Artemisia annua spirochetane-type sesquiterpene Modhephene synthase gene AaTPS25 and its mutants and applications

Abstract

This invention discloses the Modhephene synthase gene of Artemisia annua spirochetane-type sesquiterpenoids. AaTPS25 Its mutants and applications; the Modhephene synthase gene was first identified and cloned from Artemisia annua. AaTPS25 The study clarified that the encoded protein can catalyze the synthesis of Modhephene from FPP, laying the foundation for the study of the biosynthesis and cyclization mechanism of this compound. Through site-directed mutagenesis, the AaTPS25 mutant with enhanced activity and the AaTPS26-A295E / Q and AabrSPS-A295E / Q mutants with Modhephene synthase activity were obtained, enriching the Modhephene synthase gene resources and providing key evidence for improving the biosynthetic yield of Modhephene and studying its catalytic mechanism.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biotechnology, specifically to a Modhephene synthase gene of the spiropropane-type sesquiterpene in Artemisia annua. AaTPS25 The invention relates to its mutants and their applications in the catalytic production of Modhephene from farnesyl pyrophosphate. Background Technology

[0002] Modhephene (C 15 H 24 Modhephene is a sesquiterpene compound with a [3.3.3]spiroalkyl skeleton, first isolated from nature in 1978. Its structure consists of three five-membered rings fused together by a common spirocarbon atom, exhibiting a highly compact three-dimensional spatial configuration. This unique ring system endows modhephene with physicochemical properties unlike conventional chain, monocyclic, or bicyclic sesquiterpenes, such as higher volatility and significant configurational rigidity. Modhephene exists in two epimers, with the naturally dominant configuration (modhephene) playing a dominant role in biosynthetic and chemical synthesis research, while epi-modhephene is its minor isomer.

[0003] Due to the high complexity and uniqueness of its skeletal structure, modhephene is considered an important synthetic precursor for the study of polycyclic terpenoids, chiral molecules, and spiroalkyl derivatives. Its unique spatial arrangement not only simplifies the synthetic routes for target products but also shows significant potential in improving stereoselectivity and constructing novel spiroalkyl molecules, providing structural templates for the development of high-value fine chemicals. Although modhephene has been chemically synthesized since 1981, its rigid skeletal structure and cumbersome synthetic steps still face bottlenecks in large-scale preparation, limiting its widespread application. Therefore, elucidating its biosynthetic mechanism, especially identifying key enzymes in its synthetic pathway, has become a crucial breakthrough in overcoming these bottlenecks.

[0004] In plant sesquiterpene metabolic pathways, the vast majority of sesquiterpene compounds use farnesene pyrophosphate (FPP) as a direct precursor, which is then catalyzed by sesquiterpene synthases to produce structurally diverse terpene products. This provides important clues for exploring key enzymes in modhephene biosynthesis. Artemisia annua L., also known as Qinghao, is a medicinal plant with Chinese characteristics, and its medicinal value has been recorded in ancient texts such as the *Shennong Bencao Jing* and *Compendium of Materia Medica*. The artemisinin isolated and identified from Artemisia annua by Tu Youyou and her team has saved the lives of millions of malaria patients worldwide, making Artemisia annua a hot topic in global medicinal plant research. Recent studies have shown that, in addition to artemisinin, modhephene is also present in the roots of Artemisia annua and its closely related plants such as Artemisia abrotanum L., especially in root tissues where it exhibits high synthetic abundance. This suggests that plants such as Artemisia annua may carry modhephene synthase genes, possessing the potential to elucidate its catalytic mechanism and functional sites.

