Atractylodes lancea alpha-eudesmol synthase gene AlTPS alpha, and coding product and application thereof

Abstract

The invention discloses an Atractylodes lancea alpha-eudesmol synthase gene AlTPS alpha as well as a coding product and application thereof. The nucleotide sequence of the atractylis lancea alpha-eudesmol synthase is shown as SEQ NO.1, and the coded amino acid sequence of the atractylis lancea alpha-eudesmol synthase is shown as SEQ NO.2. The alpha-eudesmol has the activity of generating a terpenoid alpha-eudesmol by taking farnesyl pyrophosphate (FPP) as a substrate. The alpha-eudesmol is a volatile pharmacological active component of various plants and has the effects of tranquilizing and allaying excitement and the like. The alpha-eudesmol synthase coding gene provided by the invention can be used for efficiently producing alpha-eudesmol through a microbial engineering technology.

Description

Technical Field

[0001] This invention relates to the fields of plant genetic engineering and natural product biotechnology, specifically to the AlTPSα gene of Atractylodes lancea α-eucalyptol synthase, its encoded product, and its applications. Background Technology

[0002] Terpenes are among the most diverse and structurally varied secondary metabolites found in natural products, with wide applications in medicine, fragrances, food, and agriculture. Terpene synthases (TPS) are key enzymes in the biosynthetic pathway of terpenes, catalyzing the formation of structurally diverse basic terpene skeletons from precursors such as geraniylgeraniyl pyrophosphate (GGPP) or farnesyl pyrophosphate (FPP), which then modify the enzyme to form various bioactive terpenes. Currently, terpene synthase genes from various plants have been cloned and functionally identified, but their catalytic specificity, efficiency, and expression regulation mechanisms still vary considerably. Therefore, identifying terpene synthase genes with specific catalytic functions and characterizing their functions is crucial for elucidating the terpene biosynthetic pathway and achieving efficient and targeted production of target bioactive components.

[0003] α-Cineole is a component of Atractylodes lancea (…). Atractylodes lancea One of the main active components of *Atractylodes lancea* (Thunb.) DC., it belongs to the sesquiterpenoid class of compounds and possesses various pharmacological activities, including anti-inflammatory, antibacterial, and neuroprotective effects. Currently, the production of α-cineole mainly relies on plant extraction, but this is limited by factors such as the long plant growth cycle, low content, and complex extraction processes, making it difficult to meet market demand. Constructing efficient cell factories for α-cineole production using synthetic biology strategies is an important approach to achieving its sustainable large-scale production, and cloning and obtaining a highly efficient and specific α-cineole synthase gene is a core prerequisite for realizing this strategy. However, there are currently no reports on the cloning and functional verification of the α-cineole synthase gene from *Atractylodes lancea*, and its catalytic properties and application potential as an encoded enzyme protein still require further investigation. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides an Atractylodes lancea α-cineole synthase gene AlTPSα, its encoded product, and its applications. The encoding gene of Atractylodes lancea α-cineole synthase provided by the present invention enables the sustainable large-scale production of α-cineole.

[0005] In one aspect, the present invention provides a gene encoding Atractylodes lancea α-eucalyptol synthase, the encoded nucleotide sequence of which is shown in SEQ NO.1.

[0006] In another aspect, the present invention provides an Atractylodes lancea α-eucalyptol synthase, the amino acid sequence of which is shown in SEQ NO.2.

[0007] In another aspect, the present invention provides a recombinant expression vector comprising the aforementioned coding gene. Preferably, the expression vector is pET-28a.

[0008] In another aspect, the present invention provides a recombinant strain containing the aforementioned encoding gene.

[0009] In another aspect of the present invention, a host cell comprising the aforementioned encoding gene is provided.

