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WO2019200318A1 - Modified organisms for improved flavor and aroma - Google Patents

Modified organisms for improved flavor and aroma 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
Prior art date
Application number
PCT/US2019/027322
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French (fr)
Inventor
Sean Raspet
Original Assignee
Sean Raspet
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.)
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Publication date
Application filed by Sean Raspet filed Critical Sean Raspet
Priority claimed from US16/383,086 external-priority patent/US11352618B2/en
Publication of WO2019200318A1 publication Critical patent/WO2019200318A1/en

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    • 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, e.g. protozoa; 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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Abstract

The present disclosure provides for genetically modified organisms that provide numerous health benefits but also have an improved flavor profile and a more palatable aroma for the consumer of the organism.

Description

MODIFIED ORGANISMS FOR IMPROVED FLAVOR AND AROMA
Inventor:
SEAN RASPET
Claim of Priority
This application is a PCT International non-provisional application and claims priority to U.S. Application 16/383,086 filed on April 12, 2019 and to U.S. Application 62/657,410 filed on April 13, 2018, the contents of which are herein fully incorporated by reference in its entirety.
Field of the Embodiments
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.
Background of the Embodiments
Presently, there's an ever-growing need for additional food sources, particularly food sources that are inexpensive to produce and nutritious. Moreover, the global reliance on meat products, as the staple of many diets, contributes significantly to the release of greenhouse gases. Thus, there is a need for new foodstuffs that are palatable and nutritious while also being ecologically efficient with a low greenhouse gas footprint.
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.
The above described algal uses do have drawbacks. For example, 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. When formulated into food products or as nutritional supplements, these algal powders impart an unpleasant fishy, muddy, or seaweed-like flavor and odor.
Thus, there remains a need for to produce foodstuffs from algae cheaply and efficiently, at large scale, that are both tasty and nutritious. Such modification may be accomplished via genetic modification of the organism. The present invention and its embodiments meet these and other needs.
Summary of the Embodiments
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. In one instance, 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.
Some have endeavored to grow various organisms in media having additional nitrogen or copper or a combination thereof to remove/reduce the presence of any of the compounds listed herein and other compounds not explicitly listed herein. While growing conditions and the fertilizer/nutrient profile can alter the flavor of the organism, it also reduces the flexibility of producers to optimize their growth conditions towards efficiency and nutrition. 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.
In one embodiment of the present invention there is a method of genetic modification of an organism, the method 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 thereof; and editing the at least one genomic location of the organism by knocking out the at least one genomic location or in the alternative, upregulating or downregulating the at least one genomic location.
In yet another embodiment of the present invention there is 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.
In one embodiment of the present invention the organism is algae. In one embodiment of the present invention, the editing step, as described herein, is performed by CRISPR, TALENs, or zinc fingers.
Description of the Preferred Embodiments
Reference will now be made in detail to each embodiment of the present invention. Such embodiments are provided by way of explanation of the present invention, which is not intended to be limited thereto. In fact, those of ordinary skill in the art may appreciate upon reading the present specification and viewing the present drawings that various modifications and variations can be made thereto.
In general, 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) is an economically useful algae cyanobacteria that contains a high level of beneficial nutrients and protein that also has an expedient growth rate. This makes spirulina a sustainable and healthy food source. Despite these benefits, the flavor of spirulina and other algae has prevented it from widespread consumer adoption. The present application identified herein sources of the undesirable flavors associated with spirulina and other algae. In particular, 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. Several approaches to genome editing have been developed and are becoming widely utilized. 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.
Further, 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.
Similarly, 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. One advantage of TALENs is that these 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. For example, geosmin (shown below) is an organic compound that imparts an earthy, muddy flavor and aroma into organisms in which it is produced. Particularly, problematic is that 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.
Figure imgf000005_0001
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.
Figure imgf000006_0001
2-methylisoborneol
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.
Figure imgf000006_0002
putrescine
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 -one, Octan-3-one, Benzene, Naphthalene, Hexanal, Pentan-3-one, Styrene, Octanal, Decanal, Nonanal, Undecanal, histamine, Thioesters, Uric acids, Uronic acids, Purines, Pinene, Limonene, Bomeol, Fenchol, Indole, Skatole, Polyunsaturated fatty acids, and Free fatty acids may be targeted through the genomic editing processes described herein to obtain a more palatable organism.
In a preferred embodiment, 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,
Nannochloropsis occulate, the genus Nannochloropsis, Haematococcus Pluvialis, the genus Ulva, the genus Enteromorpha, the genus Spirulina,“red algae”,“brown algae”,“green algae”, “blue-green algae”, Parachlorella kessleri, Parachlorella beijerinckii, Neochloris oleabundans, Bracteacoccus, including B. grandis, B. cinnabarinas, and B. aerius, Bracteococcus sp. or Scenedesmus rebescens. 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,
A.c. taylori, A.c. tenuis, A.c. delicatissima, A.c. delicatissima capitata; Anabaena;
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.
medionucleatus; Carteria; Chaetoceros, including C. gracilis, C. muelleri, and C. muelleri subsalsum; Chlorococcum, including C. infusionum; Chlorogonium; Chlorophyceae;
Chlorophyta; Chroomonas; Chrysosphaera; Chrysophyceae; Cricosphaera; Crypthecodinium cohnii; Cryptomonas; Cryptophyceae; Cyclotella, including C. cryptica and C. meneghiniana; Cyanophyceae; Cyanobacteria; Dunaliella, including D. bardawil, D. bioculata, D. granulate, D. maritime, D. minuta, D. parva, D. peircei, D. primolecta, D. salina, D. terricola, D. tertiolecta, and D. 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.
pseudotenelloides, N. pelliculosa, and N. saprophila; 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;
Pyrobotrys; Rhodococcus opacus; Rhodophyceae; Sarcinoid chrysophyte; Scenedesmus, including S. armatus and S. rubescens; Schizochytrium; Spirogyra; Spirulina platensis;
Stichococcus; Synechococcus; Tetraedron; Tetraselmis, including T. suecica; Thalassiosira weissflogii; and Viridiella fridericiana. In a preferred embodiment the algae is spirulina
(arthrospira platensis). Various combinations of the aforementioned algae and other not specifically named herein may further be utilized under the purview of the present invention.
In other embodiments, 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.
Additionally, other applications of the techniques described herein could include altering the flavor profile of nitrogen fixing species of bacteria or cyanobacteria such that they can be used as fertilizer without imparting a muddy taste to the organism grown with the aid of the fertilizer.
Although this invention has been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.