[0005] Therefore, screening and identifying sesquiterpene synthase genes from plants rich in modhephene, such as Artemisia annua, and conducting in-depth research on the structure-function relationship and catalytic mechanism of their encoded proteins, is of great theoretical and applied value for elucidating the biosynthetic mechanism of spiroalkyl sesquiterpenes, breaking through the bottleneck of modhephene biomanufacturing, and expanding their application in polycyclic terpene synthesis and chiral molecule construction. Summary of the Invention

[0006] In view of this, one of the objectives of the present invention is to provide a Modhephene synthase gene for Artemisia annua. AaTPS25The second objective of this invention is to provide a mutant protein capable of catalyzing the production of Modhephene from farnesyl pyrophosphate; the third objective of this invention is to provide a nucleic acid encoding the mutant protein capable of synthesizing Modhephene; the fourth objective of this invention is to provide a recombinant vector containing the nucleic acid; the fifth objective of this invention is to provide a host cell containing the nucleic acid or the recombinant vector; the sixth objective of this invention is to provide the application of the mutant protein, the nucleic acid, the recombinant vector, or the host cell in the preparation of spiroalkyl sesquiterpenes Modhephene; the seventh objective of this invention is to provide a recombinant vector containing the nucleic acid shown in SEQ ID NO. 6 and a host cell in the preparation of spiroalkyl sesquiterpenes Modhephene or windowane sesquiterpenes Silphiperfol-5-ene, Silphinene, α-Isocomene, β-Isocomene, and / or caryophyllene sesquiterpenes β-Caryophyllene; the eighth objective of this invention is to provide a method for preparing spiroalkyl sesquiterpenes Modhephene; and the ninth objective of this invention is to provide a method for improving the quality of Artemisia annua. Methods for determining the activity of Modhephene synthase AaTPS25.

[0007] To achieve the above objectives, the present invention provides the following technical solution: 1. Modhephene synthase gene of Artemisia annua AaTPS25 The Artemisia annua Modhephene synthase gene AaTPS25 The nucleotide sequence is shown in SEQ ID NO.3.

[0008] 2. A mutant protein capable of catalyzing the production of Modhephene from farnesyl pyrophosphate, said mutant protein being obtained by site-directed mutation of amino acid residue 295 of the amino acid sequence of Modhephene synthase AaTPS25 from Artemisia annua (SEQ ID NO.4) to glutamine, or by site-directed mutation of amino acid residue 295 of the amino acid sequence of β-Caryophyllene synthase AaTPS26 from Artemisia annua (SEQ ID NO.8) to glutamic acid or glutamine, or by site-directed mutation of amino acid residue 295 of the amino acid sequence of 7-epi-silphiperfol-5-ene synthase AabrSPS from Artemisia abrotanum (SEQ ID NO.11) to glutamic acid or glutamine.

[0009] 3. A nucleic acid encoding the mutant protein that catalyzes the formation of Modhephene from farnesyl pyrophosphate.

[0010] Preferably, the nucleotide sequence of the nucleic acid is as shown in SEQ ID NO.6, SEQ ID NO.13-14, or SEQ ID NO.15-16.

[0011] 4. A recombinant vector containing the said nucleic acid.

[0012] 5. A host cell containing the nucleic acid or the recombinant vector.

[0013] 6. The application of the mutant protein, the nucleic acid, the recombinant vector, or the host cell in the preparation of the propane-type sesquiterpene Modhephene.

[0014] 7. The application of recombinant vectors and host cells containing the nucleic acid shown in SEQ ID NO.6 in the preparation of spiroalkyl sesquiterpenes Modhephene or windowane sesquiterpenes Silphiperfol-5-ene, Silphinene, α-Isocomene, β-Isocomene and / or caryophyllene sesquiterpenes β-Caryophyllene.

[0015] 8. A method for preparing the spiroalkyl sesquiterpene Modhephene, comprising the following steps: expressing the nucleic acid in a host cell possessing the MVA synthesis pathway and a farnesyl pyrophosphate synthase gene to generate the mutant protein, and catalyzing the farnesyl pyrophosphate to generate Modhephene.

