Application of centella glycosyl transferase gene in catalyzing glycosylation of asiatic acid and madecassic acid

Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a centella asiatica glycosyl transferase gene in catalyzing glycosylation of asiatic acid and madecassic acid. The nucleotide sequence of the gene is shown as SEQ ID No.1, and the protein coded by the gene can catalyze the C-28 site of asiatic acid and madecassic acid to perform glucose group modification so as to generate asiatic acid monoglucoside and madecassic acid monoglucoside, and preferentially catalyzes madecassic acid. By constructing a metabolic regulation network, the genes are widely and accurately screened, and the glycosylation process of catalyzing asiatic acid and madecassic acid is analyzed; an important reference basis is provided for improving the glycosylation efficiency of asiatic acid and madecassic acid and the yield of asiaticoside and madecassoside in the in-vitro synthesis and industrial production process of the centella asiatica triterpenoid saponin, and an important theoretical support is provided for large-scale production of the centella asiatica active saponin.

Description

Technical Field

[0001] The 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 Centella asiatica and madecassic acid. Background Art

[0002] Centella asiatica Centella asiatica Centella asiatica (L.) Urban) is a perennial herb of the genus Centella in the family Apiaceae. Centella asiatica can produce new roots at the stem nodes when it is creeping, so it has a strong regeneration ability and mainly reproduces asexually. Centella asiatica is cold in nature, bitter and spicy in taste, and has the effects of clearing away heat and dampness, detoxifying and reducing swelling. It is often used to treat damp-heat jaundice, carbuncles, sores, and injuries from falls. People in southern my country often drink Centella asiatica as a cold tea to dispel wind and dampness, relax muscles and activate blood circulation, and can also treat skin inflammation. Some foreign countries also use Centella asiatica as a traditional herbal medicine to treat skin diseases and leprosy. In modern medicine, Centella asiatica is often used to treat skin burns.

[0003] A variety of chemical substances have been detected in Centella asiatica, mainly triterpenes, volatile oils, polyacetylenes, flavonoids, alkaloids and other components, in addition to other compounds such as sterols and polyphenols. Centella asiatica is rich in a variety of pentacyclic triterpenes with physiological activity, mainly asiaticoside, asiatic acid, madecassoside, madecassic acid, etc. Asiaticoside and madecassoside belong to ursane-type saponins, which play an important role in neuroprotection, myocardial cell protection, prevention of cell aging and acute kidney injury, and are important physiologically active substances in Centella asiatica. Therefore, the research on Centella asiatica extracts is common in the pharmaceutical and cosmetic industries. In addition, studies have found that asiaticoside has good antibacterial activity, can protect cells from pathogen infection, and is considered to be a plant antibiotic against bacteria, fungi and parasites.

[0004] The biosynthetic pathway of Centella asiatica triterpenoid saponins includes three stages: precursor supply, synthesis of a pentacyclic triterpenoid skeleton, and multi-step glycosylation modification. Asiatic acid and madecassoside are the synthetic precursors of asiaticoside and madecassoside, respectively. The final asiaticoside and madecassoside can be produced by three consecutive glycosylation modifications (glucose-glucose-rhamnose, GGR) at the C-28 position of asiatic acid and madecassoside. This step requires the catalysis of a series of glycosyltransferases. At present, two subfamilies of glycosyltransferases involved in the glycosylation of asiatic acid and madecassoside have been identified. One subfamily is involved in the first step of glucoseylation. 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, these studies have only involved the functional verification of Centella asiatica saponin glycosyltransferase, and have not yet verified the UGT73s Study on the functional differences and substrate preferences of tandemly repeated genes. Summary of the invention

[0005] The purpose of the present invention is to explore the involved factors in the synthesis of triterpenoid saponins in Centella asiatica by constructing a metabolic regulation 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: The first aspect of the present invention provides a use of a protein 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.

[0007] The protein is named CaUGT73L69 and can be any of the following: A1) a protein having an amino acid sequence of SEQ ID No. 3; A2) A fusion protein having the same function as A1) is obtained by connecting a tag to the N-terminus and / or C-terminus of A1).

[0008] 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.

[0009] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) 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.

[0010] The second aspect of the present invention provides the use of biomaterials 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.

