Application of a Centella asiatica glycosyltransferase gene in catalyzing the glycosylation of asiatic acid and madecassic acid
By screening and verifying the functions of UGT73s tandem repeat gene clusters of Centella Asakusa, especially CaUGT73L69, the glycosylation efficiency of Centella Asakusa and hydroxycentella Asakusa is improved, the problem of low synthesis efficiency of Centella Asakusa triterpene saponin in the prior art is solved, and the industrial production of Centella Asakusa active ingredients is promoted.
Patent Information
- Application Number
- CN202510474508.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-16
- Publication Date
- 2025-08-26
- Estimated Expiration
- 2045-04-16
AI Technical Summary
The prior art has not yet conducted in-depth research on the functional differences and substrate preferences of glycosyltransferases of Centella asiatic acid and hydroxycentella asiatic acid, which has affected the synthesis efficiency and industrial production of Centella asiatic acid triterpene saponins.
By constructing the metabolic regulation network of bioactive substances of Centella asiatica, UGT73s tandem repeat gene clusters were screened out, and the functions of CaUGT1 and CaUGT73L69, especially the efficient catalytic ability of CaUGT73L69 to hydroxycentella asiatic acid were verified, and an in vitro enzyme catalytic system was constructed.
The glycosylation efficiency of Centella asoxic acid and hydroxycentella asoxic acid has been improved, the yield of Centella asoxic acid and hydroxycentella asoxic acid has been enhanced, and an important theoretical basis for the in vitro synthesis and industrial production of Centella asoxic triterpene saponins.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of a Centella asiatica glycosyltransferase gene in catalyzing the glycosylation of asiatic acid and madecassic acid. Background Art
[0002] Centella asiatica Centella asiatica Centella asiatica (L.) Urban) is a perennial herbaceous plant of the genus Centella in the family Apiaceae. Centella asiatica can produce new roots at the nodes of its stems during its prostrate growth, resulting in a strong regenerative ability and primarily asexual reproduction. Centella asiatica is cold in nature, bitter, and pungent in flavor. It has the effects of clearing heat and dampness, detoxifying, and reducing swelling. It is commonly used to treat damp-heat jaundice, carbuncles, sores, and injuries from falls. In southern my country, people often drink Centella asiatica as a cold tea to dispel wind and dampness, relax muscles and activate blood circulation, and treat skin inflammation. Some countries abroad also use Centella asiatica as a traditional herbal remedy for skin diseases and ailments such as leprosy. In modern medicine, it is often used to treat skin burns.
[0003] A variety of chemical substances have been detected in Centella asiatica, primarily triterpenes, volatile oils, polyacetylenes, flavonoids, and alkaloids, as well as other compounds such as sterols and polyphenols. Centella asiatica is rich in a variety of physiologically active pentacyclic triterpenes, primarily asiaticoside, asiatic acid, madecassoside, and madecassic acid. Asiaticoside and madecassoside are ursane-type saponins, playing important roles in neuroprotection, cardiomyocyte protection, and prevention of cell aging and acute kidney injury. They are important physiologically active substances in Centella asiatica. Therefore, research on Centella asiatica extracts is common in the pharmaceutical and cosmetic industries. Furthermore, studies have shown that asiaticoside exhibits excellent antimicrobial activity, protecting cells from pathogenic infection and is considered a phytoanthin against bacteria, fungi, and parasites.
[0004] The biosynthetic pathway of Centella asiatica triterpenoid saponins includes three stages: precursor supply, synthesis of pentacyclic triterpenoid skeleton, and multi-step glycosylation modification. Centella asiatica and madecassoside are the synthetic precursors of Centella asiatica and madecassoside, respectively. The final Centella asiatica and madecassoside can be produced by three consecutive glycosylation modifications (glucose-glucose-rhamnose, GGR) at the C-28 position of Centella asiatica and madecassoside. This step requires the catalysis of a series of glycosyltransferases. At present, two subfamilies of glycosyltransferases involved in the glycosylation of Centella asiatica and madecassoside have been identified. One subfamily is involved in the first step of glucose sylation. UGT73sThe second type is responsible for the catalysis of the second D-glucose and the third L-rhamnose in the GGR sugar chain. UGT94s However, all of them only involve the functional verification of Centella asiatica saponin glycosyltransferase, and have not yet UGT73s Study on functional differences and substrate preferences of tandemly repeated genes. Summary of the Invention
[0005] The purpose of the present invention is to explore the factors involved in the synthesis of triterpenoid saponins in Centella asiatica by constructing a metabolic regulatory network of bioactive substances in Centella asiatica. UGT73s Tandemly repeated gene clusters and the genes reported therein CaUGT1 and new genes CaUGT73L69 In vitro functional validation was performed to further demonstrate the functional differences of the tandem genes in the synthesis of triterpenoid saponins in Centella asiatica, providing a new genetic basis for optimizing the in vitro synthesis and industrial production of triterpenoid saponins in Centella asiatica.
