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WO2025143773A1 - Variant de saccharose synthase, variant d'udp-glucosyltransférase et utilisations associées pour la production de glycosides de stéviol - Google Patents

Variant de saccharose synthase, variant d'udp-glucosyltransférase et utilisations associées pour la production de glycosides de stéviol Download PDF

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WO2025143773A1
WO2025143773A1 PCT/KR2024/021074 KR2024021074W WO2025143773A1 WO 2025143773 A1 WO2025143773 A1 WO 2025143773A1 KR 2024021074 W KR2024021074 W KR 2024021074W WO 2025143773 A1 WO2025143773 A1 WO 2025143773A1
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amino acid
udp
substitution
acid corresponding
variant
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문준호
장병관
고혁진
김진하
박부수
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Samyang Corp
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin

Definitions

  • the present invention relates to sucrose synthase mutants, UDP-glucosyltransferase mutants and their use in producing steviol glycosides.
  • Stevia extract is a natural sweetener that can be extracted from the perennial shrub, Stevia rebaudiana. Stevia extracts of various levels of refinement are used as a high-potency flavoring in foods and blends, or are sold on their own as a table sweetener.
  • Extracts of the stevia plant contain rebaudioside and other steviol glycosides that contribute to the sweetness, but existing commercial products are primarily rebaudioside (Reb) A, with smaller amounts of other glycosides such as Reb C, D, and F.
  • Stevia extracts from the plant may contain contaminants such as derived compounds that cause off-flavors. These off-flavors can be problematic in many cases, depending on the food system or application chosen.
  • the stevia extract or composition comprising the stevia extract can vary greatly depending on the soil and climate in which the plant is grown. Depending on the source plant, climate conditions, and extraction process, the amount of Reb A in a commercial manufacturing process has been reported to vary from 20 to 97% of the total steviol glycoside content. Other steviol glycosides are present in varying amounts in stevia extracts.
  • Stevia extracts produced from stevia plants contain various steviol glycosides and off-flavor causing compounds, and their recovery and purification are labor intensive and inefficient. Therefore, a recombinant production system capable of accumulating desired steviol glycosides such as Reb D and Reb M in high yields is still required. For this purpose, the development of efficient enzymes is still required. In addition, the improvement of the production of Reb D for the production of Reb M in a recombinant host for commercial use is still required.
  • Patent Document 1 Korean Publication No. 10-2021-0114899
  • Another object of the present invention is to provide a fusion protein comprising the UDP-glucosyltransferase variant and sucrose synthase.
  • Another object of the present invention is to provide a vector comprising the polynucleotide.
  • Another object of the present invention is to provide a microorganism comprising the polynucleotide or a vector comprising the same.
  • Another object of the present invention is to provide a composition for producing steviol glycosides, comprising at least one selected from the group consisting of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.
  • Another object of the present invention is to provide a method for producing steviol glycosides, comprising the step of reacting (i) stevioside, rebaudioside, or a mixture thereof, and (ii) at least one selected from the group consisting of UDP, UDP-glucose, and sucrose with the composition.
  • Another object of the present invention is to provide a use for producing steviol glycosides of a composition comprising at least one selected from the group consisting of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.
  • Another object of the present invention is to provide a use for producing a composition for producing steviol glycosides of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; or an extract thereof.
  • sucrose synthase mutant comprising at least one substitution of an amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 with glutamic acid (E) and at least one substitution of an amino acid corresponding to position 456 with lysine (K).
  • sucrose synthase is an enzyme that produces fructose and UDP-glucose from sucrose and UDP. Therefore, when sucrose and UDP are supplied together with SUS to a reaction in which UDP-glucosyltransferase transfers glucose from UDP-glucose to a steviol glycoside (e.g., a reaction in which Reb A is converted into Reb D or a reaction in which stevioside is converted into Reb E), SUS produces UDP-glucose from sucrose and UDP and provides it to UDP-glucosyltransferase (at this time, UDP-glucose may be supplied instead of or together with UDP).
  • SUS glucose synthase
  • SUS in the conversion reaction of the UDP-glucosyltransferase is more economical than supplying UDP-glucose separately.
