EP1711609A1 - Polynucleotides et polypeptides isoles dans i lactobacillus rhamnosus hn001 /i , matieres les contenant et leurs methodes d'utilisation - Google Patents

Polynucleotides et polypeptides isoles dans i lactobacillus rhamnosus hn001 /i , matieres les contenant et leurs methodes d'utilisation

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Publication number
EP1711609A1
EP1711609A1 EP03781132A EP03781132A EP1711609A1 EP 1711609 A1 EP1711609 A1 EP 1711609A1 EP 03781132 A EP03781132 A EP 03781132A EP 03781132 A EP03781132 A EP 03781132A EP 1711609 A1 EP1711609 A1 EP 1711609A1
Authority
EP
European Patent Office
Prior art keywords
altered
genetic
seq
protein
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03781132A
Other languages
German (de)
English (en)
Inventor
Matthew Glenn
Ilkka Havukkala
Mark William Lubbers
James Dekker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Genesis Research and Development Corp Ltd
Fonterra Cooperative Group Ltd
Original Assignee
Genesis Research and Development Corp Ltd
Fonterra Cooperative Group Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genesis Research and Development Corp Ltd, Fonterra Cooperative Group Ltd filed Critical Genesis Research and Development Corp Ltd
Publication of EP1711609A1 publication Critical patent/EP1711609A1/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • exopeptidases include: • Aminopeptidases - release a free amino acid from the N-terminus of a peptide chain; • dipeptidyl-peptidase (also known as dipeptidyl-aminopeptidases) - release a dipeptide from the N-terminus of a peptide chain; • tripeptidyl-peptidases (also known as tripeptidyl-aminopeptidases) - release a tripeptide from the N-terminus of a peptide chain); • carboxypeptidases - release a free amino acid from the C-terminus of a peptide chain; • peptidyl-dipeptidase - release a dipeptide from the C-terminus of a peptide chain; • dipeptidases - release two free amino acids from a dipeptide; and • tripeptidases - release a free amino acid and a dipeptide from a tripeptide.
  • Polynucleotides identified using sequencing techniques may be partial or full-length genes, and may contain open reading frames, or portions of open reading frames, that encode polypeptides. Putative polypeptides may be identified based on polynucleotide sequences and further characterized. The sequencing data relating to polynucleotides thus represents valuable and useful information. Polynucleotides and polypeptides may be analyzed for varying degrees of novelty by comparing identified sequences to sequences published in various public domain databases, such as EMBL.
  • Example 6 shows the results of UV light exposure assay measuring relative viability in response to increasing doses of UV light in AMI ' HNOOl strain ( ⁇ ) and wild-type HNOOl ( ⁇ ). Results indicate that the AMI ' HNOOl mutant strain showed enhanced survival to exposure to UV light compared to wild-type HNOOl .
  • the polynucleotides disclosed herein were isolated by high throughput sequencing of DNA libraries from the lactic acid bacteria Lactobacillus rhamnosus as described below in Example 1.
  • PlnG displays strong similarities and/or protein, fusion protein to the proposed transport proteins of production, genetic modification, several other bacteriocins and to mutagenesis amplification of proteins implicated in the signal- genetic material or for other sequence-independent export of genetic or protein manipulations.
  • Escherichia coli hemolysin, PlnH is Altered survival characteristics: its accessory protein (Huhne et al, survival of industrial processes, Microbiol. 142:1437-1448, 1996). growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer gut health). Modified antibiotic resistance. Improved antimicrobial properties.
  • bacteria may use galC production, genetic modification, to release sugars for metabolism, the mutagenesis amplification of by-products, including ceramide, asct genetic material or for other as signalling molecules in eukaryotic genetic or protein manipulations. cells and can lead to apoptosis or Altered survival characteristics: differentiation. Therefore, glaC plays survival of industrial processes, a role in probiotic effects and survival growth or storage in product in the gut environment. formats, persistence in gut environment. Altered metabolic properties. Altered probiotic attributes. Modified health properties (including immunoregulatory, anticancer, gut health, apoptosis). Modified antibiotic resistance. SEQ SEQ ⁇ D
  • TagE Altered cell wall or cell surface Homologue of tagE, encoding characteristics, structures or poly(glycerol-phosphate) alpha- functions.
  • glucosyltransferase (EC 2.4.1.52) Modified adhesion to human or also called uridine diphosphate- animal cells or cell lines. glucose poly-(glycerol phosphate) Production of desirable flavors. alpha-glucosyl transferase.
  • TagE is Modified flavor, aroma and/or involved in techoic acid synthesis. texture attributes.
  • Techoic acid is one component of the Construction of genetic vectors thick peptidoglycan layers in the cell for controlled expression of RNA wall of Gram-positive bacteria and is and/or protein, fusion protein susceptible to the enzyme lysozyme production, genetic modification, and to penicillin, mutagenesis amplification of genetic material or for other genetic or protein manipulations
  • Altered survival characteristics survival of industrial processes, growth or storage in product formats, persistence in gut environment.
  • Altered metabolic properties. Altered probiotic attributes. Modified health properties (mcluding immunoregulatory, anticancer, gut health). Modified antibiotic resistance. Improved fermentation properties or other industrially useful processes.
  • Myo-inositol is for controlled expression of RNA abundant in nature, especially in soil. and/or protein, fusion protein
  • RNA abundant in nature especially in soil. and/or protein, fusion protein
  • Various microorganisms are able to production, genetic modification, grow on myo-inositol as the sole mutagenesis amplification of carbon source.
  • the expression of the genetic material or for other iol operon is under glucose repression genetic or protein manipulations. (Miwa and Fujita, J. Bacteriol. Altered survival characteristics: 183:5877-5884, 2001). survival of industrial processes, growth or storage in product formats, persistence in gut environment. Altered metabolic properties. Modified carbohydrate levels or functional properties. Altered cell wall or cell surface characteristics, structures or functions. Modified adhesion to human or animal cells or cell lines. Altered probiotic attributes. Organisms or materials with improved health properties (including immunoregulatory, anticancer, gut health).
  • Lactocepin genetic material or for other is a type I membrane protein, located genetic or protein manipulations. in the cell wall and belongs to Altered survival characteristics: peptidase family S8; also known as survival of industrial processes, the Subtilase Family. Lactocepin is growth or storage in product responsible for the hydrolysis of formats, persistence in gut casein in milk and specificity environment. differences between lactocepins from Altered metabolic properties. different starter strains may be partly Altered probiotic attributes. responsible for imparting different Modified health properties flavor qualities to cheese (Broadbent (including immunoregulatory, et al, Appl. Environ. Microbiol. anticancer, gut health). 68:1778-1785, 2002). Modified antibiotic resistance. Improved fermentation properties or other industrially useful processes.