[0006] In order to achieve the above object, the present invention provides the following technical solutions:
[0007] The first aspect of the present invention provides a use of a protein in catalyzing one or more of the following reactions:
[0008] E1) catalyzes asiatic acid to produce asiatic acid monoglucoside;
[0009] E2) catalyzes madecassic acid to produce madecassic acid monoglucoside.
[0010] The protein is named CaUGT73L69 and can be any of the following:
[0011] A1) a protein having an amino acid sequence of SEQ ID No. 3;
[0012] A2) A fusion protein with the same function as A1) is obtained by connecting a tag to the N-terminus and / or C-terminus.
[0013] In order to facilitate purification or detection of the protein in A1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 3 in the sequence listing.
[0014] The tag protein includes but is not limited to: GST (glutathione sulfhydryl transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.
[0015] The second aspect of the present invention provides the use of biomaterials in catalyzing one or more of the following reactions:
[0016] E1) catalyzes asiatic acid to produce asiatic acid monoglucoside;
[0017] E2) catalyzes madecassic acid to produce madecassic acid monoglucoside.
[0018] The biological material is any one of the following D1) to D4):
[0019] D1) an expression cassette containing a nucleic acid molecule encoding the protein CaUGT73L69;
[0020] D2) a recombinant vector containing a nucleic acid molecule encoding the protein CaUGT73L69, and a recombinant vector containing the expression cassette described in D1);
[0021] D3) a recombinant microorganism containing a nucleic acid molecule encoding the protein CaUGT73L69, a recombinant microorganism containing the expression cassette described in D1), or a recombinant microorganism containing the recombinant vector described in D2);
[0022] D4) A genetically engineered host cell containing a nucleic acid molecule encoding the protein CaUGT73L69, a genetically engineered host cell containing the expression cassette described in D1), or a genetically engineered host cell containing the recombinant vector described in D2).
[0023] The nucleic acid molecule is a DNA molecule whose nucleotide sequence is shown in SEQ ID No. 1. The nucleotide sequence shown in SEQ ID NO. 1 is the nucleotide sequence of the gene (CDS) encoding protein CaUGT73L69, and has a length of 1488 bp.
[0024] The third aspect of the present invention provides the use of the above-mentioned protein CaUGT73L69 and its encoding gene and the above-mentioned biological material in preparing enzyme catalysts in one or more of the following reactions:
[0025] E1) catalyzes asiatic acid to produce asiatic acid monoglucoside;
[0026] E2) catalyzes madecassic acid to produce madecassic acid monoglucoside.
[0027] The fourth aspect of the present invention provides the use of the above-mentioned protein CaUGT73L69 and its encoding gene and the above-mentioned biological material in the preparation of Centella asiatica monoglucoside or Madecassic acid monoglucoside.
[0028] The fifth aspect of the present invention provides an in vitro glycosylation method, comprising the following steps: in the presence of a glycosyltransferase, transferring the glycosyl of a glycosyl donor to the C-28 position of asiatic acid or madecassic acid, thereby forming asiatic acid monoglucoside or madecassic acid monoglucoside; wherein the glycosyltransferase is selected from the glycosyltransferase shown in SEQ ID No. 3.
[0029] The specific process of realizing the present invention is as follows:
[0030] The present invention utilizes the metabolic regulatory network of bioactive substances in Centella asiatica to screen multiple reporter genes and potential candidate genes related to the metabolic accumulation of Centella asiatica saponins. At the same time, the metabolic regulatory network reveals that a tandem gene cluster of glycosyltransferases on chromosome 8 is involved in the biosynthesis of Centella asiatica saponins. The genes in this gene cluster belong to UGT73 family, and through tissue expression analysis, it was found that these genes were highly expressed in the aboveground tissues (leaves and stems) of Centella asiatica. CaUGT73L69 In vitro functional validation was performed and the reporter gene in the gene cluster was compared CaUGT1 Comparison of catalytic abilities was performed. CaUGT73L69 and CaUGT1 The nucleotide sequences of the genes are shown in SEQ ID No. 1 and SEQ ID No. 2, both of which have a sequence length of 1488 bp. The protein sequences encoded by the genes are shown in SEQ ID No. 3 and SEQ ID No. 4, both of which have 495 amino acid residues.
