Glycosyltransferase, nucleic acid fragment, recombinant expression vector, host cell and application thereof

Abstract

The invention relates to glycosyltransferase, a nucleic acid fragment, a recombinant expression vector, a host cell and application of the glycosyltransferase. The amino acid sequence of the glycosyl transferase is as shown in SEQ ID NO: 1. According to the application, an enzyme for converting 3-oxo-ionol glycosyl into 3-oxo-ionol glucoside is found from tobacco, namely, a novel glycosyl transferase is provided, and the function of the enzyme in biosynthesis of related glycosyl transferase genes is cloned and verified for the first time; in vitro, the recombinant protein can be used for efficiently converting a 3-oxo-ionol substrate into corresponding glycoside. The invention further provides a recombinant plasmid containing the glycosyl transferase gene, glucoside can be massively synthesized through a bioengineering method, meanwhile, the invention further provides application of the recombinant plasmid capable of massively synthesizing 3-oxo-ionol glucoside, more basic elements are provided for synthetic biology of natural products, and the recombinant plasmid can be used for preparing a large amount of 3-oxo-ionol glucoside. In addition, guidance and basis are provided for rational design of the enzymes, and the method has good industrialization prospects.

Description

Technical Field

[0001] This application belongs to the field of biotechnology, and specifically relates to glycosyltransferases, nucleic acid fragments, recombinant expression vectors, host cells, and their applications. Background Technology

[0002] Glycosides also play an indispensable role in tobacco, serving as important precursors to tobacco aroma compounds. During the processing, aging, or combustion of tobacco, glycosides undergo decomposition or transformation, releasing numerous aroma components that play a crucial role in aroma perception. These components greatly enrich the aroma profile of tobacco and have a significant impact on its aroma quality.

[0003] Currently, the preparation of tobacco flavorings and fragrances mainly relies on two methods: direct extraction from natural products or traditional chemical synthesis. However, both methods have significant limitations: natural product extraction is limited by the scarcity of raw material resources, making it difficult to achieve large-scale and stable production; traditional chemical synthesis faces problems such as lengthy routes and harsh reaction conditions, and also carries the risk of heavy metal pollution.

[0004] Therefore, traditional technologies still need improvement. Summary of the Invention

[0005] Based on this, this application provides a glycosyltransferase that can efficiently transfer glycosyl groups, glycosylation 3-oxoionol into 3-oxoionol glucoside.

[0006] The specific proposal of this application is as follows:

[0007] The first aspect of this application provides a glycosyltransferase having the amino acid sequence shown in SEQ ID NO:1.

[0008] A second aspect of this application provides a nucleic acid fragment encoding the aforementioned glycosyltransferase.

[0009] In some embodiments, the nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO:2.

[0010] A third aspect of this application provides a PCR primer pair that can detect the aforementioned nucleic acid fragments via PCR amplification.

[0011] In some embodiments, an upstream primer with a nucleotide sequence as shown in SEQ ID NO:3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:4 are included.

[0012] A fourth aspect of this application provides a recombinant expression vector comprising the aforementioned nucleic acid fragment.

[0013] The fifth aspect of this application provides a host cell that expresses the above-described glycosyltransferase, or that includes the above-described nucleic acid fragment or the above-described recombinant expression vector.

[0014] The sixth aspect of this application provides a method for preparing a glycosyltransferase, comprising: transforming a host cell with a recombinant expression vector containing a nucleic acid fragment encoding the glycosyltransferase, culturing the transformed host cell, and preparing the glycosyltransferase.

[0015] The seventh aspect of this application provides a method for preparing 3-oxoionol glucoside, comprising the following steps:

[0016] 3-O-ionol, a glycosyl donor, and the glycosyltransferase were mixed and reacted to prepare 3-oxoionol glucoside.

[0017] In some embodiments, the concentration of 3-oxoionol in the reaction system is 0.01 mM to 100 mM;

[0018] And / or, the concentration of the glycosyl donor in the reaction system is 0.01 mM to 100 mM;

[0019] And / or, the reaction temperature is 16℃~60℃, the reaction time is 0.1h~1h, and the reaction pH is 5~9.

[0020] This application discovered an enzyme in tobacco that converts 3-oxoionol glycosyls to 3-oxoionol glucoside, thus providing a novel glycosyltransferase. During the research, the applicant not only cloned the gene encoding this enzyme but also clarified its core role as a key glycosyltransferase in the biosynthetic pathway through functional verification experiments. The protein recombinantly expressed by this gene can efficiently convert 3-oxoionol substrates and directionally generate the target glycoside product.

