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WO2025091009A3 - Overexpression of lysozyme in t7 rna polymerase-expressing host cells - Google Patents

Overexpression of lysozyme in t7 rna polymerase-expressing host cells Download PDF

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Publication number
WO2025091009A3
WO2025091009A3 PCT/US2024/053215 US2024053215W WO2025091009A3 WO 2025091009 A3 WO2025091009 A3 WO 2025091009A3 US 2024053215 W US2024053215 W US 2024053215W WO 2025091009 A3 WO2025091009 A3 WO 2025091009A3
Authority
WO
WIPO (PCT)
Prior art keywords
host cells
rna polymerase
overexpression
lysozyme
expressing host
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/US2024/053215
Other languages
French (fr)
Other versions
WO2025091009A2 (en
Inventor
Daniel James GROFF
Jacquelyn Marie BLAKE-HEDGES
Wilson Lee FOO
Dawei Yuan
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.)
Sutro Biopharma Inc
Original Assignee
Sutro Biopharma 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 Sutro Biopharma Inc filed Critical Sutro Biopharma Inc
Publication of WO2025091009A2 publication Critical patent/WO2025091009A2/en
Publication of WO2025091009A3 publication Critical patent/WO2025091009A3/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2462—Lysozyme (3.2.1.17)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1247—DNA-directed RNA polymerase (2.7.7.6)
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07006—DNA-directed RNA polymerase (2.7.7.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01017—Lysozyme (3.2.1.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01088—Peptide deformylase (3.5.1.88)
    • 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
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Landscapes

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

Abstract

Disclosed herein are host cells engineered for enhanced cell health and improved productivity in T7 RNAP overexpression. Host cells, polynucleotides, polypeptides, and related methods for co-expressing lysozyme with T7 RNA polymerase are provided. Also disclosed are methods for preparing a bacterial cell extract with T7 RNA polymerase for use in cell-free protein synthesis.
PCT/US2024/053215 2023-10-27 2024-10-28 Overexpression of lysozyme in t7 rna polymerase-expressing host cells Pending WO2025091009A2 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202363546075P 2023-10-27 2023-10-27
US63/546,075 2023-10-27
US202463565189P 2024-03-14 2024-03-14
US63/565,189 2024-03-14

Publications (2)

Publication Number Publication Date
WO2025091009A2 WO2025091009A2 (en) 2025-05-01
WO2025091009A3 true WO2025091009A3 (en) 2025-05-30

Family

ID=93563553

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/053215 Pending WO2025091009A2 (en) 2023-10-27 2024-10-28 Overexpression of lysozyme in t7 rna polymerase-expressing host cells

Country Status (1)

Country Link
WO (1) WO2025091009A2 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998003664A1 (en) * 1996-07-19 1998-01-29 Monsanto Company DEFORMYLATION OF f-MET PEPTIDES IN BACTERIAL EXPRESSION SYSTEMS
WO2008073746A2 (en) * 2006-12-08 2008-06-19 New England Biolabs, Inc. Expression of toxic genes in vivo in a non-natural host
WO2022253962A1 (en) * 2021-06-02 2022-12-08 Danmarks Tekniske Universitet Mutant rnase e for enhancing recombinant protein expression

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5168062A (en) 1985-01-30 1992-12-01 University Of Iowa Research Foundation Transfer vectors and microorganisms containing human cytomegalovirus immediate-early promoter-regulatory DNA sequence
US5866755A (en) 1993-06-14 1999-02-02 Basf Aktiengellschaft Animals transgenic for a tetracycline-regulated transcriptional inhibitor
ATE445699T1 (en) 2002-08-19 2009-10-15 Univ Leland Stanford Junior IMPROVED METHODS FOR IN VITRO PROTEIN SYNTHESIS
US7351563B2 (en) 2005-06-10 2008-04-01 The Board Of Trustees Of The Leland Stanford Junior University Cell-free extracts and synthesis of active hydrogenase
ES2415635T3 (en) 2006-06-29 2013-07-26 The Board Of Trustees Of The Leland Stanford Junior University Acellular synthesis of proteins containing unnatural amino acids
EP2379578A4 (en) 2009-01-12 2012-05-02 Sutro Biopharma Inc Dual charging system for selectively introducing non-native amino acids into proteins using an in vitro synthesis method
US8697359B1 (en) 2012-12-12 2014-04-15 The Broad Institute, Inc. CRISPR-Cas systems and methods for altering expression of gene products
DK3242931T3 (en) 2015-01-08 2021-05-10 Sutro Biopharma Inc FORMULATIONS FOR STABILIZING BACTERIA CELL EXTRACTS

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998003664A1 (en) * 1996-07-19 1998-01-29 Monsanto Company DEFORMYLATION OF f-MET PEPTIDES IN BACTERIAL EXPRESSION SYSTEMS
WO2008073746A2 (en) * 2006-12-08 2008-06-19 New England Biolabs, Inc. Expression of toxic genes in vivo in a non-natural host
WO2022253962A1 (en) * 2021-06-02 2022-12-08 Danmarks Tekniske Universitet Mutant rnase e for enhancing recombinant protein expression

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DES SOYE BENJAMIN J ET AL: "A Highly Productive, One-Pot Cell-Free Protein Synthesis Platform Based on Genomically Recoded Escherichia coli", CELL CHEMICAL BIOLOGY, ELSEVIER, AMSTERDAM, NL, vol. 26, no. 12, 6 November 2019 (2019-11-06), pages 1743, XP085965118, ISSN: 2451-9456, [retrieved on 20191106], DOI: 10.1016/J.CHEMBIOL.2019.10.008 *
ELROY-STEIN O ET AL: "CYTOPLASMIC EXPRESSION SYSTEM BASED ON CONSTITUTIVE SYNTHESIS OF BACTERIOPHAGE T7 POLYMERASE IN MAMMALIAN CELLS", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (PNAS), NATIONAL ACADEMY OF SCIENCES, vol. 87, no. 17, 1 September 1990 (1990-09-01), pages 6743 - 6747, XP000563742, ISSN: 0027-8424, DOI: 10.1073/PNAS.87.17.6743 *
INVITROGEN: "BL21 Star (TM) (DE3) One Shot BL21 Star (TM) (DE3)pLysS One Shot Chemically Competent Cells", 17 September 2002 (2002-09-17), XP055965965, Retrieved from the Internet <URL:https://wiki.duke.edu/download/attachments/12027076/BL21%20STAR%20DE3%20CELLS.pdf> [retrieved on 20220928] *
XIAO LIANG ET AL: "Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid", ACS SYNTHETIC BIOLOGY, vol. 7, no. 5, 2 April 2018 (2018-04-02), Washington DC ,USA, pages 1424 - 1435, XP055725189, ISSN: 2161-5063, DOI: 10.1021/acssynbio.8b00055 *

Also Published As

Publication number Publication date
WO2025091009A2 (en) 2025-05-01

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