Oxo-isopentenyl transferase participating in synthesis of sesquiterpene coumarin and application of oxo-isopentenyl transferase

Abstract

The invention discloses an oxygen isopentenyl transferase participating in synthesis of sesquiterpene coumarin and an application of the oxygen isopentenyl transferase. Belongs to the technical field of gene engineering. The invention discloses an open reading frame sequence of a Xinjiang ferula oxygen isopentenyl transferase FsPT9 gene and an amino acid sequence coded by the Xinjiang ferula oxygen isopentenyl transferase FsPT9 gene. The FsPT9 gene is obtained by screening and cloning based on combined analysis of multi-omics data of ferula sinkiang, a yeast expression vector is constructed, and a catalytic mechanism of the FsPT9 is identified in vitro by taking 7-hydroxycoumarin and farnesyl pyrophosphate as substrates. A yeast heterologous expression system proves that the FsPT9 can catalyze 7-hydroxycoumarin and farnesyl pyrophosphoric acid to generate umbelliferone. The invention discloses a key enzyme for initial synthesis of sesquiterpene coumarin for the first time, and the key enzyme is an oxygen isopentenyl transferase which is reported for the first time and is used for catalyzing three isopentenyl units at the same time, so that theoretical guidance is provided for synthesis and transformation of pharmacodynamic substances of asafetida sesquiterpene coumarin.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to an oxoisopentenyltransferase involved in the synthesis of sesquiterpene coumarin and its application. Background Technology

[0002] Approximately 370 sesquiterpene coumarins have been isolated and characterized from various plants. About 79.2% and 17.3% of these are found in plants of the Apiaceae (Ferula) and Asteraceae families, respectively, exhibiting a variety of biological activities. Sesquiterpene coumarins structurally consist of a sesquiterpene unit and a coumarin unit. Based on the specific chemical structure formed by the connection between the coumarin core and the sesquiterpene unit, sesquiterpene coumarins can be systematically divided into two main types: O-isopentenylated and C-isopentenylated sesquiterpene coumarins. Of all sesquiterpene coumarins, approximately 84% of the coumarin unit is derived from 7-hydroxycoumarin (also known as umbelliferone, chemical formula C9H6O3, CAS number 93-35-6). Notably, over 70% of sesquiterpene coumarins (comprising more than 260 individual compounds) are derivatives of umbelliferone (C24H30O3, CAS No. 23838-17-7), establishing its position as a major biosynthetic precursor for this type of natural product. However, almost nothing is known about the biosynthetic enzymes involved, such as O-farnesyltransferase. Therefore, elucidating the crucial biosynthetic step of the transfer of farnesyl from farnesyl pyrophosphate (FPP, C15) to the C-7-O of 7-hydroxycoumarin is essential.

[0003] Ferula assa-foetida, a traditional Chinese medicinal herb with distinctive ethnic characteristics, is listed as a critically endangered plant under national second-class protection. It is mainly distributed in Kashgar Township, Yining County, Xinjiang. Ferula assa-foetida has a long history of medicinal use, dating back to the Tang Dynasty, and is renowned for its insecticidal, detoxifying, and anti-inflammatory effects. Modern research has identified various metabolites from Ferula assa-foetida gum, its roots, seeds, and aerial parts, including sesquiterpenoid coumarins, sterol esters, phenylpropanoids, aromatic acids, sesquiterpenes, monoterpenes, lignans, thioalkyl groups, and coumarin compounds. Therefore, using Ferula assa-foetida as experimental material to analyze the key enzymes in the initial synthesis of sesquiterpenoid coumarins can provide theoretical guidance for the synthesis and modification of pharmacologically active sesquiterpenoid coumarins from Ferula assa-foetida. Summary of the Invention

[0004] In view of this, the present invention provides an oxoisopentenyltransferase involved in the synthesis of sesquiterpene coumarin and its application.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: An oxoisopentenyltransferase involved in the synthesis of sesquiterpene coumarin has the amino acid sequence shown in SEQ ID No. 2.

