Glycosyltransferase ugt-x01 and use thereof in the production of rebaudioside u

Abstract

The application belongs to the technical field of biology and relates to a glycosyltransferase UGT-X01 and application thereof in production of rebaudioside U. The application provides a glycosyltransferase UGT-X01, and the amino acid sequence of the glycosyltransferase is any one of the following (1) and (2): (1) an amino acid sequence as shown in SEQ ID NO. 1; (2) a protein having an amino acid sequence with homology of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% to the amino acid sequence in (1) and having the same or similar activity of the glycosyltransferase UGT-X01. The glycosyltransferase has catalytic activity of catalyzing rebaudioside A to generate rebaudioside U, and has high catalytic efficiency. The glycosyltransferase UGT-X01 is used to realize efficient enzymatic catalysis of rebaudioside U by taking rebaudioside A as a substrate and UDP-xylose as a glycosyl donor.

Description

Technical Field

[0002] This invention belongs to the field of biotechnology and relates to a glycosyltransferase UGT-X01 and its application in the production of rebaudioside U. Background Technology

[0004] Steviosides are a series of natural sweeteners extracted from stevia rebaudiana. Due to their high sweetness, low calorie content, and minimal impact on blood sugar and insulin levels, they have become an important alternative to sucrose and synthetic sweeteners in the global food and beverage industry. Currently known structures of steviol glycosides include rebaudioside A, rebaudioside B, rebaudioside D, rebaudioside M, rebaudioside N, rebaudioside J, and rebaudioside U.

[0005] Among them, Reb U is a novel, high-value steviol glycoside identified in recent years, with the molecular formula C0. 49 H 78 O 27 Currently, the production of rebaudioside U mainly relies on plant extraction and enzymatic conversion methods. For example, Chinese patent 202211104357.5 discloses a method for obtaining rebaudioside F and rebaudioside U from stevia mother liquor, which can effectively separate rebaudioside F and rebaudioside U from stevia mother liquor. However, the content of rebaudioside U in stevia leaves is low, which requires processing large amounts of plant raw materials to obtain trace amounts of the target product, resulting in high production costs. Furthermore, the separation and purification of rebaudioside U is difficult, hindering large-scale industrial production. Current enzymatic conversion methods use glycosyltransferases to transfer xylose, a glycosyl donor, to the substrate to generate rebaudioside U. However, the catalytic efficiency of these enzymes is low, resulting in a low conversion rate of rebaudioside U.

[0006] Therefore, developing a glycosyltransferase that can efficiently, specifically, and cost-effectively convert readily available steviol glycosides into rebaudioside U is of great significance for overcoming the production bottleneck of rebaudioside U, enriching the steviol glycoside product system, and meeting the market demand for novel natural sweeteners. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides a glycosyltransferase UGT-X01 and its application in the production of rebaudine U. This glycosyltransferase possesses catalytic activity for the conversion of rebaudine A to rebaudine U, and exhibits high catalytic efficiency. Utilizing this glycosyltransferase UGT-X01, rebaudine A is used as a substrate, and UDP-xylose is used as a glycosyl donor to achieve highly efficient enzymatic catalytic production of rebaudine U. Furthermore, the reaction system can achieve the synthesis of expensive UDP-xylose from sucrose through the cascade coordination of sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3, significantly reducing production costs.

[0009] The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

[0010] The first aspect of the present invention provides a glycosyltransferase UGT-X01, wherein the glycosyltransferase is any one of the following (1) or (2):

[0011] (1) It has the amino acid sequence shown in SEQ ID NO.1;

[0012] (2) is a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the amino acid sequence described in (1), and having the same or similar activity as the glycosyltransferase UGT-X01.

[0013] The second aspect of the present invention provides a gene encoding the glycosyltransferase UGT-X01 as described in the first aspect, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0014] A third aspect of the present invention provides a recombinant vector or recombinant strain comprising the coding gene described in the second aspect.

