Method for preparing high-intensity sweeteners

Abstract

The methods provided herein include methods of preparing mogroside compounds (e.g., Compound 1), compositions (e.g., host cells) for preparing mogroside compounds, and mogroside compounds prepared by the methods disclosed herein, as well as compositions (e.g., cell lysates) and recombinant cells comprising mogroside compounds. Also provided herein are novel cucurbitadienol synthases and uses thereof.

Description

[0001] The present invention is a divisional application of invention patent application No. 201880044909.3 filed on May 2, 2018, entitled “Method for Preparing High Intensity Sweetener”. Technical Field

[0002] The present disclosure relates to methods, systems, and compositions for producing sweet compounds, and compositions comprising the same. Background Art

[0003] The taste system provides sensory information about the chemical composition of the outside world. Taste transduction is one of the most complex forms of chemically induced sensation in animals. Taste signals are found throughout the animal kingdom, from simple 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 flavor).

[0004] For centuries, various natural and non-natural ingredients and / or compounds have been added to ingestible compositions (including foods and beverages) and / or orally administered medicinal ingredients to improve their taste. Although it has long been understood that there are only a few basic types of "tastes", the biological and biochemical basis of taste is poorly understood, and most taste-enhancing or taste-modifying agents have been discovered primarily through a simple process of trial and error.

[0005] For sweet taste, diabetes and cardiovascular disease are health problems that are increasing worldwide, but they are developing at an alarming rate in the United States. Sugar and calories are key components that can be limited to obtain a positive nutritional effect on health. High-intensity sweeteners can provide the sweetness of sugar and have a variety of taste qualities. Because they are many times sweeter than sugar, much less sweetener is needed to replace sugar.

[0006] High intensity sweeteners have a variety of different chemical structures and therefore have different properties, such as, but not limited to, smell, flavor, mouthfeel and aftertaste. These properties, especially flavor and aftertaste, are generally considered to change over the time of tasting, making each time curve (temporal profile) sweetener specific.

[0007] There has been significant progress recently in identifying useful natural flavoring agents, such as sweeteners, such as sucrose, fructose, glucose, erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, certain known natural terpenes, flavonoids, or protein sweeteners. See, for example, Kinghom et al., "Noncariogenic Intense Natural Sweeteners", Med. Res. Rev. 18 (5) 347-360 (1998) (discussing natural substances found to be much sweeter in intensity than common natural sweeteners such as sucrose, fructose, etc.). Similarly, progress has recently been made in the identification and commercialization of new artificial sweeteners, such as aspartame, saccharin, acesulfame potassium, cyclamate, sucralose, etc. See, for example, Ager et al., Angew. Chem. Int. Ed. 37, 1802-1817 (1998). The entire disclosures of the above-mentioned documents are incorporated herein by reference in their entirety.

[0008] Sweeteners such as saccharin and 6-methyl-1,2,3-oxathiazine-4(3H)-one-2,2-dioxide potassium salt (acesulfame potassium) are generally characterized as having a bitter and / or metallic aftertaste. Products prepared using 2,4-dihydroxybenzoic acid are said to exhibit a reduced undesirable aftertaste associated with sweeteners, and this is true at concentrations below those at which their own taste is perceptible. In addition, high-intensity sweeteners such as sucralose and aspartame are reported to have sweetness delivery problems, i.e., delayed onset and residual sweetness. See SG Wiet et al., J. Food Sci., 58(3):599-602,666(1993).

[0009] There is a need for new sweetening compounds, sweetness enhancers, and compositions containing such compounds and enhancers having improved taste and delivery properties. In addition, there is a need for food products containing new sweetening compounds and / or sweetness enhancers having such desirable properties. Summary of the invention

[0010] The present invention provides a method for producing compound 1 having the following structure:

[0011]

[0012] 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, the method includes contacting mogroside IIIE with a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In certain embodiments, the mogroside IIIE contacted with the first enzyme comprises mogroside IIIE contacted with a recombinant host cell comprising a first gene encoding the first enzyme. For example, the first gene can be an exogenous gene of the recombinant host cell.

[0013] In some embodiments, mogroside IIIE is contacted with a first enzyme in a recombinant host cell comprising a first polynucleotide encoding the first enzyme. Mogroside IIIE can, for example, be provided to a recombinant cell, be present in a recombinant host cell, be produced by a recombinant host cell, or be produced in any combination thereof. In some embodiments, the method includes culturing a recombinant host cell in a culture medium under conditions of expressing the first enzyme. The first enzyme can be, for example, one or more of UDP glycosyltransferases, cyclomaltodextrin glucanotransferases (CGTase), glycosyltransferases, dextransucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and dextranases.

[0014] In some embodiments, the first enzyme is CGTase. In some embodiments, the CGTase comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers with the sequence of any one of SEQ ID NOs: 1, 3, 78-101, 148 and 154. In some embodiments, the CGTase comprises any one of SEQ ID NOs: 1, 3, 78-101, 148 and 154. In some embodiments, the CGTase consists of the amino acid sequences in SEQ ID NOs: 1, 3, 78-101, 148 and 154.

[0015] In some embodiments, the first enzyme is a dextran sucrase. For example, the dextran sucrase may comprise an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers with any sequence shown in SEQ ID NO: 2, 103, 106-110, 156, 159-162 and 896. In some embodiments, the dextran sucrase is encoded by a nucleic acid sequence having at least 70% sequence identity with any one of SEQ ID NO: 104, 105, 157, 158 and 895.

[0016] In some embodiments, the first enzyme is a transglucosidase. For example, the transglucosidase may comprise an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any of SEQ ID NOs: 163-291 and 723. In some embodiments, the transglucosidase comprises an amino acid sequence of any one of SEQ ID NOs: 163-291 and 723.

[0017] In some embodiments, the first enzyme is a β-glucosidase β-. For example, the β-glucosidase can comprise an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 102, 292, 354-374, and 678-741, or a range between any two of these numbers.

[0018] In some embodiments, the method includes contacting mogroside IIA with an enzyme capable of catalyzing mogroside IIA to produce mogroside IIIE. In some embodiments, contacting mogroside IIA with the enzyme includes contacting mogroside IIA with a recombinant host cell to produce mogroside IIIE, wherein the recombinant host cell comprises a gene encoding an enzyme capable of catalyzing mogroside IIA to produce mogroside IIIE. Mogroside IIA can, for example, be provided to the recombinant host cell, produced by the recombinant host cell, present in the recombinant host cell, or any combination thereof. The enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA can be, for example, one or more of a UDP glycosyltransferase, a CGTase, a glycosyltransferase, a glucansucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase.

[0019] In some embodiments, the second enzyme is a uridine diphosphate-glucosyltransferase (UGT). For example, the UGT can be UGT73C3 (SEQ ID NO: 4), UGT73C6 (SEQ ID NO: 5), UGT 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). In some embodiments, the UGT comprises an amino acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% 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, and 444, or a range between any two of these numbers. The UGT is encoded by a nucleic acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to the 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), SEQ ID NOs: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, and 445, or a range between any two of these numbers.

[0020] In some embodiments, the method includes contacting mogroside IIIE and / or IIE from mogroside with one or more enzymes capable of catalyzing the production of mogroside IIIE and / or IIE from mogroside. In some embodiments, contacting mogroside with one or more enzymes includes contacting mogroside with a recombinant host cell to produce mogroside IIIE and / or mogroside IIE, wherein the recombinant host cell comprises one or more genes encoding one or more enzymes capable of catalyzing the production of mogroside IIIE and / or mogroside IIE. For example, mogroside can be provided to the recombinant host cell, produced by the recombinant host cell, present in the recombinant host cell, or any combination thereof.

[0021] In some embodiments, at least one of the one or more enzymes capable of catalyzing mogroside IIE and / or mogroside IIIE from mogroside alcohol comprises a sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% identity to any one of SEQ ID NOs: 315, 316, 420, 422, 424, 426, 430, 431, 446, 871, 845-949, and 951-1012, or is encoded by a sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers. NOs: 315, 316, 420, 422, 424, 426, 430, 431, 446, 871, 845-949 and 951-1012 have or have at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or any two of these numbers in the range of identity. In some embodiments, the enzyme capable of catalyzing the production of mogroside IIE and / or mogroside IIIE from mogroside alcohol includes one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. In some embodiments, at least one of the one or more enzymes is uridine diphosphate-glucosyltransferase (UGT). For example, the UGT can be UGT73C3, UGT73C6, 85C2, UGT73C5, UGT73E1, UGT98, UGT1495, UGT1817, UGT5914, UGT8468, UGT10391, UGT1576, UGT SK98, UGT430, UGT1697, or UGT11789, or the UGT comprises an amino acid sequence that is identical to SEQ ID Any of NOs: 4-9, 15-19, 125, 126, 128, 129, 293-307, 405, 406, 407, 409, 411, 413, 439, 441 and 444 has or has a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers.

[0022] In some embodiments, the method includes contacting a mogroside compound with one or more enzymes capable of catalyzing the production of mogroside IIIE from a mogroside compound to produce mogroside IIIE, 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, and siamenoside. In some embodiments, contacting a mogroside compound with one or more enzymes capable of catalyzing the production of mogroside IIIE products includes contacting a mogroside compound with a recombinant host cell, wherein the recombinant host cell comprises one or more genes encoding the one or more enzymes capable of catalyzing the production of mogroside IIIE from a mogroside compound. The mogroside compound can, for example, be provided to a recombinant host cell, produced by a recombinant host cell, present in a recombinant host cell, and any combination thereof. In some embodiments, the one or more enzymes capable of catalyzing the production of mogroside IIIE of a mogroside compound include one or more of UDP glycosyltransferase, CGTase, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and glucanase. In some embodiments, the mogroside compound is mogroside IIE.

[0023] In some embodiments, one or more enzymes capable of catalyzing a mogroside compound to produce mogroside IIIE comprise an amino acid sequence having or 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, 106-109, 147, 154, 163-303, 405, 411, 354-405, 447-723, 770, 776, and 782. In some embodiments, the mogroside compound is mogroside IIA or mogroside IIE. In some embodiments, contacting with one or more enzymes produces one or more of mogroside IIIA, mogroside IVE, and mogroside V.

[0024] In some embodiments, the one or more enzymes comprise an amino acid sequence having or 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: 304, 405, 411, 872, 874, 978, 880, 882, 884, 886, 888, 890, 892, 894 and 896. In some embodiments, the one or more enzymes are encoded by a sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 305, 406, 412, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, and 895, or a range between any two of these numbers.

[0025] In some embodiments, the method comprises contacting mogroside IA1 with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a UGT98 or UGT SK98 enzyme. In some embodiments, the UGT98 or UGT SK98 enzyme comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 9, 407, 16 or 306. In some embodiments, UGT98 is encoded by the sequence shown in SEQ ID NO: 307. In some embodiments, contacting results in the production of mogroside IIA in the cell.

[0026] In some embodiments, the method comprises contacting 11-hydroxy-24,25-epoxycucurbitadienol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding an epoxide hydrolase. In some embodiments, 11-hydroxy-24,25-epoxycucurbitadienol is provided to the recombinant host cell, present in the recombinant host cell, produced by the recombinant host cell, and any combination thereof.

[0027] In some embodiments, the method comprises contacting 11-hydroxycucurbitacinol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a cytochrome P450 or an epoxide hydrolase. The 11-hydroxycucurbitacinol can be provided to the recombinant host cell, produced by the recombinant host cell, and / or present in the recombinant host cell.

[0028] In some embodiments, the method includes contacting 3,24,25-trihydroxycucurbitacinol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a cytochrome P450. In some embodiments, 3,24,25-trihydroxycucurbitacinol is provided to the recombinant host cell, present in the recombinant host cell, produced by the recombinant host cell, or any combination thereof. The contacting results in the production of mogrosanol in the recombinant host cell. In some embodiments, the cytochrome P450 comprises an amino acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 20, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 889, and 892; or the cytochrome P450 is encoded by a nucleic acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 20, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 889, and 892; The sequence of any one of NOs:31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 890 and 891 has or has a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers.

[0029] In some embodiments, the epoxide hydrolase comprises an amino acid sequence that has, or has 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; or the epoxide hydrolase is encoded by a nucleic acid sequence that has, or has 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: 114 and 115.

[0030] In some embodiments, the method includes contacting cucurbitacinol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a cytochrome P450. In some embodiments, the contact results in the production of 11-hydroxycucurbitacinol. In some embodiments, cucurbitacinol is provided to a recombinant host cell, produced by a recombinant host cell and / or present in a recombinant host cell. In some embodiments, cytochrome P450 is encoded by a nucleic acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889 and 892. In some embodiments, the cytochrome P450 comprises an amino acid sequence having or 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, 31, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890 and 891. In some embodiments, the method further comprises contacting one or more of 2,3-squalene oxide, squalene dioxide and diepoxysqualene with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a polypeptide having cucurbitadienol synthase activity.

[0031] In some embodiments, the method includes contacting the mogroside intermediate with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a polypeptide having cucurbitadienol synthase activity. In some embodiments, the polypeptide having cucurbitadienol synthase activity is a fusion protein comprising one or more fusion domains fused to the cucurbitadienol synthase. In some embodiments, the fusion protein comprises a fusion domain fused to the N-terminus, the C-terminus or both of the cucurbitadienol synthase. In some embodiments, the fusion domain is about 3 to about 1000 amino acids long. In some embodiments, the fusion domain is about 5 to about 50 amino acids long. In some embodiments, the fusion domain is the main part or the entire sequence of the functional protein.

[0032] In some embodiments, the fusion polypeptide having cucurbitadienol synthase activity comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NOs:70-73, 75-77, 319, 321, 323, 325, 327-333, 417, 420, 422, 424, 426, 446, 902, 904, or 906, or a range between any two of these numbers. In some embodiments, the cucurbitadienol synthase is encoded by a nucleic acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to 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.

[0033] In some embodiments, contacting results in the production of cucurbitadienol. In some embodiments, 2,3-squalene oxide and diepoxysqualene are provided to, produced by and / or present in a recombinant host cell. In some embodiments, one or more of 2,3-squalene oxide and diepoxysqualene are produced by an enzyme comprising a sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers with SEQ ID NO: 898 or 900. In some embodiments, one or more of 2,3-oxidosqualene, diepoxysqualene, and diepoxysqualene are produced by an enzyme encoded by a nucleic acid sequence having or 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. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NO: 70-73, 75-77, 319, 321, 323, 325, 327, 329-333, 420, 422, 424, 426, 446, 902, 904, and 906. In some embodiments, the polypeptide comprising cucurbitadienol synthase activity is encoded by a gene comprising a nucleic acid sequence having or 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: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905.

[0034] In some embodiments, 11-hydroxycucurbitadienol is provided to, produced by, and / or present in a recombinant host cell. In some embodiments, 11-hydroxycucurbitadienol is expressed in a cell (e.g., a recombinant host cell) comprising a gene encoding a CYP87D18 and / or SgCPR protein. In some embodiments, the CYP87D18 or SgCPR protein comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers with SEQ ID NO: 872 or 874. In some embodiments, the CYP87D18 or SgCPR protein is encoded by a nucleic acid sequence that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 871 or 873, or a range between any two of these numbers.

[0035] In some embodiments, the method includes contacting squalene with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding squalene epoxidase. In some embodiments, 2,3-oxidized squalene is produced by contacting. In some embodiments, squalene is provided to a recombinant host cell, produced by a recombinant host cell and / or present in a recombinant host cell. In some embodiments, squalene epoxidase comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity with any one of SEQ ID NO:50-56, 60, 61, 334 or 335. In some embodiments, squalene epoxidase is encoded by a nucleic acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity with any two of these numbers with SEQ ID NO:335.

[0036] In some embodiments, the method includes contacting farnesyl pyrophosphate with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding squalene synthase. In some embodiments, the production of squalene is caused by contact. In some embodiments, farnesyl pyrophosphate is provided to a recombinant host cell, produced by a recombinant host cell and / or present in a recombinant host cell. In some embodiments, squalene synthase comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any one of SEQ ID NO:69 and 336. In some embodiments, squalene synthase is encoded by a sequence comprising a nucleic acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with SEQ ID NO:337.

[0037] In some embodiments, the method includes contacting geranyl-PP with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a farnesyl-PP synthase. In some embodiments, the production of farnesyl-PP is caused by contacting. In some embodiments, geranyl-PP is provided to a recombinant host cell, produced by a recombinant host cell, and / or present in a recombinant host cell. In some embodiments, the farnesyl-PP synthase comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity with SEQ ID NO: 338. In some embodiments, the farnesyl-PP synthase is encoded by a nucleic acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity with SEQ ID NO: 339.

[0038] In some embodiments, one or more genes are operably linked to a heterologous promoter, the gene encoding (1) a first enzyme capable of catalyzing the production of compound I from mogroside IIIE, (2) an enzyme capable of catalyzing the production of mogroside IIIE from mogroside IIA, (3) an epoxide hydrolase, (4) a cytochrome P450, (5) a polypeptide having cucurbitadienol synthase activity, (6) a squalene epoxidase, (7) a farnesyl-PP synthase. In some embodiments, the heterologous promoter is 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, repressible or constitutive promoter. In some embodiments, the production of one or more of pyruvate, acetyl-CoA, citrate, and TCA cycle intermediates has been upregulated in the recombinant host cell. In some embodiments, cytoplasmic localization has been upregulated in the recombinant host cell. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth genes comprises at least one sequence encoding a 2A self-cleaving peptide.

[0039] In some embodiments, the recombinant host cell is a cell of a plant, bivalve, fish, fungus, bacteria, or mammal. 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, 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, 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, Sacccharaomyces, 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 recombinant host cell is a Saccharomyces cerevisiae cell or a Yarrowia lipolytica cell. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes are codon-optimized genes for expression in bacteria, mammals, plants, fungi and / or insect cells. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes include functional mutations that increase the activity of the encoded enzyme. In some embodiments, cultivating the recombinant host cell includes monitoring the pH, dissolved oxygen level, nitrogen level or a combination thereof of the culture conditions.

[0040] In some embodiments, the method includes isolating compound 1. In some embodiments, isolating compound 1 includes lysing recombinant host cells, and / or isolating compound 1 from culture medium. In some embodiments, the method includes purifying compound 1. Purifying compound 1 includes HPLC, solid phase extraction, or a combination thereof. In some embodiments, purification includes obtaining recombinant host cells, preserving supernatant, and lysing recombinant host cells. In some embodiments, lysis includes subjecting cells to shear force or detergent washing to obtain lysate. Shear force can, for example, come from ultrasonic treatment, high pressure cell lysis (french pressurized cells), or beads. In some embodiments, the lysate is filtered and purified. In some embodiments, the lysate is filtered and purified by solid phase extraction.

[0041] In some embodiments, the method further comprises contacting the first mogroside IIIE with one or more hydrolases to produce mogroside IIIE before contacting the first mogroside IIIE with the first enzyme. For example, the hydrolase can be a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1 or EXG2. In some embodiments, the hydrolase comprises an amino acid sequence having or having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a sequence identity of any two of SEQ ID NOs: 292, 366-368, 372, 376-398, 447-520, 524-845, 1013, 1014, and 1023.

[0042] In some embodiments, the first mogroside is mogroside IV, mogroside V, mogroside VI, or a combination thereof. In some embodiments, the first mogroside is mogroside V, simenoside I, mogroside IVE, mogroside VI, mogroside IVA, or a combination thereof.

[0043] In some embodiments, contacting the first mogroside with one or more hydrolases comprises contacting the first mogroside with a host cell comprising a gene encoding a hydrolase. In some embodiments, the first mogroside contacts one or more hydrolases in a host cell comprising a gene encoding a hydrolase and a gene encoding an enzyme capable of catalyzing the production of compound 1. The host cell may be a recombinant host cell comprising a first gene encoding a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the host cell is not a recombinant host cell comprising a first gene encoding a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the first mogroside is provided to the host cell, produced by the host cell and / or present in the host cell. The gene encoding the hydrolase may be heterologous or homologous to the host cell. In some embodiments, the gene encoding the hydrolase is expressed at normal levels in the host cell. In some embodiments, the gene encoding the hydrolase is overexpressed in the host cell.

[0044] In some embodiments, the recombinant host cell comprises an oxidized squalene cyclase, such as a cycloartenol synthase or a β-amyrin synthase, or a nucleic acid sequence encoding an oxidized squalene cyclase, such as a cycloartenol synthase or a β-amyrin synthase β-, and wherein the oxidized squalene cyclase, cycloartenol synthase, or β-amyrin synthase β- is modified to produce cucurbitadienol or epoxycucurbitadienol. The oxidized squalene cyclase may, for example, comprise or consist of 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: 341, 343, and 346-347.

[0045] In some embodiments, the recombinant host cell comprises a cytochrome P450 reductase or a gene encoding a cytochrome P450 reductase. In some embodiments, the cytochrome P450 reductase comprises or consists of a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to SEQ ID NO: 318, or a range between any two of these numbers.

[0046] The present invention discloses a compound having the structure of compound 1,

[0047]

[0048] wherein the compound is produced by any of the methods disclosed herein.

[0049] The present invention discloses a cell lysate, which comprises a compound 1 having the following structure:

[0050]

[0051] The present invention also discloses a recombinant cell, which comprises: a compound 1 having the following structure:

[0052]

[0053] and a gene encoding an enzyme capable of catalyzing mogroside IIIE to produce compound 1. In some embodiments, the gene is a heterologous gene of the recombinant cell.

[0054] The present invention includes a recombinant cell comprising a first gene encoding a first enzyme capable of catalyzing the production of compound 1 having the following structure from mogroside IIIE:

[0055]

[0056] The first enzyme can be, for example, one or more of a UDP glycosyltransferase, a cyclomaltodextrin glucanotransferase (CGTases), a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase. In some embodiments, the first enzyme is a CGTase. For example, the CGTase can comprise an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a sequence identity of 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 an amino acid sequence of any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the first enzyme is a glucan sucrase. For example, the glucansucrase can comprise 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 of the sequences shown in SEQ ID NOs: 2, 103, 106-110, 156 and 896. In some embodiments, the glucansucrase 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 sequence identity to any of SEQ ID NOs: 104, 105, 157, 158 and 895. In some embodiments, the first enzyme is a transglucosidase. For example, the transglucosidase may comprise, for example, an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any sequence of SEQ ID NOs: 163-291 and 723. In some embodiments, the transglucosidase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 3, 95-102, 163-291 and 723. In some embodiments, the first enzyme is a β-glucosidase. In some embodiments, the β-glucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any sequence of SEQ ID NOs: 102, 292, 354-376 and 678-741.

[0057] In some embodiments, the 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% 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, and 444, or a range between any two of these numbers. In some embodiments, the UGT 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: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, and 445. In some embodiments, the UGT is encoded by the sequence shown in UGT 1495 (SEQ ID NO: 10), UGT 1817 (SEQ ID NO: 11), UGT 5914 (SEQ ID NO: 12), UGT 8468 (SEQ ID NO: 13), or UGT 10391 (SEQ ID NO: 14). In some embodiments, the cell further comprises a third gene encoding UGT 98 or UGT SK98. In some embodiments, UGT 98 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, 16, or 306. In some embodiments, UGT 98 is encoded by the nucleic acid sequence set forth in SEQ ID NO: 307.

