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WO2025090082A1 - Synthèse évolutive, économique de la sélénonéine et de ses analogues - Google Patents

Synthèse évolutive, économique de la sélénonéine et de ses analogues Download PDF

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Publication number
WO2025090082A1
WO2025090082A1 PCT/US2023/077824 US2023077824W WO2025090082A1 WO 2025090082 A1 WO2025090082 A1 WO 2025090082A1 US 2023077824 W US2023077824 W US 2023077824W WO 2025090082 A1 WO2025090082 A1 WO 2025090082A1
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WIPO (PCT)
Prior art keywords
protein
sena
selenoneine
microorganism
cell
Prior art date
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PCT/US2023/077824
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English (en)
Inventor
Mohammad R. Seyedsayamdost
Chase M. KAYROUZ
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Princeton University
Original Assignee
Princeton University
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Publication date
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Priority to PCT/US2023/077824 priority Critical patent/WO2025090082A1/fr
Priority to US19/124,615 priority patent/US20250327105A1/en
Publication of WO2025090082A1 publication Critical patent/WO2025090082A1/fr
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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography

Definitions

  • a process for producing selenoneine or an analog thereof may be provided.
  • the process may include providing a recombinant microorganism (such as an E. coli strain) configured to express a SenA protein [SEQ ID NO: 1] or analog thereof.
  • the process may include preparing a cell-free lysate of the modified microorganism.
  • the cell-free lysate may include the SenA protein in a fluid (such as a neutral-pH aqueous buffer).
  • the SenA protein may be soluble in the fluid.
  • the process may include purifying the selenoneine (e.g., via chromatography).
  • the process may include chemically derivatizing seleno-containing substrates and products with a reactant (such as monobromobimane).
  • a reactant such as monobromobimane
  • an engineered microorganism such as a strain of E. coli
  • the microorganism may include a DNA sequence configured to express a SenA protein [SEQ ID NO: 1] or an analog thereof.
  • the microorganism may be free of sequences configured to express a SenB protein [SEQ ID NO: 2] or an analog thereof and sequences configured to express a SenC protein [SEQ ID NO: 3] or an analog thereof.
  • an intermediate composition may be provided.
  • the intermediate composition may include a cell-free lysate including a SenA protein [SEQ ID NO: 1] or an analog thereof in a fluid.
  • the SenA protein may be soluble in the fluid.
  • the cell-free lysate Princeton - 91876 may be free of SenB protein [SEQ ID NO: 2] or analogs thereof and SenC protein [SEQ ID NO: 3] or analogs thereof.
  • an intermediate composition may be provided.
  • the intermediate composition may include hercynine and a selenosugar.
  • the intermediate composition may include a reductant. A ratio of concentrations of hercynine to selenosugar to reductant may be about 2:2:1 in the intermediate composition.
  • Figure 1 is a flowchart of a method.
  • Figure 2 is a graph showing the result of a typical reaction containing 20 mM hercynine, 20 mM 1-seleno-N-acetyl- ⁇ -D-glucosamine (monomer basis), and 10 mM dithiothreitol in 0.1 mL of cell-free E. coli lysate containing recombinant SenA.
  • analog refers to a molecule which is structurally similar or shares similar or corresponding attributes with another molecule.
  • analog [of a protein] refers to any polypeptide that is structurally similar to the protein in question, and that shares the biochemical or biological activity of the protein in question upon which the analog is based.
  • Various references disclose modification of polypeptides by polymer conjugation or glycosylation.
  • analogs of the instant invention may comprise a linker or polymer, wherein the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid, or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine. encompasses the conventional and well-known naturally occurring amino acids, as well as all synthetic variations and derivatives thereof.
  • the analogs comprise alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine substitutions.
  • analogs comprise N-methylated ⁇ - amino acids, hydroxylated amino acids, and aminooxy acids.
  • analogs Princeton - 91876 comprise N-alkyl amino acids (such as N-methyl glycine), hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, nor-valine, nor-leucine, and ornithine.
  • amino acid refer to a molecule containing both an amino group and a carboxyl group bound to a carbon which is designated the ⁇ -carbon.
  • Suitable amino acids include, without limitation, both the D- and L- isomers of the naturally-occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic routes.
  • a single “amino acid” might have multiple sidechain moieties, as available per an extended aliphatic or aromatic backbone scaffold.
  • amino acid as used herein, is intended to include amino acid analogs including non-natural analogs.
  • the myriad cytoprotective properties of selenoneine have been known for some time. Methods have been developed for the chemical production of selenoneine. However, these utilize harsh chemicals as the reactive element selenium has to be incorporated in a specific fashion. Moreover, these methods are very low-yielding and not suitable for scalable production of selenoneine.
  • Princeton - 91876 100 may include providing 110 a modified microorganism (e.g., a recombinant microorganism) configured to express a SenA protein [SEQ ID NO: 1] or analog thereof.
  • the microorganisms may have already been prepared.
