WO2023064845A1 - Ingénierie rationnelle reposant sur la conception d'enzymes tomo par mutagenèse dirigé vers un site pour la biosynthèse du résorcinol - Google Patents

Ingénierie rationnelle reposant sur la conception d'enzymes tomo par mutagenèse dirigé vers un site pour la biosynthèse du résorcinol Download PDF

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Publication number
WO2023064845A1
WO2023064845A1 PCT/US2022/078025 US2022078025W WO2023064845A1 WO 2023064845 A1 WO2023064845 A1 WO 2023064845A1 US 2022078025 W US2022078025 W US 2022078025W WO 2023064845 A1 WO2023064845 A1 WO 2023064845A1
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seq
nucleotide sequence
amino acid
sequence
acid sequence
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PCT/US2022/078025
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English (en)
Inventor
Haiyun PAN
Jacob Edward VICK
Oliver YU
Ilya TIKH
Brendon DUSEL
David Nunn
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Conagen Inc.
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Publication of WO2023064845A1 publication Critical patent/WO2023064845A1/fr

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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)

Definitions

  • the present invention provides a recombinant polypeptide comprising an alpha hydroxylase (TouA) subunit of a toluene-o-xylene monooxygenase (ToMO) polypeptide, wherein the TouA subunit is expressed from a gene having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% identical sequence identity with the nucleotide sequence of SEQ ID NO: 2, wherein the TouA subunit expressed comprises at least one mutation compared to the amino acid sequence of SEQ ID NO: 1, wherein said at least one mutation is selected from the group consisting of F205A, F205C, F205D, F205E, F205K, F205M, F205N, F205P, F205Q, F205R, F205T, and F205V.
  • TouA alpha hydroxylase
  • ToMO toluene-o-xylene monooxygenase
  • the host cell is selected from the group consisting of a bacterium, a yeast, a filamentous fungus, a cyanobacterial alga and a plant cell.
  • the host cell may be selected from the group consisting of Escherichia; Salmonella; Bacillus; Acinetobacter; Streptomyces; Corynebacterium; Methylosinus; Methylomonas; Rhodococcus; Pseudomonas; Rhodobacter; Synechocystis; Saccharomyces; Zygosaccharomyces; Kluyveromyces; Candida; Hansenula; Debaryomyces; Mucor; Pichia; Torulopsis; Aspergillus; Arthrobotlys; Brevibacteria; Microbacterium; Arthrobacter; Citrobacter; Escherichia; Klebsiella; Pantoea; Salmonella Corynebacterium; Clostridium; and Clostridium
  • the recombinant polypeptide comprises an alpha hydroxylase (TouA) subunit of a toluene-o-xylene monooxygenase (ToMO) polypeptide, wherein the TouA subunit is expressed from a gene having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity with the nucleotide sequence of SEQ ID NO: 2, wherein the TouA subunit expressed comprises at least one mutation compared to the amino acid sequence of SEQ ID NO: 1, wherein said at least one mutation is selected from the group consisting of F205A, F205C, F205D, F205E, F205K, F205M, F205N, F205P, F205Q, F205R, F205T, and F205V.
  • TouA alpha hydroxylase
  • ToMO toluene-o-xylene monooxygenase
  • FIG. 2 is a vector map DVK(Kan)ToMO used for induction expression of wild-type ToMO and variants.
  • the six genes coding for ToMO are touABE (three-component hydroxylase, A2B2E2), touC (ferredoxin), touD (mediating protein), and touF (NADH- ferredoxin oxidoreductase), Kanamycin resistance gene (KanR), and the PTac promoter are shown.
  • FIG. 3 is a bar graph comparing the rate of dihydroxybenzene product formation from phenol in the instance of DVK(Kan)ToMO expressing the triple mutant I100F/E103P/F205V to that reported for single mutant F205G.
  • a yeast is a eukaryotic, single-celled microorganism classified as a member of the fungus kingdom.
  • Yeasts are unicellular organisms which evolved from multicellular ancestors but with some species useful for the current disclosure being those that have the ability to develop multicellular characteristics by forming strings of connected budding cells known as pseudohyphae or false hyphae.
  • DNA fragments of different lengths may have identical promoter activity.
  • operably linked refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it can affect the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • recombinant when used herein in connection with a polypeptide or amino acid sequence, means a polypeptide or amino acid sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • recombinant DNA segments can be expressed in a host cell to produce a recombinant polypeptide.
  • the triple I100F/E103P/F205V mutant of ToMO (hereinafter “ToMOm3”) is capable of producing resorcinol at previously unreported rates, an improvement which renders it applicable to industrial settings where economic and convenient large-scale production of resorcinol is desired.
  • the relative amount of product resorcinol as measured against all product 1,3-benzenediols increased seven-fold, from 8% in the F205G single mutant to the 55.7 % that was achieved with ToMOm3.
  • the disclosure provides a mutant ToMOm3 enzyme comprising the amino acid sequence of SEQ ID NO: 3.
  • the disclosure provides variant recombinant polypeptides comprising an amino acid sequence having at least 90% (e.g., 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%) identity to SEQ ID NO: 3.
  • the recombinant polypeptide has the same or substantially the same activity as a mutant ToMO enzyme as described herein, e.g., the recombinant ToMOm3 enzyme comprising the amino acid sequence of SEQ ID NO: 3.
  • the host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.
  • Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art.
  • the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration.
  • Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.
  • Initiation control regions or promoters which are useful to drive expression of the recombinant polypeptide in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the subject technology including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOXI (useful for expression in Pichidy, and lac, trp, JPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli).
  • any combination of any promoter and any terminator capable of inducing expression of a coding region may be used in the vector sequences of the subject technology.
  • Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes.
  • One type of efficient plant promoter that may be used is a high-level plant promoter. Such promoters, in operable linkage with an expression vector of the subject technology should be capable of promoting the expression of the vector.
  • the present disclosure provide a host cell comprising a vector capable of producing a recombinant ToMO enzyme comprising the amino acid sequence of SEQ ID NO: 3.
  • the disclosure provides a host cell comprising a vector capable of producing a recombinant ToMO enzyme comprising the amino acid sequence as set forth in SEQ ID NO: 3.
  • the host cell is selected from the group consisting of a bacterium, a yeast, a filamentous fungus, a cyanobacterial alga and a plant cell.
  • BLAST Basic Local Alignment Search Tool
  • Polynucleotide molecules that have the activity of the ToMOm3 sequence of the current disclosure are capable of directing the production of a variety of dihydroxyphenyl compounds and have a substantial percent sequence identity to the polynucleotide sequences provided herein and are encompassed within the scope of this disclosure.
  • the vector plasmids were transformed into T7 Express E. coli cells which were subsequently cultured at 37 °C overnight. The following morning, 2 ml of each subculture were combined with 60 ml LB broth in a 250 ml flask to form cultures with an initial ODeoo reading of 0.05 which were incubated at 37 °C until the ODeoo reached a value of 1.2. Protein expression was induced by addition of 1 mM isopropyl P-D-l -thiogalactopyranoside (IPTG) and the culture was further grown at 25 °C for 4 hours.
  • IPTG isopropyl P-D-l -thiogalactopyranoside
  • This disclosure has applicability in the food, feed, beverage, and pharmacological industries.
  • This disclosure relates generally to a method for the biosynthetic production of resorcinol and optionally hydroquinone, e.g., via a modified microbial strain.

