Methods for making high intensity sweeteners

Abstract

To provide methods of producing new sweetening compounds improved in taste and delivery characteristics, and recombinant cells.SOLUTION: A method of producing a compound 1 comprises: contacting mogroside IIIE with a recombinant host cell that comprises a first enzyme catalyzing the production of the compound 1 from the mogroside IIIE, where the recombinant host cell comprises a cytochrome P450, a glycosylating enzyme and / or an epoxide hydrolase of specific sequences, and where the first enzyme is one or more of cyclomaltodextrin glucanotransferases, dextransucrases, and dextranases.SELECTED DRAWING: Figure 42

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 757,141, filed November 11, 2018, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] background Field The present disclosure relates to methods, systems, and compositions for producing sweet tasting compounds, as well as compositions comprising sweet tasting compounds.

[0003] Background information The gustatory system provides sensory information about the chemical composition of the external world. Gustatory transduction is one of the most sophisticated forms of chemically evoked sensations in animals. Taste signaling is observed throughout the animal kingdom, from the simplest metazoans to the most complex vertebrates. Mammals are thought to have five basic taste modalities: sweet, bitter, sour, salty, and umami (the taste of monosodium glutamate, also known as savory taste).

[0004] For centuries, a variety of natural and non-natural compositions and / or compounds have been added to ingestible compositions, including foods and beverages, and / or orally administered pharmaceutical compositions to improve their taste. Although it has long been known that there are only a few basic types of "taste," the biological and biochemical basis of taste perception remains poorly understood, and most taste improvers or taste modifiers have been discovered, often by a simple trial-and-error process.

[0005] With regard to sweetness, diabetes and cardiovascular disease are increasing health concerns worldwide, but at an alarming rate in the United States. Sugar and calories are important factors that can be limited to have a positive nutritional effect on health. High-intensity sweeteners can provide the sweetness of sugar with a range of taste qualities. Because they are many times sweeter than sugar, much smaller amounts of sweetener are needed to replace sugar.

[0006] High-intensity sweeteners have a wide range of chemically distinct structures and therefore have a variety of properties, including but not limited to odor, flavor, mouthfeel, aftertaste, etc. It is well known that these properties, especially flavor and aftertaste, change over the time of tasting, so that each time profile is unique for each sweetener.

[0007] In recent years, there has been significant progress in identifying useful natural flavoring agents, such as sweeteners such as sucrose, fructose, glucose, erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, and certain known natural terpenoids, flavonoids, or protein sweeteners. See Kinghom, et al., "Noncariogenic Intense Natural Sweeteners," Med. Res. Rev. 18 (5) 347-360 (1998) (discussion of discovered natural materials that are much sweeter than common natural sweeteners such as sucrose and fructose). Similarly, in recent years, there has been progress in identifying certain new commercialized artificial sweeteners, such as aspartame, saccharin, acesulfame K, cyclamate, and sucralose. See, e.g., Ager, et al., Angew. Chem. Int. Ed. 37, 1802-1817 (1998). The entire contents of the above identified references are hereby incorporated by reference in their entirety.

[0008] Sweeteners such as saccharin and 6-methyl-1,2,3-oxathiazin-4(3H)-one-2,2-dioxide potassium salt (acesulfame potassium) are generally characterized by a bitter and / or metallic aftertaste. Products made with 2,4-dihydroxybenzoic acid are claimed to exhibit a reduction in the undesirable aftertaste associated with sweeteners at concentrations lower than those at which their taste is perceptible. High-intensity sweeteners such as sucralose and aspartame have also been reported to have sweetness delivery problems, i.e., delayed onset and prolonged sweetness. See SG Wiet, et al., J. Food Sci., 58(3):599-602, 666 (1993).

[0009] There is a need for novel sweetening compounds, sweetness enhancers, and compositions containing such compounds and enhancers that have improved taste and delivery properties. Additionally, there is a need for food products that contain novel sweetening compounds and / or sweetness enhancers that have such desirable properties.

[0010] overview As used herein, the following structure: [ka] The method for preparing compound 1 having the formula: E Mogroside III Eand / or (2) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1025, 1026, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (3) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (4) a glycosylation enzyme comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047, 2048, 2049, 2050, 2 and / or (5) an epoxide hydrolase comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) an epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1072.

[0011] In some embodiments, the recombinant host cell comprises a cytochrome P450 comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1039, 1041, 1042, 1043, 1044, 1045, 1046, 1047, and 1049; 36, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a cytochrome P450 encoded by a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1087, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2040, 1042, 104 88, 1090, 1092, and 1094-1149; and / or (4) a glycosylation enzyme comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 10 93; and / or (4) an epoxide hydrolase comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) an epoxide hydrolase encoded by a nucleic acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1072.In some embodiments, the recombinant host cell comprises a cytochrome P450 comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1039, 1041, 1042, 1043, 1044, 1045, 1046, 1047, and 1049; 36, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a cytochrome P450 encoded by a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1087, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035, 2036, 2037, 2038, 2040, 1042, 104 88, 1090, 1092, and 1094-1149; and / or (4) a glycosylation enzyme comprising an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 10 93; and / or (5) an epoxide hydrolase comprising an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) an epoxide hydrolase encoded by a nucleic acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO: 1072.

[0012] In some embodiments, the recombinant host cell comprises a first gene encoding a first enzyme. For example, the first gene may be heterologous to the recombinant host cell. In some embodiments, the first gene encodes a first enzyme, such as mogroside III. Eis present in and / or produced by a recombinant host cell. In some embodiments, the method includes culturing a recombinant host cell in a medium under conditions in which a first enzyme is expressed. In some embodiments, the first enzyme is one or more of UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The CGTase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. The dextransucrase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of the sequences shown in SEQ ID NOs: 2, 103, 106-110, 156, 159-162, and 896, or the dextransucrase is encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 104, 105, 157, 158, and 895. The transglucosidase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 163-291 and 723. The β-glucosidase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 102, 292, 354-374, and 678-741.

[0013] In some embodiments, the method further comprises contacting mogroside IIA with a recombinant host cell to produce mogroside III. Ewherein the recombinant host cell comprises a second enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA. In some embodiments, the recombinant host cell comprises a second gene encoding the second enzyme. In some embodiments, mogroside IIA is produced by and / or present in the recombinant host cell. In some embodiments, the second enzyme is one or more of uridine diphosphate glucosyltransferase (UGT), CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. UGTs include, for example, UGT73C3 (SEQ ID NO: 4), UGT73C6 (SEQ ID NO: 5), UGT85C2 (SEQ ID NO: 6), UGT73C5 (SEQ ID NO: 7), UGT73E1 (SEQ ID NO: 8), UGT98 (SEQ ID NO: 9 or 407), UGT1576 (SEQ ID NO: 15), UGT SK98 (SEQ ID NO:16), UGT430 (SEQ ID NO:17), UGT1697 (SEQ ID NO:18), UGT11789 (SEQ ID NO:19), or comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:4-9, 15-19, 125, 126, 128, 129, 293-307, 407, 409, 411, 413, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149.UGTs can also be encoded by a nucleic acid sequence that contains at least 70% sequence identity to any one of the sequences set forth in, for example, UGT1495 (SEQ ID NO:10), UGT1817 (SEQ ID NO:11), UGT5914 (SEQ ID NO:12), UGT8468 (SEQ ID NO:13), UGT10391 (SEQ ID NO:14), and SEQ ID NOs:116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, 445, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093.

[0014] In some embodiments, the method includes contacting mogrol with a recombinant host cell, the recombinant host cell comprising one or more enzymes capable of catalyzing the production of mogroside IIE and / or IIIE from mogrol. In some embodiments, the recombinant host cell comprises one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIE and / or IIIE from mogrol. Mogrol can be, for example, produced by and / or present in a recombinant host cell. In some embodiments, the one or more enzymes capable of catalyzing the production of mogroside IIE and / or IIIE from mogrol comprise one or more of UGT, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. UGTs include, for example, UGT73C3, UGT73C6, 85C2, UGT73C5, UGT73E1, UGT98, UGT1495, UGT1817, UGT5914, UGT8468, UGT10391, UGT1576, UGT SK98, UGT430, UGT1697, or UGT11789, or comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4-9, 15-19, 125, 126, 128, 129, 293-307, 405, 406, 407, 409, 411, 413, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149.

[0015] In some embodiments, the method includes contacting a mogroside compound with a recombinant host cell, the recombinant host cell including one or more enzymes capable of catalyzing the production of mogroside IIIE from a mogroside compound to produce mogroside IIIE, the mogroside compound being one or more of mogroside IA1, mogroside IE1, mogroside IIA1, mogroside IIE, mogroside IIA, mogroside IIIA1, mogroside IIIA2, mogroside III, mogroside IV, mogroside IVA, mogroside V, and siamenoside. In some embodiments, the recombinant host cell includes one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE from a mogroside compound. In some embodiments, the mogroside compound is produced by and / or is present in the recombinant host cell. In some embodiments, the one or more enzymes capable of catalyzing the production of mogroside IIIE from a mogroside compound include one or more of UGT, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the mogroside compound is mogroside IIE. In some embodiments, the mogroside compound is morgroside IIA, or mogroside IIE, and contact with the one or more enzymes produces one or more of mogroside IIIA, mogroside IVE, and mogroside V. In some embodiments, the method includes contacting mogroside IA1 with a recombinant host cell, the recombinant host cell comprising a gene encoding a UGT98 or UGT SK98 enzyme comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9, 407, 16, or 306. In some embodiments, the contacting causes mogroside IIA to be produced in the cell.

[0016] In some embodiments, the method includes contacting 11-hydroxy-24,25 epoxy cucurbitadienol with a recombinant host cell, the recombinant host cell including a third gene encoding an epoxide hydrolase. The 11-hydroxy-24,25 epoxy cucurbitadienol can be present in and / or produced by the recombinant host cell.

[0017] In some embodiments, the method includes contacting 11-hydroxycucurbitadienol with a recombinant host cell, the recombinant host cell including a fourth gene encoding a cytochrome P450 or an epoxide hydrolase. In some embodiments, the 11-hydroxycucurbitadienol is produced by and / or present in the recombinant host cell. In some embodiments, the method includes contacting 3,24,25-trihydroxycucurbitadienol with a recombinant host cell, the recombinant host cell including a fifth gene encoding a cytochrome P450. The 3,24,25-trihydroxycucurbitadienol can be present in and / or produced by the recombinant host cell. In some embodiments, the contact results in mogrol being produced in the recombinant host cell. In some embodiments, the epoxide hydrolase comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:21-30, 309-314, and 1073, or the epoxide hydrolase is encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:114, 115, and 1072.

[0018] In some embodiments, the method includes contacting cucurbitadienol with a recombinant host cell. In some embodiments, the contact produces 11-hydroxycucurbitadienol. For example, cucurbitadienol can be produced by and / or present in the recombinant host cell.

[0019] In some embodiments, the method includes contacting one or more of 2,3-oxidosqualene, dioxidosqualene, and diepoxysqualene with a recombinant host cell, the recombinant host cell comprising a seventh gene encoding a polypeptide exhibiting cucurbitadienol synthase activity. In some embodiments, the polypeptide exhibiting cucurbitadienol synthase activity is a fusion protein comprising a fusion domain fused to cucurbitadienol synthase. In some embodiments, the contacting can produce cucurbitadienol and / or 24,25-epoxycucurbitadienol. In some embodiments, one or more of 2,3-oxidosqualene, dioxidosqualene, and diepoxysqualene are produced by and / or present in the recombinant host cell. In some embodiments, the recombinant host cell comprises a gene encoding a CYP87D18 or SgCPR protein.

[0020] In some embodiments, the method includes contacting squalene with a recombinant host cell, the recombinant host cell including an eighth gene encoding a squalene epoxidase. The contacting can result in the production of, for example, 2,3-oxidosqualene. In some embodiments, the squalene is produced by and / or present in the recombinant host cell.

[0021] In some embodiments, the method includes contacting farnesyl pyrophosphate with a recombinant host cell, the recombinant host cell including a ninth gene encoding squalene synthase. The contact can, for example, produce squalene. In some embodiments, the farnesyl pyrophosphate is produced by and / or is present in the recombinant host cell. The squalene synthase can, for example, include an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 69 and 336, or is encoded by a sequence including a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 337.

[0022] In some embodiments, the method includes contacting geranyl-PP with a recombinant host cell, the recombinant host cell including a tenth gene encoding a farnesyl-PP synthase. In some embodiments, the contact produces farnesyl-PP. The geranyl-PP can be produced by and / or present in the recombinant host cell. The farnesyl-PP synthase can include, for example, an amino acid sequence having at least 70% sequence identity to SEQ ID NO:338 or is encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:339.

[0023] In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene are operably linked to a heterologous promoter.The promoter can be, for example, an inducible promoter, a repressible promoter, or a constitutive promoter.The recombinant host cell can be, for example, a plant cell, a bivalve cell, a fish cell, a fungal cell, a bacterial cell, or a mammalian cell. In some embodiments, the fungus is selected from the group consisting of Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histophagus, and the like. The fungus is selected from the group consisting of Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, and Lipomyces. For example, the fungus is Aspergillus nidulans, Yarrowia lipolytica, or Rhodosporin toruloides. In some embodiments, the recombinant host cell is a yeast cell.In some embodiments, the recombinant host cell is a Saccharomyces cerevisiae cell, or a Yarrowia lipolytica cell.

[0024] In some embodiments, the method includes isolating compound 1. In some embodiments, isolating compound 1 includes disrupting recombinant host cells and / or isolating compound 1 from culture medium. The method can include purifying compound 1 using, for example, HPLC, solid phase extraction, or a combination thereof.

[0025] In some embodiments, the method includes contacting a first mogroside with a recombinant host cell to produce mogroside IIIE and then contacting mogroside IIIE with a first enzyme, the recombinant host cell including a hydrolase. The hydrolase can be, for example, a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1 or EXG2. In some embodiments, the first mogroside is mogroside IV, mogroside V, mogroside VI, siamenoside I, mogroside IV E , Mogroside IV A and combinations thereof. In some embodiments, the recombinant host cell comprises a gene encoding a hydrolase. In some embodiments, the first mogroside is produced by and / or present in the recombinant host cell.

[0026] In some embodiments, the recombinant cell further comprises an oxidosqualene cyclase, or a nucleic acid sequence encoding an oxidosqualene cyclase, and the oxidosqualene cyclase is modified to produce cucurbitadienol or epoxycucurbitadienol. The oxidosqualene cyclase can be, for example, cycloartenol synthase or β-amyrin synthase. In some embodiments, the recombinant cell comprises a cytochrome P450 reductase, or a gene encoding a cytochrome P450 reductase.

[0027] The present specification also describes Compound 1. [ka] Also disclosed is a compound having the structure: wherein the compound is produced by any of the methods disclosed herein.

[0028] Also provided herein is a recombinant cell comprising the following structure: [ka] and a gene encoding an enzyme capable of catalyzing the production of compound 1 from mogroside IIIE, and a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 105 3, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094 to 1149; and / or (4) a glycosylation enzyme encoding a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093; and / or Also disclosed are recombinant cells comprising one or more of: (5) an epoxide hydrolase comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:1073; and / or (6) an epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:1072.

[0029] Also provided is a recombinant cell, comprising a mogroside IIIE having the following structure: [ka] and (1) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1031, 1032, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; , 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2034, 2036, 1038, 1040, 6, 1088, 1090, 1092, and 1094 to 1149; and / or (4) any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093. Also disclosed are recombinant cells comprising one or more of: (5) a glycosylation enzyme encoding a nucleic acid sequence having at least 80% sequence identity to one of the sequences of SEQ ID NO:1073; and / or (6) an epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO:1072.

[0030] In some embodiments, the first enzyme is one or more of UGT, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The CGTase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of the sequences set forth in SEQ ID NOs: 1, 3, 78-101, 148, and 154. The dextransucrase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of the sequences set forth in SEQ ID NOs: 2, 103, 106-110, 156, and 896, or the dextransucrase is encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 104, 105, 157, 158, and 895. The transglucosidase can include, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3, 95 to 102, 163 to 291, and 723. The β-glucosidase can include, for example, an amino acid sequence having at least 70% sequence identity to SEQ ID NOs: 102, 292, 354 to 376, and 678 to 741.

[0031] In some embodiments, the cell comprises a second gene encoding a uridine diphosphate glucosyltransferase (UGT). The UGT may be an enzyme having at least 70% sequence identity to any one of the sequences set forth in, for example, SEQ ID NOs: 4-9, 15-19, 125, 126, 128, 129, 293-307, 407, 409, 411, 413, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149. The UGT can comprise an amino acid sequence or be encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, 445, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093. In some embodiments, the UGT is encoded by the sequence set forth in UGT1495 (SEQ ID NO: 10), UGT1817 (SEQ ID NO: 11), UGT5914 (SEQ ID NO: 12), UGT8468 (SEQ ID NO: 13), or UGT10391 (SEQ ID NO: 14).

[0032] In some embodiments, the cell comprises a third gene encoding UGT98, or UGT SK98, comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9, 407, 16, or 306, or UGT98 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 307. In some embodiments, the cell comprises a fourth gene encoding an epoxide hydrolase. The epoxide hydrolase can comprise, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 21-30, and 309-314, or is encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 114 and 115.

[0033] In some embodiments, the cell comprises a fifth gene encoding a P450. The P450 may be, for example, any one of SEQ ID NOs: 20, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 891, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049. The nucleic acid sequence may comprise an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 31-48, 316, 318, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 892, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048.

[0034] In some embodiments, the cell comprises a sixth gene encoding a polypeptide that exhibits cucurbitadienol synthase activity. In some embodiments, the polypeptide that exhibits cucurbitadienol synthase activity is a fusion protein. For example, polypeptides exhibiting cucurbitadienol synthase activity are those represented by SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 417, 420, 422, 424, 426, 446, 902, 904, 906, 851, 854, 856, 1024, 859, 862, 865, 867, 915, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 959, 964, 967, 971, 975, 979, 983, 987, 9 91, 995, 999, 1003, 1007, and 1011, or a polypeptide exhibiting cucurbitadienol synthase activity may be encoded by a nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905.

[0035] In some embodiments, the cell comprises a seventh gene encoding a squalene epoxidase. The squalene epoxidase can comprise, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 50-56, 60, 61, 334, and 335, or the squalene epoxidase is encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 335.

[0036] In some embodiments, the cell comprises an eighth gene encoding a squalene synthase. The squalene synthase can comprise, for example, an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 69 or 336, or the squalene synthase is encoded by a sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 337. In some embodiments, the cell comprises a ninth gene encoding a farnesyl-PP synthase. The farnesyl-PP synthase can comprise, for example, an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 338, or is encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO: 339. The cell can be, for example, a mammalian cell, a plant cell, a bacterial cell, a fungal cell, or an insect cell. Fungi include, for example, Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, m), Diaporthe, Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, or Metarhizium.In some embodiments, the cell is a yeast cell, and the yeast is selected from the group consisting of Candida, Saccharomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, and Microboryomycetes. In some embodiments, the recombinant cell comprises a gene encoding at least one hydrolase capable of hydrolyzing mogroside V. In some embodiments, compound 1 exhibits resistance to hydrolases in recombinant cells, and the hydrolases exhibit the ability to hydrolyze mogroside VI, mogroside V, and mogroside IV to mogroside IIIE. In some embodiments, the recombinant cells further comprise an oxidosqualene cyclase, or a nucleic acid sequence encoding an oxidosqualene cyclase, and the oxidosqualene cyclase is modified such that cucurbitadienol or epoxycucurbitadienol is produced. The oxidosqualene cyclase can comprise, for example, an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 341, 343, and 346-347. In some embodiments, the cells comprise a cytochrome P450 reductase, or a gene encoding a cytochrome P450 reductase. In some embodiments, the cells comprise a gene encoding a hydrolase capable of hydrolyzing the first mogroside to produce mogroside IIIE. For example, the hydrolase can be a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1 or EXG2.In some embodiments, the first mogroside is selected from the group consisting of mogroside IV, mogroside V, mogroside VI, siamenoside I, mogroside IVE, mogroside IVA, and combinations thereof. In some embodiments, the cell is a yeast cell. For example, the cell can be Saccharomyces cerevisiae, or Yarrowia lipolytica.

[0037] The present specification also relates to compound (1): [ka] Also disclosed is a compound having the structure: in isolated and purified form.

[0038] The present specification relates to compound (1): [ka] A composition is disclosed that includes a compound having the structure:

[0039] In some embodiments, the composition contains less than 1%, less than 0.5%, or less than 0.1% by weight of mogroside III. E In some embodiments, the composition comprises less than 0.3%, less than 0.1%, less than 0.05%, or less than 0.01% by weight of 11-oxomogroside III. EIn some embodiments, the composition comprises less than 1%, less than 0.5%, or less than 0.1% by weight of all isomers of mogroside I, mogroside II, and mogroside III. In some embodiments, the composition comprises less than 0.1% by weight of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, and mogroside IIE. In some embodiments, the composition comprises less than 1%, less than 0.5%, or less than 0.1% by weight of 11-oxomogrol. In some embodiments, the composition comprises more than 70%, more than 80%, or more than 90% by weight of the compound. In some embodiments, the composition is in an isolated form.

[0040] The present specification also relates to compound (1): [ka] Also disclosed is a composition comprising a solution of a compound having the structure:

[0041] In some embodiments, the concentration of the compound in the solution of the above embodiments is greater than 500 ppm, greater than 0.1%, greater than 0.5%, greater than 1%, greater than 5%, or greater than 10%. In some embodiments, the composition contains less than 100 ppm, less than 50 ppm, less than 20 ppm, or less than 5 ppm of mogroside III. EThe composition of any one of claims 1 to 4, comprising a concentration of 11-oxomogroside IIIE that is less than 30 ppm, less than 10 ppm, less than 1 ppm, or less than 0.1 ppm. In some embodiments, the composition of any one of claims 1 to 4, comprising a combined concentration of all isomers of mogroside I, mogroside II, and mogroside III that is less than 1%, less than 0.5%, less than 0.1%, less than 500 ppm, or less than 100 ppm. In some embodiments, the composition comprises a combined concentration of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, and mogroside IIE that is less than 500 ppm, or less than 100 ppm. In some embodiments, the composition comprises a concentration of 11-oxomogrol that is less than 0.5% or less than 100 ppm.

[0042] The present specification relates to a filler and a compound (1): [ka] and one or more compounds having the structure:

[0043] In some embodiments, the composition comprises more than 0.5%, more than 1%, or more than 2% by weight of the compound. In some embodiments, the composition comprises more than 30%, more than 50%, more than 70%, more than 90%, or more than 99% by weight of the filler. In some embodiments, the composition comprises the compound and at least one additional sweetener and / or sweetness modifier.