  • the above SUS can be provided by itself or in the form of a fusion protein with UDP-glucosyltransferase to produce steviol glycosides.
  • the sucrose synthase may be a polypeptide having sucrose synthase activity that is modified to produce the sucrose synthase variant of the present invention. Specifically, it may be a naturally occurring polypeptide or a wild-type polypeptide, and may include a variant or functional fragment thereof, but is included without limitation as long as it can be a parent of the sucrose synthase variant of the present invention.
  • the sucrose synthase of the present invention may be, but is not limited to, a sucrose synthase derived from a plant of the genus Glycine (e.g., Glycine max ), a plant of the genus Arabidopsis (e.g., Arabidopsis thaliana ), a plant of the genus Solanum (e.g., Solanum lycopersicum ), a plant of the genus Nicotiana (e.g., Nicotiana tabacum ), or a microorganism of the genus Thermosynechococcus (e.g., Thermosynechococcus elongatus ).
  • Glycine e.g., Glycine max
  • a plant of the genus Arabidopsis e.g., Arabidopsis thaliana
  • Solanum e.g., Solanum lycopersicum
  • Nicotiana e.g
  • sucrose synthase of the present invention can be obtained from a known database such as NCBI's GenBank.
  • homology or “identity” means the degree of relationship between two given amino acid sequences or base sequences, and may be expressed as a percentage. In the present invention, homology and identity may be used interchangeably.
  • the "corresponding amino acid” refers to an amino acid residue at a corresponding position in a polypeptide, or an amino acid residue that is similar, identical, or homologous to the amino acid residue at the corresponding position. Identifying the amino acid at the corresponding position may be determining a specific amino acid of a sequence that references a specific sequence.
  • the "corresponding position" in the present invention generally refers to a similar or corresponding position in an amino acid sequence of a related protein or a reference sequence. For example, any amino acid sequence may be aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence may be numbered by referring to the numerical position of the amino acid residue corresponding to the amino acid residue in SEQ ID NO: 3.
  • the position of the corresponding amino acid, or the position where a modification such as a substitution, insertion, or deletion occurs may be identified by comparing it with a query sequence (also referred to as a "reference sequence") through a sequence alignment algorithm known in the art.
  • a query sequence also referred to as a "reference sequence”
  • sequence alignment algorithm known in the art.
  • the sucrose synthase variant comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology or identity to the amino acid sequence of SEQ ID NO: 3 and has sucrose synthase activity, wherein 'the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with glutamic acid (E), and/or 'the amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is substituted with lysine (K).
  • E glutamic acid
  • K lysine
  • the sucrose synthase variant comprises an amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or homology thereto, wherein 'the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is replaced with glutamic acid (E), and/or 'the amino acid corresponding to position 456 from the N-terminus of the amino acid sequence of SEQ ID NO: 3' is replaced with lysine (K).
  • E glutamic acid
  • K lysine
  • sucrose synthase variant may be, but is not limited to, one in which serine (S) at position 11 in the amino acid sequence of SEQ ID NO: 3 is substituted with glutamic acid (E), and/or arginine (R) at position 456 is substituted with lysine (K).
  • polypeptide or protein having an amino acid sequence having a deletion, modification, substitution (e.g., conservative substitution) or addition of a portion of the sequence is also included within the scope of the present invention, provided that the amino acid sequence has the homology or identity described herein and exhibits an activity corresponding to that of the polypeptide or protein.
  • a "conservative substitution” means replacing an amino acid residue with an amino acid residue having a similar side chain without causing any loss of biological or biochemical function of the polypeptide or protein.
  • Classes of amino acid residues having similar side chains are well known and defined in the art.
  • amino acids having basic side chains e.g., lysine, arginine, histidine
  • amino acids having acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids having uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • amino acids having nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • amino acids having beta-branched side chains e.g., threonine, valine, isoleucine
  • amino acids having aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • variants of the present invention may comprise deletions or additions of amino acids having minimal effects on the properties and secondary structure of the polypeptide, for example, the N-terminus of the variant may be conjugated with a signal (or leader) sequence involved in co-translationally or post-translationally translocation of the protein.
  • variants of the present invention may be conjugated to other sequences or linkers so that they can be identified, purified, or synthesized.