[0058] 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 with any one of SEQ ID NOs: 21-30 and 309-314; or 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 with any one of SEQ ID NOs: 114 and 115.

[0059] In some embodiments, the cell comprises a fifth sequence encoding P450. In some embodiments, P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 20, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, and 891, or a range between any two of these numbers; or is encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 20, 49, 308, 315, 430, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, and 891, or a range between any two of these numbers; NOs:31-48,316,431,871,873,875,877,879,881,883,885,887,889 and 892 have at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or the sequence identity of the range between any two of these numbers. In some embodiments, P450 is encoded by a gene comprising or consisting of the sequence shown in any one of SEQ ID NOs:31-48,316 and 318.

[0060] In some embodiments, the cell comprises a sixth sequence encoding a polypeptide having cucurbitadienol synthase activity. In some embodiments, the polypeptide having cucurbitadienol synthase activity is a fusion protein. In some embodiments, the fusion protein comprises one or more fusion domains fused to the N-terminus, C-terminus, or both of the cucurbitadienol synthase. The length of the fusion domain can vary, for example, from about 3 to about 1000 amino acids long, or from about 5 to about 50 amino acids long. In some embodiments, the fusion domain is a major portion or the entire sequence of a functional protein. In some embodiments, the polypeptide having cucurbitadienol synthase activity comprises an amino acid sequence that is identical to SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 417, 420, 422, 424, 426, 446, 902, 9 04, 906, 906, 851, 854, 856, 1024, 859, 862, 865, 867, 915, 920, 924, 928, 932, 936, 94 991, 995, 999, 1003, 1007, and 1011. In some embodiments, the polypeptide having cucurbitadienol synthase activity is 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.

[0061] In some embodiments, the cell further comprises a seventh gene encoding 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 squalene epoxidase 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 sequence identity to SEQ ID NO: 335.

[0062] In some embodiments, the cell further comprises an eighth gene encoding a squalene synthase. 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. In some embodiments, the squalene synthase is encoded by a sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 337.

[0063] 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 with SEQ ID NO: 338. In some embodiments, the farnesyl-PP synthase 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 sequence identity with SEQ ID NO: 339.

[0064] In some embodiments, the cell is a cell of a mammal, a plant, a bacterium, a fungus or an insect. For example, the cell can be a yeast cell. In some embodiments, the yeast is selected from Candida, Sacccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium 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, 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 Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, or Metarhizium.

[0065] In some embodiments, the cell comprises a gene encoding at least one hydrolase capable of hydrolyzing mogroside V. In some embodiments, Compound 1 exhibits resistance to the hydrolase in the recombinant cell, wherein the hydrolase exhibits the ability to hydrolyze mogroside VI, mogroside V, mogroside IV to mogroside IIIE.

[0066] In some embodiments, the recombinant cell comprises an oxidized squalene cyclase, such as cycloartenol synthase or β-amyrinol synthase β-, or a nucleic acid sequence encoding an oxidized squalene cyclase, such as cycloartenol synthase or β-amyrinol synthase, wherein the oxidized squalene cyclase, cycloartenol synthase or β-amyrinol synthase β- is modified to produce cucurbitadienol or epoxycucurbitadienol.

[0067] In some embodiments, the oxidosqualene cyclase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any one of SEQ ID NOs: 341, 343, and 346-347. In some embodiments, the cell comprises a cytochrome P450 reductase or a gene encoding a cytochrome P450 reductase. In some embodiments, the cytochrome P450 reductase regenerates cytochrome P450 activity. In some embodiments, the cytochrome P450 reductase comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with SEQ ID NOs: 318.

[0068] In some embodiments, the cell comprises a sequence as set forth in any one of SEQ ID NOs: 897, 899, 909, 911, 913, 418, 421, 423, 425, 427, 871, 873, 901, 903, and 905. In some embodiments, the cell comprises an enzyme comprising a sequence as set forth in SEQ ID NOs: 315, 316, 420, 422, 424, 426, 430, 431, 446, 871, 845-949, and 951-1012, or encoded by a sequence as set forth in any one of SEQ ID NOs: 315, 316, 420, 422, 424, 426, 430, 431, 446, 871, 845-949, and 951-1012.

[0069] In some embodiments, the cell comprises a gene encoding a hydrolase capable of hydrolyzing the first mogroside to produce mogroside IIIE. In some embodiments, the hydrolase is a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1 or EXG2. In some embodiments, the hydrolase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a sequence identity of any two of the ranges between these numbers with any of SEQ ID NOs: 292, 366-368, 372, 376-398, 447-520, 524-845, 1013, 1014 and 1023.

[0070] In some embodiments, the first mogroside is mogroside IV, mogroside V, mogroside VI, or a combination thereof. In some embodiments, the first mogroside is mogroside V, simenoside I, mogroside IVE, mogroside VI, mogroside IVA, or a combination thereof.

[0071] The gene encoding the lytic enzyme can be heterologous or homologous to the recombinant host cell. The gene encoding the lytic enzyme can be expressed or overexpressed with normal levels in the recombinant host cell. In some embodiments, the cell is a yeast cell. In some embodiments, the cell is Saccharomyces cerevisiae or Yarrowia lipolytica.

[0072] The present invention also discloses a method for producing compound 1 having the following structure:

[0073]

[0074] In some embodiments, the method includes: contacting a first mogroside with one or more hydrolases to produce mogroside IIIE; contacting mogroside IIIE with an enzyme capable of catalyzing the production of Compound 1 from mogroside IIIE.

[0075] In some embodiments, the hydrolase is a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1, e.g., EXG1 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 with SEQ ID NO: 1012 or 1014. In some embodiments, the hydrolase is EXG2, e.g., EXG2 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 with SEQ ID NO: 1023.

[0076] In some embodiments, the first mogroside is mogroside IV, mogroside V, mogroside VI, or a combination thereof. In some embodiments, the first mogroside is mogroside V, simenoside I, mogroside IVE, mogroside VI, mogroside IVA, or a combination thereof.

[0077] In some embodiments, contacting the first mogroside with one or more hydrolases comprises contacting the first mogroside with a recombinant host cell comprising a first gene encoding a hydrolase and a second gene encoding an enzyme capable of catalyzing the production of Compound 1. In some embodiments, the first mogroside is contacted with one or more hydrolases in a recombinant host cell comprising a first gene encoding a hydrolase and a second gene encoding an enzyme capable of catalyzing the production of Compound 1. In some embodiments, the first mogroside is produced by a recombinant host cell. In some embodiments, the first gene is native to the recombinant host cell. In some embodiments, the first gene is expressed at a normal level. In some embodiments, the first gene is heterologous to the recombinant host cell. In some embodiments, the first gene is overexpressed. In some embodiments, the second gene is heterologous to the recombinant host cell. In some embodiments, the enzyme capable of catalyzing the production of Compound 1 is one or more of UDP glycosyltransferases, cyclomaltodextrin glucanotransferases (CGTases), glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0078] The present invention discloses a recombinant cell, which comprises: a first gene encoding a hydrolase capable of hydrolyzing a first mogroside to produce mogroside IIIE; and a second gene encoding an enzyme capable of catalyzing the production of compound 1 from mogroside IIIE, wherein compound 1 has the following structure:

[0079]

[0080] In some embodiments, the hydrolase is a β-glucan hydrolase. For example, the hydrolase can be EXG1 or EXG2. In some embodiments, the EXG2 protein comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers to SEQ ID NO: 1023. In some embodiments, the first mogroside is mogroside IV, mogroside V, mogroside VI, or a combination thereof. In some embodiments, the first mogroside is mogroside V, simenoside I, mogroside IVE, mogroside VI, mogroside IVA, or a combination thereof. The first gene can be heterologous or homologous to the recombinant host cell. The first gene can be overexpressed or normally expressed in the host cell. The cell can be, for example, a yeast cell. In some embodiments, the cell is Saccharomyces cerevisiae or Yarrowia lipolytica.

[0081] The present invention also discloses a fusion polypeptide having cucurbitadienol synthase activity, wherein the fusion polypeptide comprises a fusion domain fused to the cucurbitadienol synthase. The fusion domain can be fused to the N-terminus, the C-terminus, or both of the cucurbitadienol synthase. The fusion domain can be, for example, about 3 to about 1000 amino acids long, or about 5 to about 50 amino acids long. In some embodiments, the fusion domain comprises a major portion or the entire sequence of a functional protein. In some embodiments, the fusion domain is a major portion or the entire sequence of a functional protein. In some embodiments, the fusion domain comprises a portion or the entire sequence of a yeast protein. In some embodiments, the fusion domain is a portion or the entire sequence of a yeast protein. In some embodiments, the fusion polypeptide comprises an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 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, or a range between any two of these numbers. In some embodiments, the fusion polypeptide comprises or consists of the 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 that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 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, or a range between any two of these numbers.In some embodiments, the fusion domain of the fusion polypeptide comprises or consists of any of the following sequences: SEQ ID NO: 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.

[0082] 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 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 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327, 329-329, 420, 422, 424, 426, 446, 902, 904 and 906. 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% or a range between any two of these numbers 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 is encoded by a gene comprising or consisting of any one of the nucleic acid sequences shown in SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905.

[0083] The present invention discloses a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding any fusion polypeptide disclosed in the present invention and having cucurbitadienol synthase activity. The present invention discloses a recombinant cell comprising any fusion polypeptide disclosed in the present invention and having cucurbitadienol synthase activity and / or any recombinant nucleic acid molecule disclosed in the present invention and encoding a fusion polypeptide having cucurbitadienol synthase activity. The present invention also discloses a method, which uses any fusion polypeptide with cucurbitadienol synthase activity disclosed herein. The method may include contacting a substrate of cucurbitadienol synthase with a fusion polypeptide having cucurbitadienol synthase activity. In some embodiments, the contact results in the production of curcurbitadienol, 24,25-epoxycurcurbitadienol, or a combination thereof. In some embodiments, the substrate of cucurbitadienol synthase comprises one or more of 2,3-oxidized squalene, squalene dioxide, and diepoxysqualene. In some embodiments, contacting comprises contacting a substrate with a recombinant host cell comprising a nucleic acid sequence encoding a fusion polypeptide. The recombinant host cell can, for example, express a fusion polypeptide. In some embodiments, the substrate is provided to, present in, and / or produced by a recombinant host cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 HPLC data and mass spectrometry data of compound 1 produced after treatment of mogroside IIIE with CGTase are shown (inset).

[0085] Figure 2 showed that the use of Streptococcus mutans ( Streptococcus mutans ) HPLC data and mass spectrometry data of compound 1 produced after treatment of mogroside IIIE by Clarke ATCC 25175 glucanotransferase (inset).

[0086] Figure 3 HPLC and mass spectrometry data of the glycosylation reaction of mogrosides after treatment with Celluclast in the presence of xylan are shown (inset).

[0087] Figure 4 and Figure 5 HPLC and mass spectrometry data of the glycosylation reaction of mogroside after treatment with UDP-glycosyltransferase are shown (inset).

[0088] Figure 6 HPLC data and mass spectrometry data of mogroside I after treatment with UDP-glycosyltransferase UGT73C6 are shown (inset).

[0089] Figure 7-Figure 9HPLC data and mass spectrometry data of mogroside I, mogroside IIA and two different mogroside III products after treatment with UDP-glycosyltransferase (338) (SEQ ID NO: 405) are shown (inset).

[0090] Fig.10 and Fig.11 HPLC and mass spectrometry data for treatment of mogroside IIIE with UDP-glycosyltransferase to produce simenoside I and mogroside V products are shown (inset).

[0091] Figure 12-14 HPLC data and mass spectrometry data of treating mogroside, simanoside I or compound 1 with UDP-glycosyltransferase (339) (SEQ ID NO: 409) to produce mogroside I, isomogroside V and compound 1 derivatives, respectively, are shown (inset).

[0092] Figure 15-Figure 20 HPLC data and mass spectrometry data of treating mogroside IIA, mogroside IIE, mogroside IIIE, mogroside IVA or mogroside IVE with UDP-glycosyltransferase (330) (SEQ ID NO: 411) to produce mogroside IIIA, mogroside IVE, mogroside V, respectively, are shown (inset).

[0093] Fig.21 and Fig. 22 The mass spectra of the reaction products, mogroside IVE and mogroside V, are shown.

[0094] Fig.23 The production of cucurbitadienols using cucurbitadiene synthase (SgCbQ) (SEQ ID NO: 417) is shown.

[0095] Fig.24 The production of cucurbitadienol using the enzyme Cpep2 (SEQ ID NO: 420) is shown.

[0096] Fig.25 The production of cucurbitadienol using the enzyme Cpep4 (SEQ ID NO: 422) is shown.

[0097] Fig.26 The production of dihydroxycucurbitadienols catalyzed by epoxide hydrolase (SEQ ID NO: 428) is shown.

[0098] Fig.27A -B shows the resistance of compound 1 to hydrolysis by microbial enzymes.

[0099] Fig.28Shown is the UPLC chromatogram of the α-mogroside isomer mixture from Hilic_80_20_method.

[0100] Fig.29 The purity of the samples analyzed by UPLC on the Hilic_80_20_method is shown.

[0101] Fig.30 A flow chart of a non-limiting exemplary pathway for producing Compound 1 is shown.

[0102] Fig.31 Show Fig.30 The UV absorbance of the diepoxide of Step 1 is shown to enhance squalene oxide.

[0103] Fig.32 A method for producing cucurbitadienol in step 2 using enzymes from Cucumis melo and Cucurbita maxima is shown.

[0104] Fig.33 The production of cucurbitadienols in step 2 using enzymes from pea (Pisum sativum) is shown.

[0105] Fig.34 The production of cucurbitadienol in step 2 using an enzyme from Dictyostelium sp. is shown.

[0106] Fig.35 Shows Fig.30 Intermediate in step 3 of the indicated route.

[0107] Fig.36 Shows Fig.30 Mass spectral data of the intermediate product in step 4 of the route shown, i.e., the synthesis of mogrosanol.

[0108] Fig.37 Shows Fig.30 Step 7 of the route shown is an intermediate in the synthesis of compound 1.

[0109] Fig.38 is a schematic diagram showing the production of highly glycosylated mogrosides by glycosylases, which can then be hydrolyzed back to mogroside IIIE.

[0110] Fig.39 is a schematic diagram showing how hydrolysis is used to hydrolyze highly glycosylated mogroside to produce mogroside IIIE, which is then converted to compound 1.

[0111] Fig.40A-B shows that after 2 days of incubation, essentially all of the mogrosides were converted to mogroside IIIE in S. cerevisiae or Y. lipolytica.

[0112] Fig.41 It was shown that no hydrolysis products from Compound 1 were detected from S. cerevisiae or Y. lipolytica.

[0113] Fig.42 The production of compound 1 in S. cerevisiae modified to overexpress dextran sucrase (DexT) is shown.

[0114] Fig.43 The enzymatic reaction catalyzed by the 311 enzyme (UDP-glycosyltransferase) is shown. DETAILED DESCRIPTION

[0115] definition

[0116] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. All patents, applications, published applications and other public texts are incorporated by reference in their entirety. In the case where there are multiple definitions for the terms herein, the definitions in this section shall prevail unless otherwise stated.

[0117] "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.

[0118] As used herein, "sweetener," "sweet flavoring," "sweet flavoring entity," "sweet compound," or "sweet tasting compound" refers to a compound or a physiologically acceptable salt thereof that induces a perceptible sweet taste in a subject.

[0119] As used herein, the term "operably connected" is used to describe the connection between a regulatory element and a gene or its coding region. Typically, gene expression is placed under the control of one or more regulatory elements, such as, but not limited to, constitutive or inducible promoters, tissue-specific regulatory components, and enhancers. A gene or coding region is referred to as "operably connected" or "operably connected" or "operably associated with" a regulatory element, which means that the gene or coding region is controlled or affected by the regulatory element. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably connected to the coding sequence. A gene or coding region is "operably connected" or "can be operably connected" or "operably associated" with a regulatory element, meaning that the gene or coding region is controlled or affected by the regulatory element. For example, if a promoter affects the transcription or expression of a coding sequence, it is said to be operably connected to the coding sequence.

[0120] The terms "regulatory element" and "expression control element" are used interchangeably and refer to nucleic acid molecules that can affect the expression of operably linked coding sequences in a particular host organism. These terms are widely used to encompass and cover 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 start sites, termination sequences, polyadenylation sequences, trans (intrans), core elements required for the basic interaction of RNA polymerase and transcription factors, upstream elements, enhancers, response elements (see, e.g., Lewin, "Genes V" (Oxford, Oxford University Press), pp. 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 may include, but are not limited to, transcriptional and translational control sequences, such as promoters, enhancers, splicing signals, polyadenylation signals, terminators, protein degradation signals, internal ribosome entry elements (IRES), 2A sequences, which provide and / or regulate the expression of coding sequences and / or the production of encoded polypeptides in host cells, etc. In some embodiments herein, the recombinant cells described herein comprise a gene operably linked to a regulatory element.

[0121] As used herein, a 2A sequence or element refers to a small peptide that is introduced as a link between two proteins, thereby allowing autonomous intraribosomal self-processing of a polyprotein (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004); de Felipe et al. Traffic 5:616-626 (2004)). These short peptides allow co-expression of multiple proteins 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, those from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine parvovirus 1 (P2A) as described in U.S. Patent Publication No. 20070116690.

[0122] As used herein, the term "promoter" is a nucleotide sequence that allows RNA polymerase to bind and direct gene transcription. Typically, a promoter is located in the 5' non-coding region of a gene, near the transcription start site of the gene. The sequence elements that play a role in transcription initiation in a promoter are generally characterized by a consistent nucleotide sequence. 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. In response to certain changes in culture conditions (e.g., temperature changes), an inducible promoter initiates an increase in the level of DNA transcription under its control.

[0123] As used herein, the term "enhancer" refers to a regulatory element that increases transcription efficiency, regardless of the enhancer's distance or orientation relative to the transcription start site.

[0124] As used herein, the term "transgenic" 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 transgenic comprises a polynucleotide encoding a target protein. The polynucleotide encoding the protein is usually operably linked to other sequences that can be used to obtain the desired expression of the target gene, such as a transcriptional regulatory sequence. In some embodiments, the transgenic may additionally include a nucleic acid or other molecule that is used to mark the integration of the chromosome.

[0125] In this article, "percent (%) sequence identity" for polynucleotide or polypeptide sequences is used as the percentage of bases or amino acid residues in the candidate sequence that are identical to the bases or amino acid residues in the other sequence after the two sequences are aligned. If necessary, gaps can be introduced into the sequence alignment to achieve the maximum percentage of sequence identity. Conservative substitutions are not considered part of sequence identity. For the purpose of determining the percentage (%) sequence identity, alignment can be achieved in various ways within the technical scope of the art, for example, using publicly available computer methods and programs, such as BLAST, BLAST-2, ALIGN, FASTA (Genetics Computing Group (GCG) software package in Madison, Wisconsin, USA) or Megalign (DNASTAR). Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0126] For example, the percentage value of the amino acid sequence can be obtained using the WU-BLAST-2 computer program (described in, for example, 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, certain adjustable parameters can be set using the following values: overlap span = 1, overlap score = 0.125, word threshold (t) = 11, and scoring matrix = BLOSUM62. When using WU-BLAST-2, the amino acid sequence identity % value is determined in the following manner: the number of identical amino acid residues matched between the amino acid sequence of (a) the first target protein and the amino acid sequence of the second target protein is divided by the total number of amino acid residues of (b) the first target protein determined by WU-BLAST-2.

[0127] The amino acid sequence homology percentage can also be determined using the sequence comparison program NCBI-BLAST2 (e.g., described in Altschul et al., Nucleic Acids Research, 1997, 25: 3389-3402). The NCBI-BLAST2 sequence comparison program can be downloaded from http: / / www.ncbi.nlm.nih.gov or obtained from the National Institute of Health in Bethesda, Maryland. NCBI-BLAST2 uses multiple adjustable search parameters. Some of the default values ​​of these adjustable search parameters are, for example, unmask=yes, strand=all, expected occurrences=10, minimum low complexity length=15 / 5, multi-pass E-value=0.01, multi-normality=25, drop-off of final gap alignment=25 and scoring matrix=BLUSOM62.

[0128] In the case of using NCBI-BLAST2 for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or to a given amino acid sequence B (which may also be referred to as the percentage of a given amino acid sequence A that has or contains the same amino acid sequence as a given amino acid sequence B) is calculated as follows:

[0129] 100 times X / Y

[0130] Where X is the number of amino acid residues scored as identical matches in the alignment of A and B according to the sequence alignment program NCBI-BLAST2, and where Y is the total number of amino acid residues in B. When the length of amino acid sequence A is not equal to the length of amino acid sequence B, it is understood that the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A.

[0131] As used herein, "isolated" means that the indicated compound has been separated from its natural environment so that one or more other compounds or biological agents with which the compound is found in nature are no longer present.

[0132] As used herein, "purified" means that the compound is present in a higher amount relative to other compounds (e.g., in its natural environment) that are usually present with the compound. In some embodiments, the relative amount of the purified compound increases by more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400% or 1000%. In some embodiments, the purified compound is present in a weight percent level greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.5% relative to other compounds combined with the purified compound. In some embodiments, Compound 1 produced by the embodiments herein is present at a weight percent level of greater than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 99.5% relative to other compounds associated with the purified compound.

[0133] "Purification" as described herein can refer to a method of extracting Compound 1 from a cell lysate and / or supernatant, wherein the cells are secreting the product Compound 1. As described herein, "lysate" includes the cell contents after the cell wall and cell membrane are disrupted, and can include, for example, proteins, sugars, and mogrosides. Purification can include ammonium sulfate precipitation to remove proteins, salting out to remove proteins, hydrophobic separation (HPLC), and the use of affinity columns. Considering the products produced by the methods herein, the use of affinity media is considered when removing specific mogrosides with adsorption resins.

[0134] As used herein, "HPLC" refers to liquid chromatography, which can be used to separate compounds dissolved in a solution. Without limitation, an HPLC instrument may include a mobile phase reservoir, a pump, a syringe, a separation column, and a detector. The compounds can then be separated by injecting the sample mixture into a chromatographic column. Due to the different distribution behaviors between the mobile liquid phase and the stationary phase, different components in the mixture can pass through the chromatographic column at different rates. There are several columns that can be used. The column can be a normal phase column, a reverse phase column, a size exclusion type column, and an ion exchange column, without limitation.

[0135] Also contemplated is the use of solid phase extraction and fractionation, which can be used to desalt protein and sugar samples. Other methods may include analyzing samples using HPLC, liquid chromatography, and liquid-liquid extraction as described by Auréa Andrade-Eiroa et al. (TrAC Trends in Analytical Chemistry, Vol. 80, June 2016, pp. 641-654); the entire text of the document is incorporated herein by reference.