[0021] Based on this, this application further provides a recombinant expression vector containing the glycosyltransferase gene: with the help of this vector, 3-oxoionol glucoside can be synthesized on a large scale and at low cost through bioengineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis. Attached Figure Description

[0022] Figure 1 The diagram shows the structure of the NUGT2-pET28a recombinant plasmid in Example 1;

[0023] Figure 2 This is a schematic diagram of the reaction process of 3-oxoionol glucoside in Example 1;

[0024] Figure 3The image shows the chromatographic results of 3-oxo-ionol glucoside in Example 1. The horizontal axis represents retention time in minutes, and the vertical axis represents signal intensity. Detailed Implementation

[0025] To facilitate understanding of this application, a more comprehensive description of the application will be provided below with reference to embodiments, and preferred embodiments of the application are given below. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. These embodiments are provided to provide a more thorough and complete understanding of the disclosure of this application. It should be understood that experimental methods in the following embodiments, unless specific conditions are specified, are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used reagents used in the embodiments are commercially available products.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0027] The terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element preceded by the phrase “comprising one…” does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. The indefinite articles “a” and “an” preceding an element or component in this application are not restrictive in terms of the quantity (i.e., the number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and singular elements or components also include plural forms, unless the quantity clearly refers only to the singular. “A plurality” means at least two, such as two, three, etc., unless otherwise expressly specified.

[0028] The terms "first," "second," and "third," etc., are used only for distinguishing descriptive purposes and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly specifying the importance or quantity of the indicated technical features. Moreover, "first," "second," etc., serve only a non-exhaustive enumeration purpose and should be understood not to constitute a closed limitation on quantity.

[0029] A "vector" is a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0030] "Host cell" refers to a cell that can be used to introduce the vector, including but not limited to animal cells such as Escherichia coli, fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0031] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the weights mentioned in the embodiments of this application can be well-known units of mass in the chemical industry, such as μg, mg, g, and kg.

[0032] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0033] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0034] One embodiment of this application provides a glycosyltransferase, the amino acid sequence of which is shown in SEQ ID NO:1.

[0035] Specifically, the amino acid sequence shown in SEQ ID NO:1 is: MGQLHIFFFPMMAHGHMIPTLDMAKLFASRGVKATIITTPLNESVFSKAIQRNKHLGIEIEIRLIKFPAVENDLPEECERLDQIPSDEKLPNFFKAVAMMQEPLEKLIQECRPNCLVSDMFLPWTTDSAAKFNIPRIVFHGTSFFALCVENSVRLNKPFKNVSSDSETFVVPNLPHEIKLTRTQVSPFERSGEETAMTRMIKTVRESDSKSYGVVFNSFYELETDYVEHYT KVLGRRAWAIGPLSMCNRDIVDKAERGKKSSIDKHECLKWLDSKKPSSVVYICFGSVANTASQLHELAMGIEASGQEFIWVVRTELDNEDWLPEGFEERTKEKGLIIRGWAPQVLILDHESV GAFVTHCGWNSTLEGVSGGVPMVTWPVFAEQFFNEKLVTVVLKTGAGVGSIQWKRSASEGVKREAIAKAVKRVMVSEEADGFRNRAKAYKEMATKAIEKGGSSYTGLTTLLEDISTYSSTDH.

[0036] In addition, one embodiment of this application provides a nucleic acid fragment for encoding the above-mentioned glycosyltransferase for secretion and expression in host cells.

[0037]

[0038] In some of these embodiments, the aforementioned nucleic acid fragments can be prepared by conventional chemical synthesis or other methods.

[0039] In some embodiments, the aforementioned nucleic acid fragments may be added to a recombinant expression vector to mediate the secretory expression of glycosyltransferases.

[0040] One embodiment of this application provides a PCR primer pair capable of amplifying the aforementioned nucleic acid fragments.

[0041] As is understandable, a PCR primer pair consists of two artificially synthesized oligonucleotide sequences. One primer is complementary to one DNA template strand at one end of the target gene, and the other primer is complementary to the other DNA template strand at the other end of the target gene. In PCR (polymerase chain reaction) technology, given a known nucleotide sequence of the target gene, primers are synthesized based on this sequence. Using PCR amplification technology, the target gene DNA is denatured by heat and breaks down into single strands. The primers bind to the corresponding complementary sequences on the single strands. Then, extension is performed under the action of a thermostable DNA polymerase. This cycle is repeated, and the product obtained after extension can also bind to the primers.

[0042] In one specific example, the PCR primer pair described above includes an upstream primer with the nucleotide sequence shown in SEQ ID NO:3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO:4. It is understood that PCR primer pairs capable of amplifying the above nucleic acid fragments are not limited to these, and PCR primer pairs with other sequences can be designed according to design rules.

