Rhamnosyltransferase mutants, their encoding genes and use in the synthesis of dioscin

Abstract

The present application belongs to the technical field of dioscin biosynthesis, and particularly relates to a rhamnose transferase mutant, a coding gene thereof and application thereof in dioscin A synthesis. The amino acid sequence of the rhamnose transferase mutant is selected from at least one of SEQ ID NO. 2-19 or a fusion protein obtained by fusing a protein tag to the carboxy terminus or amino terminus of the foregoing sequence. The rhamnose transferase mutant obtained by combining rational modification, machine learning modification, combinatorial mutation modification and the like can use UDP-rhamnose as a glycosyl donor, use sibirienoside as a receptor, catalyze dioscin A to be generated, and compared with wild type DzGT1, the activity is increased by more than 20%, which provides valuable enzyme resources and efficient biological catalysts for synthesis of dioscin A and steroidal saponins, and has important industrial application value and potential in the field of biological enzyme catalysis of dioscin A and steroidal saponins.

Description

Technical Field

[0001] This invention relates to the field of diosgenin A biosynthesis technology, and particularly to a rhamnosyltransferase mutant, its encoding gene, and its application in diosgenin A synthesis. Background Technology

[0002] Dioscorea zingiberensis, also known as yellow yam, is a perennial herbaceous vine belonging to the genus Dioscorea in the family Dioscoreaceae. Its rhizomes are rich in steroidal saponins, making them an important industrial raw material for the synthesis of steroidal hormone drugs. Among these steroidal saponins, diosgenin is the main active ingredient. In recent years, diosgenin has attracted widespread attention due to its diverse pharmacological activities, including antitumor, anti-inflammatory, anti-hyperuricemia, antifungal, antiviral, and cardioprotective, renal, and hepatic protective effects.

[0003] The biosynthesis of dioscin involves a complex glycosylation process. Enzymatic glycosylation using glycosyltransferases has become a shortcut for its industrial production because: glycosyltransferase-catalyzed transglycosylation reactions are mild, have a unique glycosylation site, and can avoid glycosyl protection and deprotection. The aglycone of dioscin is diosgenin. The conversion from diosgenin to dioscin requires the sequential addition of a glucose and two rhamnose groups to its C-3 hydroxyl group. This process is synergistically completed by glucosyltransferases and rhamnosetransferases. Currently, the UDP-glucosyltransferase DzS3GT, which catalyzes the conversion of diosgenin to the C-3 glycosylation product—tribulin—has been cloned and identified from *Dioscorea zingiberensis*. Recent research has further explored and identified the first UDP-rhamnosyltransferase (UGT) DzGT1 in Dioscorea zingiberensis. This enzyme can specifically catalyze the production of prosapogenin A using triterpenoid as a substrate, filling the enzymatic gap in the key rhamnosylation step of the diosgenin biosynthesis pathway and laying a key foundation for the efficient biosynthesis of steroidal saponins through enzyme catalysis.

[0004] However, the wild-type DzGT1 exhibits low catalytic activity, making it difficult to meet the industrial production requirements of high-value steroidal saponins. Therefore, conducting molecular modification research on DzGT1 to improve its catalytic activity has significant theoretical and practical value. Summary of the Invention

[0005] To address the issue of low activity of the rhamnosyltransferase DzGT1 in existing technologies, this invention provides a novel rhamnosyltransferase mutant with an activity more than 1.2 times that of the wild type, offering a valuable enzyme resource and a highly efficient biocatalyst for the synthesis of diosgenin A and steroidal saponins. This invention also provides the nucleic acid molecule encoding this rhamnosyltransferase mutant, its expression vector, and recombinant bacterial strain, further demonstrating the application of this rhamnosyltransferase mutant in the synthesis of diosgenin A.

[0006] This invention is specifically achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a rhamnosyltransferase mutant selected from (a)-(b):

[0008] (a) The amino acid sequence is selected from at least one of SEQ ID NO.2-19;

[0009] (b) Fusion proteins obtained by attaching a fusion protein tag to the carboxyl or amino terminus of the amino acid sequence shown in SEQ ID NO.2-19.

[0010] Further, the amino acid sequence is selected from at least one of SEQ ID NO.3, 5-19; even further, the amino acid sequence is selected from at least one of SEQ ID NO.7-8, 10-19; and still further, the amino acid sequence is selected from at least one of 10-19.

[0011] Furthermore, the protein tag includes a purification tag and / or a solubilization tag, wherein the purification tag is a histidine tag and the solubilization tag is a small ubiquitin-like modified protein tag.

[0012] A second aspect of the present invention provides a nucleic acid molecule that encodes a rhamnosyltransferase mutant as described above.

[0013] A third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described above.

[0014] Furthermore, the expression vector is pET23a.

[0015] A fourth aspect of the present invention provides a recombinant strain comprising the expression vector described above.

[0016] Furthermore, the recombinant strain originated from Escherichia coli.

[0017] The fifth aspect of the present invention provides the application of the rhamnosyltransferase mutant described above in the synthesis of diosgenin A.