  • UDP-glucosyltransferase (UGT) variant comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) substitutions of:
  • steviol glycosides include, but are not limited to, stevioside, rebaudioside (Reb A, Reb B, Reb C, Reb D, Reb E, Reb F, and Reb M, etc.), or mixtures thereof.
  • the sequence of the UDP-glucosyltransferase of the present invention can be obtained from a known database such as NCBI's GenBank.
  • polypeptide or protein having an amino acid sequence in which some of the sequences are deleted, modified, substituted or added is also included within the scope of the present application, provided that it is an amino acid sequence having such homology or identity and exhibiting an activity corresponding to the polypeptide or protein.
  • the UDP-glucosyltransferase variant may be a variant in which 'an amino acid corresponding to one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) positions from the N-terminus of the amino acid sequence of SEQ ID NO: 1' is substituted as described above in the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or homology thereto.
  • the UDP-glucosyltransferase variant may comprise substitutions of amino acids corresponding to the following two or more positions of the amino acid sequence of SEQ ID NO: 1:
  • the amino acid corresponding to position 31 is isoleucine (I).
  • the amino acid corresponding to position 97 is arginine (R).
  • the amino acid corresponding to position 336 is threonine (T).
  • the amino acid corresponding to position 362 is threonine (T).
  • the amino acid corresponding to position 378 is serine (S).
  • the amino acid corresponding to position 434 is threonine (T).
  • the UDP-glucosyltransferase variant may further comprise one or more of a substitution of an amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 to serine (S) and a substitution of an amino acid corresponding to position 202 to leucine (L).
  • amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of sequence number 1 may be threonine (T), and the amino acid corresponding to position 202 may be valine (V).
  • Another aspect of the present invention provides a fusion protein comprising UDP-glucosyltransferase and sucrose synthase.
  • the supply may be, for example, separately adding or adding UDP-glucose when producing steviol glycosides.
  • the supply may include, but is not limited to, contacting a medium containing a starting material with the UDP-glucose in order to produce steviol glycosides.
  • UDP-glucose when UDP-glucose is supplied, glucose is transferred from UDP-glucose to steviol glycoside to generate UDP, and the sucrose synthase of the fusion protein produces UDP-glucose from the generated UDP. Therefore, steviol glycoside can be produced even without supplying UDP when producing steviol glycoside using the fusion protein of the present invention. That is, the fusion protein of the present invention can produce steviol glycoside without the addition of UDP-glucose or UDP.
  • the fusion protein of the present invention may comprise a sucrose synthase variant according to the present invention and a UDP-glucosyltransferase or a variant thereof.
  • the UDP-glucosyltransferase or a variant thereof is as described above.
  • the C-terminus or N-terminus of the sucrose synthase variant may be linked directly or via a linker to the N-terminus or C-terminus of the UDP-glucosyltransferase or a variant thereof.
  • the fusion protein of the present invention may comprise a UDP-glucosyltransferase variant according to the present invention and a sucrose synthase or a variant thereof.
  • the sucrose synthase or a variant thereof is as described above.
  • the C-terminus or N-terminus of the sucrose synthase or a variant thereof may be linked directly or via a linker to the N-terminus or C-terminus of the UDP-glucosyltransferase variant.
  • the UDP-glucosyltransferase or a variant thereof can be fused directly or via a linker to the sucrose synthase or a variant thereof.
  • the linker can be a peptide consisting of 4 to 15 amino acids, and specifically can comprise 4 to 15 amino acids of at least one amino acid selected from the group consisting of G (Gly), S (Ser) and P (Pro), but is not limited thereto.
  • it can be GGGS (SEQ ID NO: 8), GGGGS (SEQ ID NO: 9), GGGSGGGGS (SEQ ID NO: 10), GGGGPSPGGGGS (SEQ ID NO: 11), or GGGGSGGGGSGGGGS (SEQ ID NO: 7).
  • the fusion protein of the present invention comprises:
  • substitution in (b) above may include substitution of amino acids corresponding to the following two or more positions in the amino acid sequence of SEQ ID NO: 5:
  • the amino acid corresponding to position 31 is isoleucine (I).
