Compositions and methods for the production of siamenoside i

Abstract

The present disclosure provides novel exoglucanase compositions and methods of making same, and method of using the novel exoglucanase compositions for producing siamenoside I.

Description

TITLE OF THE INVENTIONCOMPOSITIONS AND METHODS FOR THE PRODUCTION OF SIAMENOSIDE I CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U. S. C. §119(e) of U. S. provisional application Serial No. 63 / 719,394. filed on November 12, 2024, which is herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] A sequence listing containing the file named " ELSS020WO_ST26.xml" which is 119,531 bytes (measured in MS-Windows®) and created on October 20, 2025. and comprises 39 sequences, is incorporated herein by reference in its entirety.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of enzyme compositions and methods for producing siamenoside I. The present disclosure also relates to a novel exoglucanase that can be used to produce siamenoside I and novel ingredients (e.g., extracts, purified and partially purified fractions containing siamenoside I) for foods and beverages, and novel foods and beverages (and other compositions of matter) resulting therefrom.BACKGROUND OF THE INVENTION

[0004] Low or non-caloric sweeteners, in particular natural low or non-caloric sweeteners, as an alternative to traditional high calorie sweeteners and artificial sweeteners, are becoming increasingly important to the food and beverage industry, in addition to other industries. These alternative sweeteners are used as a substitute for artificial sweeteners or high calorie sweeteners comprising sucrose, fructose, and glucose. Like some artificial sweeteners, some of these alternative sweeteners provide a greater sweetening effect than comparable amounts of caloric sweeteners, and therefore smaller amounts of these alternative sweeteners are required to achieve sweetness comparable to that of sugar. However, some low-calorie sweeteners can be expensive to produce and / or possess unfavorable taste characteristics and / or off-tastes, including, but not limited to, sweetness linger, delayed sweetness onset, negative mouth feel, and bitter, metallic, cooling, astringent, and licorice-like tastes.

[0005] A few natural plants produce low or non-calorie sweeteners. For example, mogrosides, an important class of natural sweeteners, are chemically a class of triterpene1US_ACTIVE\131477155\V-1glycosides or mogrol glycosides naturally produced by monkfruit (also known as luohan guo; scientific name: Siraitia grosvenorii). Mogrosides contain “zero” calories (less than 5 calories per 8 oz. serving) and are 100–600 times sweeter than sucrose. Mogrosides have also been reported to have a variety of important pharmacological effects. However, although plants like Siraitia grosvenorii make mogrosides, production of mogrosides from these plants is limited and expensive due to the limited natural or agricultural production of these plants. Also, Siraitia grosvenorii prefers to grow in subtropical mountainous regions and requires laborious pollination to set fruits. In addition, production of mogrosides in vitro or in yeast has been attempted, but due to extensive processing and other issues has not proven to be economically feasible.

[0006] Siamenoside I (SI) is a cucurbitane glycoside, a natural sweetener from the fruit of Siraitia grosvenorii. SI is the sweetest cucurbitane glycoside, with a relative sweetness that is 563 times higher than sucrose. The discovery of SI is of great interest to the food and beverage industry because SI has superior sweetness to mogroside V and its mouthfeel properties are nearly identical to sucrose. Nevertheless, SI only represents about 2-5% of the sweet mogrosides in a typical Monk Fruit extract. Attempts to convert mogroside V into siamenoside I through chemical hydrolysis, enzyme treatments, or microbial fermentation have had limited success due to the complexity of the glucose branching sidechains in mogrosides.

[0007] Therefore, developing efficient and economical methods to produce a highly purified preparation of SI would be of high commercial value.SUMMARY

[0008] The present disclosure solves these and other problems in the art by providing novel compositions and methods for the efficient and cost-effective production siamenoside I.

[0009] The present disclosure provides a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 or SEQ ID NO:39 operably linked to a heterologous promoter. In certain embodiments, the nucleic acid sequence has at least 95% sequence identity to SEQ ID NOT or SEQ ID NO:38. In some embodiments, the nucleic acid comprises the sequence of SEQ ID NO:20 or SEQ ID NO:21.

[0010] The present disclosure also provides a recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter. In particular embodiments, the nucleic acid sequence comprises SEQ ID NO:22.2US_ACTIVE\131477155\V-1

[0011] The present disclosure additionally provides a plant cell comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter. In certain embodiments, the plant cell further comprises a recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0012] The present disclosure further provides a bacterial or yeast cell comprising a recombinant nucleic acid construct comprising anucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter. In some embodiments, the yeast cell further comprises a recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0013] The present disclosure also provides a plant comprising a plant cell comprising a recombinant nucleic acid construct comprising anucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 operably linked to a heterologous promoter. In certain embodiments, the plant further comprises a plant cell comprising a recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0014] Additionally, the present disclosure provides a method for producing siamenoside I, comprising contacting a composition comprising mogroside V with a protein having at least 95% identity to SEQ ID NO:2. In certain embodiments, the composition comprising mogroside V is produced by a transgenic cell. In some embodiments, the transgenic cell is a transgenic plant cell, for example a transgenic lettuce, cucumber or tomato plant cell. In other embodiments, the transgenic cell is a transgenic yeast or filamentous fungi cell, for example a transgenic Saccharomyces cerevisiae, Pichia pastoris yeast cell, or Aspergillus or Trichoderma fungi cell. In yet other embodiments, the transgenic cell is a transgenic bacterial cell, for example a transgenic E. coli or Bacillus cell. In still other embodiments, the transgenic cell is a transgenic algae cell.

[0015] In further embodiments, the protein having at least 95% sequence identity to SEQ ID NO:2 is produced by a transgenic cell comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter. In still further 3US_ACTIVE\131477155\V-1embodiments, the protein having at least 95% sequence identity to SEQ ID NO:2 and the composition comprising mogroside V are produced by a single transgenic cell. In yet other embodiments, the single transgenic cell a transgenic plant cell comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter and a second recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter. In yet other embodiments, the single transgenic cell is a transgenic yeast cell comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter and a second recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0016] In certain additional embodiments, the method for producing siamenoside I comprises contacting an extract from a transgenic plant comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter is with an extract from a transgenic plant comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter. In yet other embodiments, the exoglucanase is purified from a native fungus that makes the exoglucanase, for example Wickerhamomyces ciferrit. Eremothecium gossypii, Kazachstania africana, Kazachstania naganishii, Lachancea dasiensis, Ogcitaea angusta, Pichia kudriavzevii, Saccharomyces cerevisiae, Brettanomyces bruxellensis or Pichia pastoris.

[0017] In some embodiments, the protein having at least 95% sequence identity to SEQ ID NO:2 is purified from a transgenic plant or yeast cell comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter. In certain embodiments, the purified protein is contacted with an extract from a transgenic plant comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0018] In additional embodiments, the method for producing siamenoside I comprises a protein having at least 95% sequence identity to SEQ ID NO:2 is from yeast cells comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2 operably linked to a 4US_ACTIVE\131477155\V-1heterologous promoter grown in a broth, removing the yeast cells from the broth to produce a yeast cell-free broth, and contacting the yeast cell-free broth with an extract of a plant that produces mogroside V. In some embodiments, the plant that produces mogroside V comprises at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0019] In further embodiments, the method for producing siamenoside I comprises contacting yeast cells comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 operably linked to a heterologous promoter with an extract of a plant that produces mogroside V. In certain embodiments, the plant that produces mogroside V comprises at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

[0020] The present disclosure also provides a method for producing 11-oxo siamenoside I, comprising contacting a composition comprising 11-oxo mogroside V with a protein having at least 95% identity to SEQ ID NO:2. In certain embodiments, the composition comprising 11-oxo mogroside V is produced by a transgenic cell. In some embodiments, the transgenic cell is atransgenic plant cell. In other embodiments, the transgenic plant cell is a transgenic lettuce, cucumber or tomato plant cell. In additional embodiments, the transgenic cell is a transgenic bacterial or yeast cell. In further embodiments, the transgenic yeast cell is a transgenic Saccharomyces cerevisiae or Pichia pastoris yeast cell.BRIEF DESCRIPTION OF THE SEQUENCES

[0021] SEQ ID NO:1: Nucleic acid sequence of exoglucanase from Wickerhamomyces ciferrii.

[0022] SEQ ID NO:2: Amino acid sequence of exoglucanase from Wickerhamomyces ciferrii.

[0023] SEQ ID NO:3: Nucleic acid sequence of exoglucanase from Eremothecium gossypii.

[0024] SEQ ID NO: 4: Amino acid sequence of exoglucanase from Eremothecium gossypii.

[0025] SEQ ID NO:5: Nucleic acid sequence of exoglucanase from Kazachstania africana.

[0026] SEQ ID NO:6: Amino acid sequence of exoglucanase from Kazachstania africana.

[0027] SEQ ID NO:7: Nucleic acid sequence of exoglucanase from Kazachstania naganishii.5US_ACTIVE\131477155\V-1

[0028] SEQ ID NO: 8: Amino acid sequence of exoglucanase from Kazachstania naganishii.

[0029] SEQ ID NO:9: Nucleic acid sequence of exoglucanase from Lachancea dasiensis.

[0030] SEQ ID NO: 10: Amino acid sequence of exoglucanase from Lachancea dasiensis.

[0031] SEQ ID NO: 11: Nucleic acid sequence of exoglucanase from Ogataea angusta.

[0032] SEQ ID NO: 12: Amino acid sequence of exoglucanase from Ogataea angusta.

[0033] SEQ ID NO: 13: Nucleic acid sequence of exoglucanase horn Pichia kudriavzevii.

[0034] SEQ ID NO: 14: Amino acid sequence of exoglucanase from Pichia kudriavzevii.

[0035] SEQ ID NO: 15: Nucleic acid sequence of exoglucanase from Saccharomyces cerevisiae with amino acidN361W mutation.

[0036] SEQ ID NO: 16: Amino acid sequence of exoglucanase from Saccharomyces cerevisiae with amino acidN361W mutation.

[0037] SEQ ID NO: 17: Nucleic acid sequence of exoglucanase from Brettanomyces bruxellensis.

[0038] SEQ ID NO: 18: Amino acid sequence of exoglucanase from Brettanomyces bruxellensis.

[0039] SEQ ID NO: 19: Nucleic acid sequence of His Tagged exoglucanase from Wickerhamomyces ciferrii optimized for Pichia expression.

[0040] SEQ ID NO:20: Amino acid sequence of His Tagged exoglucanase from Wickerhamomyces ciferrii.

[0041] SEQ ID NO:21: Nucleic acid sequence of plant expression vector for exoglucanase from Wickerhamomyces ciferrii.

[0042] SEQ ID NO:22: Nucleic acid sequence of yeast expression vector for exoglucanase from Wickerhamomyces ciferrii.

[0043] SEQ ID NO:23: Nucleic acid sequence of SP5425 expression vector.

[0044] SEQ ID NO:24: Amino acids 359-368 (active site loop) of exoglucanase from Saccharomyces cerevisiae.

[0045] SEQ ID NO:25: Amino acids of active site loop of exoglucanase from Wickerhamomyces ciferrii.

[0046] SEQ ID NO:26: Amino acids 359-368 (active site loop) of exoglucanase from Kazachstania naganishii.

[0047] SEQ ID NO:27: Amino acids 359-368 (active site loop) of exoglucanase from Kazachstania africana.6US_ACTIVE\131477155\V-1

[0048] SEQ ID NO:28: Amino acids 359-368 (active site loop) of exoglucanase from Lachancea dasiensis.

[0049] SEQ ID NO:29: Amino acids 359-368 (active site loop) of exoglucanase from Eremothecium gossypii.

[0050] SEQ ID NO:30: Amino acids 359-368 (active site loop) of exoglucanase from Ogataea angusta.

[0051] SEQ ID NO:31: Amino acids 359-368 (active site loop) of exoglucanase from Pichia kudriavzevii.

[0052] SEQ ID NO:32: Amino acids 359-368 (active site loop) of exoglucanase from Brettanomyces bruxellensis.

[0053] SEQ ID NO:33: Nucleic acid sequence of exoglucanase from Wickerhamomyces ciferrii. with His tag optimized for expression in Saccharomyces cerevisiae.

[0054] SEQ ID NO:34: Nucleic acid sequence of exoglucanase from Wickerhamomyces ciferrii. with alpha factor optimized for expression in Saccharomyces cerevisiae.

[0055] SEQ ID NO: 35: Amino acid sequence of exoglucanase from Wickerhamomyces ciferrii. with alpha factor.

[0056] SEQ ID NO:36: Nucleic acid sequence of exoglucanase from Wickerhamomyces ciferrii. with alpha factor and His tag optimized for expression in Saccharomyces cerevisiae.

[0057] SEQ ID NO: 37: Amino acid sequence of exoglucanase from Wickerhamomyces ciferrii. with alpha factor and His tag.

[0058] SEQ ID NO: 38: DNA sequence of Exoglucanase mature peptide from Pichia pastoris.

[0059] SEQ ID NO:39: Amino acid sequence of Exoglucanase mature peptide from Pichia pastoris identified by homology analysis.DETAILED DESCRIPTION

[0060] The present disclosure generally describes compositions and methods for making siamenoside I. The following sections provide embodiments that describe the subject matter in greater detail.I. Siamenoside I

[0061] Siamenoside I (SI) is a cucurbitane glycoside, a natural sweetener from the fruit of Siraitia grosvenorii. SI is the sweetest cucurbitane glycoside, with a relative sweetness that is 563 times higher than sucrose. The discovery of SI is of great interest to the food and beverage7US_ACTIVE\131477155\V-1industry because SI has superior sweetness to mogroside V and its mouthfeel properties are nearly identical to sucrose. Nevertheless, SI only represents about 2-5% of the sweet mogrosides in a typical Monk Fruit extract. Attempts to convert mogroside V into siamenoside I through chemical hydrolysis, enzyme treatments, or microbial fermentation have had limited success due to the complexity of the glucose branching sidechains in mogrosides.

[0062] Provided herein are exemplary nucleic acid and protein sequences for certain exoglucanase enzymes for conversion of mogroside V to siamenoside I. In certain embodiments, vectors comprising one or more of the disclosed exoglucanase genes are cotransformed with one or more vectors comprising one or more mogroside synthesis genes, which can produce mogroside V. In such embodiments, the mogroside V thus produced is converted to siamenoside I by the exoglucanase enzyme(s).

[0063] The enzymes of the mogroside pathway include, but are not limited to, squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxy or epoxide hydrolase (EPH), various cytochrome P450 enzymes (CYP), including, but not limited to, CYP72 and CYP87, uridine phosphorylase dependent glycosyltransferase enzymes (UGT). including, but not limited to, UGT720, UGT94 and UGT74, and can additionally include 3 hydroxy-3-methylglutaryl-CoA reductase (HMGR), or truncated versions thereof, NADPH: cytochrome P450 reductase (CPR2), and various upstream terpenoid biosynthetic enzymes (HMGS and CPS).II. Recombinant Host Cells and Species

[0064] Host cells and organisms are envisioned for use in certain embodiments of the present disclosure. As used herein, the term “recombinant host cell'’ is intended to refer to any host cell whose genome has been engineered to include at least one of the presently disclosed exoglucanase sequences (and in certain embodiments mogroside biosynthetic pathway nucleic acid sequences), which in certain embodiments encode one or more polypeptides. These sequences include, but are not limited to, nucleic acid or amino acid sequences that are not naturally present in the host cell or organism, DNA sequences that are not normally transcribed into RNA or translated into a protein ("expressed"), and other sequences that have been altered from those normally present in the host cell, for example by increasing the copy number of the DNA sequence or altering the expression patterns or expression levels.

[0065] A number of prokaryotes and additional eukaryotes are suitable for use as recombinant hosts in different aspects of the present disclosure. The recombinant host cells may be a bacteria, yeast or fungi or plant cells. A host cell or species selected for exoglucanase8US_ACTIVE\131477155\V-1compound production can be analyzed to determine if any exoglucanase biosynthetic pathway genes are endogenous to the host cell or species.

[0066] Exemplary prokaryotic and eukaryotic species useful in certain aspects of the present disclosure include, but are not limited to, Agaricus, Aspergillus, Auxanochlorella, Bacillus, Candida, Chlorella, Corynebacterium, Escherichia, Fusarium / Gibberella, Kluyveromyces, Laeliporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitella, Prototheca. Rhodotorula, Saccharomyces. Sphaceloma. Schizosaccharomyces, Xanthophyllomyces and Yarrowia, in addition to a variety of plant species. In some embodiments, a recombinant host may be a microorganism, for example Pichia pastoris, Schizosaccharomyces pombe, Aspergillus niger, or Saccharomyces cerevisiae. In some embodiments, a recombinant host may be a microorganism such as Escherichia coli or Agrobacterium tumefaciens. It will be appreciated that certain microorganisms can be used to screen and test genes of interest in a high throughput manner, while other microorganisms with desired productivity or growth characteristics can be used for large-scale production of exoglucanase compounds. In certain embodiments food grade microorganisms may be useful for large-scale production purposes.III. Nucleic Acid and Polypeptide Sequences

[0067] Certain embodiments of the current disclosure concern nucleic acid sequences (polynucleotides) and the corresponding amino acid sequences (proteins or polypeptides) for exoglucanase genes. Complements to any nucleic acid or protein sequences described herein are also provided.