[0010] In another aspect, the present invention provides a method for expressing Atractylodes lancea α-cineole synthase, comprising: transforming a suitable strain for expression with the aforementioned recombinant vector to obtain a recombinant strain; culturing the recombinant strain to induce the expression of Atractylodes lancea α-cineole synthase; the recombinant strain simultaneously synthesizing an FPP substrate, and the α-cineole synthase simultaneously using the FPP to synthesize α-cineole; and, after the culture is completed, recovering the synthesized α-cineole product.

[0011] In another aspect, the present invention provides the use of the aforementioned encoding gene, or recombinant vector, or recombinant strain, or host cell in the preparation of terpenoid compounds. Preferably, the terpenoid compound product is α-cineole.

[0012] The beneficial effects of this invention are as follows: A terpenoid synthase gene from Atractylodes lancea was obtained, encoding a protein with the activity of generating the terpenoid compound α-cineole using FPP as a substrate. This invention also provides a method for directly preparing terpenoid compounds from recombinant strains. The gene and preparation method provided by this invention enable the sustainable large-scale production of α-cineole. Attached Figure Description

[0013] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0014] Figure 1 The image shows the pET28a-MBP-AlTPSα recombinant plasmid.

[0015] Figure 2 Comparison of total ion chromatograms of FPP products catalyzed by unloaded pET28a and pET28a-MBP-AlTPSα.

[0016] Figure 3 The mass spectrum of the empty pET28a peak at retention time 28.409 min is shown.

[0017] Figure 4 The mass spectrum of the product of pET28a-MBP-AlTPSα catalyzed FPP at retention time 28.409 min is shown.

[0018] Figure 5The mass spectrum of α-eucalyptol is shown in the NIST17 database. Detailed Implementation

[0019] The present invention will now be described in detail with reference to specific accompanying drawings and embodiments. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is only for explaining the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention.

[0020] The technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0021] Example 1: Synthesis of Atractylodes lancea α-Cephalotaxic acid synthase gene AlTPSα and construction of recombinant expression vector Based on the genome and transcriptome data of Atractylodes lancea, a full-length terpene synthase gene, AlTPSα, was obtained. Its encoding nucleotide sequence is shown in SEQ NO.1 of the sequence listing. A tag encoding MBP (maltose-binding protein, 40 kDa) was fused before this gene fragment. The entire gene was synthesized by Shanghai Sangon Biotech Co., Ltd., and ligated into the pET28a expression vector to obtain the recombinant prokaryotic expression plasmid pET28a-MBP-AlTPSα. The plasmid map is shown below. Figure 1 As shown.

[0022] Example 2 Production of α-Cineole by Engineered Bacteria The recombinant plasmid pET28a-MBP-AlTPSα and FPP substrate were used to produce the plasmid pBbA5c-MevT(CO)-T1-MBIS(CO, ispA), which was then co-transformed into E. coli BL21 (DE3) competent cells to screen for positive E. coli BL21 (DE3) strains. This strain contains highly efficient expression of the AlTPSα recombinant gene and simultaneously produces FPP substrate. Positive clones were inoculated at a ratio of 1:100 into NZY medium containing kanamycin and chloramphenicol resistance, and cultured at 37 °C with shaking at 200 rpm until A600 = 0.4 ~ 0.6. IPTG to a final concentration of 0.1 mM and an appropriate amount of dodecane were added as extraction buffer, and the cells were induced at 16 °C. The pET28a empty vector was treated under the same conditions as a blank control. After 36 h, the dodecane extract was collected for GC-MS analysis of the product.

