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WO2026030345A1 - Variantes de séquence de signal et de région promotice pour la production améliorée de protéase dans des cellules de bacillus - Google Patents

Variantes de séquence de signal et de région promotice pour la production améliorée de protéase dans des cellules de bacillus

Info

Publication number
WO2026030345A1
WO2026030345A1 PCT/US2025/039697 US2025039697W WO2026030345A1 WO 2026030345 A1 WO2026030345 A1 WO 2026030345A1 US 2025039697 W US2025039697 W US 2025039697W WO 2026030345 A1 WO2026030345 A1 WO 2026030345A1
Authority
WO
WIPO (PCT)
Prior art keywords
substitution
modified
protease
seq
signal peptide
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.)
Pending
Application number
PCT/US2025/039697
Other languages
English (en)
Inventor
Cristina Bongiorni
Frits Goedegebuur
Yunlong Li
Mai DU
Rei OTSUKA
Harm Mulder
Andrew Abel PRIOR
Laurens LAMMERTS
Sina Pricelius
Viktor Yuryevich Alekseyev
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.)
Genencor International BV
Danisco US Inc
Original Assignee
Genencor International BV
Danisco US Inc
Genencor International Inc
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 Genencor International BV, Danisco US Inc, Genencor International Inc filed Critical Genencor International BV
Publication of WO2026030345A1 publication Critical patent/WO2026030345A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • 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
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21062—Subtilisin (3.4.21.62)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Landscapes

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

Abstract

La présente divulgation concerne de manière générale des séquences d'acide nucléique (ADN) codant pour de nouvelles séquences peptidiques signaux, des séquences d'acide nucléique (ADN) codant pour de nouvelles séquences de régions promotrices, des polynucléotides recombinants (par exemple, vecteurs, plasmides, cassettes d'expression, etc.) codant pour des protéases précurseurs, des cellules de Bacillus sp. recombinantes (modifiées) Bacillus sp. exprimant une protéase précurseur et sécrétant la protéase mature dans le bouillon de fermentation lorsqu'elles sont fermentées dans des conditions appropriées, et similaires.
PCT/US2025/039697 2024-07-29 2025-07-29 Variantes de séquence de signal et de région promotice pour la production améliorée de protéase dans des cellules de bacillus Pending WO2026030345A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463676764P 2024-07-29 2024-07-29
US63/676,764 2024-07-29

Publications (1)

Publication Number Publication Date
WO2026030345A1 true WO2026030345A1 (fr) 2026-02-05

Family

ID=96946523

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/039697 Pending WO2026030345A1 (fr) 2024-07-29 2025-07-29 Variantes de séquence de signal et de région promotice pour la production améliorée de protéase dans des cellules de bacillus

Country Status (1)

Country Link
WO (1) WO2026030345A1 (fr)

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5185258A (en) 1984-05-29 1993-02-09 Genencor International, Inc. Subtilisin mutants
US5204015A (en) 1984-05-29 1993-04-20 Genencor International, Inc. Subtilisin mutants
WO1995010615A1 (fr) 1993-10-14 1995-04-20 Genencor International, Inc. Variants de subtilisine
WO2002014490A3 (fr) 2000-08-11 2003-02-06 Genencor Int Transformation de bacille, transformants et bibliotheques de mutants
WO2003083125A1 (fr) 2002-03-29 2003-10-09 Genencor International, Inc. Expression proteinique amelioree dans bacillus
WO2015038792A1 (fr) * 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2018169780A1 (fr) * 2017-03-15 2018-09-20 Dupont Nutrition Biosciences Aps Procédés d'utilisation d'une sérine protéase d'archaea
WO2019108599A1 (fr) 2017-11-29 2019-06-06 Danisco Us Inc Variants de subtilisine à stabilité améliorée
WO2019245705A1 (fr) 2018-06-19 2019-12-26 Danisco Us Inc Variants de subtilisine
WO2020112599A1 (fr) 2018-11-28 2020-06-04 Danisco Us Inc Variants de subtilisine à stabilité améliorée
WO2023192953A1 (fr) * 2022-04-01 2023-10-05 Danisco Us Inc. Mutations de pro-région améliorant la production de protéines dans des cellules bactériennes à gram positif
WO2024050503A1 (fr) * 2022-09-02 2024-03-07 Danisco Us Inc. Nouvelles mutations de promoteur et de région non traduite 5' améliorant la production de protéines dans des cellules à gram positif