[0068] " Identity," as is well understood in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as determined by the match betw een strings of such sequences. Methods to determine "identity" are designed to give the largest match between the sequences tested. Moreover, methods to determine identity are codified in publicly available programs. " Identity" can be readily calculated by any of the many methods known to those of skill in the art. Computer programs can be used to determine "identity" betw een two sequences. These programs include, but are not limited to, GCG; suite of five BLAST programs, three designed for nucleotide sequences queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, NCBI NLM NIH, Bethesda,9US_ACTIVE\131477155\V-1Md. 20894). The well-known Smith Waterman algorithm can also be used to determine identity.

[0069] In accordance with the present disclosure, a polynucleotide or polypeptide sequence as described herein may exhibit at least from about 34%, 40%, 50%, 60%, 62% or 70% to about 100% sequence identity to at least one of the sequences set forth herein. For example, in one embodiment, an exoglucanase gene as described herein may comprise, for example, 34%, 35%, 36%. 37%. 38%. 39%. 40%. 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs:l, 3, 5. 7, 9, 11, 13, 15, 17, 19, 33, 34, 36 or 38, or a complement thereof. In other embodiments, an exoglucanase enzyme or protein as described herein may comprise for example, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%. 58%. 59%. 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%. 72%. 73%. 74%. 75%. 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity' to a sequence selected from SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 35, 37 or 39.

[0070] Parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch (J. Mol. Biol. 48:443-453, 1970); Comparison matrix: BLOSUM62 from Henikoff and Henikoff, (Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program that can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison WI. The above parameters along with no penalty for end gap may serve as default parameters for peptide comparisons.

[0071] Parameters for nucleic acid sequence comparison include the following: Algorithm: Needleman and Wunsch (supra)-, Comparison matrix: matches=+10; mismatches=0; Gap Penalty: 50; and Gap Length Penalty: 3. A program that can be used with these parameters is publicly available as the "gap" program from Genetics Computer Group, Madison Wis. The above parameters may serve as the default parameters for nucleic acid comparisons.

[0072] As used herein, "hybridization," "hybridizes," or "capable of hybridizing" is understood to mean the forming of a double- or triple-stranded molecule or a molecule with partial double- or triple-stranded nature. Such hybridization may take place under relatively 10US_ACTIVE\131477155\V-1high-stringency conditions, including low salt and / or high temperature conditions, such as provided by a wash in about 0.02 M to about 0.15 M NaCl at temperatures of about 50°C to about 70°C for 10 min. In one embodiment of the present disclosure, the conditions are 0.15 M NaCl and 70°C. Stringent conditions tolerate little mismatch between a nucleic acid and a target strand. Such conditions are well-known to those of ordinary' skill in the art and are preferred for applications requiring high selectivity. Non-limiting applications include isolating a nucleic acid, such as a gene or a nucleic acid segment thereof or detecting at least one specific mRNA transcript or a nucleic acid segment thereof, and the like. Also included may be a protein or polypeptide, or fragment thereof, such as any of those set forth herein.

[0073] " Fragment", with respect to the nucleic acid sequences disclosed herein, refers to any part of a polynucleotide molecule that retains a usable, functional characteristic. Useful fragments include oligonucleotides and polynucleotides that may be used as probes or primers in hybridization or amplification technologies or in the regulation of replication, transcription or translation.fide fragment re fers to any subs iquence of a polynucleotide, typically, of at least about 15 consecutive nucleotides, at least about 16 consecutive nucleotides, at least about 17 consecutive nucleotides, at least about 18 consecutive nucleotides, at least about 19 consecutive nucleotides, at least about 20 consecutive nucleotides, at least about 21 consecutive nucleotides, at least about 22 consecutive nucleotides, at least about 23 consecutive nucleotides, at least about 24 consecutive nucleotides, at least about 25 consecutive nucleotides, at least about 30 consecutivenucleotides, at 1 last about 40 coileotides, at least about 45 consecutive nucleotides, or at least about 50 nucleotides or more, of any of the nucleic acid sequences provided herein.

[0074] Fragments may also include subsequences of polypeptides and protein molecules, or a subsequence of the polypeptide, as disclosed herein. Fragments may have antigenic potential or may be a subsequence of the polypeptide that performs at least one biological function of the intact polypeptide in substantially the same manner, or to a similar extent, as does the intact polypeptide. Fragments can vary in size from as few as 5 amino acids to the full length of the intact polypeptide, but are preferably at least about 10 amino acids in length, at least about 15 amino acids in length, at least about 20 amino acids in length, at least about 25 amino acids in length, at least about 30 amino acids in length, at least about 35 amino acids in length, at least about 40 amino acids in length, at least about 45 amino acids in length, at least about 50 amino acids in length, at least about 55 amino acids in length, or at least about 60 amino acids in length or more, of any of the ammo acid sequences provided herein.11US_ACTIVE\131477155\V-1

[0075] The nucleic acids provided herein as SEQ ID NOs:l, 3, 5, 7, 9, 11, 13, 15, 17, 19, 33, 34, 36 or 38, and amino acids provided herein as SEQ ID NOs: 2, 4. 6, 8, 10, 12, 14, 16, 18, 20, 35, 37 or 39 may be from any source, e.g., identified as naturally occurring, or synthesized, e.g., by mutagenesis of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 33, 34, 36 or 38, for example to create a coding sequence with a G / C content more like the G / C content of naturally occurring genes from a particular plant, fungi or algae. The naturally occurring sequence may be from any plant, fungal or algal species, as described herein.IV. Transformation Constructs

[0076] Vectors used for transformation of other host cells or organisms, may include, for example, plasmids, cosmids. YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes) or any other suitable cloning system, as well as fragments of DNA therefrom. Thus, when the term “vector” or “expression vector” is used, all of the foregoing types of vectors, as well as nucleic acid sequences isolated therefrom, are included. It is contemplated that utilization of cloning systems with large insert capacities will allow introduction of large DNA sequences comprising more than one selected gene. In accordance with the present disclosure, this could be used to introduce genes corresponding to an entire biosynthetic pathway into a host cell or organism. Introduction of such sequences may be facilitated by use of bacterial or yeast artificial chromosomes (BACs or YACs, respectively), or even plant artificial chromosomes. For example, the use of BACs for Agrobacterium-mediated transformation was disclosed by Hamilton et al. (Proc. Natl. Acad. Sci. USA 93:9975-9979, 1996).

[0077] Particularly useful for transformation are expression cassettes that have been isolated from such vectors. DNA segments used for transforming plant, fungal or algal cells will, of course, generally comprise the cDNA, gene or genes that one desires to introduce into and have expressed in the host cells. These DNA segments can further include structures such as promoters, enhancers, polylinkers, terminators or even regulatory genes as desired. The DNA segment or gene chosen for cellular introduction will often encode a protein that will be expressed in the resultant recombinant cells resulting in a screenable or selectable trait and / or that will impart an improved phenotype to the resulting transgenic cells. However, this may not always be the case, and the present disclosure also encompasses transgenic organisms incorporating non-expressed transgenes. As previously discussed, in addition to plant cells, the host cells in certain aspects of the present disclosure may be a bacterial cell, such as Escherichia colt or Agrobacterium tumefaciens, yeast cells, fungal, algal or cyanobacterial12US_ACTIVE\131477155\V-1cells. The skilled artisan is aware of the genetic elements that must be present on a vector in order to successfully transform, select and propagate host cells containing a sequence of interest. Components that may be included with vectors used in the current disclosure are as follows.A. Promoters and Other Regulatory Elements

[0078] In certain embodiments, the presently disclosed expression cassettes further comprise one or more promoters. In addition to the promoters disclosed herein, other exemplary promoters for expression of a nucleic acid sequence include a plant promoter such as the CaMV 35S promoter (Odell et al., Nature 313:810-812, 1985), or others such as CaMV 19S (Lawton et al.. Plant Mol. Biol. 9:315-324, 1987), nos (Ebert et al., Proc. Natl. Acad. Sci. USA 84:5745-5749, 1987), Adh (Walker et al., Proc. Natl. Acad. Sci. USA 84:6624-6628, 1987), sucrose synthase (Yang and Russell, Proc. Natl. Acad. Sci. USA 87:4144-4148, 1990), a-tubulin, actin (Wang et al., Mol. Cell Biol. 12:3399-3406, 1992), cab (Sullivan et al., Mol. Gen. Genet. 215:431-440, 1989), PEPCase (Hudspeth and GnAa, Plant Mol. Biol. 12:579-589, 1989) or those associated with the R gene complex (Chandler et al.. Plant Cell 1:1175-1183, 1989). In one embodiment of the present disclosure, the native promoter of an exoglucanase gene is used. In some embodiments, the promoter is a strong promoter or a w eak promoter.

[0079] The DNA sequence between the transcription initiation site and the start of the coding sequence, i.e., the untranslated leader sequence, can also influence gene expression. One may thus wish to employ a particular leader sequence with a transformation construct of the present disclosure. Leader sequences are contemplated to include those that comprise sequences predicted to direct optimum expression of the attached gene, i.e., to include a consensus leader sequence that may increase or maintain mRNA stability and prevent inappropriate initiation of translation. The choice of such sequences will be known to those of skill in the art in light of the present disclosure. Sequences that are derived from genes that are highly expressed in plants may be desirable.B. Terminators

[0080] In certain embodiments, the presently disclosed expression cassettes further comprise one or more terminators. Transformation constructs prepared in accordance with the present disclosure will typically include a 3' end DNA sequence that acts as a signal to terminate transcription and allow for the polyadenylation of the mRNA produced by coding sequences operably linked to a promoter. In one embodiment of the present disclosure, the13US_ACTIVE\131477155\V-1native terminator of an exoglucanase coding sequence is used. Alternatively, a heterologous 3’ end may enhance the expression of sense or antisense exoglucanase coding sequences. In addition to the terminator sequences disclosed herein, further examples of terminators that are deemed to be useful in this context include native terminators, the terminators from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3’ end) (Bevan et al., Nucl. Acids Res. 11:369-385. 1983), the terminator for the T7 transcript from the octopine synthase gene of Agrobacterium tumefaciens, and the 3' end of the protease inhibitor I or II genes from potato or tomato. Regulatory elements such as an Adh intron (Callis et al., Genes Dev. 1:1183-1200, 1987), sucrose synthase intron (Vasil et al.. Plant Physiol. 91:1575-1579, 1989) or TMV omega element (Gallie and Kado, Proc. Natl. Acad. Sci. USA 86:129-132, 1989), may further be included where desired.C. Transit or Signal Peptides

[0081] In certain embodiments of the present disclosure transit or signal sequences may be incorporated into the exoglucanase coding sequences. Sequences that are joined to the coding sequence of an expressed gene, which are removed post-translationally from the initial translation product and that facilitate the transport of the protein into or through intracellular or extracellular membranes, are termed transit (usually into vacuoles, vesicles, plastids and other intracellular organelles) and signal sequences (usually to the endoplasmic reticulum, golgi apparatus and outside of the cellular membrane). By facilitating the transport of the protein into compartments inside and outside the cell, these sequences may increase the accumulation of gene product protecting them from proteolytic degradation. These sequences also allow for additional mRNA sequences from highly expressed genes to be attached to the coding sequence of the genes. Since mRNA being translated by ribosomes is more stable than naked mRNA, the presence of translatable mRNA in front of the gene may increase the overall stability of the mRNA transcript from the gene and thereby increase synthesis of the gene product. Since transit and signal sequences are usually post-translationally removed from the initial translation product, the use of these sequences allows for the addition of extra translated sequences that may not appear on the final polypeptide. It further is contemplated that targeting of certain proteins may be desirable in order to enhance the stability of the protein (U. S. Patent No. 5,545,818, incorporated herein by reference in its entirety).

[0082] Additionally, vectors may be constructed and employed in the intracellular targeting of a specific gene product within the cells of a transgenic organism or in directing a protein to the extracellular environment. This generally will be achieved by joining a DNA14US_ACTIVE\131477155\V-1sequence encoding a transit or signal peptide sequence to the coding sequence of a particular gene. The resultant transit, or signal, peptide will transport the protein to a particular intracellular, or extracellular destination, respectively, and will then be post-translationally removed.D. Marker Genes

[0083] By employing a selectable or screenable marker protein, one can provide or enhance the ability to identify transformants. “Marker genes” are genes that impart a distinct phenotype to cells expressing the marker protein and thus allow such transformed cells to be distinguished from cells that do not have the marker. Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait that one can “select” for by chemical means, z.e., through the use of a selective agent (e.g, a herbicide, antibiotic, or the like), or whether it is simply a trait that one can identify' through observation or testing, i.e., by “screening” (e.g, the green fluorescent protein). In addition to the marker genes described above, many additional examples of suitable marker proteins are known to the art and can be employed in the practice of the present disclosure.

[0084] Included within the terms “selectable” or “screenable” markers also are genes that encode a “secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that are secretable antigens that can be identified by antibody interaction, or even secretable enzymes that can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, e.g., by ELISA; small active enzymes detectable in extracellular solution (e.g, a-amylase, (B-lactamase, phosphinothricin acetyltransferase); and proteins that are inserted or trapped in the cell wall (e.g, proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR S).

[0085] Many selectable marker coding regions are known and could be used with the present disclosure including, but not limited to, neo (Potrykus etal., Mol. Gen. Genet. 199:169-177, 1985), which provides kanamycin resistance and can be selected for using kanamycin, G418, paromomycin, etc:, bar, which confers bialaphos or phosphinothricin resistance; a mutant EPSP synthase protein conferring glyphosate resistance; a nitrilase such as bxn from Klebsiella ozaenae, which confers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314, 1988); a mutant acetolactate synthase (ALS), which confers resistance to imidazolinone. sulfonylurea or other ALS inhibiting chemicals (European Patent Application 154,204, 1985); a methotrexate resistant DHFR (Thillet et al., J. Biol. Chem. 263:12500-12508,15US_ACTIVE\131477155\V-11988), a dalapon dehalogenase that confers resistance to the herbicide dalapon; a mutated anthranilate synthase that confers resistance to 5-methyl tryptophan, sequences that confer resistance to dicamba; or a bleomycin binding protein (ble) that confers resistance to antibiotics such as bleomycin, phleomycin, and Zeocin.

[0086] An illustrative embodiment of selectable marker capable of being used in plant systems to select transformants are those that encode the enzyme phosphinothricin acetyltransferase, such as the bar gene from Streptomyces hygroscopicus or the pat gene from Streptomyces viridochromogenes. The enzyme phosphinothricin acetyltransferase (PAT) inactivates the active ingredient in the herbicide bialaphos, phosphinothricin (PPT). PPT inhibits glutamine synthetase, causing rapid accumulation of ammonia and cell death.

[0087] Screenable markers that may be employed include a [3 glucuronidase (GUS) or uidA gene, which encodes an enzyme for which various chromogenic substrates are known; an R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues; a P lactamase gene (Sutcliffe, Proc. Natl. Acad. Sci. USA 75:3737-3741, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl. Acad. Sci. USA 80:1101-1105, 1983), which encodes a catechol dioxygenase that can convert chromogenic catechols; an a-amylase gene (Ikuta et al., Biotechnology 8:241-242, 1990); a tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714, 1983), which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone, which in turn condenses to form the easily-detectable compound melanin; a galactosidase gene, which encodes an enzyme for which there are chromogenic substrates; a luciferase (lux) gene (Ow et al., Science 234:856-859, 1986), which allows for bioluminescence detection; an aequorin gene (Prasher et al., Biochem. Biophys. Res. Commun. 126:1259-1268, 1985), which may be employed in calcium-sensitive bioluminescence detection; or a gene encoding for green fluorescent protein (GFP; Sheen et al., Plant J. 8:777-784, 1995; Haseloff et al., Proc. Nad. Acad. Sci. USA 94:2122-2127, 1997; Reichel et al., Proc. Natl. Acad. Sci. USA 93:5888-5893, 1996; WO 97 / 41228) is also contemplated as a useful reporter gene. Expression of green fluorescent protein and other related fluorescent proteins such as mCherry may be visualized in a cell as fluorescence following illumination by particular wavelengths of light.V. Gene Editing

[0088] One method for producing the transgenic bacterial, fungal or plant organisms of the present disclosure is through genome modification using site-specific integration or genome16US_ACTIVE\131477155\V-1editing. Targeted modification of genomes through the use of genome editing methods can be used to create improved strains through modification of genomic DNA. As used herein ■‘site-directed integration” refers to genome editing methods that enable targeted insertion of one or more nucleic acids of interest into a genome. Suitable methods for altering a wild-type DNA sequence or a preexisting transgenic sequence or for inserting DNA into a genome at a pre-determined chromosomal site include any method known in the art. Exemplary methods include the use of sequence specific nucleases, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucl eases, or RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 system, a CRISPR / Cpfl system, a CRISPR / CasX system, a CRISPR / CasY system, or a CRISPR / Cascade system). Several embodiments relate to methods of genome editing by using single-stranded oligonucleotides to introduce precise base pair modifications in a genome. Methods of genome editing to modify, delete, or insert nucleic acid sequences into genomic DNA are known in the art.

[0089] In certain embodiments, the present disclosure provides modification or replacement of an existing coding sequence, such as an existing transgenic insert, within a genome with a sequence encoding a different protein, or an expression cassette comprising such a protein. Several embodiments relate to the use of a known genome editing methods, such as zinc-finger nucleases, engineered or native meganucleases, TALE-endonucleases, or an RNA-guided endonucleases (for example, a Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 system, a CRISPR / Cpfl system, a CRISPR / CasX system, a CRISPR / CasY system, or a CRISPR / Cascade system).

