A horse chestnut tiglyl-CoA ligase gene AcCCL3, AcCCL3 protein and its applications

Abstract

This invention provides a horse chestnut tiglyl-CoA ligase gene. AcCCL3 This invention relates to the AcCCL3 protein and its applications, belonging to the field of gene technology. For the first time, this invention has identified and verified the key enzyme gene responsible for the biosynthesis of tigrazol-CoA in horse chestnut. AcCCL3 This invention provides a complete analysis of its biosynthetic pathway, filling a knowledge gap in this field. It can be based on... AcCCL3 This gene, through synthetic biology strategies and an engineered system, has achieved the synthesis of tiglyl-CoA, completely eliminating reliance on traditional, complex, and demanding chemical synthesis routes. This significantly reduces production costs from the source, enabling large-scale production and successfully solving the core challenge of large-scale, stable preparation and supply of tiglyl-CoA. It also breaks through the key precursor constraints that have hindered the biosynthetic research, drug development, and industrial production of a series of high-value aescin compounds, represented by aescin A.

Description

Technical Field

[0001] This invention relates to the field of gene technology, and more particularly to a horse chestnut tiglyl-CoA ligase gene. AcCCL3 AcCCL3 protein and its applications. Background Technology

[0002] Horse chestnut (Aesculus hippocastanum) is a traditional and precious medicinal plant in my country. Its dried, mature seeds, after processing, become the Chinese medicinal material "Saluozi". This medicinal material is rich in aescin, a compound that is the core active substance of horse chestnut. These compounds have clearly defined pharmacological activities, including anti-inflammatory and swelling-reducing effects, improvement of microcirculation, and neuroprotective properties. They show broad application prospects in the treatment of cardiovascular and cerebrovascular diseases, the relief of edema, and the development of related health products. Currently, more than 40 homologues of aescin have been identified from horse chestnut, exhibiting significant structural diversity.

[0003] Tigrolyl-CoA is a key precursor and acyl donor in the biosynthesis of aescin compounds. It not only provides the essential acyl side chain for the synthesis of aescin A, but is also a conserved structural unit shared by all known aescin homologues, playing an irreplaceable core role in maintaining the structural stability, spatial conformation, and biological activity of these compounds. Furthermore, the function of tigrolyl-CoA is not limited to aescin synthesis; it is also widely involved in the biosynthesis of various alkaloids, acyl sugars, some antibiotics, and volatile esters in plants, playing an important role in plant secondary metabolism and physiological responses. Isobutyryl-CoA synthases derived from microorganisms (such as *Pseudomonas aeruginosa*) have been shown to efficiently catalyze the in vitro synthesis of tigrolyl-CoA, confirming the existence of its synthetic pathway from an enzymatic perspective.

[0004] Despite the high biological value and application potential of tiglyl-CoA, its biosynthetic pathway in plants remains unclear. Currently, no research reports, either domestically or internationally, have detailed the key enzyme genes responsible for tiglyl-CoA synthesis in plants, the specific catalytic reaction steps, or their regulatory mechanisms. This lack of information on this crucial metabolic pathway severely hinders in-depth analysis of the biosynthetic networks of important bioactive natural products such as aescin, and has become a core bottleneck for the efficient artificial synthesis and industrialization of these compounds using synthetic biology strategies.

[0005] Currently, the acquisition of tiglyl-CoA mainly relies on chemical synthesis. This method generally suffers from problems such as cumbersome synthetic routes, demanding reaction conditions, and high raw material costs. Furthermore, tiglyl-CoA is chemically extremely unstable, readily degradable at room temperature, and requires extremely stringent storage and transportation conditions, making large-scale production and stable supply difficult. These factors combined result in an extremely high market price for chemically derived tiglyl-CoA (currently as high as 350,000 RMB per gram), severely limiting its accessibility for research on the biosynthetic mechanism of aescin, discovery of drug lead compounds, and applications in synthetic biology.

[0006] In the context of biosynthesis, coenzyme A ligases are generally considered key enzymes that catalyze the binding of carboxylic acids (such as tigacic acid) to coenzyme A, generating the corresponding acyl-CoA (such as tigacyl-CoA). However, among plant-derived coenzyme A ligases, which isoenzyme can specifically and efficiently catalyze the synthesis of tigacyl-CoA, its enzymatic properties, tissue expression patterns, and regulatory mechanisms in metabolic pathways are currently unknown. This further highlights the urgency and significant application value of identifying key enzymes in the synthesis of plant-derived tigacyl-CoA and elucidating its complete biosynthetic pathway.