[0015] Furthermore, the expression plasmids used in the recombinant vector include, but are not limited to: pET28a, pCDFDuet-1, pETDuet-1, pESC-URA, pESC-His, etc.

[0016] Furthermore, the recombinant vector uses the expression plasmid pET28a.

[0017] Furthermore, the hosts used for the recombinant strains include, but are not limited to: Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Yersinia lipolytica, Pichia pastoris, etc.

[0018] Furthermore, the host used for the recombinant strain is Escherichia coli;

[0019] Furthermore, the *Escherichia coli* species include, but are not limited to, *E. coli* BL21.

[0020] The fourth aspect of the present invention provides the use of the recombinant vector or recombinant strain described in the third aspect in the production of the glycosyltransferase UGT-X01 described in the first aspect.

[0021] The fifth aspect of this invention provides the use of the glycosyltransferase UGT-X01 described in the first aspect or the recombinant strain described in the third aspect in the production of rebaudioside U.

[0022] The sixth aspect of the present invention provides an engineered bacterium for producing rebaudioside U, wherein the engineered bacterium, in addition to expressing the glycosyltransferase UGT-X01 described in the first aspect in a host, also expresses one or more of sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3.

[0023] A seventh aspect of the present invention provides a method for producing rebaudine U, wherein a starting composition comprising rebaudine A is contacted with a biocatalyst to obtain rebaudine U.

[0024] Furthermore, the biocatalyst comprises any one or more of the following: the glycosyltransferase UGT-X01 described in the first aspect, the recombinant strain or its culture described in the third aspect, and the engineered bacteria or its culture described in the sixth aspect;

[0025] Furthermore, when the starting composition contains UDP-xylose, the biocatalyst is one of the glycosyltransferase UGT-X01 described in the first aspect, the recombinant strain described in the third aspect, or a culture thereof.

[0026] Furthermore, when the starting composition contains sucrose, the biocatalyst is the engineered bacteria or its culture as described in the sixth aspect, or the biocatalyst is a mixture of the glycosyltransferase UGT-X01 described in the first aspect with sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3.

[0027] The beneficial effects of this invention are:

[0028] 1. The glycosyltransferase UGT-X01 of the present invention has catalytic activity in converting rebaudioside A to rebaudioside U, and its catalytic efficiency is high. Using this glycosyltransferase UGT-X01 or its mutant, with Reb A as substrate and UDP-xylose as glycosyl donor, the enzymatic catalytic production of rebaudioside U is achieved with high efficiency. The Reb U production method of the present invention is free from the limitation of plant raw materials, realizes the high-efficiency and low-cost enzymatic production of Reb U, breaks through the production bottleneck of rebaudioside U, and is conducive to the industrial production of Reb U.

[0029] 2. The glycosyltransferase UGT-X01 cascade of sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3 of the present invention enables the production of Reb U using Reb A and sucrose as substrates. This allows for the efficient synthesis and recycling of expensive UDP-xylose using readily available and inexpensive sucrose as the starting carbon source, significantly reducing the raw material cost of glycosyl donors and further reducing production costs. Attached Figure Description

[0031] Figure 1 This is the Reb U biotransformation roadmap.

[0032] Figure 2 This is the Reb U biotransformation coupled sucrose cycle route diagram of the present invention.

[0033] Figure 3 This is a nucleic acid gel image of the glycosyltransferase UGT-X01 of the present invention.

[0034] Figure 4 This is a protein electrophoresis diagram of the glycosyltransferase UGT-X01 of this invention.

[0035] Figure 5 This is an UPLC-MS result of Reb U obtained from the reaction in Example 2 of this invention. Detailed Implementation

[0037] The principles and features of the present invention are described below (in conjunction with the accompanying drawings). The examples given are for illustrative purposes only and are not intended to limit the scope of the invention.

[0038] This invention discloses a glycosyltransferase UGT-X01 and its application in the production of rebaudioside U. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0039] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0040] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0041] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0042] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0043] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0044] In this article, amino acids are represented by single-letter or three-letter codes, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Proline (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Valine (valine).

