WO2025133003A2 - Lactoglobuline bêta bovine non animale - Google Patents

Lactoglobuline bêta bovine non animale Download PDF

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Publication number
WO2025133003A2
WO2025133003A2 PCT/EP2024/087733 EP2024087733W WO2025133003A2 WO 2025133003 A2 WO2025133003 A2 WO 2025133003A2 EP 2024087733 W EP2024087733 W EP 2024087733W WO 2025133003 A2 WO2025133003 A2 WO 2025133003A2
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WO
WIPO (PCT)
Prior art keywords
blg
cell
host
modified
seq
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PCT/EP2024/087733
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English (en)
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WO2025133003A3 (fr
Inventor
José Arnau MARTINEZ
Jan Lehmbeck
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21stBio AS
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21stBio AS
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Publication of WO2025133003A3 publication Critical patent/WO2025133003A3/fr
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4717—Plasma globulins, lactoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/38—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/69—Aspergillus oryzae

Definitions

  • the! BLG! comprises! at! least! one! substitution! of! a! non- essential!and/or!non-branched!amino!acid!with!an!essential!and/or!branched!amino!acid!compared! to!a!parent!unmodified!BLG.! [0006] In!another!aspect!this!disclosure!describes!a!modified!nucleotide!sequence!which!is!at!least!
  • polynucleotide ! ⁇ substitution ⁇ ! or! ⁇ substituted ⁇ ! refers! to! modification! of! the! polynucleotide! by! replacing!one!nucleotide!with!another,!for!instance!the!replacement!of!a!cytosine!with!a!thymine!in!a! polynucleotide!sequence!is!a!nucleotide!substitution.! [0038]
  • The! term!expression”! includes!any!step! involved! in! the!production!of!a!polypeptide! (e.g.,! encoded! enzyme)! including,! but! not! limited! to,! transcription,! post-transcriptional!
  • the! modified! BLG! may! contain! a! Histidine! and! a! Valine! at! positions! corresponding!to!position!59!and!118!respectively!in!anyone!of!the!BLG ⁇ s!comprised!in!SEQ!ID!NO:!1,! 2!or!3.!Such!a!modified!BLG!can!be!a!BLG!comprising!an!amino!acid!sequence!which!is!at!least!50%! ! ! !
  • the! heterologous! gene! encoding! the! BLG! in! the! host! cell! further! comprises!a!moiety!coding!for!a!signal!peptide!fused!to!the!BLG.!This!signal!peptide!and!the!moiety!
  • promoters! include! engineered! synthetic! promoters.! The! promoter! is! preferably! an! inducible!promoter,!inducible!e.g.!by!nutrients!such!as!starch,!sugars,!maltose,!glucose!and!the!like.!In! some!embodiments!the!promoter! is!an!amylase!promoter.!Examples!of!such!useful!promoters!are!
  • the! host! cell! can! advantageously!be!modified!by!one!or!more!further!genetic!modifications!improving!the!expression! and/or!secretion!of!the!BLG!selected!from! a ) attenuation,! disruption! and/or! deletion! the! expression! of! one! or! more! genes! which! are! endogenous/native!to!the!host!cell;!and! b) mutation!of!one!or!more!genes!native!to!the!host!cell.! [0081] In! some!
  • the! attenuated,! disrupted! and/or! deleted! native! genes! encodes! a! protease!capable!of!digesting!BLG!or!its!signal!peptide.! [0082] It! has! been! found! that! the! attenuation,! disruption! and/or! deletion! of! native! genes! advantageously!include!genes!that!encode!proteases!capable!of!digesting!BLG!or!its!signal!peptide,! ! ! Case Ref.
  • The!host!cell! is!preferably!a!eukaryotic,!a!prokaryotic!or!an!algal!cell.!
  • the! method! includes! a! recovery! and/or! isolation! step! comprising!separating!a!liquid!phase!of!the!cell!or!cell!culture!from!a!solid!phase!of!the!cell!or!cell! culture!to!obtain!a!supernatant!comprising!the!BLG!and/or!subjecting!the!supernatant!to!one!or!more! steps!selected!from:! a )! separating! the! supernatant! from! the! solid! phase!of! the! cell! culture,! such! as! by! filtration!or!
  • the!modified!BLG! comprises! at! least!one! amino! acid! substitution! compared! to! anyone! of! bovine! BLG! A,! bovine! BLG! B! and! bovine! BLG! C,! optionally! at! least! two! substitutions.!In!some!embodiments!the!modified!BLG!is!a!modified!bovine!BLG!C!comprising!at!least!
  • the! modified! BLG! is! a! modified! BLG! A! comprising! at! least! one! ! ! Case Ref. P238WO IPTector® substitution!of!Glutamine!with!Histidine!at!a!position!corresponding!to!position!59!of!the!BLG!A!in!SEQ!