[0018] The sixth aspect of this invention provides a method for synthesizing diosgenin A, comprising the following steps:

[0019] Rhamnosyltransferase mutant, triterpenoid glycoside, and UDP-rhamnose were added to a buffer solution and reacted at pH 5-8 and 10-40. o The catalytic reaction is carried out under C conditions.

[0020] Furthermore, at pH 6.0 and 30 o The catalytic reaction is carried out under C conditions.

[0021] Furthermore, the content of the rhamnosyltransferase mutant is 20-40 μg / mL, the concentration of triterpenoid glycoside is 100-400 μM, and the concentration of UDP-rhamnose is 400-800 μM.

[0022] The advantages and positive effects of this invention are as follows:

[0023] This invention utilizes a combination of rational modification, machine learning modification, and combinatorial mutagenesis to obtain a rhamnosyltransferase mutant that can use UDP-rhamnose as a glycosyl donor and triterpenoid as an acceptor to catalyze the production of diosgenin A. Compared to the wild-type DzGT1, its activity is increased by more than 20%, providing valuable enzyme resources and efficient biocatalysts for the synthesis of diosgenin A and steroidal saponins. This is beneficial for improving substrate conversion and product yield, and has significant industrial application value and potential in the field of bio-enzymatic catalysis of diosgenin A and steroidal saponins. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a three-dimensional model diagram of wild-type DzGT1 in an embodiment of the present invention, wherein the left side is the predicted structure diagram of DzGT1 and the right side is the inferred trifolin binding pocket diagram.

[0026] Figure 2 This is a vector map of the recombinant expression vector pET23a-DzGT1 in an embodiment of the present invention;

[0027] Figure 3 This is a graph showing the comparison of enzyme activity between wild-type DzGT1 and DzGT1 fusion protein containing the His-SUMO tag in an embodiment of the present invention.

[0028] Figure 4This is a graph showing the optimal temperature and pH results for the wild-type DzGT1 fusion protein in an embodiment of the present invention.

[0029] Figure 5 This is a graph showing the comparison of enzyme activity between wild-type DzGT1 and its mutants 1-7 of this invention.

[0030] Figure 6 The image shows the comparison results of enzyme activity between wild-type DzGT1 and its mutants 8-10 of this invention.

[0031] Figure 7 The diagram shows the comparison results of enzyme activity of wild-type DzGT1 and its mutants 11-18 in this invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0033] Based on the information contained in this application, various changes to the precise description of the invention can be readily made by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the invention is not limited to the defined processes, properties, or components, as these embodiments and other descriptions are merely illustrative of specific aspects of the invention. In fact, various modifications to embodiments of the invention that will be apparent to those skilled in the art or related fields are covered within the scope of the appended claims.

[0034] To better understand the invention and not to limit its scope, all figures indicating amounts, percentages, and other numerical values ​​used in this application should, in all cases, be understood to be modified by the word "approximately." Therefore, unless specifically stated otherwise, the numerical parameters listed in the specification and appended claims are approximate values ​​and may vary depending on the desired properties being sought. Each numerical parameter should at least be considered as obtained based on reported significant figures and through conventional rounding methods.

[0035] Additionally, it should be noted that, unless otherwise defined, the scientific and technical terms used in the context of this invention should have the meanings commonly understood by those skilled in the art.

[0036] The terms “including,” “contains,” “includes,” “has,” and similar words are non-restrictive and can include other steps and other components that do not affect the result.

[0037] The term “and / or” should be regarded as referring to a specific disclosure of each of the two specified features or components, with or without the other. For example, “A and / or B” would be regarded as including (i) A, (ii) B, and (iii) A and B.

[0038] The term "gene" is the complete nucleotide sequence required to produce a polypeptide chain or functional RNA. A gene can contain several operatively linked nucleic acid segments, such as a 5' non-coding region, a coding sequence, and a 3' non-coding region containing polyadenylation sites. Non-coding regions generally have regulatory functions on gene expression, such as promoters and terminators.

[0039] The term "fusion gene" has the same meaning and refers to any gene that is not a natural gene, containing regulatory and coding sequences that are not naturally occurring together. Therefore, a fusion gene may include regulatory and coding sequences originating from different organisms, or regulatory and coding sequences originating from the same organism but arranged in a manner different from their natural origin, such as the rhamnosyltransferase expression cassette of this invention.

[0040] The term "vector" refers to a self-replicating DNA molecule that transfers a target gene into a host cell, and it is often in the form of a circular double-stranded DNA molecule. A vector containing a foreign gene is called a recombinant vector.

[0041] The term "expression vector" allows a target gene inserted into a vector to be expressed in a host cell, containing regulatory elements for expression in the designated host cell, such as promoters and / or terminators. An expression vector is introduced into an appropriate host cell to enable it to express the inserted target gene.

[0042] The terms "import" or "transfer" refer to the transfer of a target gene into a host cell, resulting in stable genetic inheritance. The imported nucleic acid molecule can be in plasmid form retained in the host cell or can be integrated into the host cell genome. Nucleic acid molecules and / or vectors can be transferred into host cells via methods such as "transfection," "transformation," or "transduction." Host cells containing the imported nucleic acid molecule are referred to as "transgenic," "recombinant," "transformed," or "engineered" organisms. Vector introduction into host cells can be performed using conventional techniques well known to those skilled in the art.