Claims

Claims What is claimed is:
1. A method of genetic modification of an organism, the method comprising:
identifying at least one genomic location of the organism,
wherein the at least one genomic location corresponds to a geosmin synthase gene, a germacradienol synthase gene, MIB synthase gene or 2- MIB synthase gene, 2-pentylfuran synthase gene or 2-pentylfuran producing gene, spermidine synthase gene, spermine synthase gene, theospermine synthase gene, diamine oxidase gene, histamineN-methyltransferase gene, beta-ionone synthase gene, or any combination thereof; and
editing the at least one genomic location of the organism by knocking out or making non-functional at least one genomic location or in the alternative, upregulating or downregulating the at least one genomic location.
2. The method of claim 1 wherein the organism is algae.
3. The method of claim 1 wherein the editing step is performed using a CRISPR system, TALENs, or zinc fingers.
4. A method of producing a better tasting organism comprising: editing the genome of the organism to target production of at least one of the following molecules: geosmin, 2-methylisobomeol, 2-pentylfuran, putrescine, cadaverine, spermine, spermindine, thermospermine, and polyamines having a molecular weight below 350 Daltons.
5. The method of claim 4 wherein the organism is algae.
6. The method of claim 5 wherein the algae is a microalgae or cyanobacteria.
7. The method of claim 4 wherein a targeted gene related to the production of the molecule is knocked-out, made non-functional or removed from the genome of the organism.
8. The method of claim 4 wherein a targeted gene related to the production of the molecule is upregulated or downregulated as a result of the editing.
9. The method of claim 4 wherein the editing step is performed via a CRISPR system, TALENs, or zinc fingers.
10. 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: Beta-cyclocitral, l -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, Dimethyl disulfide,
Dimethyltrisulfide, Methyl ethanethiolate, Methyl propanethiolate, Methyl 2- methylpropanethiolate, Methyl 3-methylbutanethiolate, Pentanal, Heptanal,
Dimethyltetrasulfide, 1, 2, 4-Trithiolane, Oct-l-en-3-o-l, Octan-l-ol, Oct-2-en-l-ol, 1- octen-3-one, Octan-3-one, Benzene, Naphthalene, Hexanal, Pentan-3-one, Styrene, Octanal, Decanal, Nonanal, Undecanal, histamine, Thioesters, Uric acids, Uronic acids, Purines, Pinene, Limonene, Borneol, Fenchol, Indole, Skatole, Polyunsaturated fatty acids, and Free fatty acids
11. The method of claim 10 wherein the organism is algae.
12. The method of claim 11 wherein the algae is a microalgae or cyanobacteria.
13. The method of claim 10 wherein a targeted gene related to the production of the molecule is knocked-out, made non-functional or removed from the genome of the organism.
14. The method of claim 10 wherein a targeted gene related to the production of the molecule is upregulated or downregulated as a result of the editing.
15. The method of claim 10 wherein the editing step is performed via a CRISPR system, TALENs, or zinc fingers.
16. The method of claim 2 wherein the algae is a microalgae or cyanobacteria
PCT/US2019/027322 2018-04-13 2019-04-12 Modified organisms for improved flavor and aroma WO2019200318A1 (en)

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