[0136] As described herein, "solid phase extraction" (SPE) for purification refers to a sample preparation process that separates a compound dissolved or suspended in a liquid mixture from other compounds in the mixture based on its physical and chemical properties. For example, analytical laboratories can use solid phase extraction to concentrate and purify samples for analysis. Solid phase extraction can also be used to separate analytes of interest from a variety of matrices, such as urine, blood, water, beverages, soil, and animal tissues. In embodiments herein, Compound 1 located in a cell lysate or cell medium can be purified by solid phase extraction.

[0137] SPE uses the affinity of solutes dissolved or suspended in a liquid (called the mobile phase) for solids to pass the sample through the solid (called the stationary phase), thereby separating the mixture into the desired components and the required components. SPE can also be used directly in gas-solid and liquid-solid phases, or indirectly applied to solid samples by using, for example, thermal desorption and subsequent chromatographic analysis. This may result in the desired target analyte or undesirable impurities in the sample being retained on the stationary phase. This may result in the desired analyte or unwanted impurities in the sample being retained on the stationary phase. The portion that passes through the stationary phase can be collected or discarded, depending on whether it contains the desired analyte or unwanted impurities. If the portion retained on the stationary phase includes the desired analyte, it can be removed from the stationary phase for collection in an additional step, in which the stationary phase is rinsed with an appropriate eluent.

[0138] The method of solid phase extraction is not limited. Without limitation, it can include: normal phase solid phase extraction, reverse phase solid phase extraction, ion exchange solid phase extraction, anion exchange solid phase extraction, cation exchange solid phase extraction and solid phase microextraction. Solid phase extraction is described in Sajid et al. and PotkaWasylka J et al. (Anal Chim Acta. 2017 May 1; 965: 36-53, Crit Rev Anal Chem. 2017 Apr 11: 1-11), and the entire document is incorporated herein by reference.

[0139] In some embodiments, Compound 1 produced by cells is purified by solid phase extraction. In some embodiments, the purity of Compound 1, e.g., purified by solid phase extraction, is 70%, 80%, 90% or 100%, or any purity level defined by any of the above values.

[0140] As used herein, "fermentation" refers broadly to the growth of host cells in a host culture medium in large quantities to produce a specific product. In the embodiments herein, the final product produced is compound 1. This can also include methods implemented with or without air, for example, in an anaerobic environment. The whole cell (recombinant host cell) can be in a fermentation broth or a reaction buffer.

[0141] Compound 1 and the intermediate mogroside compound used to produce compound 1 can be separated by collecting the intermediate mogroside compound and compound 1 from the recombinant cell lysate or supernatant. The lysate can be obtained after collecting cells and lysing the cells by shear force (high pressure cell lysis (French press cell) or ultrasound) or by detergent treatment. The lysate is then filtered and treated with ammonium sulfate to remove protein, and separated on C18 HPLC (5x 10cm Atlantis PrepT3OBD column, 5um, Waters), and injected using an A / B gradient of 10→30% B (A=water B=acetonitrile) within 30 minutes, washed with 95% B, and then rebalanced at 1% (total running time=42 minutes). It can be collected at a speed of 30ml / fraction in a tared test tube (12 fractions / plate, 3 plates each time). The lysate can also be centrifuged to remove solids and particulate matter.

[0142] The plate was then dried in a Geneva HT12 / HT24. It is expected that the target compound will elute in fraction 21 along with other isomers. The combined fractions can be further fractionated in 47 runs on a fluorophenyl HPLC column (3x 10cm, Xselect fluorophenyl OBD column, 5um, Waters) using an A / B gradient (A=water, B=acetonitrile) of 15→30% B in 35 minutes, washed with 95% B, then rebalanced at 15% (total run time=45 minutes). Each time 30 ml / fraction was collected in 12 tared test tubes (12 fractions / plate, 1 plate each time). The fractions containing the desired peak and the desired purity can be combined according to UPLC analysis and dried under reduced pressure to obtain a white powdery solid. The pure compound can be resuspended / dissolved in 10 mL of water and then lyophilized to obtain a purity of at least 95%.

[0143] As used herein, "glycosidic bond" refers to a covalent bond that connects two furanose and / or pyranose groups together. Typically, a glycosidic bond is a bond between the anomeric carbon of one furanose or pyranose moiety and the oxygen of another furanose or pyranose moiety. Glycosidic bonds are named using the numbering of the carbon atoms connected and the α / β orientation. Alpha-glycosidic bonds and β-glycosidic bonds are distinguished based on the relative stereochemistry of the anomeric position farthest from C1 in the ring and the stereocenter. For example, sucrose is a disaccharide consisting of one molecule of glucose and one molecule of fructose connected by an α1-2 glycosidic bond, as shown below.

[0144]

[0145] Examples of β1-4 glycosidic bonds can be found in cellulose:

[0146]

[0147] It must be noted that, 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 "an aromatic compound" includes a mixture of aromatic compounds.

[0148] Typically, ranges are described herein as from "about" one particular value, and / or to "about" another particular value. When such ranges are described, another embodiment includes from a particular value and / or to another particular value. Similarly, when values ​​are described as approximate values ​​by using the prefix "about", it is understood that the particular value forms another embodiment. It is also understood that the endpoints of each range are significant to, and independent of, the other endpoint.

[0149] As described herein, "codon optimization" refers to the design process of changing codons to codons known to increase the efficiency of maximum protein expression. In some alternatives, codon optimization for expression in cells is described, wherein codon optimization can be performed using algorithms known to those skilled in the art to produce synthetic genetic transcripts optimized for high mRNA and protein yields in humans. For example, codons can be optimized to express proteins in bacterial cells, mammalian cells, yeast cells, insect cells, or plant cells. Programs containing codon optimization algorithms for humans are readily available. Such programs can include, for example, OptimumGene TM or Algorithms. In addition, codon-optimized sequences can be commercially available, for example, from Integrated DNA Technologies. In some embodiments herein, the recombinant cells used to produce Compound 1 comprise genes encoding enzymes for synthesis, wherein the genes are codon-optimized for expression. In some embodiments, the genes are codon-optimized for expression in bacteria, yeast, fungi, or insect cells.

[0150] As used herein, the terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are interchangeable and refer to any nucleic acid, whether composed of phosphodiester bonds or modified bonds, such as phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetimidate, carbamate, thioether, bridged phosphoramide, bridged methylenephosphonate, bridged phosphoramide, bridged phosphoramide, bridged methylenephosphonate, phosphorothioate, methylphosphonate, bridged phosphorodithioate, bridged phosphorothioate, or sultone bonds, and combinations of these bonds. The terms "nucleic acid" and "polynucleotide" also specifically include nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil).

[0151] Non-limiting examples of polynucleotides include fragments produced by deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, polymerase chain reaction (PCR), and any connection, cutting, endonuclease action, and exonuclease action. Nucleic acid molecules can be composed of monomers of natural nucleotides (such as DNA and RNA) or analogs of natural nucleotides (such as enantiomeric forms of natural nucleotides) or a combination of the two. Modified nucleotides can be changed in sugar moieties and / or pyrimidine or purine base moieties. Sugar modifications include, for example, replacement of one or more hydroxyls with halogens, alkyls, amines, and azidos, or sugars can be functionalized as ethers or esters. In addition, the entire sugar moiety can be replaced with spatially and electronically similar structures, such as azasugars and carbocyclic sugar analogs. Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes. Nucleic acid monomers can be connected by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, selenophosphates, diselenosenophosphates, aniline phosphorothioates, phosphoroanilidate, phosphoramidate, etc. The term "nucleic acid molecule" also includes so-called "peptide nucleic acids", which include naturally occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be 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 includes any one of SEQ ID NO: 1-1023.

[0152] As used herein, "coding for" or "encoding" refers to the property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA or mRNA, used as a template for the synthesis of other macromolecules (e.g., a defined amino acid sequence). Therefore, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene encodes a protein. In some embodiments herein, a recombinant cell is provided, wherein the recombinant cell comprises a gene encoding an enzyme, such as a glucan sucrase, a UDP glycosyltransferase, a CGTase, a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase and a glucanase and / or a UGT. In some embodiments, the transglucosidase comprises an amino acid sequence shown in any one of SEQ ID NOs: 163-290 and 723. In some embodiments, the CGTase is encoded by any one of SEQ ID NOs: 1, 3, 78-101, 147 and 154 or has a sequence thereof. In some embodiments, genes encoding enzymes such as glucansucrase, UDP glycosyltransferase, CGTase, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase and / or UGT are codon optimized for expression in a host cell. A "nucleic acid sequence encoding a polypeptide" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence.

[0153] Can also be optimized to reduce the appearance of secondary structure in polynucleotide.In some alternatives of this method, can also optimize the sequence in carrier to reduce total GC / AT ratio.Strict codon optimization may cause unwanted secondary structure, or cause the GC content of secondary structure to be too high.Like this, secondary structure affects transcription efficiency.After codon usage optimization, can use program such as GeneOptimizer to avoid secondary structure and GC content optimization.These additional programs can be used for further optimization and troubleshooting after initial codon optimization, to limit the secondary structure that may occur after first round of optimization.Optimized alternative programs are easy to obtain.In some alternatives of this method, carrier comprises sequence optimized for avoiding secondary structure and / or sequence optimized for reducing total GC / AT ratio and / or sequence optimized for expressing in bacteria or yeast cell.

[0154] A "vector," "expression vector," or "construct" is a nucleic acid used to introduce a heterologous nucleic acid into a cell, which has regulatory elements to provide for 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 used to express proteins in bacterial systems such as E. coli. In some alternatives, the vector is used to express proteins in bacterial systems such as E. coli. In some alternatives, the vector is used to express proteins in yeast systems. In some embodiments, the vector used for expression is a viral vector. In some embodiments, the vector is a recombinant vector comprising a promoter sequence for upregulating gene expression. A "regulatory element" may refer to a nucleic acid having a nucleotide sequence that can affect the initiation of transcription or translation and the rate, stability, and mobility of the transcription or translation product.

[0155] As used herein, a "recombinant host" or "recombinant host cell" is a host whose genome has been enhanced by at least one incorporated DNA sequence. The incorporated DNA sequence may 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 are desired to be introduced into a non-binding host. In some embodiments, recombinant host cells are used to prevent expression problems such as codon bias. There are commercial hosts for expressing proteins, such as BL21-CodonPlus. TM Cells, tRNA-complemented host strains for heterologous gene expression, Rosetta for enhanced protein expression TM (DE3) competent strains and commercial yeast expression systems in the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, and Yarrowia.

[0156] The recombinant host may be a commercially available cell, such as Rosetta cells for expressing enzymes that may have rare codons.

[0157] In some embodiments, the recombinant cell comprises a recombinant gene for producing a cytochrome P450 polypeptide comprising an amino acid sequence of any one of CYP533CYP937, 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 in a gene comprising a sequence shown in any one of SEQ ID Nos: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885887, and 891.

[0158] 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 shown in SEQ ID NO: 112. In some embodiments, the recombinant host cell further comprises a gene encoding CPR4497, wherein the amino acid sequence of amino acid CPR4497 is shown in SEQ ID NO: 113.

[0159] In some embodiments where the recombinant host cell is a yeast cell, the recombinant cell has a deletion of the EXG1 gene and / or the EXG2 gene to prevent a reduction in glucanase activity, which can result in deglycosylation of mogrosides.

[0160] The type of host cell can vary. For example, the host cell can be selected from the group consisting of Agaricus, Aspergillus, Bacillus, Candida, corynebacterium, Escherichia, Fusarium / Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomycetes, Sphaceloma, Xanthophyllmoyces, Yarrowia, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomycetes, Sphaceloma, Xanthophyllmoyces, Yarrowia, Lentinus tigrinus), Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Physcomitrella patens, Rhodoturula glutinis, Rhodoturula mucilaginosa, Phaffia rhodozyma, Xanthophyllomyces dendrorhous, Fusarium fujikuroi / Gibberella fujikuroi, Candida utilisutilis), Yarrowialipolytica, Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Acanthopanax Eleutherococcus, Aralia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchan tia and Morus, Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histoplasma Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, Metarhizium, Aspergillus, Yarrowia, Lipomyces, Aspergillus nidulans, Yarrowia lipolytica, Rhodosporintoruloides, Candida, Sacccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes, Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Saccharomyces cerevisiae), Escherichia coli, Rhodobacter sphaeroides, and Rhodobacter capsulatus. Using Saccharomyces cerevisiae as an example, methods for increasing product yield are described. Methods for making recombinant microorganisms are well known.

[0161] WO2014086842 describes a method for preparing recombinant host cells from Aspergillus spp., which is incorporated herein by reference in its entirety. The nucleotide sequence of the genome can be obtained through publicly available gene databases and allows for rational design and modification of pathways to increase and improve product yields.

[0162] As used herein, "medium" may be a nutrient-rich broth for the growth and maintenance of cells during their production phase. Yeast cultures used to maintain and propagate various strains may require specific formulations of complex media for cloning and protein expression, and are understood by those skilled in the art. For example, commercially available medium may be obtained from ThermoFisher. The medium may be YPD broth or may have yeast nitrogen base. Yeast may be grown in YPD or synthetic medium at 30°C.

[0163] Lysogeny broth (LB) is commonly used for bacterial cells. Bacterial cells used for enzyme and mogroside growth can have antibiotic resistance to prevent growth and contamination of other cells in the culture medium. These cells can have antibiotic gene cassettes that are resistant to antibiotics such as chloramphenicol, penicillin, kanamycin and ampicillin.

[0164] As described herein, "fusion protein" is a protein produced by connecting two or more nucleic acid sequences together, which originally encode a part or all of the amino acid sequence of the isolated protein. For example, the fusion protein can include a functional protein (e.g., an enzyme (including but not limited to cucurbitadienol synthase)) and one or more fusion domains. As described herein, the fusion domain can be the full length or part / fragment of a protein (e.g., a functional protein including but not limited to an enzyme, a transcription factor, a toxin, and a translation factor). The position of one or more fusion domains in the fusion protein can vary. For example, one or more fusion domains can be in the N- and / or C- terminal region (e.g., N- and / or C- terminal) of the fusion protein. One or more fusion domains can also be in the central region of the fusion protein. The fusion domain does not need to be located at the end of the fusion protein. The fusion domain 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 multi- (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 fused thereto. As a non-limiting example, the fusion protein may include a full-length protein A and a fusion domain fused to the N-terminal region and / or the C-terminal region of the full-length protein A. In some examples, the fusion protein includes a partial protein A sequence and a fusion domain (e.g., the N-terminal and C-terminal of a partial sequence of protein A) fused to the N-terminal region and / or the C-terminal region. The fusion domain can be, for example, a portion or all of the sequence of protein A, or a portion or all of the sequence of a protein different from protein A. In some embodiments, one or more enzymes suitable for use in the methods, systems, and compositions disclosed herein may be fusion proteins. In some embodiments, the fusion protein is encoded by a nucleic acid sequence having at least 70%, 80%, 90%, 95%, or 99% sequence identity with one of the nucleic acid sequences listed in Table 1. In some embodiments, the fusion protein includes an amino acid sequence having at least 70%, 80%, 90%, 95%, or 99% sequence identity with one of the amino acid sequences listed in Table 1. In some embodiments, the fusion protein comprises an amino acid protein sequence having at least 80%, 90%, 95% or 99% sequence identity to one of the amino acid sequences listed in Table 1, and a fusion domain at the N-, C- or both terminal regions of the fusion protein. In some embodiments, the fusion protein comprises one of the amino acid protein sequences listed in Table 1, and a fusion domain at the N-, C- or both terminal regions of the fusion protein.

[0165] 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 or any two of these numbers. In some embodiments, the length of the fusion domain is about 3, 4, 5, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50 or any two of these numbers. In some embodiments, the fusion domain is a major portion or 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 having cucurbitadienol synthase activity.

[0166] It is also considered to optimize cell growth and protein expression technology in culture medium. For growth in culture medium, cells such as yeast may be sensitive to low pH values ​​(Narendranath et al., Appl Environ Microbiol. 2005 May; 71 (5): 2239-2243; the entire document is incorporated herein by reference). During growth, yeast must maintain a constant intracellular pH. During growth and metabolism, there are many enzymes at work in yeast cells. Each enzyme works best at its optimal pH, and since yeast itself has an acidophilic nature, the enzyme is acidic. When the extracellular pH deviates from the optimal level, yeast cells need to invest energy to pump in or out hydrogen ions to maintain the optimal intracellular pH. In this way, a culture medium containing a buffer for controlling pH will be optimal. Alternatively, if the monitored pH value is very high, the cells can also be transferred to a new culture medium.

[0167] Optimization of bacterial and yeast cell growth can also be achieved by adding nutrients and supplements to the culture medium. Alternatively, the culture can be grown in fermenters designed for temperature, pH control and controlled aeration rates. Dissolved oxygen and nitrogen can be flowed into the culture medium when necessary.

[0168] The term "operably linked" as used herein refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in the expression of the latter.

[0169] "Mogroside" and "mogroside compound" are used interchangeably herein and refer to a family of triterpene glycosides. Non-limiting illustrative examples of mogrosides include, for example, mogroside V, simoneoside 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 are identified from the fruit of Siraitia grosvenorii (Swingle) and are responsible for the sweetness of the fruit. In embodiments herein, the mogroside intermediates can be used in vivo, in vitro or in vitro to produce compound 1 having the following structure:

[0170]

[0171] In some embodiments, the recombinant cells for producing compound 1 further produce mogroside and contain genes encoding enzymes for producing mogroside. Recombinant cells capable of producing mogroside are further described in WO2014086842, and the entire document is incorporated herein by reference. In some embodiments, the recombinant cells are grown in a culture medium to allow the expression of enzymes and the production of compound 1 and mogroside intermediates. In some embodiments, compound 1 is obtained by lysing cells with shear force (i.e., high-pressure cell lysis or ultrasonic treatment) or by a detergent lysis method. In some embodiments, precursor molecules such as mogroside alcohol are supplemented to cells in the growth medium to promote the production of compound 1.

[0172] As used herein, "promoter" refers to a nucleotide sequence that directs transcription of a structural gene. In some alternatives, the promoter is located in the 5' non-coding region of the gene, closest to the transcription start site of the structural gene. Sequence elements that play a role in transcription initiation within the promoter are generally characterized by a common nucleotide sequence. Without limitation, these promoter elements can include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993); incorporated herein by reference in its entirety), cyclic AMP response elements (CREs), serum response elements (SREs; Treismanet et al., Seminars in Cancer Biol. 1:47 (1990); incorporated herein by reference in its entirety), glucocorticoid response elements (GREs), and sites for binding of other transcription factors, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992); incorporated herein by reference in its entirety), AP2 (Ye et al., J. Biol. Chem. 268:19947 (1993); incorporated herein by reference in its entirety). et al., J. Biol. Chem. 269:257-28 (1994); the entire text of the document is incorporated herein by reference), SP1, cAMP response element binding protein (CREB; Loeken et al., Gene Expr. 3:253 (1993); the entire text of the document is incorporated herein by reference), and octamer factors (see generally Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; the entire text of the document is incorporated herein by reference)), and Lemaigre and Rousseau, Biochem. J. 303:1 (1994); the entire text of the document is incorporated herein by reference). As used herein, promoters can be constitutively active, repressible or inducible. If a promoter is an inducible promoter, the transcription rate will increase in response to an inducing agent. In contrast, if the promoter is a constitutive promoter, the transcription rate is not regulated by the inducing agent.

[0173] As described herein, "ribosome skipping (skip) sequence" refers to a sequence that forces the ribosome to "jump" the ribosome skipping sequence during translation and translates the region after the ribosome skipping sequence without forming a peptide bond. For example, some viruses have a ribosome skipping sequence that allows sequential translation of several proteins on a single nucleic acid without the need to connect proteins by peptide bonds. As described herein, this is a "joint" ("linker") sequence. In some alternatives of nucleic acids provided herein, nucleic acids include a ribosome skipping sequence between the sequences of the genes of the enzymes described herein so that the proteins are co-expressed and not connected by peptide bonds. In some alternatives, the ribosome skipping sequence is a T2A sequence.

[0174] Compound 1

[0175] As disclosed herein, Compound 1 is a compound having the following structure (1) or a salt thereof:

[0176]

[0177] Compound 1 is a high intensity sweetener that can be used in a variety of products that require sweetness. Compound 1 has a low calorie advantage compared to other sweeteners such as sucrose or fructose.

[0178] In some embodiments, Compound 1 is in isolated and purified form. In some embodiments, Compound 1 is present in a composition in which Compound 1 is substantially purified.

[0179] In some embodiments, compound 1 or its salt 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 lyophilized form. In some embodiments, compound 1 is isolated and in a buffer.

[0180] Those skilled in the art will recognize that some of the structures described herein may be resonance forms or tautomers of compounds, even though they may be reasonably represented kinetically by other chemical structures; those skilled in the art recognize that such structures may represent only a small fraction of such compound samples. Such compounds are considered to be within the scope of the described structures, even though such resonance forms or tautomers are not shown herein.

[0181] Isotopes may be present in compound 1. Each chemical element represented by a compound structure may include any isotope of the element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in a compound. Any position where a hydrogen atom may be present in a compound, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, unless the context otherwise clearly indicates, the compounds mentioned herein encompass all potential isotopic forms. In some embodiments, the compounds described herein are enriched in one or more isotopes relative to the natural prevalence of such isotopes. In some embodiments, the compounds described herein are enriched in deuterium. In some embodiments, greater than 0.0312% of hydrogen atoms in the compounds described herein are deuterium. In some embodiments, greater than 0.05%, 0.08%, or 0.1% of hydrogen atoms in the compounds described herein are deuterium.

[0182] In some embodiments, Compound 1 is capable of forming acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto.

[0183] In some embodiments, Compound 1 is substantially isolated. In some embodiments, Compound 1 is substantially purified. In some embodiments, the compound is in lyophilized form. In some embodiments, the compound is crystalline. In some embodiments, the compound is amorphous.

[0184] Product Composition

[0185] In some embodiments, the product composition does not contain undesirable compounds, or contains less than a certain amount of undesirable compounds. In some embodiments, the composition does not contain one or more isomers of mogroside I, mogroside II and mogroside III. In some embodiments, the composition contains less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm or 100ppm of all isomers of mogroside I, mogroside II and mogroside III by weight. In some embodiments, the composition does not contain one or more of mogroside IIIE, 11-oxo-mogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE and 11-oxo-mogroside alcohol. In some embodiments, the composition contains less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm or 100ppm by weight of one or more of mogroside IIIE, 11-oxo-mogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE and 11-oxo-mogroside alcohol. In some embodiments, the composition contains less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm or 100ppm by weight of mogroside IIIE. In some embodiments, the composition contains less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm or 100ppm by weight of 11-oxo-mogroside IIIE. In some embodiments, the composition contains 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-oxo-mogrool.

[0186] In some embodiments, the product composition is a solid form, which can be crystalline or amorphous. In some embodiments, the composition is a particle form. Any suitable technology can be used to produce the solid form of the composition, and the suitable technology includes but is not limited to recrystallization, filtration, solvent evaporation, grinding, milling, spray drying, spray agglomeration, fluidized bed agglomeration, wet granulation or dry granulation and combination thereof. In some embodiments, a flowable particle composition is provided so as to be further used in the food manufacturing process. In some such embodiments, a particle diameter of 50 μm to 300 μm, 80 μm to 200 μm or 80 μm to 150 μm is obtained.

