PRODUCTION OF ISOSTEVIOL BY ACID HYDROLYSIS

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/169,979, filed on April 2, 2021 and entitled “PRODUCTION OF ISOSTEVIOL BY ACID HYDROLYSIS,” the entire contents of which are incorporated herein by reference.

FIELD OF THE INVENTION

The field of the invention relates to methods and processes useful in the production of isosteviol via acid hydrolysis.

BACKGROUND

Isosteviol is a stable, aglycon of several steviol glycosides that has been shown to exhibit several pharmaceutical and medicinal features. However, the extent to which isosteviol achieves these desired effects is limited by the method by which isosteviol is synthesized and isolated.

Generally, isosteviol is produced by conversion from a steviol glycoside in the presence of concentrated hydrochloric acid or hydrobromic acid. These strong acids remove sugar groups via hydrolysis from steviol glycosides, resulting in the aglycon steviol. Steviol further undergoes a Wagner-Meerwein rearrangement to form the stable isosteviol. The production of isosteviol from steviol glycosides via acid hydrolysis requires the use of highly corrosive and toxic materials and often results in impure final product and toxic by-products.

SUMMARY

A need exists in the field for the development of a novel method for the large-scale production of isosteviol that can be performed economically and conveniently (e.g., without the use of highly corrosive and toxic materials) to further enable the application of isosteviol for pharmaceutical and medicinal purposes. The present disclosure, in some aspects, relate to methods of producing isosteviol from various steviol glycosides (e.g., rubusoside, stevioside, and/or rebaudiosides) via acid hydrolysis using sulfuric acid. In some embodiments, the molar ratio between sulfuric acid and the steviol glycoside is optimized for efficient production of isosteviol. Some aspects of the present disclosure provide methods of producing isosteviol, the method comprising:

(i) preparing a reaction mixture comprising:

(a) a steviol glycoside;

(b) sulfuric acid; and

(c) an organic solvent; and

(ii) incubating the reaction mixture for a sufficient time to produce isosteviol; wherein the isosteviol has the structure of:

In some embodiments, the reaction mixture comprises 2%-10% (v/v) of sulfuric acid, optionally wherein the reaction mixture comprises 5% (v/v) of sulfuric acid.

In some embodiments, the organic solvent is selected from the group consisting of: methanol and ethanol. In some embodiments, the organic solvent is methanol.

In some embodiments, the steviol glycoside is selected from the group consisting of: rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside M, and combinations thereof.

In some embodiments, the steviol glycoside is rubusoside having the structure of:

In some embodiments, the molar ratio between rubusoside and the sulfuric acid is between 1:3 and 1:5. In some embodiments, the molar ratio between rubusoside and sulfuric acid is 1:3.8.

In some embodiments, the steviol glycoside is stevioside having the structure of:

In some embodiments, the molar ratio between stevioside and the sulfuric acid is between 1:3 and 1:5. In some embodiments, the molar ratio between stevioside and sulfuric acid is 1:3.8.

In some embodiments, the steviol glycoside is rebaudioside A having the structure of:

In some embodiments, the molar ratio between rebaudioside A and the sulfuric acid is between 1:3 and 1:5. In some embodiments, the molar ratio between rebaudioside A and sulfuric acid is 1:3.8.

In some embodiments, the reaction mixture is incubated at a temperature between 60°C and 80°C for at least one hour. In some embodiments, the reaction mixture is incubated at 75°C for at least one hour. In some embodiments, the methods further comprise collecting the isosteviol. In some embodiments, at least 70% of the steviol glycoside is converted to isosteviol. In some embodiments, the reaction mixture comprises at least 70% (w/v) isosteviol.

Further provided herein is a composition comprising a steviol glycoside, sulfuric acid, and organic solvent, and isosteviol. In some embodiments, the organic solvent is methanol. In some embodiments, the steviol glycoside is selected from the group consisting of: rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside M, and combinations thereof. In some embodiments, the isosteviol has a purity of a least 70% (w/v). In some embodiments, the isosteviol is for medicinal use.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In the drawings, each identical or nearly identical component that is illustrated in various figures is represented by a like numeral. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

FIGs. 1A-C. Production of isosteviol by acid hydrolysis. FIG. 1A shows the production of isosteviol from rubusoside by acid hydrolysis. FIG. IB shows the production of isosteviol from stevioside by acid hydrolysis. FIG. 1C shows the production of isosteviol from rebaudioside A by acid hydrolysis.

