Novel Mogroside Manufacturing System and Method

Abstract

This disclosure presents a solution for producing mogrosides and low- or no-calorie mogroside-based sweeteners. Using recombinant genetic and plant transformation techniques, non-native genes encoding mogroside pathway enzymes are introduced / implemented into the genome of a plant, thereby forming a transgenic plant that, due to its native genome, is not naturally capable of producing mogrosides prior to transformation. Such transgenic plants and their progeny are enabled to produce the non-native mogrol precursor, mogrol, mogroside, and / or its metabolites or derivatives.

Description

Technical Field

[0001] The present application is filed as an international PCT patent application on March 17, 2021, and claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 990,802, filed on March 17, 2020, the entire disclosure of which is incorporated herein by reference in its entirety.

[0002] Pursuant to 37 C.F.R. § 1.821(c) or (e), a file containing an ASCII text version of the Sequence Listing is submitted herewith as part of the present application, the content of which is incorporated herein by reference. Background Art

[0003] Introduction With increasing awareness of healthy diets and the potential risks of obesity and diabetes in countries around the world, low-calorie or non-calorie sweeteners as alternatives to conventional high-calorie sweeteners have become increasingly important for the food and beverage business and other industries. These alternative natural sweeteners are used to replace artificial sweeteners as well as high-calorie sweeteners including sucrose, fructose and glucose. Some of them, like several artificial sweeteners, provide a higher sweetening effect than sweeteners with an equivalent amount of calories; therefore, a smaller amount of these alternatives is required to achieve a sweetness equivalent to that of sugar. However, several low-calorie sweeteners can be costly to manufacture and / or may have unfavorable taste characteristics and / or off-notes including, but not limited to, residual aftertaste, delayed sweetness onset, unpleasant mouthfeel, bitter, metallic, cooling, astringent and licorice-like flavors.

[0004] There are few natural plants that biosynthetically produce low-calorie or calorie-free sweeteners. For example, mogrosides, an important class of natural sweeteners, are chemically a type of triterpene glycoside or mogrol glycoside naturally produced by Siraitia grosvenorii (Luo Han Guo). Mogrosides contain no calories and are 100 to 400 times sweeter than sucrose. Mogrosides have also been reported to possess various important pharmacological effects.

[0005] Mogrosides are highly stable molecules based on a triterpene skeleton, formed by various numbers of glucose units (1-6) bonded to carbon 24 and / or carbon 3 (Figure 1). Various mogrosides and their structures are shown in Figure 2. Mogrosides may also include non-glucose moieties, such as Grosmomoside I.

[0006] Generally, the natural biosynthesis of mogrosides is available only in Siraitia grosvenorii. Extracts from both fresh and dried Siraitia grosvenorii yield a powder that is approximately 80% mogroside, with mogroside V being the main component. All enzymes involved in the mogrool / mogroside biosynthesis pathway and the step from squalene to mogroside V were identified by genome sequencing and transcriptome analysis at each growth stage of the fruit. Figure 1 shows the mogroside V biosynthesis pathway (Seki et al Bioscience, Biotechnology, and Biochemistry, 2018 VOL. 82, NO. 6, 927-934).

[0007] Plants such as Siraitia grosvenorii produce natural low-calorie and calorie-free sweeteners, but the production of sweeteners from these plants is limited to their limited natural or agricultural production. More importantly, sweeteners produced in plants tend to be more acceptable to consumers than those chemically or biochemically synthesized in vitro. Furthermore, sweeteners produced in plants can be useful for a variety of reasons. Such fruits or plants can be used not only as low-calorie and calorie-free sweeteners, but also to produce juices, including flavorings, extracts, or fillings for beverages, for use in foods and beverages that offer calorie reduction and other nutritional benefits.

[0008] Furthermore, while the mass production of mogrosides in vitro or microbially has been conceptually proven, it may require large-scale processing and is therefore not economically viable. Biosynthetic pathways producing mogrosides have been attempted in microorganisms for fermentation. However, small sweet molecules like mogrosides have not been fully developed.

[0009] Patron's U.S. Patent Application Publication 2019 / 0071705 provides a method for producing mogroside IIIE in recombinant host cells, comprising culturing recombinant host cells in a culture medium under specific conditions, wherein the genes of the recombinant host cells express an enzyme that catalyzes the production of mogroside IIIE.

[0010] Houghton-Larson International Publication No. 2018 / 229283 provides recombinant host cells capable of producing one or more mogroside compounds in cell culture, comprising recombinant genes encoding heterologous or endogenous polypeptides capable of catalyzing mogroside production.

[0011] Itkin's International Publication No. 2016 / 038617 relates to a method for biosynthetically producing and isolating a mogroside-producing enzyme, and to a method for producing mogol precursor, mogol, and mogroside in recombinant host cells.

[0012] Liu's U.S. Patents 9932619 and 9920349 both relate to in vitro methods and materials for the enzymatic synthesis of mogroside compounds, and to methods for producing mogrol using a cytochrome P450 enzyme and glycosylation of mogrol using uridine-5'-diphospho (UDP)-dependent glucosyltransferase (UGT) to produce various mogroside compounds. [Overview of the Initiative] [Problems that the invention aims to solve]

[0013] Thus, there is a need for new methods and biological systems for the efficient production of mogrosides, and this disclosure presents advantages and advances that address this need in light of the above background. [Means for solving the problem]

[0014] Novel Mogroside Manufacturing System and Method Summary of Disclosure This disclosure presents a solution for producing mogol, mogrosides, and mogroside-based sweeteners that are low in calories or calorie-free. By using recombinant genes and plant transformation techniques, non-natural genes encoding morgol-producing and mogroside-producing enzymes are introduced / implemented into the genome of a natural plant, thereby forming a transgenic plant, which, due to its natural genome, could not naturally produce mogol or mogrosides before transformation. Such a transgenic plant is then enabled to produce non-natural mogol precursors, mogol, mogrosides, and / or their metabolites or derivatives.

[0015] The proposed solution offers significant advantages. Firstly, the production of mogrol, low-calorie, or non-calorie mogrosides through the cultivation and regeneration of genetically modified plants may be more techno-economically viable due to mature agricultural technology. Furthermore, the solution enables mogroside production not only within the fruit but also across a wider range of plant parts, thereby increasing the overall nutritional and economic value of the genetically modified plant. Moreover, implementing mogrol and / or mogroside-producing genes into fast-growing or fast-maturing plants / crops may improve the efficiency of mogroside production and processing, providing cost-effective advantages. The proposed solution, through the incorporation of these genetically modified plants and materials or parts thereof, may enable novel low-calorie or non-calorie foods and beverages.

[0016] It should be noted that previous disclosures have primarily focused on methods based on genetically modified microorganisms (mostly yeast) for producing mogrosides. This disclosure explicitly describes genetically modified plants that are enabled to produce mogrol / mogrosides. While yeast-based methods are conceptually capable of synthesizing mogrosides, they offer little to no economic advantage. More importantly, plant-derived sweeteners and food products offer a clear improvement in consumer acceptance. The genetically modified plants described herein enable the production of fruit juices or plant extracts or plant materials or other derived consumables with lower calorie-to-sweet ratios that can be used with less processing or in conjunction with desirable additional flavor characteristics and profiles.

[0017] It is important to note that genetically modified organisms containing mogroside-producing genes rarely have the ability to produce fruits and / or seeds. Surprisingly, the genetically modified plants according to this disclosure produce various tissues, including fruits and seeds, and all of these tissues, including fruits and seeds, contain mogrosides. The mogroside-containing fruits of these genetically modified plants can be used as a source for various food and beverage products, thus providing techno-economic advantages in the food and consumables industries. Furthermore, the seed-producing genetically modified plants of this disclosure may benefit the large-scale, cost-effective production of mogrosides through seed propagation and the agricultural regeneration of genetically modified plants using various plant breeding techniques.

[0018] As an example of its application, watermelon fruit has great potential for the production of low-calorie and / or zero-calorie sweeteners due to its large size and popular taste. To design genome editing or cisgenic strategies for pathway engineering, it is crucial to identify watermelon fruit-specific promoters that enable the optimal expression of gene payloads, such as mogroside-producing sequences. Identifying these promoters requires high-resolution transcriptome datasets from which a list of genes specifically expressed in the edible portion of watermelon fruit can be generated. The methods and systems described herein advantageously provide effective techniques for tissue-specific expression of target genes at different growth stages.

[0019] This disclosure generally describes gene-modified plants and mogol and / or mogroside-producing enzymes in their tissues or parts, as well as gene-modified plants and their biosynthetic systems for producing mogol / mogroside, and methods for producing such gene-modified plants.

[0020] In some embodiments, the disclosure relates to a transgenic plant comprising a genomic transformation event, wherein the genomic transformation event results in the expression or concentration of a moglol and / or mogroside-producing enzyme, and the transgenic plant biosynthetically produces a non-natural moglol precursor, moglol, mogroside and / or its metabolites or derivatives. In certain embodiments of such a transgenic plant, the genomic transformation event comprises an expression cassette, the expression cassette comprising one or more nucleotide sequences shown in SEQ ID NOs: 1-31. In other embodiments, such an expression cassette comprises one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1-31.

[0021] In other embodiments, the disclosure relates to a genetically modified plant comprising a non-natural moglol precursor and / or moglol, wherein the genetically modified plant biosynthetically produces moglol, mogrosides and / or their metabolites or derivatives. In certain embodiments, such a genetically modified plant comprises an expression cassette comprising one or more nucleotide sequences shown in SEQ ID NOs: 1-31. In other embodiments, such an expression cassette comprises one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1-31.

[0022] In certain embodiments, the transgenic plant of the present disclosure includes obtainable parts thereof, including but not limited to organs, tissues, leaves, stems, roots, flowers or flower parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic sites, callus tissues, seeds, cuttings, cell or tissue cultures, or any other parts or products of the transgenic plant, wherein said parts comprise mogrol precursors, mogrol, mogrosides and / or metabolites or derivatives thereof.

[0023] In other embodiments, the transgenic plant of the present disclosure is cultivable and propagable. Progeny or ancestors of a transgenic plant are a source of non-natural enzymes that allow the progeny and ancestors to produce mogrol, mogrosides and / or metabolites or derivatives thereof. Propagation of seeds of a transgenic plant results in viable progeny thereof, and the progeny produce mogrol, mogrosides and / or metabolites or derivatives thereof.

[0024] In some embodiments, the transgenic plant is a diploid plant. In some embodiments, the transgenic plant is a Cucurbitaceae / cucurbit plant. In certain embodiments, the transgenic plant is a transgenic watermelon (Citrullus lanatus).

[0025] The present disclosure also relates to food or beverage products obtained from a transgenic plant, comprising mogrol, mogrosides or mogroside-based sweeteners. In some embodiments, the present disclosure relates to a mogroside-based sweetener extracted or purified from the transgenic plant or a part thereof according to the present disclosure. In certain embodiments, the method for extracting and / or purifying a mogroside-based sweetener from a transgenic plant is immersion, chromatography or absorption chromatography.

[0026] The present disclosure relates to a method for producing a transgenic plant that produces non-natural mogroside, comprising combining a plant with a genome conversion event, thereby forming a transgenic plant, wherein the genome conversion event results in non-natural expression or concentration of mogrol-producing and / or mogroside-producing enzymes. Combining a plant with a genome conversion event is generally performed using one or more of the following methods: use of liposomes, use of electroporation, use of chemicals that increase uptake of free DNA, use of direct injection of DNA into plants, use of particle gun bombardment, use of transformation using viruses or pollen, use of microprojection, or use of Agrobacterium-mediated transformation.

[0027] In some embodiments, the present disclosure relates to a biosynthetic method for producing non-natural mogrol precursors, mogrol and mogroside in a transgenic plant, comprising: (a) a step of combining a plant with a genome conversion event, thereby forming a transgenic plant, wherein the genome conversion event results in non-natural expression or concentration of mogrol-producing and / or mogroside-producing enzymes; (b) a step of cultivating and regenerating a population of transgenic plants; (c) a step of selecting a transgenic plant that produces mogroside; and (d) a step of collecting mogroside. In certain embodiments, the biosynthetic method further comprises: preparing / providing a plasmid comprising an expression cassette that expresses a non-natural mogrol-producing and / or mogroside-producing enzyme; transforming a host cell with the plasmid; and transfecting a plant with the plurality of transformed host cells.

[0028] Definition and Interpretation of Terms The following definitions or interpretations of technical terms are used throughout this disclosure. Technical terms used herein are generally given the meanings commonly applied to them in the relevant fields of plant biology, molecular biology, bioinformatics, and plant breeding. All of the following definitions apply throughout this application. As used herein and in the claims, “one (a)” or “one (an)” should be understood to include one or more, depending on the context in which they are used. For example, a reference to “cell” may mean that at least one cell may be available.

[0029] Terms related to characteristics or values, such as "basically," "about," and "approximately," also define the very characteristic or the very value, respectively. In relation to a given number or range, the term "about" specifically refers to a value or range that is within 20%, 10%, or 5% of the given value or range. As used herein, the term "including" also includes the term "consisting of."