[0187] Some embodiments provide product compositions in the form of solutions. For example, in some embodiments, the solution produced by one of the production methods described herein is used without further purification. In some embodiments, the concentration of the desired compound in the solution is greater than 300ppm, 500ppm, 800ppm, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% or 20% by weight. In some embodiments, the concentration of all isomers of mogroside I, mogroside II and mogroside III is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm or 100ppm. In some embodiments, the concentration of one or more of mogroside IIIE, 11-oxo-mogroside IIIE, mogroside IIIA2, mogroside IE, mogroside IIE, and 11-oxo-mogroalcohol is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm, 100ppm, 50ppm, 30ppm, 20ppm, 10ppm, 5ppm, 1ppm, or 0.1ppm. In some embodiments, the concentration of mogroside IIIE is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm, 100ppm, 50ppm, 30ppm, 20ppm, 10ppm, 5ppm, 1ppm, or 0.1ppm. In some embodiments, the concentration of 11-oxo-mogroside IIIE is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm, 100ppm, 50ppm, 30ppm, 20ppm, 10ppm, 5ppm, 1ppm or 0.1ppm. In some embodiments, the concentration of 11-oxo-mogroside IIIE is less than 5%, 3%, 2%, 1%, 0.5%, 0.3%, 0.1%, 800ppm, 500ppm, 200ppm, 100ppm, 50ppm, 30ppm, 20ppm, 10ppm, 5ppm, 1ppm or 0.1ppm.

[0188] Method for producing compound 1 and intermediate mogroside compound

[0189] In some embodiments, compound 1 is produced by contacting various starting and / or intermediate compounds with one or more enzymes. The contact can be in vivo (e.g., in a recombinant cell) or in vitro. The starting compounds and intermediate compounds used to produce compound 1 may include, but are not limited to, mogroside V, mogroside IIE, mogroside IIIE, mogroside I, mogroside VI isomers, mogroside IIA, mogroside IVE, or mogroside IVA.

[0190] In some embodiments, compound 1 as described herein is produced in a recombinant host cell in vivo, or by adjusting these methods. Methods for adjusting the methods include, inter alia, temperature, solvents, reagents, etc., known to those skilled in the art. The methods shown and described herein are merely illustrative and are not intended or should be interpreted as limiting the scope of the claims in any way. Those skilled in the art will be able to identify adjustments to the disclosed methods and design alternative routes based on the disclosure herein; all such adjustments and alternative routes are within the scope of the claims.

[0191] In some embodiments, compound 1 disclosed herein is obtained by purification and / or separation from recombinant bacterial cells, yeast cells, plant cells or insect cells. In some embodiments, the recombinant cells are from Siraitia grosvenorii. In some such embodiments, the extract obtained from Siraitia grosvenorii is fractionated using suitable purification techniques. In some embodiments, the extract is fractionated using HPLC, and appropriate fractions are collected to obtain the desired compound in an isolated and purified form.

[0192] 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 contact may be in vivo (e.g., in a recombinant cell) or in vitro. The starting and intermediate compounds for preparing compound 1 include, but are not limited to, mogroside V, mogroside IIE, mogroside IIIE, mogroside I, mogroside VI isomers, mogroside IIA, mogroside IVE, or mogroside IVA. One or more of these compounds may be prepared in vivo. Enzymes suitable for producing the compounds described herein may include, but are not limited to, pectinase, β-galactosidase (e.g., Aromase), cellulase (e.g., Celluclast), cyclophosphamide transferase (e.g., Toruzyme), invertase, glucose transferase (e.g., UGT76G1), dextranase, lactase, arabanse, xylanase, hemicellulose, amylase, or a combination thereof. In some embodiments, the enzyme is Toruzyme, which comprises the amino acid sequence shown in any one of SEQ ID NOs: 89-94.

[0193] Some embodiments provide methods for preparing compound 1,

[0194]

[0195] The method comprises separating an extract of Siraitia grosvenorii on an HPLC column and collecting the eluted fractions containing Compound 1.

[0196] Some embodiments provide a method for preparing Compound 1,

[0197]

[0198] Wherein the method comprises treating mogroside IIIE with glucose transferase UGT76G1. In some embodiments, UGT76G1 is encoded by the sequence shown in SEQ ID NO: 440. In some embodiments, UGT76G1 comprises the amino acid sequence shown in SEQ ID NO: 439.

[0199] Various mogroside compounds can be used as intermediates for producing compound 1. A non-limiting example of such a mogroside compound is compound 3 having the following structure:

[0200]

[0201] In some embodiments, the method for preparing compound 3 comprises contacting mogroside IIIE with a cell (e.g., a recombinant host cell) expressing one or more cyclomaltodextrin glucanotransferases. In some embodiments, the cyclomaltodextrin glucanotransferase comprises the amino acid sequence shown in SEQ ID NO:95.

[0202] Various mogroside compounds can be used as intermediates for producing compound 1. A non-limiting example of such a mogroside compound is compound 12 having the following structure:

[0203]

[0204] In some embodiments, methods for producing Compound 12 include contacting mogroside VI with a cell (eg, a recombinant host cell) expressing one or more converting enzymes.

[0205] Various mogroside compounds can be used as intermediate compounds for producing compound 1. A non-limiting example of such a mogroside compound is compound 5 having the following structure:

[0206]

[0207] In some embodiments, the method of producing compound 5 comprises contacting mogroside IIIE with a cell (eg, a recombinant host cell) expressing one or more cyclomaltodextrin glucanotransferases. In some embodiments, the method is performed in the presence of starch.

[0208] Various mogroside compounds can be used as intermediate compounds for producing compound 1. A non-limiting example of such a mogroside compound is compound 4 having the following structure:

[0209]

[0210] In some embodiments, the method of producing Compound 4 comprises contacting mogroside IIIE with a cell (eg, a recombinant host cell) expressing one or more cyclomaltodextrin glucanotransferases. In some embodiments, the method is performed in the presence of starch.

[0211] Hydrolysis of high-glycosyl mogroside

[0212] In some embodiments, one or more highly glycosylated mogrosides are hydrolyzed into mogrosides by contacting with one or more hydrolases. In some embodiments, the highly glycosylated mogrosides are selected from mogroside IV, mogroside V, mogroside VI, and combinations thereof. In some embodiments, the highly glycosylated mogrosides are selected from mogroside V, simone I, mogroside IV, and combinations thereof. E, isomogroside V, 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A 、11-Oxomogroside VI、11-Oxomogroside IV E and combinations thereof.

[0213] It was surprisingly found that Compound 1 exhibited resistance to hydrolysis by certain hydrolases, even though such enzymes exhibited the ability to hydrolyze highly glycosylated mogrosides to mogroside IIIE. The α-linked glycosides present in Compound 1 provide a unique advantage over other mogrosides (e.g., β-linked glycosides) due to their resistance to hydrolysis. In some embodiments, during microbial production of Compound 1, the microbial host hydrolyzes the unwanted β-linked mogrosides back to mogroside IIIE. This will improve the purity of Compound 1 for the following reasons: 1) reducing the levels of unwanted mogroside VI, mogroside V, and mogroside IV, and 2) hydrolysis will increase the amount of mogroside IIIE that can be used as a precursor for producing the compound.

[0214] Fig.38 It is shown that highly glycosylated mogrosides produced by glycosylases can then be hydrolyzed back to mogroside IIIE. The result is a mixture of mogrosides with a lower yield than the desired highly glycosylated mogrosides. This hydrolase can be removed, but a mixture of mogrosides will still be obtained and the life span of the producing organism may be shortened. However, since compound 1 is resistant to hydrolysis, hydrolysis can be used to convert highly glycosylated mogrosides to mogroside IIIE, which can then be converted to compound 1 (e.g. Fig.39 shown).

[0215] In some embodiments, the hydrolase is a β-glucan hydrolase. In some embodiments, the hydrolase is EXG1. The EXG1 protein may comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity with SEQ ID NO: 1013 or 1014. In some embodiments, the EXG1 protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 1013 or 1014. In some embodiments, the hydrolase is EXG2. The EXG2 protein may comprise an amino acid sequence having at least 70%, 80%, 90%, 95%, 98%, 99% or more sequence identity with SEQ ID NO: 1023. In some embodiments, the EXG2 protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 1023. The hydrolase may be, for example, any of the hydrolases disclosed herein.

[0216] Production of Compound 1 from Mogroside IIIE

[0217] Compound 1 can be produced from mogroside IIIE by contacting with one or more enzymes capable of converting mogroside IIIE to compound 1. In some embodiments, the enzyme capable of catalyzing the production of compound 1 is one or more of UDP glycosyltransferases, cyclomaltodextrin glucanotransferases (CGTases), glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0218] 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 with any one of SEQ ID NOs: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises an 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 glucanase. In some embodiments, the glucanase 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 with any one of SEQ ID NOs: 2, 103, 106-110, 156, and 896. In some embodiments, the glucanase comprises or consists of an amino acid sequence of any of the following sequences: SEQ ID NO: 2, 103, 106-110, 156 and 896. In some embodiments, the glucanase is encoded by a nucleic acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers to any one of SEQ ID NO: 104, 105, 157, 158 and 895. In some embodiments, the glucanase is encoded by a nucleic acid sequence comprising or consisting of any one of the following sequences: SEQ ID NO: 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 of sequence identity between any two of these numbers to any one of SEQ ID NOs: 163-290 and 723. In some embodiments, the transglucosidase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 163-292 and 723. Sequence identity can be determined using the CLUSTALW software of BLAST search (ncbi.nih.gov). The use of these programs can determine the identity between protein homologs.

[0219] In some embodiments, the enzyme capable of catalyzing the production of Compound 1 is a uridine diphosphate-glucosyltransferase (UGT). The UGT may comprise, for example, an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of the sequences with any of SEQ ID NOs: 4-9, 15-19, 125, 126, 128, 129, 293-307, 407, 409, 411, 413, 439, 441, and 444. In some embodiments, the UGT comprises or consists of an amino acid sequence of any of the following sequences: SEQ ID NOs: 4-9, 10-14, 125, 126, 128, 129, 293-304, 306, 407, 409, 411, 413, 439, 441, and 444. In some embodiments, the UGT is encoded by a nucleic acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers, sequence identity to 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, and 445. In some embodiments, the UGT is encoded by a nucleic acid sequence comprising 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 any one of SEQ ID NOs: 116-124, 127, 130, 408, 410, 412, 414, 440, 442, 443, and 445. In some embodiments, the enzyme may be UGT98 or UGT SK98. For example, as described herein, a recombinant host cell capable of producing Compound 1 may comprise a third gene encoding UGT98 and / or UGT SK98. In some embodiments, UGT98 or UGT SK98 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: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 numbers, 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), UGTSK98 (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 that has 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 that comprises or consists of any one 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 of the UGT enzymes disclosed herein.In addition, a recombinant host cell capable of producing Compound 1 may comprise an enzyme comprising or consisting of a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or higher sequence identity to any UGT enzyme disclosed herein. In some embodiments, the recombinant host cell comprises an enzyme comprising or consisting of any of the UGT enzymes disclosed herein.

[0220] In some embodiments, the method of producing Compound 1 comprises treating mogroside IIIE with a glucose transferase UGT76G1, such as the UGT76G1 of SEQ ID NO: 439 and the UGT76G1 encoded by the nucleic acid sequence of SEQ ID NO: 440.

[0221] Enzymes for producing mogroside compounds and compound 1

[0222] As described herein, the enzymes of UDP glycosyltransferase, cyclomaltodextrin glucanotransferase (CGTases), glycosyltransferase, glucan sucrase, cellulose, beta-glucosidase, amylase, transglucosidase, pectinase and glucanase can comprise the amino acid sequence described in this paper sequence table, and can also be encoded by the nucleic acid sequence described in the sequence table. In addition, these enzymes can also include functional homologues with at least 70% sequence identity to the amino acid sequence described in the sequence table. The parameters for determining the sequence identity percentage can be performed using the ClustalW software of Blast retrieval (ncbi.nih.gov). The use of these programs can determine the consistency between protein homologues.

[0223] 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 enzyme 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 or is encoded by a nucleic acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 163-290 and 723, or a range between any two of these numbers.

[0224] The methods herein also include incorporating genes into 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 for producing compound 1. Gruchattka et al. and Rodriguez et al. (PLoS One. 2015 Dec 23; 10 (12; Microb Cell Fact. 2016 Mar 3; 15: 48; the entire text of the document is incorporated herein by reference) introduced methods for increasing squalene content and are incorporated by reference.

[0225] Further contemplated is the expression of enzymes that produce squalene oxide and diepoxysqualene. Enzymes that produce squalene oxide and diepoxysqualene can be used to facilitate squalene synthesis using squalene synthase and / or squalene epoxidase. For example, Suet et al. described a gene encoding SgSQS, a 417 amino acid protein of squalene synthase from Siraitia grosvenorii (Biotechnol Lett. 2017 Mar 28; the entire text of the document is incorporated herein by reference). Recombinant cells are genetically engineered to express HMG CoA reductase, which can also be used 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.; the entire text of the document is incorporated herein by reference). In some embodiments, 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme comprising a sequence corresponding to SEQ ID NO:898 or 900 has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers in sequence identity. In some embodiments, 2,3-oxidosqualene or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers in sequence identity with SEQ ID NO:897 or 899.

[0226] The expression of enzymes for preparing cucurbitadienols / epoxycucurbitadienols is also contemplated. It is also contemplated that cucurbitadienol synthases from C PEPO, S grosvenorii, C melo, C moschata and C maxim are used to enter recombinant cells via expression vectors. It is also contemplated that oxidative squalene cyclases for triterpene biosynthesis expressed in recombinant cells are also contemplated, which will result in the cyclization of acyclic substrates into 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 document is incorporated herein by reference).

[0227] The expression of enzymes that exhibit epoxide hydrolase activity to prepare hydroxy-cucurbitadienols is also contemplated. In some embodiments herein, the recombinant cells used to produce Compound 1 also include genes encoding enzymes that exhibit epoxide hydrolase activity, which are provided to prepare hydroxycyclases. Such enzymes are described by Itkin et al., which are incorporated herein by reference in their entirety. Sequence Table 1 provided herein provides enzymes described by Itkin et al. Ikin et al. also describe enzymes for preparing key mogrosides, UGS families, glycosyltransferases and hydrolases, which can be genetically modified for reverse reactions such as glycosylation.

[0228] Also contemplated is the expression of enzymes for hydroxylating mogroside compounds in recombinant cells to produce mogroside alcohol. These enzymes may include proteins of the CAZY family, UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, transglucosidases, pectinases and glucanases, yeast and fungal hydrolases. Such enzymes may be used, for example, to hydrolyze mogroside V to mogroside IIIE, wherein mogroside IIIE may be further processed, for example, to produce compound 1 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 of sequence identity between any two sequences in these numbers to any one of SEQ ID NOs: 678-722.

[0229] In some embodiments, a precursor of mogroside alcohol such as squalene or oxidosqualine, mogroside alcohol or mogroside is prepared. The precursor can be used as a precursor in the production of compound 1. Squalene can be produced from famesyl pyrophosphate using squalene synthase, and oxidosqualine can be produced from squalene using squalene epoxidase. Squalene synthase can be a squalene synthase from the Cucurbitaceae plant Gynostemmapentaphyllum (protein accession number: C4P9M2). The squalene synthase may also include squalene synthases from Arabidopsis thaliana (protein accession number C4P9M3), Brassica napus, Citrus macrophylla, Euphorbiatirucalli (protein accession number B9WZW7), Glycine max, Glycyrrhiza glabra (protein accession number Q42760, Q42760), Glycyrrhiza glabra (protein accession number Q42760), uralensis) (Protein Accession Nos. D6QX40, D6QX41, D6QX42, D6QX43, D6QX44, D6QX45, D6QX47, D6QX39, D6QX55, D6QX38, D6QX53, D6QX37, D6QX35, B5AID5, B5AID4, B5AID3, C7EDD0, C6KE07, C6KE08, C7EDC9), Lotus japonicas (Protein Accession No. Q84LE3), Medicago truncatula (Protein Accession No. Q8GSL6), Pisum sativum, Ricinus communis (Protein Accession No. B9RHC3). Various squalene synthases are described in WO 2016 / 050890, the contents of which are incorporated herein by reference.

[0230] Recombinant host cells

[0231] Any enzyme disclosed herein can be produced in vitro, in vitro or in vivo. For example, the nucleic acid sequence encoding the enzyme (including but not limited to UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases, cytochrome P450, epoxide hydrolases, cucurbitadienol synthases, squalene epoxidases, squalene synthases, hydrolases and oxidized squalene cyclases) 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 by, for example, standard transformation techniques (e.g., thermal transformation) or by transfection. The expression system can produce enzymes for the production of mogrosides and compound 1 so as to produce compound 1 in cells in vivo. Useful expression systems include but are not limited to bacteria, yeast and insect cell systems. For example, an insect cell system can be infected with a recombinant viral expression system for expressing an enzyme of interest. In some embodiments, the gene is codon optimized to be expressed in a specific cell. In some embodiments, the gene is operably linked to a promoter to drive transcription and translation of the enzyme protein. As described herein, codon optimization can be achieved and the optimized sequence can then be designed into a vector for transforming a recombinant host cell.

[0232] The expression vector may further comprise a transcriptional or translational regulatory sequence, a coding sequence of a transcriptional or translational factor or various promoters (eg, GPD1 promoter) and / or enhancers to promote the transcription of the target gene in yeast cells.

[0233] In some embodiments, a recombinant cell as described herein is genetically modified to produce Compound 1 in vivo. In addition, a mogroside precursor or a mogroside precursor may be provided to the cell during or after cell growth to increase the rate of production of a specific intermediate in a pathway for producing Compound 1 in vivo. The cell may be suspended or fixed. The cell may be in a fermentation broth or in a reaction buffer. In some embodiments, an osmotic agent is used to transfer the mogroside precursor or a mogroside precursor into the cell. In some embodiments, the mogroside precursor or a mogroside precursor may be provided in a purified form or as part of a composition or extract.

[0234] The recombinant host cell can be, for example, a cell of a plant, a bivalve, a fish, a fungus, a bacterium, or a mammal. For example, the plant can be selected from the group consisting of Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Aralia (Aralia), Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia and Morus. The fungus may be selected from the group consisting of Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, 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, Sacccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, Yarrowia, and Microboryomycetes. In some embodiments, the bacteria is selected from Frankia, Actinobacteria, Streptomyces, and Enterococcus. In some embodiments, the bacterium is Enterococcus faecalis.

[0235] In some embodiments, the recombinant gene is codon optimized for expression in cells of bacteria, mammals, plants, fungi or insects. In some embodiments, one or more genes comprise functional mutations that increase the activity of the encoded enzyme. In some embodiments, cultivating the recombinant host cell comprises monitoring the pH, dissolved oxygen level, nitrogen level or a combination thereof of the culture conditions.

[0236] Production of Mongosan Alcohol from Squalene

[0237] Some embodiments of the methods of producing Compound 1 include producing an intermediate for producing Compound 1. Compound 1 having the following structure is produced in vivo in a recombinant host.

[0238]

[0239] In some embodiments, the compound is secreted into the medium in which the recombinant cell is grown, or both are grown in the medium in which they are grown. In some embodiments, the recombinant cell further produces intermediates such as mogroside compounds in vivo. Under conditions where the genes disclosed herein are expressed, the recombinant cell can be grown in a culture medium. Some embodiments of methods of growing cells are described herein.

[0240] In some embodiments, the intermediate is or comprises at least one of squalene, squalene oxide, cucurbitadienol, mogroside and mogroside. In some embodiments, mogroside is mogroside IIE. As described herein, mogroside is a family of mogrosides that can be naturally isolated, for example, from plants or fruits. As described herein, mogroside can be produced by a recombinant host cell.

[0241] In some alternatives of the methods described herein, the recombinant host cell comprises a polynucleotide or sequence comprising one or more of:

[0242] a gene encoding squalene epoxidase;

[0243] a gene encoding cucurbitadienol synthase;

[0244] Genes encoding cytochrome P450;

[0245] A gene encoding a cytochrome P450 reductase; and

[0246] Gene encoding epoxide hydrolase.

[0247] 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 comprises a sequence from Arabidopsis thaliana (protein accession number: Q9SM02, 065403, 065402, 065404, 081000 or Q9T064), Brassica napus (Brassica napus) (protein accession number: 10 065727, 065726), Euphorbia tirucalli (protein accession number: A7VJN1), Medicago truncatula (Medicago truncatula) (protein accession number: Q8GSM8, Q8GSM9), Pisum sativum and Ricinus communis (Ricinus communis) communis) (Protein Accession Nos.: B9R6VO, B9S7W5, B9S6Y2, B9TOY3, B9S7TO and B9SX91) and functional homologs of any one of the above having 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 thereto. In some embodiments, the squalene epoxidase comprises or consists of the following amino acid sequence: the amino acid sequence has 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: 50-56, 60, 61, 334 or 335.

[0248] 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 of sequence identity between any two of these numbers with SEQ ID NO: 111. In some embodiments, the P450 polypeptide is encoded in a gene comprising a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with the sequence of any one of claims 31-48. In some embodiments, these sequences can be separated by ribosomal skipping sequences to prepare isolated proteins.

[0249] In some embodiments, the recombinant host cell comprises a gene encoding a polypeptide having cucurbitadienol synthase activity. In some embodiments, the polypeptide having cucurbitadienol synthase activity comprises an amino acid sequence as shown 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 having cucurbitadienol synthase activity comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of the sequences 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 polypeptide having cucurbitadienol synthase activity comprises a C-terminal portion comprising the sequence shown in SEQ ID NO: 73. In some embodiments, the gene encoding the polypeptide having cucurbitadienol synthase activity is codon optimized. In some embodiments, the codon optimized gene comprises the nucleic acid sequence shown in SEQ ID NO: 74.

[0250] In some embodiments, the polypeptide with cucurbitadienol synthase activity is a fusion polypeptide comprising a fusion domain fused to cucurbitadienol synthase. The fusion domain can be fused to, for example, the N-terminal or C-terminal of cucurbitadienol synthase. The fusion domain can be located at, for example, the N-terminal region or the C-terminal region of the fusion polypeptide. The length of the fusion domain can vary. For example, the fusion domain can be or be about 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or the range length between any two amino acids in these numbers. In some embodiments, the fusion domain is 3 to 1000 amino acids long. In some embodiments, the fusion domain is 5 to 50 amino acids long. In some embodiments, the fusion domain comprises the main part or all of the sequence of the functional protein. In some embodiments, the fusion domain comprises a portion or all of the sequence of a yeast protein. For example, a fusion polypeptide having cucurbitadienol synthase activity may 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 the amino acid sequence set forth in any of the following sequences: SEQ ID Nos: 851, 854, 856, 1024, 859, 862, 865, 867, 915, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 959, 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 that has or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:866, 870, 917, 921, 925, 929, 933, 937, 941, 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 the amino acid sequence shown in any of the following sequences: SEQ ID NO: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 that is or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% 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 the 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 that is or has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical 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 shown in any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 897, 901, 903 and 905. The present invention includes a recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a fusion polypeptide having cucurbitadienol synthase activity. A recombinant cell is also disclosed, comprising a fusion polypeptide having cucurbitadienol synthase activity or a recombinant nucleic acid molecule encoding the fusion polypeptide.