[0006] The nucleotide sequence of the gene encoding the aforementioned oxyisopentenyltransferase is shown in (a), (b), or (c): (a) The nucleotide sequence shown in SEQ ID No. 1; Or (b) a nucleotide sequence that can hybridize with the complementary nucleotide sequence of SEQ ID No. 1 under stringent hybridization conditions, wherein the protein encoded by the nucleotide still has the function or activity of oxyisopentenyltransferase; Or (c) a nucleotide sequence that has at least 80% homology with the nucleotide sequence of SEQ ID No. 1, and the protein encoded by the nucleotide still has the function or activity of oxyisopentenyltransferase.

[0007] Chimeric genes or expression cassettes containing the above-mentioned coding genes.

[0008] Recombinant expression vectors containing the above-mentioned coding genes or containing the above-mentioned chimeric genes or expression cassettes.

[0009] Recombinant host cells containing the recombinant expression vector described above.

[0010] The above-mentioned oxyisopentenyltransferase is used as a catalytic enzyme in the biosynthesis of sesquiterpene coumarin.

[0011] Furthermore, the sesquiterpene coumarin is an umbelliferous ether.

[0012] Furthermore, the substrates are 7-hydroxycoumarin and farnesyl pyrophosphate.

[0013] The application of the aforementioned coding genes, chimeric genes, or expression cassettes in the biosynthesis of sesquiterpene coumarins.

[0014] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for screening and functional identification of the plant oxoisopentenyltransferase FsPT9, which catalyzes the formation of umbelliferone from 7-hydroxycoumarin and farnesyl pyrophosphate. This invention reveals for the first time a key enzyme in the initiation of sesquiterpene coumarin synthesis in plants. Furthermore, this enzyme is the first reported oxoisopentenyltransferase catalyzing three isopentenyl units, providing theoretical guidance for the synthesis and modification of pharmacologically active sesquiterpene coumarins. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0016] Figure 1 This invention provides an example of the identification and evolutionary analysis of the FsPT family members of the oxoisopentenyltransferase based on the Xinjiang Ferula genome in Example 1 of this invention.

[0017] Figure 2 This figure shows the gene expression levels of the Xinjiang Ferula foetida FsPT gene in different environments and tissues in Example 1 of this invention. The gene expression values ​​have been standardized by column, with rows representing genes and columns representing samples. The left side shows the cluster analysis results of gene expression levels, and the right side shows the scale bar. The colors in the figure range from turquoise to medium purple, representing relative expression levels from low to high (see the color bar on the right). Wild represents the wild environment, Cul represents the cultivated environment, FL represents flowering plants, UFL represents non-flowering plants, L represents leaves, F represents flowers, and R represents roots.

[0018] Figure 3 This invention relates to the FsPT9 enzyme catalytic function in the synthesis of sesquiterpene coumarin from Xinjiang in Example 2 of this invention. In this example, A: FsPT9 catalyzes the reaction of 7-hydroxycoumarin and farnesyl pyrophosphate to generate umbelliferone. Empty vector represents the empty control. PT9 is an isopentenyl transferase. Umbelliferone represents the substrate 7-hydroxycoumarin. Umbelliprenin represents the synthesized product umbelliferone. B: The umbelliferone synthesis pathway.

[0019] Figure 4 This is a systematic evaluation of the selectivity of the FsPT9 enzyme isopentenyl donors (DMAPP / GPP / FPP) in Example 3 of the present invention. Empty vector represents empty control, PT9 isopentenyl transferase, Umbelliferone represents isopentenyl acceptor 7-hydroxycoumarin, DMAPP / GPP / FPP represent different isopentenyl donors, and Umbelliprenin represents the isopentenyl donor umbelliferone. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 Based on genomic and transcriptomic data, we screened for oxopentenyltransferase genes involved in the synthesis of sesquiterpene coumarins from Ferula assa-foetida in Xinjiang. FsPT9 (1) Experimental methods Based on the genome data of *Ferula asperata* from Xinjiang, a hidden Markov model of UbiA PT was extracted from the NCBI database, and *FsPTs* of *Ferula asperata* from Xinjiang were identified at the whole-genome level using OrthoFinder software. Protein sequences were aligned using MUSCLE, and an ML phylogenetic tree was constructed with bootstrap selection and 1000 replicates. RNA-Seq transcriptome data of floral organs at six different developmental stages of *Ferula asperata* from Xinjiang were aligned to the *Ferula asperata* genome using HiSAT2, and gene expression FPKM values ​​were calculated using Cufflinks.