[0044] Also disclosed herein is the use of any of the compositions disclosed herein, such as compositions comprising Compound (1), to convey, enhance, modify, or improve the sweetness perception of a consumable product. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows HPLC and mass spectrometry data (inset) of the preparation of compound 1 after treatment of mogroside IIIE with CGTase. [Diagram 2] FIG. 1 shows HPLC and mass spectrometry data (inset) of the production of compound 1 after treatment of mogroside IIIE with Streptococcus mutans Clarke ATCC 25175 dextransucrase. [Diagram 3] FIG. 13 shows HPLC and mass spectrometry data (inset) of mogroside glycosylation reactions following treatment with Celluclast in the presence of xylan. [Figure 4] FIG. 1 shows HPLC and mass spectrometry data (inset) of mogroside glycosylation reactions after treatment with UDP-glycosyltransferase. [Diagram 5] FIG. 1 shows HPLC and mass spectrometry data (inset) of mogroside glycosylation reactions after treatment with UDP-glycosyltransferase. [Figure 6] FIG. 1 shows HPLC and mass spectrometry data (inset) of mogrol after treatment with UDP-glycosyltransferase UGT73C6 to produce mogroside I. [Figure 7] FIG. 4 shows HPLC and mass spectrometry data (inset) of mogrol after treatment with UDP-glycosyltransferase (338) (SEQ ID NO:405) to produce the product mogroside I. [Figure 8] FIG. 4 shows HPLC and mass spectrometry data (inset) of mogrol after treatment with UDP-glycosyltransferase (338) (SEQ ID NO: 405) to produce the product mogroside IIA. [Figure 9] FIG. 4 shows HPLC and mass spectrometry data (inset) of mogrol after treatment with UDP-glycosyltransferase (338) (SEQ ID NO:405) to produce two different mogroside III products. [Figure 10]FIG. 13 shows HPLC and mass spectrometry data (inset) of mogroside IIIE after treatment with UDP-glycosyltransferase to produce siamenoside I product. [Figure 11] FIG. 13 shows HPLC and mass spectrometry data (inset) of mogroside IIIE after treatment with UDP-glycosyltransferase to produce the mogroside V product. [Figure 12] FIG. 1 shows HPLC and mass spectrometry data (inset) of the products of the reaction of mogrol after treatment with UDP-glycosyltransferase (339) (SEQ ID NO: 409) to produce mogroside I. [Figure 13] FIG. 1 shows HPLC and mass spectrometry data (inset) of the products of the reaction of siamenoside I after treatment with UDP-glycosyltransferase (339) (SEQ ID NO: 409) to produce isomogroside V. [Figure 14] FIG. 1 shows HPLC and mass spectrometry data (inset) of the products of the reaction of Compound 1 after treatment with UDP-glycosyltransferase (339) (SEQ ID NO: 409) to produce Compound 1 derivatives. [Figure 15] FIG. 4 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IIA with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 16] FIG. 411 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IIE with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 17] FIG. 411 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IIIE with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 18]FIG. 4 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IVA with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 19] FIG. 4 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IVE with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 20] FIG. 411 shows HPLC and mass spectrometry data (inset) of mogroside IIIA, mogroside IVA, and mogroside V, produced by treating mogroside IIA, mogroside IIE, mogroside IIIE, mogroside IVA, or mogroside IVE, respectively, with UDP-glycosyltransferase (330) (SEQ ID NO:411). [Figure 21] FIG. 1 shows the mass spectrometry profiles of the reaction products of mogroside IVE and siamenoside I. [Figure 22] FIG. 1 shows the mass spectrometry profiles of the reaction products of mogroside IVE and siamenoside I. [Diagram 23] FIG. 1 shows the production of cucurbitadienol using cucurbitadienol synthase (SgCbQ) (SEQ ID NO: 417). [Figure 24] FIG. 1 shows the production of cucurbitadienol using enzyme Cpep2 (SEQ ID NO: 420). [Diagram 25] FIG. 1 shows the production of cucurbitadienol using enzyme Cpep4 (SEQ ID NO: 422). [Figure 26] FIG. 1 shows the production of dihydroxycucurbitadienol from catalysis by epoxide hydrolase (SEQ ID NO: 428). [Figure 27A] FIG. 1 shows the resistance of compound 1 to hydrolysis by microbial enzymes. [Figure 27B] FIG. 1 shows the resistance of compound 1 to hydrolysis by microbial enzymes. [Figure 28]Figure 2 shows the UPLC chromatogram of α-mogroside isomer mixture from the Hilic_80_20_ process. [Figure 29] Diagram showing purity of samples from UPLC analysis with the Hilic_80_20_ method. [Diagram 30] FIG. 1 shows a flow chart depicting non-limiting exemplary routes for the preparation of compound 1. [Diagram 31] FIG. 31 shows UV absorbance of diepoxysqualene from step 1 shown in FIG. 30 for boosting oxidosqualene. [Diagram 32] Diagram showing the production of cucurbitadienol in step 2 using enzymes from Cucumis melo and Cucurbita maxima. [Diagram 33] Diagram showing the production of cucurbitadienol in step 2 using an enzyme derived from Pisum sativum. [Diagram 34] FIG. 1 shows the production of cucurbitadienol in step 2 using an enzyme derived from Dictyostelium sp. [Diagram 35] A diagram showing an intermediate of Step 3 of the pathway shown in Figure 30. [Diagram 36] 31 shows mass spectrometry data for intermediates in the synthesis of mogrol, step 4 of the pathway shown in FIG. 30. [Figure 37] FIG. 31 shows an intermediate in the synthesis of compound 1, which is step 7 of the pathway shown in FIG. 30. [Figure 38] Schematic diagram showing the production of hyperglycosylated mogroside by glycosylation enzymes, which can then be hydrolyzed back to mogroside IIIE. [Figure 39] Schematic showing how a hydrolysis reaction can be used to hydrolyze hyperglycosylated mogrosides to produce mogroside IIIE, which can then be converted to compound 1. [Figure 40A]FIG. 1 shows that after 2 days of incubation, virtually all mogroside was converted to mogroside IIIE in S. cerevisiae. [Figure 40B] FIG. 1 shows that after 2 days of incubation, virtually all mogrosides were converted to mogroside IIIE in Y. lipolytica. [Diagram 41] FIG. 1 shows that no hydrolysis products from compound 1 were detected in S. cerevisiae or Y. lipolytica. [Diagram 42] FIG. 1 shows the production of compound 1 in S. cerevisiae modified to overexpress dextransucrase (DexT). [Diagram 43] 311Diagram showing the enzymatic reaction catalyzed by the enzyme (UDP-glycosyltransferase. [Diagram 44] Scheme showing the preparation of 11-OH cucurbitadienol. [Figure 45A] Diagram showing the production of mogroside IA. [Figure 45B] Diagram showing the production of mogrosides IA and IE. [Diagram 46] FIG. 1 shows the manufacture of Mogrol.

[0046] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.All patents, applications, published applications, and other publications are incorporated herein by reference in their entirety.If there are multiple definitions for a term in this specification, those in this section shall prevail unless otherwise stated.

[0047] "Solvate" refers to a compound formed by the interaction of a solvent with a compound described herein or a salt thereof. Suitable solvates are physiologically acceptable solvates, including hydrates.

[0048] As used herein, "sweetener", "sweet flavoring agent", "sweet flavor entity", "sweet compound" or "sweet compound" refers to a compound or a physiologically acceptable salt thereof that induces a detectable sweet flavor in a subject. As used herein, "sweet taste modifier" refers to a compound or a physiologically acceptable salt thereof that enhances, alters, or improves the perception of sweetness.

[0049] As used herein, the term "operably linked" is used to describe the connection between a regulatory element and a gene or its coding region. Typically, gene expression is under the control of one or more regulatory elements, such as, but not limited to, a constitutive or inducible promoter, a tissue-specific regulatory element, and an enhancer. A gene or coding region is described as "operably linked" or "operably linked" or "operably associated" with a regulatory element, which means that the gene or coding region is controlled or influenced by the regulatory element. For example, a promoter is operably linked to a coding sequence if the promoter effects the transcription or expression of the coding sequence.

[0050] The terms "regulatory element" and "expression control element" are used interchangeably and refer to nucleic acid molecules that can affect the expression of an operably linked coding sequence in a particular host organism. These terms are used broadly to encompass all elements that promote or regulate transcription, including promoters, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, intracellular sequences, core elements required for basic interaction of RNA polymerase with transcription factors, upstream elements, enhancers, response elements (see Lewin, "Genes V" (Oxford University Press, Oxford) pages 847-873), and any combination thereof. Exemplary regulatory elements in prokaryotes include promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells can include, but are not limited to, transcriptional and translational control sequences, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, proteolytic signals, internal ribosome entry elements (IRES), 2A sequences, etc., that provide and / or regulate expression of a coding sequence and / or production of an encoded polypeptide in a host cell. In some embodiments herein, the recombinant cells described herein contain a gene operably linked to a regulatory element.

[0051] As used herein, a 2A sequence or element refers to a small peptide introduced as a linker between two proteins that allows autonomous self-processing of a polyprotein in the ribosome (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004); deFelipe et al. Traffic 5:616-626 (2004)). These short peptides allow multiple proteins to be co-expressed from a single vector. Many 2A elements are known in the art. Examples of 2A sequences that can be used in the methods and systems disclosed herein include, but are not limited to, 2A sequences from foot and mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A), as described in U.S. Patent Publication No. 2007016690.

[0052] As used herein, the term "promoter" is a nucleotide sequence that allows binding of RNA polymerase and directs transcription of a gene. Usually, promoters are located in the 5' non-coding region of a gene and are proximal to the transcription start site of the gene. Sequence elements within a promoter that function in initiating transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans). Promoters can be inducible, repressible, and / or constitutive. An inducible promoter initiates increased levels of transcription from DNA under its control in response to some change in culture conditions, such as a change in temperature.

[0053] As used herein, the term "enhancer" refers to a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.

[0054] As used herein, the term "transgene" refers to any nucleotide or DNA sequence that is integrated into one or more chromosomes of a target cell by human intervention. In some embodiments, the transgene comprises a polynucleotide that encodes a protein of interest. The polynucleotide that encodes the protein is generally operatively linked to other sequences that are useful for obtaining the desired expression of the gene of interest, such as transcriptional regulatory sequences. In some embodiments, the transgene can further comprise a nucleic acid or other molecule that is used to mark the chromosome when it is integrated.

[0055] As used herein, the "percentage of sequence identity" in relation to a polynucleotide or polypeptide sequence is used as the percentage of bases or amino acid residues of a candidate sequence that are identical to the bases or amino acid residues of the other sequence after the alignment of the two sequences. If necessary, gaps can be introduced into the sequence alignment to achieve the maximum percent sequence identity. Conservative substitutions are not considered as part of sequence identity. Alignment for the purpose of determining percent sequence identity can be achieved in various ways at the discretion of the skilled person, for example, using publicly available computer methods and programs such as BLAST, BLAST-2, ALIGN, FASTA (available in the package of Genetics Computing Group (GCG), Madison, Wisconsin, USA), or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0056] For example, the percent (%) value of amino acid sequence identity can be obtained by using the WU-BLAST-2 computer program described, for example, in Altschul et al., Methods in Enzymology, 1996, 266:460-480. Many search parameters in the WU-BLAST-2 computer program can be adjusted by those skilled in the art. For example, some of the adjustable parameters can be set with the following values: overlap span=1, overlap fraction=0.125, word threshold (T)=11, scoring matrix=BLOSUM62. When WU-BLAST-2 is used, the percent value of amino acid sequence identity is determined by (a) dividing the number of identical amino acid residues that match between the amino acid sequence of the first protein of interest and the amino acid sequence of the second protein of interest, as determined by WU-BLAST-2, by (b) the total number of amino acid residues of the first protein of interest.

[0057] The percent amino acid sequence identity can also be determined using the sequence comparison program NCBI-BLAST2, described, for example, in Altschul et al., Nucleic Acids Res., 1997, 25:3389-3402. The NCBI-BLAST2 sequence comparison program can be downloaded from http: / / www.ncbi.nlm.nih.gov and is also available from the National Institutes of Health, Bethesda, Maryland. NCBI-BLAST2 uses several adjustable search parameters. The default values ​​of some of these adjustable search parameters are, for example, unmask=yes, strand=all, expected frequency of occurrence=10, minimum low complexity length=15 / 5, multipass e-value=0.01, multipass constant=25, final gap alignment dropoff=25, and scoring matrix=BLOSUM62.

[0058] In situations where NCBI-BLAST2 is used for comparing amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (or which may be expressed as a given amino acid sequence A having or containing a given % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: Fraction X / Y×100 where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program NCBI-BLAST2, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A.

[0059] As used herein, "isolated" means that the indicated compound has been separated from its natural environment, such that one or more other compounds or biological agents that exist with the compound in its natural state are no longer present.

[0060] As used herein, "purified" means that the indicated compound is present in greater amounts than other compounds that are typically found with the indicated compound (e.g., in its natural environment). In some embodiments, the relative amount of the purified compound is increased by more than 1%, more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 80%, more than 90%, more than 100%, more than 120%, more than 150%, more than 200%, more than 300%, more than 400%, or more than 1000%. In some embodiments, the purified compound is present at a weight percent level of more than 1%, more than 5%, more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 98%, more than 99%, or more than 99.5% relative to other compounds combined with it. In some embodiments, compound 1 produced from embodiments herein is present at a weight percent level of greater than 1%, greater than 5%, greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, greater than 98%, greater than 99%, or greater than 99.5% relative to other compounds with which it is combined after production.

[0061] "Purification" as described herein can refer to methods of extracting compound 1 from cell lysates and / or supernatants, where the cells excrete the production of compound 1. "Lysate" as described herein includes the cellular contents of cells following disruption of the cell wall and cell membrane, and can include, for example, proteins, sugars, and mogrosides. Purification can include ammonium sulfate precipitation to remove proteins, salt treatment to remove proteins, hydrophobic separation (HPLC), and the use of affinity columns. In view of the products produced by the methods described herein, affinity media using adsorbent resins to remove specific mogrosides are contemplated.

[0062] "HPLC" as described herein is a form of liquid chromatography that can be used to separate compounds dissolved in a solution. Without being limited thereto, an HPLC instrument can consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector. Compounds can then be separated by injecting a sample mixture into the column. Due to differences in partitioning behavior between the liquid mobile phase and the stationary phase, various components of the mixture pass through the column at different rates. There are several columns that can be used. Without being limited thereto, the columns can be normal phase columns, reverse phase columns, size exclusion type columns, and ion exchange columns.

[0063] Also contemplated is the use of solid phase extraction and fractionation, which is useful for desalting protein and sugar samples.Other methods can include the use of HPLC, liquid chromatography for sample analysis, and liquid-liquid extraction, as described in Aurea Andrade-Eiroa et al. (TrAC Trends in Analytical Chemistry Volume 80, June 2016, Pages 641-654; the entire contents of which are incorporated herein by reference).

[0064] "Solid-phase extraction" (SPE) for purposes of purification described herein refers to a sample preparation process in which compounds dissolved or suspended in a liquid mixture are separated from other compounds in the mixture according to their physical and chemical properties. For example, in analytical laboratories, solid-phase extraction can be used to concentrate and purify samples for analysis. Solid-phase extraction can also be used to separate analytes of interest from a wide variety of matrices, such as urine, blood, water, beverages, soil, and animal tissue. In the embodiments described herein, compound 1 in cell lysates or cell culture medium can be purified by solid-phase extraction.

[0065] SPE separates a mixture into desired and unwanted components by utilizing the affinity between solutes dissolved or suspended in a liquid (called the mobile phase) and a solid (called the stationary phase) through which the sample passes. SPE can be used and applied directly in the gas-liquid phase or indirectly to solid samples, for example by using thermal desorption followed by chromatographic analysis. This allows either the desired analytes of interest in the sample or the unwanted impurities to be retained in the stationary phase. The portion that passes through the stationary phase can be collected or discarded depending on whether it contains the desired analytes or the unwanted impurities. If the portion retained in the stationary phase contains the desired analytes, they can be removed from the stationary phase for recovery in an additional step, in which the stationary phase is rinsed with an appropriate eluent.

[0066] The method that can perform solid phase extraction is not limited.It can include, but is not limited to, the following techniques: normal phase SPE, reverse phase SPE, ion exchange SPE, anion exchange SPE, cation exchange, and solid phase microextraction.Solid phase extraction is described in Sajid et al., and Plotka-Wasylka J et al. (Anal Chim Acta. 2017 May 1;965:36-53, Crit Rev Anal Chem. 2017 Apr 11:1-11; the entire contents of which are incorporated herein by reference).

[0067] In some embodiments, Compound 1 produced by the cells is purified by solid phase extraction. In some embodiments, Compound 1 purified, for example by solid phase extraction, is 70%, 80%, 90%, or 100% pure, or any level of purity defined by any of the foregoing values.

[0068] "Fermentation" as described herein broadly refers to the bulk growth of host cells in a host medium to produce a particular product. In the embodiments described herein, the end product produced is Compound 1. This includes processes that can occur with or without air, for example, in an anaerobic environment. Whole cells (recombinant host cells) can be in a fermentation broth or in a reaction buffer.

[0069] Compound 1, and intermediate mogroside compounds for the production of compound 1, can be isolated by recovering intermediate mogroside compounds or compound 1 from recombinant cell lysates or supernatants. Lysates can be obtained after harvesting cells and subjecting them to disruption by shear forces (French press cell sonication) or detergent treatment. Lysates are then filtered, treated with ammonium sulfate to remove proteins, and fractionated by injection on a C18 HPLC (5 x 10 cm Atlantis prep T3 OBD column, 5 um, Waters) with an A / B gradient (A=water, B=acetonitrile) from 10 to 30% B over 30 minutes, washed with 95% B, and then re-equilibrated at 1% (total time of analysis=42 minutes). These analytes can be collected at 30 mL / fraction in tared tubes (12 fractions / plate, 3 plates per analysis). Lysates can also be centrifuged to remove solids and particulate matter.

[0070] The plate can then be dried in a Genevac HT12 / HT24. The desired compound is expected to elute in fraction 21 along with other isomers. The pooled fractions are further fractionated on a fluorophenyl HPLC column (3 x 10 cm, Xselect fluoro-phenyl OBD column, 5 um, Waters) using an A / B gradient of 15 to 30% B over 35 min (A=water, B=acetonitrile) for 47 runs, washed with 95% B, and then re-equilibrated at 15% (total run time = 45 min). Each analyte was collected at 30 mL / fraction in 12 tared tubes (12 fractions / plate, one plate per run). Fractions containing the desired peak with the desired purity are pooled based on the UPLC analysis and dried under vacuum to obtain a whitish powdery solid. The pure compound can be resuspended / dissolved in 10 mL water and lyophilized to obtain a purity of at least 95%.

[0071] As used herein, "glycosidic bond" refers to a covalent bond that connects two furanose and / or pyranose groups together. In general, a glycosidic bond is a bond between the anomeric carbon of one furanose or pyranose moiety and the oxygen of the other furanose or pyranose moiety. Glycosidic bonds are named using the position number and α / β orientation of the bonded carbon atom. α- and β-glycosidic bonds are differentiated based on the anomeric position and the relative stereochemistry of the stereocenter furthest from C1 of the ring. For example, sucrose is a disaccharide composed of one molecule of glucose and one molecule of fructose linked together via an α-1-2 glycosidic bond, as shown below.

[0072] [ka]

[0073] An example of a β-1-4 glycosidic bond can be seen in cellulose: [ka]

[0074] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "aromatic compounds" includes a mixture of aromatic compounds.

[0075] Often, ranges are expressed herein as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0076] "Codon optimization" as described herein refers to a design process that changes codons to codons known to increase maximum protein expression efficiency. In some alternatives, codon optimization for expression in cells is described, where codon optimization can be performed by using algorithms known to those skilled in the art to produce synthetic genetic transcripts optimized for high mRNA and protein yields in humans. Codons can be optimized for protein expression in, for example, bacterial cells, mammalian cells, yeast cells, insect cells, or plant cells. Programs that include algorithms for human codon optimization are readily available. Such programs can include, for example, the OptimumGene™ or GeneGPS® algorithms. Additionally, codon-optimized sequences can be commercially obtained, for example, from Integrated DNA Technologies. In some embodiments described herein, the recombinant cells for producing Compound 1 include genes encoding enzymes for synthesis, and the genes are codon-optimized for expression. In some embodiments, the genes are codon-optimized for expression in bacterial cells, yeast cells, fungal cells, or insect cells.

[0077] As used herein, the terms "nucleic acid", "nucleic acid molecule" and "polynucleotide" are interchangeable and refer to any nucleic acid, whether it is composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphoramidate, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate, or sultone bonds, and combinations of such bonds.The terms "nucleic acid" and "polynucleotide" also include nucleic acids that are composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0078] Non-limiting examples of polynucleotides include deoxyribonucleic acid (DNA), or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by either ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers that are naturally occurring nucleotides (e.g., DNA and RNA), or analogs of naturally occurring nucleotides (e.g., enantiomeric forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have mutations in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azide groups, or the sugar can be functionalized as an ether or ester. Additionally, the entire sugar moiety can be replaced with sterically and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. The nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and the like. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids," which contain naturally occurring or modified nucleic acid bases linked to a polyamide backbone. The nucleic acid can be either single-stranded or double-stranded. In some alternatives, a nucleic acid sequence encoding a fusion protein is provided. In some alternatives, the nucleic acid is RNA or DNA. In some embodiments, the nucleic acid comprises any one of SEQ ID NOs: 1-1023.

[0079] "Encode" or "code" as used herein refers to the property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, that serves as a template for the synthesis of another macromolecule, such as a given amino acid sequence. Thus, a gene encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. In some embodiments described herein, a recombinant cell is provided that includes a gene encoding an enzyme, such as, for example, dextransucrase, UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, dextranase, and / or UGT. In some embodiments, the transglucosidase includes an amino acid sequence set forth in any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the CGTase is encoded by or has the sequence of any one of SEQ ID NOs: 1, 3, 78-101, 147, and 154. In some embodiments, genes encoding enzymes such as, for example, dextransucrase, UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, dextranase, and / or UGT are codon-optimized for expression in a host cell. "Nucleic acid sequences encoding a polypeptide" include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0080] Optimization can also be performed to reduce the occurrence of secondary structures in polynucleotides. In some alternatives of the method, sequence optimization in vectors can also be performed to reduce overall GC / AT ratio. Strict codon optimization can lead to undesired secondary structures or undesirable high GC content that leads to secondary structures. Thus, secondary structures affect transcription efficiency. After optimizing codon usage, programs such as GeneOptimizer can be used to avoid secondary structures and optimize GC content. These additional programs can be used for further optimization and troubleshooting after the initial codon optimization to limit secondary structures that may occur after the initial optimization. Alternative programs for optimization are readily available. In some alternatives of the method, the vector contains sequences optimized to avoid secondary structures, and / or the sequences are optimized to reduce overall GC / AT ratio, and / or the sequences are optimized for expression in bacterial or yeast cells.

[0081] A "vector", "expression vector", or "construct" is a nucleic acid used to introduce a heterologous nucleic acid into a cell that has regulatory elements to provide expression of the heterologous nucleic acid in the cell. Vectors include, but are not limited to, plasmids, minicircles, yeast, and viral genomes. In some alternatives, the vector is a plasmid, minicircle, yeast, or genome. In some alternatives, the vector is for protein expression in bacterial systems such as E. coli. In some alternatives, the vector is for protein expression in bacterial systems such as E. coli. In some alternatives, the vector is for protein expression in yeast systems. In some embodiments, the vector for expression is a viral vector. In some embodiments, the vector is a recombinant vector that includes a promoter sequence for upregulation of expression of a gene. A "regulatory element" can refer to a nucleic acid that has a nucleotide sequence that can affect initiation and rate of transcription or translation, stability and mobility of the transcription or translation product.

[0082] A "recombinant host" or "recombinant host cell" as described herein is a host whose genome is augmented by at least one integrated DNA sequence. This integrated DNA sequence can be a heterologous nucleic acid encoding one or more polypeptides. Such DNA sequences include, but are not limited to, non-naturally occurring genes, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other genes or DNA sequences that one desires to introduce into a non-recombinant host. In some embodiments, recombinant host cells are used to prevent expression problems such as codon bias. There are commercially available hosts for protein expression, such as BL21-CodonPlus™ cells, tRNA-supplemented host strains for heterologous gene expression, Rosetta™ (DE3) competent strains for enhanced protein expression, and commercially available yeast expression systems for the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia.

[0083] The recombinant host can be a commercially available cell such as Rosetta cells for expression of enzymes that may have rare codons.

[0084] In some embodiments, the recombinant cell comprises a recombinant gene for the production of a cytochrome P450 polypeptide comprising any one of the amino acid sequences of CYP533, CYP937, CYP1798, CYP1994, CYP2048, CYP2740, CYP3404, CYP3968, CYP4112, CYP4149, CYP4491, CYP5491, CYP6479, CYP7604, CYP8224, CYP8728, CYP10020, and CYP10285. In some embodiments, the P450 polypeptide is encoded by a gene comprising any one of the sequences set forth in SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891.

[0085] In some embodiments, the P450 enzyme is assisted by at least one CYP activator, such as CPR4497. In some embodiments, the recombinant host cell further comprises a gene encoding CPR4497, wherein the gene comprises the nucleic acid sequence set forth in SEQ ID NO: 112. In some embodiments, the recombinant host cell further comprises a gene encoding CPR4497, wherein the amino acid sequence of CPR4497 is set forth in SEQ ID NO: 113.

[0086] In some embodiments, the recombinant host cell is a yeast cell, and the recombinant cell has a deletion of the EXG1 and / or EXG2 genes to prevent a reduction in glucanase activity that can lead to deglucosylation of mogrosides.

[0087] The host cell type may vary. For example, the host cell may be selected from the group consisting of Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Escherichia, Fusarium / Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, and the like. affia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces, Yarrowia, Lentinus tigrinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Fusarium fujikuroi / Gibberella fujikuroi, Candida utilis utilis, Yarrowia lipolytica, Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis,Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Aralia, Morus, Medicago, Betula, Astragalus us, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia, and Morus, Trichophyton, Sanghuan gporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histoplasma, Coccidioides ioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, Lipomyces, Aspergillus nidulans, Yarrowia lipolytica,Rhodosporin toruloides toruloides, Candida, Sacccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes, Gibberella fujikuroi. The recombinant microorganism may be selected from the group consisting of S. fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Saccharomyces cerevisiae, Escherichia coli, Rhodobacter sphaeroides, and Rhodobacter capsulatus. For example, a method for increasing the yield of a product in S. cerevisiae has been described. Methods for producing recombinant microorganisms are known.

[0088] Methods for producing recombinant host cells from Aspergillus species are described in International Publication No. WO2014086842, the entire contents of which are incorporated herein by reference. The nucleotide sequences of the genomes can be obtained through publicly available genetic data libraries, allowing for the rational design and modification of pathways to enhance and improve product yields.

[0089] The "medium" described herein can be a rich nutrient broth for the growth and maintenance of cells during the production stage of cells. Yeast cultures for maintaining and growing various strains may require specific formulations of complex media for use in cloning and protein expression, which can be understood by those skilled in the art. For example, media commercially available from ThermoFisher can be used. The medium can be YPD broth or have a yeast nitrogen base. Yeast can be grown at 30°C in YPD or synthetic medium.

[0090] Lysogeny broth (LB) is typically used for bacterial cells. The bacterial cells used for growth of enzymes and mogrosides can have antibiotic resistance to prevent growth and contamination of other cells in the medium. The cells can have antibiotic gene cassettes for resistance to antibiotics such as, for example, chloramphenicol, penicillin, kanamycin, and ampicillin.