[0031] The gene of the present invention was amplified from the cDNA obtained by reverse transcription of the mRNA of Centella asiatica using PCR technology. CaUGT1 and CaUGT73L69 , both sequences can be constructed into pDonr207 ( Figure 5 and Figure 6 ) entry vector and pGEX-6p-1 ( Figure 7 and Figure 8 ) prokaryotic expression vector, and by expressing and purifying the protein, the in vitro enzyme activity verification analysis of the two genes was obtained.
[0032] In vitro enzyme activity experiments showed that both CaUGT1-pEGX-6P-1 and CaUGT73L69-pEGX-6P-1 recombinant proteins could catalyze the glucosyl modification of asiatic acid and madecassic acid once. Among them, CaUGT1 showed a catalytic preference for asiatic acid, while CaUGT73L69 had a higher catalytic efficiency for madecassic acid.
[0033] Compared with the prior art, the present invention has the following effects:
[0034] 1. By constructing a metabolic regulatory network, we extensively and accurately screened candidate genes involved in the metabolic synthesis of triterpenoid saponins in Centella asiatica.
[0035] 2. In vitro enzyme activity verification confirmed that CaUGT1 and CaUGT73L69 can catalyze the initial glucosylation modification of asiaticoside precursors asiatic acid and hydroxymadecassic acid.
[0036] 3. Based on the above advantages, the present invention can provide an important reference for the in vitro synthesis of Centella asiatica triterpenoid saponins and for improving the glycosylation efficiency of asiatic acid and madecassic acid and the yield of asiaticoside and madecassinoside during industrial production, and provide important theoretical support for the large-scale production of Centella asiatica active saponins.
[0037] The present invention will be further described below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 To screen and locate the metabolic network of asiaticoside and madecassoside triterpenoids by constructing CaUGT1 and CaUGT73L69 Gene: Figure 1 A in the figure is the metabolic network construction analysis result of asiaticoside and madecassoside triterpenoid compounds; Figure 1 B in the figure is an analysis of multiple tandem repeats in the chromosome 8 region where the located gene is located and the tissue expression of the tandem repeat genes.
[0039] Figure 2 for CaUGT1 Genes and CaUGT73L69 The purification results of the protein corresponding to the gene, the five lanes on the left are CaUGT73L69 The purification results of the gene-synthesized protein are shown in the five lanes on the right. CaUGT1 Purification results of gene synthesized proteins.
[0040] Figure 3 The results of in vitro enzyme activities of CaUGT1 and CaUGT73L69 using asiatic acid as substrate are shown in Figure 1. Figure 3 A in the reaction is asiatic acid (1), which is catalyzed by glycosyltransferases CaUGT1 and CaUGT73L69 to form asiatic acid monoglucoside (2); Figure 3 Figure B is the in vitro enzyme activity chromatogram of CaUGT1 and CaUGT73L69. The yellow solid line is the CaUGT1 chromatogram, the green solid line is the CaUGT73L69 chromatogram, and the purple solid line is the blank control chromatogram.
[0041] Figure 4 The in vitro enzyme activity results of CaUGT1 and CaUGT73L69 using madecassic acid as substrate: Figure 4A in the reaction is madecassic acid (3), which is catalyzed by glycosyltransferases CaUGT1 and CaUGT73L69 to form madecassic acid monoglucoside (4); Figure 4 B in the figure is the in vitro enzyme activity chromatogram of CaUGT1 and CaUGT73L69; the yellow solid line is the chromatogram of CaUGT1, the green solid line is the chromatogram of CaUGT73L69, and the purple solid line is the chromatogram of the blank control group.
[0042] Figure 5 This is the plasmid map of the entry vector CaUGT1-pDonr207.
[0043] Figure 6 This is the plasmid map of the entry vector CaUGT73L69-pDonr207.
[0044] Figure 7 This is the plasmid map of the prokaryotic expression vector CaUGT1-pGEX-6p-1.
[0045] Figure 8 This is the plasmid map of the prokaryotic expression vector CaUGT73L69-pGEX-6p-1. DETAILED DESCRIPTION
[0046] The following examples define the present invention and describe the invention in isolating clones comprising CaUGT1 and CaUGT73L69 DNA fragments of the complete coding region, and verification CaUGT1 and CaUGT73L69 The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The experimental materials used in the following examples, unless otherwise specified, were purchased from commercial channels.