[0022] (2) Results and Analysis Sixteen PT genes were screened from the genome of Ferula ferox from Xinjiang, including eight genes ( FsPT1 , FsPT3 , FsPT4 , FsPT7 , FsPT8 , FsPT9 , FsPT10 and FsPT11 ) aggregates with known coumarin PT genes in the same branch, such as Figure 1 Further transcriptome data analysis revealed that among the eight genes... FsPT9 It has a high expression level in Ferula assa-foetida from Xinjiang, such as Figure 2 Therefore, it is speculated that it is a key enzyme gene involved in the synthesis of sesquiterpene coumarin in Xinjiang.

[0023] FsPT9 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0024]

[0025] coding FsPT9 The amino acid sequence of the gene is shown in SEQ ID No. 2.

[0026] MVQTHTLREGSSVNKRTRDRSQIGQIFVATLEDEFDLQPKDDNNKVTVQSDLWMKWDVFVRFCRLHSVTGTVTGIISVSLLPLTSFRDLSPAFFVGLLKAVIPFVFVNIYVVTLNQLVDVGIDKINKPHLVLTSGEYTMGQGKAITSAFGIMCLAMAIMFQSPPLLYGIIIHFLVGTA YSVELPLLRWKTKPFLAALGIGLYAFNFQLAVFCHIQKYVLGRPLVHTKSFGFVLIFFILFGTVLGVFKDIPDVAGDQAFGNRTYSVRHGKKRVFSICIFILLIDYGFGVVTGALLSSLLLSKLVAVVGHCTLASLLWRRARSLNLDDDSSVESLYMFLWKLFTAEYVLIQFIR, SEQ ID No.2.

[0027] Example 2 Validation of the catalytic function of oxyisopentenyltransferase FsPT9 (1) Experimental methods In vivo yeast expression: Yeast expression was achieved using the ClonExpress Ultra One-Step Cloning Kit (Vazyme, Nanjing, China). FsPT9 The recombinant plasmid was cloned into the pESC-HIS vector. The resulting recombinant plasmid was transformed into *Saccharomyces cerevisiae* strain WAT11, and transformants were screened on histidine-deficient SD-HIS plates. Single-clone yeast cells were picked for PCR verification, and positive single-clone yeast colonies were then inoculated into 4 mL of SD-His liquid medium and cultured at 30°C and 230 rpm for 24 hours with shaking. Subsequently, 200 μL of the pre-culture was transferred to 2 mL of SD-His medium and cultured under the same conditions (30°C, 230 rpm) for another 24 hours until the stationary phase.

[0028] In vivo yeast feeding: Collect 400 μL of stationary phase culture and centrifuge at 1,000×g to collect cells. Resuspend the pellet in 400 μL of YPA induction medium (containing 1% yeast extract, 1% bacterial peptone, and 2% galactose) to rapidly obtain a large number of yeast strains containing positive clones, and supplement with 1 mM MgCl2 (to provide the key cofactor Mg required for isopentenyltransferase). 2+0.1 mM isoprene acceptor (7-hydroxycoumarin) and 0.1 mM isoprene donor (farnesyl pyrophosphate, FPP) were used. The mixture was cultured at 30 °C and 220 rpm with shaking for 48 hours. After induction and reaction, 400 μL of ethyl acetate solution containing 10% (v / v) methanol was added for vortex extraction, which was repeated three times. The combined organic phases were dried under vacuum, and the residue was reconstituted with 150 μL of methanol for subsequent analysis.