[0030] The terms “peptide,” “oligopeptide,” “polypeptide,” “protein,” or “enzyme” are used interchangeably herein and refer to polymeric amino acids of any length linked by peptide bonds, unless otherwise specified herein.

[0031] The terms “gene sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleotide sequence,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably herein and refer to any length of unbranched, multimeric nucleotides that are either ribonucleotides or deoxyribonucleotides, or a combination of both.

[0032] Gene transfer / Transgene / Recombinant gene For the purposes of this disclosure, “transfer,” “transgene,” or “recombination” means, for example, with respect to nucleic acid sequences, expression cassettes, gene constructs, or vectors containing nucleic acid sequences, or organisms transformed by nucleic acid sequences, expression cassettes, or vectors according to this disclosure, all constructs brought about by recombinant methods, in which (a) a sequence of nucleic acid or a portion thereof, or (b) a gene regulatory sequence, such as a promoter, operably linked to the nucleic acid sequence according to this disclosure, or (c) a combination of (a) and (b) is not present in their natural genetic environment, or is modified by recombinant methods, such as artificially modified and / or inserted by genetic engineering methods.

[0033] As used herein, the term “gene transfer” refers to an organism, for example, a genetically modified plant, which preferably contains exogenously the nucleic acids, constructs, vectors, or expression cassettes or parts thereof described herein, introduced by a non-biological process, preferably by Agrobacterium-mediated transformation or particle bombardment, such as a plant, plant cell, callus, plant tissue, or plant part. Therefore, a genetically modified plant for the purposes of this disclosure is understood to mean, as stated above, that the nucleic acids described herein are not present in, nor arise from, the genome of the plant, or are present in the genome of the plant but not in its natural genetic environment within the genome of the plant, and that the nucleic acids can be expressed both homogeneously and heterogeneously. However, as stated above, gene transfer also means that the nucleic acids used in the nucleic acids or methods disclosed herein are in their natural position in the genome of the plant, but the sequence has been modified with respect to the natural sequence and / or the regulatory sequences of the natural sequence have been modified. Gene transfer is understood to preferably mean the expression of nucleic acid sequences that are naturally present in the plant in a non-natural genetic environment within the genome, i.e., homogeneous expression or heterogeneous expression of nucleic acid sequences that are not naturally present in the plant.

[0034] Plants / Genetically modified plants / Natural plants As used herein, the term "plant" encompasses the entire plant, its ancestors and offspring, and plant parts including seeds, buds, stems, leaves, roots (including tubers), flowers, and tissues and organs, each of which contains the target gene / nucleic acid. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic tissues, gametophytes, sporophytes, pollen, and microspores, each of which also contains the target gene / nucleic acid.

[0035] As used herein, a genetically modified plant refers to a plant in which one or more transgenes from another species have been introduced into the plant genome using genetic engineering techniques. The introduced transgenes encode and express non-natural proteins or enzymes, thereby enabling the genetically modified plant to have novel characteristics, such as producing non-natural enzyme pathway products that were not naturally present in the plant before the introduction of the transgenes. A genetically modified plant is in contrast to a natural plant, which is a natural product without artificial human intervention. As used herein, a natural plant refers to an untransformed / non-transformed plant used as a control to characterize a wild-type plant, a plant not genetically engineered by humans, or a genetically modified plant produced in accordance with this disclosure.

[0036] endogenous / natural "Endogenous" or "natural" nucleic acids and / or proteins refer to the nucleic acids and / or proteins of interest that exist in the plant in their natural form (i.e., without human intervention such as recombinant DNA manipulation), but also refer to the same gene (or substantially homologous nucleic acid / gene) in an isolated form that is subsequently (re)introduced into the plant (transgene). Genetically modified plants containing such transgenes may or may not experience a significant decrease in transgene expression and / or a significant decrease in the expression of endogenous genes.

[0037] exogenous The term “exogenous” (in contrast to “endogenous”) nucleic acid or gene refers to nucleic acids introduced into plants by recombinant DNA technology. “Exogenous” nucleic acids may either not exist naturally in the plant in their native form, be different from the nucleic acid of interest that exists naturally in the plant, or be identical to the nucleic acid that exists naturally in the plant but not integrated into its natural genetic environment. The corresponding meaning of “exogenous” applies in relation to protein expression. For example, a transgene containing a transgene, i.e., an exogenous nucleic acid, may, as a whole, experience a significant increase in the expression of each gene or protein compared to the expression of endogenous genes. The transgenes according to this disclosure contain one or more exogenous nucleic acids integrated at any locus, and optionally, the plant may also contain endogenous genes within its natural genetic background.

[0038] Expression Cassette As used herein, “expression cassette” is a vector DNA capable of being expressed in a host cell. The arrangement of DNA, a portion of DNA, or genetic factors forming an expression cassette may be artificial. Those skilled in the art will know the genetic factors that must be present in an expression cassette to produce expression without issue. An expression cassette comprises a target sequence to be expressed, operably ligated to one or more regulatory sequences (at least to a promoter) as described herein. Additional regulatory elements may include transcriptional and translational enhancers. Those skilled in the art will know terminator and enhancer sequences that may be suitable for use in carrying out the present invention. Intron sequences may also be added to the 5' untranslated region (UTR) or coding sequence to increase the amount of mature message accumulating in the cytosol as described in the definition section of increased / overexpression. Other regulatory sequences (besides promoters, enhancers, silencers, intron sequences, 3'UTR and / or 5'UTR regions) may be protein and / or RNA stabilizing elements. Such sequences are known to those skilled in the art or readily obtainable by those skilled in the art.

[0039] The expression cassette can be integrated into the genome of a host cell and amplified together with the genome of the host cell.

[0040] vector A vector or vector construct is DNA (not limited to plasmids, viral DNA, and chromosomal vectors, etc.) that is artificial in part or as a whole, or whose arrangement of contained genetic factors is artificial, and which is capable of replicating in a host cell and used to introduce a target DNA sequence into a host cell or host organism. A vector may be a construct or may consist of at least one construct. A vector, for example, a plasmid vector in a bacterial host cell, may replicate without integrating with the host cell's genome, or it may integrate part or all of its DNA with the host cell's genome, thereby resulting in the replication and expression of its DNA. The host cell of the present invention may be any cell selected from bacterial cells such as Escherichia coli or Agrobacterium species cells, yeast cells, fungal, algal or cyanobacterial cells, or plant cells. Those skilled in the art know the genetic factors that must be present on a gene construct in order to successfully transform, select, and grow host cells containing the target sequence. Typically, a vector contains at least one expression cassette. One or more target sequences are operably ligated (at least to promoters) to one or more control sequences described herein. Additional regulatory elements may include transcriptional and translational enhancers. Those skilled in the art will know of terminator and enhancer sequences that may be suitable for use when carrying out the techniques disclosed herein.

[0041] Operablely connected The terms “operatably linked” or “functionally linked” are used interchangeably and refer to a functional linkage between a promoter sequence and a target gene, as used herein, such that the promoter sequence can direct the transcription of the target gene.

[0042] Promoter / Plant promoter / Strong promoter / Weak promoter A “promoter” or “plant promoter” comprises a regulatory element that mediates the expression of a coding sequence segment in a plant cell. A “plant promoter” may be derived from a plant cell, for example, from a plant transformed with the nucleic acid sequence to be expressed in the system and as described herein. This also applies to other “plant” regulatory signals, such as plant terminators. Upstream promoters of nucleotide sequences useful in the methods of this disclosure may be modified by one or more nucleotide substitutions, insertions, and / or deletions without interfering with the function or activity of any 3'-regulatory region, such as a promoter, open reading frame (ORF), or terminator, or any other 3'-regulatory region located away from an ORF. The activity of promoters may be increased by the modification of their sequences, or they may be completely replaced by more active promoters, even promoters derived from other organisms. For expression in a plant, the nucleic acid molecule must be operably ligated to or include a suitable promoter that expresses the gene having the correct time and required spatial expression pattern, as described herein.

[0043] The promoters used herein broadly include constitutive promoters, ubiquitous promoters, embryologically controlled promoters, inductive promoters, organ-specific promoters, tissue-specific promoters, seed-specific promoters, green tissue-specific promoters, and meristematic-specific promoters. A "ubiquitous promoter" is active in substantially all tissues or cells of an organism.

[0044] To identify functionally homogeneous promoters, the promoter strength and / or expression patterns of candidate promoters can be analyzed, for example, by operably ligating the promoter to a reporter gene and assaying the expression levels and patterns of the reporter gene in various plant tissues. Generally, a “weak promoter” is intended to be a promoter that promotes the expression of coding sequences at a low level. “Low level” is intended to be at levels of approximately 1 / 10,000 transcripts, 1 / 100,000 transcripts, or 1 / 500,000 transcripts per cell. Conversely, a “strong promoter” promotes the expression of coding sequences at a high level or at approximately 1 / 10 transcripts, 1 / 100 transcripts, or 1 / 1,000 transcripts per cell. Generally, a “medium-strength promoter” is intended to be a promoter that promotes the expression of coding sequences at a lower level than a strong promoter.

[0045] Terminator The term "terminator" encompasses regulatory sequences, which are DNA sequences at the ends of transcription units that signal the 3' processing and polyadenylation of primary transcripts, as well as the termination of transcription. Terminators can be derived from native genes, from various other plant genes, or from T-DNA. The added terminators can be derived, for example, from nopalin synthase or octopine synthase genes, or from another plant gene instead, or, less preferably, from any other eukaryotic gene.

[0046] Reporter gene A “selection marker,” “selection marker gene,” or “reporter gene” includes any gene that confers a phenotype to the cells on which it is expressed, thereby facilitating the identification and / or selection of cells translocated or transformed by the nucleic acid construct of the present invention. These marker genes enable the identification of successful propagation of nucleic acid molecules by a set of different principles. Preferred markers may be selected from markers that confer antibiotic or herbicide resistance, markers that introduce novel metabolic characteristics, or markers that enable visual selection. Examples of selection marker genes include genes that confer resistance to antibiotics such as kanamycin (KAN) or hygromycin (Hyg). Expression of visual marker genes results in the formation of fluorescence (green fluorescent protein, GFP; red fluorescent protein, RFP; and their derivatives). This list represents only a few possible markers. Those skilled in the art will be familiar with such markers. Different markers are preferred depending on the organism and the selection method.

[0047] Expression / Gene Expression The term "expression" or "gene expression" means the transcription of a specific gene or a group of specific genes or a specific gene construct. More specifically, the term "expression" or "gene expression" means the translation of RNA and the synthesis of the protein / enzyme encoded thereby, i.e., protein / enzyme expression.

[0048] Identity / Homologousity Percentage As used herein, sequence identity, homology, or “identity percentage” means the degree to which two optimally aligned DNA or protein segments are invariant across the entire alignment window of their components, for example, nucleotide or amino acid sequences. The “identity percentage” of aligned segments of a test sequence and a reference sequence is the number of identical components shared by the sequences of the two aligned segments divided by the total number of sequence components in the reference segment across the smaller of the alignment window of the entire test sequence and the entire reference sequence. The “identity percentage” (“identity %”) is 100 times the identity percentage.

[0049] Introduction / Implementation / Transformation As used herein, the terms “introduction,” “implementation,” or “transformation” encompass the transfer of exogenous polynucleotides into host cells, regardless of the method used for propagation.

[0050] Plant tissues capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed by the gene constructs of the present invention and the entire plant regenerated therefrom. The specific tissues selected will vary depending on the specific species being transformed and the optimal clonal propagation system. Exemplary tissue targets include leaflets, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristems (e.g., apical meristem, axillary buds, and root meristems) and induced meristems (e.g., cotyledonous meristems and hypocotyl meristems). Polynucleotides can be introduced into host cells transiently or stably and maintained without integration, for example, as a plasmid. Alternatively, they can be integrated into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in ways known to those skilled in the art. Alternatively, plant cells that cannot be regenerated into plants can be selected as host cells, i.e., the resulting transformed plant cells lack the ability to regenerate into a plant (whole).

[0051] The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly standardized technique. Advantageously, any of several transformation methods can be used to introduce the target gene into a suitable progenitor cell. The methods described with respect to transformation and the regeneration of plants from plant tissue or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into plants, particle gambolization, transformation using viruses or pollen, and microprojection. Genetically modified plants, including genetically modified crops, are preferably produced by Agrobacterium-mediated transformation. An advantageous transformation method is transformation within the plant body. For this purpose, for example, it is possible to act on plant seeds with Agrobacterium or to inoculate plant meristems with Agrobacterium. It has been found particularly advantageous according to the present invention to act on a suspension of transformed Agrobacterium onto a whole plant or at least a floral primordium. Subsequently, the plants are grown until seeds of the treated plants are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). The nucleic acid or construct to be expressed is preferably cloned into a vector suitable for transforming Agrobacterium tumefaciens, such as pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). The Agrobacteria transformed with such a vector can then be used in known methods of plant transformation, such as by immersing wounded or chopped leaves in an Agrobacteria solution and then culturing them in a suitable medium, for example, in plants used as a model, such as Arabidopsis thaliana (Arabidopsis thaliana is not considered a crop and is within the scope of this invention), or in crops such as tobacco plants.Plant transformation by Agrobacterium tumefaciens is known, for example, from Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877, or, more specifically, from FF White, Vectors for Gene Transfer in Higher Plants in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. SD Kung and R. Wu, Academic Press, 1993, pp. 15-38.