[0043] Unless otherwise stated, terms such as "nucleotide," "nucleic acid," "nucleic acid molecule," and "nucleic acid fragment" are interchangeable in the context of this invention. The terms "gene," "nucleic acid sequence," "nucleotide sequence," or "nucleic acid molecule" as used in this invention refer to a polymeric form of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides; this term refers only to the primary structure of the molecule.

[0044] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below.

[0045] Prosapogenin A is a natural steroidal saponin with significant pharmacological activity and an important intermediate in the synthesis of dioscin. Its natural abundance in plants is usually low, making direct extraction difficult. Enzymatic synthesis can solve the problems of low plant content and extraction difficulties.

[0046] In a 2021 study by the inventors, “Li J, Mosongo I, Li H, et al. Identification and Characterization of a Trillin Rhamnosyltransferase From Dioscoreazingiberensis[J]. Frontiers in Plant Science, 2021, 12. DOI:10.3389 / fpls.2021.713036,” a key rhamnosyltransferase—DzGT1—was first identified and characterized in Dioscoreazingiberensis. In vitro experiments showed that it can specifically transfer rhamnosyl groups to trillin using UDP-rhamnose as a glycosyl donor. The discovery of DzGT1 confirms that the pathway from trillin (CAS number: 14144-06-0) to prosapogenin A (CAS number: 19057-67-1) is a key branch point in the biosynthesis of diosgenin, filling a crucial gap in the biosynthetic pathway of diosgenin in Dioscorea peltatum and providing valuable gene elements for the biosynthesis of high-value steroidal saponins such as prosapogenin A.

[0047] The structural formula of trillin:

[0048] .

[0049] The structural formula of prosapogenin A is as follows:

[0050] .

[0051] To improve enzyme activity, this invention uses the wild-type DzGT1 sequence as a basis and employs MEGA11 software for multiple sequence alignment to analyze amino acid conservation, providing guidance for mutating amino acid residues into target amino acids. Subsequently, AlphaFold2 software is used to predict the three-dimensional structure of the target protein. Using structural visualization software such as Pymol, combined with AutoDock molecular docking software, the functional domains of DzGT1 and the spatial interactions between the target amino acid and the triterpenoid substrate binding pocket and key functional domains are analyzed. Based on the molecular docking results, amino acid residues within 5 Å of the substrate are selected as mutation targets. Through the aforementioned rational modification, further machine learning modification (method described in patent "CN119724342A") is performed to obtain multiple single-point mutants. These single mutants are then combined with mutated mutants. After fusing the genes of these candidate mutants with a histidine tag (10×His, for purification) and a small ubiquitin-like modified protein (SUMO, for solubilization) tag, they are heterologously expressed in an E. coli system (vector pET23a, host bacterium BL21(DE3)), and purified to obtain pure enzymes.

[0052] By comparing the catalytic activities of wild-type and mutant enzymes, several superior mutants were screened, including single mutants N200V, I203V, Y236F, R162G, N211A, N211V, D240A, F19W, R140G, and R140N, as well as combined mutants F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A. The amino acid sequences are shown in SEQ ID NO.2-19. In the mutant, the numbers represent amino acid sites, with wild-type amino acids on the left and mutant amino acids on the right. For example, N200V means that the 200th amino acid of wild-type DzGT1 is mutated from N to V.

[0053] Based on this, the present invention provides a rhamnosyltransferase mutant selected from (a)-(b):

[0054] (a) The amino acid sequence is selected from at least one of SEQ ID NO.2-19;

[0055] (b) Fusion proteins obtained by attaching a fusion protein tag to the carboxyl or amino terminus of the amino acid sequence shown in SEQ ID NO.2-19.

[0056] This invention utilizes a combination of rational modification, machine learning modification, and combinatorial mutagenesis to obtain more than ten rhamnosyltransferase mutants with enhanced enzyme activity. These mutant enzymes can catalyze the rhamnosylation of triterpenoids to generate diosgenin A using UDP-rhamnose as a glycosyl donor and triterpenoid as an acceptor. Compared to the wild-type DzGT1, their activity is increased by more than 20%, providing valuable enzyme resources and efficient biocatalysts for the synthesis of diosgenin A and steroidal saponins. This is beneficial for improving substrate conversion and product yield, and has significant industrial application value and potential in the field of bio-enzymatic catalysis of diosgenin A and steroidal saponins. The implementation of this invention also provides a reference strategy for the protein engineering modification of steroidal glycosyltransferases.

[0057] The preferred rhamnosyltransferase mutants are I203V, R162G, N211A, N211V, D240A, F19W, R140G, R140N, F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A, with amino acid sequences as shown in SEQ ID NO. 3 and 5-19, respectively. These mutants exhibit enzyme activity that is more than 1.6 times that of the wild type.

[0058] More preferably, the rhamnosyltransferase mutants are N211V, D240A, R140G, R140N, F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A, with amino acid sequences as shown in SEQ ID NO. 7-8 and 10-19, respectively. The enzyme activity of these mutants is more than 1.9 times that of the wild type.