FIGs. 2A-2D. Total ion chromatogram (TIC) profile for isosteviol. FIG2A shows the TIC profile for the isosteviol standard. FIG. 2B shows the TIC profile for isosteviol converted by acid hydrolysis from stevioside. FIG. 2C show the TIC profile for isosteviol converted by acid hydrolysis from rebaudioside A. FIG. 2D shows the TIC profile for isosteviol converted by acid hydrolysis from rubusoside.

FIGs. 3A-3D. LC-MS profile for isosteviol. FIG. 3A shows the LC-MS profile for the isosteviol standard. FIG. 3B shows the LC-MS profile for isosteviol converted by acid hydrolysis from stevioside. FIG. 3C show the LC-MS profile for isosteviol converted by acid hydrolysis from rebaudioside A. FIG. 3D shows the LC-MS profile for isosteviol converted by acid hydrolysis from rubusoside.

FIG. 4. The structure of isosteviol.

FIG. 5. ’H-NMR spectrum for isosteviol. FIG. 5 shows a ID ’H-NMR spectrum for isosteviol, with TMS as an internal standard, using standard pulse sequences. This analysis was performed in CDCI3.

FIG. 6. ¹³ C-NMR spectrum for isosteviol. FIG. 6 shows a ID ¹³ C-NMR spectrum for isosteviol, with TMS as an internal standard, using standard pulse sequences. This analysis was performed in CDCI3.

FIG. 7. HSQC spectrum for isosteviol. FIG. 7 shows an HSQC spectrum for isosteviol, with TMS as an internal standard, using standard pulse sequences. This analysis was performed in CDCI3.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present disclosure, the preferred materials and methods are described below.

The terms “incubating” and “incubation” as used herein refers to a process of mixing two or more chemical or biological entities (such as a chemical compound and an enzyme) and allowing them to interact under conditions favorable for producing a steviol glycoside composition. The term “medicinal use” as used herein refers to uses as an antibacterial, antifungal, antibiofilm, antioxidant, and anticancer agent.

As used herein, the term “w/v” refers to the weight of a compound (in grams) for every 100 ml of a liquid or aqueous solution containing such compound. As used herein, the term “NIN” refers to the volume of a compound (in milliliters) for every 100 ml of a liquid or aqueous solution containing such compound.

As used herein, the singular forms "a, an" and "the" include plural references unless the content clearly dictates otherwise.

To the extent that the term "include," "have," or the like is used in the description or the claims, such term is intended to be inclusive in a manner similar to the term "comprise" as "comprise" is interpreted when employed as a transitional word in a claim.

The word "exemplary" is used herein to mean "serving as an example, instance, or illustration”. Any embodiment described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

DETAILED DESCRIPTION

The present disclosure relates, at least in part, to the synthesis and production of isosteviol from various steviol glycosides (e.g., rubusoside, stevioside, and/or rebaudiosides) via acid hydrolysis using sulfuric acid. In some embodiments, the molar ratio between sulfuric acid and the steviol glycoside is optimized for efficient production of isosteviol.

Acid hydrolysis of steviol glycosides

Steviol glycosides are the chemical compounds responsible for the sweet taste of the leaves of the South American plant Stevia rebaudiana (Asteraceae') and in the plant Rubus chingii (Rosaceae'). These compounds are glycosylated diterpenes. Specifically, their molecules can be viewed as a steviol molecule, with its hydroxyl hydrogen atom replaced by a glucose molecule to form an ester, and a hydroxyl hydrogen with combinations of glucose and rhamnose to form an acetal. Isosteviol is a derivative of steviol glycosides that can be used for a variety of pharmaceutical and medicinal purposes.

Some aspects of the present disclosure provide methods of producing isosteviol, the method comprising:

(i) preparing a reaction mixture comprising:

(a) a steviol glycoside; (b) sulfuric acid; and

(c) an organic solvent; and

(ii) incubating the reaction mixture for a sufficient time to produce isosteviol.