[0052] Ploidy / Ploidy level / Chromosome ploidy / Polyploidy Ploidy, or chromosomal ploidy, refers to the number of complete sets of chromosomes present in the nucleus of a cell. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present ("ploidy levels"): haploid (1 set), diploid (2 sets), triploid (3 sets), tetraploid (4 sets), pentaploid (5 sets), hexaploid (6 sets), heptaploid (7 sets), and so on. The genetic term polyploidy is used herein to describe cells that have three or more sets of chromosomes.

[0053] adjustment The term "regulation" refers to the process by which the expression level changes in relation to expression or gene expression compared to a control plant, and the expression level may increase or decrease. Original, unregulated expression may be any type of expression of structural RNA (rRNA, tRNA) or mRNA having subsequent translation. For the purposes of this application, original, unregulated expression may also be no expression at all. The term "regulating activity" or "regulating expression" means any change in the expression of a target nucleic acid sequence and / or encoded protein that results in increased or decreased yield-related characteristics and / or increased or decreased plant growth, including but not limited to increased or decreased seed yield. Expression may increase from zero (no expression or unmeasurable expression) to a specific amount, or decrease from a specific amount to an unmeasurable amount or zero.

[0054] Generally, after transformation, plant cells or cell populations are selected for the presence of one or more markers encoded by genes expressible in plants, which were transmitted simultaneously with the target gene, and then the transformed material is regenerated throughout the plant. To select transformed plants, the plant material obtained in transformation is typically subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, seeds obtained by the method described above may be planted and, after the initial growth period, subjected to suitable selection by spraying. A further possibility is to grow the seeds on an agar plate using a suitable selector agent, after sterilization where appropriate, so that only transformed seeds can grow into plants. Alternatively, transformed plants are screened for the presence of selection markers, such as those described herein.

[0055] After DNA propagation and regeneration, plants presumed to have been transformed can also be evaluated for the presence, copy number, and / or genomic mechanism of the target gene.

[0056] The resulting transformed plants can be propagated by various means, such as clonal propagation or classical propagation techniques. For example, first-generation (or T1) transformed plants may be self-pollinated, homozygous second-generation (or T2) transformants may be selected, and then the T2 plants may be further propagated by classical propagation techniques. The resulting transformed organisms can take various forms. For example, they may be chimeras of transformed and untransformed cells; clonal transformants (e.g., all cells transformed to contain expression cassettes); or grafts of transformed and untransformed tissues (e.g., transformed rootstock grafted onto an untransformed scion in a plant).

[0057] Throughout this application, a plant, plant part, seed, or plant cell transformed with a construct, or interchangeably transformed by a construct, or transformed with or by a nucleic acid, shall be understood to mean a plant, plant part, seed, or plant cell having the construct or the nucleic acid as a transgene as a result of the introduction of the construct or the nucleic acid by biotechnological means. Accordingly, a plant, plant part, seed, or plant cell includes the expression cassette, the recombinant construct, or the recombinant nucleic acid.

[0058] Mogroside biosynthesis pathway Figure 1 shows the mogroside biosynthesis pathway from Siraitia grosvenorii (Itkin et al., Proc Nat Acad Sci USA, 2016; 113:E7619-E7628; Seki et al., Bioscience, Biotechnology, and Biochemistry, 2018 VOL. 82, NO. 6, 927-934). The enzymes squalene epoxidase (SQE), cucurbitadienol synthase (CDS), and epoxyhydrolase (EPH) are involved in the sequential steps of producing mogrool precursors and converting them to mogrool. Mogrol precursors as intermediate products in the enzymatic pathway include, but are not limited to, 2,3-oxidesqualene, 2,3;22,23-dioxidesqualene, 24,25-epoxycucurbitadienol, and 24,25-dihydroxycucurbitadienol. One characteristic of the mogroside biosynthesis pathway is that the oxidesqualene cyclase CDS uses 2,3;22,23-diepoxysqualene as its substrate to produce 24,25-epoxycucurbitadienol. Genomic analysis revealed that S. grosvenorii possesses five genes capable of encoding squalene epoxidase (SQE). Two of these, along with CDS, cytochrome P450 (CYP87D18), and epoxy hydrolase (EPH), are predicted to be strongly expressed during the early stages of fruit development, catalyzing subsequent steps and participating in the production of 2,3;22,23-diepoxysqualene. The S. grosvenorii genome contains eight genes encoding epoxide hydrolases that catalyze the conversion of 24,25-epoxycucurbitadienol to 24,25-dihydroxycucurbitadienol. Importantly, the enzymatic pathway of mogrosides according to this disclosure is not limited to the mechanism shown in Figure 1a. Other terpene structures, mogrool precursors, and enzyme-catalyzed reactions or conversion mechanisms are also possible. Certain enzymes may catalyze two or more reactions.For example, cytochrome P450 enzymes catalyze the conversion of cucurbitadienol to 11-hydroxy-cucurbitadienol or 11-oxo-cucurbitadienol, and the conversion of 24,25-epoxycucurbitadienol to 11-hydroxy-24,25-epoxycucurbitadienol. In some relevant embodiments, non-monk fruit cucurbitaceous plants can produce mogrol-like tetracyclic triterpenoid compounds because at least one intermediate, such as a triterpene, is present in the cellular pathway. Furthermore, given that the relevant pathways for modifying tetracyclic triterpenoids require relevant enzymes such as reductases, these relevant plants already express these relevant network enzymes. In other relevant embodiments, further genes may be introduced into non-cucurbitaceous or natural enzymes with functionality that can be upregulated to enable the production of intermediate metabolites or related enzymes to yield mogrol, mogrosides, and mogroside-type sweeteners.

[0059] Following the formation of mogrol, a series of glycosylations occur, adding glucose molecules at the C-3 and C-24 positions to yield mogrosides I-VI with varying degrees of glycosylation. The Roman numerals I, II, III, IV, V, and V represent the number of glucose units in the corresponding glycosylated mogroside, isomogroside, or oxomogroside, respectively. Two uridine phosphorylase-dependent glycosyltransferases (UGTs) have been shown to contribute to these steps. One of them, UGT720-269-1, is strongly expressed in the early stages of fruit development and transfers one glucose molecule to the hydroxyl groups at the C-24 and C-3 positions of mogrol, respectively, resulting in mogroside I-A1 (C-24 glucosylation) as an intermediate, and then mogroside IIE (C-3 and C-24 glucosylation). The second UGT is UGT94-289-3, which is strongly expressed after fruit development and adds sugars to other sugars already present on the acceptor molecule. UGT94-289-3 has been shown to add one glucose molecule to the C-2' and C-6' positions, respectively, of the C-24 glucose of mogroside IIE, which has been previously added by UGT720-269-1. UGT94-289-3 also adds a glucose molecule to the C-6' position of the glucose bonded to the C-3 position of mogroside IIE, thereby catalyzing three or more glycosyltransfer reactions in sequence, resulting in a mogroside with five or more glucose units.

[0060] Other natural plants may also express enzymes that produce mogrol precursors through their natural genomes, but it is important to note that these plants do not naturally produce all the enzymes required for mogloside production in a systematic manner. For example, as shown in Figure 1b, plants such as cucumbers, melons, and watermelons naturally express cucurbitadienol synthase, which is capable of producing cucurbitadienol, a common precursor to mogrol from silaitia or cucurbitacin from melon. However, other enzymes, such as cytochrome P450 enzymes that can alter the cucurbitadienol scaffold (Banerjee et al., Phytochem. Rev. 2018, 17:81-111), redirect this intermediate to other terpene derivatives. These non-Siraitia plants possess other cytochrome P450, hydrolase, epoxidase, and glycosylase genes, and their enzyme products may be heterogeneous in activity and may not be expressed in an organized manner for the mogroside pathway; therefore, such plants may possess enzymes for the mogroside pathway. Consequently, alterations to the genome of these non-Siraitia plants by recombination, gene editing, or other modern plant breeding techniques may enable these non-Siraitia plants to begin producing mogrol and mogrosides.

[0061] As used herein, the term “mogroside pathway enzyme” encompasses any enzyme capable of catalyzing or promoting the biosynthetic reactions that produce mogol precursors, mogol, mogrosides, and their metabolites and / or derivatives. Such mogroside pathway enzymes include, but are not limited to, the enzyme families of CDS, SQE, EPH, cytochrome P450, and UGT.

[0062] Mogrol precursors include, but are not limited to, 2,3-oxidesqualene, 2,3;22,23-dioxidesqualene, 24,25-epoxycucurbitadienol and 24,25-dihydroxycucurbitadienol, cucurbitadienol, 11-hydroxycucurbitadienol, and 11-oxocucurbitadienol, and broadly encompass all possible terpene derivatives and intermediates toward the production of the mogrol product via the enzymatic pathway shown in Figures 1a and 1b. The mogrosides as disclosed herein include, but are not limited to, siamenoside I, silatose (a stereoisomer of siamenoside I), mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside IIE, mogroside IIA, mogroside IE, and mogroside IA, and refer to all possible glycosylation products of mogrool. Some of these structures are shown in Figure 2. Other examples of mogrosides include, but are not limited to, mogroside IIB, 7-oxomogroside IIE, 11-oxomogroside A1, mogroside III A2, 11-deoxymogroside III, 11-oxomogroside IVA, 7-oxomogroside V, and 11-oxo-mogroside V. The mogroside metabolites and derivatives described herein refer to any close variants of mogrosides resulting from metabolic reactions, naturally occurring reactions, or non-naturally occurring reactions. Mogroside derivatives may involve the deletion, modification, or addition of atoms or functional groups compared to standard mogrosides. However, mogroside metabolites and derivatives retain substantially the same functions and properties as standard mogrosides. [Brief explanation of the drawing]