  • myo-inositol as the sole carbon Altered survival characteristics: source.
  • the expression of the iol survival of industrial processes, operon is under glucose repression growth or storage in product (Miwa and Fujita, J. Bacteriol. formats, persistence in gut 183:5877-5884, 2001). environment.
  • Altered metabolic properties Modified carbohydrate levels or functional properties.
  • 54 146 Altered cell wall or cell surface Homologue of mga4, a positive characteristics, structures or regulatory protein that acts as a functions.
  • Altered survival characteristics 183:5877-5884, 2001) survival of industrial processes, growth or storage in product formats, persistence in gut environment.
  • Altered metabolic properties Modified carbohydrate levels or functional properties.
  • the term "; -mer,” with reference to a specific value of "x,” refers to a polynucleotide comprising at least a specified number (" ") of contiguous residues of any of the polynucleotides identified as SEQ ID NOS: 1-80.
  • the value of* may be from about 20 to about 600, depending upon the specific sequence.
  • the present invention provides isolated polypeptides encoded, or partially encoded, by the above polynucleotides.
  • such polypeptides comprise a sequence selected from the group consisting of SEQ ID NO: 81-183, and variants thereof.
  • polypeptides may encode naturally occurring polypeptides, portions of naturally occurring polypeptides, or other variants thereof.
  • polypeptides are provided that comprise at least a functional portion of a polypeptide having an amino acid sequence encoded by a polynucleotide of the present invention.
  • a "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants.
  • the active site may be made up of separate portions present on one or more polypeptide chains and wUl generally exhibit high binding affinity.
  • Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms.
  • Polynucleotides may also be analyzed using the BLASTX algorithm, which compares the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The percentage identity of polypeptide sequences may be examined using the BLASTP algorithm.
  • the BLASTN, BLASTX and BLASTP programs are avaUable on the NCBI anonymous FTP server and from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894, USA.
  • the following running parameters are prefened for determination of alignments and similarities using BLASTP that contribute to the E values and percentage identity of polypeptide sequences: blastall -p blastp -d swissprottrembledb -e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value (E) [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line descriptions (v) [Integer]; -b Number of alignments to show (b) [Integer]; -I Query File [File In]; -o BLAST report Output File [File Out] Optional.
  • variant polynucleotides and polypeptides preferably comprise sequences producing an E value of 0.01 or less when compared to the polynucleotide or polypeptide of the present invention.
  • polynucleotides comprising sequences that differ from the inventive polynucleotide sequences or complements, reverse complements, or reverse sequences as a result of deletions, and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention.
  • polypeptides comprising sequences that differ from the inventive polypeptide sequences as a result of amino acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence length are contemplated by and encompassed v thin the present invention, provided the variant polypeptide has similar activity to the inventive polypeptide.
  • Genomic or cDNA libraries are prepared using techniques well known in the art.
  • the resulting target DNA is then labeled with a suitable marker, such as a radiolabel, chromophore, fluorophore or chemiluminescent agent, using protocols well known for those skilled in the art.
  • a solution of the labeled target DNA is contacted with the surface of the a ⁇ ay and incubated for a suitable period of time. The surface of the a ⁇ ay is then washed free of unbound target DNA and the probes to which the target DNA hybridized are determined by identifying those regions of the array to which the markers are attached.
  • the marker is a radiolabel, such as 2 P
  • autoradiography is employed as the detection method.
  • Sensitivity in assaying the presence of probe can be usefully increased by using branched oligonucleotides, as described by Horn et al., Nucleic Acids Res. 25(23):4842-4849, 1997, enabling detection of as few as 50 DNA molecules in the sample.
  • Polynucleotides of the present invention may also be used to specifically suppress gene expression by methods that operate post-transcriptionally to block the synthesis of products of targeted genes, such as RNA interference (RNAi), and quelling.
  • RNAi RNA interference
  • traditional methods of gene suppression employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of interest such that binding interferes with subsequent cellular processes and therefore blocks synthesis of the conesponding protein.
  • the resulting pBEryl construct encoding the HNOOl relA GTP pyrophosphokinase AMI gene was transformed into competent HNOOl cells and grown anaerobicaUy for 48 hrs at 37 °C on MRS lactobacilli agar (Difco, Detroit MI) containing 2.5 ⁇ g ml Em.
  • Em-resistant HNOOl were checked for integration of the plasmid construct into the relA gene by PCR using vector-specific (T3 or T7) and AMI internal fragment-specific primers. Colonies giving PCR patterns consistent with the insertional inactivation of the endogenous HNOOl relA GTP pyrophosphokinase AMI gene, were assessed for increased resistance to UV inadiation as described in Example 2.
  • GTP pyrophosphokinase or (EC 2.7.6.5) produces guanosine 3 '-diphosphate 5'- triphosphate, a marker of the "stringent response", a regulatory state induced in bacteria by nutrient starvation and other environmental stresses (reviewed in Chatterji and Ojha, Curr Opin Microbiol. 4:160-5, 2001).
  • GTP pyrophosphokinase relA gene expression improved the resistance to a number of stress conditions including UV light exposure, as well as high salt, pH and temperature, in Lactococcus lactis (Duwat et al, Int J. Food Microbiol. 55:83-6, 2000).
  • Applications for HNOOl GTP pyrophosphokinase AMI include: • methods of enhanced survival of industrial processes; • improved colonization of human intestinal environment; and • improved survival of Lactobacilli to multiple stress conditions.
  • SEQ ID NOS: 1-187 are set out in the attached Sequence Listing. The codes for nucleotide sequences used in the attached Sequence Listing, including the symbol "n," conform to WLPO Standard ST.25 (1998), Appendix 2, Table 1.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des nouveaux polynucléotides isolés dans lactobacillus rhamnosus, ainsi que des sondes oligonucléotidiques et des amorces, des constructions génétiques comprenant les polynucléotides, des matières biologiques, dont des plantes, des micro-organismes et des organismes multicellulaires comprenant les polynucléotides, les polypeptides exprimés par les polynucléotides, ainsi que des méthodes d'utilisation des polynucléotides et polypeptides.
EP03781132A 2003-12-15 2003-12-15 Polynucleotides et polypeptides isoles dans i lactobacillus rhamnosus hn001 /i , matieres les contenant et leurs methodes d'utilisation Ceased EP1711609A1 (fr)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/NZ2003/000278 WO2005056801A1 (fr) 2003-12-15 2003-12-15 Polynucleotides et polypeptides isoles dans lactobacillus rhamnosus hn001, matieres les contenant et leurs methodes d'utilisation