The term “isosteviol,” as used herein, refers to a tetracyclic diterpenoid (enZ-16- oxobeyran- 19-oic acid) derived from the aglycon steviol following acid hydrolysis. In some embodiments, the isosteviol produced using the method described herein has the structure of:

An “aglycon,” as used herein, refers to a non-sugar compound remaining after replacement of the glycosyl group from a glycoside by a hydrogen atom.

A “reaction mixture,” as used herein, refers to a mixture of reactants in a suitable solvent such that a chemical reaction can occur to completion or nearly to completion.

In some embodiments, the method described herein comprises preparing a reaction mixture comprising: (a) a steviol glycoside; (b) sulfuric acid; and (c) an organic solvent.

In some embodiments, the steviol glycoside is selected from the group consisting of: rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside M, and combinations thereof. In some embodiments, the reaction mixture comprises one or more (e.g., 1, 2, 3, 4 ,5, 6, 7, 8, or more) steviol glycosides. In some embodiments, the reaction mixture comprises one or more (e.g., 1, 2, 3, 4 ,5, 6, 7, 8, or more) steviol glycosides selected from the group consisting of: rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside M.

“Sulfuric acid,” as used herein, refers to a mineral acid composed of the elements sulfur, oxygen, and hydrogen, with the molecular formula H2SO4. Sulfuric acid is a strong acid that complete dissociates in a suitable solvent such that the elemental hydrogen atoms dissociate from the molecule resulting in freely available hydrogen protons.

An “organic solvent,” as used herein, refers to a carbon-based substance that dissolves reactants resulting in a solution or reaction mixture. Organic solvents facilitate the interaction between reactants in a reaction mixture allowing for the completion or near completion of a chemical reaction. Suitable organic solvents for use in the reaction mixture described herein include, without limitation: methanol, ethanol. In some embodiments, the organic solvent is methanol.

In some embodiments, the reaction mixture comprises 2% to 10% (v/v) of sulfuric acid. For example, the reaction mixture comprises 2% to 10% (v/v), 2% to 8% (v/v), 2% to 5% (v/v), 5% to 10% (v/v), 5% to 8% (v/v), or 8% to 10% (v/v) of sulfuric acid. In some embodiments, the reaction mixture comprises 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v/v) of sulfuric acid. In some embodiments, the reaction mixture comprises 5% (v/v) of sulfuric acid.

In some embodiments, in the reaction mixture, the molar ratio between the steviol glycoside and the sulfuric acid is between 1:1 and 1:10. For example, the molar ratio between the steviol glycoside and the sulfuric acid in the reaction mixture is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the molar ratio between the steviol glycoside and the sulfuric acid in the reaction mixture is 1:4.

In some embodiments, the steviol glycoside is rubusoside having the structure of:

In some embodiments, in the reaction mixture, the molar ratio between the rubusoside and the sulfuric acid is between 1:1 and 1:10. For example, the molar ratio between the rubusoside and the sulfuric acid in the reaction mixture is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the molar ratio between the rubusoside and the sulfuric acid in the reaction mixture is 1:4.

In some embodiments, the steviol glycoside is stevioside having the structure of:

In some embodiments, the molar ratio between the stevioside and the sulfuric acid is between 1:1 and 1:10. For example, the molar ratio between the stevioside and the sulfuric acid is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the molar ratio between the stevioside and the sulfuric acid is 1:4.

In some embodiments, in the reaction mixture, the steviol glycoside is rebaudioside A having the structure of:

In some embodiments, the molar ratio between the rebaudioside A and the sulfuric acid in the reaction mixture is between 1:1 and 1:10. For example, the molar ratio between the rebaudioside A and the sulfuric acid in the reaction mixture is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In some embodiments, the molar ratio between the rebaudioside A and the sulfuric acid in the reaction mixture is 1:4.

In some embodiments, the reaction mixture is incubated at 50°C to 120°C. For example, the reaction mixture is incubated at 50°C to 120°C, 55°C to 115°C, 60°C to 110°C, 65°C to 105°C, 70°C to 100°C, 75°C to 95°C, or 80°C to 90°C. In some embodiments, the reaction mixture is incubated at 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 105°C, 110°C, 115°C, or 120°C. In some embodiments, the reaction mixture is incubated at 75°C.