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente divulgation concerne une cellule hôte microbienne génétiquement modifiée exprimant un ou plusieurs gènes hétérologues codant pour une bêta-lactoglobuline modifiée (BLG), produisant ainsi ladite BLG modifiée, la BLG modifiée comprenant au moins une substitution d'un acide aminé non essentiel et/ou non ramifié avec un acide aminé essentiel et/ou ramifié par rapport à une BLG non modifiée parente.
PCT/EP2024/087733 2023-12-19 2024-12-19 Lactoglobuline bêta bovine non animale Pending WO2025133003A2 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP23218277.4 2023-12-19
EP23218277 2023-12-19
EP24163561 2024-03-14
EP24163561.4 2024-03-14
EP24198998.7 2024-09-06
EP24198998 2024-09-06

Publications (2)

Publication Number Publication Date
WO2025133003A2 true WO2025133003A2 (fr) 2025-06-26
WO2025133003A3 WO2025133003A3 (fr) 2025-10-02

Family

ID=94227696

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2024/087733 Pending WO2025133003A2 (fr) 2023-12-19 2024-12-19 Lactoglobuline bêta bovine non animale

Country Status (1)

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WO (1) WO2025133003A2 (fr)

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO2000039322A1 (fr) 1998-12-23 2000-07-06 Novozymes A/S Procedes permettant de produire des polypeptides dans des cellules mutantes d'aspergillus
WO2005121333A1 (fr) 2004-06-14 2005-12-22 Novozymes A/S Peptide signal pour production d'un polypeptide
WO2012160093A1 (fr) 2011-05-23 2012-11-29 Novozymes A/S Intégrations simultanées spécifiques d'un site de multiples copies géniques dans un champignon filamenteux
US8586330B2 (en) 2006-07-14 2013-11-19 Novozymes A/S Methods for producing secreted polypeptides having biological activity
WO2013178674A1 (fr) 2012-05-31 2013-12-05 Novozymes A/S Sélection améliorée dans les champignons
WO2015025055A1 (fr) 2013-08-23 2015-02-26 Novozymes A/S Expression de pepc régulé
WO2020112911A1 (fr) 2018-11-28 2020-06-04 Novozymes A/S Cellules hôtes fongiques filamenteuses améliorées
WO2020219596A1 (fr) 2019-04-22 2020-10-29 Perfect Day, Inc. Protéines de lait de recombinaison et compositions les comprenant
WO2021168343A2 (fr) 2020-02-19 2021-08-26 Perfect Day, Inc. Protéines de lait recombinantes hypoallergéniques et compositions les comprenant

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023122280A2 (fr) * 2021-12-23 2023-06-29 Perfect Day, Inc. Compositions et procédés de traitement de défauts cutanés
WO2023150696A2 (fr) * 2022-02-03 2023-08-10 Perfect Day, Inc. Compositions et procédés d'administration d'agents bioactifs hydrophobes