[0016] 9. A method for improving the activity of Modhephene synthase AaTPS25 from Artemisia annua, comprising site-directedly mutating the 295th amino acid residue of Modhephene synthase AaTPS25 from Artemisia annua as shown in SEQ ID NO.4 from glutamic acid to glutamine to obtain a mutant with improved Modhephene synthesis activity.

[0017] The beneficial effects of this invention are as follows: This invention is the first to identify and clone the Modhephene synthase gene from Artemisia annua. AaTPS25This invention clarifies that the encoded protein can catalyze the synthesis of Modhephene from FPP, laying the foundation for research on the biosynthesis and cyclization mechanism of this compound. The AaTPS25 mutant obtained through site-directed mutagenesis provides crucial evidence for improving Modhephene biosynthetic yield and studying its catalytic mechanism. Furthermore, this invention also obtained AaTPS26-A295E / Q and AabrSPS-A295E / Q mutants through site-directed mutagenesis, both of which can transform non-Modhephene-producing enzymes into Modhephene synthases, enriching the Modhephene synthase gene resources and further promoting the development of the theory of spiroalkyl sesquiterpene biosynthesis.

[0018] The Modhephene biosynthesis method established in this invention has advantages such as simple steps and green environmental protection. It can replace the traditional lengthy chemical synthesis process, laying a technical foundation for the large-scale industrial production of Modhephene, and also laying a solid foundation for further exploration of the biological functions and drug applications of Modhephene. Attached Figure Description

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 For in vitro functional validation of AaTPS25 protein (A: GC-MS detection results; B: Mass spectrometry identification results); Figure 2 For in vivo functional validation of AaTPS25 protein (A: GC-MS detection results; B: Percentage of product after 36 hours of fermentation); Figure 3 In vitro functional validation of AaTPS25-E295A / D / Q mutant and wild-type proteins (A: In vitro enzyme activity product content analysis of wild-type and mutant AaTPS25-E295A / D protein; B: In vitro enzyme activity product content analysis of wild-type and mutant AaTPS25-E295A / Q protein). Figure 4 In vitro functional validation of AaTPS26-A295E / Q mutant protein and wild-type protein (GC-MS detection results of in vitro enzyme activity of AaTPS26 wild-type and A295E / Q mutant proteins). Figure 5 In vitro functional validation of AabrSPS-A295E / Q mutant protein and wild-type protein (GC-MS results of in vitro enzyme activity of AabrSPS wild-type and A295E / Q mutant proteins). Detailed Implementation

[0020] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0021] Example 1: Artemisia annua Modhephene synthase gene AaTPS25 Cloning and Identification Based on the genome and transcriptome data of Artemisia annua, a systematic screening and bioinformatics analysis of its terpene synthase (AaTPS) gene family was conducted. Through conserved domain identification, sequence alignment, and evolutionary relationship analysis, the gene expression patterns were correlated with metabolite accumulation patterns. Genes significantly overexpressed in the roots were experimentally verified, and a gene designated AaTPS25 was identified. This gene catalyzes the cyclization of farnesyl pyrophosphate (FPP) to form the spiroalkyl sesquiterpene Modhephene.

[0022] The specific primers are designed as follows: AaTPS25-F: 5'-ATGTCTGCACAAGAGAATGTTATACGTC-3' (SEQ ID NO. 1); AaTPS25-R: 5'-TCATGCCATTATAGGATCAACGACAAG-3' (SEQ ID NO. 2).

