertion of spec
The yoqM gene in B. subtilis Α164Δ1 1 was inactivated by insertion of spec by preparing chromosomal DNA from AEB1766, obtained in example 2, and transforming it into B. subtilis Α164Δ1 1. By selecting for resistance towards spectinomycin, strains in which the spec gene was inserted in the chromosome could be isolated. Correct insertion of spec in yoqM was verified by PCR on chromosomal DNA from spectinomycin resistant transformants with appropriate primers. One correct strain was named AEB1779.
Example 4. YoqM is absent in supernatant of strain AEB1779
Expression constructs were prepared of four bacterial genes, representing four different enzyme classes: a bacterial amylase (glycoside hydrolase family 13), a peptidase (family M28 leucine aminopeptidase), a phytase (histidine acid phosphatase) and a kappa-carrageenase (glycoside hydrolase family 16). All the constructs were designed so that His-tags were fused to the expressed enzymes. Gene expression cassettes were inserted by homologous recombination in the pectate lyase locus of the two Bacillus subtilis hosts Α164Δ1 1 and AEB1779 under the control of a triple promoter system as described in W099/43835. The gene coding for chloramphenicol acetyltransferase was used as a marker (as described in Diderichsen et al., 1993, Plasmid 30:312-315).
Recombinant B. subtilis clones containing the integrated expression construct were grown in liquid culture in CAL 18-2 medium (WOOO/075344). Supernatants were analysed for presence of expressed protein by SDS-PAGE. Visual inspection of the resulting gels shows that for all four bacterial genes the approximately 8 kDa protein band corresponding to YoqM that is present in strains constructed in Α164Δ1 1 is absent in strains derived from AEB1779 (amylase, protease, and phytase are shown in Figure 2. NB: The kappa-carageenase is not shown). Example 5. YoqM absent from peptidase Ni-NTA column eluate from AEB1779
The yoqM wildtype and yoqM deleted strains both expressing a His-tagged leucine aminopeptidase as described in Example 4 were fermented in CAL 18-2 medium and culture supernatants were purified by filtering through a Nalgene 0.2μηι filtration unit in order to remove the rest of the Bacillus host cells. The pH of the 0.2μηι filtrate was adjusted to pH 7.5 with 3M Tris-base and applied to a Ni-NTA column (Ni-NTA Superflow from Qiagen) equilibrated in 50mM HEPES, 5mM Imidazole, 500mM NaCI, pH 7.5. After washing the column extensively with the equilibration buffer loosely bound proteins were eluted by a wash with 50mM HEPES, 15mM Imidazole, 500mM NaCI, pH 7.5. Finally, the strongly bound proteins were eluted with 50mM HEPES, 500mM Imidazole, pH 7.5. Fractions were collected during elution and the purity of the fractions was analysed by SDS-PAGE (Figure 3).
When the peptidase was produced in the Α164Δ1 1 strain YoqM was co-purified on the Ni-NTA-column and was clearly visible as a ca. 8 kDa band on the SDS-PAGE. However, when the peptidase was produced in the yoqM deleted strain AEB1779 this specific band was absent, resulting in a purer peptidase eluent product.
Example 6. Absence of YoqM leads to improved yields on Ni-NTA columns
The yoqM wt and yoqM deleted strains expressing the His-tagged amylase described in
Example 4 were fermented in CAL 18-2 medium and culture supernatants were purified essentially as described in Example 5, and fractions were analysed by SDS-PAGE (Figure 4). Expression of the amylase was similar in both strains (Fig. 4, lanes 1 and 9) as was also found in Example 4, but the yield of amylase in the Ni-NTA-column eluent was clearly higher from the yoqM deleted host.
The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

Claims
1 . A mutant Bacillus host cell which comprises an inactivation of a polynucleotide encoding a YoqM polypeptide selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having a least 70% sequence identity with the nucleotide sequence of SEQ ID NO: 1 ; and
(c) a polypeptide encoded by a polynucleotide that hybridizes under medium stringency conditions with a polynucleotide having the nucleotide sequence of SEQ ID NO: 1 .
2. The mutant host cell of claim 1 , which is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus fastidiosus, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus megaterium, Bacillus methanolicus, Bacillus pumilus, Bacillus sphaericus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis host cell.
3. The mutant host cell of claim 1 or 2, which produces a polyhistidine-tagged polypeptide of interest.
4. The mutant host cell of any of claims 1 - 3, wherein the polyhistidine-tagged polypeptide of interest is homologous or heterologous to the host cell; preferably it is an enzyme; more preferably the enzyme is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase; and most preferably the enzyme is an alpha-glucosidase, aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, glucocerebrosidase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, urokinase, or xylanase.
5. The mutant host cell of any of claims 1 - 4, wherein the polyhistidine-tagged polypeptide of interest comprises at least six consecutive histidine residues, (H)6 in its amino acid sequence or at least three alternating histidine residues in a region of six amino acid sequences, (HX)3, preferably (HQ)3.
6. The mutant host cell of any of claims 1 - 5, wherein the at least six consecutive histidine residues or the at least three alternating histidine residues in a region of six amino acid sequences are located in the N-terminal or C-terminal end of the polypeptide.
7. The mutant host cell of any of claims 1 - 6, wherein the YoqM polypeptide is capable of binding to an affinity resin comprising bound bivalent nickel or cobalt ions; preferably sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt.
8. The mutant host cell of any of claims 1 - 7, wherein the polynucleotide inactivation is a disruption or a partial or full deletion of the YoqM-en coding polynucleotide.
9. A method of producing a polyhistidine-tagged polypeptide of interest, comprising the steps of:
(a) cultivating a host cell of any of claims 1 - 8 under conditions conducive for production of the polypeptide; and, optionally
(b) recovering the polypeptide.
10. The method of claim 9, wherein the polypeptide is recovered by affinity purification in a resin comprising bound bivalent nickel or cobalt ions; preferably the resin is sepharose or agarose functionalised with iminodiacetic acid (Ni-IDA) or nitrilotriacetic acid (Ni-NTA) for nickel and carboxyl-methylaspartate (Co-CMA) for cobalt.
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