[0059] More preferably, the rhamnosyltransferase mutant contains F19W and / or R140G / N mutation sites, including: R140G, R140N, F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A, with amino acid sequences as shown in SEQ ID NO.10-19, respectively. In a preferred embodiment, the enzyme activity is selected from combined mutants containing the F19W and R140G / N mutation sites (SEQ ID NO.12-19), which are more than 2.2 times that of the wild type.

[0060] Optionally, the protein tag includes a purification tag, wherein the purification tag is a histidine tag.

[0061] Optionally, the protein tag includes a solubilization tag, which is a small ubiquitin-like modified protein (SUMO) tag.

[0062] Another embodiment of the present invention provides a nucleic acid molecule that encodes a rhamnosyltransferase mutant as described above.

[0063] The advantages of the nucleic acid molecule over the prior art are the same as those of the rhamnosyltransferase mutant over the prior art as described above, and will not be repeated here.

[0064] Nucleic acid molecules include DNA molecules (such as genomic DNA or cDNA) and / or RNA molecules (such as mRNA), and the molecules can be single-stranded or double-stranded.

[0065] The sequence of a nucleic acid molecule can be derived from the amino acid (AA) sequence of a rhamnosyltransferase mutant using conventional methods such as codon coding rules. The full-length sequence of a nucleic acid molecule or a fragment thereof can usually be obtained using PCR amplification, recombination, or artificial synthesis.

[0066] Another embodiment of the present invention provides an expression vector comprising the nucleic acid molecules described above.

[0067] The advantages of the expression vector over the prior art are the same as those of the rhamnosyltransferase mutant over the prior art as described above, and will not be repeated here.

[0068] Optionally, the expression vector includes a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector (such as lentivirus or adenovirus). Correspondingly, the recipient cells transformed or transfected by the expression vector can be prokaryotic cells or eukaryotic cells, selected according to the type of expression vector. For example, when using a prokaryotic expression vector, prokaryotic cells are selected as recipient cells. Common examples of prokaryotic cells include Escherichia coli, Bacillus, Corynebacterium, Fermentosum motilityis, and Streptomyces. When using a eukaryotic expression vector, eukaryotic cells are selected as recipient cells. Common examples of eukaryotic cells include Saccharomyces cerevisiae, Pichia pastoris, Saccharomyces cerevisiae, and filamentous fungi.

[0069] Typical vectors include plasmids (such as the pUC series, pET series, pWB series, pGEX series, pDXW series, pBR series, pEZ15a, pTZ28a, pMA5, pPIC series, pHIL-S1, pSET152, pGAPZa, pA0815), viral vectors, bacteriophages (such as λgt4λB, λ-Charon, λΔz1, and M13), entrapments, and mini-chromosomes. Plasmids are the most commonly used vectors, and in the context of this invention, unless otherwise specified, plasmids and vectors are used interchangeably.

[0070] In a preferred embodiment, the expression vector is pET23a. The above-mentioned nucleic acid molecule is inserted between the multiple cloning sites of the pET23a expression vector to obtain a recombinant expression vector. This vector is then transformed into E. coli and cultured using conventional methods to express rhamnosyltransferase in the cells.

[0071] In another embodiment of the present invention, a recombinant strain is provided, the recombinant strain containing the nucleic acid molecule as described above or the expression vector as described above.

[0072] Optionally, the recombinant strain originates from Escherichia coli, specifically BL21(DE3).

[0073] Recombinant strains can be transformed into target gene nucleic acid molecules or expression vectors using various methods known in the art, including: CaCl2 transformation, calcium phosphate-DNA coprecipitation, electroporation, gene gun bombardment, microinjection, conjugation transfer, liposome transfection, liposome fusion, and protoplast fusion, etc.

[0074] This invention also provides a method for preparing a rhamnosyltransferase mutant, comprising the following steps:

[0075] The recombinant strain described above was cultured, the fermentation broth was centrifuged, the cell pellet was collected, the cells were lysed, the cell lysate was collected, and the rhamnosyltransferase mutant was obtained by affinity purification using a nickel column.

[0076] The present invention further provides the application of the rhamnosyltransferase mutant as described above in the synthesis of diosgenin A and the corresponding synthesis method.

[0077] The synthesis method includes: adding rhamnosyltransferase mutant, triterpenoid glycoside, and UDP-rhamnose to a buffer solution, and reacting at pH 5-8 and 10-40. o The catalytic reaction is carried out under C conditions.

[0078] Preferably, the pH value is 6.0 and the temperature is 30°C. o C.

[0079] Optionally, in the reaction system, the content of the rhamnosyltransferase mutant is 20-40 μg / mL, the concentration of triterpenoid glycoside is 100-400 μM, and the concentration of UDP-rhamnose is 400-800 μM.

[0080] The present invention will be further illustrated below with reference to specific embodiments. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions, such as those described in *Molecular Cloning: A Laboratory Manual (Fourth Edition)* published by Cold Spring Harbor Laboratory, or generally under the conditions recommended by the manufacturer.

[0081] In the following embodiments, the main biomaterials include:

[0082] 1) Biomaterials: Escherichia coli BL21 (DE3) and pET23a vector, commercially available.

[0083] 2) Culture medium: LB medium: 10 g / L sodium chloride, 5 g / L yeast extract and 10 g / L peptone.