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (fr) 1986-01-30 1990-11-27 Cetus Corp
WO2000039322A1 (fr) 1998-12-23 2000-07-06 Novozymes A/S Procedes permettant de produire des polypeptides dans des cellules mutantes d'aspergillus
WO2005121333A1 (fr) 2004-06-14 2005-12-22 Novozymes A/S Peptide signal pour production d'un polypeptide
US8586330B2 (en) 2006-07-14 2013-11-19 Novozymes A/S Methods for producing secreted polypeptides having biological activity
WO2012160093A1 (fr) 2011-05-23 2012-11-29 Novozymes A/S Intégrations simultanées spécifiques d'un site de multiples copies géniques dans un champignon filamenteux
WO2013178674A1 (fr) 2012-05-31 2013-12-05 Novozymes A/S Sélection améliorée dans les champignons
WO2015025055A1 (fr) 2013-08-23 2015-02-26 Novozymes A/S Expression de pepc régulé
WO2020112911A1 (fr) 2018-11-28 2020-06-04 Novozymes A/S Cellules hôtes fongiques filamenteuses améliorées
WO2020219596A1 (fr) 2019-04-22 2020-10-29 Perfect Day, Inc. Protéines de lait de recombinaison et compositions les comprenant
WO2021168343A2 (fr) 2020-02-19 2021-08-26 Perfect Day, Inc. Protéines de lait recombinantes hypoallergéniques et compositions les comprenant

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
"Current protocols in Molecular Biology", 1995, JOHN WILEY AND SONS
"Handbook Of Experimental Immunology", vol. 1-4, 1986, COLD SPRING HARBOR LABORATORY PRESS
"Immunochemical Methods In Cell And Molecular Biology", 1987, R. I. FRESHNEY, ALAN R. LISS, INC.
ARNAU ET AL.: "Grand challenges in fungal biotechnology, grand challenges in biology and biotechnology", 2020, article "Strategies and challenges for the development of industrial enzymes using fungal cell factories"
B. PERBAL: "A Practical Guide To Molecular Cloning", 1984
CHRISTENSEN ET AL.: "High-level expression of recombinant genes in Aspergillus oryzae", BIOTECHNOLOGY, vol. 6, 1988, pages 1419 - 1422, XP000003462, DOI: 10.1038/nbt1288-1419
EUR. J. BIOCHEM., 1985
FREDERICK M. AUSUBEL: "Current Protocols in Molecular Biology", 2000, WILEY AND SON INC, LIBRARY OF CONGRESS
LIU ET AL.: "High-yield production of acidic pectin lyase pnlzj5b for juice processing", LETT APPL MICROBIOL, vol. 75, 2022, pages 1055 - 1062
MYGIND ET AL.: "Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus", NATURE, vol. 437, 2005, pages 975 - 980, XP002365945
NAKAMURA ET AL.: "Highly efficient gene targeting in aspergillus oryzae industrial strains under ligd mutation introduced by genome editing: strain-specific differences in the effects of deleting ecdr, the negative regulator of sclerotia formation", J. GEN. APPL. MICROBIOL., vol. 63, 2017, pages 172 - 178
PUNT ET AL.: "Characterization of the aspergillus niger prtt, a unique regulator of extracellular protease encoding genes", FUNGAL GENET BIOL, vol. 45, 2008, pages 1591 - 1599, XP025678185, DOI: 10.1016/j.fgb.2008.09.007
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular cloning: A laboratory manual", vol. 154-155, 1989, COLD SPRING HARBOR LAB.
SIWECKA: "the structure, activation and signaling of ire1 and its role in determining cell fate", BIOMEDICINES, vol. 9, 2021, pages 156
TANAKA ET AL.: "effects of codon optimization on the mrna levels of heterologous genes in filamentous fungi", APPL MICROBIOL BIOTECHNOL, vol. 98, 2014, pages 3859 - 3867, XP035328382, DOI: 10.1007/s00253-014-5609-7
VANEGAS ET AL.: "a mad7 system for genetic engineering of filamentous fungi", J. FUNGI, vol. 9, 2023, pages 16
YOON ET AL.: "Disruption of ten protease genes in the filamentous fungus aspergillus oryzae highly improves production of heterologous proteins", APPL. MICROBIOL. BIOTECH., vol. 89, 2011, pages 747 - 759, XP055872360
ZHOU ET AL.: "Functional and transcriptomic analysis of the key unfolded protein response transcription factor hacA in aspergillus oryzae", GENE, vol. 593, 2016, pages 143 - 153, XP029735545, DOI: 10.1016/j.gene.2016.08.018

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