[0251] The fusion polypeptide with cucurbitadienol synthase activity disclosed herein can be used to catalyze the enzymatic reaction of cucurbitadienol synthase. For example, the substrate of 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 cucurbitatedienol, 24,25-epoxycucurbitadienol and any combination thereof. Non-limiting examples of the substrate of cucurbitadienol synthase include 2,3-oxidized squalene, squalene dioxide, diepoxysqualene and any combination thereof. In some embodiments, the substrate can be contacted with a recombinant host cell, and the recombinant host cell comprises a nucleic acid sequence encoding one or more fusion polypeptides with cucurbitadienol synthase activity. The substrate can be provided to the recombinant host cell, present in the recombinant host cell, produced by the recombinant host cell, or any combination thereof.

[0252] In some embodiments, the cytochrome P450 is CYP5491. In some embodiments, the cytochrome P540 comprises an amino acid sequence having or 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: 44 and / or SEQ ID NO: 74. In some embodiments, the P450 reductase polypeptide comprises an amino acid sequence having or 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 or having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 100% sequence identity to any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891, or a range between any two of these numbers.

[0253] In some embodiments, the epoxide hydrolase comprises a sequence having or 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 or 40. In some embodiments, the epoxide hydrolase comprises or consists of the sequence shown in SEQ ID NO: 38 or 40.

[0254] Several methods for producing squalene for producing mogrosanol

[0255] Squalene is a natural 30-carbon organic molecule that can be produced in plants and animals and is a biochemical precursor of the steroid family. In addition, squalene can be used as a precursor for the synthesis of mogrosanol in host recombinant cells. Oxidation of one of the terminal double bonds of squalene (by squalene monooxygenase) produces 2,3-squalene oxide, which undergoes enzyme-catalyzed cyclization to provide lanosterol, which is then refined into cholesterol and other steroids. As described by Gruchattkaet 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 document is incorporated herein by reference), the synthesis of squalene can be prepared from the precursors of the glycolytic cycle. In turn, squalene can be upregulated by overexpression of ATP-citrate lyase, thereby increasing the yield of squalene. Some embodiments disclosed herein include enzymes for producing squalene and / or promoting squalene production in recombinant host cells (e.g., recombinant yeast cells). It is found in yeast that ATP citrate lyase can also mediate the synthesis of acetyl-CoA that can be used for the preparation of squalene and mevalonate, which can be found in yeast Saccharomyces cerevisiae (S.cerevisiae) (Rodrigues et al. "ATP citrate lyasemediated cytosolic acetyl-CoA biosynthesis increases mevalonate production inSaccharomyces cerevisiae" Microb Cell Fact. 2016; 15: 48.; the document is incorporated herein by reference). An example of a gene encoding an enzyme for mediating acetyl-CoA synthesis is listed in SEQ ID NO: 130. In some embodiments herein, the recombinant cell comprises a sequence for mediating acetyl-CoA synthesis.

[0256] Some embodiments disclosed herein provide methods for producing Compound 1 having the structure:

[0257]

[0258] 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 producing Compound 1 comprises at least one enzyme capable of converting dioxidized squalene to 24,25-epoxycucurbitadienol, converting oxidized squalene to cucurbitadienol, catalyzing the hydroxylation of 24,25-epoxycucurbitadienol to 11-hydroxy-24,25-epoxycucurbitadienol, catalyzing the hydroxylation of cucurbitadienol to 11-hydroxy-cucurbitadienol, epoxidizing cucurbitadienol to 24,25-epoxycucurbitadienol, catalyzing the epoxidation of 11-hydroxy-cucurbitadienol to produce 11-hydroxy-24,25-epoxycucurbitadienol, converting 11-hydroxy-cucurbitadienol to 11-hydroxy-24,25-epoxycucurbitadienol, catalyzing the conversion of 11-hydroxy-24,25-epoxycucurbitadienol to produce mogroside and / or catalyzing the glycosylation of a mogroside precursor to produce a mogroside compound. In some embodiments, the enzyme for glycosylation is encoded by the sequence shown in any one of SEQ ID NOs: 121, 122, 123, and 124.

[0259] In some embodiments, the enzyme used to catalyze the hydroxylation of 24,25-epoxycucurbitadienol to form 11-hydroxy-24,25-epoxycucurbitadienol is CYP5491. In some embodiments, CYP5491 comprises the sequence shown in SEQ ID NO: 49. In some embodiments, squalene epoxidase comprises a sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with the sequence of SEQ ID NO: 54.

[0260] In some embodiments, the enzyme capable of epoxidizing 11-hydroxy-cucurbitadienol comprises the amino acid sequence shown in SEQ ID NO:74.

[0261] In some alternatives, the recombinant cell comprises a gene expressing an enzyme capable of converting squalene dioxide to 24,25-epoxycucurbitadienol, converting squalene oxide to cucurbitadienol, hydroxylating 24,25-epoxycucurbitadienol to 11-hydroxy-24,25-epoxycucurbitadienol, hydroxylating cucurbitadienol to produce 11-hydroxy-cucurbitadienol, epoxidation of cucurbitadienol to produce 24,25-epoxycucurbitadienol, and / or epoxidation of 11-hydroxycucurbitadienol to produce 11-hydroxy-24,25-epoxycucurbitadienol. In these embodiments herein, the intermediates and mogrosides are produced in vivo.

[0262] In some embodiments, the method of producing Compound 1 further comprises producing one or more mogroside compounds and intermediates, such as squalene oxide, squalene dioxide, cucurbitadienol, 24,25-epoxycucurbitadienol, 11-hydroxy-cucurbitadienol, 11-hydroxy-24,25-epoxycucurbitadienol, mogroside alcohol, and mogroside compounds.

[0263] Production method of mogroside compound

[0264] Methods for producing mogroside compounds are described herein, for example, one of the mogroside compounds is described in WO2014086842 (incorporated herein by reference). The mogroside compounds can be used as intermediates by cells to further produce Compound 1 disclosed herein.

[0265] Recombinant hosts, such as microorganisms, plant cells, or plants, can be used to express polypeptides useful for the biosynthesis of mogroside (a triterpene core) and various mogroside glycosides (mogrosides).

[0266] In some embodiments, the production method may include one or more of the following steps in any order:

[0267] (1) Enhance the level of squalene oxide

[0268] (2) Enhance the level of squalene dioxide

[0269] (3) Squalene oxide->Cucurbitadienol

[0270] (4) Squalene dioxide -> 24,25-epoxycucurbitadienol

[0271] (5) Cucurbitacadienol->11-Hydroxycucurbitacadienol

[0272] (6) 24,25-Epoxycucurbitadienol->11-Hydroxy-24,25-Epoxycucurbitadienol

[0273] (7) 11-Hydroxycucurbitadienol->Mogrosanol

[0274] (8) 11-Hydroxy-24,25-epoxycucurbitadienol->Mogrosanol

[0275] (9) Mogroside alcohol -> various mogroside compounds.

[0276] In the embodiments herein, squalene oxide, squalene dioxide, cucurbitacinol, 24,25-epoxycucurbitacinol or mogroside alcohol can also be produced by recombinant cells. The method can include growing a recombinant microorganism in a culture medium in which one or more enzyme catalytic steps of the method of the present invention are expressed, such as synthases, hydrolases, CYP450s and / or UGTs. The recombinant microorganism can be cultured in batches or continuously. Typically, the recombinant microorganism is grown in a fermenter at a specified temperature for a desired period of time to increase the yield of compound 1.

[0277] In some embodiments, whole cells can be used to produce mogroside compounds, and the whole cells are provided with raw materials containing precursor molecules to increase the yield of compound 1. The raw materials can be provided during cell growth or after cell growth. The whole cells can be suspended or fixed. The whole cells can be in a fermentation broth or in a reaction buffer.

[0278] In some embodiments, the recombinant host cell may comprise a heterologous nucleic acid encoding an enzyme or a mixture of enzymes capable of catalyzing the oxidation of -squalene to cucurbitadienol, the conversion of cucurbitadienol to 11-hydroxycucurbitadienol, the conversion of 11-hydroxy-cucurbitadienol to mogroside and / or mogroside to mogroside. In some embodiments, the cell may further comprise a heterologous nucleic acid encoding an enzyme or a mixture of enzymes capable of catalyzing the oxidation of squalene to 24,25-epoxycucurbitadienol, 24,25-epoxycucurbitadienol to hydroxy-24,25-epoxycucurbitadienol, 11-hydroxy-24,25-epoxycucurbitadienol to mogroside, and / or mogroside to mogroside.

[0279] The host cell may comprise a recombinant gene encoding a cucurbitadienol synthase and / or a recombinant gene encoding a cytochrome P450 polypeptide.

[0280] In some embodiments, the cells comprise 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 of SEQ ID NOs: 70-73, 75-77, 319, 321, 323, 325, 327-333, 420, 422, 424, 426, 446, 902, 904, and 906 (cucurbitadienol synthase).

[0281] In some embodiments, the conversion of oxidized-squalene 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 thereof, the homologue having 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 with the above synthase. In some embodiments, the cucurbitadienol synthase polypeptide comprises a C-terminal portion comprising the sequence shown 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 shown in SEQ ID NO: 74.

[0282] In some embodiments, the conversion of cucurbitadienol to 11-hydroxycucurbitadienol is catalyzed by CYP5491 of SEQ ID NO: 49 or a functional homolog thereof, CYP5491 having 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 with its functional homolog.

[0283] In some embodiments, the conversion of 11-hydroxy-cucurbitadienol to mogrohol 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 thereof, wherein the above hydrolases have 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 with their homologs. 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 shown in SEQ ID NO: 114 or 115.

[0284] In some embodiments, the epoxide hydrolase comprises an amino acid sequence as set forth in SEQ ID NOs: 21-28 (Itkin et al., incorporated herein by reference).

[0285] In some embodiments, in the host recombinant cell, one or more UGTs catalyze the conversion of mogroside to mogroside, and the UGT is selected from: UGT1576 of SEQ ID NO: 15, UGT98 of SEQ ID NO: 9, UGTSK98 of SEQ ID NO: 68, and functional homologs thereof having 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.

[0286] In some embodiments, the host recombinant cell comprises a recombinant gene encoding a cytochrome P450 polypeptide encoded by any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887 and 891.

[0287] In some embodiments, the host recombinant cell comprises a recombinant gene encoding a squalene epoxidase polypeptide comprising the sequence in SEQ ID NO:50.

[0288] 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 shown 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 shown in SEQ ID NO: 74.

[0289] Production of Mogroside Compounds from Mogroside Alcohol

[0290] In some embodiments, the method of producing compound 1 comprises 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, wherein the recombinant cell comprises a gene encoding a first enzyme capable of catalyzing the production of compound 1 from mogroside IIIE. In some embodiments, the cell further comprises a gene encoding an enzyme capable of catalyzing the production of mogroside IE1 from mogroside alcohol. In some embodiments, the enzyme comprises a sequence as shown in any one of SEQ ID NOs: 4-8.

[0291] In some embodiments, the cell further comprises an enzyme that converts mogroside IIE into mogroside IV, mogroside V, 11-oxomogroside V, and simanoside I. In some embodiments, the enzyme for converting mogroside IIE into mogroside IV, mogroside V, 11-oxomogroside V, and simanoside I is encoded by a gene comprising the nucleic acid sequence shown in SEQ ID NOs: 9-14 and 116-120. In some embodiments, the method of producing Compound 1 comprises treating mogroside IIIE with glucose transferase UGT76G1.

[0292] In some embodiments, the method comprises separating a lysate from recombinant cells on an HPLC column and collecting the eluted fractions comprising Compound 1.

[0293] In some embodiments, the method includes contacting mogroside IIIE with a first enzyme capable of catalyzing the production of compound 1 by 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 the recombinant host cell, and the recombinant host cell comprises 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 culture medium under conditions of expressing the first enzyme. In some embodiments, the first enzyme is one or more of a UDP glycosyltransferase, a cyclomaltodextrin glucanotransferase (CGTases), a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase. In some embodiments, the first enzyme 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 with the sequence of 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 shown in any one of SEQ ID NOs: 1, 3, 78-101, 148 and 154. In some embodiments, the first enzyme is a glucan sucrase. 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 with the sequence of any one of SEQ ID NOs: 2, 103, 106-110, 156 and 896. In some embodiments, DexT comprises the amino acid sequence of any one of SEQ ID NOs: 2, 103, 106-110, 156 and 896. In some embodiments, DexT comprises the nucleic acid sequence shown in SEQ ID NOs: 104 or 105. In some embodiments, the dextran sucrase comprises the amino acid sequence of SEQ ID NOs: 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 of sequence identity between any two of these numbers to any sequence 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 as shown 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 of SEQ ID NOs: 1, 3, 78-101 and 154.

[0294] In some embodiments, the method includes contacting mogroside IIA with a recombinant host cell to produce mogroside IIIE, wherein the recombinant host cell further 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 produced by a recombinant host cell. In some embodiments, the second enzyme is one or more of a UDP glycosyltransferase, a CGTase, a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase. In some embodiments, the second enzyme is a uridine diphosphate-glucosyltransferase (UGT). In some embodiments, the transglucosidase comprises an amino acid sequence shown in any one of SEQ ID NOs: 163-290 and 723. In some embodiments, the gene encodes a CGTase, and the CGTase comprises an amino acid sequence shown 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, and 444. In some embodiments, the UGT is encoded by the 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).

[0295] In some embodiments, the method includes contacting mogroside IIIE with a recombinant host cell, 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 mogroside. In some embodiments, mogroside is produced by a recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferases), CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. In some embodiments, the second enzyme is 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.

[0296] In some embodiments, the method includes contacting a mogroside compound 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 a mogroside compound, wherein the mogroside compound is 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 simenoside. In some embodiments, the mogroside compound is produced by a recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferase, CGTase, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase, and glucanase. In some embodiments, the transglucosidase comprises an amino acid sequence as shown in any one of SEQ ID NOs: 163-290 and 723. In some embodiments, the gene encodes a CGTase comprising an amino acid sequence as shown 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, wherein the recombinant host cell comprises 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 of sequence identity between any two of these numbers to SEQ ID NOs: 9, 407 or 16. In some embodiments, contacting results in the production of mogroside IIA in the cell. In some embodiments, the one or more enzymes comprise the amino acids 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.

[0297] In some embodiments, the method further comprises contacting 11-hydroxy-24,25-epoxycucurbitadienol with a recombinant host cell, wherein the recombinant host cell further comprises a third gene encoding an epoxide hydrolase. In some embodiments, 11-hydroxy-24,25-epoxycucurbitadienol is produced by a recombinant host cell. In some embodiments, the method further comprises contacting 11-hydroxy-cucurbitadienol with a recombinant host cell, wherein the recombinant host cell comprises a fourth gene encoding a cytochrome P450 or an epoxide hydrolase. In some embodiments, the P450 polypeptide is encoded in a gene comprising any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887 and 891, and in some embodiments, the 11-hydroxy-cucurbitadienol is produced by a recombinant host cell.

[0298] In some embodiments, the method further comprises contacting 3,24,25-trihydroxycucurbitacinol with a recombinant host cell, wherein the recombinant host cell further comprises a fifth gene encoding a cytochrome P450. In some embodiments, the P450 polypeptide is encoded by a gene comprising a sequence shown in any one of SEQ ID NOs: 31-48, 316, and 318. In some embodiments, 3,24,25-trihydroxycucurbitacinol is produced by a recombinant host cell. In some embodiments, contacting results in the production of mogrosanol in a recombinant host cell. In some embodiments, cytochrome P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range of sequence identity between any two of these numbers with a sequence of SEQ ID NOs: 20, 308, or 315. In some embodiments, the P450 polypeptide is encoded in 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 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.

[0299] In some embodiments, the method further comprises contacting cucurbitacinol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a cytochrome P450. In some embodiments, contacting results in the production of 11-cucurbitacinol. In some embodiments, 11-hydroxycucurbitacinol is expressed in cells comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, CYP87D18 or SgCPR comprises a sequence listed in SEQ ID NO: 315, 872 or 874. In some embodiments, CYP87D18 or SgCPR is encoded by SEQ ID NO: 316, 871 or 873. In some embodiments, cucurbitacinol is 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% sequence identity to any one of SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887, and 891, or a range between any two of these numbers. In some embodiments, the cytochrome P450 comprises an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range between any two of these numbers to SEQ ID NOs: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887 and 891. In some embodiments, the P450 polypeptide is encoded in a gene that comprises 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.

[0300] In some embodiments, the method further comprises contacting 2,3-oxidized squalene 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 shown in 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 any one of the sequences shown in SEQ ID NO: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903 and 905. In some embodiments, contacting produces cucurbitadienol. In some embodiments, 2,3-oxidized squalene is produced by the recombinant host cell. In some embodiments, 2,3-squalene oxide or diepoxysqualene is produced by an enzyme comprising the sequence shown in SEQ ID NO: 898 or 900. In some embodiments, 2,3-squalene oxide or diepoxysqualene is produced by an enzyme encoded by a nucleic acid sequence shown in SEQ ID NO: 897 or 899.

[0301] In some embodiments, the cucurbitadienol synthase is encoded by a gene comprising a sequence as shown in SEQ ID NO: 74. In some embodiments, the cucurbitadienol synthase is encoded by a gene comprising any of the following nucleic acid sequences: SEQ ID NO: 74, 320, 322, 324, 326, 328, 418, 421, 423, 425, 427, 897, 899, 901, 903 and 905. In some embodiments, the cell produces 11-hydroxycucurbitadienol. In some embodiments, 11-hydroxycucurbitadienol is expressed in a cell comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, CYP87D18 or SgCPR comprises a sequence in SEQ ID NO: 315, 872 or 874. In some embodiments, 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 a sequence as 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 a nucleic acid sequence as set forth in SEQ ID NO: 74. In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence having at least 70% sequence identity with 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 (e.g., including cucurbitadienol 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 comprises an amino acid comprising a polypeptide from Lotus japonicas (BAE53431), Populus trichocarpa (XP_002310905), Actaea racemosa (ADCWO84219), Betula platyphylla (BAB83085), Glycyrrhiza glabra (BAA76902), Vitis vinifera (XP_002264289), Centella asiatica (AAS01524), Panax ginseng (BAA33460), and Betula platyphylla (BAB83086), as described in WO 2016 / 050890, the entire contents of which are incorporated herein by reference.

[0302] In some embodiments, the method includes contacting squalene with a recombinant host cell, wherein the recombinant host cell comprises an eighth gene encoding a squalene epoxidase. In some embodiments, contacting produces 2,3-oxidized squalene. In some embodiments, squalene is produced by a 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 of sequence identity between any two of these numbers with SEQ ID NO:50-56, 60, 61, 334 or 335.

[0303] In some embodiments, the method includes contacting farnesyl pyrophosphate with a recombinant host cell, wherein the recombinant host cell comprises a ninth gene encoding a squalene synthase. In some embodiments, contacting results in the production of squalene. In some embodiments, farnesyl pyrophosphate is produced by a 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 sequence identity between any two of these numbers with SEQ ID NO: 69 or 336.

[0304] In some embodiments, the method further comprises contacting the geranyl-PP with a recombinant host cell, wherein the recombinant host cell comprises a tenth gene encoding a farnesyl-PP synthase. In some embodiments, the contacting results in the production of farnesyl-PP. In some embodiments, the 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 of sequence identity with SEQ ID NO: 338. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes are operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is CMV, EF1a, SV40, PGK1, human β-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, repressible or constitutive promoter. In some embodiments, the production of one or more of pyruvate, acetyl-CoA, citric acid and TCA cycle intermediates in the recombinant host cell is upregulated. In some embodiments, cytoplasmic localization has been upregulated in the recombinant host cell. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes contain 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. and similar terms do not infer a specific order and / or the requirement for the presence of an earlier number. For example, the recombinant host cell described herein may comprise the first gene and the third gene, but not the second gene. As another example, the recombinant host cell may comprise the first gene, the fifth gene, and the tenth gene, but not the second gene, the third gene, the fourth gene, the sixth gene, the seventh gene, the eighth gene, and the ninth gene. The recombinant host cell may be, for example, a plant, a bivalve, a fish, a fungus, a bacterium, or a mammalian cell.For example, the plant is selected from the group consisting of Siraitia, Momordica, Gynostemma, Cucurbita, Cucumis, Arabidopsis, Artemisia, Stevia, Panax, Withania, Euphorbia, Medicago, Chlorophytum, Eleutherococcus, Aralia, 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, 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, Sacccharaomyces, 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, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes have been codon optimized to express in cells of bacteria, mammals, plants, fungi or insects. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes include functional mutations that increase the activity of the encoded enzyme. In some embodiments, cultivating recombinant host cells includes monitoring the pH, dissolved oxygen level, nitrogen level or a combination thereof of the culture conditions. In some embodiments, the method includes separating compound 1. In some embodiments, separating compound 1 includes cracking recombinant host cells. In some embodiments, separating compound 1 includes separating compound 1 from culture medium. In some embodiments, the method includes purifying compound 1. In some embodiments, purifying compound 1 includes HPLC, solid phase extraction or a combination thereof. In some embodiments, purification includes harvesting recombinant cells, preserving supernatant and lysing cells. In some embodiments, lysis includes subjecting cells to shear force or detergent washing, thereby obtaining lysates. In some embodiments, the shear force is from ultrasound, high pressure cell lysis (french pressurized cells) or beads. In some embodiments, the lysate is filtered and purified. In some embodiments, the lysate is filtered and purified by solid phase extraction.

[0305] In some embodiments, a compound 1 having the following structure (1) is provided:

[0306]

[0307] wherein the compound is produced by any of the alternative methods provided herein.

[0308] In some embodiments, the cell lysate comprises Compound 1 having the following structure (1):

[0309]

[0310] In some embodiments, the recombinant cell comprises Compound 1 having the following structure (1):

[0311]

[0312] 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 of the recombinant cell.