[0045] In this invention, the glycosyltransferase UGT-X01 is derived from stevia (Stevia rebaudiana), and its amino acid sequence is shown in SEQ ID NO.1:

[0046] MLNDHKQLHVAMFPWLAFGHMIPFLELSKFIAEKGHKVSFLSTTRNIQRLPTIPSNLSPLINLVKLTLPRVQELPEDAEATIDVHTHDVHHLKKAFDGLQPEVTRFLEKESPDWIIYDFAPYWLPSVAAGLRISRAFYSNFNAWFIAFLGASTDDLISGSGYDHRTRVENLMTPPKWVPFPTDVCYRKYEAVRMVGNTSANASGISSVYRVGMILKGSDCMFIRHSYEFEPQWLTLLEKLHHLPVVPVGLLPPEKPTNIEDGNDETWDTVKMWLDGQQKGHVVYVAFGSEVTLSRSELAELALGLELSGLPFFWALRKPVASTESKLVELPDGFLDRTSDRGLVGTSWAPQLQILSHESVGGFLTHCGWSSIVEAMMFGHPLIMLPCLADQGLNARVMVDKKVGIEIPRNGEDGSFHKESVARSVWVVVADDEGKIYKKNAMELSQLFGDTEMGKKYINQFIDYLEKKRRTLTV (SEQ ID NO. 1);

[0047] The nucleotide sequence of the present invention is shown in SEQ ID NO. 2:

[0048]

[0049] Proteins that have 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more homology with the amino acid sequence shown in SEQ ID NO.1 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or even 99.9% or more homology) and have the same or similar activity as glycosyltransferase UGT-X01 are also within the scope of protection of this invention.

[0050] Homology refers to the "sequence identity" between two amino acid sequences, that is, the percentage of identical amino acids between the sequences.

[0051] In this invention, the sucrose synthase SUS1 is derived from Arabidopsis thaliana, with its amino acid sequence as shown in SEQ ID NO.3 and its nucleotide sequence as shown in SEQ ID NO.4.

[0052] In this invention, the UDP-glucose dehydrogenase UGDH is derived from Arabidopsis thaliana, with the amino acid sequence shown in SEQ ID NO.5 and the nucleotide sequence shown in SEQ ID NO.6.

[0053] In this invention, the UDP-glucuronide decarboxylase UXS3 is derived from Arabidopsis thaliana, with its amino acid sequence as shown in SEQ ID NO.7 and its nucleotide sequence as shown in SEQ ID NO.8.

[0054] The glycosyltransferase UGT91D6, derived from stevia rebaudiana, has the amino acid sequence shown in SEQ ID NO. 9:

[0055] MDDHKQLHVAMFPWLAFGHILPFFELSKFITKNGHKVSFLSPTGNIQRLPSSNLSPLMNLVKLTLPRVQELPQNASATTDLHADDVQYLKQAFDGLQPEVTRFLEQESPDWIIYDFAPYWLPAVATSLGISRGFFSIFNAWTVSFFGSSPDDIINGTDDRKTADDFLTPPKWFPFPSKVCYRKHEANLIFADNISVNSSGVSDLYRLGMVIKGSDCMFIRHCHEFEPQWLTLLEKLHQLPVVPVGLLPPEPPTSTGDPWVTIKKWLDGQPIGHVVYVAFGSEGTMSQSELAELALGLELSGLPFFWVLRKPVGSGNSVELPEGFLERTRDRGLVWTSWVPQLQILSHESVCGFLTHSGWSSFVEAMMFGHPLIMLPLSVDQGLNARVMADNQVGIEIPRNDEDGSFTKESVARSLRLVLVDDEGKIYKAKAMELSQRFGDSKPENKYINPFIDYLEQKGRVVAIEHEL (SEQ ID NO. 9);

[0056] The nucleotide sequence thereof is shown as SEQ ID NO. 10:

[0057]

[0058] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0059] In this invention, Reb A was purchased from Guilin Rhine Biotechnology Co., Ltd., batch number SRE10-24052408, with a purity of 97%; UDP-xylose was purchased from Yuanye Biotechnology.