[0091] As described herein, a "fusion protein" is a protein produced by the joining of two or more nucleic acid sequences that originally encoded part or all of the amino acid sequence of separate proteins. For example, a fusion protein can include a functional protein (e.g., an enzyme, including but not limited to cucurbitadienol synthase) and one or more fusion domains. The fusion domains described herein can be full length or a portion / fragment of a protein (e.g., functional proteins, including but not limited to enzymes, transcription factors, toxins, and translation factors). The location of the one or more fusion domains in a fusion protein can vary. For example, the one or more fusion domains can be present in the N-terminal and / or C-terminal regions (e.g., N-terminus and / or C-terminus) of the fusion protein. The one or more fusion domains can also be present in the central region of the fusion protein. The fusion domains need not be located at the ends of the fusion protein. The fusion domains can be selected to confer desired properties. For example, the fusion domain can affect (e.g., increase or decrease) the enzymatic activity of the enzyme to which it is fused or affect (e.g., increase or decrease) the stability of the protein to which it is fused. The fusion domain can be a multimerization (e.g., dimerization and tetramerization) domain and / or a functional domain. In some embodiments, the fusion domain can enhance or reduce the multimerization of the protein to which it is fused. As a non-limiting example, the fusion protein can include a full-length protein A and a fusion domain fused to the N-terminal and / or C-terminal regions of the full-length protein A. In some examples, the fusion protein includes a subsequence of protein A and a fusion domain fused to the N-terminal and / or C-terminal regions (e.g., N-terminus and C-terminus) of the subsequence of protein A. The fusion domain can be, for example, a part or the entire sequence of protein A, or a part or the entire sequence of a protein different from protein A.In some embodiments, one or more of the enzymes suitable for use in the methods, systems, and compositions disclosed herein can be a fusion protein.

[0092] The length of the fusion domain can vary, for example, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500 amino acids, or a range between any two of these numbers. In some embodiments, the fusion domain can be about 3, about 4, about 5, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 40, about 50 amino acids, or a range between any two of these numbers. In some embodiments, the fusion domain is a substantial portion or the entire sequence of a functional protein (e.g., an enzyme, a transcription factor, or a translation factor). In some embodiments, the fusion protein is a protein that exhibits cucurbitadienol synthase activity.

[0093] Optimization of cell growth and protein expression techniques in medium is also contemplated. When growing in medium, cells such as yeast may be sensitive to low pH (Narendranath et al., Appl Environ Microbiol. 2005 May; 71(5): 2239-2243; the entire contents of which are incorporated herein by reference). During growth, yeast needs to maintain a constant intracellular pH. There are many enzymes that function in yeast cells during growth and metabolism. Each enzyme works best at its optimum pH, which is acidic due to the acidophilic nature of yeast itself. When the extracellular pH is out of the optimum level, yeast cells need to consume energy to pump in or out hydrogen ions to maintain the optimum pH inside the cell. Therefore, a medium containing a buffer to control pH would be optimal. Alternatively, if the monitored pH is high, the cells can be transferred to a new medium.

[0094] Optimization of bacterial and yeast cell growth can also be achieved by the addition of nutrients and supplements to the medium. Alternatively, cultures can be grown in fermentors designed for temperature, pH control, and controlled aeration rates. Dissolved oxygen and nitrogen can be added to the medium as needed.

[0095] As used herein, the term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the heterologous nucleic acid sequence.

[0096] "Mogroside" and "mogrosidic compounds" are used interchangeably herein and refer to a family of triterpene glycosides. Non-limiting examples of mogrosides include, for example, mogroside V, siamenoside I, mogroside IV, E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 , and mogroside III, which were identified in the fruit of Siraitia grosvenorii (Swingle), which is responsible for the sweetness of the fruit. In embodiments described herein, the mogroside intermediates include the following: [ka] The recombinant cells can be used for in vivo, ex vivo, or in vitro production of compound 1 having the structure of: In some embodiments, the recombinant cells for producing compound 1 further comprise genes that produce mogrosides and encode enzymes for producing mogrosides. Recombinant cells capable of producing mogrosides are further described in WO2014086842, the entire contents of which are incorporated herein by reference. In some embodiments, the recombinant cells are grown in a medium to allow expression of the enzymes and production of compound 1 and mogroside intermediates. In some embodiments, compound 1 is obtained by disrupting the cells with shear forces (i.e., French press cell sonication) or by detergent disruption methods. In some embodiments, the cells are supplemented with precursor molecules such as mogrol in the growth medium to promote production of compound 1.

[0097] As used herein, "promoter" refers to a nucleotide sequence that directs the transcription of a structural gene. In some alternatives, the promoter is located in the 5' non-coding region of the gene, proximal to the transcription start site of the structural gene. Sequence elements within the promoter that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include, but are not limited to, RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993); expressly incorporated herein by reference in its entirety), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman et al., Seminars in Cancer Biol. 1:47 (1990); expressly incorporated herein by reference in its entirety), glucocorticoid response elements (GREs), and binding sites for other transcription factors, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992); expressly incorporated herein by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1992); (1994); the contents of which are expressly incorporated herein by reference in their entirety), SP1, cAMP response element binding protein (CREB; Loeken et al., Gene Expr. 3:253 (1993); the contents of which are expressly incorporated herein by reference in their entirety), and octamer factor (see generally, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; the contents of which are expressly incorporated herein by reference in their entirety), and Lemaigre and Rousseau, Biochem. J. 303:1 (1994); the contents of which are expressly incorporated herein by reference in their entirety). As used herein, a promoter can be constitutively active, repressible, or inducible.If a promoter is an inducible promoter, the rate of transcription increases in response to an inducing agent. In contrast, if a promoter is a constitutive promoter, the rate of transcription is not regulated by an inducing agent.

[0098] The "ribosome skipping sequence" described herein refers to a sequence that, during translation, causes the ribosome to "skip" the ribosome skipping sequence and translate the region following the ribosome skipping sequence without forming a peptide bond. For example, some viruses have ribosome skipping sequences that allow several proteins to be translated sequentially on a single nucleic acid without linking the proteins via peptide bonds. As described herein, this is a "linker" sequence. In some alternatives of the nucleic acids provided herein, the nucleic acid includes a ribosome skipping sequence between the sequences of the genes of the enzymes described herein so that the proteins are co-expressed and are not linked by peptide bonds. In some alternatives, the ribosome skipping sequence is a P2A, T2A, E2A, or F2A sequence. In some alternatives, the ribosome skipping sequence is a T2A sequence.

[0099] compound 1 As disclosed herein, Compound 1 is: [ka] or a salt thereof.

[0100] Compound 1 is a high-intensity sweetener that can be used in a wide variety of products where sweetness is desired. Compound 1 offers a low-calorie advantage over other sweeteners such as sucrose and fructose.

[0101] In some embodiments, compound 1 is in an isolated and purified form. In some embodiments, compound 1 is present in a composition in which compound 1 is substantially purified.

[0102] In some embodiments, Compound 1 or a salt thereof is isolated and in solid form. In some embodiments, the solid form is amorphous. In some embodiments, the solid form is crystalline. In some embodiments, the compound is in the form of a lyophilizate. In some embodiments, Compound 1 is isolated and in a buffer.

[0103] Those skilled in the art will recognize that some structures described herein may be resonance forms or tautomers of compounds that may be appropriately represented, even kinetically, by other chemical structures. The skilled artisan will recognize that such structures may represent only a small portion of a sample of such compounds. Although such resonance forms or tautomers are not represented herein, such compounds are considered to be within the scope of the depicted structures.

[0104] Compound 1 may be isotopes. Each chemical element represented in the compound structure may include any isotope of that element. For example, in the compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. At any position of the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, reference to a compound herein encompasses all potential isotopic forms, unless the context clearly dictates otherwise. In some embodiments, the compounds described herein are enriched in one or more isotopes compared to the natural prevalence of such isotopes. In some embodiments, the compounds described herein are enriched in deuterium. In some embodiments, more than 0.0312% of the hydrogen atoms in the compounds described herein are deuterium. In some embodiments, more than 0.05%, more than 0.08%, or more than 0.1% of the hydrogen atoms in the compounds described herein are deuterium.

[0105] In some embodiments, compound 1 is capable of forming acid and / or base salts by virtue of the presence of amino and / or carboxyl groups or groups similar thereto.

[0106] In some embodiments, Compound 1 is substantially isolated. In some embodiments, Compound 1 is substantially purified. In some embodiments, the compound is in the form of a lyophilizate. In some embodiments, the compound is crystalline. In some embodiments, the compound is amorphous.

[0107] manufacturing composition In some embodiments, the composition of manufacture does not contain any undesirable compounds or contains less than a certain amount. In some embodiments, the composition contains or does not contain one or more isomers of mogroside I, mogroside II, and mogroside III. In some embodiments, the composition contains less than 5% by weight, less than 3% by weight, less than 2% by weight, less than 1% by weight, less than 0.5% by weight, less than 0.3% by weight, less than 0.1% by weight, less than 800 ppm by weight, less than 500 ppm by weight, less than 200 ppm by weight, or less than 100 ppm by weight of all isomers of mogroside I, mogroside II, and mogroside III. In some embodiments, the composition contains or does not contain one or more of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE, and 11-oxomogrol. In some embodiments, the composition comprises less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, or 100 ppm by weight of one or more of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE, and 11-oxomogrol. In some embodiments, the composition comprises less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, or 100 ppm by weight of mogroside IIIE. In some embodiments, the composition comprises less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, or 100 ppm by weight of 11-oxomogroside IIIE. In some embodiments, the composition comprises less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, or 100 ppm by weight of 11-oxomogrol.

[0108] In some embodiments, the composition produced is in solid form, which can be crystalline or amorphous. In some embodiments, the composition is in particulate form. The solid form of the composition can be produced using any suitable technique, including, but not limited to, recrystallization, filtration, solvent evaporation, grinding, milling, spray drying, spray agglomeration, fluidized bed agglomeration, wet or dry granulation, and combinations thereof. In some embodiments, a flowable particulate composition is provided to facilitate use in further food production methods. In some such embodiments, particle sizes of 50 μm to 300 μm, 80 μm to 200 μm, or 80 μm to 150 μm are produced.

[0109] In some embodiments, a manufactured composition is provided that includes compound 1 in solution form. For example, in some embodiments, the solution manufactured by one of the manufacturing methods described herein is used without further purification. In some embodiments, the concentration of compound 1 in the solution is greater than 300 ppm by weight, greater than 500 ppm by weight, greater than 800 ppm by weight, greater than 0.1% by weight, greater than 0.5% by weight, greater than 1% by weight, greater than 2% by weight, greater than 3% by weight, greater than 4% by weight, greater than 5% by weight, greater than 6% by weight, greater than 7% by weight, greater than 8% by weight, greater than 9% by weight, greater than 10% by weight, greater than 15% by weight, or greater than 20% by weight. In some embodiments, the concentration of all isomers of mogroside I, mogroside II, and mogroside III is less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 800 ppm, less than 500 ppm, less than 200 ppm, or less than 100 ppm. In some embodiments, the concentration of one or more of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE, and 11-oxomogrol in the manufactured composition is less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 800 ppm, less than 500 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm, or less than 0.1 ppm of one or more of mogroside IIIE, 11-oxomogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE, and 11-oxomogrol. In some embodiments, the concentration of mogroside IIIE is less than 5%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 800 ppm, less than 500 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 30 ppm, less than 20 ppm, less than 10 ppm, less than 5 ppm, less than 1 ppm, or less than 0.1 ppm.In some embodiments, the concentration of 11-oxomogroside IIIE is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, 100 ppm, 50 ppm, 30 ppm, 20 ppm, 10 ppm, 5 ppm, 1 ppm, or less than 0.1 ppm. In some embodiments, the concentration of 11-oxomogrol is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800 ppm, 500 ppm, 200 ppm, 100 ppm, 50 ppm, 30 ppm, 20 ppm, 10 ppm, 5 ppm, 1 ppm, or less than 0.1 ppm.

[0110] Methods for preparing compound 1 and intermediate mogroside compounds In some embodiments, compound 1 is produced by contacting various starting and / or intermediate compounds with one or more enzymes. The contacting can be in vivo (e.g., in a recombinant cell) or in vitro. Starting and intermediate compounds for producing compound 1 include, but are not limited to, mogroside V, mogroside IIE, mogroside III, mogroside IV ... E , siamenoside I, mogroside VI isomer, mogroside II A , Mogroside IV E , or mogroside IV A may be included.

[0111] In some embodiments, compound 1 disclosed herein is produced in vivo in a recombinant host cell as described herein or by modifications of these methods. Modifications of the methodology include, among others, temperature, solvents, reagents, etc., known to those of skill in the art. The methods shown and described herein are merely exemplary and are not intended to, and should not be construed to, limit the scope of the claims in any way. Those of skill in the art will recognize modifications of the disclosed methods and be able to devise alternative routes based on the disclosure herein. Any such modifications and alternative routes are within the scope of the claims.

[0112] In some embodiments, compound 1 disclosed herein is obtained by purification and / or isolation from recombinant bacterial cells, recombinant yeast cells, recombinant plant cells, or recombinant insect cells. In some embodiments, the recombinant cells are derived from Siraitia grosvenorii. In some such embodiments, an extract obtained from Siraitia grosvenorii can be fractionated using appropriate purification techniques. In some embodiments, the extract is fractionated using HPLC and appropriate fractions are collected to obtain the desired compound in isolated and purified form.

[0113] In some embodiments, compound 1 is produced by enzymatic modification of a compound isolated from Siraitia grosvenorii. For example, in some embodiments, compound 1 isolated from Siraitia grosvenorii is contacted with one or more enzymes to obtain the desired compound. The contacting can be in vivo (e.g., in a recombinant cell) or in vitro. Starting and intermediate compounds for producing compound 1 include, but are not limited to, mogroside V, mogroside IIE, mogroside III, mogroside VII, mogroside VIII ... E , siamenoside I, mogroside VI isomer, mogroside IIA , Mogroside IV E , or mogroside IV A One or more of these compounds can be produced in vivo. Enzymes suitable for use in producing the compounds described herein can include, but are not limited to, pectinase, β-galactosidase (e.g., allomases), cellulases (e.g., celluclasts), cyclochromato dextrin glucanotransferase (e.g., Toruzyme), invertase, glucose transferase (e.g., UGT76G1), dextransucrase, lactase, arabinose, xylanase, hemicellulose, amylase, or combinations thereof. In some embodiments, the enzyme is Toruzyme and comprises an amino acid sequence set forth in any one of SEQ ID NOs: 89-94.

[0114] In some embodiments, compound 1 [ka] The present invention provides a method for preparing compound 1, the method comprising fractionating an extract of Siraitia grosvenorii on an HPLC column and collecting eluted fractions containing compound 1.

[0115] In some embodiments, compound 1 [ka] The method includes the steps of: E with the glucose transferase enzyme UGT76G1. In some embodiments, UGT76G1 is encoded by the sequence set forth in SEQ ID NO: 440. In some embodiments, UGT76G1 comprises the amino acid sequence set forth in SEQ ID NO: 439.

[0116] Various mogroside compounds can be used as intermediate compounds for producing Compound 1. One non-limiting example of such a mogroside compound is the following: [ka] In some embodiments, a method for producing compound 12 includes contacting mogroside VI with a cell (e.g., a recombinant host cell) that expresses one or more invertases.

[0117] Various mogroside compounds can be used as intermediate compounds for producing Compound 1. One non-limiting example of such a mogroside compound is the following: [ka] In some embodiments, the method for producing compound 5 comprises the step of: E with a cell (e.g., a recombinant host cell) expressing one or more cyclomaltodextrin glucanotransferases. In some embodiments, the method is performed in the presence of starch.

[0118] Various mogroside compounds can be used as intermediate compounds for producing Compound 1. One non-limiting example of such a mogroside compound is the following: [ka] In some embodiments, the method for producing compound 4 comprises the steps of: E with a cell (e.g., a recombinant host cell) expressing one or more cyclomaltodextrin glucanotransferases. In some embodiments, the method is performed in the presence of starch.

[0119] Hydrolysis of highly glycosylated mogrosides In some embodiments, one or more highly glycosylated mogrosides are hydrolyzed by contact with one or more hydrolase enzymes to produce mogroside III. E In some embodiments, the highly glycosylated mogroside is selected from mogroside IV, mogroside V, mogroside VI, and combinations thereof. In some embodiments, the highly glycosylated mogroside is selected from mogroside V, siamenoside I, mogroside IV, mogroside V, mogroside VI ... E , Isomogroside V, 11-Deoxymogroside V, 11-Oxomogroside V, Mogroside VI, Mogroside IV A , 11-oxomogroside VI, 11-oxomogroside IV E and combinations thereof.

[0120] Surprisingly, it has been found that compound 1 is resistant to hydrolysis by certain hydrolases, even though such enzymes exhibit the ability to hydrolyze highly glycosylated mogrosides to mogroside IIIE. The α-linked glycosides present in compound 1 offer a unique advantage over other mogrosides (e.g., β-linked glycosides) because they are resistant to hydrolysis. In some embodiments, during the microbial production of compound 1, the microbial host hydrolyzes the unwanted β-linked mogrosides back to mogroside IIIE. This improves the purity of compound 1 because: 1) the levels of unwanted mogroside VI, mogroside V, and mogroside IV are reduced; and 2) the amount of available mogroside IIIE that can be used as a precursor for the production of compound 1 is increased by hydrolysis.

[0121] FIG. 38 shows the production of hyperglycosylated mogrosides by glycosylase enzymes, which can then be hydrolyzed back to mogroside IIIE. This results in a lower yield of the mixture of mogrosides than is desired for hyperglycosylated mogrosides. The hydrolase enzymes can be removed, but still yield a mixture of mogrosides and may shorten the life span of the producing organism. However, because compound 1 is resistant to hydrolysis, hydrolysis can be used to drive hyperglycosylated mogrosides to mogroside IIIE, which can then be converted to compound 1 (as shown in FIG. 39).

[0122] In some embodiments, the hydrolase is a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1. The EXG1 protein can comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity to SEQ ID NO: 1013 or 1014. In some embodiments, the EXG1 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1013 or 1014. In some embodiments, the hydrolase is EXG2. The EXG2 protein can comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity to SEQ ID NO: 1023. In some embodiments, the EXG2 protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1023. The hydrolase can be, for example, any one of the hydrolases disclosed herein.

[0123] Preparation of Compound 1 from Mogroside IIIE Compound 1 can be produced from mogroside IIIE by contact with one or more enzymes capable of converting mogroside IIIE to compound 1. In some embodiments, the enzymes capable of catalyzing the production of compound 1 are one or more of UDP glycosyltransferase (also known as UGT), cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0124] In some embodiments, the enzyme capable of catalyzing the production of compound 1 is a CGTase. In some embodiments, the CGTase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises or consists of the amino acid sequence of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the enzyme capable of catalyzing the production of compound 1 is a dextransucrase. In some embodiments, the dextransucrase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the dextransucrase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the dextransucrase is encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 104, 105, 157, 158, and 895. In some embodiments, the dextransucrase comprises or consists of any one of SEQ ID NOs: 104, 105, 157, 158, and 895. In some embodiments, the enzyme capable of catalyzing the production of Compound 1 is a transglucosidase. In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 163-290, and 723.In some embodiments, the transglucosidase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 163-292, and 723. The parameters for determining the percent sequence identity can be performed by Blast search with ClustalW software (ncbi.nih.gov). These programs can be used to determine conservation between protein homologs.

[0125] In some embodiments, the enzyme capable of catalyzing the production of Compound 1 is a uridine diphosphate glucosyltransferase (UGT). The UGT can include, for example, amino acids having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these values, sequence identity to any one of SEQ ID NOs: 4-9, 15-19, 125, 126, 128, 129, 293-307, 407, 409, 411, 413, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149. In some embodiments, the UGT comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 4-9, 10-14, 125, 126, 128, 129, 293-304, 306, 407, 409, 411, 413, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149. In some embodiments, the UGT is selected from the group consisting of UGT1495 (SEQ ID NO: 10), UGT1817 (SEQ ID NO: 11), UGT5914 (SEQ ID NO: 12), UGT8468 (SEQ ID NO: 13), UGT10391 (SEQ ID NO: 14), and SEQ ID NOs: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, 445, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093, having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these figures.In some embodiments, the UGT is encoded by a nucleic acid sequence comprising any one of the nucleic acid sequences of UGT1495 (SEQ ID NO: 10), UGT1817 (SEQ ID NO: 11), UGT5914 (SEQ ID NO: 12), UGT8468 (SEQ ID NO: 13), UGT10391 (SEQ ID NO: 14), SEQ ID NOs: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, 445, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093. In some embodiments, the enzyme can be UGT98 or UGT SK98. For example, a recombinant host cell capable of producing Compound 1 as described herein can include a third gene encoding UGT98 and / or UGT SK98. In some embodiments, UGT98 or UGT SK98 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers of sequence identity to SEQ ID NO: 9 or 16. In some embodiments, the UGT comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these figures, sequence identity to UGT73C3 (SEQ ID NO:4), UGT73C6 (SEQ ID NO:5), UGT85C2 (SEQ ID NO:6), UGT73C5 (SEQ ID NO:7), UGT73E1 (SEQ ID NO:8), UGT98 (SEQ ID NO:9), UGT1576 (SEQ ID NO:15), UGT SK98 (SEQ ID NO:16), UGT430 (SEQ ID NO:17), UGT1697 (SEQ ID NO:18), and UGT11789 (SEQ ID NO:19).In some embodiments, the UGT comprises or consists of the amino acid sequence of any one of UGT73C3 (SEQ ID NO:4), UGT73C6 (SEQ ID NO:5), 85C2 (SEQ ID NO:6), UGT73C5 (SEQ ID NO:7), UGT73E1 (SEQ ID NO:8), UGT98 (SEQ ID NO:9), UGT1576 (SEQ ID NO:15), UGT SK98 (SEQ ID NO:16), UGT430 (SEQ ID NO:17), UGT1697 (SEQ ID NO:18), and UGT11789 (SEQ ID NO:19). In some embodiments, the UGT is encoded by a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity to UGT1495 (SEQ ID NO:10), UGT1817 (SEQ ID NO:11), UGT5914 (SEQ ID NO:12), UGT8468 (SEQ ID NO:13), or UGT10391 (SEQ ID NO:14). In some embodiments, the UGT is encoded by a nucleic acid sequence comprising or consisting of any one of the sequences of UGT1495 (SEQ ID NO:10), UGT1817 (SEQ ID NO:11), UGT5914 (SEQ ID NO:12), UGT8468 (SEQ ID NO:13), and UGT10391 (SEQ ID NO:14). As disclosed herein, an enzyme capable of catalyzing the production of compound 1 can comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to any one of the UGT enzymes disclosed herein. Additionally, a recombinant host cell capable of producing compound 1 can comprise an enzyme that comprises or consists of a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to any one of the UGT enzymes disclosed herein. In some embodiments, a recombinant host cell comprises an enzyme that comprises or consists of the sequence of any one of the UGT enzymes disclosed herein.

[0126] In some embodiments, the method for producing compound 1 comprises the step of: Ewith a glucose transferase enzyme UGT76G1, such as the UGT76G1 of SEQ ID NO: 439, and the UGT76G1 encoded by the nucleic acid sequence of SEQ ID NO: 440.

[0127] Mogroside compounds and enzymes for producing compound 1 As described herein, the enzymes UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase can comprise the amino acid sequences set forth in the sequence listing herein, and can be encoded by the nucleic acid sequences set forth in the sequence listing. In addition, the enzymes can also comprise functional homologs having at least 70% sequence identity to the amino acid sequences set forth in the sequence listing. The parameters for determining the percentage of sequence identity can be performed by Blast search with ClustalW software (ncbi.nih.gov). These programs can be used to determine the conservation between protein homologs.

[0128] In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the CGTase comprises or consists of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 1, 3, 78-101, and 154. In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 163-290, and 723, or is encoded by such a nucleic acid sequence.

[0129] The methods herein also include incorporating genes into the recombinant cells to produce intermediates such as pyruvate, acetyl-CoA, citrate, and other TCA intermediates (citric acid cycle). The intermediates can be further used to produce mogroside compounds to produce compound 1. Methods for increasing squalene content are described in Gruchattka et al. and Rodriguez et al. (PLoS One. 2015 Dec 23; 10(12; Microb Cell Fact. 2016 Mar 3; 15:48; the contents of which are incorporated herein by reference in their entirety).

[0130] Expression of enzymes for producing oxidosqualene and diepoxysqualene is further contemplated. The use of enzymes to produce oxidosqualene and diepoxysqualene can facilitate squalene synthesis via squalene synthase and / or squalene epoxidase. For example, Su et al. describe a gene encoding SgSQS, a 417 amino acid protein from Siraitia grosvenorii, for squalene synthase (Biotechnol Lett. 2017 Mar 28; the entire contents of which are incorporated herein by reference). Engineering recombinant cells for expression of HMG CoA reductase is also useful for squalene synthesis (Appl Environ Microbiol. 1997 Sep; 63(9):3341-4.; Front Plant Sci. 2011 Jun 30; 2:25; FEBS J. 2008 Apr; 275(8):1852-9; both of which are incorporated herein by reference in their entireties. In some embodiments, 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 898 or 900. In some embodiments, 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 897 or 899.