[0313] In some embodiments, the recombinant cell comprises a first gene encoding a first enzyme capable of catalyzing the production of Compound 1 having the following structure (1) from mogroside IIIE:

[0314]

[0315] 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 of sequence identity between any two of these numbers with the sequence of SEQ ID NO: 1, 3, 78-101, 148 or 154 (CGTase). In some embodiments, the first enzyme comprises an amino acid sequence of SEQ ID NO: 1, 3, 78-101, 148 or 154 (CGTase). In some embodiments, the first enzyme is a dextran sucrase. In some embodiments, the dextran sucrase comprises or consists of an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any one of SEQ ID NO: 2, 103, 106-110, 156 and 896. In some embodiments, the glucansucrase comprises or consists of the amino acid sequence of SEQ ID NO:2, 103, 104 or 105. In some embodiments, the glucansucrase comprises or consists of the amino acid sequence of any one of SEQ ID NO:2, 103-110 and 156-162 and 896. In some embodiments, DexT comprises the nucleic acid sequence shown 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 of sequence identity between any two sequences in these numbers with 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 of sequence identity between any two sequences of SEQ ID NO: 4, 5, 6, 7, 8, 9, 15, 16, 17, 18, and 19. In some embodiments, the UGT comprises or consists of any one of SEQ ID NO: 4, 5, 6, 7, 8, 9, 15, 16, 17, and 18. In some embodiments, the UGT is encoded by the sequence shown 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 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 with the sequence of 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 with the sequence of any one of SEQ ID NO: 21-30 and 309-314. In some embodiments, the cell comprises a fifth sequence encoding a P450. In some embodiments, P450 comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with a sequence of SEQ ID NO: 20, 49, 308, 315 or 317. In some embodiments, P450 is encoded by a gene comprising a sequence of any one of SEQ ID NO: 31-48, 316, 431, 871, 873, 875, 877, 879, 881, 883, 885, 887 and 891. In some embodiments, a sixth sequence encoding a cucurbitadienol synthase is further included. 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 of sequence identity between any two of the sequences 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 shown 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 shown in SEQ ID NO: 74. In some embodiments, the cell further comprises a seventh gene encoding 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 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 of sequence identity between any two of these numbers with a sequence of 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 of sequence identity between any two of these numbers with a sequence of SEQ ID NO:338. In some embodiments, the cell is a cell of a mammal, a bacterium, a fungus or an insect. In some embodiments, the cell is a yeast cell. Non-limiting examples of yeast include Candida, Sacccharaomyces, 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. , Aralia, Morus, Medicago, Betula, Astragalus, Jatropha, Camellia, Hypholoma, Aspergillus, Solanum, Huperzia, Pseudostellaria, Corchorus, Hedera, Marchantia and Morus. In some embodiments, the fungus is Trichophyton, Sanghuangporus, Taiwanofungus, Moniliophthora, Marssonina, Diplodia, Lentinula, Xanthophyllomyces, Pochonia, Colletotrichum, Diaporthe, Histoplasma, Coccidioides, Histoplasma, Sanghuangporus, Aureobasidium, Pochonia, Penicillium, Sporothrix, or Metarhizium.

[0316] In some embodiments, the cell comprises the sequence of an enzyme set forth in any one of SEQ ID NOs: 897, 899, 909, 911, 913, 418, 421, 423, 425, 427, 871, 873, 901. In some embodiments, the enzyme comprises a sequence in or encoded by SEQ ID NOs: 420, 422, 424, 426, 446, 872, 874-896, 898, 900, 902, 904, 906, 908, 910, 912, and 951-1012.

[0317] In some embodiments, DNA can be obtained by gene synthesis. For example, this operation can be performed by Genescript or IDT. DNA can be cloned into overexpression vectors by standard molecular biology techniques, such as: pQE1, pGEX-4t3, pDest-17, pET series, pFASTBAC. Escherichia coli host strains can be used to produce enzymes (i.e., Top10 or BL21 series + / - codon plus), induced using 1mM IPTG at OD600 of 1. Escherichia coli strains can be propagated at 37°C, 250rpm and switched to room temperature or 30°C (150 rpm) during induction. When indicated, pFASTBAC and optimized MOI can also be used to express certain enzymes through SF9 insect cell lines. Crude extracts containing enzymes can be produced by ultrasonic treatment and used for reactions described herein. All UDP-glycosyltransferase reactions contain sucrose synthase, which can be obtained from Arabidopsis thaliana by gene synthesis and expressed in Escherichia coli.

[0318] Hydrolysis of highly glycosylated mogrosides to produce compound 1

[0319] In some embodiments, the highly glycosylated mogrosides can be hydrolyzed to produce Compound 1. Non-limiting examples of highly glycosylated mogrosides include mogroside V, simenoside 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-Oxo Momordica VI、11-Oxo Momordica III E 、11-Oxo Monk Fruit IV E , Mogroside I E , mogroside, 11-oxomogroside, mogroside II E , Mogroside IIIA2 and mogroside III. Enzymes capable of catalyzing the hydrolysis process to produce compound 1 can be, for example, CGTase (e.g., showing no hydrolysis of starch), cellulase, β-glucosidase, transglucosidase, amylase, pectinase, glucanase, and fungal lactase. Table 1 lists the amino acid sequences of some of these enzymes and the nucleic acid sequences encoding some of these enzymes.

[0320] In some embodiments, Compound 1 exhibits resistance to hydrolases in recombinant cells, wherein the hydrolases exhibit the ability to hydrolyze mogroside VI, mogroside V, and mogroside IV to mogroside IIIE. Due to its resistance to hydrolysis, the α-linked glycosides present in Compound 1 have unique advantages over other mogrosides (β-linked glycosides). During the microbial production of Compound 1, recombinant host cells (e.g., microbial host cells) can hydrolyze unwanted β-linked mogrosides back to mogroside IIIE. Without being bound by any particular theory, we believe that the hydrolysis by the host cell can increase the purity of Compound 1 due to the following reasons: 1) reducing the levels of unwanted mogroside VI, mogroside V, and mogroside IV, and / or 2) hydrolysis will increase the amount of mogroside IIIE that can be used to produce the precursor of Compound 1.

[0321] Purification of Mogroside Compounds

[0322] Some embodiments include isolating mogroside compounds, such as Compound 1. In some embodiments, isolating Compound 1 includes lysing recombinant host cells. In some embodiments, isolating Compound 1 includes isolating Compound 1 from a 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, purification includes harvesting recombinant cells, preserving the supernatant, and lysing the cells. In some embodiments, lysis includes subjecting the cells to shear forces or detergent washing to obtain a lysate. In some embodiments, the shear force comes from sonication, high pressure cell lysis, or beads. In some embodiments, the lysate is subjected to filtering and purification steps. In some embodiments, the lysate is filtered and purified by solid phase extraction. The lysate can then be filtered and treated with ammonium sulfate to remove proteins and filtered through a C18 HPLC (5X 10 cm Atlantis prep T3OBD column, 5um, Waters), injected using an A / B gradient (A=water B=acetonitrile) of 10→30% B over 30 minutes and washed with 95% B, then re-equilibrated at 1% (total run time = 42 minutes). It can be collected in tared tubes at a rate of 30 mL / fraction (12 fractions / plate, 3 plates each). The lysate can also be centrifuged to remove solids and particulate matter. The plate is then dried in a Geneva HT12 / HT24. The desired compound is expected to elute in fraction 21 along with other isomers. The combined fractions can be further fractionated in 47 runs on a fluorophenyl HPLC column (3 x 10 cm, Xselect fluorophenyl OBD column, 5 um, Waters) using an A / B gradient (A = water, B = acetonitrile) of 15 → 30% B in 35 minutes, washed with 95% B, and then re-equilibrated at 15% (total run time = 45 minutes). Each run was collected in 12 tared test tubes (12 fractions / plate, 1 plate at a time) at 30 mL / fraction. The fractions containing the desired peak and the desired purity can be combined according to UPLC analysis and dried under reduced pressure to obtain a white powdery solid. The pure compound can be resuspended / dissolved in 10 mL of water and then lyophilized to obtain a purity of at least 95%.

[0323] In order to purify compound 1, in some embodiments, the compound can be purified by solid phase extraction, and HPLC may not be required. Compound 1 can be purified to, for example, a purity of about 70%, 80%, 90%, 95%, 98%, 99% or 100% or any purity within the range described by any of the above two values. In some embodiments, the purity of compound 1 purified by solid phase extraction is the same or substantially the same as that of HPLC purification. In some embodiments, the method includes separating a lysate from a recombinant cell on an HPLC column, and collecting an eluted fraction containing compound 1.

[0324] Fermentation

[0325] The host cells can be fermented as described herein to produce Compound 1. This can also include, for example, methods performed with or without air, and can be performed in, for example, an anaerobic environment. The whole cells (eg, recombinant host cells) can be in a fermentation broth or in a reaction buffer.

[0326] Monk fruit (Siraitia grosvenorii) extract can also be used to contact cells to produce Compound 1. In some embodiments, a method of producing Compound 1 is provided. The method can include contacting a monk fruit extract with a first enzyme capable of catalyzing the production of Compound 1 from mogrosides. Mogrosides such as mogroside V, simenoside 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-Mogroside III E 、11-Mogroside IV E , Mogroside I E , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2and mogroside III. In some embodiments, contacting includes contacting the mogroside alcohol extract with a recombinant host cell comprising a first gene encoding a first enzyme. In some embodiments, the first gene is heterologous to the recombinant host cell. In some embodiments, the mogroside alcohol 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 in the mogroside alcohol extract. In some embodiments, mogroside IIIE is also produced by a recombinant host cell. In some embodiments, the method further includes culturing the recombinant host cell in a culture medium under conditions of expressing the first enzyme. In some embodiments, the first enzyme is one or more of a UDP glycosyltransferase, a cyclomaltodextrin glucanotransferase (CGTases), a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase. In some embodiments, the first enzyme is a CGTase. For example, the CGTase may 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 NO: SEQ ID NO: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises an amino acid sequence of any one of SEQ ID NO: SEQ ID NO: 1, 3, 78-101, 148, and 154. In some embodiments, the CGTase comprises an amino acid sequence of any one of SEQ ID NO: 78-101. In some embodiments, the first enzyme is a glucan sucrase. In some embodiments, the glucansucrase 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 with any sequence shown in SEQ ID NO: 2, 103, 106-110, 156 and 896. In some embodiments, the glucansucrase comprises an amino acid sequence of any one of SEQ ID NO: 2, 103, 106-110, 156 and 896. 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 with any one of SEQ ID NO: 163-290 and 723. In some embodiments, the transglucosidase comprises the amino acid sequence of any one of SEQ ID NOs: 163-290 and 723. In some embodiments, the first enzyme is a β-glucosidase.In some embodiments, the β-glucosidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers to SEQ ID NO: 292. In some embodiments, the monk fruit alcohol extract comprises mogroside IIA, and the recombinant host cell comprises a second gene encoding a second enzyme capable of catalyzing mogroside IIA to produce mogroside IIIE. In some embodiments, mogroside IIA is also produced by a recombinant host cell. In some embodiments, the second enzyme is one or more of a UDP glycosyltransferase, a CGTase, a glycosyltransferase, a glucan sucrase, a cellulase, a β-glucosidase, an amylase, a transglucosidase, a pectinase, and a glucanase. 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), or comprises an amino acid sequence that is similar to 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). 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) have a sequence identity of at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100%, or a range between any two of these numbers.In some embodiments, the UGT is encoded by a gene 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 monk fruit extract comprises mogroside alcohol. In some embodiments, the method further comprises contacting the mogroside alcohol of the monk fruit extract, 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 mogroside alcohol. In some embodiments, mogroside alcohol is also produced by the recombinant host cell. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. 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 that has 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 the above UGTs. 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 a 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 simenoside. In some embodiments, the recombinant host cell also produces a mogroside compound. In some embodiments, the one or more enzymes include one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. In some embodiments, the mogroside compound is mogroside IIE. In some embodiments, the one or more enzymes comprise the amino acids set forth in any one of SEQ ID NOs: 293-303.In some embodiments, the mogroside compound is mogroside IIA or mogroside IIE, wherein contacting the monk fruit extract with a recombinant cell expressing one or more enzymes can produce mogroside IIIA, mogroside IVE, and mogroside V. In some embodiments, the one or more enzymes comprise the amino acids set forth in SEQ ID NO:304. In some embodiments, the one or more enzymes are encoded by the sequence set forth in SEQ ID NO:305. In some embodiments, the monk fruit extract comprises mogroside IA1. In some embodiments, the method further comprises contacting the monk fruit extract with a recombinant host cell, wherein the recombinant host cell comprises 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, UGT98 is encoded by the sequence shown in SEQ ID NO: 307. In some embodiments, contacting results in the production of mogroside IIA in the cell. In some embodiments, the monk fruit extract comprises 11-hydroxy-24,25 epoxycucurbitadienol. In some embodiments, the method further comprises contacting the monk fruit extract with a recombinant host cell, wherein the recombinant host cell also comprises a third gene encoding an epoxide hydrolase. In some embodiments, the recombinant host cell also produces 11-hydroxy-24,25 epoxycucurbitadienol. In some embodiments, the method further comprises contacting the monk fruit extract with a recombinant host cell, wherein the recombinant host cell comprises a fourth gene encoding a cytochrome P450 or an epoxide hydrolase. In some embodiments, the 11-hydroxy-cucurbitadienol is also produced by the recombinant host cell. In some embodiments, the monk fruit extract comprises 3,24,25 trihydroxycucurbitadienol. In some embodiments, the method further comprises contacting the monk fruit extract with a recombinant host cell, wherein the recombinant host cell further comprises a fifth gene encoding a cytochrome P450. In some embodiments, 3,24,25 trihydroxycucurbitadienol is also produced by a recombinant host cell. In some embodiments, contact with a monk fruit alcohol extract results in the production of monk fruit alcohol in a 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 of sequence identity between any two of these numbers to SEQ ID NO: 20 or 308.In some embodiments, the epoxide hydrolase has at least 70%, 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any of SEQ ID NOs: 21-30 and 309-314. In some embodiments, the monk fruit extract comprises cucurbitacinol. In some embodiments, the method further comprises contacting cucurbitacinol with a recombinant host cell, wherein the recombinant host cell comprises a gene encoding a cytochrome P450. In some embodiments, the contacting results in the production of 11-hydroxycucurbitacinol. In some embodiments, the 11-hydroxycucurbitacinol is expressed in a cell comprising a gene encoding a CYP87D18 or SgCPR protein. In some embodiments, CYP87D18 or SgCPR comprises a sequence in SEQ ID NOs: 315, 872 or 874. In some embodiments, CYP87D18 or SgCPR is encoded by SEQ ID NO: 316, 871 or 873. In some embodiments, 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 of sequence identity between any two of these numbers to any one of SEQ ID NO: 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 NO: 20 or 49. In some embodiments, the monk fruit extract comprises 2,3-oxidized squalene. In some embodiments, the method further comprises contacting the 2,3-oxidized squalene of the monk fruit extract 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 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 monk fruit extract comprises a mogroside intermediate, such as mogroside V, simenoside I, mogroside IV. E , isomogroside V, mogroside III E , 11-deoxymogroside V, 11-mogroside V, mogroside VI, mogroside IV A , Mogroside II A , Mogroside II A1 , Mogroside II A2 , Mogroside I A 、11-Mogroside VI、11-Oxomogroside III E 、11-Oxomogroside IV E , Mogroside I E , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2and 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. The sequence has at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with 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, contacting produces cucurbitadienol. In some embodiments, 2,3-squalene oxide and diepoxysqualene are also produced by the recombinant host cells. 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, or comprising the sequence set forth in 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%, 99%, 100% or a range between any two of these numbers sequence identity to SEQ ID NO: 897 or 899, or encoded by a nucleic acid set forth in SEQ ID NO: 897 or 899.

[0327] In some embodiments, the cucurbitadienol synthase comprises an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% identical 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 cucurbitadienol synthase from C pepo, S grosvenorii, C sativus, C melo, C moschata, or C maxim. 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%, or a range between two of these numbers, sequence identity to any one of SEQ ID NOs: 74, 320, 322, 324, 326, 328, 418, 418, 421, 423, 425, 427, 897, 899, 901, 903, and 905, or a nucleic acid sequence as 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 a cell comprising 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% sequence identity to SEQ ID NO: 315, 872 or 874, or a range between any two of these numbers, or comprises the sequence shown 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 two of these numbers, sequence identity to or as set forth in SEQ ID NOs: 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% or more sequence identity to SEQ ID NO: 898 or 900, or produced by an enzyme encoded by 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 of sequence identity to SEQ ID NO: 897 or 899, or an enzyme encoded by SEQ ID NO: 897 or 897. In some embodiments, the method further comprises contacting squalene with a recombinant host cell, wherein the recombinant host cell comprises an eighth gene encoding a squalene epoxidase. In some embodiments, contacting produces 2,3-oxidosqualene. In some embodiments, squalene is also produced by recombinant host cells. In some embodiments, squalene epoxidase comprises an amino acid sequence having at least 70%, 80%, 85%, 90%, 95%, 98%, 99%, 100% or a range of sequence identity between any two of these numbers with any one of SEQ ID NOs: 50-56, 60, 61, 334 or 335. In some embodiments, squalene epoxide is encoded by the nucleic acid sequence listed in SEQ ID NO: 335. In some embodiments, monk fruit extract comprises farnesyl pyrophosphate. In some embodiments, the method further comprises contacting farnesyl pyrophosphate with a recombinant host cell, wherein the recombinant host cell comprises a ninth gene encoding squalene synthase. In some embodiments, contacting produces squalene. In some embodiments, farnesyl pyrophosphate is also produced by a 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 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 monk fruit extract comprises geranyl-PP. In some embodiments, the method further comprises contacting the geranyl-PP with a recombinant host cell, wherein the recombinant host cell comprises a tenth gene encoding a farnesyl-PP synthase. In some embodiments, the contacting results in the production of farnesyl-PP. In some embodiments, the 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 shown in SEQ ID No: 339. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes are operably linked to a heterologous promoter. In some embodiments, the heterologous promoter is CMV, EF1A, SV40, PGK1, human β-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, repressible or constitutive promoter. In some embodiments, the production of one or more of pyruvate, acetyl-CoA, citrate, and TCA cycle intermediates is upregulated in the recombinant host cell. In some embodiments, the cytoplasmic localization in the recombinant host cell is upregulated. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, and tenth genes comprise at least one sequence encoding a 2A self-cleaving peptide. In some embodiments, the recombinant host cell is a cell of a plant, a bivalve, a fish, a fungus, a bacterium, or a mammal. In some embodiments, the plant is selected from the genus Siraitia, the genus Momordica, the genus Gynostemma, the genus Cucurbita, the genus Cucumis, the genus Arabidopsis, the genus Artemisia, the genus Stevia, the genus Panax, the genus Withania, the genus Euphorbia, the genus Medicago, the genus Chlorophytum, the genus Eleutherococcus, the genus Aralia 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, 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, Sacccharaomyces, Saccharomycotina, Taphrinomycotina, Schizosaccharomycetes, Komagataella, Basidiomycota, Agaricomycotina, Tremellomycetes, Pucciniomycotina, Aureobasidium, Coniochaeta, Rhodosporidium, and Microboryomycetes. In some embodiments, the bacteria 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, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes have been optimized to be expressed in bacteria, mammals, plants, fungi or insect cells. In some embodiments, one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth and tenth genes include functional mutations that increase the activity of the encoded enzyme. In some embodiments, cultivating recombinant host cells includes monitoring the pH, dissolved oxygen level, nitrogen level or combination of culture conditions of the culture. In some embodiments, the method includes separating compound 1. In some embodiments, separating compound 1 includes cracking recombinant host cells. In some embodiments, separating compound 1 includes separating compound 1 from culture medium. In some embodiments, the method also includes purifying compound 1. In some embodiments, purifying compound 1 includes HPLC, solid phase extraction or a combination thereof. In some embodiments, purification further harvests recombinant cells, preserves the supernatant of treated cells and lyses cells. In some embodiments, dissolving includes subjecting cells to shear force or detergent washing, thereby obtaining lysates. In some embodiments, the shear force is from ultrasound, french pressurized cells, or beads. In some embodiments, the lysate is subjected to a filtration and purification step. In some embodiments, the lysate is filtered and purified by solid phase extraction. In some embodiments, the method further comprises adding the monk fruit extract to the growth medium of the recombinant host cells a second or third time. In addition, the method can be performed by contacting the monk fruit extract with a recombinant cell lysate, wherein the recombinant cell lysate comprises an expressed enzyme listed herein.

[0328] In general, the compounds disclosed and described herein, alone or in combination, can be provided in compositions such as ingestible compositions. In one embodiment, compounds as disclosed and described herein, alone or in combination, can provide sweetness to ingestible compositions. In other embodiments, compounds disclosed and described herein can be used as sweeteners alone or in combination to enhance the sweetness of another sweetener. By combining one or more compounds disclosed and described herein with one or more other sweeteners in a sweetener composition, the compounds disclosed herein give the sweetener composition a temporal characteristic and / or flavor characteristic that is more similar to sugar. In another embodiment, compounds as disclosed and described herein, alone or in combination, can be contacted with a modified composition by its composition as disclosed and described herein to form a modified composition, thereby increasing or enhancing the sweetness of the composition. In another embodiment, compounds as disclosed and described herein, alone or in combination, can be used in a composition that modulates sweet receptors and / or their ligands expressed in the body other than taste buds.

[0329] As used herein, "ingestible compositions" include any substance that is intended to be used or not for consumption and can be taken orally alone or with another substance. Ingestible compositions include both "food or beverage products" and "non-food products". "Food or beverage products" means any food product intended for human or animal consumption, including solid, semi-solid or liquid (e.g., beverages), and also includes functional foods (e.g., any fresh or processed food that claims to have health-promoting and / or disease-preventing properties, whose nutritional functions exceed the basic nutritional function of providing nutrition). The term "non-food product" or "non-food composition" includes any product or composition that can be taken by humans or animals for purposes other than consumption or as food or beverage. For example, non-food products or non-food compositions include supplements, nutritional supplements, pharmaceuticals and over-the-counter drugs, oral care products (such as toothpaste and mouthwash) and chewing gum.

[0330] Composition containing mogroside compounds

[0331] The present disclosure also includes compositions, such as ingestible compositions, comprising one or more of the mogroside compounds disclosed herein, including but not limited to Compound 1 and Fig.43In some embodiments, the ingestible composition can be a beverage. For example, the beverage can be selected from an enhanced sparkling beverage, cola, lemon-lime flavored sparkling beverage, orange flavored sparkling beverage, grape flavored sparkling beverage, strawberry flavored sparkling beverage, pineapple flavored sparkling beverage, ginger beer, root beer, fruit juice, fruit-flavored juice, fruit juice beverage, honey juice beverage, vegetable juice, vegetable-flavored juice, sports drink, energy drink, fortified water beverage, vitamin-fortified water, near water beverage, coconut water, tea beverage, coffee, cocoa beverage, beverage containing milk component, beverage containing cereal extract and smoothie. In some embodiments, the beverage can be a soft drink.

[0332] An "ingestible ingredient" is a substance suitable for oral administration that can be combined with the compounds described herein to form an ingestible composition. Depending on the intended use of the product, the ingestible ingredient can be in any form, such as solid, semi-solid, liquid, paste, gel, emulsion, cream, foam, suspension, solution or any combination thereof (e.g., liquid containing solid ingredients). Ingestible ingredients can be artificial or natural. Ingestible ingredients include many common food ingredients, such as water, fruit or vegetable juice, vinegar, marinade, 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 propylene glycol oligomers, fatty acid glycerides and dispersions or emulsions of such hydrophobic substances in aqueous media, salts (such as sodium chloride), wheat flour, solvents (such as ethanol), solid edible diluents (such as plant powder or flour) or other liquid carriers; dispersion or suspension aids; surfactants; isotonic agents; thickeners or emulsifiers, preservatives; solid adhesives; lubricants, etc.