[0090] Several embodiments may therefore relate to a recombinant DNA construct comprising an expression cassette(s) encoding a site-specific nuclease and, optionally, any associated protein(s) to carry out genome modification. These nuclease-expressing cassette(s) may be present in the same molecule or vector as a donor template for templated editing. Several methods for site-directed integration are known in the art involving different sequencespecific nucleases (or complexes of proteins or guide RNA or both) that cut the genomic DNA to produce a double strand break (DSB) or nick at a desired genomic site or locus. As understood in the art, during the process of repairing the DSB or nick introduced by the nuclease enzyme, the donor template DNA, transgene, or expression cassette may become integrated into the genome at the site of the DSB or nick. The presence of the homology arm(s) in the DNA to be integrated may promote the adoption and targeting of the insertion sequence into the genome during the repair process through homologous recombination, although an 17US_ACTIVE\131477155\V-1insertion event may occur through non-homologous end joining (NHEJ). As used herein, the term “double-strand break inducing agent” refers to any agent that can induce a double-strand break (DSB) in a DNA molecule. In some embodiments, the double-strand break inducing agent is a site-specific genome modification enzyme.

[0091] As used herein, the term “site-specific genome modification enzyme” refers to any enzyme that can modify a nucleotide sequence in a sequence-specific manner. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a single-strand break. In some embodiments, a site-specific genome modification enzyme modifies the genome by inducing a double-strand break. In some embodiments, a site-specific genome modification enzyme comprises a cytidine deaminase. In some embodiments, a sitespecific genome modification enzyme comprises an adenine deaminase. Site-specific genome modification enzymes include endonucleases, recombinases, transposases, deaminases, helicases and any combination thereof. In some embodiments, the site-specific genome modification enzyme is a sequence-specific nuclease.

[0092] In one aspect, the endonuclease is selected from a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (nonlimiting examples of Argonaute proteins include Thermits thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), and Natronobcicterium gregoryi Argonaute (NgAgo)), an RNA-guided nuclease, such as a CRISPR associated nuclease (non-limiting examples of CRISPR associated nucleases include, but are not limited to, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2. Csf3, Csf4, Cpfl, CasX, CasY, homologs thereof, or modified versions thereof).

[0093] In some embodiments, the site-specific genome modification enzyme is a recombinase. Non-limiting examples of recombinases include a ty rosine recombinase attached to a DNA recognition motif and is selected from the group consisting of a Cre recombinase, a Gin recombinase, a Flp recombinase, and a Tnpl recombinase. In one aspect, a Cre recombinase or a Gin recombinase is tethered to a zinc-finger DNA-binding domain, or a TALE DNA binding domain, or a Cas9 nuclease. In another aspect, a serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. In another aspect, a DNA transposase18US_ACTIVE\131477155\V-1attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.

[0094] Any DNA of interest as provided herein can be integrated into a target site of a chromosome sequence by introducing the DNA of interest and the disclosed site-specific genome modification enzymes. Any method provided herein can utilize any site-specific genome modification enzyme disclosed herein.VI. Antisense and RNAi Constructs

[0095] Antisense and RNAi treatments represent one way of altering exoglucanase gene activity or genes in competing pathways in accordance with the present disclosure (e.g., by down regulation of genes or transcription factors that inhibit expression of such genes).

[0096] Techniques for RNAi are well known in the art and are described in, for example, Lehner et al., (Brief Funct. Genomic Proteomic 3:68-83, 2004) and Downward (BMJ 328: 1245-1248, 2004). The technique is based on the fact that double stranded RNA is capable of directing the degradation of messenger RNA with sequence complementary to one or the other strand (Fire et al., Nature 391:806-811, 1998). Therefore, by expression of a particular coding sequence in sense and antisense orientation, either as a fragment or longer portion of the corresponding coding sequence, the expression of that coding sequence can be downregulated.

[0097] Antisense, and in some aspects RNAi, methodology takes advantage of the fact that nucleic acids tend to pair with “complementary” sequences. By complementary, it is meant that polynucleotides are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G: C) and adenine paired with either thymine (A: T) in the case of DNA, or adenine paired with uracil (A: U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

[0098] Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense oligonucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and / or stability. Antisense and RNAi constructs, or DNA encoding such RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo. In certain embodiments of the present disclosure, such an oligonucleotide may comprise any unique portion of a nucleic19US_ACTIVE\131477155\V-1acid sequence provided herein. In certain embodiments of the present disclosure, such a sequence comprises at least 18, 20, 25, 30, 50, 75 or 100 or more contiguous nucleic acids of a nucleic acid sequence of interest, and / or complements thereof, which may be in sense and / or antisense orientation. By including sequences in both sense and antisense orientation, increased suppression of the corresponding coding sequence may be achieved.

[0099] Constructs may be designed that are complementary to all or part of the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective constructs may include regions complementary to intron / exon splice junctions. Thus, it is proposed that one embodiment includes a construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

[0100] As stated above, “complementary” or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very' few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences that are completely complementary will be sequences that are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology' also are contemplated. For example, an RNAi or antisense construct that has limited regions of high homology, but also contains a non-homologous region (e.g, ribozyme) could be designed. Methods for selection and design of sequences that generate RNAi are well known in the art (e g, Reynolds et al., Nat. Biotechnol. 22:326-330, 2004). These molecules, though having less than 50% homology', would bind to target sequences under appropriate conditions.

[0101] It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone may be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence. Constructs useful for20US_ACTIVE\131477155\V-1generating RNAi may also comprise concatemers of sub-sequences that display gene regulating activity.VII. Transformation

[0102] In some embodiments, transgenic bacterial, fungal, algal or plant organisms of the present disclosure are created by transforming the selected natural bacterial, fungal, algal or plant organisms with one or more of the expression cassettes disclosed herein. The natural bacterial, fungal, algal or plant organisms prior to transformation do not naturally produce the desired exoglucanase enzyme(s).

[0103] Suitable methods for transformation of bacterial, fungal, algal or plant cells for use with the current disclosure are believed to include virtually any method by which DNA can be introduced into a cell, such as by direct delivery of DNA such as by PEG-mediated transformation of protoplasts (Omirulleh et al., Plant. Mol. Biol. 21:414-428, 1993), by desiccation / inhibition-mediated DNA uptake (Potrykus et al., Mol. Gen. Genet. 199:169-177, 1985), by electroporation (U. S. Patent No. 5,384,253, specifically incorporated herein by reference in its entirety), by agitation with silicon carbide fibers (U. S. Patent Nos. 5,302,523 and 5,464,765, specifically incorporated herein by reference in their entirety), by Agrobacterium-mediated transformation (U. S. Patent No. 5,591,616 and U. S. Patent No.5,563,055; both specifically incorporated herein by reference in their entirety) and by acceleration of DNA coated particles (U. S. Patent No. 5,550.318; U. S. Patent No. 5,538,877; and U. S. Patent No. 5,538,880; each specifically incorporated herein by reference in their entirety), etc. Through the application of techniques such as these, the cells of virtually any species may be stably transformed, and these cells developed into transgenic organisms. A. Agrobacterium-mediated Transformation

[0104] Agrobacterium-mediated transfer is a widely applicable system for introducing genes into plant cells. See, for example, the methods described by Fraley et al.. (Proc. Natl. Acad. Sci. USA 80:4803-4807, 1985), and U. S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety.21US_ACTIVE\131477155\V-1

[0105] Modem Agrobacterium transformation vectors are capable of replication in E. coli as well as Agrobacterium, allowing for convenient manipulations. Moreover, recent technological advances in vectors for Agrobacterium-mediated gene transfer have improved the arrangement of genes and restriction sites in the vectors to facilitate the construction of vectors capable of expressing various polypeptide coding genes. The vectors have convenient multi-linker regions flanked by a promoter and a polyadenylation site for direct expression of inserted polypeptide coding genes and are suitable for present purposes. In addition, Agrobacterium containing both armed and disarmed Ti genes can be used for the transformations.B. Electroporation

[0106] In this technique, one would partially degrade the cell walls of the chosen cells by exposing them to pectin-degrading enzymes (pectolyases) or mechanically wounding in a controlled manner. Examples of some plant species that have been transformed by electroporation of intact cells include maize (U. S. Patent No. 5,384,253, incorporated herein by reference in its entirety; Rhodes et al., Methods Mol. Biol. 55:121-131, 1995; D’Halluin et al., Plant Cell 4:1495-1505, 1992), wheat (Zhou et al., Plant Cell Rep. 12:612-616, 1993), tomato (Tsukada et al., Plant Cell Physiol. 30:599-603, 1989), soybean (Christou et al., Proc. Nall. Acad. Sci. USA 84:3962-3966, 1987) and tobacco (Riggs and Bates, Proc. Natl. Acad. Sci. USA 83:5602-5606, 1986).

[0107] One also may employ protoplasts for electroporation transformation of plants (Bates, Mol. Biotechnol. 2:135-145, 1994; Lazzeri, Methods Mol. Biol. 49:95-106, 1995) and algal cells (Caisova and Jobe, Plant Methods 15, 74 (2019). https: / / doi.org / 10.1186 / sl3007-019-0460-6). For example, the generation of transgenic soybean plants by electroporation of cotyledon-derived protoplasts is described in WO 9217598 (specifically incorporated herein by reference). Other examples of species for which protoplast transformation has been described include barley (Lazzeri, supra), sorghum (Battraw etal., Theor. Appl. Genet. 82:161-168, 1991). maize (Rhodes et al.. Science 240:204-207, 1988). wheat (He etal., Plant Cell Rep.14: 192-196, 1994) and tomato (Tsukada, supra).C. Microprojectile Bombardment

[0108] Another method for delivering transforming DNA segments to plant, fungal, algal or bacterial cells in accordance with the present disclosure is microprojectile bombardment (U. S. Patent No. 5,550,318; U. S. Patent No. 5,538,880; U. S. Patent No. 5,610,042; and PCT22US_ACTIVE\131477155\V-1Application WO 94 / 09699; each of which is specifically incorporated herein by reference in its entirety). In this method, particles may be coated with nucleic acids and delivered into cells by a propelling force. Exemplary particles include those comprised of tungsten, platinum, and often, gold. It is contemplated that in some instances DNA precipitation onto metal particles would not be necessary for DNA delivery to a recipient cell using microprojectile bombardment. However, it is contemplated that particles may contain DNA rather than be coated with DNA. Hence, it is proposed that DNA-coated particles may increase the level of DNA delivery via particle bombardment but are not, in and of themselves, necessary.

[0109] For the bombardment, cells in suspension are concentrated on filters or solid culture medium. The cells to be bombarded are positioned at an appropriate distance below the macroprojectile stopping plate.

[0110] An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System, which can be used to propel particles coated with DNA or cells through a screen, such as a stainless steel or Nytex screen, onto a filter surface covered with monocot plant cells cultured in suspension. The screen disperses the particles so that they are not delivered to the recipient cells in large aggregates. Microprojectile bombardment techniques are widely applicable. Examples of plant species that have been transformed by microprojectile bombardment include monocot species such as maize (PCT Application WO 95 / 06128), barley (Ritala et al., Plant Mol. Biol. 24:317-325, 1994; Hensgens et al.. Plant Mol. Biol. 22:1101-1127. 1993). wheat (U. S. Patent No.5,563,055, specifically incorporated herein by reference in its entirety), rice (Hensgens et al., supra), oat (Torbet et al.. Crop Science 38:226-231, 1998), rye (Hensgens et al., supra), sugarcane (Bower et al., Plant J. 2:409-416, 1992), and sorghum (Casas et al., Proc. Natl. Acad. Sci. USA 90:11212-11216, 1993; Hagio et al.. Plant Cell Rep. 10:260-264, 1991); as well as a number of dicots including tobacco (Tomes et al., PlantMol. Biol. 14:261-268, 1990), soybean (U. S. Patent No. 5,322,783, specifically incorporated herein by reference in its entirety), sunflower (Knittel et al., Plant Cell Rep. 14:81-86, 1994), peanut (Singsit et al., Transgenic Res. 6:169-176, 1997), cotton (McCabe and Martinell, Nat. Biotechnol. 11:596-598, 1993). tomato (VanEck et al., Plant Cell. Rep. 14:299-304, 1995). switchgrass (Richards et al.. Plant Cell Rep. 20:48-54, 2001), and legumes in general (U. S. Patent No. 5,563,055, specifically incorporated herein by reference in its entirety).23US_ACTIVE\131477155\V-1D. Other Transformation Methods

[0111] Transformation of protoplasts can be achieved using methods based on calcium phosphate precipitation, polyethylene glycol treatment, electroporation, and combinations of these treatments see, e.g, Potrykus et al., supra,' Omirulleh et al., supra,''). Application of these systems to different organisms depends upon the ability to regenerate that particular organism from protoplasts. Illustrative methods for the regeneration of cereals from protoplasts have been described (Toriyama et al., Nat. Biotechnol. 6:1072-1074, 1988; Abdullah et al., Nat. Biotechnol. 4:1087-1090. 1986; Omirulleh et al., supra, and U. S. Patent No. 5,508,184; each specifically incorporated herein by reference in its entirety). Examples of the use of direct uptake transformation of cereal protoplasts include transformation of rice (Ghosh-Biswas et al., J. Biotechnol. 32:1-10, 1994), sorghum (Battraw etal., supra), barley (Lazzeri, supra), oat, and maize (Omirulleh et al., supra).VIII. Production and Characterization of Stably Transformed Cells

[0112] After effecting delivery of exogenous DNA to recipient cells, the next steps generally concern identifying the transformed cells for further culturing and scale-up. In order to improve the ability to identify transformants, one may desire to employ a selectable or screenable marker gene with a transformation vector prepared in accordance with the present disclosure. In this case, one would then generally assay the potentially transformed cell population by exposing the cells to a selective agent or agents, or one would screen the cells for the desired marker gene trait.A. Selection

[0113] It is believed that DNA is introduced into only a small percentage of target cells in any one study. In order to provide an efficient system for identification of those cells receiving DNA and integrating it into their genomes one may employ a means for selecting those cells that are stably transformed. One exemplary embodiment of such a method is to introduce into the host cell, a marker gene that confers resistance to some normally inhibitory agent, such as an antibiotic or herbicide. Examples of antibiotics that may be used include the aminoglycoside antibiotics neomycin, kanamycin and paromomycin, or the antibiotic hygromycin. Resistance to the aminoglycoside antibiotics is conferred by aminoglycoside phosphotransferase enzymes such as neomycin phosphotransferase II (NPT II) or NPT I, whereas resistance to hygromycin is conferred by hygromycin phosphotransferase.24US_ACTIVE\131477155\V-1

[0114] Potentially transformed cells then are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA.

[0115] One herbicide that constitutes a desirable selection agent is the broad spectrum herbicide bialaphos. Bialaphos is a tripeptide antibiotic produced by Streptomyces hygr os copious and is composed of phosphinothricin (PPT), an analogue of L-glutamic acid, and two L-alanine residues. Upon removal of the L-alanine residues by intracellular peptidases, the PPT is released and is a potent inhibitor of glutamine synthetase (GS), a pivotal enzyme involved in ammonia assimilation and nitrogen metabolism (Ogawa et al., Sci. Rep. Meiji Seiko 13:42-48, 1973). Synthetic PPT, the active ingredient in the herbicide Liberty™ also is effective as a selection agent. Inhibition of GS in plants by PPT causes the rapid accumulation of ammonia and death of the plant cells.

[0116] The organism producing bialaphos and other species of the genus Streptomyces also synthesizes an enzyme phosphinothricin acetyl transferase (PAT), which is encoded by the bar gene in Streptomyces hygroscopicus and the pat gene in Streptomyces viridochromogenes. The use of the herbicide resistance gene encoding phosphinothricin acetyl transferase (PAT) is referred to in DE 3642 829 A, wherein the gene is isolated from Streptomyces viridochromogenes. In the bacterial source organism, this enzyme acetylates the free amino group of PPT preventing auto-toxicity (Thompson et al., EMBO J. 6:2519-2523, 1987). The bar gene has been cloned (Thompson et al., supra) and expressed in transgenic tobacco, tomato, potato (De Block et al., EMBO J. 6:2513-2518, 1987) Brassica (De Block et al., Plant Physiol.91:694-701, 1989) and maize (U. S. Patent No. 5,550,318, incorporated herein by reference in its entirety).

[0117] Another example of a herbicide that is useful for selection of transformed cell lines in the practice of the present disclosure is the broad spectrum herbicide glyphosate. Glyphosate inhibits the action of the enzyme EPSPS, which is active in the aromatic amino acid biosynthetic pathway. Inhibition of this enzyme leads to starvation for the amino acids phenylalanine, tyrosine, and tryptophan and secondary metabolites derived thereof. U. S. Patent No. 4,535,060 (incorporated herein by reference in its entirety) describes the isolation of EPSPS mutations that confer glyphosate resistance on the Salmonella typhimurium gene for EPSPS, aroA. The EPSPS gene was cloned from Zea mays and mutations similar to those found in a glyphosate resistant aroA gene were introduced in vitro. Mutant genes encoding25US_ACTIVE\131477155\V-1glyphosate resistant EPSPS enzymes are described in, for example, International Patent WO 97 / 4103.