[0029] Detection of catalytic synthesis products: Qualitative and quantitative analysis of enzyme reaction products was performed using a Waters Acquity UPLC system (Waters, Milford, USA) equipped with a photodiode array (PDA) detector. The separation process used a C18 reversed-phase column (ACQUITY UPLC BEH C18, 130 Å, 1.7 μm, 2.1 mm × 100 mm; Waters, Milford, Massachusetts, USA). Gradient elution was performed using an aqueous solution containing 0.1% (v / v) formic acid as mobile phase A and acetonitrile solution as mobile phase B: the proportion of mobile phase B increased from 5% to 95% over 45 minutes, held for 5 minutes, then decreased to 5% over another 5 minutes and held for another 5 minutes. The flow rate was set at 0.2 mL / min. Enzyme reaction products were detected and quantitatively analyzed at 325 nm using umbelliferone standards as a reference.

[0030] (2) Results and Analysis A yeast strain containing a homozygous expression plasmid of FsPT9 was obtained, and in vivo yeast feeding experiments demonstrated that FsPT9 catalyzes the formation of umbelliferone from 7-hydroxycoumarin and farnesyl pyrophosphate. Figure 3 .

[0031] Example 3 Substrate specificity of oxyisopentenyltransferase FsPT9: isopentenyl donor selection analysis (1) Experimental methods Yeast microsome preparation: 500 µL of the positive monoclonal yeast preculture prepared in Example 2 was diluted 100-fold to 50 mL of SD-His medium and cultured at 220 rpm and 30 °C until the OD600 reached approximately 1.7. Cells were collected by centrifugation at 1,000 × g, and the pellet was resuspended in 50 mL of YPA induction medium (containing 1% yeast extract, 1% bacterial peptone, and 2% galactose) and cultured overnight at 30 °C. Microsomes were extracted according to the method described by Meng et al. (Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta).

[0032] In vitro catalysis using yeast microsomes: The catalytic reaction was carried out in a 100 μL system. The reaction mixture contained microsomes, 0.4 mM isopentenyl acceptor (7-hydroxycoumarin), and 4 mM MgCl2 (providing the key cofactor Mg required by isopentenyltransferase). 2+ The reaction mixture contained 0.4 mM isopentenyl donors (dimethylallyl pyrophosphate DMAPP, geranyl pyrophosphate GPP, or farnesyl pyrophosphate FPP) and 100 mM Tris-HCl buffer (pH 9.0). After incubation at 30°C for 2 hours, the product was extracted twice with ethyl acetate (200 μL each time). The organic phases were combined and evaporated to dryness. The residue was reconstituted with 150 μL of methanol and analyzed by UPLC.

[0033] The detection method for catalytic synthesis products is the same as in Example 2.

[0034] (2) Results and Analysis In vitro experiments using yeast microsomes demonstrated that the oxyisopentenyltransferase FsPT9 exhibits substrate specificity and isopentenyl donor specificity in selecting FPP, such as... Figure 4 .

[0035] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0036] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An oxylprenyltransferase involved in the synthesis of a sesquiterpene coumarin, characterized in that, The amino acid sequence of which is shown as SEQ ID No.

2.

2. The gene encoding the oxylpentenyl transferase according to claim 1, characterized in that, The nucleotide sequence of which is shown as (a), (b) or (c): (a) the nucleotide sequence shown as SEQ ID No. 1; (b) a nucleotide sequence capable of hybridizing to the complementary nucleotide sequence of SEQ ID No. 1 under stringent hybridization conditions, the protein encoded by the nucleotide sequence still having the function or activity of an oxidosqualene transferase; or (c) a nucleotide sequence having at least 80% homology with the nucleotide sequence of SEQ ID No. 1, and the protein encoded by the nucleotide sequence still having the function or activity of an oxidosqualene transferase.

3. A chimeric gene or expression cassette containing the coding gene of claim 2.

4. A recombinant expression vector containing the coding gene of claim 2 or containing the chimeric gene or expression cassette of claim 3.

5. A recombinant host cell containing the recombinant expression vector of claim 4.

6. Use of the oxidosqualene transferase of claim 1 for catalyzing the biosynthesis of a sesquiterpene coumarin. The sesquiterpene coumarin is umbelliferone.

7. Use according to claim 6, wherein The substrates are 7-hydroxycoumarin and farnesyl pyrophosphate.

8. Use according to claim 7, wherein the compound is ###0002### 9. Use of the coding gene of claim 2, the chimeric gene or expression cassette of claim 3 for the biosynthesis of a sesquiterpene coumarin. ​

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