  • the microorganisms may be provided by first providing 112 a base microorganism strain, and then introducing 114 DNA which encodes a polypeptide sequence configured to express the SenA protein into the base microorganism strain. Such techniques are well understood in the art. While E. coli was in various experiments disclosed herein, it will be understood that various other microorganisms may be readily utilized to express SenA.
  • Useful microorganisms include algae, yeast and bacteria.
  • the recombinant microorganism may be a gram-negative bacterium, e.g., E. coli, Salmonella, Rhodobacter, Synechtocystis, or Erwinia.
  • Other gram- negative bacteria include members from the Methylococcaceae and Methylocystaceae families; Thermotoga hypogea, Thermotoga naphthophila, Thermotoga subterranean, Petrotoga halophila, Petrotoga mexicana, Petrotoga miotherma, and Petrotoga mobilis.
  • the recombinant microorganism may be a gram-positive bacterium, e.g., B. subtilis or Clostridium.
  • the recombinant microorganism may be a fungus such as Saccharomyces cerevisae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Scizosacchromyces pombe or Trichoderma reesei or other yeast species of genus Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia, Trichoderma or Scizosacchromyces.
  • the recombinant microorganism may also be a eukaryote such as an algae.
  • Example of algae include Chlamydomonas.
  • the SenA protein or analog thereof is preferably a SenA protein [SEQ ID NO: 1], 426 amino acids in length. However, in some embodiments, a protein with at least a 99% sequence identity to the SenA protein [SEQ ID NO: 1] may be utilized. In some embodiments, a single addition, substitution, or deletion mutation may exist in the analog. In some embodiments, two or fewer addition, substitution, or deletion mutations may exist in the analog. In some Princeton - 91876 embodiments, three or fewer addition, substitution, or deletion mutations may exist in the analog. In some embodiments, four or fewer addition, substitution, or deletion mutations may exist in the analog. In some embodiments, there are no mutations in positions 1-50.
  • the SenA protein or analog thereof may be fused to one or more tags.
  • the tag(s) may include a purification tag.
  • purification tag preferably refers to an additional amino acid sequence (a peptide or polypeptide) which allows for purification of the SenA protein or analog thereof.
  • Non-limiting examples of purification tags include polyhistidine tag, polyarginine tag, glutathione-S-transferase (GST), maltose binding protein (MBP), influenza virus HA tag, thioredoxin, staphylococcal protein A tag, the FLAGTM epitope, and the c-myc epitope.
  • the purification tag is a polyhistidine tag.
  • the polyhistidine tag comprises at least 6 consecutive histidine residues.
  • the tag(s) may include a fluorescent protein or tag.
  • Non-limiting examples of fluorescent proteins include green fluorescent proteins (e.g., GFP, eGFP, GFP-2, tagGFP, turboGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., BFP, EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T- sapphire), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi- Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem
  • the process may include incubating 120 the modified microorganism, during which time the SenA may be expressed by the microorganism. Such techniques are well understood, and appropriate growth media and incubation conditions are well known.
  • the process may include preparing 130 a cell-free lysate of the modified microorganism. Such techniques are well known in the art.
  • the process may include lysing 132 the microorganism. Lysing techniques are well known in the art, and may include, e.g., sonication, homogenization, freeze-thaw lysis, high-temperature lysis, enzymatic lysis, and/or chemical lysis. This may include centrifugation. Princeton - 91876 For example, 6xHis ⁇ tagged SenA was produced in E.
  • coli BL21(DE3) cells grown in two 4 L flasks, each containing 2 L Terrific Broth supplemented with 50 mg/L kanamycin at 37 °C / 170 rpm.
  • Small cultures were prepared by inoculating 40 mL of LB medium containing 50 mg/L Kan with a single colony of E. coli BL21(DE3) carrying a desired plasmid. After overnight growth at 37 °C / 170 rpm, 4 L of TB medium plus 50 mg/L Kan were inoculated with the 40 mL small culture and incubated at 37 °C / 170 rpm.
  • deoxyribonuclease I from Alfa Aesar
  • 0.1 mg/mL deoxyribonuclease I from Alfa Aesar
  • the cells were lysed by the addition of 5 mg/mL lysozyme followed by sonication using 30% power ( ⁇ 150 W) in 15 s on/15 s off cycles for a total of 4 min. This process was repeated twice.
  • the lysate was then clarified by centrifugation (17,000g, 15 min, 4 °C) and loaded onto a 5 mL Ni ⁇ NTA column pre ⁇ equilibrated in lysis buffer.
  • the cell-free lysate may include the SenA protein in a fluid.
  • the SenA is preferably soluble in the fluid.
  • the fluid may be a buffer, such as an aqueous buffer, such as a neutral- pH aqueous buffer.
  • SenA is a remarkable enzyme that can be used to join N,N,N-trimethyl-L-histidine (hercynine) with a selenosugar to generate selenoneine or an analog thereof.
  • the process may include enzymatically generating 140 selenoneine or an analog thereof by adding additional materials to the cell-free lysate.
  • the additional materials may include hercynine and a selenosugar.
  • the selenosugar may be acetylated or non-acetylated 1-selenosugar.
  • the sugar may be a monosaccharide or a polysaccharide.
  • the sugar may be an aminosugar.
  • Non-limiting examples of non-acetylated selenosugars include 1-selenoglucose, 1-selenomannose, 1- selenogalactose, 1-selenoribose, 1-selenomaltose and 1-selenofucose.
  • Non-limiting examples of acetylated selenosugars include 1-seleno-N-acetyl- ⁇ -D-glucosamine, 1 ⁇ -Methylseleno-N- acetyl-D-galactosamine.
  • the additional materials may include a reductant.