[0063] Brief explanation of the drawing [Figure 1A] The reported enzymatic pathways for the production of mogrol and mogroside in Siraitia grosvenorii are shown. (Seki et al Bioscience, Biotechnology, and Biochemistry, 2018 VOL. 82, NO. 6, 927-934). [Figure 1B]This shows the enzymatic pathway for mogol precursor production in several natural plant species. (Banerjee et al., Phytochem. Rev. 2018, 17:81-111). [Figure 2] The structures of mogrol and selected moglosides derived therefrom are shown. [Figure 3] This paper describes the design of various expression cassettes containing nucleotide sequences encoding mogroside pathway enzymes. [Figure 4] The results of ultra-high-performance liquid chromatography-time-of-flight mass spectrometry (UPLC-TOFMS) of mogroside standards are shown. [Figure 5] The following shows the results of UPLC-TOFMS (retention time) analysis of mogroside II detection in the leaves of the genetically modified plant Nicotiana bentamiana, transformed with expression cassettes pBing008 and pBing024, compared to the control plant p019. [Figure 6] This shows the results of UPLC-TOFMS (retention time) analysis of mogroside II detection in the leaves of transgenic Nicotiana bentamiana co-transformed with expression cassettes pBing003 and pBing007, and in the leaves of transgenic Nicotiana bentamiana co-transformed with expression cassettes pBing006 and pBing015, compared to the control plant p019. [Figure 7] This shows the UPLC-TOFMS (retention time) results for detecting mogroside II in the leaves of transgenic Nicotiana bentamiana transformed with the expression cassette pBing008. [Figure 8-1] This shows the UPLC-TOFMS (MS spectrum) results for detecting mogroside II in the leaves of the transgenic plant Nicotiana bentamiana transformed with the expression cassette pBing008. [Figure 8-2]This shows the UPLC-TOFMS (MS spectrum) results for detecting mogroside II in the leaves of the transgenic plant Nicotiana bentamiana transformed with the expression cassette pBing008. [Figure 9] This shows photographic images of the dissection of a watermelon and its various fruit parts. [Figure 10] The expression of the chromogenic gene PSY1 in various fruit tissues according to Example 4 is shown. [Figure 11] Table 5 shows the expression of eight identified tissue-specific genes in various tissues of sugar baby watermelon according to Example 4. [Figure 12] Table 5 shows the expression of eight identified tissue-specific genes in various tissues of Charles Thorngrace squid according to Example 4. [Figure 13] This shows the results of protein detection analysis in various genetically modified watermelon samples (transformed with pBing008). [Figure 14] This shows the chemiluminescence results of protein detection in genetically modified watermelons produced by transformation using the expression cassette pBing008. [Figure 15] This shows the results of protein detection analysis in various tissues of the genetically modified watermelon sample 008SBE4-1, which was produced by transformation using the expression cassette pBing008. [Figure 16] This shows the results of protein detection analysis in various tissues of the genetically modified watermelon sample 008SBE5-4, which was produced by transformation using the expression cassette pBing008. [Figure 17] This shows the results of protein detection in various tissues of the genetically modified watermelon sample 008CHE4-13, which was produced by transformation using the expression cassette pBing008. [Figure 18] This shows the results of protein detection in various tissues of the genetically modified watermelon sample 008CHE4-16, which was produced by transformation using the expression cassette pBing008. [Figure 19] The results of UPLC-TOFMS comparing genetically modified watermelons containing the expression cassette pBing008 with a control are shown. [Figure 20] The UPLC-TOFMS (MS spectrum) results for gene-modified watermelons containing the expression cassette pBing008 are shown. [Figure 21A] The results of UPLC-TOFMS of sample extracts from the fruit of genetically modified watermelon 008CH4-19 are shown. [Figure 21B] The UPLC-TOFMS results for a control sample compared to a fruit extract sample of genetically modified watermelon 008CH4-19 are shown. The control sample is an extract of wild-type, unmodified fruit prepared and spiked with 100 ng / ml mogroside IIE. [Figure 22-1] Both images show the ULC-TOFMS results of seed coats from genetically modified watermelon samples 008SBE5-2 and 008CHE4-5, which contain the expression cassette pBing008. [Figure 22-2] Both images show the ULC-TOFMS results of seed coats from genetically modified watermelon samples 008SBE5-2 and 008CHE4-5, which contain the expression cassette pBing008. [Figure 23] This shows a comparison of CDS gene expression levels in 31 gene-modified watermelon leaves and fruits. CDS expression levels were analyzed by RT-PCR and standardized to 10% of actin expression (set to 1). Orange bars represent expression levels in fruits, and blue bars represent expression levels in leaves. [Figure 24] This shows a comparison of CYP87 gene expression levels in 31 gene-modified watermelon leaves and fruits according to Example 6. CYP87 expression levels were analyzed by RT-PCR and standardized to 10% of actin expression (set to 1). Orange bars represent expression levels in fruits, and blue bars represent expression levels in leaves. [Figure 25] This shows a comparison of SQE gene expression levels in 31 gene-modified watermelon leaves and fruits according to Example 6. SQE expression levels were analyzed by RT-PCR and normalized to 10% of actin expression (set to 1). Orange bars represent expression levels in fruits, and blue bars represent expression levels in leaves. [Figure 26]This shows a comparison of EPH gene expression levels in 31 gene-modified watermelon leaves and fruits according to Example 6. EPH expression levels were analyzed by RT-PCR and standardized to 10% (set to 1) of actin expression. Orange bars represent expression levels in fruits, and blue bars represent expression levels in leaves. [Figure 27] This shows a comparison of EPH gene expression levels in 31 gene-modified watermelon leaves and fruits according to Example 6. EPH expression levels were analyzed by RT-PCR and standardized to 10% (set to 1) of actin expression. Orange bars represent expression levels in fruits, and blue bars represent expression levels in leaves. [Figure 28] The results of UPLC-MS analysis of standard mogroside IIE are shown. On the left is a superposition of three chromatograms of mogroside IIE at three different concentrations: 1000 pg / ml, 500 pg / ml, and 250 pg / ml. On the right are the characteristic ion peaks of the fragmented mogroside IIE standard. [Figure 29] The results of the detection of mogroside IIE in the metabolite extract of the T0 watermelon fruit sample 008CHE4-19 according to Example 6 are shown. The LC-MS extracted ion chromatogram (m / z 423.36) is shown on the left. The ionic intensity of the mass spectrometry fragment is shown on the right. [Figure 30] The results of CDS gene expression in T1 gene-modified watermelon samples according to Example 6 are shown. DNA from 32 gene-modified plant samples was amplified using multiplex-PCR. The identity and genotyping conclusions for all samples are listed in the tables on the left and right. [Figure 31] The results of the detection of mogroside IIE in the metabolite extract of the T1 watermelon fruit sample 008DLE11-4-S4 according to Example 6 are shown. The LC-MS extracted ion chromatogram (m / z 423.36) is shown on the left. The ionic intensity of the mass spectrometry fragment is shown on the right. [Figure 32]The analytical results for the detection of mogroside IIE in the metabolite extract of the T1 watermelon fruit sample 008DLE11-2-S1 according to Example 6 are shown. The LC-MS extracted ion chromatogram (m / z 423.36) is shown on the left. The ionic intensity of the mass spectrometry fragment is shown on the right. [Figure 33] The results of the detection of mogroside IIE in the metabolite extract of the T1 watermelon fruit sample 008DLE11-9-S3 according to Example 6 are shown. The LC-MS extracted ion chromatogram (m / z 423.36) is shown on the left. The ionic intensity of the mass spectrometry fragment is shown on the right. [Modes for carrying out the invention]

[0064] Detailed explanation This specification generally describes genetically modified plants and their biosynthetic systems for producing mogrol / mogroside pathway enzymes and mogrosides, as well as methods for producing such genetically modified plants. The following sections provide embodiments that illustrate the subject matter in detail.

[0065] Construction of expression cassettes and vectors In some embodiments, the disclosure describes a transgenic plant comprising a genomic transformation event, wherein the genomic transformation event results in the non-natural expression or concentration of mogroside pathway enzymes, and the transgenic plant biosynthetically produces non-natural mogol precursors, mogol, mogrosides and / or their metabolites or derivatives.

[0066] In other related embodiments, the disclosure describes a gene-edited plant comprising a genome transformation event, wherein the genome transformation event results in the non-natural expression or concentration of mogroside pathway enzymes, and the gene-edited plant biosynthetically produces non-natural mogol precursors, mogol, mogrosides and / or their metabolites or derivatives. In at least these embodiments, various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and meganucleases (MNs) can be used to obtain a desired plant having non-natural mogol precursors, mogol, mogrosides and / or their metabolites or derivatives.

[0067] As further described herein, gene-edited plants may include SEQ ID NOs: 1-31. In some exemplary embodiments, gene-edited plants include an expression cassette or transformation event comprising one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1-31.

[0068] In some embodiments, the disclosure describes genetically modified plants containing non-natural moglol precursors and / or moglol, which biosynthetically produce moglol, mogrosides and / or their metabolites or derivatives.

[0069] In some embodiments, the transgenic plants according to the Disclosure have a genome transformation event comprising an expression cassette containing one or more nucleotide sequences shown in SEQ ID NOs: 1 to 31. In some embodiments, the expression cassette of the transgenic plant contains one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1 to 31.

[0070] The expression cassettes described herein were designed and constructed using recombinant gene techniques suitable for plant transformation.

[0071] In some embodiments of the genetically modified plant, the nucleotide sequences shown in SEQ ID NO: 1; SEQ ID NO: 2; SEQ ID NO: 3; SEQ ID NO: 4; or SEQ ID NO: 5 in the expression cassette can encode at least one enzyme selected from the group consisting of CDS, cytochrome P450, EPH, SQE, UGT, and combinations thereof.

[0072] In some embodiments, the expression cassette further comprises one or more components selected from the group consisting of a promoter, a target nucleotide sequence, an epitope tag, a terminator, a spacer, and combinations thereof.

[0073] In certain embodiments, the expression cassette further comprises one or more promoters. In some embodiments, one or more promoters are strong promoters. In other embodiments, one or more promoters are weak promoters. In yet another embodiment, one or more promoters have one or more nucleotide sequences shown in SEQ ID NOs: 6-17. In yet another embodiment, one or more promoters have one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 6-17.

[0074] In certain embodiments, the expression cassette further comprises one or more epitope tags having one or more nucleotide sequences shown in SEQ ID NOs: 18-22. In other embodiments, one or more epitope tags have one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 18-22.

[0075] In certain embodiments, the expression cassette further comprises one or more terminators having one or more nucleotide sequences shown in SEQ ID NOs: 23-27. In other embodiments, one or more terminators have one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 23-27.

[0076] Figure 3 shows designs of some non-limiting examples of expression cassettes according to this disclosure. Each of the target genes, for example, a nucleotide sequence encoding CDS, is operably ligated to a promoter sequence and a nucleotide sequence encoding an epitope tag, and the epitope is operably ligated to a terminator sequence, thereby forming an expressible gene as promoter-CDS-epitope tag-terminator. Such “expressible genes” can be further modified by being operably ligated by spacer sequences to alter the enzyme products expressed or produced by the “expression cassette.” In some embodiments, an expression cassette of a transgenic plant comprises one or more expressible genes and one or more spacers, each expressible gene comprising a target gene sequence selected from the group consisting of a nucleotide sequence encoding CDS, a nucleotide sequence encoding cytochrome P450 (CYP87D18), a nucleotide sequence encoding EPH, a nucleotide sequence encoding SQE, a nucleotide sequence encoding UGT720, and combinations thereof.

[0077] In some embodiments, the expression cassette of the present disclosure further comprises one or more reporter gene sequences encoding and expressing one or more reporter proteins. Reporter proteins include, but are not limited to, kanamachine resistance protein (KAN), hygromycin resistance protein (Hyg), green fluorescent protein (GFP), and green fluorescent protein (RFP). In some embodiments, one or more reporter genes have one or more nucleotide sequences shown in SEQ ID NOs. 28-31. In other embodiments, one or more reporter genes have one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs. 28-31. In some embodiments, nucleotide SEQ ID NO 28 can encode KAN, nucleotide SEQ ID NO 29 can encode Hyg, nucleotide SEQ ID NO 30 can encode GFP, and nucleotide SEQ ID NO 31 can encode RFP. In certain embodiments, the expression cassette includes at least one reporter gene selected from the group consisting of nucleotide sequences shown in SEQ ID NOs. 28-31. In other embodiments, the expression cassette of the transgenic plant includes at least two reporter genes selected from the group consisting of nucleotide sequences shown in SEQ ID NOs. 28-31.

[0078] Table 1 shows various non-limiting examples representing the expression cassette of the present invention. Table 2 shows the components of the expression cassette pBing008, which includes five promoter sequences, five protein tags, five terminator sequences, and five target transgenes.

[0079] [Table 1]

[0080] As an example embodiment, the expression cassette pBing008 includes a promoter sequence, a nucleotide sequence encoding a mogroside pathway enzyme, a nucleotide sequence encoding an epitope tag, a reporter gene encoding GFP and Hyg, and a terminator sequence.

[0081] In pBing008, nucleotide sequence number 1 codes for CDS; nucleotide sequence number 2 codes for cytochrome P450 (CYP87D18); nucleotide sequence number 3 codes for EPH; nucleotide sequence number 4 codes for SQE; nucleotide sequence number 5 codes for UGT720; nucleotide sequence number 6 represents promoter TCTP; nucleotide sequence number 7 represents promoter Fsgt-PFlt; nucleotide sequence number 8 represents promoter CsVMV; nucleotide sequence number 9 represents promoter HLV H12; nucleotide sequence number 10 represents promoter PCLSV; nucleotide sequence number 11 represents promoter MMV; nucleotide sequence number 12 represents promoter CaMV The nucleotide in SEQ ID NO: 18 represents e35S; the nucleotide in SEQ ID NO: 19 represents the protein tag HSV; the nucleotide in SEQ ID NO: 20 represents the protein tag FLAG; the nucleotide in SEQ ID NO: 21 represents the protein tag HA; the nucleotide in SEQ ID NO: 22 represents the protein tag V5; the nucleotide in SEQ ID NO: 23 represents the terminator CaMV 35S; the nucleotide in SEQ ID NO: 24 represents the terminator UBQ3; the nucleotide in SEQ ID NO: 25 represents the terminator HSP18.2; the nucleotide in SEQ ID NO: 26 represents the terminator Pea3A; the nucleotide in SEQ ID NO: 27 represents the terminator E9; the nucleotide in SEQ ID NO: 28 codes for reporter protein Hyg; and the nucleotide in SEQ ID NO: 29 codes for reporter protein GFP.

[0082] In particular, the expression cassette pBing008 contains the following seven expressible genes: Nucleotide sequence encoding promoter TCTP-CDS - Nucleotide sequence encoding epitope tag MYC - Terminator CaMV 35S; Promoter Fsgt-PFlt-nucleotide sequence encoding cytochrome P450 (CYP87D18) - epitope tag nucleotide sequence encoding HSV - terminator UBQ3; Nucleotide sequence encoding promoter CsVMV-EPH3 - Nucleotide sequence encoding epitope tag FLAG - Terminator HSP18.2; Nucleotide sequence encoding promoter HLV H12-SQE - Nucleotide sequence encoding epitope tag HA - Terminator Pea3A; Nucleotide sequence encoding promoter PCLSV-UGT720 - Nucleotide sequence encoding epitope tag V5 - Terminator E9; Nucleotide sequence encoding promoter MMV-GFP; The nucleotide sequence encoding the promoter CaMV e35S-Hyg, Here, the seven expressible genes mentioned above are operably linked by spacers to form an integrated expression cassette pBing008.

[0083] [Table 2]

[0084] In some embodiments, the expression cassette is delivered by a plasmid to enable enzyme production by the host cell. In other embodiments, the expression cassette is delivered by a vector that allows integration into a chromosome, thereby enabling enzyme expression from the chromosome.