[0131] The expression of the enzyme for producing cucurbitadienol / epoxycucurbitadienol is also contemplated. For the manipulation into recombinant cells by vector for expression, examples of curvitadienol synthase from C. pepo, S. grosvenorii, C. sativus, C. melo, C. moschata, and C. maxim are contemplated. For the expression in recombinant cells, oxidosqualene cyclase for triterpene biosynthesis is also contemplated, which leads to the cyclization of acyclic substrates to various polycyclic triterpenes, which can also be used as intermediates for producing compound 1 (Org Biomol Chem. 2015 Jul 14;13(26):7331-6; the entire contents of which are incorporated herein by reference).

[0132] Expression of an enzyme exhibiting epoxide hydrolase activity for producing hydroxycucurbitadienol is also contemplated. In some embodiments herein, the recombinant cell for producing compound 1 further comprises a gene encoding an enzyme exhibiting epoxide hydrolase activity for producing hydroxycucurbitadienol. Such enzymes are provided in Itkin et al., the entire contents of which are incorporated herein by reference. Itkin et al. also describe enzymes for producing major mogrosides, UGS family, glycosyltransferases, and hydrolases that can be genetically modified for reverse reactions such as glycosylation.

[0133] Expression of enzymes in recombinant cells to hydroxylate mogroside compounds to produce mogrol is also contemplated. These enzymes include CAZY family proteins, UDP glycosyltransferases, CGTases, glycotransferases, dextransucrase, cellulases, β-glucosidases, transglucosidases, pectinases, dextranases, yeast, and fungal hydrolases. Such enzymes can be used, for example, to hydrolyze mogroside V to mogroside IIIE, which can be further processed to produce compound 1, for example, in vivo. In some embodiments, the fungal lactase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 678-722.

[0134] In some embodiments, squalene or a mogrol precursor, such as oxidosqualine, mogrol, or mogroside, is produced. The mogrol precursor can be used as a precursor in the production of compound 1. Squalene can be produced from famecil pyrophosphate using squalene synthase, and oxidosqualene can be produced from squalene using squalene epoxidase. The squalene synthase can be, for example, squalene synthase from Gynostemma pentaphyllum (protein accession number C4P9M2), a plant of the Cucurbitaceae family. Squalene synthase has been reported in Arabidopsis thaliana (protein accession number C4P9M3), Brassica napus, Citrus macrophylla, Euphorbia tirucalli (protein accession number B9WZW7), Glycine max, Glycyrrhiza glabra (protein accession numbers Q42760, Q42761), Glycyrrhiza uralensis (protein accession number Q42762), and Brassica napus (protein accession number C4P9M2). uralensis (protein accession numbers D6QX40, D6QX41, D6QX42, D6QX43, D6QX44, D6QX45, D6QX47, D6QX39, D6QX55, D6QX38, D6QX53, D6QX37, D6QX35, B5AID5, B5AID4, B5AID3, C7EDD0, C6KE07, C6KE08, C7EDC9), Lotus japonicas (protein accession number Q84LE3), Medicago truncatula (protein accession number Q8GSL6), Pisum sativum, Ricinus communis Alternatively, the recombinant protein may include squalene synthase from C. communis (protein accession number B9RHC3).Various squalene synthases are described in WO 2016 / 050890, the contents of which are incorporated by reference in their entirety.

[0135] Recombinant host cells Any one of the enzymes disclosed herein can be produced in vitro, ex vivo, or in vivo. For example, a nucleic acid sequence encoding an enzyme (including, but not limited to, any one of UGT, CGTase, glycotransferase, dextransucrase, celluase, β-glucosidase, amylase, transglucosidase, pectinase, dextranase, cytochrome P450, epoxide hydrolase, cucurbitadienol synthase, squalene epoxidase, squalene synthase, hydrolase, and oxidosqualene cyclase) can be introduced into a host recombinant cell, for example, in vivo, in the form of an expression vector comprising the encoding nucleic acid sequence. The expression vector can be introduced into a host cell, for example, by standard transformation techniques (e.g., heat transformation) or transfection. The expression system can produce enzymes for producing mogrosides and compound 1 to produce compound 1 in a cell in vivo. Useful expression systems include, but are not limited to, bacterial cell systems, yeast cell systems, and insect cell systems. For example, insect cell systems can be infected with recombinant virus expression systems to express the enzyme of interest. In some embodiments, the gene is codon-optimized for expression in a particular cell. In some embodiments, the gene is operably linked to a promoter to drive the transcription and translation of the enzyme protein. As described herein, codon optimization can be obtained, and the optimized sequence can then be engineered into a vector for transforming recombinant host cells.

[0136] The expression vector may further include transcriptional or translational regulatory sequences, coding sequences for transcriptional or translational factors, or various promoters (e.g., the GPD1 promoter) and / or enhancers to facilitate transcription of the gene of interest in yeast cells.

[0137] The recombinant cells described herein, in some embodiments, are genetically modified to produce compound 1 in vivo. Additionally, the cells can be fed with mogrol or mogroside precursors during or after cell growth to increase the rate of production of certain intermediates for the pathway to produce compound 1 in vivo. The cells can be in suspension or immobilized. The cells can be in a fermentation broth or in a reaction buffer. In some embodiments, a permeabilizing agent is used to transfer the mogrol or mogroside precursors to the cells. In some embodiments, the mogrol or mogroside precursors can be provided in purified form or as part of a composition or extract.

[0138] The recombinant host cell can be, for example, a plant cell, a bivalve cell, a fish cell, a fungal cell, a bacterial cell, or a mammalian cell. For example, plants include Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Aralasia, and the like. The herb may be selected from among: Albizia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia, and Morus.The fungi include Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histoplasma The fungus is selected from Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, and Lipomyces. In some embodiments, the fungus is Aspergillus nidulans, Yarrowia lipolytica, or Rhodosporin toruloides. In some embodiments, the recombinant host cell is a yeast cell.In some embodiments, the yeast is selected from Candida, Saccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes. In some embodiments, the bacterium is selected from Frankia, Actinobacteria, Streptomyces, and Enterococcus. In some embodiments, the bacterium is Enterococcus faecalis.

[0139] In some embodiments, the recombinant gene is codon-optimized for expression in bacterial cells, mammalian cells, plant cells, fungal cells, or insect cells.In some embodiments, one or more genes contain functional mutations for increasing the activity of the encoded enzyme.In some embodiments, culturing the recombinant host cell comprises monitoring the culture for pH, dissolved oxygen level, nitrogen level, or a combination of these culture conditions.

[0140] Production of Mogrol from Squalene Some embodiments of the method for preparing Compound 1 include preparing an intermediate for use in preparing Compound 1, as follows: [ka] A compound having the structure of is produced in vivo in a recombinant host. In some embodiments, the compound is present in the recombinant host cell, secreted into the medium in which the recombinant cell is growing, or both. In some embodiments, the recombinant cell further produces an intermediate, such as a mogroside compound, in vivo. The recombinant cell can be grown in a medium under conditions in which the genes disclosed herein are expressed. Some embodiments of methods for growing cells are described herein.

[0141] In some embodiments, the intermediate is or includes at least one of squalene, oxidosqualene, curbitadienol, mogrol, and mogroside. In some embodiments, the mogroside is mogroside IIE. As described herein, mogrosides are a family of glycosides that can be naturally isolated, for example, from plants or fruits. As contemplated herein, mogrosides can be produced by recombinant host cells.

[0142] In some alternatives of the methods described herein, the recombinant host cell comprises: the gene encoding squalene epoxidase; the gene encoding cucurbitadienol synthase; genes encoding cytochrome P450; A gene encoding cytochrome P450 reductase; and Genes encoding epoxide hydrolases The present invention includes a polynucleotide or sequence comprising one or more of the following:

[0143] In some embodiments, the squalene epoxidase comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO:54. In some embodiments, the squalene epoxidase is selected from the group consisting of Arabidopsis thaliana (protein accession numbers: Q9SM02, 065403, 065402, 065404, 081000, or Q9T064), Brassica napus (protein accession numbers 10065727, 065726), Euphorbia tirucalli (protein accession number A7VJN1), Medicago truncatula (protein accession numbers Q8GSM8, Q8GSM9), Pisum sativum, and Ricinus communis. communis (protein accession numbers B9R6VO, B9S7W5, B9S6Y2, B9TOY3, B9S7TO, B9SX91), as well as functional homologs of any of the foregoing sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. In some embodiments, the squalene epoxidase comprises or consists of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NOs: 50-56, 60, 61, 334, or 335.

[0144] In some embodiments, the cell comprises a gene encoding ERG7 (lanosterol synthase). In some embodiments, the lanosterol synthase comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 111. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of claims 31 to 48. In some embodiments, the sequences can be separated by ribosomal skip sequences to produce separated proteins.

[0145] In some embodiments, the recombinant host cell comprises a gene encoding a polypeptide exhibiting cucurbitadienol synthase activity. In some embodiments, the polypeptide exhibiting cucurbitadienol synthase activity comprises an amino acid sequence set forth in any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the polypeptide exhibiting cucurbitadienol synthase activity comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the polypeptide exhibiting cucurbitadienol synthase activity comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the polypeptide exhibiting cucurbitadienol synthase activity is codon-optimized. In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74.

[0146] In some embodiments, the polypeptide exhibiting cucurbitadienol synthase activity is a fusion polypeptide comprising a fusion domain fused to cucurbitadienol synthase. The fusion domain can be, for example, fused to the N-terminus or C-terminus of cucurbitadienol synthase. The fusion domain can be, for example, located at the N-terminus or C-terminus region of the fusion polypeptide. The length of the fusion domain can vary. For example, the fusion domain can be about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 20, about 25, about 30, about 40, about 50, about 100, about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1500, about 2000 amino acids in length, or a range between any two of these numbers. In some embodiments, the fusion domain is between 3 and 1000 amino acids in length. In some embodiments, the fusion domain is between 5 and 50 amino acids in length. In some embodiments, the fusion domain comprises a substantial portion or the entire sequence of a functional protein. In some embodiments, the fusion domain comprises part or the entire sequence of a yeast protein. For example, a fusion polypeptide exhibiting cucurbitadienol synthase activity can comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs:851, 854, 856, 1024, 859, 862, 865, 867, 915, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 959, 964, 967, 971, 975, 979, 983, 987, 991, 995, 999, 1003, 1007, and 1011. In some embodiments, the fusion polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 851, 854, 856, 1024, 859, 862, 865, 867, 915, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 959, 964, 967, 971, 975, 979, 983, 987, 991, 995, 999, 1003, 1007, and 1011.In some embodiments, the fusion domain of the fusion polypeptide comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 866, 870, 917, 921, 925, 929, 933, 937, 941, 945, 949, 953, 957, 961, 968, 972, 976, 980, 984, 988, 992, 996, 1000, 1004, 1008, and 1012. In some embodiments, the fusion domain of the fusion polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 866, 870, 917, 921, 925, 929, 933, 937, 941, 945, 949, 953, 957, 961, 968, 972, 976, 980, 984, 988, 992, 996, 1000, 1004, 1008, and 1012. In some embodiments, the cucurbitadienol synthase fused to the fusion domain comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327, 329-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the cucurbitadienol synthase fused to the fusion domain comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327, 329-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the cucurbitadienol synthase fused to the fusion domain is encoded by a gene comprising a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905.In some embodiments, the cucurbitadienol synthase fused to the fusion domain is encoded by a gene comprising or consisting of a nucleic acid sequence set forth in any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905. Also disclosed herein are recombinant nucleic acid molecules comprising a nucleic acid sequence encoding a fusion polypeptide that exhibits cucurbitadienol synthase activity.

[0147] The fusion polypeptides exhibiting cucurbitadienol synthase activity disclosed herein can be used to catalyze enzymatic reactions as cucurbitadienol synthase. For example, a substrate for cucurbitadienol synthase can be contacted with one or more of these fusion polypeptides to produce a reaction product. Non-limiting examples of reaction products include curcurbitadienol, 24,25-epoxy curcurbitadienol, and any combination thereof. Non-limiting examples of substrates for cucurbitadienol synthase include 2,3-oxidosqualene, dioxidosqualene, diepoxysqualene, and any combination thereof. In some embodiments, the substrate can be contacted with a recombinant host cell comprising a nucleic acid sequence encoding one or more fusion polypeptides exhibiting cucurbitadienol synthase activity. The substrate can be provided to, present in, or produced by the recombinant host cell, or any combination thereof.

[0148] In some embodiments, the cytochrome P450 is CYP5491. In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of the sequences set forth in SEQ ID NOs: 44, 74, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049. In some embodiments, the P450 reductase polypeptide comprises an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 46. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 891, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048.

[0149] In some embodiments, the epoxide hydrolase comprises a sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 38, 40 or 1073. In some embodiments, the epoxide hydrolase comprises or consists of a sequence set forth in SEQ ID NO: 38, 40 or 1073. In some embodiments, the epoxide hydrolase is encoded by a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 1072.

[0150] Several methods for producing squalene to produce mogrol Squalene is a naturally occurring 30-carbon organic molecule that can be produced in plants and animals and is the biochemical precursor of a family of steroids. In addition, squalene can be used as a precursor for the synthesis of mogrol in vivo in host recombinant cells. Oxidation of one of the terminal double bonds of squalene (via squalene monooxygenase) produces 2,3-squalene oxide, which undergoes enzyme-catalyzed cyclization to produce lanosterol, which is then synthesized into cholesterol and other steroids. As described in Gruchattka et al. ("In Vivo Validation of In Silico Predicted Metabolic Engineering Strategies in Yeast: Disruption of α-Ketoglutarate Dehydrogenase and Expression of ATP-Citrate Lyase for Terpenoid Production." PLOS ONE December 23, 2015; the entire contents of which are incorporated herein by reference), synthesis of squalene can occur initially from precursors in the glycolytic cycle to produce squalene. Squalene can be upregulated by overexpression of ATP-citrate lyase to increase production of squalene. Some embodiments disclosed herein include enzymes for producing and / or facilitating production of squalene in a recombinant host cell, such as a recombinant yeast cell.ATP citrate lyase can also mediate acetyl-CoA synthesis, which can be used to produce squalene and mevalonate as seen in the yeast S. cerevisiae (Rodrigues et al. "ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae" Microb Cell Fact. 2016; 15: 48; the entire contents of which are incorporated herein by reference). An example of a gene encoding an enzyme for mediating acetyl-CoA synthesis is shown in SEQ ID NO: 130. In some embodiments herein, the recombinant cell comprises a sequence for mediating acetyl-CoA synthesis.

[0151] Some embodiments disclosed herein include the following: [ka] The present invention provides a method for producing compound 1 having the structure:

[0152] In some embodiments, the method further comprises producing an intermediate in a pathway for producing compound 1 in vivo. In some embodiments, the recombinant host cell that produces compound 1 comprises at least one enzyme capable of catalyzing the conversion of dioxidosqualene to produce 24,25 epoxy cucurbitadienol, the conversion of oxidosqualene to cucurbitadienol, the hydroxylation of 24,25 epoxy cucurbitadienol to 11-hydroxy-24,25 epoxy cucurbitadienol, an enzyme for catalyzing the hydroxylation of cucurbitadienol to 11-hydroxycucurbitadienol, an enzyme for catalyzing the epoxidation of cucurbitadienol to 24,25 epoxy cucurbitadienol, and an enzyme for catalyzing the epoxidation of cucurbitadienol to 24,25 epoxy cucurbitadienol. The enzymes include an enzyme for catalyzing the conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to 11-hydroxy-24,25 epoxy cucurbitadienol, an enzyme for catalyzing the conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to 11-hydroxy-24,25 epoxy cucurbitadienol, an enzyme for catalyzing the conversion of 11-hydroxy-24,25 epoxy cucurbitadienol to produce mogrol, and / or an enzyme for catalyzing the glycosylation of a mogroside precursor to produce a mogroside compound. In some embodiments, the enzyme for glycosylation is encoded by a sequence set forth in any one of SEQ ID NOs: 121, 122, 123, and 124.

[0153] In some embodiments, the enzyme for catalyzing the production of 11-hydroxy-24,25 epoxy cucurbitazinol by hydroxylation of 24,25 epoxy cucurbitadienol is CYP5491. In some embodiments, the CYP5491 comprises a sequence set forth in SEQ ID NO: 49. In some embodiments, the squalene epoxidase comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to the sequence of SEQ ID NO: 54.

[0154] In some embodiments, the enzyme capable of epoxidizing 11-hydroxycucurbitadientol comprises the amino acid sequence set forth in SEQ ID NO:74.

[0155] In some alternatives, the recombinant cells contain genes for expressing enzymes capable of converting dioxidosqualent to 24,25 epoxy cucurbitadienol, converting oxidosqualene to cucurbitadienol, hydroxylating 24,25 epoxy cucurbitadienol to 11-hydroxy-24,25 epoxy cucurbitadienol, hydroxylating cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol, epoxidizing cucurbitadienol to produce 24,25 epoxy cucurbitadienol, and / or epoxidizing 11-hydroxy-24,25 epoxy cucurbitadienol to produce 11-hydroxy-24,25 epoxy cucurbitadienol. In these embodiments herein, the intermediates and mogrosides are produced in vivo.

[0156] In some embodiments, the method for producing compound 1 further includes producing one or more of mogroside compounds and intermediates, such as oxidosqualene, dixidosqualene, cucurbitdienol, 24,25 epoxy cucurbitadienol, 11-hydrosy-cucurbitadienol, 11-hydroxy 24,25 epoxy cucurbitadienol, mogrol, and mogroside compounds.

[0157] Method for producing mogroside compounds Described herein is a method for producing a mogroside compound, such as one of the mogroside compounds described in International Publication No. WO2014086842, the contents of which are incorporated herein by reference in their entirety. The mogroside compound can be used as an intermediate by cells to further produce compound 1 as disclosed herein.

[0158] Recombinant hosts such as microorganisms, plant cells, or plants can be used to express polypeptides useful for the biosynthesis of mogrol (the triterpene core) and the various mogrol glycosides (mogrosides).

[0159] In some embodiments, the method comprises the steps of: (1) Increases oxidosqualene levels (2) Increases levels of dioxidosqualene (3) Oxidosqualene → cucurbitadienol, (4) Dioxidosqualene → 24,25 epoxy cucurbitadienol, (5) Cucurbitadienol → 11-hydroxycucurbitadienol, (6) 24,25 epoxy cucurbitadienol → 11-hydroxy-24,25 epoxy cucurbitadienol, (7) 11-hydroxycucurbitadienol → mogrol, (8) 11-hydroxy-24,25 epoxy cucurbitadienol → mogrol, (9) Mogrol - various mogroside compounds, may include one or more of in any order.

[0160] In an embodiment of the present specification, oxidosqualene, dioxidosqualene, cucurbitadienol, 24,25 epoxy cucurbitadienol, or mogrol can also be produced by recombinant cells. The method can include growing a recombinant microorganism in a medium under conditions in which one or more enzymes that catalyze the steps of the method of the present invention, such as synthases, hydrolases, CYP450s, and / or UGTs, are expressed. The recombinant microorganism can be grown in a fed-batch or continuous method. Typically, the recombinant microorganism is grown in a fermenter at a predetermined temperature for a desired period of time to improve the yield of compound 1.

[0161] In some embodiments, mogroside compounds can be produced using whole cells, which are fed with feedstocks containing precursor molecules to improve the yield of compound 1. The feedstocks can be fed during or after cell growth. The whole cells can be in suspension or immobilized. The whole cells can be in a fermentation broth or in a reaction buffer.

[0162] In some embodiments, the recombinant host cell can contain a heterologous nucleic acid encoding an enzyme or mixture of enzymes capable of catalyzing oxidosqualene to cucurbitadienol, cucurbitadienol to 11-hydroxycucurbitadienol, 11-hydroxycucurbitadienol to mogrol, and / or mogrol to mogroside. In some embodiments, the cell can further contain a heterologous nucleic acid encoding an enzyme or mixture of enzymes capable of catalyzing dioxidosqualene to 24,25 epoxy cucurbitadienol, 24,25 epoxy cucurbitadienol to hydroxy-24,25 epoxy cucurbitadienol, 11-hydroxy-24,25 epoxy cucurbitadienol to mogrol, and / or mogrol to mogroside.

[0163] The host cell can contain a recombinant gene encoding a cucurbitadienol synthase and / or a recombinant gene encoding a cytochrome P450 polypeptide.

[0164] In some embodiments, the cell comprises a protein having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, 906 (curcurbitadienol synthase).

[0165] In some embodiments, the conversion of oxidosqualene to cucurbitadienol is catalyzed by a cucurbitadienol synthase of any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906, or a functional homologue of the foregoing sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the cucurbitadienol synthase polypeptide is codon-optimized. In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74.

[0166] In some embodiments, the conversion of cucurbitadienol to 11-hydroxycucurbitadienol is catalyzed by CYP5491 of SEQ ID NO: 49, or a functional homolog thereof sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.

[0167] In some embodiments, the conversion of 11-hydroxycucurbitadienol to mogrol comprises a polypeptide selected from the group consisting of epoxide hydrolase 1 of SEQ ID NO: 29, epoxide hydrolase 2 of SEQ ID NO: 30, and functional homologs of the foregoing sharing at least 70%, such as at least 80%, such as at least 90%, such as at least 95%, such as at least 98% sequence identity therewith. In some embodiments, the genes encoding epoxide hydrolase 1 and epoxide hydrolase 2 are codon-optimized for expression. In some embodiments, the codon-optimized gene for epoxide hydrolase comprises the nucleic acid sequence set forth in SEQ ID NO: 114 or 115.

[0168] In some embodiments, the epoxide hydrolase comprises an amino acid sequence set forth in any one of SEQ ID NOs:21-28 (Itkin et al., incorporated herein by reference in its entirety).

[0169] In some embodiments, the conversion of mogrol to mogroside is catalyzed in the host recombinant cell by one or more UGTs selected from the group consisting of UGT1576 of SEQ ID NO: 15, UGT98 of SEQ ID NO: 9, UGT SK98 of SEQ ID NO: 68, and functional homologues of the foregoing sharing at least 70%, such as at least 80%, for example at least 90%, such as at least 95%, for example at least 98% sequence identity therewith.

[0170] In some embodiments, the host recombinant cell comprises a recombinant gene encoding a cytochrome P450 polypeptide encoded by any one of the sequences set forth in SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 891, 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093.

[0171] In some embodiments, the host recombinant cell comprises a recombinant gene encoding a squalene epoxidase polypeptide comprising the sequence of SEQ ID NO:50.

[0172] In some embodiments, the host recombinant cell comprises a recombinant gene encoding a cucurbitadienol synthase polypeptide of any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the cucurbitadienol synthase polypeptide is codon-optimized. In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74.

[0173] Production of mogroside compounds from mogrol In some embodiments, the method for producing compound 1 includes contacting mogroside IIIE with a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the method is performed in vivo and the recombinant cell includes a gene encoding a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the cell further includes a gene encoding an enzyme capable of catalyzing the production of mogroside IE1 from mogroside. In some embodiments, the enzyme includes a sequence set forth in any one of SEQ ID NOs: 4-8.

[0174] In some embodiments, the cells further comprise an enzyme for converting mogroside IIE to mogroside IV, mogroside V, 11-oxomogroside V, and siamenoside I. In some embodiments, the enzyme for converting mogroside IIE to mogroside IV, mogroside V, 11-oxomogroside V, and siamenoside I is encoded by a gene comprising a nucleic acid sequence set forth in SEQ ID NOs: 9-14, and 116-120. In some embodiments, the method for producing Compound 1 further comprises converting mogroside III to mogroside V. E with the glucose transferase enzyme UGT76G1.

[0175] In some embodiments, the method comprises fractionating lysates from the recombinant cells on an HPLC column and collecting elution fractions containing Compound 1.

[0176] In some embodiments, the method includes contacting mogroside IIIE with a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, contacting mogroside IIIE with the first enzyme includes contacting mogroside IIIE with a recombinant host cell comprising a first gene encoding the first enzyme. In some embodiments, the first gene is heterologous to the recombinant host cell. In some embodiments, mogroside IIIE contacts the first enzyme in a recombinant host cell comprising a first polynucleotide encoding the first enzyme. In some embodiments, mogroside IIIE is present in the recombinant host cell. In some embodiments, mogroside IIIE is produced by the recombinant host cell. In some embodiments, the method includes culturing the recombinant host cell in a medium under conditions in which the first enzyme is expressed. In some embodiments, the first enzyme is one or more of UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the first enzyme is CGTase. In some embodiments, the CGTase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the transglucosidase is encoded by any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the CGTase comprises or consists of a sequence set forth in any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the first enzyme is a dextransucrase.In some embodiments, the dextransucrase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the DexT comprises an amino acid sequence as set forth in SEQ ID NO: 2, 103, 106-110, 156, and 896. In some embodiments, the DexT comprises a nucleic acid sequence as set forth in SEQ ID NO: 104 or 105. In some embodiments, the dextransucrase comprises an amino acid sequence as set forth in SEQ ID NO: 2, or 106-110. In some embodiments, the first enzyme is a transglucosidase. In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these values ​​to any one of SEQ ID NOs: 3163-291, and 723. In some embodiments, the transglucosidase comprises an amino acid sequence of SEQ ID NOs: 163-291, and 723. In some embodiments, the transglucosidase is encoded by any one of SEQ ID NOs: 163-291, and 723. In some embodiments, the transglucosidase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the gene encodes a CGTase comprising any one of the sequences set forth in SEQ ID NOs: 1, 3, 78-101, and 154.

