WO2019200318A1 - Organismes modifiés pour une saveur et un arôme améliorés - Google Patents

Organismes modifiés pour une saveur et un arôme améliorés Download PDF

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Publication number
WO2019200318A1
WO2019200318A1 PCT/US2019/027322 US2019027322W WO2019200318A1 WO 2019200318 A1 WO2019200318 A1 WO 2019200318A1 US 2019027322 W US2019027322 W US 2019027322W WO 2019200318 A1 WO2019200318 A1 WO 2019200318A1
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Prior art keywords
organism
algae
gene
editing
synthase gene
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English (en)
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Sean Raspet
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Priority claimed from US16/383,086 external-priority patent/US11352618B2/en
Publication of WO2019200318A1 publication Critical patent/WO2019200318A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H13/00—Algae
    • 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
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome

Definitions

  • the field of the embodiments of the present invention relate to genetically modified organisms, namely algae, to impart an improved flavor and/or aroma to the organism.
  • Algae have long been looked to as a potential source of food. While certain types of algae, primarily seaweed, do indeed provide important foodstuffs for human consumption, the promise of algae as a foodstuff has not yet been fully realized. However, there are several species of algae that are used in foodstuffs today, most being macroalgae such as kelp, dulse and sea lettuce. Microalgae, such as spirulina, are grown commercially in open ponds or tanks for use as a nutritional supplement or incorporated in small amounts in various beverages. Other microalgae, including some species of chlorella, are also utilized as a nutritional supplement. Microalgae are potentially easier to cultivate and much more resource efficient than macroalgae, producing much more biomass with the same amount of inputs. Despite the use of microalgae in supplements, they have not been widely adopted as a staple food source.
  • macroalgae such as kelp, dulse and sea lettuce.
  • Microalgae such as
  • algal powders made with algae grown photosynthetically in outdoor ponds or photobioreactors are readily available but have a deep green color and a characteristic unpleasant taste.
  • these algal powders When formulated into food products or as nutritional supplements, these algal powders impart an unpleasant fishy, muddy, or seaweed-like flavor and odor.
  • the present invention relates to a platform that can be used to genetically modify a target genome in a plant or an algae or in another organism to make the organism more palatable.
  • the platform includes a CRISPR/Cas system (e.g., a type I, II, or III CRISPR/Cas system, as well as modified versions thereof, such as a CRISPR/dCas9 system), TALENs, or zinc fingers to accomplish the desired genomic editing.
  • the present invention and its embodiments makes a highly-targeted alteration to the organism to remove the source of the specific flavor volatiles, without requiring any large scale infrastructural or process changes.
  • a method of genetic modification of an organism comprising: identifying at least one genomic location of the organism, wherein the at least one genomic location corresponds to genes that code for an enzyme such as: a geosmin synthase gene, a germacradienol synthase gene, a MIB synthase gene, or 2-MIB synthase gene or 2-methylisoborneol synthase gene, a 2-pentylfuran synthase gene, a spermidine synthase gene, a spermine synthase gene, a thermospermine synthase gene, a diamine oxidase gene, lipases, lipoxygenases, agmatine imino hydroxylase, arginine decarboxylase, lysine decarboxylase, ahistamineN-methyltransferase gene, another gene related to polyamine production, a beta-ionone synthase gene, or any combination
  • an enzyme such as: a geos
  • a method of producing a better tasting organism comprising: editing a genome of the organism to target production of at least one of the following molecules: geosmin, 2-methylisoborneol, 2-pentylfuran, putrescine, cadaverine, spermine, spermindine, thermospermine, and polyamines having a molecular weight below 350 Daltons.
  • the organism is algae.
  • the editing step, as described herein, is performed by CRISPR, TALENs, or zinc fingers.
  • the present invention and its embodiments provide an algal food product for human consumption that is rich in nutrients with a satisfying taste.
  • Spirulina arthrospira platensis
  • the present application identified herein sources of the undesirable flavors associated with spirulina and other algae.
  • spirulina produces several molecules that relate to several perceived off notes from the comsumer perspective.
  • Two potent off-note molecules are geosmin and 2-methylisobomeol and other off notes include 2-pentylfuran and di-amino compounds such as cadaverine, putrescine, spermidine, spermine, thermospermine and others.
  • Genome editing also called gene editing is a group of technologies that provide the ability to change an organism's DNA. These technologies allow genetic material to be added, removed, or altered (upregulated, downregulated, etc.) at particular locations in the genome.
  • CRISPR-Cas9 One approach is known as CRISPR-Cas9.
  • the CRISPR-Cas9 system is faster, cheaper, more accurate, and more efficient than other previously-existing genome editing methods.
  • engineered zinc finger arrays may be utilized to modify an organism’s DNA.