In some embodiments, the reaction mixture is incubated for 1 hour to 10 hours. For example, the reaction mixture is incubated for 1 hour to 10 hours, 2 hours to 9 hours, 3 hours to 8 hours, 4 hours to 7 hours, or 5 hours to 6 hours. In some embodiments, the reaction mixture is incubated for at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, or at least 10 hours. In some embodiments, the reaction mixture is incubated for at least 1 hour.

In some embodiments, the reaction mixture is incubated at 75°C for at least 1 hour (e.g., 1, 2, 3, 4, 5 hours or more).

In some embodiments, the method described herein further comprises collecting the isosteviol. The term “collecting the isosteviol,” as used herein, refers to the process of isolating crystallized isosteviol from a reaction mixture or composition in some embodiments. For example, after the chemical reaction described herein, the reaction mixture is incubated at room temperature overnight. The reaction mixture is then mixed with 3 L of water and stirred overnight. The crude reaction mixture is then filtered and mixed with 25% methanol, followed by incubation at 70°C for 3 hours. After incubation, the crude mixture is dissolved in 400 ml of 100% ethanol and crystallized overnight at 5-10°C. Finally, pure, crystallized isosteviol is collected by filtration.

The term “room temperature,” as used herein, refers to the ambient temperature of 20°C to 25°C. For example, the ambient temperature of 20°C to 25°C, 21 °C to 24°C, or 22°C to 23°C. For example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C. For example, the ambient temperate of 25 °C.

The term “overnight,” as used herein, refers to 5 hours to 16 hours. For example, 5 hours to 16 hours, 6 hours to 15 hours, 7 hours to 14 hours, 8 hours to 13 hours, 9 hours to 12 hours, 10 hours to 11 hours. For example, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, or 16 hours. For example, 14 hours.

In some embodiments, 50% to 100% of the steviol glycoside is converted to isosteviol. For example, 50% to 100%, 55% to 95%, 60% to 90%, 65% to 85%, or 70% to 80% of the steviol glycoside is converted to isosteviol. In some embodiments, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the steviol glycoside is converted to isosteviol. In some embodiments, at least 70% of the steviol glycoside is converted to isosteviol. The term “converted to isosteviol,” as used herein, refers to a chemical reaction in which a reactant, such as a steviol glycoside, interacts with another reactant, such as a strong acid, to produce a product, such as isosteviol.

In some embodiments, the reaction mixture comprises 50% to 99% (w/v) isosteviol. For example, the reaction mixture comprises 50% to 99% (w/v), 55% to 95% (w/v), 60% to 90% (w/v), 65% to 85% (w/v), or 70% to 80% (w/v) isosteviol. In some embodiments, the reaction mixture comprises 50% (w/v), 55% (w/v), 60% (w/v), 65% (w/v), 70% (w/v), 75% (w/v), 80% (w/v), 85% (w/v), 90% (w/v), 95% (w/v), or 99% (w/v) isosteviol. In some embodiments, the reaction mixture comprises at least 70% (w/v) isosteviol.

Other aspects of the present disclosure provide a composition comprising a steviol glycoside, sulfuric acid, an organic solvent, and isosteviol. In some embodiments, the organic solvent is methanol, ethanol, or another suitable organic solvent. In some embodiments, the organic solvent is methanol. In some embodiments, the steviol glycoside is selected from the group consisting of: rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside M, and combinations thereof.

In some embodiments, the reaction mixture is in a flask, i.e., the method described herein is performed in a flask. For organic reactions, the strong acid can be added to the steviol glycoside substrate and organic solvent in a round-bottomed flask to create a reaction mixture.

In some embodiments, a reflux condenser is fitted to the round-bottomed flask and the flask is placed on a heating plate to raise the temperature of the reaction mixture.

In some embodiments, the reaction mixture is heated to 75°C for at least one hour, then transferred to another flask containing water and stirred overnight. In some embodiments, the reaction mixture is filtered and the crude isosteviol product is collected. In some embodiments, the crude isosteviol product is washed with 25% methanol and dried for 3 hours at 70°C. In some embodiments, the dried isosteviol product is dissolved in 400 ml of 100% ethanol and crystallized overnights at 5°C-10°C.