[0084] 1. Screening of rhamnosyltransferase mutants

[0085] 1.1 Rational modification of the DzGT1 protein sequence

[0086] (1) Analysis of conserved amino acid sites in the DzGT1 sequence

[0087] Trillin rhamnosyltransferase GT1 (DzGT1, amino acid sequence see GenBank: QZL13805.1) was sequence-aligned with enzymes possessing UDP-rhamnosyltransferase activity, including AtUGT78D1 from Arabidopsis thaliana, UGT29-24 from Panax ginseng, SGT3 from soybean (Glycine max), UGT85H2 from alfalfa (Medicago tribuloides), C12RT1 from Citrus grandis, UGT89C1 from Arabidopsis thaliana, and UGT73CE1 and UGT73YD1 from Paris polyphylla. The alignment was performed using the ClustalW algorithm in MEGA11 software with default parameters, and the conservation of amino acids was determined based on the sequence alignment results. Download the Fasta file containing DzGT1 sequence information. On Windows, download ncbi_blast_2.9.0+ and configure the environment variables. Download and unzip the SwissProt database. Use PSI-BLAST to read the amino acid sequence corresponding to each protein and generate the corresponding position-specific scoring matrix (PSSM). The matrix results provide guidance for mutating amino acid residues into target amino acids.

[0088] The amino acid sequence of wild-type DzGT1 is shown below:

[0089] MATHAGHEPRPLRVFFIPFFATGHMIPMVDIARLFAARGVDATVLVTPSNAALISRTIDDAAASGLPIRTLLYPFPSAEVGLPPGAENIASVPLADAPKVDAASLLTAGDHDRLLRLHHPDAVV SDTHFAWTTSIARDLRIPRITFHAIGLFPVCVLGSLIRNLPHLSVTDDVHPFIVPDLPHPVQMFRPELPDFLRSENTAIAQAMSALNEAEAGSLGVVVNSFAEIESAYADCYYKFDNMRTWFVG PVALADGGEKASRGAAAADAKAVANRELCMRWLDKQRERSVVYACFGSWCHFSAAQLRELALGLEAAGHPFLWVVRDDGEEWMPEGFEERLGEKGLVVRGWAPQVAVLGHDAVGGFVTHCGWNSV LEGVSSGLPMVTWPLSTEQFINEKLVVEVLGTAVRAWEGFRSTEEGEKEVVKAGDVAAAVEKVMGGGEEGEKRRVTAREFGEKAKAAVKVGGSSYEGLSRLIGEIRDWPKKINGCDAATA (see SEQ ID NO.1).

[0090] (2) Obtain the DzGT1 structural prediction model

[0091] The amino acid sequence of wild-type DzGT1 was input into the Alphafold2 online software (https: / / alphafoldserver.com / ) to predict the protein's three-dimensional structure. Five models (model_0-4) were generated, with confidence levels decreasing sequentially. The result with the highest confidence, cif_0, was selected, and the cif file was converted to a PDB file using Open Babel to obtain the three-dimensional structure predicted by DzGT1.

[0092] (3) Molecular docking of DzGT1 with trifolinine

[0093] The three-dimensional structure of DzGT1 was visualized using Pymol 3.0.0 software. DzGT1 belongs to the GT-B fold, with its N-terminal domain being a tribulus glycoside-binding domain and its C-terminus being a UDP-rhamnose-binding domain. Based on the visualization of the surface structure of DzGT1, it is inferred that there are two potential tribulus glycoside-binding pockets. Figure 1A three-dimensional model of DzGT1 is shown, with the predicted structure of DzGT1 on the left and the inferred trillin binding pocket diagram on the right. In the diagram, the UDP-Rha binding pocket represents the UDP-rhamnose binding domain, H24 represents the key amino acid of DzGT1 deprotonated trillin, Trillin binding pocket 1 represents trillin binding pocket 1, and Trillin binding pocket 2 represents trillin binding pocket 2.

[0094] Trillium glycoside was docked to binding pocket 1 using AutoDock 4.2.6 software. Docking box parameters: npts 4058 72, spacing 0.375, gridcenter 2.448 12.917 -8.576, smooth 0.5, and dielectric -0.1465. The docking results indicate that trillium glycoside may have two binding modes at binding pocket 1. The 2D interaction between DzGT1 and trillium glycoside at binding pocket 1 was visualized using Discovery Studio 2021 software. In the first binding mode, residues T389, E390, and I149 form hydrogen bonds with tribulus glycoside; residues D101, F195, I149, W296, and H24 form van der Waals interactions with tribulus glycoside; and residues W131C, A209, A130, L105, L210, A206, and H128 form alkyl / π-alkyl interactions with tribulus glycoside. In the second binding mode, residues H147 and A148 form hydrogen bonds with tribulus glycoside; residues S198, N200, D194, P193, T389, E390, H24, L210, H128, L157, and T127 form van der Waals interactions with tribulus glycoside; and residues I203 and F195 form alkyl / π-alkyl interactions with DzGT1.