  • Engineered zinc finger arrays are often fused to a DNA cleavage domain to generate zinc finger nucleases thereby creating useful reagents for manipulating genomes of various organisms. For example, by targeting a double-strand break to a desired genomic locus one can introduce frame- shift mutations into the coding sequence of a gene. If a homologous DNA“donor sequence” is also used, then the targeted genomic locus can be converted to a defined DNA sequence via the homology directed repair pathway.
  • TALENs can be used to edit genomes by inducing double-strand breaks to which cells respond to with repair mechanisms.
  • TALENs are simply restriction enzymes that can be engineered to cut specific sequences of DNA thereby allowing potential negative genes to be excised from the genome.
  • restriction enzymes can be engineered to bind to practically any DNA sequence.
  • the present invention and its embodiments are directed to utilizing the above and other genomic editing techniques to remove unwanted traits, particularly aroma and taste, from various organisms.
  • geosmin shown below
  • geosmin is an organic compound that imparts an earthy, muddy flavor and aroma into organisms in which it is produced.
  • the human nose is extremely sensitive to geosmin and is able to detect it at concentrations as low as 5 parts per trillion (the human nose is responsible for most of a food’s flavor via retronasal olfaction).
  • the human nose can often detect it at thresholds below that of standard GCMS analysis.
  • Geosmin Another compound, 2-methylisobomeol or 2-MIB (shown below), is an organic compound that gives off an unpleasant earthy odor. Many blue-green algae produce 2-MIB and geosmin which results in an undesirable odor and taste that is imparted to the blue-green algae and other 2-MIB containing organisms.
  • Other compounds include the class of polyamines, including several volatile diamino compounds such as putrescine and cadaverine, among others. These compounds can impart a fishy, fleshy and even fowl aroma and flavor. Larger polyamine molecules tend to have a less potent smell, therefore, one embodiment of the invention upregulates the genes for enzymes that convert smaller polyamines into larger polyamines, thus decreasing the proportion of smaller polyamines in the organism. Another embodiment downregulates the production of polyamines in order to reduce their overall concentration.
  • the above compounds are intended to be exemplary only and other compounds, including but not limited to Beta-cyclocitral, 1 -octen-3 -ol, Sulfides (including dimethyl sulfide and dimethyl trisulfide), Thiomethyl esters, and 2,4-decadienal, 1, 3-Octadiene, Octatrienes, 4-Methylthio-l,2- dithiolane, 5-Methylthio-l,2,3-trithiane, Dimethyldisulfide, Dimethyltrisulfide, Methyl ethanethiolate, Methyl propanethiolate, Methyl 2-methyl propanethiolate, Methyl 3- methylbutanethiolate, Pentanal, Heptanal, Dimethyl tetrasulfide, 1, 2, 4-Trithiolane, Oct-l-en-3-o- 1, Octan-l-ol, Oct-2-en-l-ol, 1 -octen-3 -
  • the organism is a microalgae. More preferably, the microalgae is a species selected from the group consisting of Arthrospira Platensis, Arthrospira Maxima, the genus Arthrospira, Chlorella Vulgaris, the genus Chlorella, the division
  • Chlorophyta Chlamydomonas reinhardtii, Dunaliella Salina, the genus Euglena,
  • Other nonlimiting examples of microalgae species include those species from the group of species and genera consisting of Achnanthes orientalis; Agmenellum;
  • Amphiprora hyaline Amphora, including A. coffeiformis including A.c. linea, A.c. punctata,
  • Ankistrodesmus including A. falcatus; Aphanizomenon flosaquae; Bacillariophyceae;
  • Boekelovia hooglandii Borodinella
  • Botryococcus braunii including B. sudeticus
  • Bracteoccocus including B. aerius, B. grandis, B. cinnabarinas, B. minor, and B.
  • viridis Dinophyceae; Eremosphaera, including E. viridis; Ellipsoidon; Euglenozoa; Franceia; Fragilaria, including F. crotonensis; Gleocapsa; Gloeothamnion; Hymenomonas;
  • Isochrysis including I. aff galbana and I. galbana; Lepocinclis; Micractinium (including EGTEC LB 2614); Monoraphidium, including M. minutum; Monoraphidium; Nannochloris;
  • Nannochloropsis including N. salina; Navicula, including N. acceptata, N. biskanterae, N.
  • Neochloris oleabundans Nephrochloris; Nephroselmis; Nitschia communis; Nitzschia, including N. alexandrina, N. communis, N. dissipata, N. frustulum, N. hantzschiana, N. inconspicua, N. intermedia, N. microcephala, N. pusilla, N. pusilla elliptica, N. pusilla monoensis, and N. quadrangular; Ochromonas; Oocystis, including O. parva and O. pusilla; Oscillatoria, including O. limnetica and O. subbrevis;
  • Parachlorella including P. beijerinckii (including strain SAG 2046) and P. kessleri (including any of SAG strains 11.80, 14.82, 21.11H9); Pascheria, including P. acidophila; Pavlova; Phagus; Phormidium; Platymonas; Pleurochrysis, including P. carterae and P. dentate; Prototheca, including P. stagnora (including UTEX 327), P. portoricensis, and P. moriformis (including UTEX strains 1441, 1435, 1436, 1437, 1439); Pseudochlorella aquatica; Pyramimonas;
  • the algae is spirulina
  • algae (and other organisms) with removed genes such as those that code for enzymes responsible for the production of geosmin, 2-methylisoborneol, and 2- pentylfuran, and other molecules described herein will also improve the flavor of animals that consume algae including farmed salmon, cows, and chickens that use algae or algae containing food stuffs as feed. Further, the principles of the present invention and its embodiments will also be useful for cellular agriculture/cultured meat applications as an off-flavor free feedstock.