[0060] Information on the culture medium used in this application:

[0061] LB agar medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride (NaCl) 10 g / L, agar 15~20 g / L.

[0062] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (NaCl).

[0063] TB medium: 12 g / L peptone, 24 g / L yeast extract, 4 ml / L glycerol, 17 mM KH2PO4 and 72 mM K2HPO4.

[0064] Table 1 shows some of the primer information involved in the embodiments of this application:

[0065] Table 1 Primer Table

[0066] pET28a-R CATGGATCCGCGACCCATTTG pET28a-F TAACTCGAGCACCACCACCACC UGT-X01-F GGGTCGCGGATCCATGCTGAACGATCACAAAC UGT-X01-R GGTGGTGCTCGAGTCACACGGTCAGAGTAC AtSUS1-F GGTCGCGGATCCATGGCAAACGCTGAAC AtSUS1-R GTGGTGCTCGAGTCAATCATCTTGTGCAAGAG AtUGDH-F GGTCGCGGATCCATGGTGAAAATCTGTTGTATTG AtUGDH-R GTGGTGGTGCTCGAGTTAGGCCAGGGCCGG AtUXS3-F GGTCGCGGATCCATGACCTTCAACGCGTAC AtUXS3-R GGTGGTGCTCGAGTTAGTTGCGCGGTACG AtSUS1-pETDuet-1-F GATATACCATGGATGGCAAACGCTGAAC AtSUS1-pETDuet-1-R CTTAAAGTTAAACAAAATTATTTCTGCTGCTCAATC UGTX01-pETDuet-1-F GTCGGTACCCTCGAGATGCTGAACGATCACAAAC UGTX01-pETDuet-1-R GGTTTCTTTACCAGATCACACGGTCAGAGTACGAC pETDuet-1-F CTCTGACCGTGTGATCTGGTAAAGAAACCGCTG pETDuet-1-R CAGCGTTTGCCATCCATGGTATATCTCCTTC AtUGDH-pCDFDuet-1-F CCATGGGGATCCATGGTGAAAATCTGTTGTATTG AtUGDH-pCDFDuet-1-R CGAGCTCGAATTCTTAGGCCAGGGCCGGC AtUXS3-pCDFDUet-1-F AAGTATAAGAAGATGACCTTCAACGCGTAC AtUXS3-pCDFDuet-1-R CAAAATTATTTCTACAGGTTAGTTGCGCGGTACG UGT91D6-F GGTCGCGGATCCATGGACGACCACAAACAG UGT91D6-R GGTGGTGCTCGAGTTACAGCTCGTGCTCG pCDFDuet-1-F CCGCGCAACTAACCTGTAGAAATAATTTTGTTTAAC pCDFDuet-1-R CAGATTTTCACCATGGATCCCCATGGTATATC

[0067] It should be noted that the strain construction methods provided in the embodiments of the present invention are exemplary and not restrictive. Those skilled in the art can use any technical means to achieve the final strain construction objective.

[0068] The present invention will be further illustrated below with reference to the embodiments.

[0069] Example 1

[0070] 1. Cloning of glycosyltransferase UGT-X01 and AtSUS1, AtUGDH, and AtUXS3 genes

[0071] UGT-X01 (SEQ ID NO.2), AtSUS1 (SEQ ID NO.4), AtUGDH (SEQ ID NO.6), and AtUXS3 (SEQ ID NO.8) were artificially synthesized to obtain the full-length DNA sequence. The electrophoresis results of UGT-X01 are shown below. Figure 3 As shown, the size is 1000~1500 bp, which is consistent with expectations.