[0095] Trillium glycoside was docked to binding pocket 2 using AutoDock 4.2.6 software. Docking box parameters: npts 6040 42, spacing 0.375, gridcenter -6.763 7.308 -6.78, smooth 0.5, and dielectric -0.1465. The 2D interaction between DzGT1 and trillium glycoside at docking pocket 2 was visualized using Discovery Studio 2021 software. Residues N211, E390, H128, and H24 form hydrogen bond interactions with tribulus glycoside; residues R162, D240, L157, T389, W296, F19, I89, A148, T127, I161, and G158 form van der Waals interactions with tribulus glycoside; and residues H147, Y236, V154, I149, L210, and M207 form alkyl / π-alkyl interactions with tribulus glycoside.

[0096] (4) Design of DzGT1 rationally modified mutants

[0097] Based on molecular docking results, amino acid residues within 5 Å of the substrate were selected as mutation targets. Referring to sequence alignment and PSSM results, mutants were designed considering pocket size, substrate channels, and interaction forces. Mutants D101G, D101L, D101V, F195W, I203V, N200V, H128N, and H128C were constructed in pocket 1 of the DzGT1 receptor; mutants N211A, N211V, H147L, H147W, Y236F, R162G, D240A, and L157Y were constructed in pocket 2 of the DzGT1 receptor.

[0098] Enzyme activity tests were performed on these mutants, yielding seven superior mutants: N200V, I203V, Y236F, R162G, N211A, N211V, and D240A, with activities more than 1.2 times that of the wild type. These mutants were named mutants 1-7, and their amino acid sequences are shown in SEQ ID NO.2-8 (Table 1).

[0099] Table 1. Amino acid sequences of mutants 1-7 (the shaded and bolded areas indicate the mutation sites).

[0100]

[0101] 1.2 Machine Learning Modification of the DzGT1 Protein Sequence

[0102] Referring to the modification model and method described in patent "CN119724342A A Method for Molecular Modification of Glycosyltransferases Based on Artificial Intelligence (Publication Date: 2025-03-28)", DzGT1 was modified using machine learning. The first round of prediction yielded the following mutants: L157Y, E229Q, D358P, H147W, L402W, I203W, L151W, F452W, A112C, W131C, H128F, S224T, E390D, E390W, G469N, D194W, R478K, S134D, A42V, G362C, E285P, L45V, F195W, R33H, Y73I, and A291V.

[0103] The mutants D358P, L402W, I203W, F452W, A112C, W131C, H128F, G469N, S134D, L45V, R33H, A291V, R478K, A42V, and E285P were characterized by enzyme kinetics. The kinetic parameters were summarized and fed back into the enzyme prediction model. After iterative optimization of the model, the following mutants were obtained: R243P, T22E, R13H, F19W, A130P, A36H, G342N, L473F, I477W, L139F, V44F, V398M, R140G, R140N, F452L, L380V, and C276W.

[0104] Using UDP-Glo TM The kit (purchased from Promega, catalog number: S39250423120333) was used to determine enzyme activity. The detection principle is as follows: the kit converts UDP, catalyzed by DzGT1, into ATP, which is then catalyzed by luciferase to generate a light signal. The enzyme activity of DzGT1 is determined by detecting the light signal value. Prepare a 25 μL reaction system according to Table 4. After the reaction, add 25 μL of UDP detection reagent, mix well, and continuously monitor the light signal value in an OMEGA instrument for 1 h. The DzGT1 enzyme activity is then determined using a constructed UDP standard curve.

[0105] Table 2. Rhamnosyltransferase catalytic reaction system

[0106]

[0107] Three superior mutants, F19W, R140G, and R140N, were generated from the iterative mutants and named mutants 8-10, respectively. Their activity was 120% higher than that of the wild type. The amino acid sequences are shown in SEQ ID NO.9-11 (Table 3).

[0108] Table 3. Amino acid sequences of mutants 8-10 (the shaded and bolded areas indicate the mutation sites).

[0109]

[0110] 1.3. Modification of DzGT1 combined mutants

[0111] The F19W and R140G / N mutants are far from the binding pocket and have little impact on substrate binding. Therefore, they were combined with the superior mutants N200V, I203V, Y236F, R162G, N211A, N211V, and D240A obtained through rational modification to obtain superior mutants F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A, which were named mutants 11-18, respectively. Their amino acid sequences are shown in SEQ ID NO.12-19 (Table 4).

[0112] Table 4. Amino acid sequences of mutants 11-18 (the shaded and bolded areas indicate the mutation sites).

[0113]

[0114] 2. Expression and purification of wild-type DzGT1 and its mutants

[0115] 2.1 Construction of rhamnosyltransferase expression vector

[0116] A 10×His and SUMO tag was fused to the N-terminus of DzGT1 to facilitate the isolation and purification of the fusion protein. The amino acid sequence of the His-SUMO tag is shown below:

[0117] HHHHHHHHHHMSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG (see SEQ ID NO. 20).

[0118] The fusion gene sequences of the DzGT1 mutant and its wild-type were synthesized by Nanjing Qingke Biotechnology Co., Ltd. The nucleotide sequence of the wild-type DzGT1 gene is shown in SEQ ID NO. 21, and the nucleotide sequence of the His-SUMO tag is shown in SEQ ID NO. 22. The gene sequences of each mutant were obtained by replacing the amino acid codons at the mutation sites of the wild-type DzGT1 gene with the codons of the mutated amino acids, and therefore will not be shown again.