[0085] Plant lineage construction and transformation In some embodiments, the method for producing genetically modified plants according to the Disclosure relates to constructing a plant line and transforming selected natural plants with an expression cassette produced according to the Disclosure.

[0086] It is generally known that the natural expression of mogol / mogroside pathway enzymes and the natural production of mogol / mogroside are available only in Siraitia grosvenorii. In some embodiments of the present application, the natural plants selected to be transformed with nucleotide sequences encoding mogol / mogroside pathway enzymes are not Siraitia grosvenorii. Specifically, the natural plants prior to transformation do not naturally produce all of the mogol / mogroside pathway enzymes, nor do they produce morgol and mogroside through their natural genomes. In some embodiments, even natural plants may, through their natural genomes, produce one or more enzymes capable of producing mogol precursors or mogol, but these plants do not naturally produce non-natural mogroside. In certain embodiments, the natural plants selected for transformation include wild-type, untransformed, or non-transformed watermelons whose natural genomes do not naturally produce detectable mogrol or mogrosides.

[0087] Transformations of fast-growing, economical fruits, vegetables, or plants that enable rapid mogroside production are of greater interest in terms of efficiency and cost. Non-limited examples of fast-growing plants include bush cherries, peaches and nectarines, apricots, radishes, plums and their related species, sour (pie) cherries, apples, pears, sweet cherries, citrus fruits, cucumbers, zucchini, peas, and turnips.

[0088] The genetically modified plants described herein are produced by combining a plant with a genome transformation event, thereby forming a genetically modified plant, where the genome transformation event results in the non-native expression or concentration of mogrol / mogroside pathway enzymes. In some embodiments, combining a plant with a genome transformation event is carried out using one or more of the following methods: the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into the plant, particle gambooming, transformation using viruses or pollen, microprojection, or Agrobacterium-mediated transformation. Preferably, the genetically modified plants are produced by an Agrobacterium-mediated transformation method. In some embodiments, Agrobacterium tumefaciens is transformed with an expression cassette to produce genetically modified Agrobacterium, which is then used to transfect a target plant, and plants that are successfully transformed are selected based on the expression of a reporter gene in the expression cassette.

[0089] In some embodiments, the genetically modified plant is Nicotiana bentamiana produced by transient transformation. First, Agrobacterium tumefaciens stain EHA105 was transformed with the expression cassette of this invention using the free-thaw method reported by Weigel et al. (Transformation of agrobacterium using the freeze-thaw method, CSH Protoc. 2006 Dec 1; 2006(7)). Briefly, chemically competent Agrobacterium was prepared. After the addition of the expression cassette, the mixture was alternately frozen in liquid nitrogen and thawed in liquid. The cells were then recovered in lysogenic broth (LB) medium and seeded on LB plates with selected antibiotics.

[0090] Secondly, Nicotiana bentamiana plants were infected. In short, transformed EHA105 Agrobacterium were grown to produce a suitable population / culture. Selected Nicotiana bentamiana plants with appropriate maturity were chosen for transformation. An appropriate amount of transformed Agrobacterium culture was loaded into the tissues of Nicotiana bentamiana plants until completion was indicated. The plants loaded with the transformed Agrobacterium culture were grown for an appropriate period before sampling and selection.

[0091] Thirdly, Nicotiana bentamiana plants that were successfully transformed were selected based on the expression of the reporter gene in the expression cassette.

[0092] In certain embodiments of the transgenic Nicotiana bentamiana plants, the expression cassette used to transform the Agrobacterium Tumefaciens stain EHA105 and yield the transgenic Nicotiana bentamiana plants was pBing008. In other embodiments, the expression cassettes used were selected from those shown in Table 1. In some embodiments, the reporter gene of the expression cassette was GFP, and the selection of the transformed Nicotiana bentamiana plants was based on their leaves having GFP expression.

[0093] In another embodiment, the genetically modified plant was genetically modified watermelon (Citrullus lanatus), which was produced by the following method. Briefly, firstly, Agrobacterium Tumefaciens stain EHA105 was transformed with the expression cassette of the present invention using the same free thawing method. Secondly, watermelon seedlings of appropriate maturity were used to prepare explants for transformation. The cotyledons were excised from the hypocotyl, harvested, and appropriately processed for transformation. The transformed Agrobacterium culture was then added to these explants. After infection, the explants were dried on sterile paper towels and transferred to plates containing Murasigeskoog (MS) medium. The plates were sealed and co-cultured for an appropriate period. After co-culture, the explants were transferred to a growth chamber and grown under a selection of threshold-enriched antibiotics.

[0094] In a specific embodiment of genetically modified watermelon, Agrobacterium Tumefaciens stain EHA105 was transformed, and the expression cassette used to produce the genetically modified watermelon was pBing008. In other embodiments, the expression cassette used was selected from those shown in Table 1. In some embodiments, the reporter gene of the expression cassette was GFP, and the selection of the transformed watermelon plant was based on its leaves having GFP expression.

[0095] In other embodiments, plants were co-transformed by infection with two or more expression cassettes, the expression cassettes used being selected from those shown in Table 1.

[0096] Protein expression in genetically modified plants and their tissues In some embodiments, the method for producing the genetically modified plants of this disclosure relates to monitoring and analyzing the expression of mogrol / mogroside pathway proteins / enzymes by expression cassettes introduced into the genetically modified plants.

[0097] In some embodiments, tissues or portions of genetically modified plants produced according to the present invention were sampled and processed to obtain samples ready for analysis. The samples were further analyzed to detect the presence and / or content of proteins expressed by the target gene in the expression cassette.

[0098] In some embodiments, leaves of genetically modified Nicotiana bentamiana plants produced according to this application were pulverized in protein extraction buffer and then centrifuged. The resulting supernatant was further diluted and then used for antibody detection. The presence of each target protein was confirmed by detection of a chemiluminescent signal produced by the binding of the corresponding antibody and by the size of the protein indicated by a protein size ladder used as a control in each measurement. In some embodiments, protein detection was carried out using a Jess instrument (Bio-Techne), which automates the immunodetection of conventional Western blotting methods for protein separation and detection. In certain embodiments, a signal-to-noise ratio (S / N ratio) greater than 3 was used as a cutoff for positive signals for analytical and selection purposes.

[0099] In some embodiments, the genetically modified plants showed the presence of all five mogrol / mogroside pathway enzymes / proteins listed below: CDS, SQE, cytochrome P450 (CYP87D18), UGT720, and EPH, as determined by protein detection results. In a specific embodiment, the genetically modified plant was genetically modified Nicotiana bentamiana. In another embodiment, the genetically modified plant was genetically modified watermelon.

[0100] Mogrol / mogroside pathway enzymes were detected in various tissues of the genetically modified plants, including but not limited to organs, tissues, leaves, stems, roots, flowers or floral parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic sites, callus tissue, seeds, cuts, cell or tissue cultures, placentas, chulmonaries, mesocarps, cartilage, epidermis, or any other parts or products of the genetically modified plants. In certain embodiments, mogrol / mogroside pathway enzymes CDS, SQE, cytochrome P450 (CYP87D18), UGT720, and EPH were detected in the placentas, chulmonaries, mesocarps, cartilage, and epidermis of the genetically modified plants. In some embodiments, the expression of mogrol / mogroside pathway enzymes was tissue-specific. In certain embodiments, the expression levels of CDS and UGT720 were lower than those of CYP87, SQE, and EPH. In other embodiments, the expression level of EPH is remarkably high compared to other mogrol / mogroside pathway enzymes, particularly in fruit tissue.

[0101] Metabolic regulation and enzyme production of mogrosides In some embodiments, the methods for producing genetically modified plants according to the Disclosure relate to analyzing the production of various non-natural mogrosides. In certain embodiments, the production of mogrosides by genetically modified plants is analyzed and, for selection purposes, compared to a corresponding control plant.

[0102] In some embodiments, tissues or portions of the genetically modified plant were extracted and / or purified to obtain samples ready for analysis. In some embodiments, UPLC coupled with TOFMS was used to analyze metabolites in the tissues of the genetically modified plant. The presence of mogrosides was determined by comparing the analytical results with those of standard mogrosides in terms of retention time and peak patterns of the MS spectrum.

[0103] In some embodiments, genetically modified plants analyzed by ULC-TOFMS showed signals from at least one mogroside, while control plants did not show the presence of mogrosides in the analysis results.

[0104] In a particular embodiment, genetically modified plants analyzed by UPLC-TOFMS showed at least one mogroside selected from the group consisting of siamenoside I, silatose, mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside II, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, mogroside I, mogroside IA, mogroside IE, or any combination thereof, while control plants did not show the presence of mogrosides in the analysis results.

[0105] In another embodiment, genetically modified plants analyzed by UPLC-TOFMS showed at least one mogroside selected from the group consisting of mogroside IA, mogroside IE, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, or any combination thereof, while control plants did not show the presence of mogrosides in the analysis results.

[0106] Mogrosides were detected in various tissues of the genetically modified plant, including but not limited to organs, tissues, leaves, stems, roots, flowers or floral parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic tissues, callus tissue, seeds, cuts, cells or tissue cultures, or any other parts or products of the genetically modified plant. In a specific embodiment of the genetically modified watermelon, mogrosides were detected in the seed coat of the fruit.

[0107] In some embodiments, the genetically modified plants of this disclosure are cultivable and reproductive. The offspring or ancestors of the genetically modified plants are sources of non-natural enzymes that enable the offspring and ancestors to produce mogrol, mogroside and / or their metabolites or derivatives. Reproduction of the seeds of the genetically modified plants yields viable offspring that produce mogrol, mogroside and / or their metabolites or derivatives.

[0108] In some embodiments, the genetically modified plant that produces non-natural mogol / mogroside is a diploid plant having a diploid set of chromosomes. In certain embodiments, the diploid genetically modified plant produces seeds, the seeds containing non-natural mogroside, and propagation of the seeds of the diploid genetically modified plant produces viable offspring, which produce morgol, mogroside and / or its metabolites or derivatives. In some embodiments, the genetically modified plant is a member of the Cucurbitaceae family / cucurbits. In some embodiments, the genetically modified plant is a genetically modified watermelon (Citrullus lanatus). In certain embodiments, the genetically modified watermelon is diploid.

[0109] Mogroside-containing sweeteners and consumables derived from genetically modified plants In some embodiments, the Disclosure generally relates to sweeteners or sweetening compositions comprising mogrosides and / or their metabolites or derivatives, wherein the sweeteners or sweetening compositions are derived from genetically modified plants that produce and contain non-natural mogrols / mogrosides. In certain embodiments, the sweeteners or sweetening compositions are derived from genetically modified plants of the mogrol / mogroside pathway produced in accordance with the Disclosure.

[0110] The mogrol / mogroside pathway gene-transformed plants of this disclosure can induce mogroside-containing sweeteners upon appropriate processing. The resulting sweeteners can be used to provide low-calorie or zero-calorie sweetness for a variety of purposes. Examples of such sweetening applications include beverages such as tea, coffee, fruit juices and fruit drinks; foods such as jams and jellies, peanut butter, pies, puddings, cereals, candies, ice cream, yogurt, and bakery products; healthcare products such as toothpaste, mouthwash, cough drops and cough syrups; chewing gum; and sugar substitutes. In certain embodiments, the sweetener is present in the fruit juice of the gene-transformed plants according to this application.

[0111] In some embodiments, the disclosure also relates to methods for producing sweeteners derived from genetically modified plants that produce non-natural moglol / mogrosides. The methods generally include, but are not limited to, steps of pretreatment washing and grinding of the genetically modified plant or part thereof, extraction of the genetically modified plant or part thereof, sedimentation and / or centrifugation, adsorption and / or separation, concentration and recovery to produce a crude sweetener, further purification, optional concentration / drying and formulation. Means of extraction include water extraction at room temperature or heating temperature or refrigeration temperature; extraction with organic solvents such as alcohol; and others. Means of separation and purification include centrifugation, immersion, gravity sedimentation, filtration, microfiltration, nanofiltration, ultrafiltration, reverse osmosis, chromatography, absorption chromatogram, and exchange resin purification.

[0112] In certain embodiments, the sweetener is obtained from the leaves of a genetically modified plant produced according to this disclosure. In other embodiments, the sweetener is obtained from the fruit of a genetically modified plant produced according to this disclosure.

[0113] In some embodiments, the sweetener is obtained from genetically modified watermelon according to the present disclosure, and the sweetener comprises a non-natural mogroside produced by the genetically modified watermelon.

[0114] The forms of mogrol / mogroside pathway gene-transformed plants and methods for producing them described herein constitute preferred embodiments of the Disclosure, but it should be understood that the Disclosure is not limited to these detailed forms. As will be apparent to those skilled in the art, the various embodiments described above can be combined to give further embodiments. The forms of the gene-transformed plants, methods and processes (including their specific components) can be modified as needed to best utilize the systems, methods, sections and components and concepts of the Disclosure. These embodiments are considered to be entirely within the scope of the invention as described in the claims. For example, the various methods described above may omit some actions, include other actions, and / or perform actions in a different order than described in the embodiments described.