[0023] Example 3 Detection of α-Cephalotaxol Products For the dodecane extract in Example 2, 200 μL was transferred to an end-capsule, and the catalytic product was detected by gas chromatography-mass spectrometry (GC-MS). The following protocol was used for GC-MS: An Agilent 7890B-5977B GC-MS instrument and a 19091S-433 column were used. The GC furnace was initially set at 85°C and held for 5 min, then increased to 185°C at a rate of 3°C / min and held at 185°C for 10 min. The furnace was then increased to 250°C at a rate of 5°C / min and held for another 5 min. The split ratio was 1:10, and the injection volume was 1 μL. The interface temperature was maintained at 250°C. ◦ C. Mass spectrometry was performed in full scan mode, with a scanning range of 33–350 amu; ion source temperature was 230 °C, and solvent delay time was 15 min. The products were then qualitatively analyzed. Figure 2 A comparison of total ion chromatograms of the products of FPP catalyzed by unloaded pET28a and pET28a-MBP-AlTPSα. Figure 3 The mass spectrum of the empty pET28a peak at retention time 28.409 min is shown. Figure 4 The mass spectrum of the FPP product catalyzed by pET28a-MBP-AlTPSα has a retention time of 28.409 min. The sample exhibits a characteristic peak at this retention time. A search and comparison with the NIST17 database indicates that the corresponding compound is α-eucalyptol. Figure 5 ).

[0024] 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.

[0025] SEQ NO.1

[0026] SEQ NO.2 MCPEQENDLRPTADFPPDIWGDQFLVYDEEEQDHGVEQVVEDLKLKVKKEILATLNVPAEHTNLLKLVDTIQRLGISYYFEEEINEALQHIYDEYGDNWTGGCPSVWFRLLRQQGFFVSCGIFNKDKDGAFKESLMNDVQGLLDLYEAAYMRVPGEVILDDALVFTRSRLDDISKDPLHGTNIDSTQIQEALTQPIVKRLPRLEALRYIPFYQRQDSHNEHLLKLAKLGFNLLQSLHKKEISQLSKWWKSYDVTNNFPYARDRLVECYFWIQGVYFEPKYFQSRILLTKNLAMASILDDTYDAYSTCEELELFTKAIDMWSITCYDVLPKYMKPLYQMVMELYKEMEETMADEGKSYLLNYLKESIKEFITGYMIEAKWRNEGYMPTLEEHVSVSLITSGYKYLITASFVGMGDVITEESFKWVSTNPPIVISSCVVGRFRDDVITHKEEQERNHVPSVIECYMKQFDVTKDHAYGLANKKVEDAWKEIIRESLTCKDVPMPLIMRVINFTRVIHVMYKDQDNYTHVGDEMKNHIRSLLIDTMST*

Claims

1. A gene encoding α-eucalyptol synthase in Atractylodes lancea, characterized in that, Its encoding nucleotide sequence is shown in SEQ NO.

1.

2. The amino acid sequence of the Atractylodes macrocephala α-eucalyptol synthase according to claim 1.

3. The amino acid sequence according to claim 2, characterized in that, Its amino acid sequence is shown in SEQ NO.

2.

4. A recombinant vector comprising the encoding gene and translated amino acids as described in claim 1, 2 or 3.

5. The recombinant vector according to claim 4, characterized in that, The carrier is pET28a.

6. A recombinant strain comprising the encoding gene and translated amino acids as described in claim 1, 2 or 3.

7. A host cell comprising the encoding gene and translated amino acids as described in claim 1, 2 or 3.

8. A method for expressing α-cineole synthase and producing α-cineole, characterized in that, The method comprises: transforming a suitable strain for expression with the recombinant vector of claim 4 or 5 to obtain the recombinant strain or host cell of claim 6 or 7; culturing the recombinant strain or host cell to induce the expression of α-eucalyptol synthase of claim 2 or 3; the recombinant strain simultaneously synthesizing FPP substrate, and the α-eucalyptol synthase simultaneously using FPP to synthesize α-eucalyptol; and, after the culture is completed, recovering the α-eucalyptol product.

9. The use of the encoding gene as described in claim 1, or the encoding amino acid sequence as described in claim 2 or 3, or the recombinant vector as described in claim 4 or 5, or the recombinant strain as described in claim 6, or the host cell as described in claim 7 in the preparation of α-eucalyptol.

10. The application according to claim 9, characterized in that, The terpene compound is α-eucalyptol.

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