[0047] Example 1: CaUGT1 and CaUGT73L69 Discovery and positioning
[0048] The applicant constructed a metabolic regulatory network of Centella asiatica physiologically active substances by using multi-omics data and discovered a subnetwork containing 8 triterpenoid compounds ( Figure 1 ). This terpenoid synthesizer network contains multiple functional genes: 5 OSC s, 2 CYP714s , 6 CYP716s , 7 UGT73s and 3 UGT94s Among them, there are several reporter genes involved in the synthesis of Centella asiatica saponins, such as CaCYP714E19 and CaCYP716C11 It can catalyze ursolic acid to produce asiatic acid. CaUGT3 、 CaUGT4 、 CaUGT73AD1 and CaUGT73C8Participates in the formation of GGR sugar chains in asiaticoside and madecassoside. Further analysis found that the UGT73s Tandemly repeated gene clusters are involved in the formation of saponin sugar chains, and genes with higher expression levels were selected. CaUGT73L69 And reported CaUGT1 Perform subsequent in vitro validation.
[0049] Example 2: Isolation of clones CaUGT1 and CaUGT73L69 Gene
[0050] In order to obtain CaUGT1 and CaUGT73L69 The applicant used TRIZOL reagent (Invitrogen) to extract total RNA from Centella asiatica (the extraction method was based on the instructions for the TRIZOL reagent) and reverse transcribed the RNA into cDNA using a reverse transcription kit Supermix (purchased from Beijing Quanshijin Company). The reaction conditions were: 42°C for 30 min, 80°C for 5 s. Using the cDNA as a template, the primers CaUGT1 F- aaaaagcaggcttaATGGCATCTAATAAGATTCAAC, CaUGT1 R-agaaagctgggtaTCAAAATTGAGTTAAATTTTGCTG and CaUGT73L69 F- aaaaagcaggcttaATGGCTACCAATATTGAGCAG, CaUGT73L69 R-agaaagctgggtaTCATGAATGACTTAAATTTTGTTG was amplified by PCR. CaUGT1 and CaUGT73L69 The full-length CDS coding sequence of the gene (1488 bp, see SEQ ID No. 2 and No. 1 in the sequence listing). PCR reaction conditions: 95°C pre-denaturation for 2 min; 94°C for 10 s, 60°C for 30 s, 72°C for 2 min, 35 cycles; 72°C extension for 5 min. The amplified PCR product was ligated into the pDonr207 entry vector via the BP reaction of the GATEWAY cloning technology. Positive clones were screened and confirmed by sequencing to obtain CaUGT1 and CaUGT73L69 The full-length cDNA was then linked into the prokaryotic expression vector PGEX-6P-1 through the LR reaction of GATEWAY cloning technology.
[0051] Example 3: CaUGT1 and CaUGT73L69 Prokaryotic expression and fusion protein purification
[0052] The prokaryotic expression and fusion protein purification methods are as follows: First, the pGEX-6P-1 positive colonies obtained in Example 2 were picked and cultured in culture medium. The expression was induced by IPTG, the protein was broken, and the crude protein was obtained by centrifugation. The protein was then purified by GST purification column and eluate ( Figure 2 ).
[0053] Specific steps: (1) Small shaking: Pick positive colonies from the dish to the shaking bottle, add 6mL of LB with the corresponding antibiotic (resistance is generally ampicillin), and culture overnight at 37℃ for 8 hours until the mixture is shaken; (2) Large shaking: In the clean bench (sterilized for at least 15 minutes in advance), add 250µL of antibiotics to a large triangular flask with LB (250mL), and then add the small shaking solution (250mL) at a ratio of 1:50. LB / 5mL bacterial solution), seal, mark, and culture in a shaker at 37℃, 220rpm for 2-3 hours until the bacterial solution becomes turbid; (3) Induce expression: When expressing GST / his tag protein, add 1M IPTG (protein expression inducer) 25µL to the triangular flask at a ratio of 1:10000, and culture in a shaker at 16℃, 180rpm for 14-16 hours; (4) Collect bacteria: Centrifuge at 4℃, 5000rpm, centrifuge for 8min; (5) Resuspend: Add 50mL Lysis buffer to each flask and vortex until there are no bacterial clumps; (6) Break cells: Add 50µL PMSF protease inhibitor and 10µL reducing agent β-mercaptoethanol to each tube of bacterial solution, and use a cell disruptor to break the protein; (9) Purify fusion protein: Centrifuge at 4℃, 10000rpm, 1 hour, collect the supernatant and transfer it to the purification column, filter twice and use Lysis Buffer was used to wash the impurities, and then the target protein was eluted with glutathione buffer (GST) and stored in aliquots at -80℃.