[0007] In conclusion, developing a plant-derived enzyme and its encoding gene that can efficiently and specifically catalyze the synthesis of tigel-CoA is of great significance for breaking through the research bottlenecks in the biosynthesis of natural products such as aescin, reducing the cost of key precursor preparation, and promoting the development of the biomanufacturing industry for related pharmaceutical and health products. Summary of the Invention

[0008] The purpose of this invention is to provide a horse chestnut tiglyl-CoA ligase gene. AcCCL3 AcCCL3 protein and its applications: identifying key genes catalyzing the synthesis of timoyl-CoA. AcCCL3 This not only enables the efficient biosynthesis of aescin, but also provides a key genetic basis for molecular design breeding and variety innovation of horse chestnut, which has significant scientific value and broad prospects for industrial application.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a horse chestnut tiglyl-CoA ligase gene. AcCCL3 The tigel-CoA ligase gene AcCCL3 The sequence is shown in SEQ ID NO.1.

[0010] The present invention also provides the above-mentioned tigel-CoA ligase gene. AcCCL3 The expressed AcCCL3 protein, the sequence of which is shown in SEQ ID NO.2.

[0011] The present invention also provides the above-mentioned tigel-CoA ligase gene. AcCCL3 Or the application of the AcCCL3 protein mentioned above in the production of aescin compounds.

[0012] The present invention also provides an application of the above-mentioned AcCCL3 protein as a tigel-CoA ligase.

[0013] The present invention also provides a gene containing the above-mentioned tigrazol-CoA ligase. AcCCL3 The products include expression cassettes, recombinant expression vectors, transgenic cell lines, or recombinant microorganisms.

[0014] The present invention also provides an application of the above-mentioned product in the production of aescin compounds.

[0015] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention is the first to identify and verify the key enzyme gene responsible for the biosynthesis of tigel-CoA in horse chestnut. AcCCL3 This invention provides a complete analysis of its biosynthetic pathway, filling a knowledge gap in this field. It can be based on... AcCCL3 By employing synthetic biology strategies and an engineered system, the gene has achieved the synthesis of tigryl-CoA, completely eliminating the reliance on traditional, complex, and demanding chemical synthesis routes. This significantly reduces production costs from the source, enables large-scale production, and successfully solves the core problem of the difficulty in large-scale, stable preparation and supply of tigryl-CoA.

[0016] (2) This invention breaks through the key precursor constraints that have hindered the biosynthesis research, drug development, and industrial production of a series of high-value aescin compounds, represented by aescin A. The obtained key genes... AcCCL3 This research provides an important target for molecular breeding of horse chestnut and lays the foundation for cultivating new varieties with high content through metabolic engineering. More importantly, it offers a new precursor supply solution for reconstructing the complete synthetic pathway of aescin in microbial cell factories, making it possible to produce this rare natural product through sustainable fermentation without relying on plant extraction. This could potentially alleviate the shortage of related traditional Chinese medicine resources. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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.

[0018] Picture 1 In Embodiment 1 of the present invention AcCCL3 Gene expression profiles in different tissue regions, where F represents flower; G represents pericarp; R represents kernel; Y represents leaf; and Z represents branch. Picture 2 In Embodiment 1 of the present invention AcCCL3 Gene cloning and vector construction gel mapping; Picture 3 This is a gel image of AcCCL3 protein purification in Example 2 of the present invention; Picture 4 The chromatogram and mass spectrum of the AcCCL3 protein catalytic product in Example 2 of this invention are shown. Picture 5 The chemical structural formulas of tigacic acid and tigacyl-CoA in Example 2 of this invention are shown. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0021] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0022] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0023] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0024] Example 1 Example 1 of this invention provides a tiglyl-CoA ligase gene. AcCCL3 The separation and detection method, with the following specific steps: (1) cDNA template preparation Transcriptome analysis of flowers, pericarps, seeds, leaves, and branches of *Aesculus chinensis* Bge. revealed... AcCCL3 Genes are specifically highly expressed in horse chestnut seeds ( Picture 1 This is consistent with the distribution pattern of aescin.

[0025] Mature seeds of horse chestnut were collected in September, and total RNA was extracted from the seeds using the Adley RN38 EASYspin plus RNA extraction kit. The purity and integrity of the extracted RNA were tested, and after the test indicators were qualified, high-quality cDNA templates were prepared by reverse transcription.

[0026] (2) Primer design Specific primers were designed based on the target gene sequence, as shown in Table 1.