[0072] 2. Construction of recombinant expression vectors

[0073] 2.1 Using pET28a as the expression vector, recombinant expression plasmids were constructed using the In-Fusion strategy. PCR amplification primers containing homologous arms were designed (see Table 1) for the clone UGT-X01 (UGT-X01-F and UGT-X01-R as primer pairs) and the linearized pET28a vector (pET28a-F and pET28a-R as primer pairs). After agarose gel electrophoresis, the corresponding DNA fragments and linearized vectors were recovered and combined for homologous recombination. After incubation at 50℃ for 20 min, the mixture was transformed into E. coli DH5α competent cells, heat-shocked, and then plated onto Kan resistance plates and cultured overnight at 37℃. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB medium containing Kan for sequencing verification. The correctly sequenced recombinant plasmid pET28a-UGT-X01 was extracted.

[0074] Recombinant expression plasmids were constructed using an in-fusion strategy. PCR amplification primers containing homologous arms (see Table 1) were designed for the clone AtSUS1 (AtSUS1-F and AtSUS1-R were primer pairs) and the linearized pET28a vector (pET28a-F and pET28a-R were primer pairs). After agarose gel electrophoresis, the corresponding DNA fragments and the linearized vector were recovered and combined for homologous recombination. The reaction was carried out at 50℃ for 20 min, followed by transformation into E. coli DH5α competent cells. After heat shock incubation, the cells were plated onto Kans resistant plates and cultured overnight at 37℃. Positive clones were screened by colony PCR, and positive clones were picked and placed in 5 mL of LB medium containing Kans resistant culture medium for sequencing verification. The correctly sequenced recombinant plasmid pET28a-AtSUS1 was extracted.

[0075] pET28a-AtUGDH and pET28a-AtUXS3 were constructed using the same method described above.

[0076] 2.2 Using pET28a-UGT-X01 and pET28a-AtSUS1 obtained in 2.1 above as templates, recombinant expression plasmids were constructed using the same method. PCR amplification primers containing homologous arms were designed to clone the UGT-X01 gene fragment (UGTX01-pETDuet-1-F and UGTX01-pETDuet-1-R as primer pairs) and the AtSUS1 gene fragment (AtSUS1-pETDuet-1-F and AtSUS1-pETDuet-1-R as primer pairs), as well as the linearized pETDuet-1 fragment (pETDuet-1-F and pETDuet-1-R as primer pairs). The corresponding fragments were recovered and multi-fragment homologous recombination was performed to obtain the recombinant plasmid AtSUS1-UGTX01-pETDuet-1 (Amp resistant).

[0077] Using pET28a-AtUGDH and pET28a-AtUXS3 obtained in 2.1 above as templates, recombinant expression plasmids were constructed using the same method. PCR amplification primers containing homologous arms were designed to clone AtUGDH gene fragments (AtUGDH-pCDFDuet-1-F and AtUGDH-pCDFDuet-1-R as primer pairs) and AtUXS3 gene fragments (AtUXS3-pCDFDUet-1-F and AtUXS3-pCDFDUet-1-R as primer pairs) and linearized pCDFDuet-1 fragments (pCDFDuet-1-F and pCDFDuet-1-R). The corresponding fragments were recovered and multi-fragment homologous recombination was performed to obtain the recombinant plasmid AtUGDH-AtUXS3-pCDFDuet-1 (SmR resistant).

[0078] 3. Construction of recombinant expression bacteria

[0079] 3.1 Construction of recombinant strains V01-V04

[0080] The recombinant plasmid pET28a-UGT-X01 obtained in 2.1 was transformed into E. coli BL21 (DE3) to obtain recombinant strain V01, which was then frozen at -80℃. The recombinant strains pET28a-AtSUS1, pET28a-AtUGDH, and pET28a-AtUXS3 obtained in 2.1 were then transformed into E. coli BL21 (DE3) in sequence to obtain recombinant strains V02-V04, which were then frozen at -80℃.