[0011] The biological material is any one of the following D1) to D4): D1) an expression cassette containing a nucleic acid molecule encoding the protein CaUGT73L69; D2) a recombinant vector containing a nucleic acid molecule encoding the protein CaUGT73L69, and a recombinant vector containing the expression cassette described in D1); 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); 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).

[0012] 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 protein CaUGT73L69 coding gene (CDS), and has a length of 1488 bp.

[0013] 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: E1) catalyzes asiatic acid to produce asiatic acid monoglucoside; E2) catalyzes madecassic acid to produce madecassic acid monoglucoside.

[0014] 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.

[0015] 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 site of asiatic acid or hydroxymadecassic acid, thereby forming asiatic acid monoglucoside or hydroxymadecassic acid monoglucoside; wherein the glycosyltransferase is selected from the glycosyltransferase shown in SEQ ID No.3.

[0016] The specific process of implementing the present invention is as follows: The present invention utilizes the metabolic regulation network of biologically active 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 regulation 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 analysis of tissue expression profiles revealed that these genes were highly expressed in the aboveground tissues (leaves and stems) of Centella asiatica. CaUGT73L69 In vitro functional validation was performed and reporter genes in the gene cluster were 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, and the sequence lengths are both 1488 bp. The protein sequences encoded by the genes are shown in SEQ ID No. 3 and SEQ ID No. 4, and both have 495 amino acid residues.

[0017] The gene of the present invention is 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.

[0018] 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. CaUGT1 showed a catalytic preference for asiatic acid, while CaUGT73L69 had a higher catalytic efficiency for madecassic acid.

[0019] Compared with the prior art, the present invention has the following effects: 1. By constructing a metabolic regulatory network, we have extensively and accurately screened candidate genes involved in the metabolic synthesis of Centella asiatica triterpenoid saponins.

[0020] 2. In vitro enzyme activity verification confirmed that CaUGT1 and CaUGT73L69 can catalyze the initial glucosylation modification of asiatic acid and asiatic acid, the precursors of asiaticoside.

[0021] 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 madecassososide in the industrial production process, and provide important theoretical support for the large-scale production of Centella asiatica active saponins.

[0022] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 To screen and locate the metabolic network of asiaticoside and madecassoside triterpenoids CaUGT1 and CaUGT73L69 Gene: Figure 1 A in the figure is the metabolic network construction and analysis result of asiaticaoside and hydroxy-madecassoside triterpenoid compounds; Figure 1 B in the figure is the analysis of multiple tandem repeats in the chromosome 8 region where the gene is located and the tissue expression of the tandem repeat genes.

[0024] 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. The five lanes on the right are CaUGT1 Purification results of gene synthesized proteins.

[0025] Figure 3 The results of in vitro enzyme activities of CaUGT1 and CaUGT73L69 using asiatic acid as substrate. Figure 3 A in the formula is asiatic acid (1), which is catalyzed by glycosyltransferases CaUGT1 and CaUGT73L69 to form asiatic acid monoglucoside (2); Figure 3 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.

[0026] Figure 4 The in vitro enzyme activity results of CaUGT1 and CaUGT73L69 using madecassic acid as substrate: Figure 4 A in the above formula 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.

[0027] Figure 5 This is the plasmid map of the entry vector CaUGT1-pDonr207.

[0028] Figure 6 This is the plasmid map of the entry vector CaUGT73L69-pDonr207.

[0029] Figure 7 This is the plasmid map of the prokaryotic expression vector CaUGT1-pGEX-6p-1.

[0030] Figure 8 This is the plasmid map of the prokaryotic expression vector CaUGT73L69-pGEX-6p-1. DETAILED DESCRIPTION

[0031] 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 are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all purchased from commercial channels unless otherwise specified.

[0032] Embodiment 1: CaUGT1 and CaUGT73L69 Discovery and positioning 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 ). There are many functional genes in this terpenoid synthesizer network: 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 CaUGT73C8 Participates in the formation of GGR sugar chains in asiaticoside and madecassoside. Further analysis found that the UGT73s Tandem repeat gene clusters are involved in the formation of saponin sugar chains and selected for the ones with higher expression levels CaUGT73L69 And the reported CaUGT1 Perform subsequent in vitro validation.