[0027] Table 1 Primer sequences used for cloning genes.

[0028] Note: Lowercase letters in the sequence are homologous arm sequences.

[0029] Using the cDNA prepared in step (1) as a template, 2 × KeyPo Master Mix (Dye Plus) high-fidelity enzyme was used for processing. AcCCL3 PCR cloning of the gene fragment yielded 1 AcCCL3 Gene fragments.

[0030] The total volume of the high-fidelity enzyme PCR reaction was 50 μL: 25 μL of 2× high-fidelity enzyme reaction solution, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of cDNA template, and the remainder was added to a final volume of 50 μL with sterile deionized water. The PCR reaction procedure is shown in Table 2, and the results are as follows. Picture 2 As shown.

[0031] Table 2. Gene Cloning PCR Reaction Procedure

[0032] (3) Preparation of recombinant bacteria The pET28a vector was digested using a double enzyme digestion method. The total digestion volume was 50 μL, with the following components: 1 μL of each component. BamH I restriction endonuclease, 1 μL SalI. Restriction endonuclease, 10 μL 5× digestion buffer, 10 μg pET28a plasmid, add sterile deionized water to a final volume of 50 μL, and obtain the linearized vector after digestion.

[0033] The ClonExpress Ultra One Step Cloning Kit was used to complete step (2). AcCCL3 The recovered gene fragment was ligated into the linearized pET28a vector. The total ligation volume was 5 μL, specifically consisting of: 2.5 μL of cloning ligase, 1 μL of the digested linearized vector, and 1.5 μL of... AcCCL3 Gene fragments were recovered, and the ligation system was incubated in a 50 ℃ metal bath for 20 min to complete the ligation reaction, yielding the pET28a-AcCCL3 recombinant plasmid. The results are as follows: Picture 2 As shown.

[0034] The above pET28a-AcCCL3 recombinant plasmid was directly transformed into DH5α. α Single colonies were picked from competent E. coli cells and identified by colony PCR.

[0035] The total volume of colony PCR was 20 μL, and the specific components were: 10 μL 2×Taq PCR Mix, 1 μL colony template, 1 μL forward primer, 1 μL reverse primer, and 7 μL sterile deionized water. The PCR reaction procedure was performed according to Table 3.

[0036] Table 3 Colony PCR reaction procedure

[0037] Clones that tested positive by PCR were selected for sequencing. Alignment of the sequencing results with the target gene's CDS sequence showed a nucleotide sequence similarity of 100%, indicating... AcCCL3 The gene was successfully cloned and correctly inserted into the pET28a vector. AcCCL3 The gene sequence and the protein sequence it encodes are as follows: AcCCL3 Gene sequence - SEQ ID NO.1:

[0038] Amino acid sequence - SEQ ID NO.2: .

[0039] Example 2 Embodiment 2 of the present invention is for the separation AcCCL3 The specific steps for verifying gene function are as follows: (1) Transform the pET28a-AcCCL3 recombinant plasmid constructed in step (3) of Example 1 into Weidi Biotechnology BL21(DE3) Escherichia coli competent cells: Thaw competent E. coli cells BL21(DE3) stored at -80℃ on ice. Add 1 μL of pET28a-AcCCL3 recombinant plasmid to 50 μL of competent cells, mix well, and place on ice for 30 min. Incubate at 42℃ for 45 s, then place on ice for 2 min. Add 600 μL of antibiotic-free LB liquid medium and activate at 37℃ and 200 rpm for 1 h. Centrifuge the activated E. coli at 4000 rpm for 1 min, remove 500 μL of supernatant, resuspend the cells in the remaining supernatant, and spread them all onto LB agar plates containing kanamycin. Incubate overnight at 37℃. Pick single colonies from the LB agar plates and incubate in 700 μL of 0.1% kanamycin liquid LB medium. Activate at 37℃ and 200 rpm for 3 h, then perform bacterial PCR (PCR system shown in Table 3).

[0040] (2) Induction, expression and purification of the target protein 1) Inoculate correctly identified positive clones into 10 mL of LB liquid medium containing 0.1% kanamycin and incubate overnight at 37°C and 200 rpm with shaking. Transfer 2 mL of the overnight culture to 100 mL of LB liquid medium containing the same antibiotic and continue incubation at 37°C and 200 rpm until OD (outlet count) is reached. 600 ≈0.4; 2) Lower the culture temperature to 16℃, add IPTG to a final concentration of 1 mM, induce expression at 16℃ and 100 rpm for 20 h, collect the cells by centrifugation at 4℃ and 6000 rpm for 5 min, and discard the supernatant; resuspend and wash the cells with 10 mL of 5 mM Tris-HCl buffer, centrifuge again and discard the supernatant, and resuspend the precipitate with 3 mL of 5 mM Tris-HCl buffer.