[0177] In some embodiments, the method includes contacting mogroside IIA with a recombinant host cell to produce mogroside IIIE, the recombinant host cell further comprising a second gene encoding a second enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA. In some embodiments, mogroside IIA is produced by the recombinant host cell. In some embodiments, the second enzyme is one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the second enzyme is a uridine diphosphate glucosyltransferase (UGT). In some embodiments, the transglucosidase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the gene encodes a CGTase comprising the amino acid sequence set forth in SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the UGT is UGT73C3 (SEQ ID NO: 4), UGT73C6 (SEQ ID NO: 5, 444, or 445), 85C2 (SEQ ID NO: 6), UGT73C5 (SEQ ID NO: 7), UGT73E1 (SEQ ID NO: 8), UGT98 (SEQ ID NO: 9, or 407), UGT1576 (SEQ ID NO: 15), UGT SK98 (SEQ ID NO:16), UGT430 (SEQ ID NO:17), UGT1697 (SEQ ID NO:18), or UGT11789 (SEQ ID NO:19), or any one of SEQ ID NOs:4, 5, 7-9, 15-19, 125, 126, 128, 129, 293-304, 306, 307, 407, 439, 441, 444, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149.In some embodiments, the UGT is encoded by a nucleic acid sequence set forth in any one of UGT1495 (SEQ ID NO:10), UGT1817 (SEQ ID NO:11), UGT5914 (SEQ ID NO:12), UGT8468 (SEQ ID NO:13), UGT10391 (SEQ ID NO:14), and SEQ ID NOs:1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093.

[0178] In some embodiments, the method includes contacting a mogroside with a recombinant host cell to produce mogroside IIIE, the recombinant host cell further comprising one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE from mogrol. In some embodiments, mogrol is produced by the recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the second enzyme is a uridine diphosphate glucosyltransferase (UGT). In some embodiments, the UGT is UGT73C3, UGT73C6, 85C2, UGT73C5, UGT73E1, UGT98, UGT1495, UGT1817, UGT5914, UGT8468, UGT10391, UGT1576, UGT SK98, UGT430, UGT1697, or UGT11789.

[0179] In some embodiments, the method includes contacting a mogroside compound with a recombinant host cell to produce mogroside IIIE, the recombinant host cell further comprising one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE from the mogroside compound, the mogroside compound being one or more of mogroside IA1, mogroside IE1, mogroside IIA1, mogroside IIE, mogroside IIIA1, mogroside IIIA2, mogroside III, mogroside IV, mogroside IVA, mogroside V, or siamenoside. In some embodiments, the mogroside compound is produced by the recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the transglucosidase comprises an amino acid sequence set forth in any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the gene encodes a CGTase comprising an amino acid sequence set forth in SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the method comprises contacting mogroside IA1 with a recombinant host cell, the recombinant host cell comprising a gene encoding UGT98 or UGT SK98. In some embodiments, the UGT98 or UGT SK98 enzyme comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 9, 407, or 16. In some embodiments, the contacting produces mogroside IIA in the cell. In some embodiments, the one or more enzymes comprise an amino acid sequence set forth in any one of SEQ ID NOs: 1, 3, 78-101, 106-109, 147, 154, 163-303, 405, 411, 354-405, 447-723, 770, 776, and 782.

[0180] In some embodiments, the method further comprises contacting 11-hydroxy-24,25 epoxy cucurbitadienol with a recombinant host cell, the recombinant host cell further comprising a third gene encoding an epoxide hydrolase. In some embodiments, the 11-hydroxy-24,25 epoxy cucurbitadienol is produced by a recombinant host cell. In some embodiments, the method further comprises contacting 11-hydroxy cucurbitadienol with a recombinant host cell, the recombinant host cell further comprising a fourth gene encoding a cytochrome P450 or epoxide hydrolase. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence set forth in any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891. In some embodiments, the 11-hydroxy cucurbitadienol is produced by a recombinant host cell.

[0181] In some embodiments, the method further comprises contacting 3,24,25 trihydroxycucurbitadienol with a recombinant host cell, the recombinant host cell further comprising a fifth gene encoding a cytochrome P450. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence set forth in any one of SEQ ID NOs: 31-48, 316, 318, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049. In some embodiments, the 3,24,25 trihydroxycucurbitadienol is produced by the recombinant host cell. In some embodiments, the contacting produces mogrol in the recombinant host cell. In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 20, 308, or 315. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence set forth in any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 891, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048. In some embodiments, the epoxide hydrolase comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs:21-30, and 309-314.

[0182] In some embodiments, the method further comprises contacting cucurbitadienol with a recombinant host cell, the recombinant host cell comprising a gene encoding a cytochrome P450. In some embodiments, the contact produces 11-cucurbitadienol. In some embodiments, the 11-hydroxycucurbitadienol is expressed in a cell comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, the CYP87D18 or SgCPR comprises a sequence set forth in SEQ ID NO: 315, 872, or 874. In some embodiments, the CYP87D18 or SgCPR is encoded by SEQ ID NO: 316, 871, or 873. In some embodiments, the cucurbitadienol is produced by the recombinant host cell. In some embodiments, the gene encoding cytochrome P450 comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891. In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 891, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence set forth in any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 891, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048.

[0183] In some embodiments, the method further comprises contacting 2,3-oxidosqualene with a recombinant host cell, the recombinant host cell comprising a seventh gene encoding a cucurbitadienol synthase. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence set forth in SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, or 906. In some embodiments, the cucurbitadienol synthase is encoded by any one of the sequences set forth in SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905. In some embodiments, the contacting produces cucurbitadienol. In some embodiments, the 2,3-oxidosqualene is produced by a recombinant host cell. In some embodiments, the 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme comprising a sequence set forth in SEQ ID NO:898 or 900. In some embodiments, the 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence set forth in SEQ ID NO:897 or 899.

[0184] In some embodiments, the cucurbitadienol synthase is encoded by a gene comprising the sequence set forth in SEQ ID NO: 74. In some embodiments, the cucurbitadienol synthase is encoded by a gene comprising the nucleic acid sequence set forth in any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905. In some embodiments, 11-hydroxycucurbitadienol is produced by a cell. In some embodiments, 11-OH cucurbitadienol is expressed in a cell comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, the CYP87D18 or SgCPR comprises the sequence set forth in SEQ ID NO: 315, 872, or 874. In some embodiments, the CYP87D18 or SgCPR is encoded by SEQ ID NO: 316, 871, or 873. In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the cucurbitadienol synthase polypeptide is codon-optimized. In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906 (including, for example, curbitadienol synthases from C. pepo, S. grosvenorii, C. sativus, C. melo, C. moschata, and C. maxim). In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the cucurbitadienol synthase polypeptide is codon optimized.In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the cucurbitadienol synthase is selected from the group consisting of Lotus japonicus (BAE53431), Populus trichocarpa (XP_002310905), Actaea racemosa (ADC84219), Betula platyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera (XP_002264289), Centella asiatica (BAA76902), and the like, as described in WO 2016 / 050890, the contents of which are incorporated by reference in their entirety. The amino acid containing polypeptides are derived from Acanthus asiatica (AAS01524), Panax ginseng (BAA33460), and Betula platyphylla (BAB83086).

[0185] In some embodiments, the method comprises contacting squalene with a recombinant host cell, wherein the recombinant host cell comprises an eighth gene encoding a squalene epoxidase. In some embodiments, the contacting produces 2,3-oxidosqualene. In some embodiments, the squalene is produced by the recombinant host cell. In some embodiments, the squalene epoxidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NOs: 50-56, 60, 61, 334, or 335.

[0186] In some embodiments, the method comprises contacting farnesyl pyrophosphate with a recombinant host cell, the recombinant host cell comprising a ninth gene encoding squalene synthase. In some embodiments, the contacting produces squalene. In some embodiments, the farnesyl pyrophosphate is produced by the recombinant host cell. In some embodiments, the squalene synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 69 or 336.

[0187] In some embodiments, the method further comprises contacting geranyl-PP with a recombinant host cell, the recombinant host cell comprising a tenth gene encoding a farnesyl-PP synthase. In some embodiments, the contacting produces farnesyl-PP. In some embodiments, geranyl-PP is produced by the recombinant host cell. In some embodiments, the farnesyl-PP synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 338. In some embodiments, one or more of the first gene, second gene, third gene, fourth gene, fifth gene, sixth gene, seventh gene, eighth gene, ninth gene, and tenth gene are operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is a CMV, EF1a, SV40, PGK1, human beta actin, CAG, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, T7, T7lac, Sp6, araBAD, trp, Lac, Ptac, pL promoter, or a combination thereof. In some embodiments, the promoter is an inducible promoter, a repressible promoter, or a constitutive promoter. In some embodiments, the production of one or more of pyruvate, acetyl-CoA, citrate, and a TCA cycle intermediate is upregulated in the recombinant host cell. In some embodiments, cytosolic localization is upregulated in the recombinant host cell. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene comprise at least one sequence encoding a 2A self-cleaving peptide. As used herein, the terms first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, etc. do not imply a requirement that a particular order and / or preceding numbers be present.For example, a recombinant host cell described herein can include a first gene and a third gene but not include a second gene. As another example, a recombinant host cell can include a first gene, a fifth gene, and a tenth gene but not include a second gene, a third gene, a fourth gene, a sixth gene, a seventh gene, an eighth gene, and a ninth gene.

[0188] The recombinant host cell can be, for example, a plant cell, a bivalve cell, a fish cell, a fungal cell, a bacterial cell, or a mammalian cell. For example, plants include Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Alaria, and the like. ralia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia, and Morus.In some embodiments, the fungus is selected from the group consisting of Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, The fungus is selected from Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, and Lipomyces. In some embodiments, the fungus is Aspergillus nidulans, Yarrowia lipolytica, or Rhodosporin toruloides. In some embodiments, the recombinant host cell is a yeast cell.In some embodiments, the yeast is selected from Candida, Saccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes. In some embodiments, the bacterium is selected from Frankia, Actinobacteria, Streptomyces, and Enterococcus. In some embodiments, the bacterium is Enterococcus faecalis. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene are codon-optimized for expression in a bacterial cell, a mammalian cell, a plant cell, a fungal cell, or an insect cell. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene comprise a functional mutation for increasing the activity of the encoded enzyme. In some embodiments, culturing the recombinant host cells includes monitoring the culture for pH, dissolved oxygen levels, nitrogen levels, or a combination of these culture conditions. In some embodiments, the method includes isolating Compound 1. In some embodiments, isolating Compound 1 includes disrupting the recombinant host cells.In some embodiments, isolating compound 1 comprises isolating compound 1 from the culture medium. In some embodiments, the method comprises purifying compound 1. In some embodiments, purifying compound 1 comprises HPLC, solid phase extraction, or a combination thereof. In some embodiments, the purification step comprises harvesting the recombinant cells, saving the supernatant, and disrupting the cells. In some embodiments, the disruption step comprises subjecting the cells to shear force or washing with a detergent to obtain a lysate. In some embodiments, the shear force is by sonication, French press treated cells, or beads. In some embodiments, the lysate is subjected to a filtration step and a purification step. In some embodiments, the lysate is filtered and purified by solid phase extraction.

[0189] In some embodiments, compound 1 [ka] wherein the compound is prepared by any one of the alternative methods provided herein.

[0190] In some embodiments, the following: [ka] A cell lysate is provided that contains compound 1 having the structure:

[0191] In some embodiments, the following: [ka] and a gene encoding an enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the gene is a heterologous gene to the recombinant cell.

[0192] In some embodiments, from mogroside IIIE: [ka] Recombinant cells are provided that include a first gene encoding a first enzyme capable of catalyzing the production of compound 1 having the structure of: In some embodiments, the first enzyme comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 1, 3, 78-101, 148, or 154 (CGTase). In some embodiments, the first enzyme comprises the amino acid sequence of SEQ ID NO: 1, 3, 78-101, 148, or 154 (CGTase). In some embodiments, the first enzyme is a dextransucrase. In some embodiments, the dextransucrase comprises or consists of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the dextransucrase comprises or consists of an amino acid sequence of SEQ ID NO: 2, 103, 104, or 105. In some embodiments, the dextransucrase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 2, 103-110, and 156-162, and 896. In some embodiments, the DexT comprises a nucleic acid sequence set forth in SEQ ID NO: 104 or 105. In some embodiments, the first enzyme is a transglucosidase. In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to the sequence of SEQ ID NO: 201 or SEQ ID NO: 291. In some embodiments, the recombinant cell further comprises a second gene encoding a uridine diphosphate glucosyltransferase (UGT).In some embodiments, the UGT comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 15, 16, 17, 18, 19, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149. In some embodiments, the UGT comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 15, 16, 17, 18, 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094-1149. In some embodiments, the UGT is encoded by UGT1495 (SEQ ID NO:10), UGT1817 (SEQ ID NO:11), UGT5914 (SEQ ID NO:12), UGT8468 (SEQ ID NO:13), or UGT10391 (SEQ ID NO:14), or a sequence set forth in any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093. In some embodiments, the cell comprises a third gene encoding UGT98 or UGT SK98. In some embodiments, UGT98 or UGT SK98 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 9, 407, or 16. In some embodiments, the cell comprises a fourth gene encoding an epoxide hydrolase.In some embodiments, the epoxide hydrolase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 21-30, and 309-314. In some embodiments, the cell comprises a fifth sequence encoding a P450. In some embodiments, the P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NOs: 20, 49, 308, 315, or 317. In some embodiments, the P450 is encoded by a gene comprising a sequence set forth in any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891. In some embodiments, the gene further comprises a sixth sequence encoding a cucurbitadienol synthase. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence set forth in SEQ ID NO: 73. In some embodiments, the gene encoding the cucurbitadienol synthase polypeptide is codon-optimized. In some embodiments, the codon-optimized gene comprises the nucleic acid sequence set forth in SEQ ID NO: 74. In some embodiments, the cell further comprises a seventh gene encoding a squalene epoxidase.In some embodiments, the squalene epoxidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 50-56, 60, 61, 334, and 335. In some embodiments, the cell further comprises an eighth gene encoding a squalene synthase. In some embodiments, the eighth gene comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 69 or SEQ ID NO: 336. In some embodiments, the cell further comprises a ninth gene encoding a farnesyl-PP synthase. In some embodiments, the farnesyl-PP synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 338. In some embodiments, the cell is a mammalian cell, a bacterial cell, a fungal cell, or an insect cell. In some embodiments, the cell is a yeast cell. Non-limiting examples of yeast include Candida, Saccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, and Microboryomycetes.In some embodiments, the plant is selected from the group consisting of Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Alaria, and the like. The plant is selected from the group consisting of Aralia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia, and Morus.In some embodiments, the fungus is a species of Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, or the like. Colletotrichum, Diaporthe, Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, or Metarhizium.

[0193] In some embodiments, the cell comprises an enzyme sequence set forth in any one of SEQ ID NOs: 897, 899, 909, 911, 913, 418, 421, 423, 425, 427, 871, 873, 901, 903, or 905. In some embodiments, the enzyme comprises or is encoded by a sequence set forth in SEQ ID NOs: 420, 422, 424, 426, 446, 872, 874-896, 898, 900, 902, 904, 906, 908, 910, 912, and 951-1012.

[0194] In some embodiments, the DNA can be obtained by gene synthesis. This can be done, for example, by either Genescript or IDT. The DNA can be cloned into overexpression vectors, such as, for example, pQE1, pGEX-4t3, pDest-17, pET series, pFASTBAC, by standard molecular biology techniques. E. coli host strains can be used to produce the enzymes (i.e., Top10 or BL21 series + / - codon plus) using 1 mM IPTG for induction at OD600=1. E. coli strains can be grown at 37C, 250 rpm and switched to room temperature or 30C (150 rpm) during induction. If necessary, some enzymes can also be expressed by SF9 insect cell line using pFASTBAC and optimized MOI. Crude extracts containing the enzymes can be produced by sonication and used in the reactions described herein. All UDP-glycosyltransferase reactions include sucrose synthase, which can be obtained by gene synthesis from A. thaliana and expressed in E. coli.

[0195] Preparation of compound 1 by hydrolysis of highly glycosylated mogrosides In some embodiments, hyperglycosylated mogrosides can be hydrolyzed to produce compound 1. Non-limiting examples of hyperglycosylated mogrosides include mogroside V, siamenoside I, mogroside IV, E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside IIE , Mogroside III A2 , and mogroside III. Enzymes capable of catalyzing the hydrolysis process for the production of compound 1 can be, for example, CGTase (e.g., exhibiting hydrolysis without starch), cellulase, β-glucosidase, transglucosidase, amylase, pectinase, dextranase, and fungal lactase.

[0196] In some embodiments, compound 1 exhibits resistance to hydrolases in recombinant cells, where the hydrolases exhibit the ability to hydrolyze mogroside VI, mogroside V, and mogroside IV to mogroside IIIE. The α-linked glycosides present in compound 1 offer a unique advantage over other mogrosides (β-linked glycosides) because they exhibit resistance to hydrolysis. During the microbial production of compound 1, the recombinant host cell (e.g., microbial host cell) can hydrolyze the unwanted β-linked mogrosides back to mogroside IIIE. Without being bound to any particular theory, it is believed that hydrolysis by the host cell can improve the purity of compound 1 because: 1) the levels of unwanted mogroside VI, mogroside V, and mogroside IV are reduced, and / or 2) the hydrolysis increases the amount of available mogroside IIIE that can be used as a precursor for the production of compound 1.

[0197] Purification of mogroside compounds Some embodiments include isolating a mogroside compound, e.g., compound 1. In some embodiments, isolating compound 1 includes disrupting recombinant host cells. In some embodiments, isolating compound 1 includes isolating compound 1 from the culture medium. In some embodiments, the method further includes purifying compound 1. In some embodiments, purifying compound 1 includes HPLC, solid phase extraction, or a combination thereof. In some embodiments, the purification step includes harvesting the recombinant cells, saving the supernatant, and disrupting the cells. In some embodiments, the disruption step includes subjecting the cells to shear force or washing with a detergent to obtain a lysate. In some embodiments, the shear force is by sonication, French press treated cells, or beads. In some embodiments, the lysate is subjected to a filtration step and a purification step. In some embodiments, the lysate is filtered and purified by solid phase extraction. The fragments are then filtered, treated with ammonium sulfate to remove protein, and fractionated by injection on a C18 HPLC (5 x 10 cm Atlantis prep T3 OBD column, 5 um, Waters) using an A / B gradient (A=water, B=acetonitrile) from 10 to 30% B over 30 min, washed with 95% B, and then re-equilibrated to 1% (total time of analysis=42 min). The analytes can be collected at 30 mL / fraction in tared tubes (12 fractions / plate, 3 plates per analysis). The fragments can also be centrifuged to remove solids and particulate matter. The plates can then be dried in a Genevac HT12 / HT24. The desired compound is expected to elute in fraction 21 along with other isomers.The pooled fractions were further fractionated on a fluorophenyl HPLC column (3 x 10 cm, Xselect fluoro-phenyl OBD column, 5 um, Waters) using an A / B gradient (A=water, B=acetonitrile) from 15 to 30% B over 35 min, washed with 95% B, and then re-equilibrated at 15% (total time of run=45 min). Each analyte was collected at 30 mL / fraction in 12 tared tubes (12 fractions / plate, one plate per run). Fractions containing the desired peak with the desired purity were pooled based on the UPLC analysis and dried under vacuum to obtain a whitish powdery solid. The pure compound can be resuspended / dissolved in 10 mL water and lyophilized to obtain a purity of at least 95%.

[0198] For the purification of compound 1, in some embodiments, the compound can be purified by solid phase extraction, which may eliminate the need for HPLC. Compound 1 can be purified, for example, to 70%, 80%, 90%, 95%, 98%, 99%, or 100% purity, or to about 70%, about 80%, about 90%, about 95%, about 98%, about 99%, or about 100% purity, or to any level of purity within the range indicated by any two of the aforementioned values. In some embodiments, compound 1 purified by solid phase extraction is identical or substantially identical to the material purified by HPLC. In some embodiments, the method includes fractionating the lysate from recombinant cells on an HPLC column and collecting the eluted fraction containing compound 1.

[0199] fermentation The host cells can be fermented as described herein for the production of Compound 1. This includes methods that can occur with or without air, e.g., in an anaerobic environment. The whole cells (e.g., recombinant host cells) can be present in a fermentation broth or in a reaction buffer.

[0200] An extract of Momordica (Siraitia grosvenorii) can also be used to contact the cells to produce Compound 1. In some embodiments, a method for producing Compound 1 is provided. The method comprises contacting the Momordica extract with mogrosides, such as mogroside V, siamenoside I, mogroside IV, or the like. E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2and a first enzyme capable of catalyzing the production of compound 1 from mogroside III. In some embodiments, the contacting step comprises contacting the mogrol fruit extract with a recombinant host cell comprising a first gene encoding the first enzyme. In some embodiments, the first gene is heterologous to the recombinant host cell. In some embodiments, the mogrol fruit extract is contacted with the first enzyme in a recombinant host cell comprising a first polynucleotide encoding the first enzyme. In some embodiments, mogroside IIIE is present in the mogrol fruit extract. In some embodiments, mogroside IIIE is also produced by the recombinant host cell. In some embodiments, the method further comprises culturing the recombinant host cell in a medium under conditions in which the first enzyme is expressed. In some embodiments, the first enzyme is one or more of UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the first enzyme is a CGTase. For example, the CGTase can comprise an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises an amino acid sequence of any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises an amino acid sequence of any one of SEQ ID NOs: 78-101. In some embodiments, the first enzyme is dextransucrase.In some embodiments, the dextran sucrase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of the sequences set forth in SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the dextran sucrase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of the sequences set forth in SEQ ID NOs: 163-290, and 723. In some embodiments, the first enzyme is a transglucosidase. In some embodiments, the transglucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to any one of the sequences set forth in SEQ ID NOs: 163-290, and 723. In some embodiments, the transglucosidase comprises an amino acid sequence of any one of SEQ ID NOs: 163-290, and 723. In some embodiments, the first enzyme is β-glucosidase. In some embodiments, the β-glucosidase comprises an amino acid sequence set forth in SEQ ID NO: 292, or an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these values ​​of sequence identity to SEQ ID NO: 292. In some embodiments, the mogrol fruit extract comprises mogroside IIA, and the recombinant host cell comprises a second gene encoding a second enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA. In some embodiments, mogroside IIA is also produced by the recombinant host cell. In some embodiments, the second enzyme is one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase, In some embodiments, the second enzyme is a uridine diphosphate glucosyltransferase (UGT).In some embodiments, the UGT is UGT73C3 (SEQ ID NO:4), UGT73C6 (SEQ ID NO:5, 444, or 445), 85C2 (SEQ ID NO:6), UGT73C5 (SEQ ID NO:7), UGT73E1 (SEQ ID NO:8), UGT98 (SEQ ID NO:9, or 407), UGT1576 (SEQ ID NO:15), UGT SK98 (SEQ ID NO:16), UGT430 (SEQ ID NO:17), UGT1697 (SEQ ID NO:18), UGT11789 (SEQ ID NO:19); The amino acid sequence has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to SK98 (SEQ ID NO: 16), UGT430 (SEQ ID NO: 17), UGT1697 (SEQ ID NO: 18), UGT11789 (SEQ ID NO: 19), or a range between any two of these numbers. In some embodiments, the UGT is encoded by a gene set forth in UGT1495 (SEQ ID NO: 10), UGT1817 (SEQ ID NO: 11), UGT5914 (SEQ ID NO: 12), UGT8468 (SEQ ID NO: 13), or UGT10391 (SEQ ID NO: 14). In some embodiments, the Momordica extract comprises mogrol. In some embodiments, the method further comprises contacting the mogrol of the Momordica extract, and the recombinant host cell further comprises one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE from mogrol. In some embodiments, mogrol is also produced by a recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the second enzyme is a uridine diphosphate glucosyltransferase (UGT).In some embodiments, the UGT is UGT73C3, UGT73C6, 85C2, UGT73C5, UGT73E1, UGT98, UGT1495, UGT1817, UGT5914, UGT8468, UGT10391, UGT1576, UGT SK98, UGT430, UGT1697, or UGT11789, or comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers of sequence identity thereto. In some embodiments, the method further comprises contacting the Monk Fruit extract with a recombinant host cell to produce mogroside IIIE, wherein the recombinant host cell further comprises one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE from the mogroside compound. wherein the mogroside compound is one or more of mogroside IA1, mogroside IE1, mogroside IIA1, mogroside IIE, mogroside IIA, mogroside IIIA1, mogroside IIIA2, mogroside III, mogroside IV, mogroside IVA, mogroside V, or siamenoside. In some embodiments, the mogroside compound is also produced by a recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. In some embodiments, the mogroside compound is mogroside IIE. In some embodiments, the one or more enzymes include an amino acid set forth in any one of SEQ ID NOs: 293-303. In some embodiments, the mogroside compound is morgroside IIA or mogroside IIE, and contacting the Monk Fruit extract with a recombinant cell expressing one or more enzymes produces mogroside IIIA, mogroside IVE, and mogroside V. In some embodiments, the one or more enzymes comprise an amino acid set forth in SEQ ID NO: 304. In some embodiments, the one or more enzymes are encoded by a sequence set forth in SEQ ID NO: 305.In some embodiments, the swingle extract comprises mogroside IA1. In some embodiments, the method further comprises contacting the swingle extract with a recombinant host cell, the recombinant host cell comprising a gene encoding UGT98 or UGT SK98. In some embodiments, the UGT98 or UGT SK98 enzyme comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 9, 407, 16, or 306. In some embodiments, the UGT98 is encoded by the sequence set forth in SEQ ID NO: 307. In some embodiments, the contacting causes the cell to produce mogroside IIA. In some embodiments, the swingle extract comprises 11-hydroxy-24,25 epoxy cucurbitadienol. In some embodiments, the method further comprises contacting the Monk Fruit extract with a recombinant host cell, the recombinant host cell further comprising a third gene encoding an epoxide hydrolase. In some embodiments, 11-hydroxy-24,25 epoxy cucurbitadienol is also produced by the recombinant host cell. In some embodiments, the method further comprises contacting the Monk Fruit extract with a recombinant host cell, the recombinant host cell further comprising a fourth gene encoding a cytochrome P450 or an epoxide hydrolase. In some embodiments, 11-hydroxy cucurbitadienol is also produced by the recombinant host cell. In some embodiments, the Monk Fruit extract comprises 3,24,25 trihydroxy cucurbitadienol. In some embodiments, the method further comprises contacting the Monk Fruit extract with a recombinant host cell, the recombinant host cell further comprising a fifth gene encoding a cytochrome P450. In some embodiments, 3,24,25 trihydroxy cucurbitadienol is also produced by the recombinant host cell. In some embodiments, mogrol fruit. The recombinant host cell is contacted with the extract to produce mogrol. In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 20 or 308. In some embodiments, the epoxide hydrolase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NO: 21-30, and 309-314. In some embodiments, the swingle extract comprises cucurbitadienol. In some embodiments, the method further comprises contacting cucurbitadienol with the recombinant host cell, the recombinant host cell comprising a gene encoding a cytochrome P450. In some embodiments, the contact produces 11-hydroxycucurbitadienol. In some embodiments, the 11-hydroxycucurbitadienol is expressed in a cell comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, the CYP87D18 or SgCPR comprises a sequence set forth in SEQ ID NO: 315, 872, or 874. In some embodiments, the CYP87D18 or SgCPR is encoded by SEQ ID NO: 316, 871, or 873. In some embodiments, the cucurbitadienol is also produced by a recombinant host cell. In some embodiments, the gene encoding cytochrome P450 comprises a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891.In some embodiments, the cytochrome P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers of sequence identity to SEQ ID NO: 20 or 49. In some embodiments, the Monk Fruit extract comprises 2,3-oxidosqualene. In some embodiments, the method further comprises contacting the Monk Fruit extract 2,3-oxidosqualene with a recombinant host cell, wherein the recombinant host cell comprises a seventh gene encoding cucurbitadienol synthase. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO:70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, or 906. In some embodiments, the cucurbitadienol synthase is encoded by a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, or 905. In some embodiments, the swingle extract contains mogroside intermediates, such as mogroside V, siamenoside I, mogroside IV. E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside IIE , Mogroside III A2, and mogroside III. In some embodiments, the method further comprises contacting the mogroside intermediate with a recombinant host cell, wherein the recombinant host cell comprises a seventh gene encoding a cucurbitadienol synthase. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, or 906. In some embodiments, the cucurbitadienol synthase is encoded by a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, or 905. In some embodiments, the contacting produces cucurbitadienol. In some embodiments, 2,3-oxidosqualene and diepoxysqualene are also produced by the recombinant host cell. In some embodiments, the 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to SEQ ID NO: 898 or 900, or is produced by an enzyme comprising a sequence set forth in SEQ ID NO: 898 or 900. In some embodiments, the 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, to SEQ ID NO: 897 or 899, or is produced by an enzyme encoded by a nucleic acid set forth in SEQ ID NO: 897 or 899.