[0043] Further, the nucleotide sequence shown in SEQ ID NO:3 is specifically: 5'-TGCCGCGCGGCAGCCATATGATGGGTCAGCTCCATATTTTCTTCTTCCC-3'; the nucleotide sequence shown in SEQ ID NO:4 is specifically: 5'-CAAGCTTGTCGACGGAGCTCTTAATGATCAGTAGAACTATATGTACTTATATCTTCC-3'.

[0044] In addition, one embodiment of this application provides a recombinant expression vector containing the above-mentioned nucleic acid fragment encoding glycosyltransferase.

[0045] In one of the optional specific examples, the vector used to express the glycosyltransferase is pET28a. The glycosyltransferase expression vector is synthesized and constructed, the vector is double-digested with restriction endonucleases NdeI and SacI, and then gel electrophoresis is performed to recover the target fragment. The target sequence of the glycosyltransferase is cloned into the restriction sites of NdeI and SacI, and the expression vector containing the glycosyltransferase is transformed into host cells to express the target protein.

[0046] It can be understood that the recombinant expression vectors mentioned above are prokaryotic expression vectors.

[0047] In a specific example, the recombinant expression vector described above is E. coli DH5α. It is understood that the specific type of vector is not limited to this and can be selected according to specific needs.

[0048] This application further provides a recombinant expression vector containing the glycosyltransferase gene: with the help of this vector, 3-oxoionol glucoside can be synthesized on a large scale and at low cost through bioengineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis.

[0049] In addition, one embodiment of this application provides a host cell whose genome contains the above-mentioned recombinant expression vector, which can promote the secretion and expression of glycosyltransferase.

[0050] In one embodiment, the host cell is *Escherichia coli*. Of course, in other embodiments, the host cell is not limited to *E. coli* and can be selected as needed.

[0051] Another embodiment of this application provides a method for preparing the above-mentioned glycosyltransferase, which includes transforming a recombinant expression vector containing a nucleic acid fragment encoding the above-mentioned glycosyltransferase into a host cell, culturing the transformed host cell, and preparing the glycosyltransferase.

[0052] After culturing transfected host cells for a period of time, the host cells or the culture medium containing the host cells were collected to prepare a primary product containing glycosyltransferase. The primary product was then purified to prepare the purified glycosyltransferase. The results showed that the glycosyltransferase synthesized in this application can efficiently convert 3-oxoionol substrates to directionally generate the target glycoside product.

[0053] One embodiment of this application also provides the use of the above-mentioned glycosyltransferase, the above-mentioned nucleic acid fragment, the above-mentioned recombinant expression vector, or the above-mentioned host cell in the preparation of 3-oxoionol glucoside.

[0054] Another embodiment of this application provides a method for preparing 3-oxoionol glucoside, comprising the following steps: mixing 3-oxoionol, a glycosyl donor, and the above-mentioned glycosyltransferase to prepare 3-oxoionol glucoside.

[0055] In some embodiments, the concentration of the 3-oxoionol in the above reaction system is 0.01 mM to 100 mM. For example, the concentration can be 0.01 mM, 0.1 mM, 1 mM, 3 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. In some examples, it can be any two of these point values ​​as the end values, and the same applies below.

[0056] In one specific example, the concentration of the above-mentioned 3-oxoionol in the above reaction system is 0.5 mM.

[0057] In some embodiments, the concentration of the glycosyl donor in the reaction system is 0.01 mM to 100 mM. For example, the concentration can be 0.01 mM, 0.1 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM.

[0058] In a specific example, the concentration of the glycosyl donor in the above reaction system is 2 mM.

[0059] In one specific example, the glycosyl donor is uridine diphosphate glucose.

[0060] In some embodiments, the concentration of the glycosyltransferase in the reaction system is 0.1 mg / L to 100 mg / L. For example, the concentration can be 0.1 mg / L, 1 mg / L, 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L.

[0061] In a specific example, the concentration of the aforementioned glycosyltransferase in the above reaction system is 50 mg / L.

[0062] In some embodiments, the reaction temperature is 16°C to 60°C, the reaction time is 0.1h to 1h, and the pH is 5 to 9. For example, the temperature can be 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, 52°C, 54°C, 56°C, 58°C, or 60°C; the time can be 0.1h, 0.5h, or 1h; and the pH can be 5, 6, 7, 8, or 9.

[0063] In a specific example, the reaction temperature was 30°C, the reaction time was 1 hour, and the pH was 7.5.

