A Stevioside Glucosyltransferase Gene, Its Encoding Product and Applications

Abstract

The present invention belongs to the field of plant genetic engineering, and relates to a steviol glycoside glycosyltransferase gene related to steviol glycoside biosynthesis, its encoded product and applications. The gene of the present invention was first cloned from Stevia rebaudiana, and its nucleotide sequence is as shown in SEQ ID NO.2, and the amino acid sequence encoded by it is as shown in SEQ ID NO.1. The steviol glycoside glycosyltransferase of the present invention has the ability to specifically glycosylate the C13 position, but cannot glycosylate the C19 position, and has a higher activity towards substrates with disaccharide glycosylation at the C13 position, and a weaker activity towards glycosides with only monosaccharide at the C13 position. Overexpression of the glycosyltransferase of the present invention in Stevia rebaudiana can provide a new direction for the improvement of Stevia rebaudiana varieties.

Description

Technical Field

[0001] The present invention relates to a gene of UDP - glycosyltransferases (UGT) related to steviol glycoside biosynthesis, and UGT genes, their encoded products and applications in the steviol glycoside biosynthesis pathway, belonging to the field of plant genetic engineering. Background Art

[0002] Steviol glycosides (SGs) are a class of diterpenoid saponin natural products extracted from Stevia rebaudiana, which have characteristics such as high sweetness and low calories. They are known as the "third sugar source in the world" after sucrose and beet sugar, and also have a wide range of biological activities, including anti - inflammatory, antibacterial and hypoglycemic effects. Due to the relatively low price and easy large - scale extraction and preparation of stevioside and its derivatives, they have been widely used in fields such as food and beverage, medicine and organic chemistry.

[0003] All SGs biosynthesis takes steviol as a precursor. Under the catalysis of different UGTs, different numbers of glucose groups, rhamnose groups and xylose groups are respectively linked to the C13 and C19 positions to form various SGs with different properties. Currently, 4 UGT - encoding genes involved in SGs biosynthesis are known, namely SrUGT85C2, SrUGT91D2, SrUGT74G1 and SrUGT76G1. SrUGT85C2 can glycosylate the hydroxyl group at the C13 position of steviol; SrUGT74G1 glycosylates the carboxyl group at the C19 position of the steviol skeleton; SrUGT91D2 is responsible for catalyzing the β - 1,2 glycosidic bond between glucose; SrUGT76G1 is responsible for catalyzing the β - 1,3 glycosidic bond between glucose. Although the donor substrate specificities of these UGTs are relatively high and can specifically utilize UDP - activated glucose, their acceptor substrate specificities are poor. One UGT can often glycosylate multiple glycoside substrates. Especially SrUGT76G1 can recognize up to 8 glycoside substrates. These UGTs play roles successively and interact with each other in the SGs biosynthesis pathway, forming a complex glycosylation network, which makes the Stevia rebaudiana contain more than 60 kinds of steviol glycosides, bringing great interference to the preparation and separation of glycosides.

[0004] Among the currently known steviol glycosides, stevioside (STV) and rebaudioside A (Reb A) are the two main glycoside components. STV accounts for 5% - 10% of the dry leaf weight of the leaves, and its sweetness is 110 - 270 times that of sucrose. After SrUGT76G1 glycosylates STV, Reb A is produced. Reb A accounts for 2% - 4% of the dry leaf weight of the leaves, and its sweetness is 180 - 300 times that of sucrose. Its taste is relatively close to that of sucrose, and there is almost no bitter aftertaste. Its taste and quality are superior to those of STV. SrUGT91D2 can glycosylate Reb A to obtain rebaudioside D (Reb D). The sweetness of Reb D is 350 times that of sucrose, with almost no bitter aftertaste and a low sweetness threshold, but its content only accounts for 0.4 - 0.5% of the dry leaf weight. At the same time, SrUGT76G1 can also continue to glycosylate Reb A to produce rebaudioside I (Reb I), and the sweetness of Reb I does not increase with the increase in the number of glycosylations, and the bitter aftertaste is more significant. Therefore, how to improve the selectivity of SrUGT76G1, reduce the synthesis of by-product glycosides, and increase the accumulation of Reb D is of great significance for the quality improvement of Stevia rebaudiana varieties and the in vitro enzymatic catalysis of steviol glycoside synthesis.