In some embodiments, the isosteviol of the composition has a purity of 50% to 100%. For example, the isosteviol of the composition has a purity of 50% to 100%, 55% to 95%, 60% to 90%, 65% to 85%, or 70% to 80%. In some embodiments, the isosteviol of the composition has a purity of 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In some embodiments, the isosteviol of the composition has a purity of at least 70%.

In some embodiments, the isosteviol of the composition is for medicinal use. The term “medicinal use,” as used herein, refers to the application of a product to treat, cure, or otherwise attenuate a medical condition or ailment. Non-limiting examples of medicinal uses include treating, curing, or otherwise attenuating a bacterial infection, an infection by biofilm, a fungal infection, a disease or ailment of aberrant oxidation, or cancer.

Steviol glycosides and Synthetic Biology

As previously stated steviol glycosides are the chemical compounds responsible for the sweet taste of the leaves of the South American plant Stevia rebaudiana (Asteraceae') and in the plant Rubus chingii (Rosaceae'). These compounds are glycosylated diterpenes. Specifically, their molecules can be viewed as a steviol molecule, with its hydroxyl hydrogen atom replaced by a glucose molecule to form an ester, and a hydroxyl hydrogen with combinations of glucose and rhamnose to form an acetal.

Steviol glycosides (e.g., rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside M, and combinations thereof) are used as substrates in the methods described herein for producing isosteviol. Steviol glycosides may be produced using biosynthetic pathways known in the art.

In some embodiments, the steviol glycosides may be produced using methods involving recombinantly expressing enzymes in a microbial system capable of producing steviol. In general, such enzymes may include: a copalyl diphosphate synthase (CPS), a kaurene synthase (KS) and a geranylgeranyl diphosphate to synthase (GGPPS) enzyme. Preferably, in some embodiments, this occurs in a microbial strain that expresses an endogenous isoprenoid synthesis pathway, such as the non-mevalonate (MEP) pathway or the mevalonic acid pathway (MVA). In some embodiments, the cell is a bacterial cell, such as E. coli, or a yeast cell, such as a Saccharomyces cell, Pichia cell, or a Yarrowia cell. In some embodiments, the cell is an algal cell or a plant cell.

Thereafter, the precursor can be recovered from the fermentation culture and used in chemical synthesis. Typically, this is steviol though it can be kaurene, or a steviol glycoside from the cell culture. In some embodiments, the steviol, kaurene and/or steviol glycosides is recovered from the gas phase while in other embodiments, an organic layer or polymeric resin is added to the cell culture, and the kaurene, steviol and/or steviol glycosides is recovered from the organic layer or polymeric resin. In some embodiments, the steviol glycoside is selected from rubusoside, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, and rebaudioside M, and combinations thereof. In some embodiments, the terpenoid produced is steviobioside or stevioside. It should also be appreciated that in some embodiments, at least one enzymatic step, such as one or more glycosylation steps, are performed ex vivo.

As described herein, the enzymes for producing steviol glycosides have UDP- glycosyltransferase activities. The substrate for producing steviol glycosides can be any natural or synthetic compound capable of being converted into a steviol glycoside compound in a reaction catalyzed by one or more UDP-glucosyltransferases. For example, the substrate can be natural stevia extract, steviol, steviol-13-O-glucoside, steviol- 19-O-glucoside, 2- bioside, rubusoside, stevioside, rebaudioside A, rebaudioside D, rebaudioside D3, or rebaudioside E. The substrate can be a pure compound or a mixture of different compounds.

In some embodiments, the methods for producing steviol glycosides involve a coupling reaction system in which the enzymes (e.g., UDP transferases) described herein can function in combination with one or more additional enzymes (e.g., sucrose synthase) to improve the efficiency or modify the outcome of the overall biosynthesis of steviol glycoside compounds. For example, the additional enzyme may regenerate the UDP-glucose needed for the glycosylation reaction by converting the UDP produced from the glycosylation reaction back to UDP-glucose (using, for example, sucrose as a donor of the glucose residue), thus improving the efficiency of the glycosylation reaction.