[0118] To use the bar-bialaphos or the EPSPS-glyphosate selective system, transformed tissue is cultured for 0-28 days on nonselective medium and subsequently transferred to medium containing from 1-3 mg / 1 bialaphos or 1-3 mM glyphosate as appropriate. While ranges of 1 -3 mg / 1 bialaphos or 1 -3 mM glyphosate may be beneficial, it is proposed that ranges of 0.1-50 mg / 1 bialaphos or 0.1-50 mM glyphosate will find utility.

[0119] An example of a screenable marker trait is the enzyme luciferase. In the presence of the substrate luciferin, cells expressing luciferase emit light that can be detected on photographic or x-ray film, in a luminometer (or liquid scintillation counter), by devices that enhance night vision, or by a highly light sensitive video camera, such as a photon counting camera. These assays are nondestructive and transformed cells may be cultured further following identification. The photon counting camera is especially valuable as it allows one to identify specific cells or groups of cells that are expressing luciferase and manipulate those in real time. Another screenable marker that may be used in a similar fashion is the gene coding for green fluorescent protein or similar sequences such as mCherry.B. Characterization

[0120] To confirm the presence of the exogenous DNA or “transgene(s)” in the transformed cells, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays, such as Southern and northern blotting and PCR™; “biochemical” assays, such as detecting the presence of a protein product, e.g., by immunological means (ELISAs and western blots) or by enzymatic function; and also, by analyzing the growth and phenotype of the recovered cells.C. DNA Integration, RNA Expression and Inheritance

[0121] Genomic DNA may be isolated from transformed cell lines to determine the presence of the exogenous gene through the use of techniques well known to those skilled in the art. Note, that intact sequences will not always be present, presumably due to rearrangement or deletion of sequences in the cell. The presence of DNA elements introduced through the methods of this disclosure may be determined, for example, by polymerase chain reaction (PCR™). Using this technique, discreet fragments of DNA are amplified and detected by gel electrophoresis. This type of analysis permits one to determine whether a gene is present in a stable transformant but does not prove integration of the introduced gene into the host cell26US_ACTIVE\131477155\V-1genome. It is typically the case, however, that DNA has been integrated into the genome of all transformants that demonstrate the presence of the gene through PCR™ analysis. In addition, it is not typically possible using PCR™ techniques to determine whether transformants have exogenous genes introduced into different sites in the genome, i.e., whether transformants are of independent origin. It is contemplated that using PCR™ techniques it would be possible to clone fragments of the host genomic DNA adjacent to an introduced gene.

[0122] Positive proof of DNA integration into the host genome and the independent identities of transformants may be determined using the technique of Southern hybridization. Using this technique specific DNA sequences that were introduced into the host genome and flanking host DNA sequences can be identified. Hence the Southern hybridization pattern of a given transformant serves as an identifying characteristic of that transformant. In addition, it is possible through Southern hybridization to demonstrate the presence of introduced genes in high molecular weight DNA, i.e., confirm that the introduced gene has been integrated into the host cell genome. The technique of Southern hybridization provides information that is obtained using PCR™, e.g, the presence of a gene, but also demonstrates integration into the genome and characterizes each individual transformant.

[0123] It is contemplated that using the techniques of dot or slot blot hybridization, which are modifications of Southern hybridization techniques, one could obtain the same information that is derived from PCR™, e.g., the presence of a gene.

[0124] PCR™ techniques also may be used for detection and quantitation of RNA produced from introduced genes. In this application of PCR™ it is first necessary to reverse transcribe RNA into DNA, using enzymes such as reverse transcriptase, and then through the use of conventional PCR™ techniques amplify the DNA. In most instances PCR™ techniques, while useful, will not demonstrate integrity of the RNA product. Further information about the nature of the RNA product may be obtained by northern blotting. This technique will demonstrate the presence of an RNA species and give information about the integrity of that RNA. The presence or absence of an RNA species also can be determined using dot or slot blot northern hybridizations. These techniques are modifications of northern blotting and will only demonstrate the presence or absence of an RNA species. Real-time or qPCR is another rapid and valuable method to quantify RNA expression levels.D. Gene Expression

[0125] While Southern blotting and PCR™ may be used to detect the gene(s) in question, they do not provide information as to whether the corresponding protein is being expressed.27US_ACTIVE\131477155\V-1Expression may be evaluated by specifically identifying the protein products of the introduced genes or evaluating the phenotypic changes brought about by their expression.

[0126] Assays for the production and identification of specific proteins may make use of physical-chemical, structural, functional, or other properties of the proteins. Unique physical-chemical or structural properties allow the proteins to be separated and identified by electrophoretic procedures, such as native or denaturing gel electrophoresis or isoelectric focusing, or by chromatographic techniques such as ion exchange or gel exclusion chromatography. The unique structures of individual proteins offer opportunities for use of specific antibodies to detect their presence in formats such as an ELISA assay. Combinations of approaches may be employed with even greater specificity such as western blotting in which antibodies are used to locate individual gene products that have been separated by electrophoretic techniques. Additional techniques may be employed to absolutely confirm the identify of the product of interest such as evaluation by amino acid sequencing following purification. Although these are among the most commonly employed, other procedures may be additionally used.

[0127] Assay procedures also may be used to identify the expression of proteins by their functionality, especially the ability of enzymes to catalyze specific chemical reactions involving specific substrates and products. These reactions may be followed by providing and quantifying the loss of substrates or the generation of products of the reactions by physical or chemical procedures. Examples are as varied as the enzyme to be analyzed and may include assays for PAT enzymatic activity by following production of radiolabeled acetylated phosphinothricin from phosphinothricin and ¹⁴C-acetyl CoA or for anthranilate synthase activity by following loss of fluorescence of anthranilate, to name two.

[0128] Very frequently the expression of a gene product is determined by evaluating the phenotypic results of its expression. These assays also may take many forms including, but not limited to, analyzing changes in the chemical composition, morphology, or growth characteristics of the transformed cells. Chemical composition may be altered by expression of genes encoding enzymes or storage proteins that change amino acid composition and may be detected by amino acid analysis, or by enzymes that change starch quantity, which may be analyzed by near infrared reflectance spectrometry.IX. Purification of Exoglucanase

[0129] In some embodiments, the present disclosure relates generally to purification of the disclosed exoglucanase proteins from a transgenic organism producing the desired28US_ACTIVE\131477155\V-1exoglucanase protein. An exoglucanase can be isolated from a biological sample of transgenic organism expressing an exoglucanase by a variety of means. The methods generally encompass steps that can include, but are not limited to, pre-treatment cleaning and homogenizing of the transgenic organisms, extraction of the transgenic organisms, sedimentation and / or centrifuge, adsorption and / or separation, concentration and recovery to produce the crude exoglucanase, further purification, optional concentration / drying, and formulation. Means of extraction encompasses water-extraction at room temperatures, or heated temperature, or refrigerated temperature; extraction via organic solvent such as alcohol, etc. Means of separation and purification encompasses centrifuge, steeping, gravity sedimentation, filtration, micro-filtration, nano-filtration, ultra-filtration, reverse osmosis, chromatography, absorption chromatogram, exchanged resin purification, etc.

[0130] In certain embodiments, purification of an exoglucanase protein can be achieved by engineering a histidine tag at the N- or C-terminus of the exoglucanase. Histidine-tagged proteins are recombinant proteins designed to include a polyhistidine tail (his-tag) that facilitates purification of the proteins. The preferential binding of the his-tag to metal chelating resins has been exploited in purifying his-tagged proteins from undesired contaminating proteins using immobilized metal affinity’ chromatography (IMAC). Metal chelating resins typically include a transition metal such as Ni or Co.

[0131] In other embodiments, an exoglucanase can be isolated via ammonium sulfate fractionation (e.g.. Kohl method of ammonium fractionation). As another example, a composition can be extracted from a biological sample, such as serum or plasma, by organic solvent(s). The composition can be extracted by a series of organic solvents. An organic solvent extract can be evaporated to produce a powder or crystalline form of the composition. A solvated composition can be further purified by evaporation of the solvent and further extraction with water. A water extract can be evaporated to produce a powder or crystalline form of the composition. A dried form of the composition can be re-solubilized in, for example, water, saline, or a pharmaceutically acceptable oil (e.g., mineral oil). In some embodiments, a composition described herein is isolated via an automatic titration machine. In some embodiments, isolation methods produce a composition having at least about 60% purity. For example, isolation methods can produce a composition having at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% purity.29US_ACTIVE\131477155\V-1X. Siamenoside I Containing Sweeteners and Consumables

[0132] In some embodiments, the present disclosure relates generally to a sweetener or sweetening composition comprising siamenoside I and / or metabolites or derivatives thereof, wherein the sweetener or sweetening composition is derived from a transgenic organism producing the desired siamenoside I. The term "sweetener", as used herein, refers to a consumable product that produces a sweet taste when consumed alone. In certain embodiments, the sweetener or sweetening composition is derived from the transgenic organisms made according to the present disclosure. In some embodiments the sweeteners are high intensity or low intensity sweeteners. Siamenoside I-containing sweeteners can be derived from the transgenic organisms of the present disclosure upon appropriate processing. The resulting sweeteners could be used to provide low or non-caloric sweetness for many purposes. Examples of such uses to provide sweetness are in beverages, such as tea, coffee, fruit juice, and fruit beverages, foods, such as jams and jellies, peanut butter, pies, puddings, cereals, candies, ice creams, yogurts, bakery products; health care products, such as toothpastes, mouthwashes, cough drops, cough syrups; chewing gums; and sugar substitutes, including powdered or granular sugar substitutes.

[0133] In certain embodiments, the sweetener is in a liquid extract from a transgenic organism according to the present disclosure. Applications for liquid extract, containing siamenoside I include, but are not limited to, as a beverage, including, for example, premixed cocktails and dairy alternatives, as an ingredient, for example to be sprayed onto bars or cereal, or used to sweeten ketchup or other common products. In such embodiments the liquid extract can be devitalized, have the protein removed or concentrated. Additionally, a concentrated liquid preparation, produced from the presently disclosed transgenic organisms, can be used to substitute for high fructose com syrup in various foods and beverages. In some embodiments of the present disclosure the siamenoside I can be used to produce a dry formulation which can then be used, for example, to produce a wide variety’ of lower calorie food products.

[0134] In some embodiments, the present disclosure also relates to methods of making a sweetener derived from the presently disclosed transgenic organisms producing siamenoside I. The methods generally encompass steps that can include, but are not limited to, pre-treatment cleaning and homogenizing of the transgenic organisms, extraction of the transgenic organisms, sedimentation and / or centrifuge, adsorption and / or separation, concentration and recovery to produce the crude sweetener, further purification, optional concentration / drying, and formulation. Means of extraction encompasses water-extraction at room temperatures, or30US_ACTIVE\131477155\V-1heated temperature, or refrigerated temperature; extraction via organic solvent such as alcohol, etc. Means of separation and purification encompasses centrifuge, steeping, gravity sedimentation, filtration, micro-filtration, nano-filtration, ultra-filtration, reverse osmosis, chromatography, absorption chromatogram, exchanged resin purification, etc.

[0135] In further embodiments the presently disclosed transgenic organisms can be processed to produce siamenoside I-containing ingredients, for example by extraction, aqueous separation of small molecules having a siamenoside I fraction, removal of residual proteins to yield an aqueous fraction free from any genetically engineered components. The resulting siamenoside I-containing ingredient(s) can be in any form, including, but not limited to, a powder, liquid, syrup, concentrate or extract. Additionally in some embodiments a whole siamenoside I-containing fruit or vegetable is the consumable.

[0136] Additionally the siamenoside I produced by the presently disclosed transgenic organisms can be blended with one or more other naturally occurring or artificial sweeteners, such as steviol glycosides, a-siamenoside I, sucrose, glucose, fructose, lactose, maltose, sorbitol, galactose, thaumtin. sucrooctate, bemadame, sucrononic acid, carrelame, lugduname, high fructose com syrup, RealSweet™ Sugarcane RebM, erythritol, xylitol, yacon syrup, allulose, saccharin, aspartame, acesulfame potassium, sucralose, neotame, advantame, cyclamates or glycyrrhizin. The ratio of siamenoside I to the other sweetener in the final formulation can be, for example, 10 / 90, 20 / 80. 30 / 70, 40 / 60 / 50 / 50, 60 / 40, 70 / 30, 80 / 20 or 90 / 10, or any other desired ratio. In one embodiment, the ratio is about 40% siamenoside I, about 40% mogroside V and about 20% 11 -oxo-mogroside V.

[0137] In certain embodiments, the one or more additional sweeteners may be a carbohydrate sweetener. Non-limiting examples of suitable carbohydrate sweeteners include sucrose, fructose, glucose, erythritol, maltitol, lactitol, sorbitol, mannitol, xylitol, tagatose, trehalose, galactose, rhamnose, cyclodextrin (e.g., a-cyclodextrin, (3-cyclodextrin, and y-cyclodextrin), ribulose, threose, arabinose, xylose, lyxose, allose, altrose, mannose, idose, lactose, maltose, invert sugar, isotrehalose, neotrehalose, palatinose or isomaltulose, ery throse, deoxyribose, gulose, idose, talose, erythrulose, xylulose, psicose, turanose, cellobiose, glucosamine, mannosamine, fucose, fuculose, glucuronic acid, gluconic acid, glucono-lactone, abequose, galactosamine, xylo-oligosaccharides (xylotriose, xylobiose and the like), gentio-oligoscaccharides (gentiobiose, gentiotriose, gentiotetraose and the like), galactooligosaccharides, sorbose, ketotriose (dehydroxy acetone), aldotriose (glyceraldehyde), nigero-oligosaccharides, fructooligosaccharides (kestose. nystose and the like), maltotetraose, maltotriol, tetrasaccharides, mannan-oligosaccharides, malto-oligosaccharides (maltotriose.31US_ACTIVE\131477155\V-1maltotetraose, maltopentaose, maltohexaose, maltoheptaose and the like), dextrins, lactulose, melibiose, raffinose, rhamnose, ribose, isomerized liquid sugars such as high fructose com / starch syrup (HFCS / HFSS) (e.g., HFCS55, HFCS42, or HFCS90), coupling sugars, soybean oligosaccharides, glucose syrup and combinations thereof. D- or L-configurations can be used when applicable. In other embodiments, the additional sweetener is a carbohydrate sweetener selected from the group consisting of glucose, fructose, sucrose and combinations thereof. In another embodiment, the additional sweetener is a carbohydrate sweetener selected from D-allose, D-psicose, L-ribose, D-tagatose, L-glucose, L-fucose, L-Arabinose, Turanose and combinations thereof.

[0138] In yet other embodiments, the one or more additional sweeteners is not directly derived from a natural extraction. Such a sweetener characteristically has a sweetness potency greater than sucrose, fructose, or glucose, yet has less calories. Non-limiting examples of such sweeteners suitable for embodiments of this disclosure include sucralose, potassium acesulfame, acesulfame acid and salts thereof, aspartame, alitame, saccharin and salts thereof, neohesperidin dihydrochalcone, cyclamate, cyclamic acid and salts thereof, neotame, advantame. glucosylated steviol glycosides (GSGs) and combinations thereof. The at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration from about 0.3 ppm to about 3,500 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration from about 0.5 ppm to about 3,000 ppm, from about 1.0 ppm to about 2,500 ppm, from about 5.0 ppm to about 2,000 ppm, from about 10 ppm to about 1,500 ppm, from about 50 ppm to about 1000 ppm, from about 100 ppm to about 800 ppm. or from about 400 ppm to about 600 ppm when present in a sweetened beverage. In another embodiment, the at least one embodiment, the at least one sweetener not directly derived from natural extraction is present in the sweetener composition in an amount effective to provide a concentration greater than about 0.3 ppm, greater than about 0.5 ppm, greater than about 1.0 ppm. greater than about 5.0 ppm, greater than about 10 ppm, greater than about 20 ppm, greater than about 50 ppm. greater than about 100 ppm, greater than about 250 ppm, greater than about 500 ppm or greater than about 1000 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage.

[0139] In still other embodiments, the additional sweetener can be a natural high potency sweetener. Suitable natural high potency sweeteners include, but are not limited to.32US_ACTIVE\131477155\V-1rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside I, rebaudioside H, rebaudioside L, rebaudioside K, rebaudioside J, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside A, dulcoside B, rubusoside, Stevia, stevioside, mogroside IV, mogroside V, Luo Han Guo, miraculin, monatin and its salts (monatin SS, RR, RS, SR), curculin, glycyrrhizic acid and its salts, thaumatin, monellin, mabinlin, brazzein. hemandulcin, phyllodulcin, glycyphyllin, phloridzin, trilobatin, baiyunoside, osladin, polypodoside A, pterocaryoside A, pterocaryoside B, mukurozioside, phlomisoside I, periandrin T, abrusoside A, steviolbioside and cyclocarioside I. The natural high potency sweetener can be provided as a pure compound or, alternatively, as part of an extract. For example, rebaudioside A can be provided as a sole compound or as part of a Stevia extract. The natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration from about 0.1 ppm to about 3,000 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration from about 0.5 ppm to about 2500 ppm, from about 1.0 ppm to about 2000 ppm, from about 5 ppm to about 1500 ppm, from about 10 ppm to about 1000 ppm, or about 25 ppm to about 500 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage. In one embodiment, the natural high potency sweetener is present in the sweetener composition in an amount effective to provide a concentration of greater than about 0.1 ppm, about 0.5 ppm, about 1.0 ppm, about 2.5 ppm, about 5.0 ppm, about 10 ppm, about 20 ppm, about 25 ppm, about 50 ppm, about 75 ppm, about 100 ppm, about 200 ppm, about 500 ppm, about 1000 ppm, about 2000 ppm, or about 300 ppm when present in a sweetened composition, such as, for example, a food, other consumable or beverage.