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

Abstract

Polypeptide recombiné comprenant une sous-unité alpha hydroxylase (TouA) d'un polypeptide toluène-o-xylène monooxygénase (ToMO), la sous-unité TouA étant exprimée à partir d'un gène possédant une identité de séquence d'au moins 95 % avec la séquence nucléotidique de SEQ ID NO : 2, la sous-unité TouA exprimée comprenant au moins une mutation par comparaison avec la séquence d'acides aminés de la SEQ ID NO : 1, ladite au moins une mutation étant choisie dans le groupe constitué par F205A, F205C, F205D, F205E, F205K, F205M, F205N, F205P, F205Q, F205R, F205T et F205V.
PCT/US2022/078025 2021-10-13 2022-10-13 Ingénierie rationnelle reposant sur la conception d'enzymes tomo par mutagenèse dirigé vers un site pour la biosynthèse du résorcinol WO2023064845A1 (fr)

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US202163255086P 2021-10-13 2021-10-13
US63/255,086 2021-10-13

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060051782A1 (en) * 2004-06-04 2006-03-09 Wood Thomas K Directed evolution of recombinant monooxygenase nucleic acids and related polypeptides and methods of use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060051782A1 (en) * 2004-06-04 2006-03-09 Wood Thomas K Directed evolution of recombinant monooxygenase nucleic acids and related polypeptides and methods of use

Non-Patent Citations (17)

* Cited by examiner, † Cited by third party
Title
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ALTSCHUL ET AL., J. MOL. BIOL., vol. 20894, 1990, pages 403 - 410
ASLANIDISDE JONG, NUCL. ACID. RES, vol. 18, 1990, pages 6069 - 74
BERRY-LOWE ET AL., J. MOLECULAR APP. GEN, vol. 1, 1982, pages 483 498
BY AUSUBEL, F. M ET AL.: "IN CURRENT PROTOCOLS MOLECULAR BIOLOGY", 1987, GREENE PUBLISHING AND WILEY-INTERSCIENCE
BY SILHAVY, T. J.BENNAN, M. LENQUIST, L. W: "Cold Spring Harbor Laboratory", 1984, COLD SPRING HARBOR, article "EXPERIMENTS GENE FUSIONS"
CORUZZI, G ET AL., THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 258, 1983, pages 1399
DUNSMUIR, P ET AL., JOURNAL OF MOLECULAR AND APPLIED GENETICS, vol. 2, 1983, pages 285
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NEEDLEMANWUNSCH: "JOURNAL MOLECULAR BIOLOGY", vol. 48, 1970, GENETICS COMPUTER GROUP, INC., article "Gap", pages: 443 - 453
REECK ET AL., CELL, vol. 50, 1987, pages 667
SAMBROOK, J.FRITSCH, E. FMANIATIS, T: "MOLECULAR CLONING: A LABORATORY MANUAL", 1989, COLD SPRING HARBOR LABORATORY: COLD SPRING HARBO
SMITH ET AL., NUCLEIC ACIDS RESEARCH, vol. 11, 1983, pages 2205 - 2220
SMITHWATERMAN: "ADVANCES IN APPLIED MATHEMATICS", vol. 2, 1981, pages: 482 - 489
VARDAR G ET AL: "Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for oxidizing nitrobenzene to 3-nitrocatechol, 4-nitrocatechol, and nitrohydroquinone", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 115, no. 2, 26 January 2005 (2005-01-26), pages 145 - 156, XP027663328, ISSN: 0168-1656, [retrieved on 20050126] *
VARDAR GÖNÜL ET AL: "Protein Engineering of Toluene- o -Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 70, no. 6, 1 June 2004 (2004-06-01), US, pages 3253 - 3262, XP093020086, ISSN: 0099-2240, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC427803/pdf/2474-03.pdf> DOI: 10.1128/AEM.70.6.3253-3262.2004 *
VARDAR, G ET AL., APPL. ENVIRON. MICROBIOL., vol. 70, no. 6, 2004, pages 3253 - 62

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