BACKGROUND OF THE INVENTION

Steviol glycosides are a class of compounds that occur naturally in plants of the species Stevia rebaudiana and that are of commercial interest due to their intense sweet taste. Extracts of S. rebaudiana are marketed as zero calorie sweeteners for use in food and beverages. These extracts contain a mixture of steviol glycosides that differ in their taste properties. All share a common aglycon (FIG. 1) and differ in the number and position of glucose residues.

Rebaudioside A (RebA) is one of the main components of commercially available stevia extracts. In RebA, the aglycon carries three glucose residues on the Rl position and one glucose residue on the R2 position. Rebaudioside D (RebD) and Rebaudioside M (RebM) have superior taste properties compared to RebA, but only occur in small amounts in stevia extracts. RebD carries two glucose residues on R2 and RebM carries three glucose residues on R2, respectively. They can be presumably synthesized from RebA in reactions that are catalyzed by UDP-glycosyl transferases (UGTs). These enzymes require UDP-glucose as a co-substrate. This compound is highly instable and costly, therefore it needs to be regenerated. Many living cells possess the ability to regenerate UDP-glucose intracellularly.

To efficiently produce RebD and/or RebM a host cell is required that can take up RebA and regenerate UDP glucose and that can be engineered to produce UGT enzymes that catalyze the required glycosylation reactions. Preferably, the species that the host cell belongs to a microbial species that has a history of safe use in food or beverage products and has the ability to grow as single cells in simple culture media.

Previously, it was unclear if any microbe has the ability to assimilate RebA. RebA is a very rare compound in nature; to date only two plant species have been discovered that produce this compound (Philippe et al., 2014). Studies on the degradation of RebA by the gut microbiome indicate that the glucose residues are cleaved off and that the remaining steviol is not metabolized further (Gardana et al., 2003). This indicates that the glucose residues are cleaved off by extracellular (secreted) enzymes and subsequently the free glucose is assimilated. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of an aglycon of steviol glycosides.

FIG. 2 is a bar graph showing the concentration of Rebaudioside A in quench, wash, and cell extract solutions of a Kluyveromyces marxianus strain.

DETAILED DESCRIPTION

Initial Screening

Candidate microbial strains were chosen based on strains belonging to species that have been described to be present in non-spoiled food or that have been previously used in biotechnological processes, and were ranked with respect to l.eukaryote or prokaryote, 2. availability of molecular biology protocols, and 3. biological safety level. Suitable genera for selecting candidate microbial strains include Candida, Cyberlindnera, Kluyveromyces, Meyerozyma, Pischia., Rhodosporidium, Zygosaccharomyces, Saccharomyces, Aspergillus, Hansenula, Humicola, Trichosporon, Brettanomyces, Pachysolen, Yarrowia, Yamadazyma, Schizosaccharomyces, Ashbya, Cyberlindnera, Pichia, Arxula, Xanthophyllomyces or Escherichia. In certain embodiments, Arthrobacter globiformis, Aspergillus niger, Aspergillus oryzae, Bacillus licheniformis, Bacillus sphaericus, Bacillus subtilis, Brevibacterium linens, Candida utilis, Candida vini, Corynebacterium glutamicum, Cyberlindnera jadinii, Cyberlindnera sp., Debaryomyces hansenii, Fusarium semitectum, Hypomyces armeniacus, Kluyveromyces lactis, Kluyveromyces marxianus, Kocuria rhizophila, Lactobacillus brevis, Lactobacillus casei, Lactobacillus pentoses, Lactobacillus plantarum, Lactobacillus reuteri, Meyerozyma guilliermondii, Microbacterium sp., Micrococcus luteus, Mucor hiemalis, Mucor racemosus, Penicillium roqueforti, Pichia guilliermondii, Pichia jadinii, Pichia pastoris, Pseudomonas fluorescens, Pseudomonas stutzeri, Rhodosporidium sp., Rhodosporidium toruloides, Rhodotorula mucilaginosa, Rhodotorula rubra, Rhodotorula sp., Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces pastorianus, Streptomyces albus, Streptomyces coelicolor, Streptomyces griseus, Streptomyces lividans, Torulaspora delbrueckii, Trichosporon laibachii, Trichosporon oleaginosus, Yarrowia lipolytica, Zygosaccharomyces rouxii, Zymomonas mobilis strains are useful. Strains were cultivated on diluted complex medium containing (per liter) 1 g yeast extract, 2 g peptone, 10 g RebA. A volume of 1 mL culture medium was inoculated with a single colony.