[0033] Example 2: Isolation of clones CaUGT1 and CaUGT73L69 Gene To obtain CaUGT1 and CaUGT73L69 The applicant used TRIZOL reagent (Invitrogen) to extract the total RNA of Centella asiatica (the extraction method was based on the instructions of the TRIZOL reagent), and used the reverse transcription kit Supermix (purchased from Beijing Quanshijin Company) to reverse transcribe the RNA into cDNA. The reaction conditions were: 42℃ 30min, 80℃ 5s. 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 list). PCR reaction conditions: 95℃ pre-denaturation for 2 min; 94℃ for 10 s, 60℃ for 30 s, 72℃ for 2 min, 35 cycles; 72℃ extension for 5 min. The amplified PCR product was connected to the pDonr207 entry vector through the BP reaction of GATEWAY cloning technology, and the positive clones were screened and sequenced 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.

[0034] Embodiment 3: CaUGT1 and CaUGT73L69 Prokaryotic expression and purification of fusion protein The prokaryotic expression and fusion protein purification method is as follows: First, the pGEX-6P-1 positive colony obtained in Example 2 is selected and cultured in the culture medium. The expression is induced by IPTG, the protein is broken, and the crude protein is obtained by centrifugation. The protein is then purified by GST purification column and eluent ( Figure 2 ).

[0035] Specific steps: (1) Small shaking: Pick positive colonies from the dish to a shaking bottle, add 6 mL of LB with the corresponding antibiotic (resistance is generally ampicillin), and culture overnight at 37°C in a shaking incubator for 8 hours until the mixture is shaken; (2) Large shaking: In a clean bench (sterilized for at least 15 minutes in advance), add 250 µL of antibiotics to a large triangular flask containing LB (250 mL), and then add the small shaking solution (250 mL) 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-tagged protein, add 1M IPTG (protein expression inducer) 25µL to the 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 lumps; (6) Disrupt cells: add 50µL PMSF protease inhibitor and 10µL reducing agent β-mercaptoethanol to each tube of bacterial solution, and use a cell disruptor to disrupt 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 glutathione buffer (GST) was used to elute the target protein, and the aliquots were stored at -80°C.

[0036] Preparation of reagents and culture medium used: 1) Preparation of LB medium

[0037] Dissolve at room temperature and make up to 1L with distilled water, seal and sterilize at high temperature.

[0038] 2) IPTG formula Weigh 1.19 g IPTG and dissolve it in 5 mL water. After dissolution, filter and sterilize, and store in aliquots at -20 °C.

[0039] 3) Lysis buffer formula Weigh 23.37 g of NaCl, take 50 mL of 1 M Tris-HCl (pH = 7.4), and add water to make up to 1 L.

[0040] 4) Glutathione buffer formula 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.

[0041] Example 4: In vitro enzyme activity assay of CaUGT1 and CaUGT73L69 Specific steps: In order to verify the function of the candidate gene, the inventors used the protein purified in Example 3 for in vitro enzyme activity assay. The protein in vitro enzyme activity reaction system is 20µL, 100mM Tris-HCl buffer (pH=7.5), 50mM MgCl2, with a final concentration of 1mM asiatic acid or hydroxy-madecassic acid as the substrate, a final concentration of 15mM UDPG as the glycosyl donor, 500ng 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 reactant 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 in vitro enzyme activity assay are as follows Figure 3 and Figure 4 As shown, and by comparing the in vitro results of CaUGT73L69 protein and CaUGT1 protein, it was found that CaUGT73L69 had substrate preference and preferred to catalyze madecassic acid.

[0042] The present invention is 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.

[0043] 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 are non-naturally encoded amino acids. 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.

[0044] The term "expression" refers to the transcription and / or translation of an endogenous gene or a transgene in a plant cell.

[0045] 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, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departing from the disclosed scope in the application, and the changes made with conventional techniques known in the art.

Claims

1. The application 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 meets the following conditions: A1) a protein having the amino acid sequence shown in SEQ ID No. 3; A2) A fusion protein having the same function as A1) is 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 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 described in claim 1 and the biomaterial described in claim 3 in preparing enzyme catalysts in 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. Use of the protein according to claim 1 and the biomaterial according to claim 3 in the preparation of asiatic acid monoglucoside or madecassic acid monoglucoside.

6. 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.

Images