[0023] Using Artemisia annua cDNA as a template and SEQ ID NO.1 and SEQ ID NO.2 as primers, PCR amplification was successfully performed to obtain the cDNA. AaTPS25 Gene. RNA extraction and cDNA preparation from *Artemisia annua* were performed according to the method described in the *Tiangen* plant RNA extraction and reverse transcription kit. TAKARA Prime STAR Max Premix (2×) was used as the DNA polymerase. The PCR amplification system and program were as follows: 2.0 μL cDNA, 1.0 μL AaTPS25-F, 1.0 μL AaTPS25-R, 25 μL Prime STAR Max Premix (2×), and ddH2O to a final volume of 50 μL; 30 cycles of 98℃ for 10 sec, 56℃ for 10 sec, and 72℃ for 20 sec. After identifying the target fragment size by 1% agarose gel electrophoresis, the target fragment was purified and recovered using the Shanghai Sangon DNA gel recovery kit, following the kit's instructions. The fragment was sent to Beijing BGI Genomics Co., Ltd. for sequencing, and the target gene sequence was obtained, as shown in SEQ ID NO.3. Analysis revealed that the *Artemisia annua* Modhephene synthase gene... AaTP25The gene is 1641 bp in length, encodes 546 amino acids, and has a molecular weight of 63.6 kDa. The amino acid sequence is shown in SEQ ID NO.4.

[0024] Example 2: Heterologous expression and in vitro functional verification of AaTPS25 protein To further identify the Modhephene synthase gene in Artemisia annua AaTP25 The functions of the above AaTPS25 After sequence optimization (see SEQ ID NO.5), the gene was forward cloned into the multiple cloning site between BamH I and HindIII of the expression vector pET28a to construct the recombinant expression plasmid pET28a-AaTPS25. The recombinant plasmid was constructed in *E. coli* DH5α cells. After confirming the transformant sequence was correct, the recombinant plasmid was extracted and transformed into *E. coli* Rosetta (DE3) competent cells for protein expression induction. The strain was cultured in LB medium supplemented with 50 mg / L kanamycin and 34 mg / L chloramphenicol at 37°C with shaking at 220 rpm until OD600 = 0.6–0.8. An inducer at a final concentration of 0.1 mM IPTG was added, and protein expression was induced at 16°C and 140 rpm for 20 h. After induction, the bacterial cells were collected by centrifugation, and the target protein (with a His-tag) was purified by sonication. The recombinant AaTPS25 protein with an N-terminal His tag was obtained by Ni-NTA affinity chromatography.

[0025] Subsequently, an in vitro reaction system for the recombinant protein was established, and the formation of the target product Modhephene was detected by GC-MS. The reaction system (100 μL) consisted of 25 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.5 mM MgCl2, and 5 μg FPP. 50 μg of recombinant protein was added, and the mixture was incubated at 30 °C for 3 h. After the reaction was complete, 200 μL of the organic solvent n-hexane was added, and the mixture was vortexed for 10 min. Then, the aqueous and organic phases were separated by centrifugation at 12000 r / min for 10 min. The organic phase was filtered through a 0.22 μm microporous membrane and added to a sample vial. The product was detected by GC-MS, and the results are as follows: Figure 1As shown, AaTPS25 can synthesize four windowane-type sesquiterpenes—silphiperfol-5-ene, silphinene, α-Isocomene, and β-Isocomene—one spiroalkyl-type sesquiterpene—Modhephene, and one caryophyllene-type sesquiterpene—β-Caryophyllene, using FPP catalysis. Modhephene is the major product of this enzyme activity (accounting for over 70% of the total sesquiterpenes), demonstrating that AaTPS25 derived from Artemisia annua is the key enzyme responsible for catalyzing the synthesis of Modhephene.