  • reductant refers to any material that is capable of either directly (chemically) or indirectly (via biological systems) of donating electrons to impact a reduction reaction as part of the reaction to generate selenoneine or an analog thereof. It will be understood that the reductant may be inorganic or organic. Non-limiting examples of a reductant include dithiothreitol (DTT), mercaptoethanol, cysteine, thioglycolate, cysteamine, glutathione, and sodium borohydride. In some embodiments, enzymatically generating selenoneine may be performed at a temperature above 25 ⁇ C.
  • an engineered microorganism (such as a strain of E. coli) may be provided.
  • the microorganism may include an algae, yeast, or bacteria.
  • the recombinant microorganism may be a gram-negative bacterium, e.g., E. coli, Salmonella, Rhodobacter, Synechtocystis, or Erwinia.
  • gram-negative bacteria include members from the Methylococcaceae and Methylocystaceae families; Thermotoga hypogea, Thermotoga naphthophila, Thermotoga subterranean, Petrotoga halophila, Petrotoga mexicana, Petrotoga Princeton - 91876 miotherma, and Petrotoga mobilis.
  • the recombinant microorganism may be a gram-positive bacterium, e.g., B. subtilis or Clostridium.
  • the recombinant microorganism may be a fungus such as Saccharomyces cerevisae, Pichia pastoris, Hansenula polymorpha, Kluyveromyces lactis, Yarrowia lipolytica, Scizosacchromyces pombe or Trichoderma reesei or other yeast species of genus Saccharomyces, Pichia, Hansenula, Kluyveromyces, Yarrowia, Trichoderma or Scizosacchromyces.
  • the recombinant microorganism may also be a eukaryote such as an algae.
  • Example of algae include Chlamydomonas.
  • the microorganism may include a DNA sequence configured to express a recombinant SenA protein [SEQ ID NO: 1] or an analog thereof.
  • the SenA protein or analog thereof is preferably a SenA protein [SEQ ID NO: 1], 426 amino acids in length.
  • an analog of SenA may have at least a 99% sequence identity to the SenA protein [SEQ ID NO: 1] may be utilized.
  • a single addition, substitution, or deletion mutation may exist in the analog.
  • two or fewer addition, substitution, or deletion mutations may exist in the analog.
  • three or fewer addition, substitution, or deletion mutations may exist in the analog.
  • the analog there are no mutations in positions 1-50. In some embodiments, there are no mutations in positions 51-100. In some embodiments, there are no mutations in positions 101-200. In some embodiments, there are no mutations in positions 201-300. In some embodiments, there are no mutations in positions 301-350. In some embodiments, there are no mutations in positions 351-426.
  • the SenA or analog thereof may be fused to one or more tags, as disclosed herein.
  • the microorganism may be free of sequences that express molecules that are configured to interfere with SenA’s interaction with hercynine and a selenosugar.
  • the microorganism may be free of genes encoding sequences involved in enzymatic synthesis of selenoneine. More specifically, the microorganism may be free of sequences configured to express a recombinant SenB protein [SEQ ID NO: 2] or an analog thereof and sequences configured to express a recombinant SenC protein [SEQ ID NO: 3] or an analog thereof. In some embodiments, the microorganism may be free of sequences configured to express a protein having at least 99% sequence identity to SenB [SEQ ID NO: 2] and sequences configured to express a protein having at least 99% sequence identity to SenC [SEQ ID NO: 3]. Princeton - 91876 In various aspects, an intermediate composition may be provided.
  • the intermediate composition may include a cell-free lysate including a recombinant SenA protein [SEQ ID NO: 1] or an analog thereof in a fluid.
  • the SenA protein or analog thereof is preferably a SenA protein [SEQ ID NO: 1], 426 amino acids in length.
  • an analog of SenA may have at least a 99% sequence identity to the SenA protein [SEQ ID NO: 1] may be utilized.
  • a single addition, substitution, or deletion mutation may exist in the analog.
  • two or fewer addition, substitution, or deletion mutations may exist in the analog.
  • three or fewer addition, substitution, or deletion mutations may exist in the analog.
  • the SenA or analog thereof may be fused to one or more tags, as disclosed herein.
  • the fluid may be a buffer, such as an aqueous buffer, such as a neutral-pH aqueous buffer.
  • the recombinant SenA protein or analog thereof may be soluble in the fluid.
  • the cell- free lysate may be free of recombinant SenB protein [SEQ ID NO: 2] or analogs thereof and recombinant SenC protein [SEQ ID NO: 3] or analogs thereof.
  • the cell-free lysate may be free of proteins having at least 99% sequence identify to SenB [SEQ ID NO: 2] and proteins having at least 99% sequence identify to SenC [SEQ ID NO: 3].
  • an intermediate composition may be provided.
  • the intermediate composition may include hercynine and a selenosugar.
  • the selenosugar may be any selenosugar as disclosed herein.
  • the intermediate composition may include a reductant as disclosed herein.
  • a ratio of concentrations of hercynine to selenosugar to reductant may be about 2:2:1 in the intermediate composition.
  • the term “about” is used to indicate that each value in the ratio may vary by ⁇ 10% (e.g., the ratio could be 2.2:1.8:1).
  • a ratio of hercynine to reductant may be at least 1.5:1.
  • a ratio of hercynine to reductant may be at least 2:1.
  • a ratio of hercynine to reductant may be 5:1 – 1.5:1.
  • a ratio of selenosugar to reductant may be at least 1.5:1.
  • a ratio of selenosugar to reductant Princeton - 91876 may be at least 2:1. In some embodiments, a ratio of selenosugar to reductant may be 5:1 – 1.5:1. In some embodiments, a ratio of hercynine to selenosugar may be 1.5:1 – 1:1.5. In various examples, the disclosed simple chemical synthesis of hercynine and the selenosugar is combined with even crude SenA to deliver selenoneine in excellent yields (e.g., at least 5 g material per liter). This is a very cost-effective technique; it is estimated that the cost of selenoneine generated in this fashion should be $100 per gram material.