  • the amino acid corresponding to position 378 is serine (S).
  • the amino acid corresponding to position 344 is valine (V).
  • the fusion protein of the present invention may further comprise at least one substitution of the amino acid corresponding to position 201 from the N-terminus of the amino acid sequence of SEQ ID NO: 5 with serine (S) and at least one substitution of the amino acid corresponding to position 202 with leucine (L).
  • Another aspect of the present invention provides a vector comprising a polynucleotide according to the present invention.
  • the vector of the present invention is not particularly limited, and any vector known in the art can be used. Examples of commonly used vectors include, but are not limited to, plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state.
  • the vector of the present invention may be an expression vector for expressing the polynucleotide in a host cell, but is not limited thereto.
  • the vector of the present invention may be a vector for intracellular chromosome insertion for inserting the polynucleotide into a chromosome, but is not limited thereto.
  • a polynucleotide encoding a target polypeptide may be inserted into a chromosome through a vector for intracellular chromosome insertion.
  • the insertion of the polynucleotide into the chromosome may be accomplished by any method known in the art, for example, homologous recombination, but is not limited thereto.
  • a selection marker for confirming whether the chromosome has been inserted may be additionally included.
  • the microorganism of the present invention includes, but is not limited to, one or more selected from the group consisting of a microorganism of the genus Escherichia (e.g., Escherichia coli , etc.), a microorganism of the genus Saccharomyces (e.g., Saccharomyces cerevisiae ), a microorganism of the genus Bacillus (e.g., Bacillus subtilis), a microorganism of the genus Pichia (e.g., Pichia pastoris ), and a microorganism of the genus Corynebacterium (e.g., Corynebacterium glutamicum ).
  • a microorganism of the genus Escherichia e.g., Escherichia coli , etc.
  • a microorganism of the genus Saccharomyces e.g., Saccharomyces cerevisiae
  • the above microorganism comprises a polynucleotide encoding a fusion protein or UDP-glucosyltransferase variant according to the present invention, and thus may be a microorganism capable of expressing or having expressed the fusion protein or UDP-glucosyltransferase variant, but is not limited thereto.
  • composition of the present invention may additionally comprise, as a substrate, at least one selected from the group consisting of (i) a steviol glycoside (e.g., stevioside, rebaudioside, or a mixture thereof) and (ii) UDP, UDP-glucose, and sucrose, but is not limited thereto.
  • a steviol glycoside e.g., stevioside, rebaudioside, or a mixture thereof
  • UDP UDP
  • UDP-glucose and sucrose
  • composition or microorganism of the present invention may further comprise sucrose synthase or a variant thereof.
  • the sucrose synthase variant is as described above, and specifically, may be one in which the amino acid corresponding to position 11 from the N-terminus of the amino acid sequence of SEQ ID NO: 3 is substituted with glutamic acid and/or the amino acid corresponding to position 456 is substituted with lysine.
  • the microorganism comprises sucrose synthase or a variant thereof
  • the microorganism comprises a polynucleotide encoding sucrose synthase or a variant thereof, and can thus express the enzyme, but is not limited thereto.
  • the composition or microorganism of the present invention may further comprise an 'enzyme that converts Reb D or Reb E into Reb M' to produce Reb M.
  • the composition may comprise a second UDP-glucosyltransferase, such as UGT76G1.
  • UGT76G1 is a UDP-glycosyltransferase derived from stevia and is known to have a wide substrate specificity, and has, for example, an activity of converting stevioside into Reb A, an activity of converting Reb A into Reb I, an activity of converting Reb E into Reb D, and/or an activity of converting Reb D into Reb M.
  • the microorganism further comprises an 'enzyme that converts Reb D or Reb E into Reb M'
  • the microorganism comprises a polynucleotide encoding the 'enzyme that converts Reb D or Reb E into Reb M', and can thus express the enzyme, but is not limited thereto.
  • the composition or microorganism comprises the fusion protein
  • the sucrose synthase of the fusion protein produces UDP-glucose
  • steviol glycosides can be produced even without externally supplying UDP-glucose when producing steviol glycosides according to the method of the present invention.
  • the supply may be, for example, separately adding or adding UDP-glucose when producing steviol glycosides.