[0026] Example 3: Heterologous expression and in vivo functional verification of AaTPS25 protein To further identify the Modhephene synthase gene in Artemisia annua AaTP25 Function, to the above AaTPS25 The gene was cloned into the expression vector pETDuet-1 to obtain the recombinant vector pETDuet-AaTPS25. An engineered *E. coli* strain possessing the heterologous MVA pathway and the FPP synthase gene IspA (Gene Accession: KP670645.1) was constructed according to the reference (Lin, GM., Voigt, CA Design of a redox-proficient *Escherichia coli* for screening terpenoids and modifying cytochrome P450s. *Nature Catalysis*, 1016-1029(2023)) to produce the substrate FPP. The recombinant vector pETDuet-AaTPS25 was then introduced into this strain, and the in vivo synthesis of Modhephene was detected. TB medium was used for fermentation. The strain was cultured to an OD600 of 0.8–1.0, and 1.0 mM IPTG was added. Fermentation was carried out at 25°C, and samples were taken at different times to further detect product synthesis. After sampling, an equal volume of n-hexane was added for extraction. Following centrifugation and filtration, GC-MS was used to detect the intracellular synthesis of Modhephene. The GC-MS results and analysis of the product synthesis percentage are shown below. Figure 2 As shown, this indicates the Modhephene synthase gene in Artemisia annua. AaTP25 Modhephene can be synthesized in Escherichia coli. The proportion and yield of the synthesis of major and minor products are limited by factors such as fermentation time. At a fermentation time of 36 h, the product type consistent with the in vitro enzymatic reaction was detected. At other times, only the synthesis of the major product could be detected.

[0027] Example 4: Rational design and mutant construction of Modhephene synthase protein The construction of mutants of the AaTPS25 gene was based on AlphaFold 3 predictions and molecular docking with the substrate FPP (performed via AutoDock). Sequence alignment was performed with reported sequences of enzymes that synthesize windowane-type tricyclic sesquiterpene synthases to screen for the key amino acid site E295 that may affect product synthesis. The GAA at positions 883-885 of the AaTPS25 gene sequence shown in SEQ ID NO.5 was mutated to GCG, GAT, and CAG, respectively, to obtain mutants AaTPS25-E295A, AaTPS25-E295D, and AaTPS25-E295Q (see SEQ ID NO.6).

[0028] To accurately assess the function of the above mutants, the mutants will be... AaTPS25 The gene was constructed in the pET28a vector and transformed into *E. coli* DH5α cells to construct a recombinant plasmid. After the transformant sequence was confirmed to be correct, the mutant recombinant plasmid was extracted and transformed into *E. coli* Rosetta (DE3) for protein expression induction. Single colonies were expanded and the protein was induced. The mutant protein was purified using a Ni column and compared with wild-type AaTPS25 by enzyme activity analysis. The protein purification steps, reaction system, and extraction method were consistent with those in Example 2. GC-MS results are shown below. Figure 3 As shown, amino acid residue 295 in the AaTPS25 protein sequence is a key site for product synthesis. The AaTPS25-E295A mutation causes the protein to lose its ability to synthesize tricyclic sesquiterpenes, with only small amounts of β-Caryophyllene and Humulene bicyclic sesquiterpenes detected. The AaTPS25-E295D mutation increases Silphiperfol-5-ene synthesis by approximately 5-fold, β-Caryophyllene synthesis by approximately 3-fold, and detects the synthesis of the new product 7-epi-Silphiperfol-5-ene, but loses the synthesis of Silphinene, Modhephene, α-Isocomene, and β-Isocomene. The AaTPS25-E295Q mutation, however, promotes the synthesis of all products, with the major product Modhephene increasing by approximately 2-fold, and the remaining products increasing by 3-17-fold, with Silphiperfol-5-ene showing the largest increase at approximately 17-fold.