[0081] The strain information is shown in Table 2 below:

[0082] Table 2. Strain Construction Information Table

[0083] V01 E. coli BL21 (DE3) pET28a-UGT-X01 V02 E. coli BL21 (DE3) pET28a-AtSUS1 V03 E. coli BL21 (DE3) pET28a-AtUGDH V04 E. coli BL21 (DE3) pET28a-AtUXS3

[0084] 3.2 Construction of engineered bacteria for producing rebaudioside U

[0085] AtSUS1-UGTX01-pETDuet-1 and AtUGDH-AtUXS3-pCDFDuet-1 were co-transformed into E. coli BL21(DE3) to obtain engineered strain U01, which was then stored at -80°C.

[0086] 4. Construction of comparative strains

[0087] Following the method in 2.1, only UGT-X01 was replaced with UGT91D6 (SEQ ID NO.10) to obtain the recombinant plasmid pET28a-UGT91D6 (Kan resistant).

[0088] pET28a-UGT91D6 was transferred into E. coli BL21 (DE3) to obtain the comparative recombinant strain V00, which was then frozen at -80°C.

[0089] Example 2: Verification of Single Enzyme Activity

[0090] 1. Enzyme expression and purification

[0091] The recombinant strains V01-V04 and V00 obtained in Example 1 were inoculated into 5-10 mL of LB medium containing Kan resistance and cultured overnight at 37°C to prepare fermentation seed culture. The seed culture was inoculated into 50 mL of TB medium at a 2% inoculation rate and cultured at 200 rpm and 37°C until OD600~0.6-0.8 was reached. 0.1 mM IPTG was added and expression was induced at 25°C for 20 h. The cells were collected by centrifugation at 10,000 rpm and 4°C for 10 min, the supernatant was removed, and the cells were stored at -20°C for later use.

[0092] The bacterial cells were resuspended in 100 mM pH 7.0 PBS buffer, followed by the addition of 1 mM PMSF and 1 mg / mL lysozyme, and stirred on ice for 30 min. The cells were then sonicated to disrupt the cell structure, and the supernatant was collected by centrifugation. The supernatant was then incubated with a Ni-NTA affinity column for 40–60 min, and the eluent was discarded. The target protein and other proteins were then eluted with 100 mM pH 7.0 PBS buffer containing different concentrations of imidazole (20 mM and 250 mM). The target protein was then subjected to buffer replacement, dialysis, desalting, and concentration using an ultrafiltration tube to obtain pure enzymes of UGT-X01, AtSUS1, AtUGDH, AtUXS3, and UGT91D6. These were then aliquoted and stored at -80°C for later use.

[0093] Electrophoresis images of UGT-X01, AtSUS1, AtUGDH, and AtUXS3 are shown below. Figure 4 As shown.

[0094] 2. Single enzyme activity verification system

[0095] 2.1 Using RA as a substrate and UDP-xylose as a glycosyl donor, RU is enzymatically generated.

[0096] 2.1.1 Reaction system: 200 μL volume includes 50 mM pH8.0 PBS buffer, 1 mM RA, 1 mM UDP-xylose and 2 μg glycosyltransferase (UGT-X01 or UGT91D6). The reaction temperature is 30℃, and the reaction time is 2 h. Each reaction condition is repeated 3 times in parallel. The sample is heated at 98℃ for 5 min to inactivate the enzyme, centrifuged at 12,000 for 10 min, and the supernatant is filtered through a 0.22 μm filter membrane for HPLC analysis to determine the amount of Reb U in the reaction sample and calculate the conversion rate.

[0097] The test results are shown in Table 3 below:

[0098] Table 3 Results of Single Enzyme Activity Verification

[0099]

[0100] As can be seen from Table 3, UGT-X01 can catalyze the conversion of Reb A to Reb U. Compared with UGT91D6 expressed by V0, the glycosyltransferase UGT-X01 of this invention has higher catalytic efficiency.

[0101] 2.1.2 Product Detection

[0102] Mass spectrometry analysis of the above-mentioned catalytic products was performed in positive ion mode, and the results are as follows: Figure 5 As shown.