  • the supply may include, but is not limited to, contacting a medium containing a starting material with the UDP-glucose in order to produce steviol glycosides.
  • steviol glycosides can be produced even without supplying UDP when producing steviol glycosides according to the method of the present invention. That is, when the composition for producing steviol glycosides of the present invention or the microorganism included therein comprises the fusion protein of the present invention, the method for producing steviol glycosides of the present invention enables production of steviol glycosides without the addition of UDP-glucose or UDP.
  • the method of the present invention can produce Reb E from stevioside and/or Reb D from Reb A.
  • composition for producing steviol glycosides according to the present invention has sucrose synthase activity together with UDP-glucosyltransferase activity
  • a steviol glycoside and UDP UDP-glucose or a mixture thereof and sucrose can be used to produce a steviol glycoside further comprising one or more glucoses, but is not limited thereto.
  • the reaction of the present invention is carried out at a temperature and pH at which the enzyme according to the present invention is active.
  • a temperature and pH can be appropriately selected by those skilled in the art, and for example, the reaction temperature can be 10 to 90°C, 20 to 80°C, 30 to 70°C, 40 to 65°C, or 45 to 60°C
  • the reaction pH can be pH 5.0 to 9.0, pH 5.5 to 8.5, pH 6.0 to 8.0, pH 6.5 to 8.0, or pH 6.5 to 7.5.
  • the method of the present invention may further comprise a step of recovering steviol glycosides produced from the reactants.
  • Another aspect of the present invention is a use for producing steviol glycosides, the composition comprising at least one selected from the group consisting of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; and extracts thereof.
  • Another aspect of the present invention is the use of the fusion protein; the UDP-glucosyltransferase variant; a microorganism comprising the fusion protein or variant; a culture of the microorganism; a lysate of the microorganism; or an extract thereof for producing a composition for producing steviol glycosides.
  • the production of the fusion protein, UDP-glucosyltransferase mutant, microorganism, culture, lysate, extract, and steviol glycosides is as described above.
  • sucrose synthase mutant, UDP-glucosyltransferase mutant and fusion enzyme using the same according to the present invention have improved thermal stability and enzyme activity, and thus can be widely applied in the field of steviol glycoside synthesis, and have excellent industrialization value and significance in that they can increase production efficiency and reduce production costs.
  • Figure 1 shows the results comparing the production of Reb E and D using TaUGT-GmSUS (template) and mutants.
  • TaUGT wild-type UDP-glucosyltransferase
  • a primary mutant TaUGT was prepared in which threonine at position 201 was substituted with serine and valine at position 202 was substituted with leucine in sequence number 1.
  • primers SEQ ID NOs: 48 and 49
  • mutant gene amplification PCR
  • the resulting mutant cloned into the pRS426 vector was named TaUGT T201S/V202L , and its amino acid sequence and base sequence are shown in SEQ ID NOs: 50 and 51, respectively.
  • the pRS426 vector manufactured in Example 1-1 was transformed into S. cerevisiae CENPK2-1c (Euroscarf) strain. Recombinant strains with inserted gene cassettes were selected through selection markers on SC-Ura solid medium (SC_Ura medium + 2% agar).
  • SC-Ura medium + 2% agar The composition of SC-Ura solid medium included YNB 6.7 g/L, Drop out mix 0.7 g/L, Glucose 50 g/L, and agar 20 g/L, and the pH was adjusted to 6.0.
  • the composition of SC_Ura liquid medium included YNB (yeast nitrogene base) 6.7 g/L, Drop-out mix g/L, Glucose g/L, and MES mM, and the pH of the medium was adjusted to 6.0 using NaOH.
  • the YPG medium composition included Bacto peptone 20 g/L, Yeast Extract 10 g/L, Galactose 20 g/L, and Phosphate buffer (sodium salt) 100 mM, and the pH of the medium was adjusted to 6.0.