[0029] To verify the crucial cyclization role of the E295 site in Modhephene synthesis, equivalent site-directed mutagenesis was performed on two homologous enzymes: β-Caryophyllene synthase AaTPS26 (wild-type nucleotide and amino acid sequences are shown in SEQ ID NO. 7-8, optimized gene sequence is shown in SEQ ID NO. 9) from Artemisia annua, which is sequence-similar to AaTPS25 but cannot synthesize Modhephene in the wild-type (WT) form; and 7-epi-Silphiperfol-5-ene synthase AabrSPS (wild-type nucleotide and amino acid sequences are shown in SEQ ID NO. 10-11, optimized gene sequence is shown in SEQ ID NO. 12) from Artemisia argyi. In both enzymes, the E295 site is A, which was mutated to E or Q to construct mutants AaTPS26-A295E / Q (mutated sequences are shown in SEQ ID NO. 13-14) and AabrSPS-A295E / Q (mutated sequences are shown in SEQ ID NO. 15-16). After sequence optimization AaTPS26 , AabrSPS Wild-type and mutant genes were constructed using the pET28a vector. The vector construction method, protein induction method, protein purification steps, reaction system, and extraction method were all consistent with those in Example 2. GC-MS results are shown below. Figure 4 and Figure 5 As shown, the changes in products between wild-type and mutant types are compared. Figure 4 The results showed that wild-type AaTPS26 catalyzed FPP to mainly synthesize β-Caryophyllene and could not synthesize Modhephene. However, after mutating A295 to E295 and Q295, the mutants could synthesize Modhephene, and the AaTPS26-A295Q mutant also achieved the synthesis of Silphinene and α-Isocomene. Figure 5 The results showed that wild-type AabrSPS catalyzed FPP mainly synthesized 7-epi-Silphiperfol-5-ene and could not synthesize Modhephene. However, mutating A295 to Q295 and E295 could convert the main product to Modhephene.

[0030] The above-described embodiments are merely preferred embodiments provided to fully illustrate the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. Modhephene synthase gene of Artemisia annua AaTPS25 Its features are: The Modhephene synthase gene of Artemisia annua AaTPS25 The nucleotide sequence is shown in SEQ ID NO.

3.

2. A mutant protein capable of catalyzing the formation of Modhephene from farnesyl pyrophosphate, characterized in that, The mutant protein is obtained by site-directed mutation of amino acid residue 295 of Modhephene synthase AaTPS25 from Artemisia annua (SEQ ID NO.4) to glutamine, or by site-directed mutation of amino acid residue 295 of β-Caryophyllene synthase AaTPS26 from Artemisia annua (SEQ ID NO.8) to glutamic acid or glutamine, or by site-directed mutation of amino acid residue 295 of 7-epi-silphiperfol-5-ene synthase AabrSPS from Artemisia abrotanum (SEQ ID NO.11) to glutamic acid or glutamine.

3. A nucleic acid encoding a mutant protein of claim 2 that can catalyze the formation of Modhephene from farnesyl pyrophosphate.

4. The nucleic acid according to claim 3, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.6 or SEQ ID NO.13-14 or SEQ ID NO.15-16.

5. A recombinant vector containing the nucleic acid of claim 3 or 4.

6. A host cell containing the nucleic acid of claim 3 or 4 or the recombinant vector of claim 5.

7. The use of the mutant protein of claim 2, the nucleic acid of claim 3 or 4, the recombinant vector of claim 5, or the host cell of claim 6 in the preparation of the propane-type sesquiterpene Modhephene product.

8. The application of recombinant vectors and host cells containing the nucleic acid shown in SEQ ID NO.6 in the preparation of spiroalkyl sesquiterpenes Modhephene or windowane sesquiterpenes Silphiperfol-5-ene, Silphinene, α-Isocomene, β-Isocomene and / or caryophyllene sesquiterpenes β-Caryophyllene.

9. A method for preparing spiroalkyl sesquiterpenes, characterized in that, Includes the following steps: The nucleic acid of claim 3 or 4 is expressed in a host cell that possesses the MVA synthesis pathway and the farnesyl pyrophosphate synthase gene to generate the mutant protein of claim 2, which catalyzes the conversion of farnesyl pyrophosphate to Modhephene.

10. A method for increasing the activity of Modhephene synthase AaTPS25 in Artemisia annua, characterized in that, The 295th amino acid residue of the Modhephene synthase AaTPS25 amino acid sequence from Artemisia annua (SEQ ID NO.4) was site-directedly mutated from glutamic acid to glutamine to obtain a mutant with enhanced Modhephene synthetic activity.

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