[0005] Stevia rebaudiana SrUGT76G1 is polymorphic. There have been many articles reporting various naturally occurring mutants of SrUGT76G1, but most of these mutants have low activity or are sequences with translation interruption. Only SrUGT76G1 (NCBI ID: AY345974) has good activity, but poor selectivity, and can catalyze different substrates to produce a variety of glycoside products in vivo and in vitro. Kim et al. once modified this enzyme by site-saturation mutagenesis, but the content of by-product glycosides in the 38 mutants produced was not significantly different from that of the wild type. Summary of the Invention

[0006] The object of the present invention is to provide a glycosyltransferase gene with selectivity at the C13 position of steviol glycosides and the protein encoded thereby. The new UGT of Stevia rebaudiana provided by the present invention can specifically connect a glucose through a β-1,3 glycosidic bond to the glucose at the C13 position of steviol glycosides, but cannot glycosylate the glucose at the C19 position. The significant change in selectivity reduces the separation cost of steviol glycoside glycosylation products. Moreover, the β-1,3 glycosyltransferase with C13 position selectivity provided by the present invention has differences in substrate recognition and the way of entering the catalytic cavity, providing a theoretical and experimental basis for the analysis of the substrate selection mechanism of SrUGT76G1. Overexpressing the glycosyltransferase of the present invention in Stevia rebaudiana provides a new direction for the variety improvement of Stevia rebaudiana.

[0007] The present invention provides a steviol glycoside glycosyltransferase, and its amino acid sequence has one or two of the following characteristics:

[0008] 1) having the amino acid sequence shown in SEQ ID NO.1;

[0009] MENKTETTVRRRRRIILFPVPFQGHINPILQLANVLYSKGFSITIFHTNFNKPKTSNYPHFTFR

[0010] FILDNDPQDERISNLPTHGPLAGMRIPIINEHGADELRRELELLMLASEEDEEVSCLITDALW

[0011] YFAQSVADSLNLRRLVLMTSSLFNFHAHVSLPQFDDLGYFDLDDKTRLEEQVIGFPMLKVK

[0012] DIKSAYSNWQVAKEIFGKMIKQTKASSGIIWNSFKELQEPEVETITRDFPTPSFLIPLPKHLTA

[0013] SSSSLLDEDRTVFTWLDQQPPNSIVYVCFGSTSEVDEKDFLEIAHGLVDSKQSFLWVVRPGF

[0014] VKGSTWVEPLPDGFLGERGRIVKWVPQQEVLAHGAIGAFWTHSGWNSTLESVCEGVPMIF

[0015] SDFGLDQPLNARYMSDVLKVGVYLENGWERGEIANAIRRVMVDEEGEYIRQNARVLKQK

[0016] ADVSLMKGGSSYESLESLVSYISSL

[0017] 2) having the amino acid residue sequence from the amino terminus of SEQ ID NO.1 at positions 1 - 458;

[0018] 3) an amino acid sequence with steviol glycoside glycosyltransferase activity formed by substituting, deleting or adding one or more than two amino acids to the amino acid sequence shown in SEQ ID NO.1.

[0019] The present invention also provides a steviol glycoside glycosyltransferase gene encoding the steviol glycoside glycosyltransferase described in claim 1.

[0020] In the above technical solution, further, its nucleotide sequence has one or more of the following characteristics:

[0021] 1) having the deoxyribonucleic acid sequence shown in SEQ ID No. 2;