[0201] In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the cucurbitadienol synthase is a curbitadienol synthase from C. pepo, S. grosvenorii, C. sativus, C. melo, C. moschata, and C. maxima. In some embodiments, the cucurbitadienol synthase is encoded by a gene comprising a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905, or a range between any two of these numbers, or is encoded by a gene comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905. In some embodiments, 11-OH cucurbitadienol is produced by the cell. In some embodiments, 11-OH cucurbitadienol is expressed in cells that contain a gene encoding CYP87D18 or SgCPR. In some embodiments, CYP87D18 or SgCPR comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers of sequence identity to SEQ ID NO: 315, 872, or 874, or comprises a sequence set forth in SEQ ID NO: 315, 872, or 874.In some embodiments, CYP87D18 or SgCPR is encoded by a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 316, 871, or 873, or is encoded by a sequence set forth in SEQ ID NO: 316, 871, or 873. In some embodiments, the Monk Fruit Extract contains squalene. In some embodiments, 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or more sequence identity to SEQ ID NO: 898 or 900. In some embodiments, the 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to SEQ ID NO: 897 or 899, or is produced by an enzyme encoded by a sequence set forth in SEQ ID NO: 897 or 899. In some embodiments, the method further comprises contacting squalene with a recombinant host cell, the recombinant host cell comprising an eighth gene encoding a squalene epoxidase. In some embodiments, the contacting results in the production of 2,3-oxidosqualene. In some embodiments, squalene is also produced by the recombinant host cell. In some embodiments, the squalene epoxidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to any one of SEQ ID NOs: 50-56, 60, 61, 334, and 335. In some embodiments, the squalene epoxide is encoded by the nucleic acid sequence set forth in SEQ ID NO: 335. In some embodiments, the swingle extract comprises farnesyl pyrophosphate.In some embodiments, the method further comprises contacting the farnesyl pyrophosphate with a recombinant host cell, the recombinant host cell comprising a ninth gene encoding squalene synthase. In some embodiments, the contact produces squalene. In some embodiments, the farnesyl pyrophosphate is also produced by the recombinant host cell. In some embodiments, the squalene synthase comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to any one of SEQ ID NOs: 69 and 336. In some embodiments, the squalene synthase is encoded by a sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 337. In some embodiments, the swingle extract comprises geranyl-PP. In some embodiments, the method further comprises contacting the geranyl-PP with a recombinant host cell, the recombinant host cell comprising a 10th gene encoding farnesyl-PP synthase. In some embodiments, the contact produces farnesyl-PP. In some embodiments, geranyl-PP is also produced by the recombinant host cell. In some embodiments, the farnesyl-PP synthase comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more sequence identity to SEQ ID NO: 338. In some embodiments, the farnesyl-PP synthase is encoded by the nucleic acid sequence set forth in SEQ ID NO: 339. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene are operably linked to a heterologous promoter.In some embodiments, the heterologous promoter is a CMV, EF1a, SV40, PGK1, human beta actin, CAG, GAL1, GAL10, TEF1, GDS, ADH1, CaMV35S, Ubi, T7, T7lac, Sp6, araBAD, trp, Lac, Ptac, pL promoter, or a combination thereof. In some embodiments, the promoter is an inducible promoter, a repressible promoter, or a constitutive promoter. In some embodiments, the production of one or more of pyruvate, acetyl-CoA, citrate, and a TCA cycle intermediate is upregulated in the recombinant host cell. In some embodiments, cytosolic localization is upregulated in the recombinant host cell. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene comprise at least one sequence encoding a 2A self-cleaving peptide. In some embodiments, the recombinant host cell is a plant cell, a bivalve cell, a fish cell, a fungal cell, a bacterial cell, or a mammalian cell.In some embodiments, the plant is selected from the group consisting of Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Amaranth, and the like. The plant is selected from Aralia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia, and Morus.In some embodiments, the fungus is selected from the group consisting of Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, The fungus is selected from Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, and Lipomyces. In some embodiments, the fungus is Aspergillus nidulans, Yarrowia lipolytica, or Rhodosporin toruloides. In some embodiments, the recombinant host cell is a yeast cell.In some embodiments, the yeast is selected from Candida, Saccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes. In some embodiments, the bacterium is selected from the group consisting of Frankia, Actinobacteria, Streptomyces, and Enterococcus. In some embodiments, the bacterium is Enterococcus faecalis. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene are codon-optimized for expression in a bacterial cell, a mammalian cell, a plant cell, a fungal cell, or an insect cell. In some embodiments, one or more of the first gene, the second gene, the third gene, the fourth gene, the fifth gene, the sixth gene, the seventh gene, the eighth gene, the ninth gene, and the tenth gene comprise a functional mutation for increased activity of the encoded enzyme. In some embodiments, culturing the recombinant host cells includes monitoring the culture for pH, dissolved oxygen levels, nitrogen levels, or a combination of these culture conditions. In some embodiments, the method includes isolating Compound 1. In some embodiments, isolating Compound 1 includes disrupting the recombinant host cells.In some embodiments, isolating compound 1 comprises isolating compound 1 from the culture medium. In some embodiments, the method further comprises purifying compound 1. In some embodiments, the method further comprises purifying compound 1. In some embodiments, purifying compound 1 comprises HPLC, solid phase extraction, or a combination thereof. In some embodiments, the purification step further comprises harvesting recombinant cells, saving the supernatant, and disrupting the cells. In some embodiments, the disruption step comprises subjecting the cells to shear force or washing with detergent to obtain lysates. In some embodiments, the shear force is by sonication, French press treated cells, or beads. In some embodiments, the lysates are subjected to filtration and purification steps. In some embodiments, the lysates are filtered and purified by solid phase extraction. In some embodiments, the method further comprises adding a second or third Momordica extract to the growth medium of recombinant host cells. Furthermore, the method can be performed by contacting Momordica extract with recombinant cell lysates, and the recombinant cell lysates comprises the expressed enzymes listed herein.

[0202] In general, the compounds disclosed and described herein can be provided individually or in combination in a composition, such as an ingestible composition. In one embodiment, the compounds disclosed and described herein can be provided individually or in combination in an ingestible composition to provide sweetness. In another embodiment, the compounds disclosed and described herein can be provided individually or in combination as a sweetness enhancer to enhance the sweetness of another sweetener. In another embodiment, the compounds disclosed herein can provide a sugar-like time profile and / or flavor profile to a sweetener composition by combining one or more of the compounds disclosed and described herein with one or more other sweeteners in a sweetener composition. In another embodiment, the compounds disclosed and described herein can be provided individually or in combination to increase or enhance the sweetness of a composition by contacting the composition with a compound disclosed and described herein to form a modified composition. In another embodiment, the compounds disclosed and described herein can be provided individually or in combination in a composition that modulates sweet receptors and / or their ligands expressed in the body other than taste buds.

[0203] As used herein, "ingestible composition" includes any composition suitable for oral ingestion, alone or together with another substance, whether or not intended for consumption. Ingestible compositions include both "food or beverage products" and "non-edible products". "Food or beverage products" means any edible product intended for human or animal consumption, including solid, semi-solid, or liquid (e.g., beverages), and includes functional foods (e.g., any fresh or processed food that is claimed to have health-promoting and / or disease-preventing properties beyond the basic nutritional function of providing nutrients). The term "non-food or beverage product" or "non-ingestible composition" includes any product or composition that can be orally ingested by humans or animals for purposes other than consumption, or as a food or beverage. For example, non-food or beverage products, or non-ingestible compositions include supplements, dietary supplements, pharmaceuticals and over-the-counter drugs, oral care products such as dentifrices and mouthwashes, and chewing gum.

[0204] In some embodiments, the compositions disclosed herein further comprise at least one additional sweetener and / or sweetness modifier. The at least one additional sweetener and / or sweetness modifier can be an artificial sweetener and / or sweetness modifier, or a natural sweetener and / or sweetness modifier. The at least one additional sweetener and / or sweetness modifier can be avidiasaponin, abrusoside, particularly abrusoside A, abrusoside B, abrusoside C, abrusoside D, acesulfame potassium, advantam, albiziasaponin, alitame, aspartame, superaspartame, bayunosides, particularly bayunosides 1, bayunosides 2, brazzein, bryoside ... Onozozuccoside, carnosifloside, karela, curculin, cyanin, chlorogenic acid, cyclamate and its salts, cyclocaryoside I, dihydroquercetin-3-acetate, dihydroflavenol, dulcoside, gaudichaudioside, glycyrrhizin, glycyrrhizic acid, gypenosides, hematoxylin, isomogrosides, especially isomogroside V, lugduname, magap, mabinlin, micra Clin, mogrosides (lo han guo), especially mogroside IV and mogroside V, monatin and its derivatives, monellin, mukurozioside, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NHDC), neotame, osladin, pentadin, periandrin I-V, perillartine, D-phenylalanine, phlomisosides, especially phlomisoside 1, phlomisoside 2, phlomisoside 3, phlomisoside 4, and floridi , phyllodalcin, polpodiosides, polypodoside A, pterocaryoside, rebaudiosides, in particular rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside M, rubusoside, saccharin and its salts and derivatives, scandenoside, selligueanin A,Siamenosides, especially siamenoside I, stevia, steviolbioside, stevioside and other steviol glycosides, strogins, especially strogin 1, strogin 2, strogin 4, suavioside A, suavioside B, suavioside G, suavioside H, suavioside I, suavioside J, sucralose, sucuronate, sucuroctate, talin, telosmoside A15, thaumatins, especially thaumatins I and II, trans-anethole, trans-cinnamaldehyde, trilobatin, D-tryptophan, erythritol, galactitol, maltitol and sorbitol syrup. The starch syrup may be selected from the group consisting of hydrogenated starch syrup including maltose, inositol, isomalt, lactitol, maltitol, mannitol, xylitol, arabinose, dextrin, dextrose, fructose, high fructose corn syrup, fructooligosaccharides, fructooligosaccharide syrup, galactose, galactooligosaccharides, glucose, glucose and (hydrogenated) starch syrup / hydrolysates, isomaltulose, lactose, hydrolyzed lactose, maltose, mannose, rhamnose, ribose, sucrose, tagatose, trehalose and xylose.

[0205] Compositions Containing Mogroside Compounds Also disclosed herein are compositions, e.g., ingestible compositions, comprising one or more mogroside compounds disclosed herein, including, but not limited to, Compound 1 and the compounds shown in FIG. 43. In some embodiments, the ingestible composition can be a beverage. For example, the beverage can be selected from the group consisting of enhanced sparkling beverages, cola, lemon-lime flavored sparkling beverages, orange flavored sparkling beverages, grape flavored sparkling beverages, strawberry flavored sparkling beverages, pineapple flavored sparkling beverages, ginger ale, root beer, fruit juice, fruit flavored juice, juice beverages, nectar, vegetable juice, vegetable flavored juice, sports drinks, energy drinks, enhanced water beverages, enhanced water with vitamins, near water beverages, coconut water, tea-type beverages, coffee, cocoa beverages, beverages containing dairy ingredients, beverages containing grain extracts, and smoothies. In some embodiments, the beverage can be a soft drink.

[0206] An "ingestible ingredient" is a substance that is suitable for oral ingestion and can be combined with a compound described herein to form an ingestible composition. The ingestible ingredient can be in any form depending on the intended use of the product, for example, a solid, semi-solid, liquid, paste, gel, lotion, cream, foam material, suspension, solution, or any combination thereof (e.g., liquid with solids). The ingestible ingredient can be artificial or natural. Ingredients that are acceptable for ingestion include many common food ingredients, such as water of neutral, acidic or basic pH, fruit or vegetable juices, vinegar, marinades, beer, wine, natural water / fat emulsions such as milk or condensed milk, edible oils and shortenings, fatty acids and their alkyl esters, low molecular weight oligomers of propylene glycol, glyceryl esters of fatty acids, and dispersions or emulsions of such hydrophobic materials in aqueous media, salts such as sodium chloride, flour, solvents such as ethanol, solid edible diluents such as vegetable powder or flour, or other liquid vehicles; dispersing or suspending aids; surfactants; isotonicity agents; thickening or emulsifying agents, preservatives; solid binders; lubricants, etc.

[0207] Additional ingredients that may be tolerated include, but are not limited to, citric acid, phosphoric acid, ascorbic acid, sodium sulfate, lactic acid, or tartaric acid; bitter ingredients, including, for example, caffeine, quinine, green tea, catechins, polyphenols, robusta coffee bean extract, coffee bean extract, whey protein isolate, or potassium chloride; caramel color, including, for example, red 40, yellow 5, yellow 6, blue 1, red 3, purple carrot, black carrot juice, purple sweet potato, vegetable juice, fruit juice, beta-carotene, turmeric curcumin, or titanium dioxide. colouring agents;preservatives including, for example, sodium benzoate, potassium benzoate, potassium sorbate, sodium metabisulfate, sorbic acid, or benzoic acid;antioxidants including, for example, ascorbic acid, calcium disodium EDTA, alpha tocopherol, mixed tocopherols, rosemary extract, grape seed extract, resveratrol, or sodium hexametaphosphate;antioxidants including, for example, resveratrol, Co-Q10, omega-3 fatty acids, theanine, choline chloride (cytocholine), fibresol, inulin (chicory root), taurine, Panax ginseng (Panax ginseng extract, guanina extract, ginger extract, L-phenylalanine, L-carnitine, L-tartrate, D-glucoronolactone, inositol, bioflavonoids, echinacea, ginkgo biloba, erbamate, flaxseed oil, garcinia cambogia husk extract, white tea extract, ribose, milk thistle extract, grape seed extract, pyrodixine HCl (vitamin B6), cyanoobalamin (vitamin B12), niacinamide (vitamin B3), biotin, calcium lactate, pantothenate Vitamins or functional ingredients including calcium acid (pantothenic acid), calcium phosphate, calcium carbonate, chromium chloride, chromium polynicotinate, copper sulfate, folic acid, ferric pyrophosphate, iron, magnesium lactate, magnesium carbonate, magnesium sulfate, monopotassium phosphate, monosodium phosphate, phosphorus, potassium iodide, potassium phosphate, riboflavin, sodium sulfate, sodium gluconate, sodium polyphosphate, sodium bicarbonate, thiamine mononitrate, vitamin D3, vitamin A palmitate, zinc gluconate, zinc lactate or zinc sulfate;Clouding agents including, for example, ester gun, brominated vegetable oil (BVO) or sucrose isobutyl acetate (SAIB); buffers including, for example, sodium citrate, potassium citrate or salts; flavors such as, for example, propylene glycol, ethyl alcohol, glycerin, gum arabic (gum acacia), maltodextrin, modified corn starch, dextrose, natural flavors, natural flavors including other natural flavors (natural flavors WONF), natural and artificial flavors, artificial flavors, silicon dioxide, magnesium carbonate or tricalcium phosphate; and stabilizers including, for example, pectin, xanthan gum, carboxymethylcellulose (CMC), polysorbate 60, polysorbate 80, medium chain triglycerides, cellulose gel, cellulose gum, sodium caseinate, modified food starch, gum arabic (gum acacia), or carrageenan;

[0208] Working Example Certain aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not intended to limit the scope of the disclosure in any way.

[0209] Example 1: Preparation of siamenoside I [ka]

[0210] As disclosed herein, siamenoside I can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, siamenoside I can be hydrolyzed to produce mogroside IIIE, which can then be used to produce compound 1. For example, a method for producing siamenoside I can include: contacting mogrol with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, a recombinant cell expressing pectinase from Aspergillus aculeatus can be used.

[0211] As another non-limiting example, a method for producing siamenoside I can include: Mogroside V, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, pectinase from Aspergillus aculeatus can be used.

[0212] Example 2: Mogroside IV E Manufacturing [ka]

[0213] As disclosed herein, mogroside IV E can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside IV from mogroside V E Compound 1 can be prepared using, for example, mogroside IV E A method for producing mogroside V can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. As another example, the recombinant cell can include a gene encoding a pectinase. The pectinase can be encoded by a gene from Aspergillus aculeatus.

[0214] Another example is mogroside IV. E The method for producing the compound may include the following: Mogroside V, Siamenoside I, Isomogroside V, Mogroside III. E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside IIE , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, pectinase from Aspergillus aculeatus can be used.

[0215] Example 3: Mogroside III E Manufacturing [ka]

[0216] As disclosed herein, mogroside III E can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside II A can be glycosylated to produce mogroside IIIE, which can then be used to produce compound 1.

[0217] Another example is mogroside III. E The method for producing mogroside V, mogroside II, A , siamenoside I, mogroside IV E , Isomogroside V, 11-Deoxymogroside V, 11-Oxomogroside V, Mogroside VI, Mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E, Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, pectinase from Aspergillus aculeatus can be encoded by a gene in the recombinant host cell.

[0218] Example 4: Mogroside IV A Manufacturing [ka]

[0219] As disclosed herein, mogroside IV A can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside IV from mogroside V A Compound 1 can be prepared using

[0220] For example, mogroside IV A The method for producing mogroside V may include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme may be, for example, β-galactosidase from Aspergillus oryzae.

[0221] Another example is mogroside IV. A The method for producing mogroside V, siamenoside I, mogroside IV may include: E , Isomogroside V, Mogroside III E, 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, β-galactosidase from Aspergillus oryzae can be used in the method.

[0222] Example 5: Mogroside II A Manufacturing [ka]

[0223] As disclosed herein, mogroside II A can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside II A A method for producing mogroside IA may include: 1 with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0224] Another example is mogroside II. AA method for producing mogroside IA may include: 1 , Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, celluclasts can also be used.

[0225] Example 6: Mogroside III from aromatase A1 Manufacturing [ka]

[0226] As disclosed herein, mogroside III A1 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside III A1 can be an intermediate for producing mogroside IVA, which can then be used as an intermediate for producing compound 1. For example, mogroside III A1A method for producing siamenoside I can include contacting siamenoside I with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, an aromatase. As another example, mogroside III can be produced by contacting siamenoside I with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, an aromatase. A1 The method for producing I can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0227] Example 8: Preparation of Compound 4 [ka]

[0228] As disclosed herein, compound 4 is produced during the production of compound 1 disclosed herein. For example, a method for producing compound 4 can also result in the production of compound 1, the method including contacting mogroside IIIE with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0229] As another example, a method for making compound 4 can include the following: mogroside V, siamenoside I, mogroside IVE, isomogroside V, mogroside IIIE, 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IVA, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IA, 11-oxomogroside VI, 11-oxomogroside IIIE, 11-oxomogroside V, 11-oxomogroside V, mogroside VI, mogroside IVA, mogroside IIA, mogroside IIA2, mogroside IA, 11-oxomogroside VI, 11-oxomogroside IIIE, 11-oxomogroside V, mogroside IV ... Contacting one or more of mogroside IVE, mogroside IE, mogrol, 11-oxomogrol, mogroside IIE, mogroside IIIA2, and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, cyclomaltodextrin glucanotransferase from Bacillus lichenformis and / or Toruzyme.

[0230] Example 9: Preparation of Compound 5 [ka]

[0231] Compound 5 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, the method for producing compound 5 can also result in the preparation of compound 1, which method produces mogroside III. E with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0232] As another example, a method for producing compound 5 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase and / or Toruzyme from Bacillus lichenformis can be used.

[0233] Example 10: Preparation of Compound 6 [ka]

[0234] As disclosed herein, compound 6 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, the method for producing compound 6 can also result in the production of compound 1, which can include the production of mogroside III. E with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0235] As another example, a method for producing compound 6 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0236] Example 11: Preparation of Compound 7 [ka]

[0237] As disclosed herein, compound 7 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, the method for producing compound 7 can also result in the production of compound 1, which method includes III. E with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0238] As another example, a method for producing compound 7 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0239] Example 12: Preparation of Compound 8 [ka]

[0240] As disclosed herein, compound 8 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, the method for producing compound 8 can also result in the production of compound 1, which can include the production of mogroside III. E with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0241] As another example, a method for producing compound 8 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside IE , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0242] Example 13: Preparation of Compound 9 [ka]

[0243] As disclosed herein, compound 9 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, a method for producing compound 9 can also result in the production of compound 1, the method including contacting mogroside IIIE with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0244] As another example, a method for making compound 9 can include the following: mogroside V, siamenoside I, mogroside IVE, isomogroside V, mogroside IIIE, 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IVA, mogroside IIA, mogroside IIA1, mogroside IIA2, mogroside IA, 11-oxomogroside VI, 11-oxomogroside IIIE, 11-oxomogroside V, 11-oxomogroside V, mogroside VI, mogroside IVA, mogroside IIA, mogroside IIA2, mogroside IA, 11-oxomogroside VI, 11-oxomogroside IIIE, 11-oxomogroside V, mo ... Contacting one or more of mogroside IVE, mogroside IE, mogrol, 11-oxomogrol, mogroside IIE, mogroside IIIA2, and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0245] Example 14: Preparation of Compound 10 [ka]

[0246] As disclosed herein, compound 10 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, the method for preparing compound 10 can also result in the preparation of compound 1, which method produces mogroside III. E with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0247] As another example, a method for producing compound 10 can include: Mogroside V, Siamenoside I, Mogroside IVE , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0248] Example 15: Preparation of Compound 11 [ka]

[0249] As disclosed herein, compound 11 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, the method for producing compound 11 can also result in the production of compound 1, which method produces mogroside III. E or 11-oxo-MIII Ewith a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0250] As another example, a method for producing compound 11 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, CGTase from Bacillus lichenformis, or Toruzyme can be used.

[0251] Example 16: Preparation of Compound 12 [ka]

[0252] As disclosed herein, compound 12 can be an intermediate mogroside compound that can be used in the production of compound 1 disclosed herein. For example, the method of producing compound 12 can also result in the production of compound 1, and the method can include contacting a mogroside VI isomer with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, invertase, and dextranase. The enzyme can be, for example, an invertase enzyme from baker's yeast.