  • Example 2 Production and cultivation of microorganisms expressing the fusion enzyme, TaUGT-GmSUS
  • reaction solution for enzyme activity evaluation, substrate RA60 (95%, Sinochem) containing 60% Rebaudioside A and 40% Stevioside, UDP, Sucrose, EDTA, and freeze-dried cells were dissolved in 50 mM phosphate buffer. A total reaction volume of 1 mL was prepared. The final reaction solution contained 10 g/L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, 750 mM Sucrose, 5 mM EDTA, and 9.3 g/L of freeze-dried cells. Enzyme reaction was performed for the prepared reaction solution under the conditions of 45°C, pH 7.2, and 150 rpm, and the degree of reaction was confirmed 1 hour after the start of the reaction.
  • the column used for HPLC analysis of the product was UG120 (C 18 250 mm x 46 mm, 5 um 110 A particle, Shiseido). The analysis was confirmed at 210 nm and the column temperature was maintained at 55°C.
  • the mobile phase was analyzed as a gradient using water containing 0.01% TFA (Trifluoroacetic acid, Sigma) and 100% acetonitrile, respectively. The mobile phase flowed at 0.8 mL/min and was confirmed through a total analysis of 34 minutes.
  • TFA Trifluoroacetic acid
  • the recombinant strain expressing the I31L, T434K, T362I, V344I, and R932K mutant enzymes showed an increased activity of up to 121% or more compared to the recombinant strain expressing the template enzyme, and when the mutations were combined (I31L-S487E-R932K-T434K-T362I-V344I-C444L-T336L-R97Q-S378M), an increased conversion activity of up to approximately 551% compared to the recombinant strain expressing the template enzyme was confirmed.
  • the whole-cell reaction was performed at a temperature higher than 45°C using the strain in which the enzyme mutation was confirmed in Example 3-2. Specifically, the reaction temperature was increased from 45°C to 50°C, 55°C, and 60°C to confirm the optimal temperature for enzyme activity.
  • a reaction solution substantially the same as that in Example 3-2 was prepared, and an enzyme reaction was performed on the prepared reaction solution under the conditions of temperatures of 45°C, 50°C, 55°C, and 60°C, pH 7.2, and 150 rpm, and the degree of reaction was confirmed 1 hour after the start of the reaction.
  • the whole-cell reaction solution was boiled at 100°C for 5 minutes to terminate the whole-cell reaction, centrifuged (13,000 rpm, 10 min), and the obtained supernatant was analyzed to identify the whole-cell reaction product.
  • the whole-cell reaction product was analyzed in the same manner as in Example 3-2. The results of the analysis are shown in Table 6 below.
  • the reaction solution for confirming the productivity of Reb E and D contained substrate RA60 (95%, Sinochem) containing 60% Rebaudioside A and 40% Stevioside, UDP, Sucrose, EDTA, and freeze-dried cells. A total reaction volume of 100 mL was prepared. The final reaction solution contained 50 g/L of substrate RA60 (95%, Sinochem), 0.3 mM UDP, and 750 mM Sucrose, 5 mM EDTA, and 15.3 g/L of freeze-dried cells. Enzyme reaction was performed on the above-mentioned prepared reaction solution at a temperature of 45°C or 55°C, pH 7.2, and 150 rpm, and the degree of reaction was checked from 30 minutes to 180 minutes after the start of the reaction.
  • the whole-cell reaction solution was boiled at 100°C for 5 minutes to terminate the whole-cell reaction, centrifuged (13,000 rpm, 10 min), and the obtained supernatant was analyzed to identify the whole-cell reaction product.
  • the analysis results are shown in Figure 1.

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Abstract

La présente invention concerne un variant de saccharose synthase, un variant d'UDP-glucosyltransférase et des utilisations associées pour la production de glycosides de stéviol. Le variant d'UDP-glucosyltransférase, le variant de saccharose synthase et une enzyme de fusion les utilisant selon la présente invention présentent une stabilité thermique et une activité enzymatique améliorées, et peuvent ainsi être largement appliqués au domaine de la synthèse de glycoside de stéviol, et présentent une valeur et une importance industrielles remarquables, dans la mesure où ils permettent d'augmenter l'efficacité de production et de réduire les coûts de production.
PCT/KR2024/021074 2023-12-26 2024-12-24 Variant de saccharose synthase, variant d'udp-glucosyltransférase et utilisations associées pour la production de glycosides de stéviol Pending WO2025143773A1 (fr)

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