[0115] Furthermore, in the genetically modified plants and methods of production taught herein, various actions may be performed in an order different from that described and printed. In light of the foregoing, these and other modifications may be made to the Systems, Methods and Articles. In general, the terms used in the following claims should not be interpreted as limiting this disclosure to the specific embodiments disclosed herein and in the claims, but rather as encompassing all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the present invention is not limited by this disclosure, and instead its scope is entirely determined by the following claims.

[0116] All publications, patents, and patent applications referenced herein represent the level of skill of those skilled in the art in which this disclosure relates.

[0117] The following examples illustrate preferred but non-limiting embodiments of the present invention. [Examples]

[0118] Examples Example 1 - Identification of nucleotide sequences related to SEQ ID NOs: 1-31 The nucleotide sequences (of full-length cDNA, EST, or genome) related to Sequence IDs 1-31 are identified using previously published non-patent literature on the mogroside pathway (Itkin et al., Proc Nat Acad Sci USA, 2016; 113:E7619-E7628) and published international patent applications, International Publication Nos. 2014086842 and International Publication Nos. 2013076577.

[0119] Example 2 - Construction of an expression cassette containing one or more nucleotide sequences selected from SEQ ID NOs: 1-31 As shown in Table 1, various expression cassettes with different combinations of nucleotide sequences encoding mogroside pathway enzymes were constructed. These expression cassettes were constructed according to standard genetic engineering methods.

[0120] In short, expression cassettes were obtained from a gene synthesis vendor (GeneWiz) and assembled by enzymatic digestion and ligation. For example, pBing008 was assembled using five synthetic gene fragments: 1) TCTP promoter / CDS coding region / c-myc epitope tag / CaMV 35S terminator; 2) N3 spacer / FSgt-PFlt promoter / CYP87D18 coding region / HSV epitope tag / At UBQ3 terminator; 3) N5 spacer / CsVMV promoter / EPH3 coding region / FLAG epitope tag / At HSP18.2 terminator; 4) N8 spacer / HLV H12 promoter / SQE1 coding region / HA epitope tag / pea 3A terminator; 5) N7 spacer / PCSLV promoter / UGT720 coding region / V5 epitope tag / E9 terminator. Expression cassettes 4 and 5 were assembled into a pCAMBIA plant binary using their unique BsaI restriction sites at their 5' and 3' ends to construct the intermediate vector pBING003. Expression cassettes 1, 2, and 3 were assembled into a pCAMBIA plant binary vector using their unique BsaI restriction sites at their 5' and 3' ends to construct the intermediate vector pBING005. Subsequently, the SbfI~SalI restriction fragments extending to the MMV promoter / eGFP gene from expression cassettes 1, 2, and 3 and intermediate vector pBING005 were subcloned to the SbfI~SalI restriction site in intermediate vector pBING003 to construct the final vector pBING008. This vector was validated by restriction digestion analysis using the enzymes SphI+PstI, and then confirmed by Sanger sequencing using a series of oligonucleotide primers designed to cover the entire T-DNA region of the binary vector.

[0121] Example 3 - Genetically modified plant Nicotiana bentamiana Expression Cassette Construction: Various expression cassettes selected from Table 1 were constructed and used to transform Nicotiana bentamiana and produce transgenic Nicotiana bentamiana plants. Expression cassette pBing008 contains all five transgenes encoding mogroside pathway enzymes, two reporter genes encoding GFP and Hyg respectively, and nucleotide sequences encoding epitope tags, weak promoters, and terminators, respectively. Expression cassettes pBing003, pBing006, pBing007, pBing015, and pBing024, which have different gene combinations, were constructed in the same manner as described in Example 2.

[0122] Preparation of transformed Agrobacterium: Agrobacterium tumefaciens stain EHA105 was transformed with one or more expression cassette plasmids (selected from Table 1) using the free thawing method (Weigel, CSH Protoc. 2006 Dec 1; 2006(7)). In short, chemically competent Agrobacterium were prepared. After plasmid addition, the mixture was alternately frozen in liquid nitrogen and thawed in a water bath at 37°C. The cells were then recovered in LB medium for approximately 1 hour and seeded on LB plates containing kanamycin.

[0123] Infection of the plant Nicotiana bentamiana: Briefly, transformed EHA105 Agrobacterium was grown overnight and then diluted until the OD600 reading reached 0.12. Six-week-old Nicotiana bentamiana plants, each with five leaves, were selected for transformation. The diluted transformed Agrobacterium culture was loaded into a 5 mL syringe without a needle, and approximately 1.5 mL was injected into the underside of the leaves until they turned dark green. The plants loaded with the transformed Agrobacterium culture were grown for a further 10 days before sampling and selection.

[0124] The four genetically modified Nicotiana bentamiana plants were each prepared by transformation using the following expression cassettes. - pBing008 (5 genes, weak promoter) - pBing024 (5 genes, strong promoter) - pBing003+pBing007 (3+2 genes, weak promoter) - pBing006+pBing015 (3+2 genes, strong promoter)

[0125] Protein expression in tissue: Approximately 50 mg of leaves were sampled into a 1.7 mL microcentrifuge tube, and 500 μl of protein extraction buffer (1X RIPA lysis buffer) was added. The leaf tissue was pulverized in the extraction buffer before centrifugation to remove debris. The supernatant was further diluted 3-fold with the extraction buffer, and 4.5 μl of the extract was used for antibody detection using a Jess instrument (Bio-Techne), which automates the immunodetection of conventional Western blotting methods for protein separation and detection. Anti-rabbit antibodies with MYC, HSV, FLAG, HA, and V5 tags were purchased from Thermo Fisher, diluted 50-fold for use in Western detection using Jess, and Western detection was performed according to the manufacturer's manual. The presence of each target protein was confirmed by detection of the chemiluminescent signal produced by the binding of the corresponding antibody and by the size of the protein indicated by the protein size ladder used as a control in each measurement.

[0126] As shown in Table 3, when Nicotiana bentamiana leaves were transformed with the expression cassette pBing008, all five target proteins could be detected within 10 days with an S / N ratio exceeding 3, which is used as the cutoff for positive signals.

[0127] [Table 3]

[0128] Metabolic regulation: Approximately 100 mg of plant tissue was extracted into 500 μl of extraction buffer (80% methanol). After centrifugation, the supernatant was filtered through a 0.22 μM filter to remove any remaining particles. A Waters Acquity UPLC connected to a Waters Xevo Quadrupole Time of Flight Tandem Mass Spectrometer was used for metabolite analysis. For UPLC separation, a Waters Acquity BEH C18 1.7 μm, 2.1 × 50 mm column was used with water and acetonitrile (both containing 1% formic acid) as solvents. 1.5 μl of sample was injected for each analysis. MS / MS with negative ESI was used to detect mogroside compounds. The collision energy was set to 30 V for the detection of mogroside II.

[0129] Figure 4 shows the UPLA analysis results for a standard mogroside. As can be seen, mogroside IIA1 and mogroside IIA eluted simultaneously with a retention time of approximately 5.9 minutes. The results also showed m / z 423 (a characteristic peak for mogroside-related compounds) across the ULC separation gradient. The mogroside standard was shown at a very high concentration (0.1 mg / ml).

[0130] Figure 5 shows the results of ULC-TOFMS (retention time) analysis of mogroside II detection in the leaves of transgenic plants Nicotiana bentamiana transformed with expression cassettes pBing008 and pBing024, respectively. As can be seen, compared to the control plant p019, which did not produce a mogroside peak, both pBing008-transgenic Nicotiana bentamiana and pBing024-transgenic Nicotiana bentamiana produced a mogroside II peak, which is confirmed by the retention time overlapping with that of the mogroside standard.

[0131] Figure 6 shows the results of ULC-TOFMS (retention time) analysis for mogroside II detection in the leaves of transgenic Nicotiana bentamiana co-transformed with expression cassettes pBing003 and pBing007, and in transgenic Nicotiana bentamiana co-transformed with expression cassettes pBing006 and pBing015. As can be seen, compared to the control plant p019, which does not produce a mogroside peak, both the pBing003+pBing007 transgenic Nicotiana bentamiana and the pBing006 and pBing015 transgenic Nicotiana bentamiana produce a mogroside II peak, which is confirmed by the retention time overlapping with that of the mogroside standard.

[0132] Figures 7 and 8 show the ULC-TOFMS results for the detection of mogroside IIA in the leaves of transgenic Nicotiana bentamiana transformed with the expression cassette pBing008. As can be seen, Nicotiana bentamiana leaves infected with the expression cassette pBing008 yield peak A and share the same characteristics as mogroside IIA in terms of retention time (Figure 6) and mass spectral pattern (Figure 7), but the transgenic Nicotiana bentamiana transformed with the expression cassette pBing008 produces and contains mogroside II.

[0133] Example 4: Assembled watermelon tissue-specific transcriptome Due to their large size and popular taste, watermelon fruits have great potential for producing a calorie-free sweetener. To design genome editing or cisgenic strategies for pathway engineering, it is crucial to identify watermelon fruit-specific promoters that enable optimal expression of gene payloads. Identifying these promoters requires high-resolution transcriptome datasets from which a list of genes specifically expressed in the edible portion of the watermelon fruit can be generated. Currently, there are no publicly available transcriptome resources that can distinguish between different parts of a watermelon fruit at different growth stages.

[0134] This study provided bioanalytical methods for detecting and quantifying the expression levels of endogenous genes in various parts of the fruit of two commercially available watermelon varieties, Charleston Gray and Sugar Baby. Watermelons were grown, and tissue and stage-specific samples were collected. High-quality RNA was extracted from all these samples, generating over 20 million RNA sequencing reads for each sample. The sequencing results and the standardized RNA expression levels of target genes in each sample were analyzed and quantified. A preliminary list of genes highly concentrated in the flesh of watermelon fruit was compiled.

[0135] Table 4 summarizes the various watermelon tissue samples collected for RNA sequencing analysis. Forty-five fruit tissue samples (5 species × 3 instars × 3 replicates = 45), six leaf samples (2 instars × 3 replicates = 6), and three root samples were collected from both the Sugar Baby and Charleston Gray varieties. Figure 9 shows the dissection of a watermelon and its various parts.

[0136] [Table 4]

[0137] Total RNA was checked using NanoDrop and BioAnalyzer. The total RNA amount per sample was approximately 1 microgram (μg) or more. Purity was set to OD260 / 280 = 1.8 to 2.2 and OD260 / 230 ≥ 2.0. RNA integrity was checked by the RIN count, which should be greater than 7, using BioAnalyzer. As a result of RNA sequencing analysis, the minimum number of reads obtained from all samples was 20.8 million, and the average number of reads was 369 million for Charleston Gray and 37.8 million for Sugarbaby, respectively.

[0138] For RNA sequencing data analysis, low-quality reads (q=30) were removed. Clean reads were aligned to the Charleston Gray reference genome (Wu et al., Genome of 'Charleston Gray', the principal American watermelon cultivar, and genetic characterization of 1,365 accessions in the US National Plant Germplasm System watermelon collection. Plant Biotechnology Journal, 2019). The resulting alignment rates ranged from 89.7 to 93.58. The number of genes in each sample was calculated. Then, the samples were standardized to explain the differences in library depth.

[0139] One notable feature of grown watermelon fruits of both Charleston Gray and Sugar Baby varieties is their pink / red flesh. Lycopene and β-carotene are responsible for the fruit, and it is well known that the production of these pigments is regulated by the phytoenzyme synthase gene PSY1 (Wang et al., Developmental Changes in Gene Expression Drive Accumulation of Lycopene and β-Carotene in Watermelon, Journal of the American Society for Horticultural Science, 2016, 141(5), 434-443). From this RNA sequencing dataset, PSY1 gene expression is highly correlated with the accumulation of pink / red color: the highest expression levels were detected in the mesocarp, placenta, and ventricle tissues of 26- and 42-day-old fruits, which are precisely the tissues that exhibit visible pink and red color, as shown in Figure 10. The correlation between PSY1 gene expression and tissue color demonstrates the biological significance of the RNA sequencing dataset produced by this project.

[0140] As the next step, the expression of all 22,545 genes from 36 sample groups was screened to identify further fruit-specific genes with expression patterns similar to PSY1. The criteria were defined as follows: (1) Gene expression was highly concentrated in the mesocarp, placenta, and ventricle tissues (referred to as "target tissues") in 26-day and 42-day-old fruits. The expression level must be more than 5 times higher than in the peel and more than 20 times higher in the root, leaf, and fruit epidermis; (2) The expression level in target tissues was low but still greater than 100 FPKM (fragments per kilobase of transcript per million mapped reads) to eliminate tissue-specific genes; and (3) Expression characteristics must meet both criteria in both varieties.

[0141] As a result, eight genes were identified as such target tissue-specific genes (using the Charleston Gray Reference Genome Identifier http: / / cucurbitgenomics.org / ). Interestingly, most of these genes are predicted to be involved in plant metabolism and are indeed expected to be concentrated during the fruit maturation stage, as shown in Table 5. The expression concentration of these genes in various tissue parts of watermelon samples, as shown in Table 4, is visualized in Figures 11-12. The gene expression dataset from this study could be a unique resource for discovering tissue-specific genes and promoters for the design and production of genetically modified plants, as well as for the analysis and quantification of target gene expression in genetically modified plants.