[0253] As another example, a method for producing compound 12 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 Contacting one or more of mogroside VI isomer, and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, invertase, and dextranase.

[0254] Example 17: Preparation of Compound 13 [ka]

[0255] As disclosed herein, compound 13 can be an intermediate mogroside produced during the preparation of compound 1 disclosed herein. For example, the method can include the preparation of mogroside III E The method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The expressed enzyme can be, for example, celluclast.

[0256] As another example, a method for producing compound 13 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, celluclasts can be used.

[0257] Example 18: Preparation of Compound 14 [ka]

[0258] As disclosed herein, compound 14 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, the method can include the preparation of mogroside III E The method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The expressed enzyme can be, for example, celluclast.

[0259] As another example, a method for producing compound 14 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, celluclasts can be used. The method can also require the presence of a sugar, such as α-lactose.

[0260] Example 19: Preparation of Compound 15 [ka]

[0261] As disclosed herein, compound 15 can be an intermediate mogroside compound that can be used to prepare compound 1 as disclosed herein. For example, the method can include the step of preparing mogroside II. A with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0262] As another example, a method for producing compound 15 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, Toruzyme can be used.

[0263] Example 20: Preparation of Compound 16 [ka]

[0264] As disclosed herein, compound 16 can be an intermediate mogroside compound that can be used to prepare compound 1 as disclosed herein. For example, the method can include the step of preparing mogroside II. A with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0265] As another example, a method for producing compound 16 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, Toruzyme can be used.

[0266] The enzyme can be, for example, Toruzyme. The recombinant cell can further comprise genes encoding, for example, cyclochromato-dextrin glucanotransferase (e.g., Toruzyme), invertase, glucose transferase (e.g., UGT76G1).

[0267] Example 21: Preparation of Compound 17 [ka]

[0268] As disclosed herein, compound 17 can be an intermediate mogroside compound for producing compound 1 as disclosed herein. For example, compound 17 can be hydrolyzed to produce mogroside IIIE, which can then be used to produce compound 1. For example, a method for producing compound 17 can include contacting siamenoside I with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, transglucosidase, sucrose synthase, pectinase, and dextranase.

[0269] As another example, a method for producing compound 17 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E, Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. For example, a UDP glycosyltransferase can be used.

[0270] Example 22: Preparation of Compound 18 [ka]

[0271] As disclosed herein, compound 18 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, compound 18 can be hydrolyzed to produce mogroside IIIE, which can then be used to produce compound 1. For example, the method for producing compound 18 can also result in the production of compound 1, which can include mogroside IIIE. E The method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzymes can be, for example, Sus1 and UGT76G1.

[0272] As another example, a method for producing compound 18 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A, 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, UGT76G1.

[0273] Example 23: Preparation of Compound 19 [ka]

[0274] As disclosed herein, compound 19 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 19 can be further hydrolyzed to produce compound 1. For example, the method for producing compound 18 can also result in the preparation of compound 1, which can include the production of mogroside III. E The method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzymes can be, for example, Sus1 and UGT76G1.

[0275] As another example, a method for producing compound 19 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IVA , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, UGT76G1. The enzyme can be, for example, sucrose synthase Sus1.

[0276] Example 24: Preparation of Compound 20 [ka]

[0277] As disclosed herein, compound 20 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 20 can be further hydrolyzed to produce compound 1. For example, the method for producing compound 20 can also result in the preparation of compound 1, which can include the production of mogroside III. E The method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzymes can be, for example, Sus1 and UGT76G1.

[0278] As another example, a method for producing compound 20 can include: Mogroside V, Siamenoside I, Mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, UGT76G1. The enzyme can be, for example, sucrose synthase Sus1. The enzyme can be, for example, sucrose synthase Sus1 and UGT76G1.

[0279] Example 25: Preparation of Compound 21 [ka]

[0280] As disclosed herein, compound 21 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 21 can be further hydrolyzed to produce compound 1. For example, the method for producing compound 21 can also result in the preparation of compound 1, which can include the production of mogroside IV. EThe method can include contacting a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzymes can be, for example, Sus1 and UGT76G1.

[0281] As another example, a method for producing compound 21 can include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, UGT76G1. The enzyme can be, for example, sucrose synthase Sus1. The enzyme can be, for example, sucrose synthase Sus1 and GT76G1.

[0282] Example 26: Preparation of Compound 22 [ka]

[0283] As disclosed herein, compound 22 can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, compound 22 can be further hydrolyzed to produce compound 1. For example, the method for producing compound 22 can also result in the production of compound 1, and the method can include contacting mogroside IVA with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzymes can be, for example, Sus1 and UGT76G1.

[0284] As another example, a method for preparing compound 22 may include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, UGT76G1. The enzyme can be, for example, sucrose synthase Sus1. The enzyme can be, for example, sucrose synthase Sus1 and GT76G1.

[0285] Example 27: Preparation of Compound 23 [ka]

[0286] As disclosed herein, compound 23 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 23 can be further hydrolyzed to produce compound 1. For example, the method for producing compound 22 can also result in the preparation of compound 1, which can produce mogroside IV. E The method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase.

[0287] As another example, a method for preparing compound 23 may include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside IIA2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a detransucrase, which is capable of isolating the highly glycosylated mogroside IV, E The isomer is hydrolyzed to the desired mogroside V isomer.

[0288] Examples 28 and 29: Mogroside II from Fungal Lactase A1 and Mogroside II A2 Manufacturing [ka]

[0289] As disclosed herein, mogroside II A1 and Mogroside II A2 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, mogroside II A1 and Mogroside II A2 can be further hydrolyzed to produce compound 1. For example, mogroside II A1 and Mogroside II A2 The method of producing the compound 1 can also result in the production of mogroside IV Ewith a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, lactase from a fungus.

[0290] Another example is mogroside II. A1 and Mogroside II A2 The method for producing mogroside V, mogroside IV, E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0291] Example 30: Mogrosides from Viscozyme IA Manufacturing [ka]

[0292] As disclosed herein, mogrosides IAcan be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, mogroside IA can be further hydrolyzed to produce compound 1. Mogroside IA The method of producing can also result in the production of compound 1, the method including contacting mogroside IIA with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a viscozyme.

[0293] Another example is mogroside I. A The method for producing mogroside V, mogroside IV, E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a viscozyme.

[0294] Example 31: Preparation of Compound 24 [ka]

[0295] As disclosed herein, compound 24 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 24 can be further hydrolyzed to produce compound 1. The method for producing compound 24 can also result in the preparation of compound 1, which can include the production of mogroside III. E The method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0296] As another example, a method for producing compound 24 can include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0297] Example 32: Preparation of Compound 25 [ka]

[0298] As disclosed herein, compound 25 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 25 can be further hydrolyzed to produce compound 1. The method for producing compound 25 can also result in the preparation of compound 1, which can include the production of mogroside III. E The method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0299] As another example, a method for producing compound 25 can include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside IIIE , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0300] Example 33: Preparation of Compound 26 [ka]

[0301] As disclosed herein, compound 26 can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, compound 26 can be further hydrolyzed to produce compound 1. The method for producing compound 26 can also result in the preparation of compound 1, which can include the production of mogroside III. E The method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0302] As another example, a method for producing compound 26 can include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A, Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, dextransucrase DexT.

[0303] Examples 34 and 35: Mogrol and Mogroside I from Pectinase E Manufacturing [ka]

[0304] As disclosed herein, mogrol and mogroside I E can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, mogrol can be used as a substrate to produce mogroside IA1, which is further hydrolyzed to produce compound 1, and mogroside I Ecan be further hydrolyzed to produce compound 1. A method for producing mogrol and mogrosides can also result in the production of compound 1, the method can include contacting mogroside V with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a pectinase enzyme from Aspergillus aculeatus.

[0305] Another example is mogrol and mogroside I. E The method for producing mogroside V, mogroside IV, E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0306] Example 36: Preparation of Mogroside IIE [ka]

[0307] As disclosed herein, mogroside IIE can be an intermediate mogroside compound produced during the production of compound 1 disclosed herein. For example, mogroside IIE can be further hydrolyzed to produce compound 1. A method for producing mogroside IIE can also result in the production of compound 1, the method can include contacting mogroside V with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a pectinase enzyme from Aspergillus aculeatus.

[0308] As another example, a method for producing Mogroside IIE can include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0309] Examples 37 and 38: Preparation of Compounds 32 and 33 [ka]

[0310] As disclosed herein, compounds 32 and 33 can be intermediate mogroside compounds produced during the production of compound 1 disclosed herein. For example, compounds 32 and 33 can be further glycosylated and hydrolyzed to produce compound 1. The method for producing compounds 32 and 33 can also result in the production of compound 1, which can include mogroside V, mogroside IV, E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, a pectinase enzyme from Aspergillus aculeatus.

[0311] As another example, a method for producing compounds 32 and 33 may include: Mogroside V, Mogroside IVE , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0312] Examples 39 and 40: Preparation of Compounds 34 and 35 [ka] [ka]

[0313] As disclosed herein, compounds 34 and 35 can be intermediate mogroside compounds produced during the production of compound 1 disclosed herein. For example, compounds 32 and 33 can be further glycosylated and hydrolyzed to produce compound 1. The method for producing compounds 34 and 35 can also result in the production of compound 1, which can be used to produce mogroside III. EThe method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, celluclast.

[0314] As another example, a method for producing compounds 34 and 35 may include: Mogroside V, Mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E , Mogrol, 11-oxomogrol, Mogroside II E , Mogroside III A2 and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0315] Examples 41 and 42: Mogroside III A2 and production of mogroside III [ka]

[0316] As disclosed herein, mogroside III A2and mogroside III can be an intermediate mogroside compound produced during the preparation of compound 1 disclosed herein. For example, mogroside III A2 And mogroside III can be further hydrolyzed to produce compound 1.

[0317] For example, mogroside III A2 And mogroside III can be contacted with a UGT to produce another mogroside compound, mogroside IVA, which can be used to produce mogroside IIIE, which can be further hydrolyzed to produce compound 1.

[0318] Mogroside III A2 The method for producing mogroside III can also result in the production of compound 1, the method comprising the steps of: E The method can include contacting the enzyme with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase. The enzyme can be, for example, celluclast.

[0319] Another example is mogroside III. A2 and methods for producing mogroside III include: mogroside V, mogroside IV E , siamenoside I, mogroside IV E , Isomogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A , 11-oxomogroside VI, 11-oxomogroside III E , 11-oxomogroside IV E , Mogroside I E, Mogrol, 11-oxomogrol, Mogroside II E and contacting one or more of mogroside III with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTase, glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

[0320] Example 43: Use of CGT-SL enzyme to produce compound 1 In a reaction volume of 1 ml, 5 mg of mogroside IIIE, 50 mg of soluble starch, 0.1 M NaOAC pH 5. 125 ul of CGT-SL enzyme (from Geobacillus thermophillus) and water were mixed and incubated at 50 C with a stir bar. Time points samples were taken for HPLC.

[0321] HPLC Data: Mass Spectrometry of the preparation of Compound 1 shown in Figure 1. In some embodiments, the CTG-SL can comprise the sequence shown in SEQ ID NO:3, 148 or 154.

[0322] Example 44: Cloning: The gene encoding the dextransucrase enzyme was PCR amplified from Leuconostoc citreum ATCC11449 and cloned into pET23a Growth conditions: BL21 Codon Plus RIL strain was grown in 2xYT at 37°C and 250 rpm to an OD600 of 1. For induction, 10 mM lactose was added and incubated overnight at room temperature at 150 rpm. Crude extracts used in the reactions were obtained by either ultrasonic or osmotic shock.

[0323] In some embodiments, the dextransucrase comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the DexT can comprise the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, the DexT comprises the nucleic acid sequence set forth in SEQ ID NO: 104 or 105.

[0324] Example 45: Preparation of Compound 1 by Reaction of Mogroside IIIE with S. mutans Clarke ATCC25175 Dextransucrase Growth conditions: The above strains were grown anaerobically with glucose supplementation as described by Wenham, Hennessey and Cole (1979) to stimulate dextransucrase production. 5 mg / ml of mogroside IIIE was added to the growth medium. Time points were taken for HPLC. The HPLC data are shown in Figure 2 as is the mass spectrometry analysis of compound 1 production.

[0325] Example 46: Reaction of mogroside IIIE with CGTase In a 1 ml reaction volume, 5 mg of mogroside IIIE, 50 mg of soluble starch, 0.1 M NaOAC pH 5.0. 25 ul of enzyme and water were mixed and incubated at 50 C with a stir bar. Time points samples were taken for HPLC. The enzyme used was CGTase. The preparation of compound 1 is shown in the HPLC and mass spectrometry data shown in Figure 1. The mass peak corresponds to the size of compound 1.

[0326] Example 47: Reaction of Mogroside IIIE with Celluclasts Xylosylation of celluclasts was carried out using mogroside IIIE and celluclasts from the native host Trichoderma reesei.

[0327] Reaction conditions: 5 mg of mogroside IIIE, 100 mg of xylan, 50 ul of celluclasts were mixed with 0.1 M sodium acetate pH 5.0 in a total volume of 1 ml and incubated with stirring at 50 C. Time point samples were taken for HPLC.

[0328] The xylosylation product is highlighted in Figure 3. The xylosylation product can be used as an intermediate in the production of compound 1. The sequence of Celluclast is disclosed herein and is used herein to produce the xylosylation product.

[0329] Example 48: Glycosyltransferase (maltotriosyltransferase) (Native host: Geobacillus sp. APC9669) In this example, glycosytransferase AGY15763.1 (Amano Enzyme USA Co., Ltd., Elgin, IL; SEQ ID NO: 434) was used. 20 mL of demineralized water, 0.6 ml of 0.5 M MES pH 6.5, 6 g of soluble starch, 150 mg of mogroside IIIE, and 3 ml of enzyme were added to a 40 ml flat-bottom screw-cap vial. The vial was sealed with a black cap, incubated at 30° C., and stirred at 500 rpm with a magnetic bar. Three more identical reactions were set up for a total of 600 mg of mogroside IIIE used as starting material. The reaction was stopped after 24 hours. Insoluble starch was removed by centrifugation (4000 rpm for 5 min, Eppendorf). The supernatant was heated to 80° C. for 30 min with stirring (500 rpm) and then centrifuged (4000 rpm for 10 min, Eppendorf). The supernatant was filtered through 250 ml 0.22 micron PES and confirmed by LC-MS (Sweet Naturals 2016-Enzymatic_2016Q4_A.SPL, line 1254) to obtain HPLC data.

[0330] The AGY15763.1 protein (SEQ ID NO:434) can be encoded by the native gDNA (SEQ ID NO:437) or a codon-optimized (for E. coli) DNA sequence (SEQ ID NO:438).

[0331] One example of an additional glycosytransferase that is expected to behave similarly is the UGT76G1 protein (SEQ ID NO:439) from Stevia rebaudiana, which can be expressed in E. coli. The native coding sequence for UGT76G1 (SEQ ID NO:439) is provided in SEQ ID NO:440.

[0332] Example 49: UDP-glycosyltransferase UGT73C5 in the presence of mogrol Mogrol was reacted with UDP-glycosyltransferase to produce mogroside I and mogroside II. 1 mg / ml of mogrol was reacted with 200 ul of crude extract containing UGT73C5 (A. thaliana) (334), 2 ul of crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, 0.5 mg / ml spectinomycin in 0.1 M Tris-HCl pH 7.0 and incubated at 30 C. Samples were taken for HPLC after 2 days. As shown in Figures 4 and 5, the reaction products were mogroside I and mogroside II from mogrol.

[0333] The protein sequence of UGT73C5 is set forth in SEQ ID NO:441, the native DNA coding sequence of UGT73C5 (SEQ ID NO:441) is set forth in SEQ ID NO:442, and the UGT73C5 coding sequence (codon-optimized for E. coli) is set forth in SEQ ID NO:443.

[0334] Example 50: Production of UDP-glycosyltransferase (UGT73C6) mogroside I in the presence of mogrol Reaction conditions: 1mg / ml mogrol was reacted with 200ul of crude extract containing UGT73C6, 2ul of crude extract containing sucrose synthase, 5mM UDP, 1x M221 protease inhibitor, 200mM sucrose, 0.5mg / ml spectinomycin in 0.1M Tris-HCl pH7.5 and incubated at 30C. Samples were taken for HPLC after 2 days. The reaction product was mogroside I from mogrol. As shown in the HPLC and mass spectrometry data in Figure 6.

[0335] The protein and gDNA sequences encoding A. thaliana UGT73C6 are shown in SEQ ID NO:444 and SEQ ID NO:445, respectively.

[0336] Example 51: Production of Mogroside I, Mogroside IIA, and Two Different Mogroside III Products by UDP-Glycosyltransferase (338) in the Presence of Mogrol Reaction conditions: 1mg / ml mogrol or mogroside IIA was reacted with 200ul of crude extract containing 338, 2ul of crude extract containing sucrose synthase, 5mM UDP, 1x M221 protease inhibitor, 200mM sucrose, 0.5mg / ml spectinomycin in 0.1M Tris-HCl pH 8.5 and incubated at 30C. Samples were taken after 2 days for HPLC.

[0337] The mogrol reaction using Bacillus sp. UDP-glycotransferase (338) (described in Pandey et al., 2014; incorporated herein by reference in its entirety) yielded the reaction products mogroside I, mogroside IIA, and two distinct mogroside III products. Figures 7-9 show the HPLC and mass spectrometry data of the products obtained after the reaction. Figure 8 shows the peak that correlates to the size of mogrol IIA.

[0338] The protein and gDNA sequences encoding UGT338 are provided in SEQ ID NO:405 and SEQ ID NO:406, respectively.

[0339] Example 52: Production of siamenoside I and mogroside V by UDP-glycosyltransferase (301(UGT98)) in the presence of mogroside IIIE Reaction conditions: 1 mg / ml of mogroside IIIE was reacted with 200 ul of crude extract containing 301, 2 ul of crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, 0.5 mg / ml spectinomycin in 0.1 M Tris-HCl pH 7.0 and incubated at 30 C. Samples were taken after 2 days for HPLC and mass spectrometry. As shown in Figures 10-11, the reaction products from mogroside IIIE were siamenoside I and mogroside V.

[0340] The protein and gDNA sequences encoding S. grosvenorii 301UGT98 are provided in SEQ ID NO:407 and SEQ ID NO:408, respectively.

[0341] Example 53: Preparation of mogroside I from mogrol, isomogroside V from siamenoside I, and compound 1 derivatives from compound 1 by UDP-glycosyltransferase (339) in the presence of mogrol, siamenoside I, or compound 1 Reaction conditions: 1 mg / ml of mogrol, siamenoside I or compound 1 was reacted with 200 ul of crude extract containing 339 (described in Itkin et al., incorporated herein by reference in its entirety), 2 ul of crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, 0.5 mg / ml spectinomycin in 0.1 M Tris-HCl pH 7.0 and incubated at 30 C. Samples were taken after 2 days for HPLC.

[0342] The reaction product from mogrol gave mogroside I, the reaction product from siamenoside I gave isomogroside V, and the reaction product from compound 1 gave compound 1 derivative (Figures 12-14).

[0343] The protein and DNA sequences encoding S. grosvenorii UGT339 are provided in SEQ ID NO:409 and SEQ ID NO:410, respectively.

[0344] Example 54: Production of Mogroside IIIA, Mogroside IVE, and Mogroside V by UDP-glycosyltransferase (330) in the presence of Mogroside IIA, Mogroside IIE, Mogroside IIIE, Mogroside IVA, or Mogroside IVE As described herein, the reaction products mogroside IIIA, mogroside IVA, and mogroside V were obtained by using UDP-glycotransferase (330) as described in Noguchi et al. 2008 (incorporated herein by reference in its entirety). The native host was Sesamum indicum and the production host was E. coli. For the reaction, 1 mg / ml of mogroside IIA, mogroside IIE, mogroside IIIE, mogroside IVA, or mogroside IVE was reacted with 200 ul of crude extract containing 330, 2 ul of crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, 0.5 mg / ml spectinomycin in 0.1 M Tris-HCl pH 7.0 and incubated at 30C. A sample was taken for HPLC after 2 days.

[0345] The reaction gave rise to the products mogroside IIIA, mogroside IV, and mogroside V. As shown in Figures 15-20, the sizes of the compounds produced correspond to mogroside IIIA, mogroside IV, and mogroside V.

[0346] The protein and gDNA sequences encoding S. grosvenorii UGT330 are provided in SEQ ID NO:411 and SEQ ID NO:412, respectively.

[0347] Example 55: Production of Mogroside IIIA, Mogroside IVE, and Mogroside V by UDP-glycosyltransferase (328) (described in Itkin et al.) in the presence of Mogroside IIA, Mogroside IIE, Mogroside IIIE, Mogroside IVA, or Mogroside IVE Reaction conditions: 1mg / ml mogroside IIIE was reacted with 200ul of crude extract containing 330, 2ul of crude extract containing sucrose synthase, 5mM UDP, 1x M221 protease inhibitor, 200mM sucrose, 0.5mg / ml spectinomycin in 0.1M Tris-HCl pH7.0 and incubated at 30C. Samples were taken after 2 days for HPLC.

[0348] The reaction products were mogroside IVE and siamenoside I. As shown in Figures 21-22, the sizes of the products from the mass spectrometry data are consistent with mogroside IVE and siamenoside I. The S. grosvenorii UGT328 protein (glycosyltransferase) and its coding sequence are provided in SEQ ID NOs: 413 and 414, respectively.

[0349] The sucrose synthase AtSus1 protein sequence and the gDNA encoding the AtSus1 protein are provided in SEQ ID NOs: 415 and 416, respectively.

[0350] Example 56: Production of mogrol in yeast DNA was obtained by gene synthesis by either Genescript or IDT. For some cucurbitadienol synthases, cDNA or genomic DNA was obtained from 10-60 day old seedlings, followed by PCR amplification using specific and degenerate primers. DNA was cloned into one of the following overexpression vectors by standard molecular biology techniques: pESC-Ura, pESC-His, or pESC-LEU. Saccharomyces cerevisiae strain YHR072 (heterozygous for erg7) was purchased from GE Dharmacon. Plasmids (pESC vectors) containing mogrol synthesis genes were transformed / co-transformed using Zymo Yeast Transformation Kit II. Strains were grown in standard medium (YPD or SC) with the appropriate selection, including 2% glucose or 2% galactose for induction of the heterologous genes, at 30C and 220 rpm. Lanosterol synthase inhibitor Ro 48-8071 (Cayman Chemicals) was added (50ug / ml) where necessary. Production of mogrol and precursors by yeast was prepared after 2 days of induction followed by disruption (Yeast Buster), ethyl acetate extraction, drying, and resuspension in methanol. Samples were analyzed by HPLC.

[0351] The production of cucurbitadienol was catalyzed by cucurbitadienol synthase S. grosvernorii SgCbQ in inhibitor-free growth conditions.

[0352] The production of cucurbitadienol is shown in HPLC and mass spectrometry data showing the indicated product mass peaks (Figure 23). The protein and DNA sequences encoding S. grosvernorii SgCbQ are provided in SEQ ID NOs: 446 and 418, respectively.

[0353] Cpep2 was also used to produce cucurbitadienol in yeast. As shown in Figure 24, the mass spectrometry profile shows peaks and characteristic fragments corresponding to cucurbitadienol. The protein sequence of Cpep2 and the DNA sequence encoding the Cpep2 protein are provided in SEQ ID NOs: 420 and 421, respectively.

[0354] Cucurbita pepo (Jack-o'-Lantern) Cpep4 was also used for the production of cucurbitadienol under inhibitor-free growth conditions. The production of cucurbitadienol is shown in the mass spectrometry data shown in Figures 24 and 25. As indicated, the peaks and fragments correspond to cucurbitadienol. The protein and DNA sequences encoding Cpep4 are provided in SEQ ID NOs: 422 and 423, respectively.

[0355] Cmax, representing the putative cucurbitadienol synthase protein sequence, was obtained from the native host, Cucurbita maxima. The deduced coding DNA sequence is used for gene synthesis and expression. The protein and DNA cucurbitadienol synthase sequences encoding cucurbitadienol synthase are shown below.

[0356] The following protein and DNA coding sequences were obtained by alignment of PCR product sequences of genomic DNA with known cucurbitadienol synthase sequences available from public databases (Pubmed). It is expected that any one of these Cmax proteins can be used in the methods, systems, compositions (e.g., host cells) disclosed herein to produce Compound 1. A non-limiting exemplary Cmax protein is Cmax1(protein) (SEQ ID NO: 424), which is encoded by Cmax1(DNA) (SEQ ID NO: 425).

[0357] Cmos1, representing a putative cucurbitadienol synthase protein sequence, was obtained from the native host, Cucurbita moschata. The predicted coding DNA sequence is used for gene synthesis and expression. The protein and DNA coding sequences shown below were obtained by alignment of PCR product sequences of genomic DNA with known cucurbitadienol synthase sequences available from public databases (Pubmed). Any one of these Cmos proteins can be used in the methods, systems, compositions (e.g., host cells) disclosed herein to produce compound 1. A non-limiting and exemplary Cmos1 protein is Cmos1(protein) (SEQ ID NO: 426), which is encoded by Cmos1(DNA) (SEQ ID NO: 427).