Sucrose synthase catalyzes the chemical reaction between UDP-glucose and D-fructose to produce UDP and sucrose. Sucrose synthase is a glycosyltransferase. The systematic name of this enzyme class is UDP-glucose:D-fructose 2-alpha-D-glucosyltransferase. Other names in common use include UDP glucose-fructose glucosyltransferase, sucrose synthetase, sucrose- UDP glucosyltransferase, sucrose-uridine diphosphate glucosyltransferase, and uridine diphosphoglucose-fructose glucosyltransferase. Addition of the sucrose synthase to the reaction mixture that includes a uridine diphospho glycosyltransferase creates a “UGT-SUS coupling system”. In the UGT-SUS coupling system, UDP-glucose can be regenerated from UDP and sucrose, which allows for omitting the addition of extra UDP-glucose to the reaction mixture or using UDP in the reaction mixture.

Suitable sucrose synthase for use in the methods described herein include Arabidopsis sucrose synthase I, an Arabidopsis sucrose synthase 3 and a Vigna radiate sucrose synthase. In some embodiments, the sucrose synthase or sucrose synthase domain is an Arabidopsis thaliana sucrose synthase I.

Suitable UDP-glycosyltransferase includes any UGT known in the art as capable of catalyzing one or more reactions in the biosynthesis of steviol glycoside compounds, such as UGT85C2, UGT74G1, HV1, UGT76G1, or the functional homologs thereof. In some embodiments, the UDP-glycotransferase is UGT76G1. In some embodiments, the UDP- glycotransferase is a UGT76Gl-sucrose synthase fusion enzyme. In some embodiments, the UDP-glycotransferase is HV1 UTG. In some embodiments, the UDP-glycotransferase is a HV1 UGT-sucrose synthase fusion enzyme.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described, for example, by Sambrook, J., Fritsch, E. F. and Maniatis, T. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1989 (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W. EXPERIMENTS WITH GENE FUSIONS; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y., 1984; and by Ausubel, F. M. et al., IN CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, published by Greene Publishing and Wiley-Interscience, 1987; (the entirety of each of which is hereby incorporated herein by reference).

Expression of proteins in prokaryotes is most often carried out in a bacterial host cell with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: (1) to increase expression of recombinant protein; (2) to increase the solubility of the recombinant protein; and (3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such vectors are within the scope of the present disclosure.

In an embodiment, the expression vector includes those genetic elements for expression of the recombinant polypeptide in bacterial cells. The elements for transcription and translation in the bacterial cell can include a promoter, a coding region for the protein complex, and a transcriptional terminator. A standard E. coli expression system was used as described herein.

A person of ordinary skill in the art will be aware of the molecular biology techniques available for the preparation of expression vectors. The polynucleotide used for incorporation into the expression vector of the subject technology, as described above, can be prepared by routine techniques such as polymerase chain reaction (PCR).

A number of molecular biology techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

In an alternative embodiment, synthetic linkers containing one or more restriction sites provide are used to operably link the polynucleotide of the subject technology to the expression vector. In an embodiment, the polynucleotide is generated by restriction endonuclease digestion. In an embodiment, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3'-single-stranded termini with their 3'-5'-exonucleolytic activities and fill-in recessed 3'-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the product of the reaction is a polynucleotide carrying polymeric linker sequences at its ends. These polynucleotides are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the polynucleotide.

Alternatively, a vector having ligation-independent cloning (LIC) sites can be employed. The required PCR amplified polynucleotide can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, NUCL. ACID. RES. 18 6069-74, (1990), Haun, et al, BIOTECHNIQUES 13, 515-18 (1992), which is incorporated herein by reference to the extent it is consistent herewith).

In an embodiment, in order to isolate and/or modify the polynucleotide of interest for insertion into the chosen plasmid, it is suitable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuclease or LIC sites, place the coding region in the desired reading frame.

In an embodiment, a polynucleotide for incorporation into an expression vector of the subject technology is prepared by the use of PCR using appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become incorporated into the amplified sequence product. In an embodiment, the amplification primers contain restriction endonuclease recognition sites, which allow the amplified sequence product to be cloned into an appropriate vector.

The expression vectors can be introduced into plant or microbial host cells by conventional transformation or transfection techniques. Transformation of appropriate cells with an expression vector of the subject technology is accomplished by methods known in the art and typically depends on both the type of vector and cell. Suitable techniques include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, chemoporation or electroporation.