[0022] ATGGAAAATAAAACGGAGACCACCGTTCGCCGGCGCCGGAGAATAATATTATTCCCGGT

[0023] ACCATTTCAAGGCCACATTAACCCAATTCTTCAGCTAGCCAATGTGTTGTACTCTAAAGG

[0024] ATTCAGTATCACCATCTTTCACACCAACTTCAACAAACCCAAAACATCTAATTACCCTCA

[0025] CTTCACTTTCAGATTCATCCTCGACAACGACCCACAAGACGAACGCATTTCCAATCTACC

[0026] GACTCATGGTCCGCTCGCTGGTATGCGGATTCCGATTATCAACGAACACGGAGCTGACG

[0027] AATTACGACGCGAACTGGAACTGTTGATGTTAGCTTCTGAAGAAGATGAAGAGGTATCG

[0028] TGTTTAATCACGGATGCTCTTTGGTACTTCGCGCAATCTGTTGCTGACAGTCTTAACCTCC

[0029] GACGGCTTGTTTTGATGACAAGCAGCTTGTTTAATTTTCATGCACATGTTTCACTTCCTCA

[0030] GTTTGATGATCTTGGTTACTTCGATCTTGATGACAAAACCCGTTTGGAAGAACAAGTGAT

[0031] TGGGTTTCCTATGCTAAAAGTGAAAGACATCAAGTCTGCGTATTCGAACTGGCAAGTAG

[0032] CCAAAGAGATATTCGGGAAGATGATAAAACAAACAAAAGCATCTTCAGGAATCATATGG

[0033] AACTCGTTTAAGGAACTCCAAGAGCCCGAGGTCGAAACGATTACCCGTGACTTCCCGAC

[0034] ACCAAGTTTCCTGATACCATTACCCAAACATTTGACAGCCTCATCAAGCAGCTTACTAGA

[0035] CGAAGATCGAACCGTTTTTACATGGTTAGACCAACAACCGCCGAATTCTATAGTTTATGT

[0036] TTGTTTTGGTAGCACTAGTGAAGTGGATGAGAAAGATTTCTTGGAAATAGCTCATGGGTT

[0037] GGTTGATAGTAAGCAGTCGTTTTTATGGGTGGTTCGACCTGGGTTTGTCAAGGGTTCGAC

[0038] ATGGGTCGAACCGTTGCCTGATGGGTTCTTGGGTGAAAGAGGACGTATTGTGAAATGGG

[0039] TTCCGCAGCAAGAAGTGCTAGCTCATGGAGCAATAGGCGCATTCTGGACTCATAGCGGA

[0040] TGGAACTCTACGTTGGAAAGCGTTTGTGAAGGTGTTCCTATGATTTTCTCGGATTTTGGG

[0041] CTCGATCAACCGTTGAATGCTAGATACATGAGTGATGTTTTGAAGGTAGGGGTGTATTTG

[0042] GAAAATGGGTGGGAAAGAGGAGAGATAGCAAATGCAATAAGAAGAGTTATGGTGGATG

[0043] AAGAAGGAGAATACATTAGACAGAATGCAAGAGTTTTGAAACAAAAGGCAGATGTTTC

[0044] TTTGATGAAGGGTGGTTCATCTTACGAATCATTAGAGTCTCTAGTTTCTTACATTTCATCG

[0045] TTGTAA

[0046] 2) A deoxyribonucleic acid sequence encoding the amino acid sequence of SEQ ID NO.1;

[0047] 3) A nucleotide sequence encoding a steviol glycoside glycosyltransferase having activity obtained by substituting, deleting or adding one or more nucleotides to the deoxyribonucleic acid sequence of SEQ ID NO.2;

[0048] 4) A deoxyribonucleic acid sequence having a homology of 80% or more with the deoxyribonucleic acid (DNA) sequence defined by SEQ ID NO.2 and capable of encoding a steviol glycoside glycosyltransferase.

[0049] The present invention also provides a recombinant expression plasmid containing the aforementioned steviol glycoside glycosyltransferase gene.

[0050] The present invention also provides a recombinant genetically engineered bacterium containing the aforementioned steviol glycoside glycosyltransferase gene.

[0051] The present invention also provides a method for preparing a steviol glycoside glycosyltransferase, which comprises cloning the aforementioned steviol glycoside glycosyltransferase gene into a recombinant expression vector, introducing it into a host cell, and obtaining a recombinantly expressed steviol glycoside glycosyltransferase;

[0052] Preferably, the expression vector for recombinantly expressing the steviol glycoside glycosyltransferase is one or more of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, a phage vector, a filamentous fungus expression vector, a plant expression vector, an insect expression vector, or a mammalian cell expression vector.

[0053] In the above technical solution, further, the host cell includes one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, an actinomycetes host cell, a filamentous fungus host cell, an insect cell, or a mammalian cell.

[0054] The present invention also provides the application of the aforementioned steviol glycoside glycosyltransferase in the production of steviol glycosides.

[0055] In the above technical solution, further, the steviol glycoside product is a steviol glycoside compound having a β-1,3 sugar chain at the C13 position; preferably, the steviol glycoside is steviolbioside D, rebaudioside B, Rebaudioside A, Rebaudioside D, Rebaudioside G, Rebaudioside E4.