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Abstract

La présente invention concerne un procédé rapide, facile, économique et hautement évolutif pour produire de la sélénonéine. Le procédé consiste à utiliser l'enzyme SenA, de la hercynine, un sélénosucre, et éventuellement un réducteur, et une étape enzymatique unique à haut rendement dans, par exemple, un tampon aqueux à pH neutre et à température ambiante. Le procédé de l'invention permet d'obtenir des rendements d'environ 5 grammes de sélénonéine par réaction de litre avec des coûts approximatifs de 100 $ par gramme de sélénonéine.
PCT/US2023/077824 2022-10-26 2023-10-26 Synthèse évolutive, économique de la sélénonéine et de ses analogues Pending WO2025090082A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/US2023/077824 WO2025090082A1 (fr) 2023-10-26 2023-10-26 Synthèse évolutive, économique de la sélénonéine et de ses analogues
US19/124,615 US20250327105A1 (en) 2022-10-26 2023-10-26 Scalable, economical synthesis of selenoneine and its analogs

Applications Claiming Priority (1)

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PCT/US2023/077824 WO2025090082A1 (fr) 2023-10-26 2023-10-26 Synthèse évolutive, économique de la sélénonéine et de ses analogues

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3808853A1 (fr) * 2018-06-15 2021-04-21 Kikkoman Corporation Procédé de production de sélénonéine
US20210317463A1 (en) * 2015-08-07 2021-10-14 Kikkoman Corporation Method for producing selenoneine
WO2023183543A1 (fr) * 2022-03-25 2023-09-28 The Trustees Of Princeton University Synthèse chimioenzymatique de sélénoéine et de ses analogues

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210317463A1 (en) * 2015-08-07 2021-10-14 Kikkoman Corporation Method for producing selenoneine
EP3808853A1 (fr) * 2018-06-15 2021-04-21 Kikkoman Corporation Procédé de production de sélénonéine
WO2023183543A1 (fr) * 2022-03-25 2023-09-28 The Trustees Of Princeton University Synthèse chimioenzymatique de sélénoéine et de ses analogues

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KAYROUZ CHASE M., HUANG JONATHAN, HAUSER NICOLE, SEYEDSAYAMDOST MOHAMMAD R.: "Biosynthesis of selenium-containing small molecules in diverse microorganisms", NATURE, SPRINGER NATURE LIMITED, LONDON, vol. 610, no. 7930, 6 October 2022 (2022-10-06), London, pages 199 - 204, XP093313087, ISSN: 0028-0836, DOI: 10.1038/s41586-022-05174-2 *

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