[0140] In still other embodiments, the additional sweetener can be chemically or enzymatically modified natural high potency sweetener. Modified natural high potency sweeteners include glycosylated natural high potency sweetener such as glucosyl-, galactosyl-, or fructosyl-derivatives containing 1-50 glycosidic residues. Glycosylated natural high potency sweeteners may be prepared by an enzymatic transglycosylation reaction catalyzed by various enzymes possessing transglycosylating activity.

[0141] When the sweetener composition contains more than one sweetener, the sweeteners may exhibit synergy when combined and have improved flavor and temporal profiles compared to each sweetener alone. As used herein, the term "temporal profile" of a composition means the intensity of sweetness perceived over time in tasting of a composition by a human. The 33US_ACTIVE\131477155\V-1term "flavor profile" or "taste profile." as generally used herein, refers to the intensity of various flavor / taste attributes of a sweetener or sweetened composition. Exemplary’ flavor / taste attributes are sweetness intensity, bitterness intensity, salty intensity, licorice intensity', cooling intensity, and licorice intensity. Methods of determining the flavor profile of a given sweetener or sweetened composition are known in the art. The term "synergistic" or "synergistic effect" refers to an effect (e.g.. flavor, temporal profile) achieved with the combination of two or more sweeteners which is greater than the sum of the effects that effect from using the particular sweeteners alone or separately. Advantageously, such synergy between the two or more sweeteners allows for the use of smaller doses of one or both sweeteners or provides greater effect at the same amounts. The amount or degree of synergism may vary.

[0142] The amount of sucrose in a reference solution may be described in degrees Brix (°Bx). One degree Brix is 1 gram of sucrose in 100 grams of solution and represents the strength of the solution as percentage by weight (% w / w) (strictly speaking, by mass). In one embodiment, a sweetener composition contains one or more of the presently disclosed sweetener compounds in an amount effective to provide sweetness equivalent from of at least about 5 degrees Brix of sugar when present in a sweetened composition, such as, for example, from at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14 or at least about 15 or more degrees Brix.

[0143] The sweetness of anon-sucrose sweetener can also be measured against a sucrose reference by determining the non-sucrose sweetener's sucrose equivalence. Typically, taste panelists are trained to detect sweetness of reference sucrose solutions containing between 1-15% sucrose (w / v). Other non-sucrose sweeteners are then tasted at a series of dilutions to determine the concentration of the non-sucrose sweetener that is as sweet as a given percent sucrose reference. For example, if a 1% solution of a sweetener is as sweet as a 10% sucrose solution, then the sweetener is said to be 10 times as potent as sucrose.

[0144] The sweetener compositions can be customized to provide the desired calorie content. For example, sweetener compositions can be "full-calorie", such that they impart the desired sweetness when added to a sweetenable composition (such as, for example, a food, other consumable or beverage) and have about 120 calories per 8 oz. serving. Alternatively, sweetener compositions can be "mid-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than about 60 calories per 8 oz. serving. In other embodiments, sweetener compositions can be "low-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than 40 calories per 34US_ACTIVE\131477155\V-18 oz. serving. In still other embodiments, the sweetener compositions can be "zero-calorie", such that they impart the desired sweetness when added to a sweetenable composition and have less than 5 calories per 8 oz. serving.

[0145] The presently disclosed the sweetener compositions can optionally include one or more additional additives. In some embodiments, the sweetener composition contains additives including, but not limited to, carbohydrates, polyols, amino acids and their corresponding salts, poly-amino acids and their corresponding salts, sugar acids and their corresponding salts, nucleotides, organic acids, inorganic acids, organic salts including organic acid salts and organic base salts, inorganic salts, bitter compounds, flavorants and flavoring ingredients, astringent compounds, proteins or protein hydrolysates, surfactants, emulsifiers, weighing agents, gums, antioxidants, colorants, flavonoids, alcohols, polymers and combinations thereof. In some embodiments, the additives act to improve the temporal and flavor profile of the sweetener to provide a sweetener composition with a taste similar to sucrose. The sweetened compositions can contain one or more functional ingredients, as detailed above. Functional ingredients include, but are not limited to, vitamins, minerals, antioxidants, preservatives, glucosamine, polyphenols and combinations thereof. Any suitable functional ingredient described herein can be used.

[0146] There is a beneficial environmental impact of the presently disclosed siamenoside I sweeteners compared to existing monkfruit production. The presently disclosed siamenoside I sweeteners enables local production, resulting in less transportation and fewer food miles as a result, compared to for example harvesting and processing only in China and shipping across the globe to food companies. The presently disclosed siamenoside I sweeteners also require minimal processing because of the ease of access to the siamenoside I sweeteners in the presently disclosed transgenic plants or organisms compared to, for example, factory processing of monkfruit sweetener in China.XI. Sweetened Compositions

[0147] The presently disclosed sweetener compositions can be incorporated in any known edible material (referred to herein as a "sweetenable composition"), such as, for example, pharmaceutical compositions, edible gel mixes and compositions, dental compositions, foodstuffs (confections, condiments, chewing gum, cereal compositions baked goods dairy products, and tabletop sweetener compositions) beverages and beverage products.

[0148] The sweetened compositions disclosed here include beverages, i.e.. ready to drink liquid formulations, beverage concentrates and the like. In certain embodiments, beverage35US_ACTIVE\131477155\V-1concentrates are prepared with an initial volume of liquid (e.g., water) to which the additional ingredients are added. Full strength beverage compositions can be formed from the beverage concentrate by adding further volumes of liquid (e.g., water) to the concentrate.

[0149] The presently disclosed siamenoside I-containing filler juice concentrate can be produced from consumer friendly organisms with sustainable domestic production, is the only sweetener for high sugar reduction while maintaining 100% j nice labeling, and is an affordable drop in solution. The filler juice may be used as single strength or concentrated to deliver clean, sweet taste across various inclusion levels. The filler juice siamenoside I concentrations can deliver the equivalent of ~10 sucrose equivalent value (SEV) when used at various formula inclusions.

[0150] Besides concentration, other juice parameters are also readily changeable, resulting in different sweetener products. For example, any natural sugars or other molecules may be partially or fully removed, the juice color and / or flavor may be minimized or removed, and the acidity may be reduced, or combinations of one or more of these parameters can be changed.

[0151] Filler juice applications include, but are not limited to, juices, nectars, fruit / flavored still drinks, energy and sports drinks, carbonated soft drinks, flavored waters, nutritional drinks, vitamins and dietary supplements or oral rehydration in the form of liquids or chews / gummies, snacks such as snack bars or fruit snacks, sugar and gum confectionary in the form of jellies and chews, dairy products such as spoonable yogurt, drinking yogurt and flavored drinks, desserts, ice cream, frozen yogurt, water-based ice pops and sorbets, breakfast cereals and other cold cereals, tabletop sweeteners, sweet spreads such as syrups and fruit spreads, sauces and seasonings such as table sauces and cooking sauces, and processed and packaged fruit and vegetables.

[0152] The presently disclosed siamenoside I-containing dry powder can be produced from organisms grown with sustainable production, results in sugar and calorie reduction with strong positive associations to health benefits, and is a fraction of monkl'ruit / parity with sucrose. The dry powder may be used across a wide range of food and drink applications to deliver the cleanest, sweetest taste at low inclusion levels. The dry powder concentrations can deliver the equivalent of ~10 SEV when used at various purity levels.

[0153] Dry powder application include, but are not limited to, wellness and functional drinks, such as energy and sports drinks, carbonated soft drinks, flavored waters, juices, nectars, fruit / flavored still drinks, protein and meal replacement drinks, drink mixes, drink concentrates, ready-to-drink tea and ready-to-drink coffee; dietary supplements and over-the-counter products, such as vitamins and dietary supplements, oral hydration, cold relief.36US_ACTIVE\131477155\V-1digestive treatments, sleep aids, pain relief in capsule, tablet, liquid, powder, chew / gummy, lozenge and other formats; snacks such as snack bars, fruit snacks, nuts, trail mixes, com rice, potato and wheat snacks; bakery products such as cookies, cakes and sweet goods, baking mixes and ingredients and breads; dairy and desserts such as spoonable and drinking yogurt, flavored drinks, creamers, ice cream and frozen yogurt, water-based ice pops and sorbets, shelfstable desserts and dessert toppings; hot and cold breakfast cereals; artificial and other natural sweeteners (tabletop sweeteners); sugar and chocolate confectionary such as jellies and chews, mints, gum, toffee and caramels, marshmallows and various chocolate formats; sweet spreads such as syrups, fruit, nut and chocolate spreads; sauces and seasonings such as table, cooking and pasta sauces, vinegar and dressings and pickled condiments; meals and processed meats such as prepared meals, meal kits, sandwiches and wraps and poultry’ and meat products; and processed and packaged fruit and vegetables.A. Beverage and Beverage Products

[0154] In one embodiment, the sweetened composition is a beverage or beverage product. " Beverage product", as used herein, is a ready-to-drink beverage, a beverage concentrate, a beverage syrup, or a powdered beverage. Suitable ready-to-drink beverages include carbonated and non-carbonated beverages. Carbonated beverages include, but are not limited to, frozen carbonated beverages, enhanced sparkling beverages, cola, fruit-flavored sparkling beverages (e.g. lemon-lime, orange, grape, strawberry and pineapple), ginger-ale. soft drinks and root beer. Non-carbonated beverages include, but are not limited to, fruit juice, fruit-flavored juice or water, juice drinks, nectars, fruit / flavored still drinks, energy’ and sports drinks, vegetable juice, vegetable-flavored juice, sports drinks, energy drinks, nutritional drinks, enhanced water drinks, enhanced water with vitamins, near water drinks (e.g., water with natural or synthetic flavorants), coconut water, tea type drinks (e.g., black tea, green tea, red tea, oolong tea), coffee, cocoa drink, beverage containing milk components (e.g, milk beverages, coffee containing milk components, cafe au lait, milk tea, fruit milk beverages), beverages containing cereal extracts and smoothies.

[0155] In certain embodiments, the beverage is a juice beverage that has been modified to remove at least some sucrose. In certain embodiments, such juice may be modified to remove at least 10%, at least 20%, at least 30%, at least 40%, at least 50% or more of the sucrose in the non-modified juice. In certain embodiments, the modification occurs through filtration of such juice to remove sucrose. In certain embodiments, sucrose in the juice is broken down to fructose and glucose, prior to adding the sweetening composition described herein.37US_ACTIVE\131477155\V-1

[0156] Beverages comprise a matrix, z.e., the basic ingredient in which the ingredients, including the compositions of the present disclosure, are dissolved. In one embodiment, a beverage comprises water of beverage quality as the matrix, such as, for example deionized water, distilled water, reverse osmosis water, carbon-treated water, purified water, demineralized water and combinations thereof, can be used. Additional suitable matrices include, but are not limited to phosphoric acid, phosphate buffer, citric acid, citrate buffer and carbon-treated water. Beverage concentrates and beverage syrups are prepared with an initial volume of liquid matrix (e.g., water) and the desired beverage ingredients. Full strength beverages are then prepared by adding further volumes of water. Powdered beverages are prepared by dry -mixing all of the beverage ingredients in the absence of a liquid matrix. Full strength beverages are then prepared by adding the full volume of water.

[0157] It is contemplated that the pH of the beverage does not materially or adversely affect the taste of the sweetener. A non-limiting example of the pH range of the beverage may be from about 1.8 to about 10. In one embodiment, the pH of the beverage is about 4. In another embodiment, the pH of the beverage is less than about 4. In a particular embodiment, the pH of the beverage is less than about 3.8, less than about 3.6, less than about 3.4, less than about 3.2, less than about 3.0, less than about 2.8, less than about 2.6, less than about 2.4 or less than about 2.2. In another embodiment, the pH of the beverage is about 3.8, about 3.6, about 3.4, about 3.2, about 3.0, about 2.8, about 2.6, about 2.4 or about 2.2 or less.B. Edible Gel Mixes and Edible Gel Compositions

[0158] In one embodiment, the sweetened composition is an edible gel or edible gel mix. Edible gels are gels that can be eaten. Non-limiting examples of edible gel compositions for use in particular embodiments include gel desserts, puddings, jellies, pastes, trifles, aspics, marshmallows, gummy candies / chews, or the like. Edible gel mixes generally are powdered or granular solids to which a fluid may be added to form an edible gel composition. Nonlimiting examples of fluids for use in particular embodiments include water, dairy fluids, dairy analogue fluids, juices, alcohol, alcoholic beverages, and combinations thereof. Non-limiting examples of dairy fluids which may be used in particular embodiments include milk, cultured milk, cream, fluid whey, and mixtures thereof. Non-limiting examples of dairy analogue fluids which may be used in particular embodiments include, for example, soy milk and non-dairy coffee whitener.38US_ACTIVE\131477155\V-1C. Confections

[0159] In one embodiment, the sweetened composition is a confection. As referred to herein, "confection" can mean a sweet, a lollie, a confectionery, or similar term. The confection generally contains a base composition component and a sweetener component. According to particular embodiments of the present disclosure, the confections may be desserts such as yogurt, jellies, drinkable jellies, puddings, Bavarian cream, blancmange, cakes, brownies, mousse and the like, sweetened food products eaten at tea time or following meals; frozen foods; cold confections, e.g., types of ice cream such as ice cream, ice milk, lacto-ice and the like, and ice confections such as sherbets, dessert ices and the like; general confections, e.g., baked confections or steamed confections such as crackers, biscuits, buns with bean-jam fdling, halvah, alfajor, and the like; rice cakes and snacks; table top products; general sugar confections such as chewing gum, hard candy, soft candy, mints, nougat candy, jelly beans, fudge, toffee, taffy, Swiss milk tablet, licorice candy, chocolates, gelatin candies, marshmallow, marzipan, divinity, cotton candy, and the like; sauces including fruit flavored sauces, chocolate sauces and the like; edible gels; cremes including butter cremes, flour pastes, whipped cream and the like; j ams including straw berry j am, marmalade and the like; and breads including sweet breads and the like or other starch products, and combinations thereof.D. Condiment Compositions

[0160] In one embodiment, the sweetened composition is a condiment composition. Condiments, as used herein, are compositions used to enhance or improve the flavor of a food or beverage. Non-limiting examples of condiments include ketchup, mustard, barbecue sauce, butter, chili sauce, chutney, cocktail sauce, curry, dips, fish sauce, horseradish, hot sauce, jellies, jams, marmalades, or preserves, mayonnaise; peanut butter, relish, remoulade, salad dressings, salsa, sauerkraut, soy sauce, steak sauce, syrups, tartar sauce, and Worcestershire sauce. Condiment bases generally comprise a mixture of different ingredients, non-limiting examples of which include vehicles (e.g., water and vinegar); spices or seasonings (e.g., salt, pepper, garlic, mustard seed, onion, paprika, turmeric, and combinations thereof); fruits, vegetables, or their products (e.g., tomatoes or tomato-based products (paste, puree), fruit juices, fruit juice peels, and combinations thereof); oils or oil emulsions, particularly vegetable oils; thickeners (e.g., xanthan gum, food starch, other hydrocolloids, and combinations thereof); and emulsifying agents (e.g., egg yolk solids, protein, gum arabic, carob bean gum, guar gum, gum karaya, gum tragacanth, carageenan, pectin, propylene glycol esters of alginic acid, sodium carboxymethyl-cellulose, polysorbates, and combinations thereof). Recipes for 39US_ACTIVE\131477155\V-1condiment bases and methods of making condiment bases are well known to those of ordinary skill in the art.E. Chewing Gum Compositions

[0161] In one embodiment, the sweetened composition is a chewing gum composition. Chewing gum compositions generally comprise a water-soluble portion and a water-insoluble chewable gum base portion. The water soluble portion dissipates with a portion of the flavoring agent over a period of time during chewing while the insoluble gum base portion is retained in the mouth. The insoluble gum base generally determines whether a gum is considered chewing gum, bubble gum, or a functional gum.

[0162] Flavoring agents may be used in either the insoluble gum base or soluble portion of the chewing gum composition. Such flavoring agents may be natural or artificial flavors. In a particular embodiment, the flavoring agent comprises an essential oil, such as peppermint oil, spearmint oil, other mint oils, clove oil, cinnamon oil, oil of wintergreen, bay, thyme, cedar leaf, nutmeg, allspice, sage, mace, and almonds. In another particular embodiment, the flavoring agent comprises an extract or an essence that is similar to the flavors extracted from apple, banana, watermelon, pear, peach, grape, strawberry, raspberry, cherry, plum, pineapple, apricot, and mixtures thereof. In still another particular embodiment, the flavoring agent comprises a citrus flavor that is similar to the oil of lemon, lime, orange, tangerine, grapefruit, citron, or kumquat.F. Cereal Compositions

[0163] In one embodiment, the sweetened composition is a cereal composition. Cereal compositions typically are eaten either as staple foods or as snacks. Non-limiting examples of cereal compositions for use in particular embodiments include ready-to-eat cereals as well as hot cereals. Ready-to-eat cereals are cereals which may be eaten without further processing (i.e., cooking) by the consumer. Examples of ready-to-eat cereals include breakfast cereals and snack bars. Breakfast cereals typically are processed to produce a shredded, flaky, puffy, or extruded form. Breakfast cereals generally are eaten cold and are often mixed with milk and / or fruit. Snack bars include, for example, energy bars, rice cakes, granola bars, and nutritional bars. Hot cereals generally are cooked, usually in either milk or water, before being eaten. Non-limiting examples of hot cereals include grits, porridge, polenta, rice, and rolled oats.