[0064] This application discovered an enzyme in tobacco that converts 3-oxoionol glycosyls to 3-oxoionol glucoside, thus providing a novel glycosyltransferase. During the research, the applicant not only cloned the gene encoding this enzyme but also clarified its core role as a key glycosyltransferase in the biosynthetic pathway through functional verification experiments. The protein recombinantly expressed by this gene can efficiently convert 3-oxoionol substrates and directionally generate the target glycoside product.

[0065] Based on this, this application further provides a recombinant expression vector containing the glycosyltransferase gene: with the help of this vector, 3-oxoionol glucoside can be synthesized on a large scale and at low cost through bioengineering technologies such as microbial fermentation and enzyme catalysis, breaking through the efficiency bottleneck of traditional natural product extraction or chemical synthesis.

[0066] Another embodiment of this application provides a method for preparing megalotrienone, comprising: heat-treating the above-mentioned 3-oxoionol glucoside or adding a deglycosylated and dehydrated substance to cigarettes to obtain megalotrienone.

[0067] In some embodiments, the temperature of the heat treatment is 110°C to 210°C.

[0068] This application expands the application scenarios of the above-mentioned technology system, which can be used for the preparation of megastigmatrienone, providing a new path for the industrial production of tobacco aroma substances and natural active terpenoids, and providing important theoretical basis and experimental reference for the rational design of such enzymes, with good industrialization prospects.

[0069] To make the objectives, technical solutions, and advantages of this application clearer and more concise, the following specific embodiments are used for illustration, but this application is by no means limited to these embodiments. The embodiments described below are merely preferred embodiments of this application and can be used to describe this application, but should not be construed as limiting the scope of this application. It should be noted that any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application. Experimental methods not specified with specific conditions in the embodiments are implemented according to conventional conditions, such as those described in documents, books, or methods recommended by the manufacturer.

[0070] The plasmid pET28a used in the examples was preserved in our laboratory. The restriction enzymes NdeI and SacI, along with the ligase, were purchased from Thermo Fisher Scientific. The competent cell preparation kit and plasmid purification kit were also purchased from Thermo Fisher Scientific. The E. coli DH5α strain and E. coli BL(21) strain used were preserved in our laboratory. Urate diphosphate glucose (UDP-Glc) was purchased from Zhongke Ruitai Company. The substrate 3-oxoionol was synthesized in our laboratory.

[0071] Example 1

[0072] 1. Construction of recombinant Escherichia coli glycosyltransferase engineered bacteria

[0073] (1) The NUGT2 gene was obtained by amplifying tobacco leaf cDNA using NUGT2-specific primers (NUGT2F and NUGT2R). Specifically, the nucleotide sequences of the specific primers NUGT2F and NUGT2R are as follows: 5'-TGCCGCGCGGCAGCCATATGATGGGTCAGCTCCATATTTTCTTCTTCCC-3'; and 5'-CAAGCTTGTCGACGGAGCTCTTAATGATCAGTAGAACTATATGTACTTATATCTTCC-3'.

[0074] (2) The NUGT2 fragment obtained by the above amplification was homologously recombined with the pET28a plasmid linearized by NdeI and SacI; the recombinant product was transformed into E. coli DH5α and screened by LB solid medium containing 50 μg / mL Kan (specific formula: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, pH 7.0-7.4).

[0075] (3) The recombinant plasmid NUGT2-pET28a obtained Figure 1The NUGT2-pET28a recombinant plasmid was transformed into E. coli BL(21) to obtain a protein expression strain. The specific transformation steps were as follows: 100 ng of plasmid was added to E. coli BL(21) competent cells, placed on ice for 30 min, heat-treated at 42℃ for 90 s, placed on ice for 3 min, 900 uL of LB liquid medium was added, and cultured at 37℃ for 1 h. The culture was then spread on LB solid medium (containing 50 μg / mL), cultured at 37℃ for 16 h, and single clones were selected for PCR verification.

[0076] PCR verification: Universal primers were T7 and T7t. T7 (SEQ ID NO:5): TAATACGACTCACTATAGGG; T7t (SEQ ID NO:6): GCTAGTATTGCTCAGCGG. The PCR amplification program was: 95℃ for 5 min; (95℃ for 30 s, 55℃ for 15 s, 72℃ for 1 min, 30 cycles), 72℃ for 5 min.

[0077] 2. Purification of glycosyltransferase

[0078] (1) The protein expression strain obtained above was inoculated into LB medium containing Kan and cultured overnight at 37°C; then transferred to 2 L of LB liquid medium containing Kan at a ratio of 1% and incubated at 37°C until the bacterial culture OD. 600 = 0.6-0.8 and placed on ice to cool; add IPTG to a final concentration of 0.2 mM and induce overnight at 16°C for 18 h.