Publications (1)

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EP1711609A1 true EP1711609A1 (fr) 2006-10-18

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EP03781132A Ceased EP1711609A1 (fr) 2003-12-15 2003-12-15 Polynucleotides et polypeptides isoles dans i lactobacillus rhamnosus hn001 /i , matieres les contenant et leurs methodes d'utilisation

Country Status (4)

Country Link
EP (1) EP1711609A1 (fr)
AU (1) AU2003288826A1 (fr)
CA (1) CA2549710A1 (fr)
WO (1) WO2005056801A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW201201820A (en) * 2010-02-15 2012-01-16 Julian Crane Use of lactic acid bacteria to treat or prevent rhinitis

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2694766B1 (fr) * 1992-08-13 1994-12-23 Agronomique Inst Nat Rech Clônage du gène de structure d'une aminopeptiddase de lactococcus lactis, production et utilisations de ladite aminopeptidase.
GB9313586D0 (en) * 1993-07-01 1993-08-18 Europ Economic Community Lys-aminopeptidase pepn from lactobacillus delbrukii ssp.lactis,nucleic acids coding for it,and its use in fermentation processes
US7026463B2 (en) * 1999-08-13 2006-04-11 Matthew Glenn Polynucleotides and polypeptides, materials incorporating them and methods for using them
US6436703B1 (en) * 2000-03-31 2002-08-20 Hyseq, Inc. Nucleic acids and polypeptides
FR2807446B1 (fr) * 2000-04-11 2005-05-06 Agronomique Inst Nat Rech Genomes de lactococcus lactis, polypeptides et utilisations

Non-Patent Citations (1)

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Title
See references of WO2005056801A1 *

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CA2549710A1 (fr) 2005-06-23
AU2003288826A1 (en) 2005-06-29
WO2005056801A1 (fr) 2005-06-23

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