[0119]

[0120] caccatcatcatcatcatcatcatcatcatagcagcggcatgtcggactcagaagtcaatcaagaagctaagccagaggtcaagccagaagtcaagcctgagactcacatcaatttaaaggtgtccgatggatcttcagagatcttcttcaagatcaaaaagaccactc ctttaagaaggctgatggaagcgttcgctaaaagacagggtaaggaaatggactccttaagattcttgtacgacggtattagaattcaagctgatcagacccctgaagatttggacatggaggataacgatattattgaggctcacagagaacagattggtgga (see SEQ ID NO.22).

[0121] The fusion gene was inserted downstream of the T7 promoter of the pET23a expression vector to obtain the recombinant vector pET23a-DzGT1. See the diagram of the recombinant vector below. Figure 2 In the figure, f1 ori represents the replication initiation site of f1 phage, AmpR represents ampicillin resistance, ori represents the replication initiator, rop represents a repetitive fragment on the plasmid DNA sequence, and P represents the replication initiator. T7 This indicates the T7 promoter.

[0122] 2.2 Expression and purification of rhamnosyltransferase

[0123] The recombinant vector pET23a-DzGT1, containing DzGT1 and its mutants, was transformed into *E. coli* BL21(DE3) using the *E. coli* BL21(DE3) expression system. Positive transformants were inoculated into LB medium and cultured overnight. The activated bacterial culture was then transferred to 1 L of LB medium (with ampicillin added at a concentration of 100 μg / mL) and incubated at 37°C. o C. Incubate at 200 rpm to allow OD 600 To reach a concentration of 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.4 mM, and then... oC. Induce at 200 rpm for 16 h. Centrifuge the induced bacterial culture at 4000 rpm for 10 min, discard the supernatant, wash the cells once with PBS buffer, then resuspend the cells in 30 mL of Tris-HCl buffer (pH 7.5), and add 300 μL of 100 mM benzyl sulfonyl fluoride (PMSF). Perform homogenization using a low-temperature continuous ultra-high pressure homogenizer. After homogenization, add 30 μL of 100 mM PMSF, and homogenize the culture at 15000 rpm for 4 h. o Centrifuge at 30°C for 30 min. Filter the supernatant after centrifugation and incubate it with a His protein purification Ni column (purchased from Sangon Biotech) at 4°C. o Incubate in a silent mixer for 1 h, then elute the recombinant protein (His-SUMO-DzGT1) with elution buffer containing gradient imidazoles (10, 20, 50, 100, 200, 300, 500 mM) (containing 350 mM sodium chloride, 20 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), and different concentrations of imidazole, pH 7.5). Collect the eluent, analyze each component by SDS-PAGE electrophoresis, and use an ultrafiltration tube with a 10 kDa cutoff at 4°C. o C. Concentration was carried out at 4000 rpm. The concentrated wild-type DzGT1 and its mutant enzyme solutions were used for subsequent enzyme activity experiments.

[0124] 3. Rhamnosyltransferase activity analysis

[0125] 3.1 Enzymatic Properties Analysis of Wild-Type Rhamnosyltransferase DzGT1

[0126] This embodiment first compares the enzyme activities of DzGT1 (without His-SUMO fusion tag) and His-SUMO-DzGT1 (with His-SUMO fusion tag, hereinafter referred to as DzGT1-SUMO). The enzyme activity was measured using UDP-Glo TM The kit was used. Results are as follows: Figure 3 As shown, the horizontal axis represents protein grouping, with DzGT1 being tested in four replicates. The vertical axis represents relative enzyme activity, with the DzGT1-SUMO fusion protease activity defined as 100%. It can be seen that the DzGT1-SUMO enzyme activity is slightly higher than that of DzGT1, but the difference is within 10%. Subsequently, wild-type DzGT1 containing the SUMO tag (still referred to as DzGT1) and its mutants were selected for enzyme activity assays.

[0127] Following the reaction systems in Table 4, the enzymatic properties of DzGT1 were studied at optimal reaction temperatures and pH values, with three replicates for each reaction group. The optimal reaction temperatures were 10, 20, 30, 40, 50, 60, and 70 °C. oThe reaction was carried out at C for 30 min, after which the light signal value was measured to determine the optimal reaction temperature of DzGT1. A citric acid-NaOH buffer system was used to control the pH of the reaction system at 3.0-6.0, and a Tris-HCl buffer system was used to control the pH of the reaction system at 7.0-9.0. The reaction systems at different pH values ​​were then subjected to a 30-minute incubation period. o The reaction was carried out under C conditions for 30 min, and then the light signal value was detected to determine the optimal reaction pH of DzGT1.