[0079] (2) Collect the above bacterial cells and resuspend them in Buffer A (20 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH=8.0) buffer. Sonicate on ice for 5 s, pause for 30 s, for a total of 4 min. Centrifuge the lysate at 24000 rpm for 2 h.

[0080] (3) Collect the supernatant, filter it through a 0.22 μm filter membrane, and load the filtrate onto a protein purification His column. Elute with 20 mL of Buffer A, and then elute with 20 mL of 250 mM imidazole buffer (Buffer A and Buffer B each 50%) (Buffer B: 500 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol, pH=8.0). Collect the protein with 280 nm absorption. Detect the purification effect by SDS-PAGE electrophoresis.

[0081] (4) The collected liquid was placed in a 10 kDa molecular sieve to concentrate the protein, centrifuged at 4000 rpm, added 500 μL of storage solution (100 mM NaH2PO4 / Na2HPO4, 10% glycerol, pH=7.0), centrifuged at 4000 rpm, and then frozen in liquid nitrogen and stored at -80℃.

[0082] 3. Enzymatic activity detection and analysis of NUGT2-pET28a recombinant protein

[0083] Experimental group: In a 100 μL system, 50 mM Tris-HCl pH=7.5, 0.5 mM substrate (3-oxoionol), 2 mM UDP-Glc, 10 μg NUGT2 enzyme, the reaction was carried out at 30℃ for 1 h. The reaction process is as follows. Figure 2 As shown, an equal volume of acetonitrile was added to stop the reaction, and the supernatant was collected by centrifugation for LC-MS analysis.

[0084] Control group (CK): No enzyme was added, and all other conditions and parameters were the same as those of the experimental group.

[0085] The detection method was as follows: the liquid chromatography-mass spectrometry (LC-MS) system was an Agilent 1290 / 6530 system, the chromatographic column was a YMC-Triart C18 (ID 4.6×250 mm), the column temperature was 30℃, the chromatographic parameters were 10-100% acetonitrile for 0-20 min, 100% acetonitrile for 20-24 min, and 10% acetonitrile for 24-28 min, and the mass spectrometer was an electrospray ionization (ESI) source, performing positive ion mode scanning.

[0086] The NUGT2 enzyme was reacted with the substrate 3-oxo-ionol. The experimental group NUGT2 showed an additional product peak compared to the control group (CK). Under analytical conditions, the retention times were 9.4 min and 9.6 min, consistent with the elution time of the standard 3-oxo-ionol glucoside. The results are as follows: Figure 3 As shown above, Figure 3 It was discovered that the recombinase NUGT2-pET28a has glycosylation activity for 3-oxoionol.

[0087] In summary, the technical solution of this application enables the enzymatic glycosylation of 3-oxoionol to 3-oxoionol glucoside for the preparation of megastigmatrienone, with high conversion rate and purity, and high preparation efficiency.

[0088] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.

Claims

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

1.

2. A nucleic acid fragment, characterized in that, It encodes the glycosyltransferase as described in claim 1.

3. The nucleic acid fragment according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid fragment is shown in SEQ ID NO:

2.

4. A PCR primer pair, characterized in that, The PCR primer pair is capable of amplifying the nucleic acid fragments described in claim 2 or 3.

5. The PCR primer pair according to claim 4, characterized in that, Includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO:3 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

4.

6. A recombinant expression vector, characterized in that, Includes the nucleic acid fragments described in claim 2 or 3.

7. A host cell, characterized in that, It expresses the glycosyltransferase of claim 1, or it comprises the nucleic acid fragment of any one of claims 2 to 3 or the recombinant expression vector of claim 6.

8. A method for preparing the glycosyltransferase according to claim 1, characterized in that, include: After transforming a host cell with a recombinant expression vector containing a nucleic acid fragment encoding the glycosyltransferase, the transformed host cell is cultured to prepare the glycosyltransferase.

9. A method for preparing 3-oxoionol glucoside, characterized in that, Includes the following steps: 3-O-ionol, a glycosyl donor, and the glycosyltransferase described in claim 1 are mixed and reacted to prepare 3-oxoionol glucoside.

10. The method for preparing 3-oxoionol glucoside according to claim 9, characterized in that, The concentration of 3-oxoionol in the reaction system is 0.01 mM to 100 mM; And / or, the concentration of the glycosyl donor in the reaction system is 0.01 mM to 100 mM; And / or, the reaction temperature is 16℃~60℃, the reaction time is 0.1h~1h, and the reaction pH is 5~9.

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