[0041] 3) The bacterial cells were disrupted using an ultrasonic cell disruptor under ice bath conditions. The power was 100 W, with a 5-second interval between cycles, for a total duration of 20 min. The cells were then centrifuged at 4℃ and 12000 rpm for 30 min, and the supernatant was collected as the crude protein extract.

[0042] 4) Add the supernatant to a nickel ion affinity chromatography column that has been fully equilibrated with 5 mM Tris-HCl buffer, and incubate at 4°C and 70 rpm for 3 h to allow the target protein to fully bind to the packing material. Elute with 2 mM imidazole washing buffer multiple times, monitoring with protein quantification reagents, until no obvious protein is detected in the eluent. Then, elute the target protein bound to the column with 50 mM imidazole elution buffer, collect the eluent fraction, and perform protein quantification.

[0043] 5) Take 10 μL of sample for 10% SDS-PAGE electrophoresis. First, perform electrophoresis at 80 V for 30 min, then at 110 V for 1.5 h. After Coomassie brilliant blue staining, a clear target band will be visible (e.g., Picture 3 As shown in the figure, the obtained protein can be used for subsequent in vitro enzymatic reactions.

[0044] (3) In vitro enzyme activity reaction and product detection of purified protein Using the target protein inactivated by boiling as a control, 50g of the purified protein obtained above was taken. μ g, 400mM Tris-HCl, 5 mM MgCl2, 2.5 mM ATP, 0.2 mM CoA and 0.4 mM tigric acid were reacted overnight at 25℃. The reaction was terminated by boiling at 100℃ for 5 min. After centrifugation at 12000 rpm for 5 min, the supernatant was collected, filtered, and analyzed by LC-MS.

[0045] LC-MS detection was performed using an Agilent LC-QTOF (1290-6546) column (XB-C18, Dim: 2.1 × 100 mm, Ultimate). The mobile phase was pure water (A) and acetonitrile (B). The elution gradient was: 0 min, 5% B; 5 min, 5% B; 10 min, 50% B; 13 min, 100% B; 15 min, 100% B; 15.5 min, 5% B; 18.5 min, 5% B. The flow rate, column temperature, and injection volume were 0.3 mL / min, 35 °C, and 1 mL / min, respectively. L. Auto MS / MS scanning was performed using the negative ion mode of an ESI ion source. The specific parameters of the ion source were designed as follows: dry gas temperature (Gas Temp) 350 ℃, dry gas flow rate (Gas Flow) 8 L / min, vaporizer temperature (Vaporizer) 350 ℃, nebulizer pressure (Nebulizer) 20 psi, capillary voltage (VCap) 4000 V, and corona current (CoronaPositive) 15 A. The Fragmentor voltage is 150 V, the Skimmer 1 voltage is 65 V, and the Collision Energy is 20 V. The mass spectrometer detector is an Agilent 6546ATOF, and the mass spectrometry scanning range is [missing information]. m / z 50-1700.

[0046] LC-MS analysis results are as follows Picture 4 As shown, the results indicate that the peak time and MS / MS chromatogram of the catalytic product are completely consistent with those of the tiglyl-CoA standard, demonstrating that the AcCCL3 protein in horse chestnut can catalyze the production of tiglyl-CoA from tiglyl acid. Picture 5 ).

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A horse chestnut tiglyl-CoA ligase gene AcCCL3 Its characteristics are, The tigryl-CoA ligase gene AcCCL3 The sequence is shown in SEQ ID NO.

1.

2. A tigenomic coenzyme A ligase gene as described in claim 1 AcCCL3 The expressed AcCCL3 protein is characterized by, The sequence of the AcCCL3 protein is shown in SEQ ID NO.

2.

3. A tigromycin-CoA ligase gene as described in claim 1 AcCCL3 Or the application of the AcCCL3 protein as described in claim 2 in the production of aescin compounds.

4. The use of the AcCCL3 protein of claim 2 as a tigenomic coenzyme A ligase.

5. A gene containing the tigel-CoA ligase as described in claim 1 AcCCL3 The product is characterized by, The products include expression cassettes, recombinant expression vectors, transgenic cell lines, or recombinant microorganisms.

6. The use of the product of claim 5 in the production of aescin compounds.

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