[0128] The results are as follows Figure 4 As shown in the figure, the horizontal axis represents temperature and pH, and the vertical axis represents relative enzyme activity, with enzyme activity measured at pH 6 as 100%. The figure shows that the optimal reaction temperature for DzGT1 is 30°C. o C. When the reaction temperature is at 10, 20, or 40 degrees Celsius... o At time C, DzGT1 maintained a speed of 30. o Under C conditions, over 90% of the activity remains; when the temperature is 60 or 70 degrees Celsius... o At time C, DzGT1 only maintained a speed of 30. o Under C conditions, the activity of DzGT1 is 40%-60%. The optimal reaction pH for DzGT1 is 6. When the pH of the buffer solution is 5, 6, 7, or 8, DzGT1 still retains more than 80% of its activity. When the pH is 3 or 9, DzGT1 essentially loses its activity. Based on these results, the subsequent enzyme activity assay conditions were determined to be a temperature of 30°C. o C. pH 6.

[0129] 3.2 Enzyme activity analysis of the rhamnosyltransferase DzGT1 mutant

[0130] The reaction system in Table 5 was reacted in the dark for 30 min. After the reaction was completed, 150 µL of methanol was added to terminate the reaction. After thorough mixing, the mixture was filtered through a 0.22 µm filter membrane to remove protein precipitate. The filtrate was then transferred to a sample vial for HPLC analysis.

[0131] Table 5 Catalytic reaction system of rhamnosyltransferase mutant

[0132]

[0133] Rhamnose transferase catalyzes the production of diosgenin A and UDP using UDP-rhamnose (UDP-Rha) as the sugar donor and tribulus glycoside as the acceptor. Tribulus glycoside and the product diosgenin A were detected using a Shimadzu high-performance liquid chromatograph (LC-2030 Plus) with an Agilent (ZORBAX SB-Aq) column. Detection wavelength: 203 nm; oven temperature: 40 °C. oC. Mobile phase: HPLC-grade acetonitrile (A) and water (B), flow rate: 0.8 mL / min, detection time: 45 min. A gradient elution program was used: 0-15 min, the proportion of mobile phase A linearly increased from 20% to 60%; 15-17 min, maintained at 60%; 17-25 min, continued to increase from 60% to 90%; 25-30 min, maintained at 90%; 30-35 min, the proportion decreased from 90% to 20%; 35-45 min, stabilized at 20%.

[0134] The enzyme activity assay results for mutants 1-18 are shown in the figure. Figure 5-7 The x-axis represents relative enzyme activity, with wild-type DzGT1 protease activity set at 100%, and the y-axis represents mutant groupings. For example... Figure 5 As shown, the activities of the superior mutants N200V, I203V, Y236F, R162G, N211A, N211V, and D240A (mutants 1-7) obtained through rational modification are 129.9%, 172.1%, 134.9%, 168.8%, 192.0%, 211.6%, and 232.1% of those of the wild-type DzGT1, respectively. Figure 6 As shown, the activities of the superior mutants F19W, R140G, and R140N (mutants 8-10) obtained through machine learning modification are 167.9%, 255.4%, and 239.2% of those of the wild-type DzGT1, respectively; Figure 7 As shown, the beneficial activities of the combined mutants F19W-R140G, F19W-R140N, F19W-R140G-N211A, F19W-R140G-D240A, F19W-R140N-N211A, F19W-R140N-D240A, F19W-R140G-N211A-D240A, and F19W-R140N-N211A-D240A (mutants 11-18) were 246.0%, 308.6%, 254.7%, 257.5%, 226.9%, 261.6%, 285.4%, and 264.1% of the wild-type DzGT1, respectively.

[0135] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rhamnosyltransferase mutant, characterized in that, Choose one of (a)-(b): (a) The amino acid sequence is selected from at least one of SEQ ID NO.2-19; (b) Fusion proteins obtained by attaching a fusion protein tag to the carboxyl or amino terminus of the amino acid sequence shown in SEQ ID NO.2-19.

2. The rhamnosyltransferase mutant according to claim 1, characterized in that, The amino acid sequence is selected from at least one of SEQ ID NO. 3, 5-19; the protein tag includes a purification tag and / or a solubilization tag, wherein the purification tag is a histidine tag and the solubilization tag is a small ubiquitin-like modified protein tag.

3. The rhamnosyltransferase mutant according to claim 1, characterized in that, The amino acid sequence is selected from at least one of SEQ ID NO. 7-8, 10-19.

4. The rhamnosyltransferase mutant according to claim 1, characterized in that, The amino acid sequence is selected from at least one of 10-19.

5. A nucleic acid molecule, characterized in that, Encodes the rhamnosyltransferase mutant as described in any one of claims 1-4.

6. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 5.

7. A recombinant bacterial strain, characterized in that, The recombinant strain includes the nucleic acid molecule as described in claim 5 or the expression vector as described in claim 6.

8. A method for synthesizing diosgenin A, characterized in that, Includes the following steps: The rhamnosyltransferase mutant as described in any one of claims 1-4, triterpenoid glycoside, and UDP-rhamnose were added to a buffer solution and the solution was prepared at pH 5-8 and 10-40. o The catalytic reaction is carried out under C conditions.

9. The method for synthesizing diosgenin A according to claim 8, characterized in that, The content of the rhamnosyltransferase mutant is 20-40 μg / mL, the concentration of triterpenoid glycoside is 100-400 μM, and the concentration of UDP-rhamnose is 400-800 μM.

10. The method for synthesizing diosgenin A according to claim 8, characterized in that, At pH 6.0, 30 o The catalytic reaction is carried out under C conditions.

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