[0103] Figure 5 UPLC-MS results of Reb U generated by UGT-X01 catalysis, from Figure 5 As can be seen, the molecular weight of the Reb U generated in the reaction is 1099, which is consistent with the theoretical molecular weight, confirming that the product generated in the reaction is Reb U.

[0104] 2.2 Using RA as a substrate, a multi-enzyme cascade reaction generates RU.

[0105] Reaction system: 200 μL volume including 50 mM pH 8.0 PBS buffer, 1 mM RA, 5 mM sucrose and a combination enzyme (2 μg AtSUS1, 2 μg AtUGDH, 2 μg AtUXS3 and 2 μg glycosyltransferase UGT-X01 or UGT91D6). The reaction temperature was 30℃, and the reaction time was 2 h. Each reaction condition was repeated 3 times in parallel. The sample was heated at 98℃ for 5 min to inactivate the enzyme, centrifuged at 12,000 for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane for HPLC analysis to determine the amount of Reb U in the reaction sample and calculate the conversion rate.

[0106] The test results are shown in Table 4 below:

[0107] Table 4 Results of Combinatorial Enzyme Activity Verification

[0108]

[0109] As can be seen from Table 4, in the reaction of RU generated by glycosyltransferase in combination with AtSUS1, AtUGDH and AtUXS3 using RA as substrate, the conversion rate of the glycosyltransferase UGT-X01 of the present invention is still higher than that of UGT91D6 in the prior art.

[0110] Example 3: Whole-cell biotransformation reaction experiment verification

[0111] 1. The engineered strain U01 obtained in Example 1 was inoculated into 5-10 mL of LB medium containing Amp and SmR resistance and cultured overnight at 37°C to prepare a fermentation seed culture. The seed culture was inoculated into 50 mL of TB medium at a 1% inoculation rate and cultured at 200 rpm at 37°C until the OD600 reached 0.6-0.8. 1 mM IPTG was added, and expression was induced at 16°C for 20 h. The cells were then collected by centrifugation at 10,000 rpm at 4°C for 10 min. The cells were resuspended in 50 mM pH 8.0 phosphate buffer and washed twice to obtain wet cells for whole-cell catalytic reactions.

[0112] The biotransformation reaction system consisted of 0.1 g / mL wet bacterial cells, 60 mM trisodium citrate, 100 mM pH 8.0 sodium phosphate buffer, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose, and 60 g / L RA substrate. The reaction temperature was 37 °C, and the reaction time was 24 h. Each reaction condition was repeated in triplicate. After the reaction, an equal volume of methanol was added to quench the reaction, and the mixture was centrifuged at 12,000 rpm for 10 min to remove the precipitate. The supernatant was filtered through a 0.22 μm filter and used for HPLC analysis to calculate the conversion rate of RU.

[0113] The test results are shown in Table 5 below:

[0114] Table 5. Validation Results of Whole-Cell Biotransformation Reaction

[0115]

[0116] 2. Optimization of the reaction system

[0117] 2.1 Effect of Temperature

[0118] The biotransformation efficiency of strain U01 was investigated at different reaction temperatures (25℃, 30℃, 37℃, 40℃, 45℃, 50℃, and 60℃). The biotransformation reaction system consisted of 0.1 g / mL wet bacterial cells (U01), 60 mM trisodium citrate, 100 mM pH 8.0 sodium phosphate buffer, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose, and 60 g / L RA substrate. Reactions were performed at each temperature for 24 h. Each reaction was repeated in triplicate. After the reaction, an equal volume of methanol was added to quench the reaction, and the mixture was centrifuged at 12,000 rpm for 10 min to remove the precipitate. The supernatant was filtered through a 0.22 μm filter and used for HPLC analysis to calculate the conversion rate of RU. The results are shown in Table 6.

[0119] Table 6 Results of the Effect of Temperature

[0120]

[0121] As can be seen from Table 6, the conversion rate of the biotransformation reaction system is highest when the temperature is controlled at 40℃, and it has a high conversion rate in the range of 37-45℃.