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5185258A (en) 1984-05-29 1993-02-09 Genencor International, Inc. Subtilisin mutants
US5204015A (en) 1984-05-29 1993-04-20 Genencor International, Inc. Subtilisin mutants
WO1995010615A1 (fr) 1993-10-14 1995-04-20 Genencor International, Inc. Variants de subtilisine
WO2002014490A3 (fr) 2000-08-11 2003-02-06 Genencor Int Transformation de bacille, transformants et bibliotheques de mutants
WO2003083125A1 (fr) 2002-03-29 2003-10-09 Genencor International, Inc. Expression proteinique amelioree dans bacillus
WO2015038792A1 (fr) * 2013-09-12 2015-03-19 Danisco Us Inc. Compositions et procédés comprenant des variants de protéase lg12-clade
WO2018169780A1 (fr) * 2017-03-15 2018-09-20 Dupont Nutrition Biosciences Aps Procédés d'utilisation d'une sérine protéase d'archaea
WO2019108599A1 (fr) 2017-11-29 2019-06-06 Danisco Us Inc Variants de subtilisine à stabilité améliorée
WO2019245705A1 (fr) 2018-06-19 2019-12-26 Danisco Us Inc Variants de subtilisine
WO2020112599A1 (fr) 2018-11-28 2020-06-04 Danisco Us Inc Variants de subtilisine à stabilité améliorée
WO2023192953A1 (fr) * 2022-04-01 2023-10-05 Danisco Us Inc. Mutations de pro-région améliorant la production de protéines dans des cellules bactériennes à gram positif
WO2024050503A1 (fr) * 2022-09-02 2024-03-07 Danisco Us Inc. Nouvelles mutations de promoteur et de région non traduite 5' améliorant la production de protéines dans des cellules à gram positif

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
"Program Manual for the Wisconsin Package", 8 August 1994, GENETICS COMPUTER GROUP
ALTSCHUL ET AL.: "Basic local alignment search tool", J. MOL. BIOL., vol. 215, no. 13, 1990, pages 403 - 410, XP002949123, DOI: 10.1006/jmbi.1990.9999
ALTSCHUL ET AL.: "Gapped BLAST and PSI BLAST a new generation of protein database search programs", NUCLEIC ACIDS RES, SET 1, vol. 25, no. 17, 1997, pages 3389 - 402, XP002905950, DOI: 10.1093/nar/25.17.3389
BEAUCAGECARUTHERS: "Deoxynucleoside phosphoramidites - A new class of key intermediates for deoxypolynucleotide synthesis", TETRAHEDRON LETT., vol. 22, 1981, pages 1859 - 1862
DATABASE Geneseq [online] 15 November 2018 (2018-11-15), "Bacillus subtilis AprE signal peptide, SEQ:16.", retrieved from EBI accession no. GSP:BFR51955 Database accession no. BFR51955 *
KARLIN ET AL.: "Applications and statistics for multiple high-scoring segments in molecular sequences", PNAS USA, vol. 90, no. 12, 1993, pages 5873 - 5787
MATTHES ET AL.: "Simultaneous rapid chemical synthesis of over one hundred oligonucleotides on a microscale", EMBO J., vol. 3, 1984, pages 801 - 805
NEEDLEMANWUNSCH: "A general method applicable to the search for similarities in the amino acid sequences of two proteins", JOURNAL OF MOLECULAR BIOLOGY, vol. 48, no. 3, 1970, pages 443 - 53
SAITOUNEI: "The neighbor-joining method: a new method for reconstructing phylogenetic trees", MOL. BIOL. EVOL., vol. 4, 1987, pages 406 - 425
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY
SCHAFFER ET AL.: "Improving the accuracy of PSI-BLAST protein database searches with composition-based statistics and other refinements", NUCLEIC ACIDS RES, vol. 29, no. 14, 2001, pages 2994 - 3005
THOMPSON ET AL.: "CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice", NUCLEIC ACIDS RES, vol. 22, no. 22, 1994, pages 4673 - 4680, XP002956304

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