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  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

La présente invention concerne des organismes génétiquement modifiés qui fournissent de nombreux avantages de santé mais ont également un profil d'arôme amélioré et un arôme plus agréable pour le consommateur de l'organisme.
PCT/US2019/027322 2018-04-13 2019-04-12 Organismes modifiés pour une saveur et un arôme améliorés Ceased WO2019200318A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201862657410P 2018-04-13 2018-04-13
US62/657,410 2018-04-13
US16/383,086 US11352618B2 (en) 2018-03-23 2019-04-12 Modified organisms for improved flavor and aroma
US16/383,086 2019-04-12

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WO2019200318A1 true WO2019200318A1 (fr) 2019-10-17

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102558660B1 (ko) * 2023-02-16 2023-07-28 대한민국 지오스민 합성 유전자 검출용 프라이머 세트
KR102558666B1 (ko) * 2023-02-16 2023-07-31 대한민국 2-메틸이소보르네올 합성 유전자 검출용 프라이머 세트
WO2023198774A1 (fr) 2022-04-14 2023-10-19 Institute Of Agricultural Resources And Regional Planning Of The Chinese Academy Of Agricultural Sciences Microalgues modifiées pour une meilleure absorption de phosphate impliquant la surexpression de psr1 et éventuellement la sous-expression de ptc1

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0567433A1 (fr) * 1992-04-23 1993-10-27 Sandoz Nutrition Ltd. Compositions d'ingestion orale ayant du goût amélioré
WO2015017866A1 (fr) * 2013-08-02 2015-02-05 Enevolv, Inc. Procédés et cellules hôtes pour ingénierie biomoléculaire, génomique et de la voie
US20160272980A1 (en) * 2012-11-16 2016-09-22 Total Marketing Services Method for targeted modification of algae genomes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0567433A1 (fr) * 1992-04-23 1993-10-27 Sandoz Nutrition Ltd. Compositions d'ingestion orale ayant du goût amélioré
US20160272980A1 (en) * 2012-11-16 2016-09-22 Total Marketing Services Method for targeted modification of algae genomes
WO2015017866A1 (fr) * 2013-08-02 2015-02-05 Enevolv, Inc. Procédés et cellules hôtes pour ingénierie biomoléculaire, génomique et de la voie

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
OU YANG ET AL.: "Activation of SAT1 engages polyamine metabolism with p53- mediated ferroptotic responses", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 113, no. 44, November 2016 (2016-11-01), pages E6806 - E6812, XP055644465 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023198774A1 (fr) 2022-04-14 2023-10-19 Institute Of Agricultural Resources And Regional Planning Of The Chinese Academy Of Agricultural Sciences Microalgues modifiées pour une meilleure absorption de phosphate impliquant la surexpression de psr1 et éventuellement la sous-expression de ptc1
KR102558660B1 (ko) * 2023-02-16 2023-07-28 대한민국 지오스민 합성 유전자 검출용 프라이머 세트
KR102558666B1 (ko) * 2023-02-16 2023-07-31 대한민국 2-메틸이소보르네올 합성 유전자 검출용 프라이머 세트

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