[0122] 2.2 Effect of pH

[0123] The biotransformation efficiency of strain U01 was investigated at different reaction pH values ​​(sodium phosphate buffer pH 6.0-8.0). The biotransformation reaction system consisted of 0.1 g / mL wet bacterial cells (U01), 60 mM trisodium citrate, 100 mM sodium phosphate buffer, 0.1 mM MgCl2, 1% (v / v) xylene, 400 g / L sucrose, and 60 g / L RA substrate. The reaction temperature was 40 °C, and the reaction time was 24 h. Each reaction condition was repeated in triplicate. After the reaction, an equal volume of methanol was added to quench the reaction, and the mixture was centrifuged at 12,000 rpm for 10 min to remove the precipitate. The supernatant was filtered through a 0.22 μm filter and used for HPLC analysis to calculate the conversion rate of RU. The results are shown in Table 7.

[0124] Table 7 Results of the effect of pH

[0125]

[0126] As can be seen from Table 7, the conversion rate is higher when the pH is controlled in the range of 7.5-8.0, while the conversion rate of RU is the highest when the pH is 8.0.

[0127] As shown in Tables 6 and 7, the optimal temperature for the biotransformation reaction system of strain U01 is 40℃ and the optimal pH is 8.0.

[0128] In summary, the glycosyltransferase UGT-X01 of this invention possesses catalytic activity for the conversion of rebaudioside A to rebaudioside U, and exhibits high catalytic efficiency. Utilizing this glycosyltransferase UGT-X01 or its mutant, with Reb A as the substrate and UDP-xylose as the glycosyl donor, a highly efficient enzymatic catalytic production of rebaudioside U is achieved. The Reb U production method of this invention overcomes the limitations of plant raw materials, realizing efficient and low-cost enzymatic production of Reb U, breaking through the bottleneck in rebaudioside U production, and facilitating the industrial production of Reb U. Furthermore, the glycosyltransferase UGT-X01 cascade with sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3 enables the production of Reb U using Reb A and sucrose as substrates. This allows for the efficient synthesis and recycling of expensive UDP-xylose using readily available and inexpensive sucrose as the starting carbon source, significantly reducing the raw material cost of the glycosyl donor.

[0129] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A glycosyltransferase UGT-X01, characterized in that, The glycosyltransferase is any one of the following (1)-(2): (1) It has the amino acid sequence shown in SEQ ID NO.1; (2) is a protein having an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology with the amino acid sequence described in (1), and having the same or similar activity as the glycosyltransferase UGT-X01.

2. The gene encoding the glycosyltransferase UGT-X01 as described in claim 1, characterized in that, The nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. A recombinant vector or recombinant strain containing the encoding gene of claim 2.

4. The recombinant strain according to claim 3, characterized in that, The host cell is one of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Yersinia lipolytica, or Pichia pastoris.

5. The use of the recombinant vector or recombinant strain according to claim 3 in the production of the glycosyltransferase UGT-X01 according to claim 1.

6. The use of the glycosyltransferase UGT-X01 of claim 1 or the recombinant strain of claim 3 in the production of rebaudioside U.

7. An engineered bacterium for producing rebaudioside U, characterized in that, In addition to expressing the glycosyltransferase UGT-X01 described in claim 1 in the host, the engineered bacteria also express one or more of sucrose synthase SUS1, UDP-glucose dehydrogenase UGDH, and UDP-glucuronide decarboxylase UXS3.

8. A method for producing rebaudioside U, characterized in that, A starting composition containing rebaudioside A is contacted with a biocatalyst to obtain rebaudioside U.

9. A method for producing rebaudioside U as described in claim 8, characterized in that, The biocatalyst comprises any one or more of the following: the glycosyltransferase UGT-X01 of claim 1, the recombinant strain or its culture of claim 3, and the engineered bacteria or its culture of claim 7.

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