All cultivations were performed in 96 square well plates on a laboratory shaker with 300 rpm and 5.1 cm shaking diameter at 28 °C for 72 h.

After cultivation, cells were separated from the culture supernatant by centrifugation at 5311 g for 5 min. The supernatant was filtered through a 0.45 μηι hPTFE filter and subsequently incubated for 48 h at 28 °C. Then the pelleted cells were re-suspended in Buffer U containing (per liter) 300 mM potassium, 50 mM phosphate, 245 mM glutamate, 20 mM sodium, 2 mM manganese, 0.5 mM calcium, adjusted to pH 6.8 with potassium hydroxide. Subsequently this suspension was centrifuged again at 5311 g for 5 min. The supernatant was discarded and the cell pellets were resuspended in buffer U. Then cells were disrupted for 2 min in a Mini Bead Beater (Biospec Produkts). Cell debris was separated from the soluble fraction by centrifugation for 4 min 4870 g. The supernatant was filtered through a 0.45 μηι HPTFE filter. Then, 70 μL· of a 2 g/L RebA solution was added to 70 μΐ of the soluble fraction before incubation at 48 h at 28 °C. Samples were taken at the beginning and at the end of the incubation and RebB was quantified by LC-MS.

20 μΐ. of samples for LC-MS was added to 980 μΐ. of a stopping solution containing 70% acetonitrile, 29.9% water and 0.1% formic acid. Stopped samples were filtered through a 0.45 μηι hPTFE filter. Five μΕ of the filtered samples were used for each injection. Separation was achieved using TSKgel Amide-80 column with dimensions 4.6 x 250mm and a isocratic flow of 70% acetonitrile, 19.9% water, 0.01% formic acid, and 10% 100 mM amonium acetate in water. RebB was detected by LCMS 2010EV mass spectrometry (Shimadzu) set to single ion monitoring (SIM) mode, negative mode, m/z = 803.50. The difference in RebB concentration between the start and the end of the incubation was calculated for each supernatant and each soluble fraction. This difference value was compared to sterile controls that were processed the same way. A large difference in the soluble fraction and a small difference in supernatant indicate that a strain might have taken up RebA and degraded it to RebB to use the thus liberated glucose molecule to support growth. Indirect Assay

Microbial strains were assayed for their ability to assimilate RebA from the culture medium by growing the strains in a defined mineral medium with RebA as the only carbon source. 19 microbial strains from Candida utilis, Cyberlindnera jadinii, Kluyveromyces lactis, Kluyveromyces marxianus, Meyerozyma guilliermondii, Pichia guilliermondii, Pichia jadinii, and Zygosaccharomyces rouxii were found to assimilate RebA .

This medium contained (per liter): 10 g RebA, 5 g (NH ₄ ) ₂ S0 ₄ , 3 g KH ₂ P0 ₄ , 0.5 g MgS0 ₄ x 7H ₂ 0,15 mg EDTA, 4.5 mg ZnS0 ₄ x 7H ₂ 0, 0.3 mg CoCl ₂ x 6H ₂ 0, 1 mg MnCl ₂ x 4H ₂ 0, 0.3 mg CuS0 ₄ x5H ₂ 0, 4.5 mg CaCl ₂ x 2H ₂ 0, 3 mg FeS0 ₄ x 7H ₂ 0, 0.4 mg NaMo0 ₄ x 2H ₂ 0, 1 mg H ₃ BO ₃ , 0.1 mg KI, 0.05 mg biotin, 1 mg calcium pantothenate, 25 mg inositol, 1 mg thiamine HCl, 1 mg pyridoxine HCl, 0.2 mg para-aminobenzoic acid. A volume of 1 mL culture medium was inoculated with 10 μΐ of an overnight culture grown.