Successfully transformed cells, that is, those cells containing the expression vector, can be identified by techniques well known in the art. For example, cells transfected with an expression vector of the subject technology can be cultured to produce polypeptides described herein. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art.

The host cells can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.

In some embodiments, the transformed cell is an animal cell, an insect cell, a plant cell, an algal cell, a fungal cell, or a yeast cell. In some embodiments, the cell is a plant cell selected from the group consisting of: canola plant cell, a rapeseed plant cell, a palm plant cell, a sunflower plant cell, a cotton plant cell, a corn plant cell, a peanut plant cell, a flax plant cell, a sesame plant cell, a soybean plant cell, and a petunia plant cell.

Microbial host cell expression systems and expression vectors containing regulatory sequences that direct high-level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct vectors for expression of the recombinant polypeptide of the subjection technology in a microbial host cell. These vectors could then be introduced into appropriate microorganisms via transformation to allow for high level expression of the recombinant polypeptide of the subject technology.

Vectors or cassettes useful for the transformation of suitable microbial host cells are well known in the art. Typically the vector or cassette contains sequences directing transcription and translation of the relevant polynucleotide, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the polynucleotide which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. It is preferred for both control regions to be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions need not be derived from the genes native to the specific species chosen as a host.

Initiation control regions or promoters, which are useful to drive expression of the recombinant polypeptide in the desired microbial host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genes is suitable for the subject technology including but not limited to CYCI, HIS3, GALI, GALIO, ADHI, PGK, PH05, GAPDH, ADCI, TRPI, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOXI (useful for expression in Pichidy, and lac, trp, JPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli).

Termination control regions may also be derived from various genes native to the microbial hosts. A termination site optionally may be included for the microbial hosts described herein.

In plant cells, the expression vectors of the subject technology can include a coding region operably linked to promoters capable of directing expression of the recombinant polypeptide of the subject technology in the desired tissues at the desired stage of development. For reasons of convenience, the polynucleotides to be expressed may comprise promoter sequences and translation leader sequences derived from the same polynucleotide. 3' non-coding sequences encoding transcription termination signals should also be present. The expression vectors may also comprise one or more introns in order to facilitate polynucleotide expression.

For plant host cells, any combination of any promoter and any terminator capable of inducing expression of a coding region may be used in the vector sequences of the subject technology. Some suitable examples of promoters and terminators include those from nopaline synthase (nos), octopine synthase (ocs) and cauliflower mosaic virus (CaMV) genes. One type of efficient plant promoter that may be used is a high-level plant promoter. Such promoters, in operable linkage with an expression vector of the subject technology should be capable of promoting the expression of the vector. High level plant promoters that may be used in the subject technology include the promoter of the small subunit (s) of the ribulose-1,5- bisphosphate carboxylase for example from soybean (Berry-Lowe et al., J. MOLECULAR AND APP. GEN., 1:483 498 (1982), the entirety of which is hereby incorporated herein to the extent it is consistent herewith), and the promoter of the chlorophyll binding protein. These two promoters are known to be light-induced in plant cells (see, for example, GENETIC ENGINEERING OF PLANTS, AN AGRICULTURAL PERSPECTIVE, A. Cashmore, Plenum, N.Y. (1983), pages 29 38; Coruzzi, G. et al., The Journal of Biological CHEMISTRY, 258: 1399 (1983), and Dunsmuir, P. et al., JOURNAL OF MOLECULAR AND APPLIED GENETICS, 2:285 (1983), each of which is hereby incorporated herein by reference to the extent they are consistent herewith).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred materials and methods are described herein.

The disclosure will be more fully understood upon consideration of the following nonlimiting Examples. It should be understood that these Examples, while indicating preferred embodiments of the subject technology, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of the subject technology, and without departing from the spirit and scope thereof, can make various changes and modifications of the subject technology to adapt it to various uses and conditions.

EXAMPLES

Example 1. Production of Isosteviol by Acid Hydrolysis

According to the methods provided herein, 50 mL of concentrated sulfuric acid (98%) was added to IL of methanol in a 2L, 3-necked round-bottomed flask and stirred. After five minutes of stirring, 201g of stevioside was added to the solution and stirred. A reflux condenser was fitted to the flask and slowly heated to 75 °C until reflux was achieved. After two hours, the reaction was cooled to room temperature overnight. The reaction mixture was transferred into a 5L flask containing 3L of water and stirred overnight. The crude isosteviol product was collected by filtration and washed with 25% ethanol. The washed isosteviol product was dried for three hours at 70°C. The dried isosteviol product was dissolved in 400mL of 100% ethanol and crystallized at 5-10°C overnight. The crystallized isosteviol product was collected by filtration. Filtration yielded pure colorless crystals of isosteviol.

