MODIFIED ORGANISMS FOR IMPROVED FLAVOR AND AROMA

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.

Claims

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-methylisoborneol, 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, 1-octen-3-ol, Sulfides (including dimethyl sulfide and dimethyl trisulfide), Thiomethyl esters, and 2,4-decadienal, 1,3-Octadiene, Octatrienes, 4-Methylthio-1,2-dithiolane, 5-Methylthio-1,2,3-trithiane, Dimethyldisulfide, Dimethyltrisulfide, Methyl ethanethiolate, Methyl propanethiolate, Methyl 2-methylpropanethiolate, Methyl 3-methylbutanethiolate, Pentanal, Heptanal, Dimethyltetrasulfide, 1,2,4-Trithiolane, Oct-1-en-3-o-1, Octan-1-ol, Oct-2-en-1-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.

Description

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] 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.

[0013] 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 consumer perspective. Two potent off-note molecules are geosmin and 2-methylisoborneol and other off notes include 2-pentylfuran and di-amino compounds such as cadaverine, putrescine, spermidine, spermine, thermospermine and others.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

##STR00001##

[0018] Another compound, 2-methylisoborneol 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.

##STR00002##

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.

##STR00003##

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-1,2-dithiolane, 5-Methylthio-1,2,3-trithiane, Dimethyldisulfide, Dimethyltrisulfide, Methyl ethanethiolate, Methyl propanethiolate, Methyl 2-methy 1propanethiolate, Methyl 3-methylbutanethiolate, Pentanal, Heptanal, Dimethyltetrasulfide, 1,2,4-Trithiolane, Oct-1-en-3-o-1, Octan-1-ol, Oct-2-en-1-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 may be targeted through the genomic editing processes described herein to obtain a more palatable organism.

[0019] 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 UTEX 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.

[0020] 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.

[0021] 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.