All cultivations were performed in 96 square well plates on a laboratory shaker with 300 rpm and 5.1 cm shaking diameter at 28 °C for 72 h. Growth was monitored by mixing 50 μΙ_, or culture volume with 50 μΙ_, ethanol in a 96 well plate and subsequently measuring the optical density at 600 nm (OD600) in a spectrophotometer (Molecular Devices). The OD600 values that were observed in wells that were inoculated were compared to the OD600 values that were observed in wells that contained culture medium only. If the OD600 exceeded that in the sterile reference wells the strain was identified to grow with RebA as the only carbon source. After cultivation, cells were separated from the culture supernatant by centrifugation at

4025 g for 5 min. The cell pellets were re-suspended in buffer U. Then cells were disrupted for 2 min using a Mini Bead Beater (Biospec Produkts). Cell debris was separated from the soluble fraction by centrifugation for 4 min at 4870 g. The supernatant was filtered through a 0.45 μπι HPTFE filter. Then, 70 μΙ_, of a 2 g/L RebA solution was added to the soluble fraction before incubation at 72 h at 28 °C. RebA was detected by LC-MS set to single ion monitoring (SFM) mode, negative mode, m/z = 965.60. The difference in RebA concentration between the start and the end of the incubation was calculated for each soluble fraction. If RebA can be degraded by the soluble fraction of the cell extract, it can be assumed that the strain can assimilate RebA from the culture medium.

Direct Assay

For one of the Kluyveromyces marxianus strains found to assimilate RebA by the indirect assay, RebA assimilation was demonstrated by quantification of intracellular RebA.

The selected microbial strain was cultivated in a defined mineral medium with RebA as the only carbon source. This medium contained (per liter): 9 g RebA, 5 g (NH ₄ )2S04, 3 g KH ₂ P0 ₄ , 0.5 g MgS0 ₄ x 7H ₂ 0, 15 mg EDTA, 4.5 mg ZnS0 ₄ x 7H ₂ 0, 0.3 mg CoCl ₂ x 6H ₂ 0, 1 mg MnCl ₂ x 4H ₂ 0, 0.3 mg CuS0 ₄ x 5H ₂ 0, 4.5 mg CaCl ₂ x 2H ₂ 0, 3 mg FeS0 ₄ x 7H ₂ 0, 0.4 mg NaMo0 ₄ x 2H ₂ 0, 1 mg H ₃ BO ₃ , 0.1 mg KI, 0.05 mg biotin, 1 mg calcium pantothenate, 25 mg inositol, 1 mg thiamine HC1, 1 mg pyridoxine HC1, 0.2 mg para-aminobenzoic acid. A volume of 20 mL culture medium was inoculated with a single colony.

All cultivations were performed in 25 mL culture medium in a 300 mL non-baffled shake flask on a laboratory shaker with 250 rpm and 2.5 cm shaking diameter at 28 °C for 72 h. The complete culture was transferred to a beaker containing 125 mL of a solution containing 45 ppm 4- Nitrophenyl β-D-glucopyranoside in 60% methanol in water that was pre-cooled to -40 °C. The mixture was then transferred to three 50 mL centrifugation tubes that were pre-cooled on dry ice and centrifuged below 0 °C at 4248 g for 10 min. Subsequently, an aliquot of the supernatant (quench) was stored at -80 °C and the rest of the supernatant was discarded, while the pellet was kept on dry ice. Then 5 mL 60% methanol in water pre-cooled on dry ice was used to re-suspend the 3 pellets. The re-suspended pellets were transferred to a single 15 mL centrifugation tube precooled on dry ice and centrifuged below 0 °C at 4248 g for 10 min. Then the supernatant was discarded and the pellet was re-suspended in fresh 5 mL 60% methanol in water pre-cooled on dry ice and the suspension was centrifuged below 0 °C at 4248 g for 10 min. Then the supernatant was discarded and the pellet was re-suspended again in fresh 5 mL 60% methanol in water pre-cooled on dry ice and the suspension was centrifuged below 0 °C at 4248 g for 10 min. Then the supernatant (wash) was separated and stored at -80 °C. Then the pellet was re- suspended in 1 mL 60% ethanol in water pre-heated to 78 °C and incubated at 78 °C for 3 minutes. Then the suspension was cooled on dry ice and transferred to a 2 mL centrifugation tube and centrifuged for 3 min at 16500 g at room temperature. The supernatant (cell extract) was separated and stored at -80 °C. The concentration of RebA in quench, wash, and cell extract was quantified by LC-MS (FIG. 2), in which the error bars indicate the standard error of mean from 2 parallel experiments. The concentration of RebA in the cell extract exceeds that in the quench and wash solutions.