[0142] [Table 5]

[0143] Example 5 - Genetically modified watermelon Expression cassette construction: Various expression cassettes were prepared according to Table 1, as in the production of gene-transfected Nicotiana bentamiana in Example 3.

[0144] Preparation of transformed Agrobacterium: Transformed Agrobacterium were prepared following the same procedure given in Example 3.

[0145] Watermelon infection: Two commercially available watermelon varieties, Charleston Gray and Sugar Baby, were used as hosts. Five-day-old watermelon seedlings were used to prepare explants for transformation. Cotyledons were excised from the hypocotyl and collected in a petri dish filled with sterile water. Two joined cotyledons were separated by cutting through the remaining hypocotyl segment, and the cotyledon explants were cut into 2 mm pieces ready for transformation. For transformation, Agrobacterium cultures were added to these explants and vacuumed for 5 minutes. After infection, the explants were dried on sterile paper towels and transferred to a filter disc in a petri dish containing MS medium. The plate was sealed and placed in the dark at 25°C for 3 days for co-incubation.

[0146] Examples of genetically modified watermelons were prepared by transformation using the following expression cassettes. - pBing008 (5 genes, weak promoter) - pBing028 (5 genes, strong promoter, Hgy and GFP reporter proteins)

[0147] [Table 6]

[0148] Protein expression in genetically modified watermelon: Protein expression in genetically modified watermelon samples was monitored and analyzed following the same procedure provided in Example 3.

[0149] Table 7 shows the ploidy, metabolite, and gene expression results for genetically modified watermelon samples. Expression levels were quantified using Q-RT-PCR from leaf RNA samples. Expression levels were standardized against a predefined standard (set to 1). For metabolite results, *** indicates abundance; ** indicates clear presence; and * indicates possible presence. As shown in Table 7, when watermelon leaves were transformed with expression cassettes pBing008 or pBing028, all five target mogroside pathway proteins could be detected in the corresponding genetically modified watermelon plants. In specific genetically modified watermelon samples, the leaves were shown to have clear or abundant mogroside IIE. It is important to note that the genetically modified watermelon 008CHE4-19, which has diploid chromosomes, produced seeds and fruits, and the leaves of 008CHE4-19 had both possible and abundant mogroside IIE, as shown in the metabolite results. In contrast, polyploid (3X) or tetraploid (4X) genetically modified watermelons only flowered, but ultimately produced neither seeds nor fruit, nor did they produce mogrosides in their leaves or other tissues. Surprisingly, these results suggest that the ability of genetically modified watermelons to produce fruit and seeds is unexpected, and that chromosomal ploidy may be an important factor in the reproductive capacity of genetically modified watermelons that produce non-natural mogrosides.

[0150] [Table 7]

[0151] Figure 13 shows the results of protein detection analysis in various genetically modified watermelon samples (transformed with pBing008). The Y-axis is set with 10% actin expression as a value of 1.0. EPH expression is higher than the range and is not shown in this graph. As can be seen, all genetically modified watermelon samples showed high expression of all five mogroside pathway transgenes. The sample 008DLE11 cluster showed high expression of all transgenes. The sample 008DLE11-8 fruit had the highest expression and produced 50 seeds.

[0152] Figure 14 shows the chemiluminescence results of protein detection in genetically modified watermelons transformed using the expression cassette pBing008. As can be seen, expression of all five target mogroside pathway enzymes was observed in the genetically modified watermelons.

[0153] To understand the differences in the ability to detect mogroside production in fruit, watermelons from various newly created plant lines were collected and cut into various fruit parts. RNA was then extracted from various fruit part samples, and the RNA expression levels of various newly integrated pathway genes were quantified using Q-RT-PCR measured using a standard protocol. The measurements showed several trends, as shown in Figures 15-18. As can be seen, all transgenes CDS, CYP87, SQE, EPH, and UGT720 were expressed in all fruit tissues, including the placenta, chulmon, mesocarp, hull, and epidermis. It is noteworthy that the expression patterns were consistent across all tissue types. Furthermore, CDS and UGT720 expression was lower than CYP87, SQE, and EPH. EPH expression was dramatically higher in some fruit parts, including the mogroside IIE-containing fruit (008CHE4-13). This may be due to the site effect of transgene insertion.

[0154] Figures 15-18 show the results of analyses of various tissues in representative genetically modified watermelon samples regarding the expression of mogroside pathway transgenes. As can be seen, all transgenes, CDS, CYP87, SQE, EPH, and UGT720, were expressed in all fruit tissues, including the placenta, chamber, mesocarp, hull, and epidermis. It is noteworthy that the expression pattern was consistent across all tissue types. Furthermore, the expression of CDS and UGT720 was lower than that of CYP87, SQE, and EPH. EPH, however, showed dramatically higher expression in some fruits.

[0155] Metabolic regulation: Metabolic regulation was monitored and metabolites of the genetically modified watermelon samples were analyzed following the same procedure as given in Example 3.

[0156] As shown in Figures 19-20, compared to the control, the transgenic watermelon containing the expression cassette pBing008 showed the presence of mogroside IIE and demonstrated successful mogroside production via the intended enzymatic pathway.

[0157] Genetically modified plants that produce mogroside IIE were evaluated for their ability to produce mogroside IIE in their fruits. At least one plant was able to produce fruit (008CHE4-13). Extracts were prepared from the fruit of this plant and analyzed by ULC-TOFMS. The fruit extracts showed the characteristic mass fingerprint of mogroside IIE (shown in Figure 21a). As a positive control, extracts from wild-type, unmodified fruit were prepared and spiked with 100 ng / ml mogroside IIE (shown in Figure 21b). These surprising results demonstrate the unexpected ability of the genetically modified plants according to this disclosure to produce fruit containing the non-natural mogroside.

[0158] Figure 22 shows the ULC-TOFMS results of seed coats from genetically modified watermelon samples 008SBE5-2 and 008CHE4-5, both containing the expression cassette pBing008. As can be seen, mogroside IIE was detected in the fruit seed coat, indicating the production of mogrosides in other tissues or parts of the genetically modified watermelon, not limited to the fruit. These surprising results demonstrate the unexpected reproductive capacity of the genetically modified plants of this disclosure.

[0159] Example 6: Expression of a mogroside-producing gene and production of mogrosides in genetically modified watermelon plants T0 and T1. Further investigations were conducted to analyze the expression of target transgenes and mogroside production in both genetically modified watermelons (T0 plants) and their offspring (T1 plants).

[0160] 1. Gene expression analysis of target genes in T0 plants Over 100 genetically modified watermelon lines were produced according to the method given in Example 5. Thirty-one plants that produced fruit were used for gene expression analysis. First, the expression levels of CDS, a key restriction enzyme in the pathway, were tested in leaf and fruit tissue using Q-RT-PCR. To compare gene expression across all samples, all gene expression values ​​were standardized to 10% of actin expression (set to 1). The results (Figure 23) showed various expression levels, confirming the presence and expression of the introduced gene CDS. While the expression fluctuations appeared random in the leaves, a more consistent pattern was observed in the fruits, with several plants derived from the two families of genetically modified lines, 008CHE4 and 008DLE11, showing the highest expression (Figure 23). 008CHE4-19 showed the highest gene expression in the fruit. Several related genetically modified fruits originally isolated from 008DLE11 exophytes also showed high CDS expression in the fruit. Significant fluctuations were observed between leaf and fruit expression. In contrast, the wild-type control (right side of Figure 23) did not show CDS expression as predicted. The expression levels of the other four target genes, CYP87, SQE, EPH, and UGT720, are shown in Figures 24-27, respectively.

[0161] 2. Metabolic screening of genetically modified watermelon T0 fruits to confirm the presence of mogroside IIE in gene transfer event 008CHE4-19. Watermelon fruits were analyzed for mogrol-derived compounds using UPLC-MS, and the results are shown in Figure 29. For control, standard mogroside IIE was also analyzed using UPLC-MS, and the results are shown in Figure 28. In comparison, both the ion chromatogram and mass spectrometry fragment results from 008CHE4-19 T0 fruits showed close agreement with those of the mogroside IIE standard, suggesting the biosynthesis and accumulation of mogroside IIE in these fruits. Interestingly, fruits derived from 008CHE4-19 also expressed the highest levels of CDS among all fruits characterized at T0, indicating a possible correlation between CDS expression and MIIE biosynthesis in T0 watermelon fruits.

[0162] 3. Characterization of events and gene expression analysis of introduced genes in offspring (T1 generation) of genetically modified watermelons. To test the inheritance of the mogroside-producing transgene and confirm that mogroside IIE can be produced in transgenic watermelons for two or more generations, seeds were collected from 008CHE4 and 008DLE11 fruits and germinated. After germination, 32 T1 plants (including 5 GFP gene transgene controls) were genotyped by PCR using DNA extracted from leaf tissue. PCR probes were designed to amplify two targets: the endogenous watermelon actin gene as a positive control and the CDS gene as an indicator of transgene integration. As shown in Figure 30, the presence of an actin product band indicates successful PCR from watermelon genomic DNA; the presence of a CDS product band indicates the presence of the transgene. The identity and genotyping conclusions for all samples are listed in the tables on the left and right. As predicted, several wild-type segregants were also identified (samples 13, 16, 18, 27, and 32 according to Figure 30). These genotyping results confirmed the presence and successful inheritance of the T1 generation of the transgene cassette in watermelon.

[0163] Leaves of the transgenic T1 plants were also sampled for Q-RT-PCR detection of all five target genes. The results are summarized in Table 8. Primers were designed to specifically amplify the transgenes, rather than using watermelon homologs (indicated by the negative control). The values ​​are the average of three independent biological replicate experiments and were standardized to 10% of the previously established actin expression standard. Plants 008CHE4-1-S3, 008CHE4-19-S5, 008CHE4-19-S10, 008DLE11-2-S5, and 008DLE11-7-S2 were found to be wild-type segregant. Consistent with the genotyping results, all wild-type segregant and negative non-transgenic plants showed virtually no expression of these targets. The 008DLE11-2 family was found to show higher overall expression of all five target genes compared to other transgenic lines, including 008CHE4-19. Of these five genes, EPH, promoted by the CsVMV promoter, consistently showed very high expression (about 10 times that of actin), suggesting strong activity of this promoter in watermelon.

[0164] [Table 8]

[0165] 4. Metabolic analysis identified three watermelon T1 fruits that produced mogrosides. Fruits from the T1 line were collected for metabolic analysis. LC-MS results, as shown in Figures 31-33, confirmed the presence of mogroside IIE in 008DLE11-4-S4, 008DLE11-2-S1, and 008DLE11-9-S3, all of which were offspring of T0 gene-transformed watermelons containing the expression cassette pBing008. Quantitatively, these three T1 samples produced approximately 30 ng, 17 ng, and 3 ng (per gram of dry weight) of mogroside IIE, respectively.

[0166] The following numbered clauses define further examples and features of the Disclosure.

[0167] 1. A plant having a genome transformation event, the genome transformation event resulting in the unnatural expression or concentration of mogroside pathway enzymes, and the plant biosynthetically producing unnatural mogol precursors, mogol, mogrosides and / or their metabolites or derivatives.

[0168] 2. Plants containing non-natural mogrol precursors and / or mogrol that biosynthetically produce mogrosides and / or their metabolites or derivatives.

[0169] 3. A genetically modified plant, wherein the genome transformation event includes an expression cassette, and the expression cassette contains one or more nucleotide sequences shown in SEQ ID NOs: 1-31, as described in Clause 1.

[0170] 4. A genetically modified plant containing an expression cassette, wherein the expression cassette contains one or more nucleotide sequences shown in SEQ ID NOs: 1-31, as described in clause 2.

[0171] 5. An expression cassette comprising one or more nucleotide sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1-31, according to any of the gene transducers in clauses 3-4.

[0172] 6. The mogroside pathway enzyme has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs: 1-31, in the genetically modified plant of Clause 2.

[0173] 7. An expression cassette comprising one or more sequences selected from the group consisting of a promoter, spacer, epitope tag, terminator, reporter gene, or combination thereof, in a gene transplant according to any of clauses 3 to 6.

[0174] 8. The mogroside pathway enzyme is selected from the group consisting of silk vitaminol synthase (CDS), squalene epoxidase (SQE), epoxy hydrolase (EPH), cytochrome P450, uridine-5'-diphospho (UDP)-dependent glucosyltransferase (UGT), or a combination thereof, in the genetically modified plant according to Clause 1.

[0175] 9. Mogrosides are genetically modified plants according to any of clauses 1 to 8, selected from the group consisting of siamenoside I, silatose, mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside II, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, mogroside I, mogroside IA, mogroside IE, or any combination thereof.

[0176] 10. A gene-transformed plant according to any of clauses 1-9, wherein the mogroside is selected from the group consisting of mogroside IA, mogroside IE, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, or any combination thereof.