Rubusoside and rubaudioside A can be converted to pure isosteviol following the same protocol (FIG. 1).

Example 2. Identification of Isosteviol by LC-MS

The LC-MS system consisted of a Dionex Ultimate UPLC 3000 system (Thermo Fisher, Germany) and a Thermo Scientific Q-Exactive high resolution hybrid Quadrupoleorbitrap mass spectrometer (Thermo Fisher, Germany). The chromatographic separation was performed using an Hypersil Gold column (1.9 pm, 2.1 mm x 50 mm, Thermo fisher) at 25 °C with mobile phase delivered at a flow rate of 0.3 mL/min. The mobile phase was 0.1% formic acid in water (MPA) and 0.1% formic acid in acetonitrile (MPB). The gradient condition for MPB was hold at 55% over 0.3 min; from 5% to 90% over 10 min; hold at 90% for 2 min, then 0.1 min back to 5%, then hold at 5% for 2min to re-equilibrate; total 14.3 min. A volume of 5 |jL of sample was injected and the full scan discovery data was acquired.

The mass spectrometer was operated in positive ESI MS mode in 70,000 full-width at half height maximum (FWHM) resolution (12 scans/second) with mass range m/z 150-1000. The operation conditions were as follows: spray voltage: 2.7 kV; capillary temperature: 300°C; S-lens RF level: 60; auxiliary gas heater temperature: 320°C. Nitrogen was used as the sheath gas, auxiliary gas and sweep gas, set at 40, 8 and 2, respectively (in arbitrary units). The external mass calibration for mass accuracy was performed according to manufacturer’s guidelines.

By total ion chromatogram, the reference isosteviol standards (FIG. 2A) showed identical retention time as products from stevioside (Fig. 2B), rebaudioside A (Fig. 2C) and rubusoside (FIG. 2D) at around 7.87 min. In the spectra of mass over charge (m/z) in positive mode, all reference standard (FIG. 3A) and products (FIG. 3B - FIG. 3D) showed an [M+H] ⁺ adduct ion at m/z 319.22.

Example 3. Structure of Isosteviol by NMR Analysis

NMR analysis was performed to confirm the molecular structure of the purified isosteviol.

NMR spectra were acquired on a Bruker Avance DRX 500 MHz instrument, with tetramethylsilane (TMS) as an internal standard, using standard pulse sequences. The ID ( ’ H and ¹³ C) and HSQC NMR spectra were performed in CDCh (FIG. 5 - FIG. 7).

The molecular formula of isosteviol product has been deduced as C20H30O3 on the basis of its positive high resolution (HR) mass spectrum which showed an [M+H] ⁺ adduct ion at m/z 319.22, which is the same as the isosteviol standard. This composition is supported by ¹³ C NMR spectral data. The ¹ H NMR spectrum of isosteviol showed the presence of three methyl singlets at 6 0.76, 0.97, and 1.24, more one than aglycon of original glycosides; eight methylene, less one than aglycon of original glycosides, and two methine protons between 6 0.93-2.64; absence of two olefinic protons of an exocyclic double bond, differently characteristic for aglycon of most of enZ-kaurane diterpenoids isolated earlier from the genus Stevia.

Compared to steviol, the ¹³ C NMR spectrum for isosteviol showed the presence of one carboxyl group singlet at 6 184.2 one more carbonyl group singlet at 223.0, and absence of two olefinic carbons of an exocyclic double bond. A comparison of the ¹ H and ¹³ C NMR spectrum of isosteviol to steviol suggested that isosteviol is one isomer of steviol (FIG. 4). The ’H and ¹³ C NMR values for protons and carbons in isosteviol were assigned on the basis of HSQC (Table 1), in agreement with the literature reported.

Table 1. ’H (500 MHz) and ¹³ C-NMR (125 MHz in CDCh) signal assignment