Patent ID: 12215312

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 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.

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.

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.

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 tri sulfide), 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-methyl propanethiolate, 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.

In a preferred embodiment, the organism is a microalgae. More preferably, the microalgae is a species selected from the group consisting ofArthrospira platensis, Arthrospira maxima, the genusArthrospira, Chlorella vulgaris, the genus Chlorella, the divisionChlorophyta, Chlamydomonas reinhardtii, Dunaliella salina, the genusEuglena, Nannochloropsis occulate, the genusNannochloropsis, Haematococcus pluvialis, the genusUlva, the genusEnteromorpha, the genusSpirulina, “red algae”, “brown algae”, “green algae”, “blue-green algae”,Parachlorella kessleri, Parachlorella beijerinckii, Neochloris oleabundans, Bracteacoccus, includingB. grandis, B. cinnabarinas, andB. aerius, Bracteococcussp. orScenedesmus rebescens. Other nonlimiting examples of microalgae species include those species from the group of species and genera consisting ofAchnanthes 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, andB. 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, includingF. crotonensis; Gleocapsa; Gloeothamnion; Hymenomonas; Isochrysis, including I. aff. galbanaandI. galbana; Lepocinclis; Micractinium(including UTEX LB 2614);Monoraphidium, includingM. 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, andN. quadrangular; Ochromonas; Oocystis, includingO. parvaandO. pusilla; Oscillatoria, including O. limneticaandO. subbrevis; Parachlorella, includingP. beijerinckii(including strain SAG 2046) andP. kessleri(including any of SAG strains 11.80, 14.82, 21.11H9);Pascheria, includingP. acidophila; Pavlova; Phagus; Phormidium; Platymonas; Pleurochrysis, includingP. carteraeandP. dentate; Prototheca, includingP. stagnora(including UTEX 327),P. portoricensis, andP. moriformis(including UTEX strains 1441, 1435, 1436, 1437, 1439);Pseudochlorella aquatica; Pyramimonas; Pyrobotrys; Rhodococcus opacus; Rhodophyceae; Sarcinoid chrysophyte; Scenedesmus, includingS. armatusandS. rubescens; Schizochytrium; Spirogyra; Spirulina platensis; Stichococcus; Synechococcus; Tetraedron; Tetraselmis, includingT. suecica; Thalassiosira weissflogii; andViridiella 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.