[0056] The present invention also provides the application of the aforementioned steviol glycoside glycosyltransferase gene in the genetic breeding of Stevia rebaudiana.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention discloses a glycosyltransferase-encoding gene SrUGT76G6 with selectivity at the C13 position of steviol glycoside and the protein encoded thereby. SrUGT76G6 has the ability to specifically glycosylate the C13 position, but cannot glycosylate the C19 position, and has a higher activity towards substrates with disaccharide glycosylation at the C13 position and a weaker activity towards glycosides with only monosaccharide at the C13 position. Compared with SrUGT76G1 without catalytic site selectivity, the use of the β-1,3 glycosyltransferase with selectivity at the C13 position provided by the present invention to prepare steviol glycosides can significantly reduce the separation cost of steviol glycoside glycosylation products. The β-1,3 glycosyltransferase with selectivity at the C13 position provided by the present invention has changed in the way of substrate recognition and substrate entry into the catalytic cavity compared with SrUGT76G1, providing a theoretical and experimental basis for the analysis of the substrate selection mechanism of β-1,3 glycosyltransferase. Overexpression of the glycosyltransferase of the present invention in Stevia rebaudiana provides a new direction for the improvement of Stevia rebaudiana varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 : Electrophoresis diagram of the ORF amplification product of the target gene SrUGT76G6.

[0059] Figure 2 : SDS-PAGE electrophoresis diagram of SrUGT76G6 protein.

[0060] Wherein: M: Protein molecular weight standard; Lane 1: Purification of SrUGT76G6 protein.

[0061] Figure 3 : In vitro catalytic liquid phase detection diagram of steviolmonoside by SrUGT76G6.

[0062] Figure 4 : In vitro catalytic liquid phase detection diagram of steviolbioside by SrUGT76G6.

[0063] Figure 5 : In vitro catalytic liquid phase detection diagram of rubusoside by SrUGT76G6.

[0064] Figure 6 : In vitro catalytic liquid phase detection diagram of STV by SrUGT76G6.

[0065] Figure 7 : In vitro catalytic liquid phase detection diagram of Reb A by SrUGT76G6.

[0066] Figure 8 : In vitro catalytic liquid phase detection diagram of Reb E by SrUGT76G6.

[0067] Figure 9 : In vitro catalytic liquid phase detection diagram of Reb D by SrUGT76G6.

[0068] Figure 10 : Glycosylation roadmap of steviol glycosides by SrUGT76G6. Detailed implementation mode

[0069] The present invention will be described in detail below with reference to examples. The examples are for better understanding of the present invention, but are not limited to the present invention. The experimental methods in the following implementation methods are all conventional methods, and the experimental reagents involved are all conventional biochemical reagents.

[0070] Example 1 Cloning and expression of SrUGT76G6 encoding gene

[0071] Design primers according to the sequence of SrUGT76G1 published in NCBI, and amplify the target gene from Stevia rebaudiana cDNA.

[0072] Full-length primers:

[0073] SrUGT76G4-F: TAAGAAGGAGATATACATATGATGGAAAATAAAACGG (SEQ ID NO.3)

[0074] SrUGT76G4-R: GTGGTGGTGGTGGTGCTCGAGTTACAACGATGAAATG (SEQ ID NO.4)

[0075] After verifying the sequence correctness by sequencing, the target gene was ligated to the pET21a vector digested with NdeI and XhoI using the Gibson Assembly method. 5 μL of the ligation product was taken to transform E. coli TOP10 competent cells, which were then spread on solid Luria-Bertani medium containing 100 μg / mL ampicillin and cultured at 37 °C for 12 - 16 h. Single colonies were picked and verified by colony PCR using universal primers. The single colonies with correct amplification were inoculated into liquid Luria-Bertani medium containing 100 μg / mL ampicillin for culture, and plasmids were extracted; the extracted plasmids were double-digested with restriction enzymes NdeI and XhoI, and the recombinant plasmids with correct results were sent to BGI for sequencing. The sequencing results showed that the candidate gene shown in SEQ ID NO 2 was inserted between the NdeI and XhoI restriction sites of pET21a, and the insertion direction was correct, proving the successful construction of the recombinant plasmid, and these recombinant plasmids were named pET21a-SrUGT76G6. The full nucleotide sequence of the steviol glycoside glucosyltransferase SrUGT76G6 gene is 1377 bp in length, and the nucleotide sequence is as shown in SEQ ID NO.2; it encodes 458 amino acids, and the amino acid sequence is as shown in SEQ ID NO.1, and the theoretical molecular weight of the protein is (52.2) kDa.