[0358] Example 57: Production of dihydroxycucurbitadienol in yeast (cucurbitadienol synthase and epoxide hydrolase) The production of dihydroxycucurbitadienol in yeast was investigated using cucurbitadienol synthase and epoxide hydrolase, the native host of which is S. grosvenorii.

[0359] Growth conditions: SgCbQ was co-expressed with epoxide hydrolase (EPH) in the presence of a lanosterol synthase inhibitor.

[0360] The possible dihydroxycucurbitadienol products are shown in FIG.

[0361] The EPH protein sequence and DNA encoding the EPH protein (codon optimized S. cerevisiae) are provided in SEQ ID NOs: 428 and 429, respectively.

[0362] Example 58: Preparation of mogrol from cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 and cytochrome P450 reductase Four enzymes, including cucurbitadienol synthase, epoxide hydrolase, cytochrome P450, and cytochrome P450 reductase, were co-expressed in S. cerevisiae. For the growth conditions, SgCbQ, EPH, CYP87D18, and AtCPR (cytochrome P450 reductase from A. thaliana) are co-expressed in the presence of lanosterol synthase inhibitor. The production of mogrol by S. cerevisiae is expected. The protein and DNA sequences encoding SgCbQ, EPH, CYP87D18, and AtCPR (cytochrome P450 reductase from A. thaliana) are: CYP87D18(protein) (SEQ ID NO:430), and CYP87D18(DNA) (SEQ ID NO:431); and AtCPR(protein) (SEQ ID NO:432), and AtCPR(DNA) (SEQ ID NO:433).

[0363] Example 59: Compound 1 is resistant to microbial hydrolysis Yeast strains Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida bombicola were incubated in YPD supplemented with 1 mg / ml of mogroside V or compound 1. After 3 days, the supernatants were analyzed by HPLC.

[0364] As shown in the HPLC data, these strains hydrolyzed mogroside V to mogroside IIIE. No hydrolysis products were observed with compound 1 (Figures 27A and 27B).

[0365] Example 60: Streptococcus mutans Clarke ATCC25175 dextransucrase Streptococcus mutans Clarke can be grown anaerobically with glucose supplementation. An example of growth conditions can be found in Wenham, Henessey and Cole (1979), for example, where the method is used to stimulate the production of dextransucrase. 5 mg / ml of mogroside IIIE was added to the growth medium. For example, time-point samples to monitor production can be taken for HPLC. Various dextransucrase sequences include dextransucrase protein sequences and nucleic acid sequences encoding dextransucrase (e.g., SEQ ID NOs: 157-162). In some embodiments, the dextransucrase comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, DexT can comprise the amino acid sequence set forth in SEQ ID NO: 103. In some embodiments, DexT comprises the nucleic acid sequence set forth in SEQ ID NO: 104 or 105. In some embodiments, a recombinant cell as referred to herein encodes a protein comprising a sequence set forth in any one of SEQ ID NOs: 156 to 162 and / or comprises a nucleic acid encoding a dextransucrase comprising a nucleic acid sequence set forth in any one of SEQ ID NOs: 157 to 162. This example is used to produce compound 1.

[0366] Example 61: Preparation procedure and sensory evaluation of compound 1 with 90% purity Mogroside III E (MIII E) with a dextransucrase / dextranase enzymatic reaction followed by SPE fractionation yields a fraction containing a mixture of three α-mogroside isomers. Based on UPLC analysis, this mixture contains three isomers, namely 11-oxo compound 1, compound 1, and mogroside V isomer, in a ratio of 5:90:5%, respectively. These three isomers are characterized by purification of the different fractions / sources by LC-MS, 1D and 2D NMR spectroscopy, and by comparison of closely related isomers of the mogroside series reported in the literature. This sample is further evaluated sensorily by comparing it with a pure compound 1 sample using the triangle test.

[0367] Enzymatic reactions and purification procedures 100 mL of 1M sodium acetate buffer, pH 5.5, 200 g of sucrose, 100 mL of dextransucrase DexT (1 mg / ml crude extract, pET23a, BL21-Codon Plus-RIL, grown in 2X YT), 12.5 g of mogroside III. E and 600 mL of water were added to a 2.8 L shake flask and the flask was shaken at 200 rpm at 30° C. The progress of the reaction was monitored periodically by LC-MS. After 72 h, the reaction was treated with 2.5 mL of dextranase (Amano) and the flask was continued to shake at 30° C. After 24 h, the reaction mixture was quenched by heating at 80° C., centrifuged at 5000 rpm for 5 min, the supernatant was filtered and loaded directly onto a 400 g C18 SPE column and diluted with MeOH:H 2 The fractionation was performed using a step gradient of O 5 / 25 / 50 / 75 / 100. Each step of the gradient was collected in six jars, each containing 225 mL. The desired product was eluted in the second jar (SPE 75_2) of the 75% MeOH fraction and dried under reduced pressure. This was then washed with 7 mL of H 2 Resuspended / dissolved in O, frozen, and the vials lyophilized for 3 days to give 1.45 g of a white solid.

[0368] According to UPLC analysis (Figures 28 and 29), the mixture contains three characterized α-mogroside isomers, namely 11-oxo compound 1, compound 1, and mogroside V isomer, in a ratio of 5:90:5%, respectively. 1 H and 13 C-NMR (pyridine-d 5 +D 2 O) Based on the analysis, no residual solvents and / or structurally unrelated impurities were observed.

[0369] Sensory evaluation Triangle testing of pure Compound 1 vs. 90% pure Compound 1 was performed on November 10, 2016. Two different compositions were tested: (1) pure Compound 1 (standard) with LSB + 175 ppm and (2) 90% pure Compound 1 with LSB + 175 ppm. Both composition samples were prepared with low sodium buffer (LSB) pH 7.1, with 0% ethanol content.

[0370] Conclusion: The panelists found that composition (1) LSB + 175 ppm pure compound 1 (standard) was not significantly different from composition (2) LSB + 175 ppm 90% pure compound 1 (test) (p>0.05). Some of the analytical results tested are shown in Tables 2-4.

[0371] [Table 1]

[0372] [Table 2]

[0373] [Table 3]

[0374] Example 62: Gene expression in recombinant yeast cells, sample processing and detection of metabolites DNA was obtained by gene synthesis by either Genescript, IDT, or Genewiz. For some cucurbitadienol synthases, cDNA or genomic DNA was obtained from 10- to 60-day-old seedlings, followed by PCR amplification using specific and degenerate primers. DNA was cloned into one of the following overexpression vectors by standard molecular biology techniques or by yeast gap repair cloning (Joska et al., 2014): pESC-Ura, pESC-His, or pESC-LEU. Gene expression was regulated by one of the following promoters: Gal1, Gal10, Tef1, or GDS. Yeast transformation was performed using the Zymo Yeast Transformation Kit II. Yeast strains were grown in standard medium (YPD or SC) with the appropriate selection, including 2% glucose or 2% galactose for induction of the heterologous genes. Yeast strains were grown at 30 °C and 140–250 rpm in shake flasks or 96-well plates. Lanosterol synthase inhibitor Ro 48-8071 (Cayman Chemicals) was added (50ug / ml) where necessary. Yeast samples producing mogrol precursor, mogrol, and mogroside were treated with either: Cucurbitadienol and 11-OH Cucurbitadienol: Samples were prepared by disrupting cell pellets (Yeast Buster) followed by ethyl acetate extraction, drying and resuspension in methanol. Mogrol and mogroside: Sample cell pellets were lysed in 50% ETOH, incubated at 80°C for 30 min, filtered, dried and resuspended in MeOH for LCMS screening.

[0375] The samples were eluted with an A / B gradient (A=H 2 O, B=acetonitrile):

[0376] To analyze diepoxysqualene, the LCMS method involved the use of a C18 2.1 x 50 mm column with 5% B for 1.5 min, a gradient from 5% to 95% B, or 5.5 min, 95% B for 6 min, 100% B for 3 min, and 5% B for 1.5, both at a flow rate of 0.3 ml / min.

[0377] To analyze cucurbitadienol, the first LCMS method involved the use of a C4 2.1 x 100 mm column with a gradient of 1% to 95% B in 6 min at a flow rate of 0.55 ml / min, and the second LCMS method involved the use of a Waters Acquity UPLC Protein BEH C4 2.1x100mM, 1.7um (with guard) with a gradient of 62% to 67% B in 2 min, 100% B in 1 min at a flow rate of 0.9 ml / min and ESI source positive single ion monitoring at 427 and 409. Enzyme activity is displayed as the LCMS peak area of ​​the product vs. the peak area of ​​the internal standard.

[0378] To analyze 11-OH cucurbitadienol, the LCMS method included the use of a C8 2.1 x 100 mm column with a gradient of 60% to 90% B in 6 min and a flow rate of 0.55 ml / min. For enzyme screening, the LCMS method included the use of a Waters Acquity UPLC Protein BEH C4 2.1x100mM, 1.7um column with a gradient of 40% to 80% B in 5 min and 95% B in 5.1-5.6 min and a flow rate of 0.55 ml / min and an ESI source positive mode single ion monitoring of 425.4. Enzyme activity is displayed as the LCMS peak area of ​​the product vs. the peak area of ​​the internal standard.

[0379] To analyze mogrol, the LCMS method involved the use of a C8 2.1 x 100 mm column with a gradient of 50% to 90% B in 6 minutes at a flow rate of 0.55ml / min. Another method used a Water Acquity HSS T3 C18 2.1X30mm, 1.8um column with 45% B isocratic for 1 minute, 100% B in 1.3 minutes at a flow rate of 1.3ml / min and APCI source positive mode single ion monitoring at 423.4.

[0380] To analyze mogroside IIIE and compound 1, the LCMS method included the use of a Fluoro-phenyl 2.1 x 100 mm column with a gradient of 15% to 30% B in 6 minutes at a flow rate of 0.55 ml / min. To analyze yeast production of mogroside I during enzyme screening, the LCMS method included the use of a Phenomenex Kinetex Polar C18, 4.6 x 100 mm, 2.6 um column with a gradient of 25% to 55% B in 3 minutes to 100% B in 3.5 minutes at a flow rate of 1.5 ml / min. Detection by ESI source negative mode single ion monitoring was 683.4 for mogroside I and 799.4 for mogroside II.

[0381] Example 63: Step 1. Boosting Oxidosqualene Availability Saccharomyces cerevisiae strain YHR072 (heterozygous for lanosterol synthase erg7) was purchased from GE Dharmacon. Expression of the active erg7 gene was reduced by replacing the promoter with that of cup1 (Peng et al., 2015). A truncated yeast HMG-CoA reductase (tHMG-CoA) under the control of the GDS promoter and yeast squalene epoxidase (erg1) under the control of the Tef1 promoter were integrated into the genome. The boost of oxidosqualene was monitored by the production of diepoxysqualene as shown by HPLC and UV absorbance (Figure 31).

[0382] tHMG-CoA (Protein) SEQ ID NO: 898 (Pathway 1) tHMG-CoA (DNA) SEQ ID NO: 897 (Pathway 1) Erg1 (protein) SEQ ID NO: 900; Erg1 (DNA) SEQ ID NO: 899

[0383] In some embodiments, the tHMG-CoA enzyme is used for the production of diepoxysqualene.

[0384] A gene encoding a putative squalene epoxidase in S. grosvenorii (Itkins et al., 2016) was selected to be tested for boosting oxidosqualene / diepoxysqualene production. The amino acid and coding sequences of three squalene epoxidases are shown in SEQ ID NOs: 50-56, 60, 61, 334, and 335. Additional sequences of squalene epoxidases suitable for use in the methods, systems, and compositions disclosed herein for the production of oxidosqualene and / or diepoxysqualene, and for boosting oxidosqualene and / or diepoxysqualene, include SQE1 (protein) SEQ ID NO: 908, SQE1 (DNA) SEQ ID NO: 909; SQE2 (protein) SEQ ID NO: 910, SQE2 (DNA) SEQ ID NO: 911; SQE3 (protein) SEQ ID NO: 912, and SQE3 (DNA) SEQ ID NO: 913.

[0385] Step 2. Preparation of Cucurbitadienol Cucurbitadienol synthase enzyme A plasmid containing the S. grosvenorii cucurbitadienol synthase gene (SgCbQ) was transformed into a yeast strain with an oxidosqualene boost. The strain was grown for 1-3 days at 30C, 150-250 rpm. The production of cucurbitadienol is shown in the HPLC and mass spectrometry data showing the indicated product mass peaks (Figure 23). The SgCbQ protein and gDNA encoding SgCbQ are provided in SEQ ID NO: 446 and SEQ ID NO: 418, respectively. The Cucurbita pepo (Jack-o'-Lantern) protein Cpep2 was also used for the production of cucurbitadienol in yeast. Figure 24 shows the mass spectrometry profile including peaks and characteristic fragments corresponding to cucurbitadienol. The Cpep2 protein and the DNA sequence encoding Cpep2 are provided in SEQ ID NO: 420 and SEQ ID NO: 421, respectively. The Cucurbita pepo (Jack-o'-Lantern) protein Cpep4 was also used to produce cucurbitadienol. The host cells were cultured under inhibitor-free growth conditions. The production of cucurbitadienol is demonstrated in the mass spectrometry data shown in FIG. 25. As shown, the peaks and fragments correspond to cucurbitadienol. The Cpep4 protein and the DNA encoding Cpep4 are provided in SEQ ID NO: 422 and SEQ ID NO: 423, respectively. The Cucurbita maxima protein Cmax was also used to produce cucurbitadienol in yeast. FIG. 32 shows the mass spectrometry profile including the peaks and characteristic fragments corresponding to cucurbitadienol. The Cmax1 protein sequence is provided in SEQ ID NO: 424, and the sequence (DNA) encoding Cmax1 is provided in SEQ ID NO: 425. The Cucumis melo protein Cmelo was also used to produce cucurbitadienol in yeast. Figure 32 shows the mass spectrometry profile including peaks and characteristic fragments corresponding to cucurbitadienol.The Cmelo protein sequence is provided in SEQ ID NO: 902 and the sequence (DNA) encoding Cmelo is provided in SEQ ID NO: 901. It is expected that the Cucurbita moschata protein Cmos1 can also be used for the production of cucurbitodienol in recombinant host cells, such as yeast cells. The Cmos1 sequences Cmos1 (protein) (SEQ ID NO: 426) and Cmos1 (DNA) (SEQ ID NO: 427) were obtained by alignment of PCR product sequences of genomic DNA with known cucurbitadienol synthase sequences available from public databases (Pubmed). It is expected that the Cmost1 protein (SEQ ID NO: 426) can be used for the production of cucurbitadienol in recombinant host cells, such as yeast cells.

[0386] Conversion of other oxidosqualene cyclases to cucurbitadienol synthase The plasmid containing the modified oxidosqualene gene was transformed into the yeast strain with oxidosqualene boost. The strain was grown at 30 C, 150-250 rpm for 1-3 days.

[0387] The protein PSX Y118L from the native host Pisum sativum was also used for the production of cucurbitadienol in yeast. Figure 33 shows the mass spectrometry profile including peaks and characteristic fragments corresponding to cucurbitadienol when tyrosine at position 118 is converted to leucine. The protein and DNA sequences encoding the modified oxidosqualene cyclase are PSXY118L (protein) (SEQ ID NO: 904) and PSXY118L (DNA, codon optimized) (SEQ ID NO: 903).

[0388] Oxidosqualene cyclase from Dictyostelium sp. was also used to produce cucurbitadienol in yeast. As shown in Figure 34, the HPLC peak of cucurbitadienol is shown when tyrosine at position 80 is converted to leucine. The protein and DNA sequences encoding the modified oxidosqualene cyclase are DdCASY80L (protein) (SEQ ID NO: 906) and DdCASY80L (DNA) (SEQ ID NO: 905).

[0389] Enhancement of cucurbitadienol synthase activity The gene encoding cucurbitadienol synthase from Cucumis melo was codon optimized (SEQ ID NO: 907) and used as a starting point to produce a library of modifications. Modifications were introduced by standard molecular biology techniques consisting of fusion peptides at the N-terminal (i.e. 5') or C-terminal (i.e. 3') ends of the enzyme. The plasmid library of modified cucurbitadienol synthase genes was transformed into a yeast strain with oxidosqualene boost. Enzyme activity was measured by the ratio of peak heights or areas of the 409 and 427 positive mass fragments at the expected retention times of cucurbitadienol vs. the internal standard using LCMS method 2 described above. Enzyme performance was recorded as the average % activity relative to the average activity of the parent enzyme (n=8). The enzymes and enzyme-encoding sequences for step 1 sequences can be found in SEQ ID NOs: 951-1012. Step 1 sequences also include fusions SS2c-G10, SS2e-A7b, SS2d-G11, SS2e-A7a, SS4d-G5, SS4d-C7, SS3b-D8, and SS2c-A10a, which are disclosed herein.

[0390] Step 3. Preparation of 11-OH cucurbitadienol CYP87D18 (CYP450, S. grosvenorii) and SgCPR (CYP450 reductase, S. grosvenorii) were expressed in a cucurbitadienol-producing S. cerevisiae strain. Using HPLC and mass spectrometry data, 11-OH cucurbitadienol (i.e., 11-hydroxycucurbitadienol) was observed (Figure 35). The S. grosvenorii CYP87D18 protein sequence is shown in SEQ ID NO:872, and the CYP87D18 (codon-optimized DNA) coding sequence is shown in SEQ ID NO:871. The S. grosvenorii SgCPR protein sequence is shown in SEQ ID NO:874, and the SgCPR1 (codon-optimized DNA) coding sequence is shown in SEQ ID NO:873.

[0391] Additional CYP450s were expressed in S. cerevisiae strains that produce cucurbitadienol and express SgCPR. The CYP450s that result in the production of 11-OH cucurbitadienol are provided in Table 5. The DNA and protein sequences of the CYP450 enzymes are shown in SEQ ID NOs: 871-890 and 1024-1049 (see Table 1). Sample LCMS data is provided in Figure 44.

[0392] [Table 4]

[0393] Step 4. Making Mogrol CYP1798 (CYP450 enzyme, S. grosvenorii) and EPH2A (epoxide hydrolase, S. grosvenorii) were expressed in a S. cerevisiae strain that produces 11-OH cucurbitadienol. Using HPLC and mass spectrometry data, mogrol was observed (Figure 36). For sequences, the DNA codes and protein sequences of the enzymes are provided in SEQ ID NOs: 891-894.

[0394] Additional EPHs were tested in yeast strains that produce the mogrol precursor, and one of the EPHs (the coding DNA and protein sequences are shown in SEQ ID NOs: 1072 and 1073, respectively) demonstrated the ability to produce mogrol (Figure 46).

[0395] Epoxidation of cucurbitadienol and / or 11-OH cucurbitadienol To test epoxidation, additional CYP450s and SQEs from S. grosvenorii and Glycyrrhiza (CYP88D6) were also expressed in S. cerevisiae strains producing cucurbitadienol or 11-OH cucurbitadienol.

[0396] For SQE, the protein and DNA coding sequences of the enzymes are provided in SEQ ID NOs: 882 to 888. For CYP450, the protein and DNA coding sequences of the enzymes are provided in SEQ ID NOs: 875 to 890.

[0397] Step 7: Preparation of compound 1 from mogroside IIIE in S. cerevisiae A S. cerevisiae strain expressing truncated dextransucrase (tDexT) was incubated in YPD (30C, 250 rpm) containing 7 mg / ml mogroside V for 1-2 days to hydrolyze it to mogroside IIIE. S. cerevisiae cells were harvested, disrupted, and mixed again with YPD supernatant containing mogroside IIIE. To initiate the dextransucrase reaction, sucrose was added to a final concentration of 200 g / L, followed by incubation at 30C, 250 rpm for 2 days. Using HPLC, the production of compound 1 was observed (Figure 37). The protein sequence of tDexT is shown in SEQ ID NO:896, and the DNA coding sequence of tDexT is shown in SEQ ID NO:895.

[0398] Example 64 S. cerevisiae or Y. lipolytica were grown in the presence of mogroside V to allow hydrolases in the yeast to produce mogroside IIIE. After 1 or 2 days, the cells were disrupted and analyzed by HPLC to measure mogroside content. After 1 day of incubation, S. cerevisiae produced a mixture of mogroside V, mogroside IV, and mogroside IIIE. After 2 days of incubation, virtually all of the mogrosides were converted to mogroside IIIE, as shown in Figure 40A.

[0399] Similarly, after 2 days of incubation, Y. lipolytica produced primarily mogroside IIIE (shown in FIG. 40B).

[0400] Example 65 S. cerevisiae or Y. lipolytica were grown in the presence of compound 1. Unlike other mogrosides (see Example 64), no hydrolysis products were observed upon hydrolysis of compound 1, as shown in FIG.

[0401] Example 66 S. cerevisiae was modified to overexpress dextransucrase (DexT). The modified strain was grown in the presence of a mogroside mixture to allow hydrolases in S. cerevisiae to produce mogroside IIIE. After 2 days of incubation, the cells were disrupted to release the DexT enzyme and supplemented with sucrose. After 24 hours, significant amounts of compound 1 were produced (see FIG. 42).

[0402] Example 67: Preparation of a fusion protein exhibiting cucurbitadienol synthase activity A collection or library of S. cerevisiae in-frame fusion polynucleotides of the cucurbitadienol synthase gene (DNA coding sequence provided in SEQ ID NO: 907, and protein sequence provided in SEQ ID NO: 902) was prepared. The in-frame fusion polynucleotides were cloned into yea...

Claims

1. The structure: 【Chemistry 1】 A method for preparing compound 1 having the formula: Mogroside III Ｅ Mogroside III Ｅ with a recombinant host cell comprising a first enzyme capable of catalyzing the production of compound 1 from (1) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a glycosylation enzyme comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094 to 1149; and / or (4) a glycosylation enzyme encoding a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093; and / or (5) an epoxide hydrolase comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) Epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1072. A method comprising:

2. The method of claim 1 , wherein the recombinant host cell comprises a first gene encoding the first enzyme.

3. Mogroside III Ｅ The method of claim 1 or 2, wherein said recombinant host cell is present in and / or produced by said recombinant host cell.

4. 4. The method of claim 1, further comprising culturing the recombinant host cell in a culture medium under conditions in which the first enzyme is expressed.

5. 5. The method of claim 1, wherein the first enzyme is one or more of UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTase), glycotransferase, dextransucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and dextranase.

6. Mogroside IIA is contacted with the recombinant host cell to produce mogroside III Ｅ 6. The method of claim 1, wherein the recombinant cell comprises a second enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA.

7. 7. The method of claim 1, comprising contacting mogrol with the recombinant host cell, the recombinant host cell comprising one or more enzymes capable of catalyzing the production of mogroside IIE and / or IIIE from mogrol.

8. 8. The method of any one of claims 1 to 7, comprising contacting mogroside IA1 with the recombinant host cell, wherein the recombinant host cell contains a gene encoding a UGT98 or UGT SK98 enzyme comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9, 407, 16, or 306.

9. 9. The method of any one of claims 1 to 8, wherein the method comprises contacting 11-hydroxy-24,25 epoxy cucurbitadienol with the recombinant host cell, the recombinant host cell comprising a third gene encoding an epoxide hydrolase.

10. 10. The method of any one of claims 1 to 9, wherein the method comprises contacting 11-hydroxycucurbitadienol with the recombinant host cell, the recombinant host cell comprising a fourth gene encoding a cytochrome P450, or an epoxide hydrolase.

11. 11. The method of any one of claims 1 to 10, wherein the method comprises contacting 3,24,25-trihydroxycucurbitadienol with the recombinant host cell, the recombinant host cell comprising a fifth gene encoding a cytochrome P450.

12. 12. The method of claim 1, wherein the method comprises contacting cucurbitadienol with the recombinant host cell.

13. 13. The method of any one of claims 1 to 12, wherein the method comprises contacting the recombinant host cell with one or more of 2,3-oxidosqualene, dioxidosqualene, and diepoxysqualene, and the recombinant host cell comprises a seventh gene encoding a polypeptide exhibiting cucurbitadienol synthase activity.

14. 1. A recombinant cell comprising the following structure: 【Chemistry 2】 and a gene encoding an enzyme capable of catalyzing the production of compound 1 from mogroside IIIE, (1) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a glycosylation enzyme comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094 to 1149; and / or (4) a glycosylation enzyme encoding a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093; and / or (5) an epoxide hydrolase comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) Epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1072. and one or more of the above.

15. A recombinant cell comprising the following structure from mogroside IIIE: 【Chemistry 3】 and a first gene encoding a first enzyme capable of catalyzing the production of compound 1 having the following: (1) a cytochrome P450 comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, and 1049; and / or (2) a cytochrome P450 encoded by a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, and 1048; and / or (3) a glycosylation enzyme comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1051, 1053, 1055, 1057, 1059, 1061, 1063, 1065, 1067, 1069, 1071, 1074, 1076, 1078, 1080, 1082, 1084, 1086, 1088, 1090, 1092, and 1094 to 1149; and / or (4) a glycosylation enzyme encoding a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1050, 1052, 1054, 1056, 1058, 1060, 1062, 1064, 1066, 1068, 1070, 1075, 1077, 1079, 1081, 1083, 1085, 1087, 1089, 1091, and 1093; and / or (5) an epoxide hydrolase comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1073; and / or (6) Epoxide hydrolase encoded by a nucleic acid sequence having at least 80% sequence identity to the sequence of SEQ ID NO: 1072. and one or more of the above.

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