[0054] Preparation of reagents and culture medium used:
[0055] 1) Preparation of LB medium
[0056]
[0057] Dissolve at room temperature and dilute to 1 L with distilled water, seal and sterilize at high temperature.
[0058] 2) IPTG formula
[0059] Weigh 1.19 g IPTG and dissolve it in 5 mL water. After dissolution, filter and sterilize the solution and store it in a -20°C refrigerator.
[0060] 3) Lysis buffer formula
[0061] Weigh 23.37 g of NaCl, add 50 mL of 1 M Tris-HCl (pH = 7.4), and add water to make up to 1 L.
[0062] 4) Glutathione buffer formula
[0063] Weigh 0.092 g of reduced glutathione, dissolve it in 20 mL of Lysis buffer, and add 300 µL of 1 M NaOH to adjust the pH to about 8.0.
[0064] Example 4: In vitro enzyme activity assay of CaUGT1 and CaUGT73L69
[0065] Specific steps: In order to verify the function of the candidate gene, the inventors used the protein purified in Example 3 to perform in vitro enzyme activity assays. The protein in vitro enzyme activity reaction system is 20µL, 100mM Tris-HCl buffer (pH=7.5), 50mMMgCl2, with a final concentration of 1mM asiatic acid or madecassic acid as a substrate, a final concentration of 15mM UDPG as a glycosyl donor, 500ng of purified recombinant protein (GST-tagged protein is also set as a negative control), incubated at 37°C for 2h, and then 80µL of pre-cooled methanol was added to terminate the reaction. The reaction was centrifuged at 4°C and 12000rpm for 10min, and the supernatant was analyzed and detected by LC-MS (LCMS-8060, Shimadzu Corporation, Japan). The results of the in vitro enzyme activity assay are as follows: Figure 3 and Figure 4 As shown, by comparing the in vitro results of CaUGT73L69 protein and CaUGT1 protein, it was found that CaUGT73L69 had substrate preference and preferred to catalyze hydroxymadecassic acid.
[0066] The present invention has been described in detail above. Definitions of Terms Related to the Present Invention Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the present invention belongs.
[0067] The term "protein" is used interchangeably herein to refer to a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid. As used herein, the term encompasses amino acid chains of any length, including full-length proteins (i.e., antigens), in which the amino acid residues are linked via covalent peptide bonds.
[0068] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.
[0069] For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, can be equivalent parameters, concentration and conditions, implement the present invention in a wide range. Although the present invention provides special embodiments, it should be understood that the present invention can be further improved. In a word, by the principle of the present invention, the application is intended to include any variation, purposes or improvements to the present invention, including departing from the disclosed range in the application, and the changes performed with conventional techniques known in the art.
Claims
1. The use of proteins in catalyzing one or more of the following reactions: E1) catalyzes asiatic acid to produce asiatic acid monoglucoside; E2) catalyzes madecassic acid to produce madecassic acid monoglucoside; The protein preferentially catalyzes madecassic acid; The protein is: A1) a protein having the amino acid sequence shown in SEQ ID No. 3; or A2) a fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1).
2. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the protein is shown in SEQ ID No.
1.
3. Use of a biomaterial related to the protein of claim 1 in catalyzing one or more of the following reactions: E1) catalyzes asiatic acid to produce asiatic acid monoglucoside; E2) catalyzes madecassic acid to produce madecassic acid monoglucoside; The biomaterial preferentially catalyzes madecassic acid; The biological material includes any one of the following D1) to D4): D1) an expression cassette containing a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 1; D2) a recombinant vector containing a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 1 or a recombinant vector containing the expression cassette described in D1); D3) a recombinant microorganism containing a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 1, a recombinant microorganism containing the expression cassette described in D1), or a recombinant microorganism containing the recombinant vector described in D2); D4) A genetically engineered host cell containing a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 1, a genetically engineered host cell containing the expression cassette described in D1), or a genetically engineered host cell containing the recombinant vector described in D2).
4. Use of the protein according to claim 1 or the biomaterial according to claim 3 in preparing an enzyme catalyst for one or more of the following reactions: E1) catalyzes asiatic acid to produce asiatic acid monoglucoside; E2) catalyzes madecassic acid to produce madecassic acid monoglucoside.
5. An in vitro glycosylation method, characterized in that The method comprises the following steps: in the presence of a glycosyltransferase, transferring the glycosyl of a glycosyl donor to the C-28 position of asiatic acid or madecassic acid, thereby forming asiatic acid monoglucoside or madecassic acid monoglucoside; wherein the glycosyltransferase is selected from the glycosyltransferase shown in SEQ ID No. 3.