Consequently, RebA has been accumulated intracellularly. The RebA concentration in the wash solution is higher than in the quench solution, which indicates that some RebA leaked from the pellet during sample processing.

In one embodiment of the invention a microbial strain capable of assimilating RebA hosts expressed genes and respective enzymes for intracellular conversion of RebA to RebD and/or Reb M.

In another embodiment the said enzymes include at least one UDP-glucosyltransferase

(UGT).

In yet another embodiment the said enzymes include at least one sucrose synthase for UDP regeneration and recycling.

In another embodiment the microbial strain is capable of excreting the intracellular Reb D and/or Reb M.

In yet another embodiment the Reb D and/or Reb M synthesized by microbial strain of this invention is recovered and purified by techniques used in steviol glycosides' extraction and purification to provide steviol glycosides compositions comprising the Reb D and/or Reb .

In another embodiment steviol glycosides compositions of present invention can be used as sweeteners, sweetness enhancers, flavors and flavor enhancers in various food and beverage products. Non-limiting examples of food and beverage products include carbonated soft drinks, including but not limited to cola flavored carbonated soft drinks, fruit flavored carbonated soft drinks, berry flavored carbonated soft drinks, ready to drink beverages, energy drinks, isotonic drinks, low-calorie drinks, zero-calorie drinks, sports drinks, teas, fruit and vegetable juices, juice drinks, dairy drinks, yoghurt drinks, alcohol beverages, powdered beverages, bakery products, cookies, biscuits, baking mixes, cereals, confectioneries, candies, toffees, chewing gum, dairy products, flavored milk, yoghurts, flavored yoghurts, cultured milk, soy sauce and other soy base products, salad dressings, mayonnaise, vinegar, frozen-desserts, meat products, fish-meat products, bottled and canned foods, tabletop sweeteners, fruits and vegetables.

Additionally the steviol glycosides compositions of present invention can be used in drug or pharmaceutical preparations and cosmetics, including but not limited to toothpaste, mouthwash, cough syrup, chewable tablets, lozenges, vitamin preparations, and the like.

The steviol glycosides compositions of present invention can be used "as -is" or in combination with other sweeteners, flavors and food ingredients.

Non-limiting examples of sweeteners include rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, rebaudioside G, rebaudioside H, rebaudioside /, rebaudioside J, rebaudioside K, rebaudioside L, rebaudioside M, rebaudioside N, rebaudioside O, dulcoside^i, steviolbioside, rubusoside, as well as other steviol glycosides found in Stevia rebaudiana plant and mixtures thereof, stevia extracts, glycosylated steviol glycosides, steviol glycosides prepared by chemical, enzymatic synthesis or by fermentation of recombinant microorganisms, Luo Han Guo extract, mogrosides, glycosylated mogrosides, high-fructose corn syrup, corn syrup, invert sugar, fructooligosaccharides, inulin, inulooligosaccharides, coupling sugar, maltooligosaccharides, maltodextrins, dextrins, limited dextrins, corn syrup solids, glucose, maltose, sucrose, lactose, allulose, tagatose, aspartame, saccharin, sucralose, sugar alcohols and mixtures thereof. Non-limiting examples of flavors include cola, lemon, lime, orange, grapefruit, banana, grape, apple, pear, pineapple, bitter almond, cinnamon, sugar, cotton candy, vanilla flavors, glycosylated steviol glycosides, NSF02 and mixtures thereof.

Non-limiting examples of other food ingredients include flavors, acidulants, organic and amino acids, coloring agents, bulking agents, modified starches, gums, texturizers, preservatives, antioxidants, vitamins, emulsifiers, stabilisers, thickeners, gelling agents caffeine, theanine, theobromine and mixtures thereof.

It is to be understood that the foregoing descriptions and specific embodiments shown herein are merely illustrative of the best mode of the invention and the principles thereof, and that modifications and additions may be easily made by those skilled in the art without departing for the spirit and scope of the invention, which is therefore understood to be limited only by the scope of the appended claims.