[0177] 11. Plant parts that can be obtained from a plant according to any of the provisions 1 to 10, including, but not limited to, plant organs, tissues, leaves, stems, roots, flowers or flower parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic tissue parts, callus tissue, seeds, cuts, cells or tissue cultures or any other parts or products, and plant parts that include mogrol precursors, mogrol, mogrosides and / or metabolites or derivatives thereof.

[0178] 12. The plant's offspring or ancestor is a source of non-natural enzymes that enable the offspring and ancestor to produce mogrol precursors, mogrol, moglosides and / or their metabolites or derivatives, according to any of clauses 1 to 11.

[0179] 13. A diploid plant, according to any of clauses 1 to 12.

[0180] 14. A plant belonging to the Cucurbitaceae family / cucurbits, according to any of the following criteria (1-13).

[0181] 15. Citrullus lanatus (watermelon), a plant according to any of clauses 1-14.

[0182] 16. A mogroside sweetener derived from a plant, wherein the plant or part thereof biosynthetically produces and contains non-natural mogrool precursors, mogrool, mogrosides and / or their metabolites or derivatives.

[0183] 17. Mogroside sweeteners, which are plant extracts, as specified in Clause 16.

[0184] 18. Mogroside sweeteners according to any of clauses 16-17, refined from plants or parts thereof.

[0185] 19. Mogroside sweeteners of Clause 18, purified by extraction, immersion, chromatography, or absorption chromatography.

[0186] 20. Foods, ingredients, flavorings, or beverages containing any of the sweeteners specified in clauses 16 to 19.

[0187] 21. A biosynthetic method for producing a non-natural moglol precursor, moglol, or mogroside, (a) A step of combining a plant with a genome transformation event to form a genetically modified plant, wherein the genome transformation event results in the non-native expression or concentration of mogrol / mogroside enzymes; (b) The process of cultivating and regenerating the population of genetically modified plants described in (a); (c) the step of selecting genetically modified plants that produce mogrosides; and (d) Process for collecting mogrosides A method that includes this.

[0188] 22. Prepare / provide plasmids containing expression cassettes that express non-natural moglol / mogroside pathway enzymes; Transforming host cells with plasmids; and Transmutation of plants using multiple transformed host cells. The methods of Article 21, further including.

[0189] 23. A method for producing a plant that produces a non-natural mogol precursor, mogol, or mogroside, comprising combining the plant with a genome transformation event to form a genetically modified plant, wherein the genome transformation event results in the non-natural expression or concentration of a mogol / mogroside pathway enzyme.

[0190] 24. Combining plants with genomic transformation events is carried out using one or more of the following methods: the use of liposomes, the use of electroporation, the use of chemicals that increase the uptake of free DNA, the use of direct injection of DNA into plants, the use of particle gambolization, the use of microprojection, or the use of Agrobacterium-mediated transformation, as described in Clause 23.

[0191] 25. Prepare / provide plasmids containing expression cassettes that express non-natural moglol / mogroside pathway enzymes; Transforming host cells with plasmids; and Transmutation of plants using multiple transformed host cells. The methods of clauses 23-24, including further.

[0192] 26. The host cell is a microorganism, according to the method of Clause 25.

[0193] 27. The microorganism is selected from the group consisting of plant cells, mammalian cells, insect cells, fungal cells, algal cells, bacterial cells, or combinations thereof, according to the method of Clause 26.

[0194] 28. The bacterial cell is a Gram-negative bacterium, according to the method of Clause 27.

[0195] 29. The Gram-negative bacterium is Agrobacterium tumefaciens, according to the method of Clause 28.

[0196] 30. Host cells are transformed with plasmids using the free thawing method, according to the biosynthesis method of Clause 29.

[0197] 31. A biosynthetic method for producing a non-natural mogol precursor, mogol, or mogroside, (a) A step of combining a plant with a genome transformation event to form a gene-edited plant, wherein the genome transformation event results in the non-natural expression or concentration of mogrol / mogroside enzymes; (b) The process of cultivating and regenerating a population of the gene-edited plants described in (a); (c) the process of selecting gene-edited plants that produce mogrosides; and (d) Process for collecting mogrosides A biosynthesis method including the following.

[0198] 32. Prepare / provide plasmids containing expression cassettes that express non-natural moglol / mogroside pathway enzymes; Transforming host cells with plasmids; and Transmutation of plants using multiple transformed host cells. The method of Article 32, which further includes the following.

[0199] 33. Foods, ingredients, flavorings, or beverages containing any of the sweeteners specified in clauses 21 to 32.

[0200] 34. A gene-edited plant according to Clause 1, wherein the genome transformation event is introduced to the plant by a method selected from the group including transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), meganucleases (MNs), and combinations thereof.

[0201] The above specification, examples, and data provide a complete description of the preparation and use of the compositions of the present invention. Since many embodiments of the present invention can be made without departing from the spirit and scope of the invention, the present invention is contained within the claims appended below.

Claims

1. A plant comprising a genome transformation event, wherein the genome transformation event comprises an expression cassette, which results in the unnatural expression of the mogroside pathway enzyme silk vitaminol synthase (CDS), squalene epoxidase (SQE), epoxy hydrolase (EPH), cytochrome P450, and uridine-5'-diphospho-dependent glucosyltransferase (UGT), wherein the expression cassette comprises a first promoter comprising SEQ ID NO: 6 or SEQ ID NO: 13, a nucleotide sequence of CDS comprising SEQ ID NO: 1, a second promoter comprising SEQ ID NO: 9 or SEQ ID NO: 16, and SQE comprising SEQ ID NO:

4. A plant comprising a nucleotide sequence of , a third promoter containing SEQ ID NO: 8 or SEQ ID NO: 15, a nucleotide sequence of EPH containing SEQ ID NO: 3, a fourth promoter containing SEQ ID NO: 7 or SEQ ID NO: 14, a nucleotide sequence of cytochrome P450 containing SEQ ID NO: 2, a fifth promoter containing SEQ ID NO: 10 or SEQ ID NO: 17, and a nucleotide sequence of UGT containing SEQ ID NO: 5, wherein the plant, and / or its offspring and / or seeds, biosynthetically produce unnatural moglol precursors, moglol, moglosides and / or their metabolites or derivatives, and the plant is a watermelon having a diploid set of chromosomes.

2. A plant containing a non-natural mogrol precursor and / or mogrol, wherein the plant and / or its offspring and / or seeds biosynthetically produce mogrosides and / or their metabolites or derivatives, the plant expresses the mogroside pathway enzymes silk vitaminol synthase (CDS), squalene epoxidase (SQE), epoxy hydrolase (EPH), cytochrome P450, and uridine-5'-diphospho-dependent glucosyltransferase (UGT), and the plant expresses a first promoter including SEQ ID NO: 6 or SEQ ID NO: 13, SEQ ID NO: 1 A plant comprising a nucleotide sequence of CDS including , a second promoter including SEQ ID NO: 9 or SEQ ID NO: 16, a nucleotide sequence of SQE including SEQ ID NO: 4, a third promoter including SEQ ID NO: 8 or SEQ ID NO: 15, a nucleotide sequence of EPH including SEQ ID NO: 3, a fourth promoter including SEQ ID NO: 7 or SEQ ID NO: 14, a nucleotide sequence of cytochrome P450 including SEQ ID NO: 2, a fifth promoter including SEQ ID NO: 10 or SEQ ID NO: 17, and a nucleotide sequence of UGT including SEQ ID NO: 5, wherein the plant is a watermelon having a diploid set of chromosomes.

3. The gene-transformed plant comprising an expression cassette, wherein the expression cassette comprises a first promoter comprising SEQ ID NO: 6 or SEQ ID NO: 13, a nucleotide sequence of CDS comprising SEQ ID NO: 1, a second promoter comprising SEQ ID NO: 9 or SEQ ID NO: 16, a nucleotide sequence of SQE comprising SEQ ID NO: 4, a third promoter comprising SEQ ID NO: 8 or SEQ ID NO: 15, a nucleotide sequence of EPH comprising SEQ ID NO: 3, a fourth promoter comprising SEQ ID NO: 7 or SEQ ID NO: 14, a nucleotide sequence of cytochrome P450 comprising SEQ ID NO: 2, a fifth promoter comprising SEQ ID NO: 10 or SEQ ID NO: 17, and a nucleotide sequence of UGT comprising SEQ ID NO: 5, as described in claim 2.

4. The plant according to claim 3, wherein the expression cassette comprises one or more sequences selected from the group consisting of a promoter, a spacer, an epitope tag, a terminator, a reporter gene, or a combination thereof.

5. The plant according to any one of claims 1 to 4, wherein the mogroside is selected from the group consisting of siamenoside I, silatose, mogroside VI, mogroside V, isomogroside V, mogroside IV, mogroside III, mogroside IIIE, mogroside II, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, mogroside I, mogroside IA, mogroside IE, or any combination thereof.

6. The plant according to any one of claims 1 to 5, wherein the mogroside is selected from the group consisting of mogroside IA, mogroside IE, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IIE, mogroside IIE2, or any combination thereof.

7. A plant part that can be obtained from a plant according to any one of claims 1 to 6, comprising organs, tissues, leaves, stems, roots, flowers or flower parts, fruits, buds, gametophytes, sporophytes, pollen, anthers, microspores, egg cells, zygotes, embryos, meristematic tissue parts, callus tissue, seeds, cuts, cells or tissue cultures of the plant, wherein the plant part comprises a mogrol precursor, mogrol, mogroside and / or its metabolites or derivatives, and the plant is a watermelon having a diploid set of chromosomes.

8. The plant according to any one of claims 1 to 7, wherein the offspring or ancestor of the plant produces a mogol precursor, mogol, mogroside and / or its metabolites or derivatives.

9. A plant belonging to the Cucurbitaceae family / gourds, as described in any one of claims 1 to 8.

10. A biosynthetic method for producing a non-natural moglol precursor, moglol, or mogroside, (a) A step of providing a plasmid comprising an expression cassette, wherein the expression cassette comprises a first promoter comprising SEQ ID NO: 6 or SEQ ID NO: 13, a nucleotide sequence of CDS comprising SEQ ID NO: 1, a second promoter comprising SEQ ID NO: 9 or SEQ ID NO: 16, a nucleotide sequence of SQE comprising SEQ ID NO: 4, a third promoter comprising SEQ ID NO: 8 or SEQ ID NO: 15, a nucleotide sequence of EPH comprising SEQ ID NO: 3, a fourth promoter comprising SEQ ID NO: 7 or SEQ ID NO: 14, a nucleotide sequence of cytochrome P450 comprising SEQ ID NO: 2, a fifth promoter comprising SEQ ID NO: 10 or SEQ ID NO: 17, and a nucleotide sequence of UGT comprising SEQ ID NO: 5; (b) A step of combining a plant with a genome transformation event including the expression cassette, thereby forming a plant, wherein the genome transformation event results in the plant, and / or its offspring and / or seeds, exhibiting unnatural expression of mogrol or mogroside pathway enzymes; (c) The process of cultivating and regenerating the group of plants in (b); (d) A step of selecting the genetically modified plant that produces mogrol or mogroside; and (e) A step of collecting mogrol or mogroside. Includes, The aforementioned plant is a watermelon having a diploid set of chromosomes, and the biosynthesis method is described.

11. Transforming host cells with the plasmid; and Transmuting the aforementioned plant with a plurality of the aforementioned transformed host cells. The method according to claim 10, further comprising:

12. A method for producing a plant that produces a non-natural mogrol precursor, mogrol, or mogroside, comprising providing a plasmid comprising an expression cassette, the expression cassette comprising a first promoter comprising SEQ ID NO: 6 or SEQ ID NO: 13, a nucleotide sequence of CDS comprising SEQ ID NO: 1, a second promoter comprising SEQ ID NO: 9 or SEQ ID NO: 16, a nucleotide sequence of SQE comprising SEQ ID NO: 4, a third promoter comprising SEQ ID NO: 8 or SEQ ID NO: 15, a nucleotide sequence of EPH comprising SEQ ID NO: 3, a fourth promoter comprising SEQ ID NO: 7 or SEQ ID NO: 14, a nucleotide sequence of cytochrome P450 comprising SEQ ID NO: 2, a fifth promoter comprising SEQ ID NO: 10 or SEQ ID NO: 17, and a nucleotide sequence of UGT comprising SEQ ID NO: 5; and a method comprising combining the plant with a genome transformation event comprising the expression cassette, the genome transformation event resulting in the plant, and / or its offspring and / or seeds, exhibiting non-natural expression of a mogrol / mogroside pathway enzyme, wherein the plant is a watermelon having a diploid set of chromosomes.

13. Transforming host cells with the plasmid; and Transmuting the aforementioned plant with a plurality of the aforementioned transformed host cells. The method according to claim 12, further comprising:

14. The method according to claim 13, wherein the host cell is Agrobacterium tumefaciens.

15. The plant according to claim 1, wherein the gene-edited plant is subjected to the genome transformation event by a method selected from the group including transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), meganucleases (MN), and combinations thereof.

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