[0076] pET21a-SrUGT76G6 was transformed into E. coli BL21(DE3), and its induced expression and purification were carried out. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to detect the expression and purification of SrUGT76G6. The purified glycosyltransferase showed a single band on the electrophoresis gel, and its position was consistent with the predicted molecular weight.

[0077] Example 2 Detection of SrUGT76G6 Glycoside Substrate

[0078] The purified glycosyltransferase protein was functionally verified in vitro according to the following reaction system (100 μL):

[0079] 20 mM Tris (pH 8.5), 100 mM sodium chloride, 20 μg purified protein, 1 mM UDP-Glucose, 1 mM receptor substrate, and the receptor substrates include: Steviolmonoside, Steviolbioside, Rubusoside, STV, Reb A, Reb E, Reb D. After reacting for 2 hours, an equal volume of anhydrous butanol was added to terminate the reaction, shaken and mixed evenly, centrifuged at 12,000 rpm for 10 minutes, the supernatant was taken and filtered through a 0.22 μm organic filter membrane, and the composition of the reaction products was qualitatively and quantitatively detected by liquid chromatography. Instrument model: Waterse2695. Injection volume: 10 μL, chromatographic column: Elite superil ODS2 (5 μm, 250×4.6 mm), column temperature: 40 °C. Chromatographic conditions: UV 210 nm, mobile phase: (A): water (containing 0.1% formic acid), (B): acetonitrile (1% formic acid), flow rate: 1 mL / min, elution program: 0 - 4 min, 20% B; 4 - 25 min, linearly increased to 30% B; 25 - 40 min, 30% B.

[0080] Liquid chromatography detection results ( Figure 3 ) showed that SrUGT76G6 could glycosylate Steviolmonoside, Steviolbioside, STV, and Reb E, but could not glycosylate Reb A and Reb D, indicating that this enzyme had the ability of specific glycosylation selection at the C13 position and could not glycosylate the C19 position. While SrUGT76G1 glycosylation of Reb A would produce Reb I, and glycosylation of Reb D would produce Reb M glycoside, increasing the complexity of the catalytic system. SrUGT76G6 had higher activity towards substrates with disaccharide chains at the C13 position (such as steviolbioside, STV, Reb E), and lower activity towards glycosides with only a single sugar chain at the C13 position (such as steviolmonoside, rubusoside). Therefore, it was speculated that in the complex steviol glycoside mixture system or in Stevia rebaudiana Bertoni, the accumulation of glycosides with lower sweetness (such as steviolbioside D, rubusoside G, Reb I) could be reduced, while the accumulation of glycosides with higher sweetness (such as Reb B, Reb A, Reb D) could be increased, thereby realizing the quality of the steviol glycoside mixture or reducing the separation cost of steviol glycosides.

[0081] For any person skilled in the art, without departing from the scope of the technical solution of the present invention, many possible variations and modifications can be made to the technical solution of the present invention by using the technical content disclosed above, or it can be modified into equivalent embodiments with equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A steviol glycoside glycosyltransferase, characterized in that Its amino acid sequence is shown in SEQ ID NO.

1.

2. A steviol glycoside glycosyltransferase gene, characterized in that, Encoding the steviol glycoside glucosyltransferase described in claim 1.

3. A recombinant expression plasmid containing the steviol glycoside glucosyltransferase gene described in claim 2.

4. A recombinant genetically engineered bacterium containing the steviol glycoside glucosyltransferase gene described in claim 2.

5. A method for preparing steviol glycoside glycosyltransferase, characterized in that, Cloning the steviol glycoside glucosyltransferase gene described in claim 2 into a recombinant expression vector, introducing it into a host cell, and obtaining a recombinantly expressed steviol glycoside glucosyltransferase; The expression vector for the recombinantly expressed steviol glycoside glucosyltransferase is one of an Escherichia coli expression vector, a yeast expression vector, a Bacillus subtilis expression vector, a Lactobacillus expression vector, a Streptomyces expression vector, and a phage vector; The host cell is one of an Escherichia coli host cell, a yeast host cell, a Bacillus subtilis host cell, a Lactobacillus host cell, and an actinomycete host cell.

6. The application of the steviol glycoside glucosyltransferase described in claim 1 in the production of steviol glycosides; the steviol glycosides are steviol glycoside products with 3-glycosylation at the C13 position.

7. The application according to claim 6, wherein The steviol glycoside products are rebaudioside B, rebaudioside A, rebaudioside D, rebaudioside G, and rebaudioside E4.

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