[0164] Cereal compositions generally comprise at least one cereal ingredient. As used herein, the term "cereal ingredient" denotes materials such as whole or part grains, whole or40US_ACTIVE\131477155\V-1part seeds, and whole or part grass. Non-limiting examples of cereal ingredients for use in particular embodiments include maize, wheat, rice, barley, bran, bran endosperm, bulgur, sorghums, millets, oats, rye, triticale, buckwheat, fonio, quinoa, bean, soybean, amaranth, teff, spelt, and kaniwa.G. Baked Goods

[0165] In one embodiment, the sweetened composition is a baked good. " Baked goods," as used herein, include ready to eat and all ready to bake products, flours, and mixes requiring preparation before serving. Non-limiting examples of baked goods include cakes, crackers, cookies, brownies, muffins, rolls, bagels, donuts, strudels, pastries, croissants, biscuits, bread, bread products, and buns.

[0166] Baked goods in accordance with particular embodiments of this disclosure generally comprise a combination of sweetener, water, fat and leavening agent. Baked goods made in accordance with many embodiments of this disclosure also contain flour in order to make a dough or a batter.

[0167] According to particular embodiments of this disclosure, leavening agents may comprise chemical leavening agents or yeast leavening agents. Non-limiting examples of chemical leavening agents suitable for use in particular embodiments of this disclosure include baking soda (e.g, sodium, potassium, or aluminum bicarbonate), baking acid (e.g., sodium aluminum phosphate, monocalcium phosphate, or dicalcium phosphate), and combinations thereof.H. Dairy Products

[0168] In one embodiment, the sweetened composition is a dairy product. Dairy products and processes for making dairy products suitable for use in this disclosure are well known to those of ordinary skill in the art. Dairy products, as used herein, comprise milk or foodstuffs produced from milk. Non-limiting examples of dairy products suitable for use in embodiments of this disclosure include milk, milk cream, sour cream, creme fraiche, buttermilk, cultured buttermilk, milk powder, condensed milk, evaporated milk, butter, cheese, cottage cheese, cream cheese, yogurt, ice cream, frozen custard, frozen yogurt, gelato, via, piima, filmjolk, kajmak, kephir, viili, kumiss, airag, ice milk, casein, ayran, lassi, khoa, or combinations thereof. The dairy products can be produced through conventional means or can be filtered or further modified to adjust the taste properties. In certain embodiments, the dairy products can be liquid dairy products from which one or more of the carbohydrate sugars (lactose or its41US_ACTIVE\131477155\V-1breakdown products galactose or glucose) are reduced as compared to milk prior to such processing, or are substantially removed and which are supplemented with the sweetening composition described herein. The reduction of carbohydrates can be about 5% or about 10% or about 20% or about 50% or about 70% or more as compared to unprocessed milk.

[0169] According to particular embodiments of this disclosure, the dairy compositions also may comprise other additives. Non-limiting examples of suitable additives include sweeteners as disclosed herein and flavorants such as chocolate, strawberry, and banana. Particular embodiments of the dairy compositions provided herein also may comprise additional nutritional supplements such as vitamins (e.g., vitamin D) and minerals (e.g, calcium) to improve the nutritional composition of the milk.I. Tabletop Sweetener Compositions

[0170] In one embodiment, the sweetened composition is a tabletop sweetener. The tabletop sweetener can further include at least one bulking agent, additive, anti-caking agent, functional ingredient or combination thereof.

[0171] Suitable "bulking agents" include, but are not limited to, maltodextrin (10 DE, 18 DE, or 5 DE), com syrup solids (20 or 36 DE), sucrose, fructose, glucose, invert sugar, sorbitol, xylose, ribulose, mannose, xylitol, mannitol, galactitol, erythritol. maltitol, lactitol, isomalt, maltose, tagatose. lactose, inulin, glycerol, propylene glycol, polyols, polydextrose, fructooligosaccharides, cellulose and cellulose derivatives, and the like, and mixtures thereof. Additionally, in accordance with still other embodiments of the present disclosure, granulated sugar (sucrose) or other caloric sweeteners such as crystalline fructose, other carbohydrates, or sugar alcohol can be used as a bulking agent due to their provision of good content uniformity without the addition of significant calories.

[0172] As used herein, the phrase "anti-caking agent" and "flow agent" refer to any composition which assists in content uniformity and uniform dissolution. In accordance with particular embodiments, non-limiting examples of anti-caking agents include cream of tartar, calcium silicate, silicon dioxide, microcrystalline cellulose (Avicel, FMC BioPolymer, Philadelphia, PA), and tricalcium phosphate. In one embodiment, the anti-caking agents are present in the tabletop sweetener composition in an amount from about 0.001 to about 3% by weight of the tabletop sweetener composition.

[0173] The tabletop sweetener compositions can be packaged in any form known in the art. Non-limiting forms include, but are not limited to. powder form, granular form, packets, tablets, sachets, pellets, cubes, solids, and liquids.42US_ACTIVE\131477155\V-1

[0174] In one embodiment, the tabletop sweetener composition is a single-serving (portion control) packet comprising a dry-blend. Dry-blend formulations generally may comprise powder or granules. Although the tabletop sweetener composition may be in a packet of any size, an illustrative non-limiting example of conventional portion control tabletop sweetener packets are approximately 2.5 by 1.5 inches and hold approximately 1 gram of a sweetener composition having a sweetness equivalent to 2 teaspoons of granulated sugar (.about.8 g). In a particular embodiment, a dry-blend tabletop sweetener formulation may contain a sweetener an amount from about 1% (w / w) to about 10% (w / w).

[0175] A tabletop sweetener composition also may be embodied in the form of a liquid, wherein a composition of the present disclosure is combined with a liquid carrier. Suitable non-limiting examples of carrier agents for liquid tabletop sweeteners include water, alcohol, polyol, glycerin base or citric acid base dissolved in water, and mixtures thereof. The sweetness equivalent of a tabletop sweetener composition for any of the forms described herein or known in the art may be varied to obtain a desired sweetness profile. For example, a tabletop sweetener composition may comprise a sweetness comparable to that of an equivalent amount of standard sugar. In another embodiment, the tabletop sweetener composition may comprise a sweetness of up to 100 times that of an equivalent amount of sugar. In another embodiment, the tabletop sweetener composition may comprise a sweetness of up to 90 times, 80 times, 70 times, 60 times, 50 times, 40 times, 30 times, 20 times, 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, and 2 times that of an equivalent amount of sugar.J. Delivery Systems

[0176] The presently disclosed sweetener compositions can also be formulated into various delivery systems having improved ease of handling and rate of dissolution. Non-limiting examples of suitable delivery systems comprise sweetener compositions co-crystallized with a sugar or a polyol, agglomerated sweetener compositions, compacted sweetener compositions, dried sweetener compositions, particle sweetener compositions, spheronized sweetener compositions, granular sweetener compositions, and liquid sweetener compositions.XII. Additional Definitions

[0177] The following definitions or interpretations of technical terms will be used throughout the present disclosure. The technical terms used herein are generally to be given the meaning commonly applied to them in the pertinent art of microbiology, phycology, plant43US_ACTIVE\131477155\V-1biology, molecular biology, and bioinformatics. All of the following term definitions apply to the complete content of this application.

[0178] To facilitate the understanding of this disclosure, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present disclosure. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one." but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and / or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

[0179] As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of' or "consisting of." As used herein, the phrase "consisting essentially of' requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method / process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method / process steps or limitation(s)) only.

[0180] The term "or combinations thereof' as used herein refers to all permutations and combinations of the listed items preceding the term. For example, " A, B, C, or combinations thereof' is intended to include at least one of: A, B, C, AB, AC, BC, or ABC. and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.44US_ACTIVE\131477155\V-1

[0181] As used herein, words of approximation such as, without limitation, "about," "substantial" or "substantially" refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skill in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as "about" may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.

[0182] The terms "peptides," "oligopeptides," "polypeptide," "protein", or "enzyme" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds, unless mentioned herein otherwise. The terms "gene sequence(s)," "polynucleotide(s)," "nucleic acid sequence(s)," "nucleotide sequence(s)," "nucleic acid(s)," "nucleic acid molecule" are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.

[0183] Endogenous. An "endogenous" or "native" nucleic acid and / or protein refers to a nucleic acid and / or protein as found in an organism in its natural form (z. e., without there being any human intervention, such as recombinant DNA engineering technology),

[0184] Exogenous. The term "exogenous" (in contrast to "endogenous") means a nucleic acid or protein that has been introduced in an organism by means of recombinant DNA technology'. An "exogenous" nucleic acid or protein can either not occur in an organism in its natural form, be different from the nucleic acid or protein as found in an organism in its natural form, be present at a higher or lower level than the nucleic acid or protein naturally present in an organism, or in the case of a nucleic acid can be identical to a nucleic acid found in an organism in its natural form, but integrated at a location different that its natural genetic environment.

[0185] Expression: The combination of intracellular processes, including transcription and translation undergone by a coding DNA molecule such as a structural gene to produce a polypeptide.

[0186] Expression Cassette. A nucleic acid sequence of interest operably linked to one or more control sequences (at least to a promoter) as described herein. An expression cassette can also include additional transcriptional and / or translational enhancers. An expression cassette can also include terminator, silencer and enhancer sequences, intron sequences added 45US_ACTIVE\131477155\V-1to the 5' untranslated region (UTR) or in the coding sequence of the nucleic acid sequence, and / or other control sequences such as protein and / or RNA stabilizing elements. An expression cassette may be integrated into the genome of a host cell and replicated together with the genome of said host cell, or transiently present in a host cell.

[0187] Genetic Transformation: A process of introducing a DNA sequence or construct (e.g., a vector or expression cassette) into a cell or protoplast in which that exogenous DNA is incorporated into a chromosome or is capable of autonomous replication.

[0188] Heterologous: A sequence that is not normally present in a given host genome in the genetic context in which the sequence is currently found. In this respect, the sequence may be native to the host genome, but be rearranged with respect to other genetic sequences within the host sequence. For example, a regulatory sequence may be heterologous in that it is linked to a different coding sequence relative to the native regulatory sequence.

[0189] Modulation. The term modulation refers to when the expression level is changed in comparison to the expression seen in a control plant or algae. Modulation refers to an expression level that is either increased or decreased.

[0190] Obtaining: When used in conjunction with atransgenic organism, obtaining means transforming anon-transgenic organism to create the transgenic organism.

[0191] Operably Linked. The term "operably linked" or "functionally linked" is used interchangeably and. as used herein, refers to a functional linkage between, for example, a promoter sequence and a nucleic acid sequence of interest, such that the promoter sequence is able to direct transcription of the nucleic acid sequence of interest, or a functional linkage between a terminator sequence and a nucleic acid sequence of interest, such that the terminator sequence is able to stop or terminate transcription of the nucleic acid sequence of interest.

[0192] Ploidy. Ploidy or chromosomal ploidy refers the number of complete sets of chromosomes occurring in the nucleus of a cell. Somatic cells, tissues, and individual organisms can be described according to the number of sets of chromosomes present (the "ploidy level"): monoploid (1 set), diploid (2 sets), triploid (3 sets), tetrapioid (4 sets), pentapioid (5 sets), hexapioid (6 sets), heptapioid or septapioid (7 sets), etc. The generic term polyploidy is used herein to describe cells with three or more chromosome sets.

[0193] Promoter: A recognition site on a DNA sequence or group of DNA sequences that provides an expression control element for a structural gene and to which RNA polymerase specifically binds and initiates RNA synthesis (transcription) of that gene.

[0194] Recombinant. A nucleic acid sequence, expression cassette, genetic construct, or vector comprising a nucleic acid sequence as disclosed herein, or an organism transformed with 46US_ACTIVE\131477155\V-1such nucleic acid sequences, expression cassettes or vectors, created by genetic engineering techniques in which either (a) the sequences of the nucleic acids or a part thereof, or (b) genetic control sequence(s) that is operably linked with the nucleic acid sequence, for example a promoter or terminator, or (c) combinations of (a) and (b), are not located in their natural genetic environment or have been modified and / or inserted artificially by genetic engineering methods.

[0195] Selected DNA: A DNA segment that one desires to introduce or has introduced into a genome by genetic transformation.

[0196] Terminator. A DNA control sequence at the end of a transcriptional unit that signals 3' processing and polyadenylation of a primary transcript and termination of transcription.

[0197] Transformation construct: A chimeric DNA molecule that is designed for introduction into a host genome by genetic transformation. Transformation constructs will often comprise all of the genetic elements necessary to direct the expression of one or more exogenous genes. In particular embodiments of the instant disclosure, it may be desirable to introduce a transformation construct into a host cell in the form of an expression cassette.

[0198] Transformed cell: A cell the DNA complement of which has been altered by the introduction of an exogenous DNA molecule into that cell.

[0199] Transgene: A segment of DNA that has been incorporated into a host genome or is capable of autonomous replication in a host cell and is capable of causing the expression of one or more coding sequences. Exemplary transgenes will provide the host cell with a novel phenotype relative to the corresponding non-transformed cell. Transgenes may be directly introduced into a cell by genetic transformation.

[0200] Vector: A DNA molecule designed for transformation into a host cell. Some vectors may be capable of replication in a host cell. A plasmid is an exemplary vector, as are expression cassettes isolated therefrom.EXAMPLES

[0201] The following examples are included to demonstrate illustrative embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the present disclosure, and thus can be considered to constitute one embodiment of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments47US_ACTIVE\131477155\V-1that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.Example 1Identification of Exoglucanase Genes

[0202] The inventors have identified a novel enzyme that can carry out selective hydrolysis of glycosidic bonds in mogroside V, converting it to Siamenoside I. The enzyme described herein that is designated “WcEXGl” predominately cleaves a single beta-l,6-linked glucose residue at the C3 position of mogroside V, whereas other related enzymes ty pically remove more than one glucose residue at various positions of mogroside V to produce smaller mogroside molecules (see below).EXG1

[0203] This type of cleavage specificity was not obvious when the inventors set out to identify exoglucanases or beta-glucosidases that could act on mogroside V. The only known example of an enzyme with similar specificity was isolated from Dekkera bruxellensis by Wang et al. (Food Chemistry 276:43-49, 2019). In contrast, the exoglucanase enzyme produced by Saccharomyces cerevisiae (ScEXGl) efficiently converts mogroside V into mogroside III E (Chiu et al., J. Agric. Food Chem. 61:7127-7134, 2013). Therefore, the inventors scanned a broad sequence space for exoglucanase genes that were assigned as orthologs with DbEXGl in OrthoDB v. 11. The amino acid sequences of all these proteins48US_ACTIVE\131477155\V-1were aligned and clustered into a phylogenetic tree. Nineteen phylogenetically diverse sequences were selected, and structures were predicted using AlphaFold v2. The predicted structures were aligned against structures predicted for DbEXGl and ScEXGl and polymorphisms around the active site were identified. Seven diverse sequences were selected based on: a) predicted structural similarity (TM) to DbEXGl, dis-similarity to ScEXGl; and b) sequence insertions relative to the predicted active site loop at residues 359 to 368 in ScEXGl (sequence WVNGDQTSSY; SEQ ID NO:24). DbEXGl had a four-residue deletion relative to ScEXGl in the amino acid alignment at this site (sequence YGGGAY; SEQ ID NO:32). The structural similarity scores ranged from 0.931 to 0.985 to DbEXGl and 0.890 to 0.951 to ScEXGl. In particular, WcEXGl was scored as 0.947 similar to DbEXGl and 0.912 similar to ScEXGl. The active site loop of WcEXGl was the same length as ScEXGl but had a distinct sequence (FNKDPSENAN; SEQ ID NO: 25). The scores and active site loop sequences for the seven proteins tested, plus DbEXGl and ScEXGl, can be found in Table 1. Note that the ScEXGl allele tested varied from the ScEXGl allele taken from OrthoDB at residues 321 (S > T) and 361 (N > W). These enzymes were then tested in vivo for their activity on mogroside V.49US_ACTIVE\131477155\V-1Table 1Organism TM to TM to Sequence Aligned SEQ ID Sequence DbEXG1 ScEXG1 to SEQ ID NO: 24 NO: Length Kazachstania 0.931 0.915 FWKNGQGSYF 26 10 naganishiiKazachstania 0.946 0.951 FFRDKNIPSDFR 27 12 africanaSaccharomyces 0.947 n / a WVWGDQTSSY 24 10 cerevisiae (Sc)Wickerhamomyces 0.947 0.912 FNKDPSENAN 25 10 ciferrii (Wc)Lachancea 0.948 0.944 FTKGSDTSSY 28 10 dasiensisEremothecium 0.95 0.926 FNKNGDKAPY 29 10 gossypiiOgataea angusta 0.974 0.895 YGGGNY 30 6 Pichia 0.979 0.897 YQNDTP 31 6 kudriavzeviiBrettanomyces n / a 0.893 YGGGAY 32 6 bruxellensis (Db)Example 2In vivo Testing of Exoglucanases in Lettuce