[0333] Other ingestion-acceptable ingredients include acids, including but 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 green coffee bean extract, green coffee bean extract, whey protein isolate, or potassium chloride; colorants, including, for example, caramel, 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; preservatives, including, for example, sodium benzoate, potassium benzoate, potassium sorbate, sodium metabisulfate, sorbitan, and sodium benzoate. Acid or benzoic acid; antioxidants including, for example, ascorbic acid, disodium EDTA, alpha-tocopherol, mixed tocopherols, rosemary extract, grape seed extract, resveratrol, or sodium hexametaphosphate; vitamins or functional ingredients including, for example, resveratrol, Co-Q10, omega 3 fatty acids, theanine, choline chloride (Citcoline), inositol, inulin (chicory root), taurine, ginseng extract, guarana extract, ginger extract, L-phenylalanine, L-carnitine, L-tartrate, D-glucuronolactone, inositol, bioflavonoids, echinacea, Ginkgo Biloba, yerba mate, flaxseed oil, Garcinia Cambogia cambogia peel extract, white tea extract, ribose, milkthistle extract, grape seed extract, pyridoxine hydrochloride (vitamin B6), cyanocobalamin (vitamin B12), niacinamide (vitamin B3), biotin, calcium lactate, calcium pantothenate (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 nitrate, vitamin D3, vitamin A palmitate, zinc gluconate, zinc lactate or zinc sulfate; clouding agents, including, for example, ester gum, brominated vegetable oil (BVO) or sucrose acetate isobutyrate (SAIB); buffers including, for example, sodium citrate, potassium citrate, or salt; flavorings including, for example, propylene glycol, ethanol, glycerin, gum arabic (acacia), maltodextrin, modified corn starch, dextrose, natural flavorings, natural flavorings with other natural flavorings (natural flavoring WONF), natural and artificial flavorings, artificial flavorings, silicon dioxide, magnesium carbonate, or tricalcium phosphate; stabilizers including, for example, pectin, xanthan gum, carboxymethyl cellulose (CMC), polysorbate 60, polysorbate 80, medium chain triglycerides, cellulose gel, cellulose gum, sodium caseinate, modified food starch, gum arabic (acacia), or carrageenan.

[0334] Example

[0335] Some aspects of the above-described embodiments are disclosed in further detail in the following examples, which are in no way intended to limit the scope of the present disclosure.

[0336] Example 1: Production of simenoside I

[0337]

[0338] As disclosed herein, simenoside I can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, simenoside I can be hydrolyzed to produce mogroside IIIE, which can then be used to prepare compound 1. For example, a method for preparing simenoside I can include contacting mogroside with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, a recombinant cell expressing a pectinase from Aspergillus aculeatus can be used.

[0339] As another example, a method for producing simenoside I may include: 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, pectinases from Aspergillus aculeatus can be used.

[0340] Example 2: Mogroside IV E Production

[0341]

[0342] As disclosed herein, mogroside IV E can be an intermediate mogroside compound used to produce Compound 1 disclosed herein. For example, mogroside IV from mogroside V can then be used E To produce compound 1. For example, for the production of mogroside IV E The method may include contacting mogroside V with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. As another example, the recombinant cell may contain a gene encoding a pectinase. The pectinase may be encoded by a gene from Aspergillus aculeatus.

[0343] As another example, for the production of mogroside IV E The method may include: making mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, pectinases from Aspergillus aculeatus can be used.

[0344] Example 3: Mogroside III E Production

[0345]

[0346] As disclosed herein, mogroside III E It can be an intermediate mogroside compound used to produce compound 1 disclosed herein. For example, mogroside II AGlycosylation was performed to produce mogroside IIIE, which was then used to produce compound 1.

[0347] As another example, for the production of mogroside III E The method may include: making mogroside V, mogroside II A , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, a pectinase from Aspergillus aculeatus can be encoded by a gene in the recombinant host cell.

[0348] Example 4: Mogroside IV A Production

[0349]

[0350] As disclosed herein, mogroside IV A can be an intermediate mogroside compound used to produce Compound 1 disclosed herein. For example, mogroside IV from mogroside V can then be used A to produce compound 1.

[0351] For example, for the production of mogroside IV A The method may include contacting mogroside V with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. As another example, the enzyme may be a β-galactosidase from Aspergillus oryzae.

[0352] As another example, for the production of mogroside IV A The method may include: making mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the above β-galactosidase from Aspergillus oryzae can be used in the method.

[0353] Example 5: Mogroside II A Production

[0354]

[0355] As disclosed herein, mogroside II A It can be an intermediate mogroside compound used to produce compound 1 disclosed herein. For example, it can be used to produce mogroside II A The method may include contacting mogroside IA1 with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0356] As another example, for the production of mogroside II A The method may include: making mogroside I A1 , Mogroside V, Simagroside I, Mogroside IV E , Mogroside V, Mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside IIA , Mogroside II A1 , Mogroside II A2 , Mogroside I A 、11-Oxomogroside VI、11-Oxomogroside III E 、11-Oxomogroside IV E , Mogroside I E , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, celluclasts may also be used.

[0357] Example 6: Production of Mogroside III from Aromase A1

[0358]

[0359] As disclosed herein, mogroside III A1 It can be an intermediate mogroside compound used to produce 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, for producing mogroside III A1 The method may include contacting simenoside I with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme may be Aromase. As another example, for producing mogroside III A1 The method may include: making mogroside V, simenoside 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, mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0360] Example 7: Production of Compound 3

[0361]

[0362] As disclosed herein, compound 3 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 3 can include contacting mogroside with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, cyclomaltodextrin glucanotransferases and / or Toruzyme from Bacillus lichenformis can be used.

[0363] As another example, a method for producing compound 3 may include: making mogroside V, simanoside 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 One or more of mogroside IVE, mogroside IIIE, mogroside IE, mogroside alcohol, 11-oxomogroside alcohol, mogroside IIE, mogroside IIIA2, and mogroside III are contacted with a recombinant host cell expressing one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, CTGase enzymes can be used.

[0364] Example 8: Production of Compound 4

[0365]

[0366] As disclosed herein, compound 4 is produced in the process of producing compound 1 disclosed herein. For example, a method of producing compound 4 can also result in the production of compound 1, which can include contacting mogroside IIIE with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0367] As another example, a method for producing compound 4 may include: making mogroside V, simanoside 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 One or more of mogroside IVE, mogroside IIIE, mogroside IE, mogroside alcohol, 11-oxomogroside alcohol, mogroside IIE, mogroside IIIA2 and mogroside III are contacted with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, the enzyme can be cyclomaltodextrin glucanotransferase and / or Toruzyme from Bacillus lichenformis.

[0368] Example 9: Production of Compound 5

[0369]

[0370] As disclosed herein, compound 5 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method of producing compound 5 can also result in the production of compound 1, the method can include contacting mogroside IIIE with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0371] As another example, a method for producing compound 5 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase and / or Toruzyme from Bacillus lichenformis can be used.

[0372] Example 10: Production of Compound 6

[0373]

[0374] As disclosed herein, compound 6 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 6 can also result in the production of compound 1, and the method can include making mogroside III E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0375] As another example, a method for producing compound 6 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside IIIA2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0376] Example 11: Production of Compound 7

[0377]

[0378] As disclosed herein, compound 7 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 7 can also result in the production of compound 1, and the method can include making mogroside III E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0379] As another example, a method for producing compound 7 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0380] Example 12: Production of Compound 8

[0381]

[0382] As disclosed herein, compound 8 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 8 can also result in the production of compound 1, and the method can include making mogroside III E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0383] As another example, a method for producing compound 8 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0384] Example 13: Production of Compound 9

[0385]

[0386] As disclosed herein, compound 9 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method of producing compound 9 can also result in the production of compound 1, which can include contacting mogroside IIIE with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0387] As another example, a method for producing compound 9 may include: making mogroside V, simanoside 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, 1 One or more of 1-oxomogroside IVE, mogroside IE, mogroside alcohol, 11-oxomogroside alcohol, mogroside IIE, mogroside IIIA2 and mogroside III are contacted with a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0388] Example 14: Production of Compound 10

[0389]

[0390] As disclosed herein, compound 10 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 10 can also result in the production of compound 1, and the method can include making mogroside III E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0391] As another example, a method for producing compound 10 may include: reacting mogroside V, simenoside I, mogroside IV E , isomogroside V, mogroside III E , 11-deoxymogroside V, 11-oxomogroside V, mogroside VI, mogroside IV A , Mogroside IIA , Mogroside II A1 , Mogroside II A2 , Mogroside I A 、11-Oxomogroside VI、11-Oxomogroside III E 、11-Oxomogroside IV E , Mogroside I E , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0392] Example 15: Production of Compound 11

[0393]

[0394] As disclosed herein, compound 11 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for producing compound 11 can also result in the production of compound 1, and the method can include making mogroside III E or 11-oxo-MIII E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0395] As another example, a method for producing compound 11 may include: preparing mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside IIE , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, CGTase or Toruzyme from Bacillus lichenformis can be used.

[0396] Example 16: Production of Compound 12

[0397]

[0398] As disclosed herein, compound 12 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, a method for 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 UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme can be an invertase in baker's yeast.

[0399] As another example, a method for producing compound 12 may include: 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, invertases, and glucanases.

[0400] Example 17: Production of Compound 13

[0401]

[0402] As disclosed herein, compound 13 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, the method can include making mogroside III E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. The expressed enzyme may also be, for example, celluclast.

[0403] As another example, a method for producing compound 13 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, celluclast can be used.

[0404] Example 18: Production of Compound 14

[0405]

[0406] As disclosed herein, compound 14 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, the method can include making mogroside III EContacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. The expressed enzyme may also be, for example, celluclast.

[0407] As another example, a method for producing compound 14 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, celluclast can be used. The method may also require the presence of a sugar such as α-lactose.

[0408] Example 19: Production of Compound 15

[0409]

[0410] As disclosed herein, compound 15 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, the method can include making mogroside II A Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0411] As another example, a method for producing compound 15 may include: making mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, Toruzyme can be used.

[0412] Example 20: Production of Compound 16

[0413]

[0414] As disclosed herein, compound 16 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, the method can include making mogroside II A Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0415] As another example, a method for producing compound 16 may include: making mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside IIIA2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, Toruzyme can be used.

[0416] For example, the enzyme may be Toruzyme. The recombinant cell may also include genes encoding cyclomaltodextrin glucanotransferase (eg, Toruzyme), invertase, glucosyltransferase (eg, UGT76G1).

[0417] Example 21: Production of Compound 17

[0418]

[0419] As disclosed herein, compound 17 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 17 can be hydrolyzed to produce mogroside III E , which can then be used to produce compound 1. For example, a method for producing compound 17 may include contacting simonoside I with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, a recombinant cell expressing a UDP glycosyltransferase may be used.

[0420] As another example, a method for producing compound 17 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, UDP glycosyltransferases can be used.

[0421] Example 22: Production of Compound 18

[0422]

[0423] As disclosed herein, compound 18 can be an intermediate mogroside compound for producing 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, a method for preparing compound 18 may include: E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzymes may be Sus1 and UGT76G1.

[0424] As another example, a method for producing compound 18 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme can be UGT76G1.

[0425] Example 23: Production of Compound 19

[0426]

[0427] As disclosed herein, compound 19 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 19 can be hydrolyzed to produce compound 1. For example, a method for preparing compound 19 may include: E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzymes may be Sus1 and UGT76G1.

[0428] As another example, a method for producing compound 19 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucan sucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. The enzyme may be, for example, UGT76G1. The enzyme may also be, for example, sucrose synthase Sus1.

[0429] Example 24: Production of Compound 20

[0430]

[0431] As disclosed herein, compound 20 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 20 can be further hydrolyzed to produce compound 1. For example, a method for producing compound 20 may include: EContacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzymes may be Sus1 and UGT76G1.

[0432] As another example, a method for producing compound 20 may include: reacting mogroside V, simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. The enzyme can be, for example, UGT76G1. The enzyme can also be, for example, sucrose synthase Sus1. The enzyme can be, for example, sucrose synthase Sus1 and UGT76G1.

[0433] Example 25: Production of Compound 21

[0434]

[0435] As disclosed herein, compound 21 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 21 can be further hydrolyzed to produce compound 1. For example, a method for producing compound 21 that can also result in the production of compound 1 can include: E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. The enzymes may be, for example, Sus1 and UGT76G1.

[0436] As another example, a method for producing compound 21 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. The enzyme may be, for example, UGT76G1. The enzyme may also be, for example, sucrose synthase Sus1. The enzymes may be, for example, sucrose synthase Sus1 and GT76G1.

[0437] Example 26: Production of Compound 22

[0438]

[0439] As disclosed herein, compound 22 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 22 can be further hydrolyzed to produce compound 1. For example, a method for producing compound 22 can also result in the production of compound 1, which can include contacting mogroside IVA with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. The enzymes can be, for example, Sus1 and UGT76G1.

[0440] As another example, a method for producing compound 22 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucan sucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. The enzyme can be, for example, UGT76G1. The enzyme can also be, for example, sucrose synthase Sus1. The enzyme can be, for example, sucrose synthase Sus1 and GT76G1.

[0441] Example 27: Production of Compound 23

[0442]

[0443] As disclosed herein, compound 23 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 23 can be further hydrolyzed to produce compound 1. For example, a method for producing compound 22 can also lead to the production of compound 1, the method comprising: reacting mogroside IV E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme can be glucansucrase.

[0444] As another example, a method for producing compound 23 may include: E , simenoside 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, Mogroside alcohol, 11-oxomogroside alcohol, Mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucan sucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. The enzyme may be, for example, glucan sucrase, which converts highly glycosylated mogroside IV E The isomers are hydrolyzed into the desired mogroside V isomer.

[0445] Examples 28 and 29: Production of Mogroside II by Fungal Lactase A1 and mogroside II A2

[0446]

[0447] As disclosed herein, mogroside II A1 and mogroside II A2 It can be an intermediate mogroside compound used to produce compound 1 disclosed herein. For example, mogroside II A1 and mogroside II A2 Further hydrolysis to produce compound 1. For example, for the production of mogroside II A1 and mogroside II A2 The method can also lead to the production of compound 1, which comprises: making mogroside IV E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. The enzyme may be, for example, lactase from a fungus.

[0448] As another example, for the production of mogroside II A1 and mogroside II A2 The method may include: making mogroside V, mogroside IV E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0449] Example 30: Production of Mogroside Compounds by Viscozyme IA

[0450]

[0451] As disclosed herein, mogroside IA It can be an intermediate mogroside compound used to produce Compound 1 disclosed herein. For example, mogroside IA Further hydrolysis to produce compound 1. For example, for the production of mogroside IA The method can also result in the production of compound 1, which may include contacting mogroside IIA with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme may be, for example, Viscozyme.

[0452] As another example, for producing mogroside IA The method may include: making mogroside V, mogroside IV E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, the enzyme can be Viscozyme.

[0453] Example 31: Production of Compound 24

[0454]

[0455] As disclosed herein, compound 24 can be an intermediate mogroside compound for producing 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 production of compound 1, which method can include: E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme can be glucansucrase DexT.

[0456] As another example, a method for producing compound 24 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme can be glucansucrase DexT.

[0457] Example 32: Production of Compound 25

[0458]

[0459] As disclosed herein, compound 25 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 25 can be further hydrolyzed to produce compound 1. A method for producing compound 25 can also result in the production of compound 1, which method can include: E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme may be glucansucrase DexT.

[0460] As another example, a method for producing compound 25 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme may be glucansucrase DexT.

[0461] Example 33: Production of Compound 26

[0462]

[0463] As disclosed herein, compound 26 can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, compound 26 can be further hydrolyzed to produce compound 1. A method for producing compound 26 can also result in the production of compound 1, the method can include:E Contacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme may be glucansucrase DexT.

[0464] As another example, a method for producing compound 26 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III are contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme may be glucansucrase DexT.

[0465] Examples 34 and 35: Production of mogroside and mogroside I using pectinase E

[0466]

[0467] As disclosed herein, mogroside and mogroside I E It can be an intermediate mogroside compound used to produce compound 1 disclosed herein. For example, mogroside alcohol can be used as a substrate to produce mogroside IA1, which is further hydrolyzed to form compound 1, and mogroside I E It can also be further hydrolyzed to produce compound 1. For the production of mogroside and mogroside I EThe method can be used to produce compound 1, which comprises contacting mogroside V with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme can be a pectinase from Aspergillus aculeatus.

[0468] As another example, for producing mogroside and mogroside I E The method may include: making mogroside V, mogroside IV E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0469] Example 36: Production of Mogroside IIE

[0470]

[0471] As disclosed herein, mogroside IIE can be an intermediate mogroside compound for producing compound 1 disclosed herein. For example, mogroside IIE can be further hydrolyzed to produce compound 1. For example, a method for producing mogroside IIE can result in the production of compound 1, the method comprising: contacting mogroside V with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucan sucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme can be a pectinase from Aspergillus aculeatus.

[0472] As another example, a method for producing mogroside IIE may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0473] Examples 37 and 38: Production of Compounds 32 and 33

[0474]

[0475] As disclosed herein, compounds 32 and 33 can be intermediate mogroside compounds for producing compound 1 disclosed herein. For example, compounds 32 and 33 can be further hydrolyzed to produce compound 1. The method for producing compounds 32 and 33 can also result in the production of compound 1, which method can include: reacting mogroside V, mogroside IV E , simenoside 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, mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and one or more of mogroside III and a recombinant host cell expressing one or more of UDP glycosyltransferase, CGTases, glycosyltransferase, glucansucrase, cellulase, β-glucosidase, amylase, transglucosidase, pectinase and glucanase. For example, the enzyme can be a pectinase from Aspergillus aculeatus.

[0476] As another example, a method for producing compounds 32 and 33 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0477] Examples 39 and 40: Production of Compounds 34 and 35

[0478]

[0479]

[0480] As disclosed herein, compounds 34 and 35 can be intermediate mogroside compounds for producing compound 1 disclosed herein. For example, compounds 32 and 33 can be further hydrolyzed to produce compound 1. A method for producing compounds 34 and 35 can also result in the production of compound 1, which method may include: EContacting with a recombinant host cell expressing one or more UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases and glucanases. For example, the enzyme may be celluclast.

[0481] As another example, a method for producing compounds 34 and 35 may include: E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0482] Examples 41 and 42: Mogroside III A2 and the production of mogroside III

[0483]

[0484] As disclosed herein, mogroside III A2 and mogroside III can be intermediate mogroside compounds used to produce compound 1 disclosed herein. For example, mogroside III A2 And mogroside III are further hydrolyzed to produce compound 1.

[0485] For example, mogroside III A2 and mogroside III can be contacted with UGT to form mogroside IVA, which is another mogroside compound used to prepare mogroside IIIE, which can be further hydrolyzed to form compound 1.

[0486] For the production of mogroside III A2and mogroside III can also lead to the production of compound 1, the method may include: making mogroside III E Contacting with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, beta-glucosidases, amylases, transglucosidases, pectinases, and glucanases. For example, the enzyme may be celluclast.

[0487] As another example, for the production of mogroside III A2 The method of preparing mogroside III may include: preparing mogroside V, mogroside IV E , simenoside 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 , mogroside, 11-oxomogroside, mogroside II E , Mogroside III A2 and mogroside III is contacted with a recombinant host cell that expresses one or more of UDP glycosyltransferases, CGTases, glycosyltransferases, glucansucrases, cellulases, β-glucosidases, amylases, transglucosidases, pectinases, and glucanases.

[0488] Example 43: Use of CGT-SL enzyme to produce compound 1

[0489] In a 1 ml reaction volume, 5 mg of mogroside IIIE, 50 mg of soluble starch, 0.1 M NaOAC pH 5.0, 125 ul of CGT-SL enzyme (from Geobaccilus thermophillus) and water were mixed with a stir bar and incubated at 50° C. Time point samples were taken for HPLC analysis.

[0490] HPLC data: Figure 1 The mass spectrum produced by compound 1 is shown. In some embodiments, CTG-SL may comprise a sequence shown in SEQ ID NO:3, 148 or 154.

[0491] Example 44: Cloning: PCR amplification from Leuconostoc citreum ATCC11449 Amplify the gene encoding glucansucrase and clone it into pET23a

[0492] Growth conditions: BL21 Codon Plus RIL strain was grown in 2xYT at 37°C, 250 rpm until OD600 was 1. 10 mM lactose was added for induction and incubated overnight at 150 rpm at room temperature. Crude extracts used for the reaction were obtained by sonication or osmotic shock.

[0493] In some embodiments, the dextran sucrase 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, DexT comprises the amino acid sequence shown in SEQ ID NO: 103. In some embodiments, DexT comprises the nucleic acid sequence shown in SEQ ID NO: 104 or SEQ ID NO: 105.

[0494] Example 45: Mogroside IIIE and Streptococcus mutans Clarke ATCC25175 Dextran Sucrose Enzymatic reaction to produce compound 1

[0495] Growth conditions: The strains were grown anaerobically with the addition of glucose to stimulate the production of glucansucrase as described by Wenham, Henessey and Cole (1979). 5 mg / ml mogroside IIIE was added to the growth medium. Samples were taken at time points for HPLC analysis. HPLC data are available at Figure 2 The mass spectrum of compound 1 is shown in FIG.

[0496] Example 46: Reaction of Mogroside IIIE and CGTase

[0497] In a 1 ml reaction volume, 5 mg of mogroside IIIE, 50 mg of soluble starch, 0.1 M NaOAC pH 5.0, 125 ul of enzyme and water were mixed with a stir bar, mixed with a stir bar, and incubated at 50°C. Time point samples were taken for HPLC analysis. The enzyme used was CGTase. Figure 1 As shown, the compound 1 product can be seen in the HPLC data and mass spectrometry data. The mass peak corresponds to the size of compound 1.

[0498] Example 47: Reaction of Mogroside IIIE and Celluclast

[0499] Celluclast glycosylation with mogroside IIIE and celluclast from the natural host Trichoderma reesei

[0500] Reaction conditions: 5 mg of mogroside IIIE, 100 mg of xylan, 50 ul of Celluclast 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. Samples were taken at time points for HPLC analysis.

[0501] Xylosylation products such as Figure 3 Products from xylosylation can be used as intermediates in the production of Compound 1. The sequence of Celluclast is contained in Table 1 and was used herein for the production of xylosylation products.

[0502] Example 48: Glycosyltransferase (maltotriosyltransferase) (Natural host: Bacillus (Geobacillus sp.) APC9669)

[0503] In this example, glycosyltransferase AGY15763.1 (Amano Enzyme USA Co., Ltd., Illinois; SEQ ID NO: 434, see Table 1) was used. 20 mL of deionized 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 using a magnetic bar. Three more identical reactions were performed using 600 mg of mogroside IIIE as a raw material. The reaction was stopped after 24 hours. Insoluble starch was removed by centrifugation (4000 rpm for 5 minutes, Eppendorf). The supernatant was heated to 80°C for 30 minutes under stirring (500 rpm) and then centrifuged (4000 rpm for 10 minutes, Eppendorf). The supernatant was filtered through 250 ml of 0.22 micron PES and checked by LC-MS (Sweet Naturals 2016-Enzymatic_2016Q4_A.SPL, line 1254) to obtain HPLC data.

[0504] The AGY15763.1 protein (SEQ ID NO:434) can be encoded by native gDNA (SEQ ID NO:437) or a codon-optimized (for E. coli) DNA sequence (SEQ ID NO:438).

[0505] An example of other glycosyltransferases with similar expected reactivity is the UGT76G1 protein from Stevia rebaudiana (SEQ ID NO: 439), which can be expressed in E. coli. SEQ ID NO: 440 provides the native coding sequence of UGT76G1 (SEQ ID NO: 439).

[0506] Example 49: UDP-Glycosyltransferase UGT73C5 in the presence of mogrosanol

[0507] Mogroside I and mogroside II were produced by the reaction of mogroside alcohol with UDP-glycosyltransferase. 1 mg / ml mogroside alcohol was reacted with 200 ul crude extract containing UGT73C5 (A. thaliana) (334), 2 ul crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, and 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 analysis. Figure 4 and Figure 5 As shown, the reaction products are from mogroside alcohol to mogroside I and mogroside II.

[0508] The protein sequence of UGT73C5 is shown in SEQ ID NO:441, the native DNA coding sequence of UGT73C5 (SEQ ID NO:441) is shown in SEQ ID NO:442, and the UGT73C5 coding sequence (codon optimized for E. coli)) is shown in SEQ ID NO:443.

[0509] Example 50: UDP-Glycosyltransferase (UGT73C6) in the presence of mogroside to produce mogroside I

[0510] Reaction conditions: 1 mg / ml of mogroside alcohol was reacted with 200 ul of crude extract containing UGT73C6, 2 ul of crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, and 0.5 mg / ml spectinomycin in 0.1 M Tris-HCl pH 7.5, and incubated at 30°C. After 2 days, samples were taken and analyzed by HPLC. The reaction product is mogroside I from mogroside alcohol. Figure 6 The HPLC data and mass spectrometry data are shown.

[0511] The protein and gDNA sequences encoding Arabidopsis thaliana UGT73C6 are shown in SEQ ID NO:444 and SEQ ID NO:445, respectively.

[0512] Example 51: UDP-glycosyltransferase (338) in the presence of mogroside to produce mogroside I, Momordica grosvenori Mogroside IIA and two different mogroside III products

[0513] Reaction conditions: 1 mg / ml of mogroside 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 30°C. Samples were taken for HPLC analysis after 2 days.

[0514] The reaction of mogroside with Bacillus sp. UDP-glycosyltransferase (338) (described in Pandey et al., 2014; incorporated herein by reference) yields the reaction products: mogroside I, mogroside IIA, and two different mogroside III products. Figure 7-9 HPLC and mass spectrometry data of the product obtained after the reaction are shown. Figure 8 A peak associated with the size of mogroside IIA is shown.

[0515] The protein and gDNA sequences encoding UGT 338 are shown in SEQ ID NO:405 and SEQ ID NO:406, respectively.

[0516] Example 52: UDP-Glycosyltransferase (301(UGT98)) in the presence of mogroside IIIE to produce simanoside I and mogroside V

[0517] 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 analysis. Figure 10-11 As shown, the reaction products of mogroside IIIE are simenoside I and mogroside V.

[0518] The protein and gDNA sequence encoding S grosvenorii 301 UGT98 are shown in SEQ ID NO: 407 and SEQ ID NO: 408, respectively.

[0519] Example 53: UDP-glycosyltransferase (339) in the presence of mogroside, simenoside I or compound 1 Producing mogroside I from hanguo alcohol, producing isomogroside V from simenoside I, and producing compound 1 derivatives from compound 1

[0520] Reaction conditions: 1 mg / ml of mogroside 1, simonoside I or compound 1 was reacted with 200 ul of crude extract containing 339 (described in Itkin et al., which is incorporated herein by reference), 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 analysis after 2 days.

[0521] The reaction product from mogroside alcohol yields mogroside I, simonoside I yields isomogroside V, and compound 1 yields a derivative of compound 1 ( Figure 12-14 ).

[0522] The protein and DNA sequence encoding S. grosvenorii UGT339 are shown in SEQ ID NO:409 and SEQ ID NO:410, respectively.

[0523] Example 54: In mogroside IIA, mogroside IIE, mogroside IIIE, mogroside IVA or mogroside UDP-glycosyltransferase (330) in the presence of IVE to produce mogroside IIIA, mogroside IVE and mogroside V

[0524] As described herein, the reaction products mogroside IIIA, mogroside IVE, and mogroside V were obtained using UDP-glycosyltransferase (330) as described in Noguchi et al., 2008 (incorporated herein by reference). The natural host was sesame (Sesamumindicum) and the production host was SF9. 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 30°C. Samples were taken after 2 days for analysis by HPLC.

[0525] The reaction produces surprising products, such as mogroside IIIA, mogroside IVE, and mogroside V. Figure 15-20 As shown, the sizes of the compounds produced correspond to mogroside IIIA, mogroside IVE and mogroside V.

[0526] The protein and the gDNA sequence encoding the S. grosvenorii UGT330 protein are shown in SEQ ID NO:411 and SEQ ID NO:412, respectively.

[0527] Example 55: In mogroside IIA, mogroside IIE, mogroside IIIE, mogroside IVA or mogroside IVE in the presence of UDP-glycosyltransferase (328) (described in Itkin et al.) to produce mogroside IIIA, mogroside IVE and Mogroside V

[0528] Reaction conditions: 1 mg / ml mogroside IIIE was reacted with 200 ul crude extract containing 330, 2 ul crude extract containing sucrose synthase, 5 mM UDP, 1x M221 protease inhibitor, 200 mM sucrose, 0.5 mg / ml spectinomycin in 0.1 ul Tris-HCl pH 7.0, and incubated at 30°C. Samples were taken for HPLC analysis after 2 days.

[0529] The reaction products are mogroside IVE and mogroside V. Figure 21-22As shown, the product sizes in the mass spectrometry data correspond to mogroside IVE and mogroside V. The S. grosvenorii UGT328 protein (glycosyltransferase) and its coding sequence are shown in SEQ ID NOs: 413 and 414, respectively.

[0530] The sucrose synthase AtSus1 protein sequence and the gDNA encoding the AtSus1 protein are shown in SEQ ID NOs: 415 and 416, respectively.

[0531] Example 56: Production of Mogroside Alcohol in Yeast

[0532] DNA was obtained by gene synthesis, either by Genescript or IDT. For some cucurbitadienol synthases, cDNA or genomic DNA was obtained from 10-60 day old seedlings and then PCR amplified using specific and degenerate primers. DNA was cloned into one of the following overexpression vectors: pESC-Ura, pESC-His or pESC-LEU by standard molecular biology techniques. Saccharomyces cerevisiae strain YHR072 (heterozygous for erg7) was purchased from GEDharmacon. The plasmid containing the mogrosanol synthesis gene (pESC vector) was transformed / co-transformed using the Zymo Yeast Transformation Kit II. The strain was cultured in an appropriately selected standard medium (YPD or SC) containing 2% glucose or 2% galactose, and heterologous genes were induced at 30°C and 220rpm. When indicated, lanosterol synthase) inhibitor Ro 48-8071 (Cayman Chemicals) (50ug / ml) was added. After 2 days of induction, the yeast producing mogrohol and its precursors were then lysed (YeastBuster), extracted with ethyl acetate, dried, and suspended in methanol. The samples were analyzed by HPLC.

[0533] Cucurbitadienol synthase SgCbQ from S. grosvernorii catalyzes the production of cucurbitadienol under growth conditions without inhibitors.

[0534] The production of cucurbitadienol was demonstrated in the HPLC and mass spectrometry data, which showed mass peaks for the indicated products ( Fig.23 The protein sequence and the DNA sequence encoding S. grosvernorii SgCbQ are shown in SEQ ID NOs: 446 and 418, respectively.

[0535] Cpep2 is also required for the production of cucurbitadienol in yeast. Fig.24As shown, the mass spectrum shows peaks and characteristic fragments corresponding to cucurbitadienol. The protein sequence of Cpep2 and the DNA sequence encoding the Cpep2 protein are shown in SEQ ID NOs: 420 and 421, respectively.

[0536] Cucurbita pepo (Jack O'Lantern) Cpep4 was also used to produce cucurbitadienols under growth conditions without inhibitors. Fig.24 and Fig.25 The mass spectrometry data shown shows the production of cucurbitadienol. As shown, the peaks and fragments correspond to cucurbitadienol. The protein sequence and the DNA sequence encoding Cpep4 are provided in SEQ ID NO: 422 and 423, respectively.

[0537] The cucurbitadienol synthase protein sequence representing Cmax was obtained from the natural host, Cmaxima. The deduced coding DNA sequence will be used for gene synthesis and expression. The cucurbitadienol synthase protein sequence and the DNA encoding the cucurbitadienol synthase are shown below:

[0538] The following protein and DNA coding sequences were obtained by aligning genomic DNA PCR product sequences with known cucurbitadienol synthase sequences available from public databases (Pubmed). It is contemplated that any 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: 425) encoded by Cmax1 (DNA) (SEQ ID NO: 424).

[0539] The putative cucurbitadienol synthase protein sequence representing Cmos1 was obtained from the natural host Chinese pumpkin (Cucurbitamoschata). The deduced coding DNA sequence was used for gene synthesis and expression. The protein and DNA coding sequences shown below were obtained by aligning the genomic DNA PCR product sequence with the known cucurbitadienol synthase sequence available from the public database (Pubmed). Any of these Cmos proteins can be used in the methods, systems, components (e.g., host cells) disclosed herein to produce compound 1. A non-limiting exemplary Cmos1 protein is Cmos1 (protein) (SEQ ID NO: 426) encoded by Cmos1 (DNA) (SEQ ID NO: 427).

[0540] Example 57: Production of dihydroxycucurbitadienols in yeast (cucurbitadienol synthase and epoxide hydrolase)

[0541] It is contemplated that dihydroxycucurbitadienols may be produced in yeast using cucurbitadienol synthase and epoxide hydrolase. The natural host for these enzymes is S. grosvenorii.

[0542] Growth conditions: SgCbQ was co-expressed with epoxide hydrolase (EPH) in the presence of a lanosterol synthase inhibitor.

[0543] Possible dihydroxycucurbitadienol products such as Fig.26 shown.

[0544] The EPH protein sequence and DNA encoding the EPH protein (codon-optimized Saccharomyces cerevisiae) are shown in SEQ ID NOs: 428 and 429, respectively.

[0545] Example 58: Cytochrome P450 synthase, epoxide hydrolase, cytochrome P450 and cytochrome P450 Mogrosanol

[0546] Four enzymes were co-expressed in Saccharomyces cerevisiae, including cucurbitadienol synthase, epoxide hydrolase, cytochrome P450 and cytochrome P450 reductase. For growth conditions, SgCbQ, EPH, CYP87D18 and AtCPR (cytochrome P450 reductase from Arabidopsis thaliana) were co-expressed in the presence of lanosterol synthase inhibitor. It is expected that mogrosanol will be produced by Saccharomyces cerevisiae. The protein sequences and DNA sequences encoding SgCbQ, EPH, CYP87D18 and AtCPR (cytochrome P450 reductase from Arabidopsis 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).

[0547] Example 59: Compound 1 is resistant to microbial hydrolysis

[0548] Yeast strains Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida bombicola were incubated in YPD supplemented with 1 mg / ml mogroside V or compound 1. After 3 days, the supernatants were analyzed by HPLC.

[0549] As shown in the HPLC data, epoxide hydrolase hydrolyzed mogroside V to mogroside IIIE. No hydrolysis product was observed for compound 1 ( Fig.37 ).

[0550] Embodiment 60: Streptococcus mutans ( Streptococcus mutans ) ClarkeATCC25175 dextran sucrase

[0551] Streptococcus mutans Clarke can be grown anaerobically by supplementing glucose. An example of growth conditions can be found in Wenham, Henessey and Cole (1979), where the method is used to stimulate the production of, for example, dextran sucrase. 5 mg / ml mogroside IIIE is added to the growth medium. For example, HPLC monitoring time point samples can be used. The sequences of various dextran sucrases can be found in Table 1, which include the protein sequence of dextran sucrase and the nucleic acid sequence encoding dextran sucrase (for example, SEQ ID NO:157-162). In some embodiments, dextran sucrase comprises or is composed of the amino acid sequence of any one of SEQ ID NO:2, 103, 106-110, 156 and 896. In some embodiments, DexT comprises the amino acid sequence shown in SEQ ID NO:103. In some embodiments, DexT comprises the nucleic acid sequence shown in SEQ ID NO:104 or 105. In some embodiments, the recombinant cell herein encodes a protein comprising a sequence as shown in any one of SEQ ID NOs: 156-162, and / or comprises a nucleic acid sequence encoding a glucansucrase.This example is used to produce Compound 1.

[0552] Example 61: Production Procedure and Sensory Evaluation of 90% Pure Compound 1

[0553] By treating mogroside III with dextran sucrase / glucanase reaction E (MIII E ), followed by SPE fractionation to obtain fractions containing a mixture of three α-mogroside isomers. Based on UPLC analysis, the mixture had three isomers in a ratio of 5:90:5%, namely 11-oxo compound 1, compound 1 and mogroside V isomers, respectively. The three isomers were characterized by purification of different fractions / sources by LC-MS, 1D and 2D NMR spectra and by comparison of closely related isomers in the mogroside series reported in the literature. The sensory properties of the sample were further evaluated by comparison with a pure sample of compound 1 using a triangle test.

[0554] Enzyme reaction and purification steps

[0555] 100 mL of pH 5.5 1 M sodium acetate buffer, 200 g of sucrose, 100 mL of dextranase DexT (1 mg / ml crude extract, pET23a, BL21-Codon Plus-RIL, grown in 2X YT), 12.5 g of mogroside III E600mL water was added to a 2.8L shaking flask, and the flask was shaken at 30°C, 200rpm. The reaction process was regularly monitored by LC-MS. After 72 hours, the reaction was treated with 2.5mL of glucanase (Amano), and the flask was continued to shake at 30°C. After 24 hours, the reaction mixture was quenched under 80°C heating, and centrifuged at 5000rpm for 5 minutes, then the supernatant was filtered, directly added to a 400g C18SPE column, and MeOH:H2O 5 / 25 / 50 / 75 / 100 gradient washing was used. Each step of the gradient was collected in 6 wide-mouth bottles, and 225mL was collected in each wide-mouth bottle. The target product was eluted with 75% MeOH fraction (SPE 75_2) in a second tank, and dried under reduced pressure. It was further suspended / dissolved in 7mL H2O, frozen and lyophilized vials for 3 days, and 1.45g of white solid was obtained.

[0556] According to UPLC analysis ( Fig.28 and Fig.29 ), the mixture has three characterized α-mogroside isomers at a ratio of 5:90:5%, respectively; 11-oxo compound 1, compound 1 and mogroside V isomer. According to 1H and 13C-NMR (pyridine-d5+D2O) analysis, no residual solvent and / or structurally unrelated impurities were observed.

[0557] Sensory evaluation

[0558] A triangulation test of pure Compound 1 versus 90% pure Compound 1 was performed on November 10, 2016. Two different compositions were tested: (1) LSB + 175 ppm pure Compound 1 (standard) and (2) LSB + 175 ppm 90% pure Compound 1. All samples of the composition were prepared with low sodium buffer (LSB) at a pH of about 7.1 and containing 0% ethanol.

[0559] Conclusion: The experts found that there was no significant difference (p>0.05) between the composition (1) LSB + 175 ppm pure compound 1 (standard) and the composition (2) LSB + 175 ppm 90% pure compound 1 (test). Tables 2-4 list some test analysis results.

[0560] Table 2. Frequency of panelists who correctly selected other samples. n=38 (19 panelists x 2 reps).

[0561] sample total mistake 24 correct 14 total 38 Correct sample selected (p-value) 0.381

[0562] Table 3. Analysis results: test day

[0563] Theoretical #(μM) Observation (μM) 175ppm (155.51uM) pure compound 1 (standard) 132.20±1.54(n＝2) 175ppm (155.56uM) 90%_pure_compound 1 (test) 157.62±0.63(n＝2)

[0564] Table 4. Analysis results: one day before the test

[0565] Theoretical #(μM) Observation (μM) 175ppm (155.51uM) pure compound 1 (standard) 134.48±7.31(n＝2) 175ppm (155.56uM) 90%_pure_compound 1 (test) 140.69±4.34(n＝2)

[0566] Example 62: Gene Expression in Recombinant Yeast Cells

[0567] DNA was obtained by gene synthesis, either by Genescript, IDT or Genewiz. For some cucurbitadiene synthases, cDNA or genomic DNA was obtained from 10-60 day old seedlings and then PCR amplified using specific and degenerate primers. DNA was cloned into one of the following overexpression vectors: pESC-Ura, pESC-His or pESC-LEU by standard molecular biology techniques or yeast gap repair cloning (Joska et al., 2014). 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) containing 2% glucose or 2% galactose as appropriate to induce heterologous genes. Yeast strains were grown in shake flasks or 96-well plates at 30°C and 140-250 rpm. When indicated, the lanosterol synthase inhibitor Ro 48-8071 (Cayman Chemicals) (50 ug / ml) was added. Yeasts that produced mogrohol and its precursors were lysed (Yeast Buster), extracted with ethyl acetate, dried, and suspended in methanol. Samples were analyzed by the following LCMS method using an A / B gradient (A=H2O, B=acetonitrile):

[0568] For analysis of diepoxysqualene, the LCMS method included using a C18 2.1 x 50 mm column, 5% B for 1.5 min, gradient 5% to 95% B or 5.5 min, 95% B for 6 min, 100% B for 3 min, 5% B for 1.5 min, all at a flow rate of 0.3 ml / min.

[0569] For the analysis of cucurbitadienols, the first LCMS method included the use of a C4 2.1x 100mm column, gradient 1 to 95% B for 6 minutes, flow rate 0.55ml / min. The second LCMS method included the use of a Waters Acquity UPLC Protein BEH C4 2.1x100mM, 1.7um, with shield, 62% to 67% B for 2 minutes, 100% B for 1 minute, flow rate 0.9ml / min.

[0570] For the analysis of 11-OH cucurbitadienol, the LCMS method involved the use of a C8 2.1 x 100 mm column with a gradient from 60% to 90% B at a flow rate of 0.55 ml / min over 6 minutes.

[0571] For the analysis of mogrosanol, the LCMS method consisted of using a C8 2.1 x 100 mm column with a gradient from 50 to 90% B at a flow rate of 0.55 ml / min for 6 minutes.

[0572] For the analysis of mogroside IIIE and compound 1, the LCMS method involved the use of a fluorophenyl 2.1 x 100 mm column with a gradient from 15% to 30% B at a flow rate of 0.55 ml / min for 6 minutes.

[0573] Embodiment 63:

[0574] Step 1: Improve the utilization of squalene oxide

[0575] 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 cup1 (Peng et al., 2015). 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 enhancement of oxidized squalene was monitored by the production of diepoxysqualene as shown by HPLC and UV absorbance ( Fig.31 ).

[0576] tHMG-CoA (protein) SEQ ID NO: 898 (pathway 1)

[0577] tHMG-CoA (DNA) SEQ ID NO: 897 (Pathway 1)

[0578] Erg1 (protein) SEQ ID NO: 900; Erg1 (DNA) SEQ ID NO: 899

[0579] In some embodiments, tHMG-CoA enzyme is used to produce diepoxysqualene.

[0580] The gene of the putative squalene epoxidase in S. grosvenorii (Itkins et al., 2016) was selected to promote the production of oxidized squalene / diepoxysqualene. The amino acid sequences and coding sequences of three squalene epoxidases are listed in Table 1 (SEQ ID NO: 50-56, 60, 61, 334 or 335). Other sequences of squalene epoxidase suitable for use in the methods, systems and compositions disclosed herein 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. The above methods, systems and compositions are used to produce oxidized squalene and / or diepoxy squalene, and to increase the production of oxidized squalene and / or diepoxy squalene.

[0581] Step 2. Production of cucurbitadienol

[0582] Cucurbitadienol synthase

[0583] The plasmid containing the S. grosvernorii cucurbitadienol synthase gene (SGCBQ) was transformed into a yeast strain that had increased squalene oxidation. The strain was grown at 30°C, 150-250 rpm for 1-3 days. The production of cucurbitadienol was shown in the HPLC and mass spectrometry data, which showed the mass peak of the product shown ( Fig.23 SEQ ID NO: 446 and SEQ ID NO: 418 provide the SgCbQ protein and the gDNA encoding SgCbQ, respectively. Cucurbita pepo (Jack O'Lantern) protein Cpep2 was also used to produce cucurbitadienol in yeast. Fig.24 A mass spectrum is shown, which contains peaks and characteristic fragments corresponding to cucurbitadienol. The Cpep2 protein and DNA 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 growth conditions without inhibitors. Fig.25The mass spectrometry data shown demonstrates the production of cucurbitadienol. 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. Cucurbita maxima protein Cmax was also used to produce cucurbitadienol in yeast. Fig.32 A mass spectrum is shown, containing peaks and characteristic fragments corresponding to cucurbitadienol. The Cmax1 protein sequence is provided in SEQ ID NO: 424, and the coding sequence for Cmax1 (D...

Claims

1. A method for producing a compound 1 having the following structure: The method includes: Mogroside III E and can catalyze the E A first enzyme contact produces Compound 1.

2. A compound having the structure of compound 1: The compound is prepared by the method according to claim 1.

3. A cell lysate comprising a compound 1 having the following structure:

4. A recombinant cell comprising: a compound 1 having the following structure: and a gene encoding an enzyme capable of catalyzing the production of compound 1 from mogroside IIIE.

5. A recombinant cell comprising a first gene encoding a first enzyme capable of catalyzing the production of compound 1 having the following structure from mogroside IIIE:

6. A method for producing a compound 1 having the following structure: The method includes: contacting the first mogroside with one or more hydrolases to produce mogroside III E ; and Mogroside III E and can catalyze the E The enzyme that produces Compound 1 is contacted.

7. A recombinant cell comprising: The first gene, which encodes a protein capable of hydrolyzing the first mogroside to produce mogroside III E hydrolases; and The second gene encodes a protein that catalyzes the conversion of mogroside III E An enzyme that produces compound 1, wherein compound 1 has the following structure:

8. A fusion polypeptide having cucurbitadienol synthase activity, wherein the fusion polypeptide comprises a fusion domain fused to cucurbitadienol synthase.

9. A recombinant nucleic acid molecule comprising a nucleic acid sequence encoding the fusion polypeptide of claim 8. 10 . A recombinant cell comprising the fusion polypeptide of claim 8 or the recombinant nucleic acid molecule of claim 9 .

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