[0204] G-blocks were ordered from IDT (Coralville, IA) corresponding to the 8 candidate exoglucanase genes (Wickerhamomyces ciferrii (SEQ ID NO: 1); Eremothecium gossypii (SEQ ID NO:3); Kazachstania africana (SEQ ID NO:5); Kazachstania naganishii (SEQ ID NO:7); Lachancea dasiensis (SEQ ID NO:9); Ogataea angusta (SEQ ID NO: 11); Pichia kudriavzevii (SEQ ID NO: 13); and Pichia kudriavzevii (SEQ ID NO: 15)) that were selected for evaluation. The exoglucanase gene from Brettanomyces bruxellensis (SEQ ID NO: 17) was used as a positive control.50US_ACTIVE\131477155\V-1

[0205] Each gene was cloned into a plant binary' vector driven by the CaMV e35S promoter. The components of this vector (SEQ ID NO:21) are shown below.Vector Coordinates Feature Name Source Description1-25 RB Agrobacterium tumefaciens T-DNA right border 26-66 Spacer NA NA67-743 CaMV e35S promoter Cauliflower mosaic virus Promoter744-809 Spacer NA NA810-2099 WcEXGl Wickerhamomyces ciferrii exo-(l, 3)-Beta-glucanase 2100-2103 Spacer NA NA2104-2589 AtHSP18.2 Terminator Arabidopsis thaliana Terminator2590-2678 Spacer NA NA2679-2704 LB Agrobacterium tumefaciens T-DNA left border 2705-2730 Spacer NA NA2731-2756 LB Agrobacterium tumefaciens T-DNA left border 2757-2966 Spacer NA NA2967-3283 SmR Pro not provided Promoter3284-4075 SmR not provided spectinomycin resistance gene 4076-4096 Spacer NA NA4097-4769 Ori pUC E. coli Bacterial origin of replication 4770-5132 Spacer NA NAAgrobacterium origin of 5133-5327 pVSl oriV Pseudomonas aeruginosa replication5328-5392 Spacer NA NAProtein required for function 6466-5393 pVSl Rep A Pseudomonas aeruginosa of oriV6467-6894 Spacer NA NAProtein required for function7524-6895 pVSl StaA Pseudomonas aeruginosa of oriV

[0206] Binary vectors containing the EXG candidate genes were transformed into EHA105 Agrobacterium cells. A binary vector, designated SP5425 (SEQ ID NO:23), containing mogroside synthesis genes squalene epoxidase (SQE), cucurbitadienol synthase (CDS), epoxy or epoxide hydrolase (EPH), cytochrome P450 72 (CYP72), cytochrome P450 87 (CYP87), uridine phosphorylase dependent glycosyltransferase 720 (UGT720), uridine phosphorylase dependent glycosyltransferase 94 (UGT94), truncated 3-hydroxy-3-methylglutaryl-CoA reductase (tHMGR), upstream terpenoid biosynthetic enzyme (HMGS), and upstream terpenoid biosynthetic enzyme (CPS) was constructed. The components of this vector are shown below.Vector Coordinates Feature Name Source Description51US_ACTIVE\131477155\V-1Agrobacterium1-25 RB tumafaciens T-DNA right border 26-70 Spacer n / a n / a71-1495 Enhanced AtEF-lA Cauliflower mosaic virus Promoter promoter and Arabidopsis thaliana1496-1496 Spacer n / a n / a1497-3083 SgSQEl GC Siraitia grosvenorii Squalene Epoxidase 3084-3087 Spacer n / a n / a3088-3648 Pea 3A terminator Pisum sativum Terminator3649-3660 Spacer n / a n / a3661-4099 DCMV Promoter Dahlia mosaic virus Promoter4100-4103 Spacer n / a n / a4104-5525 SgCYP87D18 GC Siraitia grosvenorii Cytochrome P450monoxygenase 5526-5529 Spacer n / a n / a5530-5934 AtUBQ3 terminator Arabidopsis thaliana Terminator5935-5946 Spacer n / a n / a5947-6531 FSgt PFLt chimeric Figwort mosaic virus and Promoter promoter Peanut chlorotic streakcaulimovirus6532-6535 Spacer n / a n / a6536-8815 SgCDSZm Siraitia grosvenorii Cucurbitadienol synthase 8816-8819 Spacer n / a n / a8820-9359 Gmax MYB2 Glycine max Terminator Terminator9360-9371 Spacer n / a n / aDuplic MMV9372-9992 Promoter Mirabilis mosaic virus Promoter9993-9993 Spacer n / a n / a9994-11550 SgCYP72A459vla Siraitia grosvenorii Cytochrome P450Zm monoxygenase 11551-11554 Spacer n / a n / aAtRBCS2B11555-11764 Terminator Arabidopsis thaliana Terminator11765-11776 Spacer n / a n / a11777-12518 e35S promoter Cauliflower mosaic virus Promoter12519-12519 Spacer n / a n / a12520-13944 SgUGT720-269-l GC Siraitia grosvenorii Uridine 5'-diphospho- glucuronosyltransferase 13945-13948 Spacer n / a n / aAtHSP18.213949-14434 Terminator Arabidopsis thaliana Terminator14435-14446 Spacer n / a n / a52US_ACTIVE\131477155\V-114447-15773 AtUBQlO Promoter Arabidopsis thaliana Promoter15774-15774 Spacer n / a n / a15775-17136 SgUGT94-289-l GC Siraitia grosvenorii Uridine 5'-diphospho- glucuronosyltransferase 17137-17140 Spacer n / a n / aCsHSP17.317141-17449 terminator Cucumis sativus Terminator17450-17461 Spacer n / a n / a17462-17846 FuasFSc Promoter Figwort mosaic virus Promoter17847-17847 Spacer n / a n / a17848-18798 SgEPH3 GC Siraitia grosvenorii Epoxide hydrolase 18799-18802 Spacer n / a n / aPotato Ubi318803-19216 Terminator Solanum tuberosum Terminator19217-19228 Spacer n / a n / a19229-20597 ScBV Promoter Sugarcane bacilliform Promoter badnavirus20598-20598 Spacer n / a n / a20599-21873 CltHMGR GC Citrullus lanatus HMG-CoA reductase 21874-21877 Spacer n / a n / a21878-22097 AtTub9 terminator Arabidopsis thaliana Terminator22098-22109 Spacer n / a n / a22110-22574 CmYLCV promoter Cestrum yellow leaf Promotercurling virus22575-22575 Spacer n / a n / a22576-23940 SgMGl GC Siraitia grosvenorii23941-23944 Spacer n / a n / a23945-24243 CsHSP22 terminator Cucumis sativus Terminator24244-24255 Spacer n / a n / a24256-24496 PCLSV promoter Peanut chlorotic streak Promotervirus24497-24497 Spacer n / a n / a24498-25922 SgUGT720-269-l Siraitia grosvenorii Uridine 5'-diphospho- glucuronosyltransferase 25923-25926 Spacer n / a n / a25927-26221 Pea E9 terminator Pisum sativum Terminator26222-26233 Spacer n / a n / a26234-26829 FMV Fit Promoter Figwort mosaic virus Promoter26830-26833 Spacer n / a n / a3- hydroxy- 3- methylglutaryl CoA 26834-28219 BjHMGS mutant Brassica junea synthase28220-28223 Spacer n / a n / a53US_ACTIVE\131477155\V-1AtNDUFA828224-28443 terminator Arabidopsis thaliana Terminator 28444-28451 Spacer n / a n / a28452-29955 FS4 Promoter Citrullus lanatus Promoter29956-29956 Spacer n / a n / a29957-30862 CIGPS1 Citrullus lanatus30863-30866 Spacer n / a n / a30867-31096 AtFAD2 Terminator Arabidopsis thaliana Terminator 31097-31108 Spacer n / a n / a31109-31622 CsVMV Promoter Cestrum yellow leaf Promoter curlingvirus31623-31649 SynJ 5' leader Synthetic 5' UTR31650-31650 Spacer n / a n / a31651-32679 HygR Escherichia coli Hygromycin B phosphotransferase 32680-32683 Spacer n / a n / a32684-32858 35S Terminator Cauliflower mosaic virus Terminator 32859-32965 Spacer n / a n / a Agrobacterium32966-32990 LB tumafaciens T-DNA left border 32991-33017 Spacer n / a n / a Agrobacterium33018-33042 LB tumafaciens T-DNA left border 33043-33739 Spacer n / a n / a33740-33831 AmpR promoter Escherichia coli bacterial promoter kanamycin resistance 33832-34641 KanR Escherichia coli Tn5 gene34642-34660 Spacer n / a n / a34661-35206 pl5Aori Escherichia coli Origin of replication 35207-36372 Spacer n / a n / a36373-36567 pVSl oriV Pseudomonas aeruginosa Agrobacterium origin of replication36568-36632 Spacer n / a n / a37706-36633 pVSl RepA Pseudomonas aeruginosa Protein required for function of oriV 37707-38134 Spacer n / a n / a38764-38135 pVSl StaA Pseudomonas aeruginosa Protein required forfunction of oriV

[0207] The SP5425 vector was transformed into EHA105 Agrobacterium cells, and these cells were then used for co-transformation of plant cells with the various EXG genes. Agrobacterium cultures were prepared and used for transient leaf infiltration of 2-week-old54US_ACTIVE\131477155\V-1lettuce seedlings. Leaf tissues were harvested at 6 days post-infdtration, freeze-dried, and extracted with 80% methanol for LC-MS analysis of mogroside III-E (MIII-E) content, siamenoside I (SI) content and mogroside V (MV) content. The results show that WcEXGl uniquely converts mogroside V with minimal production of the unwanted product MIII-E compared to DbEXGl and other homologous EXG1 enzymes, that WcEXGl converts mogroside V to greater levels of siamenoside than DbEXGl or other homologous EXG1 enzymes, and that WcEXGl consumes the majority of mogroside V while converting it to siamenoside.

[0208] Enzyme assays for LC-MS / MS analysis of mogrosides were prepared by mixing 25 pl of the assays into 225 pl 80% methanol in a 96-well plate. Saccharomyces cerevisiae culture samples and Pichia pastoris endogenous activity’ samples were prepared by vortexing a 200 pl of each sample with 800 pl 100% methanol and centrifuging 10 min at 21,300 ref.

[0209] The initial LC separation of the samples was done using a Waters Quaternary Solvent Manager ACQUITY UPLC H-Class PLUS connected to a Waters Sample Manager FTN-H ACQUITY UPLC and a PDA e / _ Detector ACQUITY UPLC. The LC gradients use an ACQUITY UPLC CSH C18 1.7um 2.1 mm xlOO mm column at 35°C with an acetonitrile / water gradient acidified with 0.1% formic acid. The LC system was coupled to an Xevo G2-XS mass spectrometer in sensitivity positive mode using the MSMS scan mode with a collision energy of 30 for mogrosides and 11-oxo-mogrosides with five, four, three, or two glucose and collision energy of 20 for mogrosides and 11-oxo-mogrosides with one glucose or mogrol. The following precursor and product ion pairs, expressed in m / z, were used to quantify the mogrosides with a 50 mDa window applied to the product ion: mogrosides with five glucose (1287.7 / 423.3621), mogrosides with four glucose (1125.6 / 423.3621), mogrosides with three glucose (963.6 / 423.3621), mogrosides with two glucose (801.5 / 423.3621), mogrosides with one glucose (639.4 / 423.3621), mogrol (477.4 / 423.3621), 11-Oxo-mogrosides with five glucose (1285.7 / 457.3680), 11-Oxo-mogrosides with four glucose (1123.6 / 457.3680), 11-Oxo-mogrosides with three glucose (961.5 / 457.3680), and 11-Oxo-mogrosides with two glucose (799.5 / 457.3680), and 11 -Oxo-mogrol (475.4 / 457.3680). For LockSpray mass correction, 200 pg / ml leucine-enkephalin monitoring for 556.2771 m / z was used. Data analysis was done using the Waters TargetLynx software, excel, R, and JMP

[0210] The results show that only the WcEXGl enzyme (second to last bar in each figure) and the DbEXGl positive control (first bar in each figure) produce significant quantities of siamenoside I, whereas the other candidate enzymes produce smaller amounts or no siamenoside I and large amounts of mogroside III-E. Therefore, only the WcEXGl has the 55US_ACTIVE\131477155\V-1desired mogroside V cleavage specificity of the 8 candidate enzymes that were evaluated in this assay. The mogroside III-E, siamenoside I and mogroside V chemical structures are shown below.mogroside III-Esiamenoside I56US_ACTIVE\131477155\V-1mogroside VExample 3In vivo Testing of Exoglucanases in Yeast

[0211] The JEcEXG I gene and the DAEXG I positive control gene were each cloned into a yeast expression vector behind the strong GPD promoter (WcEXGl yeast expression vector is SEQ ID NO:22). The components of this vector are shown below.Vector Coordinates Feature Name Source Description1-657 P GPD Saccharomyces cerevisiae yeast promoter658-658 Spacer NA NA659-1948 WcEXGl Wickerhamomyces ciferrii exo-(l, 3)-Beta-glucanase 1949-1955 Spacer NA NA1956-2225 3'UTR CYC1 Saccharomyces cerevisiae yeast terminator2226-2451 P URA3 Saccharomyces cerevisiae yeast promoter2452-3255 M URA3 * Saccharomyces cerevisiae URA selectable marker 3256-3333 3'UTR URA3 Saccharomyces cerevisiae yeast terminator3334-3357 Spacer NA NA3358-4684 Ori 2uni not provided yeast origin of replicatom 4685-4743 Spacer NA NA4744-4773 P Amp (short) not provided NA4774-4787 Spacer NA NA4788-4905 P Amp not provided bacterial promoter 4906-4915 Spacer NA NA4916-5776 M Ampicillin-r * not provided Ampicillin resistance gene 5777-5835 Spacer NA NA5836-6508 Ori pUC not provided bacterial origin of replication57US_ACTIVE\131477155\V-1

[0212] These vectors were transformed into an exgl- knockout strain of Saccharomyces cerevisiae using a Frozen-EZ Yeast Transformation II Kit from Zymo Research (Irvine, CA). As a negative control, wild-type Saccharomyces cerevisiae was transformed with an empty vector. Colonies that grew on Minimal SD-URA plates were grown in liquid medium at 30 degrees C in the presence of a commercial Monk Fruit Extract and then aliquots were taken at 0, 8, 24, 48. and 72 hours to monitor the conversion of mogroside V into various sweet mogrosides in Saccharomyces cerevisiae transformed with exoglucanase from Wickerhamomyces ciferrii grown in commercial Monk Fruit Extract by LC-MS. The results are shown in Table 2 (PPM w / v; MV - mogroside V; 0-MV - 11-oxo-mogroside V; SI -siamenoside I; MIV-A - mogroside IV-A; MIV - mogroside IV; Mill - mogroside III; MIII-E - mogroside III-E; MIII-A2 - mogroside III-A2; MII-E - mogroside II-E).58US_ACTIVE\131477155\V-1Table 2Treatment MV O-MV SI MIV-A MIV Mill MIII-E MIII-A2 MII-E Wild type (0 hr) 3 0.252 0.0102 0.117 <0.01 0.0151 <0.005 0.0142 <0.005 Wild type (8 hr) 2.82 0.252 0.00781 0.104 <0.01 0.0196 <0.005 0.0145 <0.005 Wild type (24 hr) 0.379 0.0165 0.203 0.0069 0.895 0.018 0.619 0.0341 0.0648 Wild type (48 hr) 0.0371 <0.005 0.0751 0.0106 0.421 0.0242 1.38 <0.005 0.0714 Wild type (72 hr) 0.0397 <0.005 0.088 0.00882 0.485 0.0207 1.45 0.015 0.0729 exgl- (0 hr) 3.06 0.282 <0.005 0.109 <0.01 0.0189 <0.005 0.0232 <0.005 exgl- (8 hr) 2.96 0.24 0.00981 0.116 <0.01 0.0166 <0.005 0.0107 <0.005 exgl - (24 hr) 3 0.241 0.00576 0.121 <0.01 0.0254 <0.005 0.0138 <0.005 exgl - (48 hr) 2.92 0.265 0.00702 0.108 0.0115 0.0135 <0.005 0.0133 <0.005 exgl - (72 hr) 3.3 0.293 0.0104 0.125 <0.01 0.0266 <0.005 0.015 <0.005 Positive control (0 hr) 3.03 0.253 0.00715 0.109 <0.01 0.024 <0.005 0.0148 <0.005 Positive control (8 hr) 2.69 0.237 0.14 0.0924 <0.01 0.0299 <0.005 0.0146 <0.005 Positive control (24 hr) 0.0247 0.016 2.02 0.0138 <0.01 0.0754 0.0118 <0.005 0.0472 Positive control (48 hr) 0.0225 0.0122 1.89 <0.005 <0.01 0.0563 0.0301 <0.005 0.0559 Positive control (72 hr) 0.0296 0.00916 2.29 <0.005 <0.01 0.0694 0.026 0.0171 0.0537 WcEXGl (0 hr) 2.98 0.281 <0.005 0.113 <0.01 0.0234 <0.005 0.0182 <0.005 WcEXGl (8 hr) 2.69 0.239 0.2 0.0906 <0.01 0.0416 <0.005 0.025 0.00514 WcEXGl (24 hr) 0.717 0.101 1.53 0.0165 0.0116 0.0527 0.025 0.0151 0.0539 WcEXGl (48 hr) 0.692 0.109 1.6 0.008 <0.01 0.0444 0.0167 0.0166 0.0601WcEXGl (72 hr) 0.786 0.13 1.75 <0.005 0.0129 0.0505 0.0214 0.0126 0.05659US_ACTIVE\131477155\V-1

[0213] Conversion of mogroside V into various sweet mogrosides in Saccharomyces cerevisiae transformed with exoglucanase from Wickerhamomyces ciferrii grown in commercial Monk Fruit Extract (WT = wild-type yeast; exgl- = EXG1 knockout mutant; PCT = DbEXGl positive control; Wc #B = WcEXGl). The results show that wild-type yeast rapidly converted the mogroside V into various other sweet mogrosides, the exgl- knockout mutant had essentially no conversion activity on mogroside V, and the PFcEXGl and DbEXGl enzymes converted mogroside V into siamenoside I with high efficiency. These two enzymes also showed negligible conversion of mogroside V into other mogroside molecules other than siamenoside I. Therefore, the JEcEXGI enzyme displays the desired cleavage specificity of mogroside V when expressed in both plants and yeast.Example 4Simultaneous MV to SI Bioconversion and Juice Desugarization

[0214] A yeast expression vector containing the ITcEXG I gene is transformed / integrated into X. cerevisiae and S. bayanus strains in which their native EXG1 genes have been rendered non-functional using any well-known gene knockout strategy. Disruption of the native EXG1 genes is necessary to prevent the undesired deglycosylation of mogroside V (MV) to mogroside III-E (Mill E) and other smaller mogrosides. Using these new strains, only the IFcEXGl will be active and convert MV to siamenoside I (SI). The use of both S. cerevisiae and S. bayanus strains favors the rapid desugarization of the juice sample, although the use of a single strain may be sufficient to break down all simple mono- and disaccharide sugars under the appropriate conditions. WcEXGl -expressing yeast strains of appropriate cell density are mixed with a concentrated juice extract containing MV and allowed to grow for the necessary amount of time (for example 48 hours at 30°C) in order to mediate maximum MV to SI bioconversion and removal of sucrose, glucose, and fructose through their normal cellular metabolism. Once these conversion processes reach the desired end point, the juice can be filtered to remove the yeast cells and other particulate matter, and additional steps can be taken to remove ethanol or other unwanted impurities. The end-product of this process is a juice that has very low caloric sugars and a pleasant, sweet taste that is imparted by non-caloric SI.60US_ACTIVE\131477155W-1Example 5MV to SI Bioconversion Using Purified WcEXGl Expressed in Yeast [002151 The / T'cEXG I gene (optimized for Pichia pastoris expression and with native signal peptide removed) was cloned into yeast expression vector pD912 behind the AOX1 methanolinducible promoter and alpha-factor secretion signal (SEQ ID 19 and 20) and transformed into Pichia pastoris strain PPS-9010 following Atum protocols. Positive colonies from Zeomycin 1000 selection plates were cultured in BMMY + Zeomycin media in 2 L flasks for 3 days at 30°C, 250 RPM, with daily 2% MeOH induction. Cultures were pelleted by centrifugation (10000 ref, 15 minutes) and supernatant was filtered through 0.2 μM filter, concentrated using Centricon 30 Da MWCO ultra centrifugal filter (5x spins, 3500 ref, 20 minutes), and recovered by inverting the filter on a recovery cup and centrifuging (1000 ref, 90 seconds). Protein was either concentrated directly into distilled water or purified on HisPur Cobalt resin, buffer exchanged, and concentrated into water or storage buffer (20 mM Sodium Citrate, 200 mM NaCl, pH 6). WcEXGl enzyme (final concentration 0.02 mg / mL) was incubated with mogroside standards (final concentration 5 mg / mL) for 60, 80, and 120 minutes. Incubations were stopped by filtration (30 Da) and samples were analyzed by LC-MS / MS according to standard methods.[00216J In vitro conversion of 11 -oxo Mogroside V (OXOV), Mogroside V (MV100), Mogroside 90 -50 mixes (MV90-50, see Table 3), Mogroside IV-A (MIVA), and Mogroside III (Mill) before incubation (NC) and over time (60, 80, 120 min) after incubating with WcEXGl enzyme purified from Pichia pastoris.Table 3% O- Mix Name % MV % SI % MIV-A % MillMV MV 100 100 0 0 0 0 MV90 90 4 1 4 1 MV70 70 12 3 11 4MV50 50 20 5 18 7

[0217] Quantification was completed according to LC-MS / MS methods. 11-oxo Siamenoside (no standard available) was deduced based on the product profile of DbEXGl with the assumption that this enzyme that specifically converts mogroside V to siamenoside would convert 11 -oxo mogroside V to 11 -oxo siamenoside. The quantification of 11 -oxo siamenoside was estimated61US_ACTIVE\131477155W-1using 11-oxo mogroside IV as a substitute standard curve. The results are shown in Table 4 (amounts presented as percentages).Table 4MIV- MIII- MIHMII- Sample ID MV O-MV SI O-SI MillA E AI E OXOV (NC) 0 98 0 0 0 0 0 0 0 OXOV (60 min) 0 8 0 90 0 0 0 0 0 OXOV (80 min) 0 5 0 94 0 0 0 0 0 OXOV (120 min) 0 2 0 97 0 0 0 0 0 MV100 (NC) 100 0 0 0 0 0 0 0 0 MV100 (60 min) 2 0 96 0 0 0 2 0 0 MV 100 (80 min) 1 0 97 0 0 0 2 0 0 MV 100 (120 min) 1 0 96 0 0 0 3 0 0 MV90 (NC) 93 3 1 0 3 1 0 0 0 MV90 (60 min) 2 1 86 6 0 0 2 0 4 MV90 (80 min) 1 0 86 6 0 0 2 0 4 MV90 (120 min) 1 0 85 7 0 0 3 0 4 MV70 (NC) 76 9 3 0 8 4 0 0 0 MV70 (60 min) 1 2 67 17 0 1 1 0 10 MV70 (80 min) 1 1 68 18 0 1 1 0 10 MV70 (120 min) 1 0 67 19 0 0 2 0 11 MV50 (NC) 58 15 5 0 15 7 0 0 0 MV50 (60 min) 1 2 50 28 0 1 1 0 16 MV50 (80 min) 1 2 50 28 0 1 1 0 17 MV50 (120 min) 0 1 50 31 0 0 2 0 16 MIVA (NC) 0 0 0 0 99 0 0 0 0 MIVA (60 min) 0 0 2 0 3 48 0 8 37 MIVA (80 min) 0 0 2 0 2 43 0 6 47 MIVA (120 min) 0 0 2 0 0 32 1 3 63 Mill (NC) 0 0 0 0 0 99 1 0 0 Mill (60 min) 0 0 1 0 1 48 1 3 47 Mill (80 min) 0 0 0 0 1 42 1 3 54Mill (120 min) 0 0 0 0 0 26 1 3 70

[0218] Enzyme activity converting Mogroside V to Siamenoside was estimated to be 1-3 μmol min-1 mg-1. Optimal pH, temperature, and potential inhibition of WcEXGl enzyme were inferred by incubating enzyme with Mogroside V in respective conditions and measuring reaction progression by glucose release (Glucose HK plate-based assay). WcEXGl activity was calculated in a range of pH conditions with 10 minute incubation. Citrate buffer was used for pH 3-6.5,US_ACTIVE\131477155W-1phosphate buffer was used for pH 6.5-8, and Tris buffer was used for pH 8-9. Glucose release was highest between pH 2.5 and 6.5, and was lower at pH values above 6.5. The impact of temperature on WcEXGl activity was measured with 1 minute incubation. Glucose increased from 20°C to 70°C and fell off sharply at temperatures above 70°C. The impact of temperature on WcEXGl stability was measured with 30 minute incubation. Glucose levels were high between 20°C and 50°C, and fell off sharply above 50°C. The impact of salts and sugars on WcEXGl activity was measured with 10 minute incubation. Raffinose is inhibitory to WcEXGl activity with an IC50 around 0.2 M. Sucrose is inhibitory to WcEXGl activity with IC50 around 1.5 M at 45°C. Potassium chloride is not inhibitory to WcEXGl activity at concentrations ranging from 0-1 M. Calcium chloride is not inhibitory to WcEXGl activity at concentrations ranging from 0-0.25 M. The impact of glucose on WcEXGl activity was measured by assessing Mogroside V to Siamenoside conversion in the presence of a range of glucose concentrations (Table 5).Table 5Treatment MV SINC: (3.75 min) 1044 3.670 M Glu: (3.75 min) 657 262.50.1 M Glu: (3.75 min) 756 211.50.5 M Glu: (3.75 min) 937 1231 M Glu: (3.75 min) 992 85.85NC: (o / n) 1090 3.44ENZ: (o / n) 0 427NC: (12 min) 1125 4.010 M Glu: (12 min) 262 586.50.1 M Glu: (12 min) 383.5 489.50.5 M Glu: (12 min) 670 3261 M Glu: (12 min) 737.5 209.5Example 6MV to SI Bioconversion by fermentation with wild-type Pichia pastoris

[0219] Wild-type Pichia pastoris strain PPS-9010 was grown in YPD broth with Mogroside V stock for 0, 3, 8, and 24 hours. Samples were taken from supernatant for mogroside analysis. Filter sterilized broth was also incubated with Mogroside V for 0 and 24h. Conversion from Mogroside V to Siamenoside was detected after 24h fermentation or incubation with broth (Table 6).Table 663US_ACTIVE\131477155W-1Treatment MV SICulture T = 0 hr 436 1.36Culture T = 3 hr 398 4.46Culture T = 8 hr 342 49.7Culture T = 24 hr 21.7 259Broth T = 0 hr 482 238Broth T = 24 hr 395 307

[0220] The exoglucanase homolog in Pichia pastoris was identified by homology analysis (SEQ ID NO:38 and SEQ ID NO: 39) and vectors were designed to knock out the native EXG enzyme. Mogroside V to Siamenoside overnight fermentation was conducted with wild-type and potential knockout strains. Several knockout strains lacked the Siamenoside conversion seen in wild-type, demonstrating that the causative exoglucanase gene was correctly identified and knocked out (Table 7).Table 7Treatment MV SIT = 0, no cells 250 0.8EXG1 knockout #1 255 0.75EXG1 knockout #2 0.65 84.6EXG1 knockout #3 253 0.85EXG1 knockout #4 263 0.6EXG1 knockout #5 252 0.8EXG1 knockout #6 256 0.55EXG1 knockout #7 0 69.4EXG1 knockout #8 0.6 83.2EXG1 knockout #9 244 0.75EXG1 knockout #10 1.05 81EXG1 knockout #11 0.75 89.6EXG1 knockout #12 241 0.6wild-type Pichia 180 33Example 7Examples of Uses of JlcEXGl

[0001] Various scenarios can be envisioned for the application of this presently disclosed technology. Examples include, but are not limited to: 1) direct production of siamenoside I (SI) in plant cells via co-expression of a highly specific EXG enzyme along with the biosynthetic enzymes necessary to produce mogroside V; 2) the EXG enzyme can be expressed at a high level 64US_ACTIVE\131477155W-1in one transgenic plant line (i.e., lettuce, cucumber, tomato, c / c ), and extracts from this plant line can be mixed with extracts from another plant tissue that has accumulated high levels of mogroside V to mediate efficient conversion to SI; 3) the EXG enzyme can be expressed and purified from a plant or microbial source, linked to beads or resin in a column or similar purification platform, and then used to treat mogroside V-containing plant extracts for the ex vivo conversion of mogroside V into SI; 4) the EXG enzyme can be expressed and secreted from a microbial source and the cell-free broth can be used to treat mogroside V-containing plant extracts for the ex vivo conversion of mogroside V into SI; and 5) yeast or other microbial cells that are expressing the EXG enzyme can be mixed with plant extracts that contain high levels of mogroside V for the efficient conversion of mogroside V into SI. The plant extract or juice would then be purified away from the microbes and solutions that carried out this conversion. An additional benefit of this approach is that the yeast / microbes could break down simple mono- and disaccharide sugars such that the final product has little or no caloric sugars and consists mainly of the non-caloric mogroside sweeteners.* * *

[0001] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and / or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. For example, all of the disclosed components of the preferred and alternative embodiments are interchangeable providing disclosure herein of many systems having combinations of all the preferred and alternative embodiment components. This application is therefore intended to cover any variations, uses, or adaptations of the disclosure using its general principles. Further, this application is intended to cover such departures from the present disclosure as come within known or customary practice in the art to which this disclosure pertains and which fall within the limits of the appended claims. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.65US_ACTIVE\131477155W-1

Claims

CLAIMS1. A recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 (WcEXGl) or SEQ ID NO:39 (Pichia) operably linked to a heterologous promoter.

2. The recombinant nucleic acid construct of claim 1, wherein the nucleic acid sequence has at least 95% sequence identity to SEQ ID NO:1 (WcEXGl) or SEQ ID NO:38 (Pichia).

3. The recombinant nucleic acid construct of claim 1, comprising the nucleic acid sequence of SEQ ID NO: 20 or SEQ ID NO:21.

4. A recombinant nucleic acid construct comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

5. The recombinant nucleic acid construct of claim 4, comprising the nucleic acid sequence of SEQ ID NO: 22.

6. A plant cell comprising the recombinant nucleic acid construct of claim 1.

7. The plant cell of claim 6, further comprising the recombinant nucleic acid construct of claim 4.

8. A yeast cell comprising the recombinant nucleic acid construct of claim 1.

9. The yeast cell of claim 8, further comprising the recombinant nucleic acid construct of claim 4.

10. A plant comprising the plant cell of claim 6.

11. A plant comprising the plant cell of claim 7.

12. A method for producing siamenoside I, comprising contacting a composition comprising mogroside V with a protein having at least 95% identity to SEQ ID NO:2.

13. The method of claim 12, wherein the composition comprising mogroside V is produced by a transgenic cell.

14. The method of claim 13, wherein the transgenic cell is a transgenic plant cell.66US_ACTIVE\131477155W-115. The method of claim 14, wherein the transgenic plant cell is a transgenic lettuce, cucumber or tomato plant cell.

16. The method of claim 13, wherein the transgenic cell is a transgenic bacterial or yeast cell.

17. The method of claim 16, wherein the transgenic yeast cell is a transgenic Saccharomyces cerevisiae or Pichia pastoris yeast cell.

18. The method of claim 12, wherein the protein having at least 95% sequence identity to SEQ ID NO:2 is produced by a transgenic cell comprising the recombinant nucleic acid construct of claim 1.

19. The method of claim 12, wherein the protein having at least 95% sequence identity to SEQ ID NO:2 and the composition comprising mogroside V are produced by a single transgenic cell.

20. The method of claim 19, wherein the single transgenic cell is the transgenic plant cell of claim 7.

21. The method of claim 19, wherein the single transgenic cell is the transgenic yeast cell of claim 9.

22. The method of claim 12, wherein an extract from a transgenic plant comprising a recombinant nucleic acid construct comprising a nucleic acid sequence encoding an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 (WcEXGl) operably linked to a heterologous promoter is contacted with an extract from a transgenic plant comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

23. The method of claim 12, wherein the protein having at least 95% sequence identity to SEQ ID NO:2 is purified from a transgenic plant or yeast cell comprising a recombinant nucleic acid construct of claim 1.

24. The method of claim 23, wherein the purified protein is contacted with an extract from a transgenic plant comprising at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

25. The method of claim 12, wherein:67US_ACTIVE\131477155W-1a) the protein having at least 95% sequence identity to SEQ ID NO:2 is secreted from yeast cells comprising the recombinant nucleic acid construct of claim 1 grown in a broth;b) the yeast cells are removed from the broth to produce a yeast cell-free broth; and c) the yeast cell-free broth is contacted with an extract of a plant that produces mogroside V.

26. The method of claim 25, wherein the plant that produces mogroside V comprises at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

27. The method of claim 12, wherein yeast cells comprising the recombinant nucleic acid construct of claim 1 are contacted with an extract of a plant that produces mogroside V.

28. The method of claim 27, wherein the plant that produces mogroside V comprises at least a second nucleic acid sequence encoding at least a first mogroside V biosynthetic protein operably linked to at least a second heterologous promoter.

29. A method for producing 11-oxo siamenoside I, comprising contacting a composition comprising 11-oxo mogroside V with a protein having at least 95% identity to SEQ ID NO:

2.

30. The method of claim 29, wherein the composition comprising 11-oxo mogroside V is produced by a transgenic cell.

31. The method of claim 30, wherein the transgenic cell is a transgenic plant cell.

32. The method of claim 31, wherein the transgenic plant cell is a transgenic lettuce, cucumber or tomato plant cell.

33. The method of claim 30, wherein the transgenic cell is a transgenic bacterial or yeast cell.

34. The method of claim 33, wherein the transgenic yeast cell is a transgenic Saccharomyces cerevisiae or Pichia pastoris yeast cell.68US_ACTIVE\131477155W-1

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