Astilbe chinensis eudesma-5, 7-diene synthase AchTPS3 and application thereof

Abstract

The invention provides an astilbe chinensis eudesma-5, 7-diene synthase AchTPS3 and application of the astilbe chinensis eudesma-5, 7-diene synthase AchTPS3, and belongs to the technical field of biosynthesis. The amino acid sequence of the astilbe eudesma-5, 7-diene synthase AchTPS3 comprises an amino acid sequence as shown in SEQ ID NO: 1, or a functional homologous sequence with at least 75% sequence similarity with the amino acid sequence as shown in SEQ ID NO: 1, or one or more amino acids are added, deleted and replaced in the amino acid sequence as shown in SEQ ID NO: 1, or the amino acid sequence as shown in SEQ ID NO: 2 is added, deleted and replaced in the amino acid sequence as shown in SEQ ID NO: 1. And the protein formed by the amino acid sequence as shown in SEQ ID NO: 1 has an amino acid sequence with the same function. The coding gene comprises a nucleotide sequence as shown in SEQ ID NO: 2 or a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence as shown in SEQ ID NO: 2. According to the invention, the coding gene of the eudesma-5, 7-diene synthase AchTPS3 is integrated into a carrier and is introduced into a host cell, so that the coding gene is expressed in the host cell, and the biosynthesis of eudesma-5, 7-diene is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and in particular to an Astilbe eudesma-5,7-diene synthase AchTPS3 and an application thereof. Background Art

[0002] Terpenoids are the most diverse and chemically rich natural products found in plants. Due to their unique structural properties and diverse activity profiles, they are widely used in pharmaceuticals, household chemicals, fuels, and other fields, generating significant market demand. However, the extraction and isolation of active compounds from plants is complex, inefficient, and unsustainable. In recent years, the production of active compounds using synthetic biology has demonstrated significant potential.

[0003] Eudesma-5,7-diene is a eudesmane-type sesquiterpenoid compound first isolated from the bark and leaf volatiles of Croton eluteria (Euphorbiaceae). It has subsequently been found in Vetiveria zizanioides (Poaceae), Guarea guidonia (Meliaceae), Preissia quadrata (Milky Way), and Plagiochila asplenioides (Plagiochila asplenioides). However, the enzyme that synthesizes eudesma-5,7-diene has not yet been identified in plants.

[0004] There are three main methods for synthesizing terpenoids: plant extraction, chemical synthesis, and biosynthesis. However, plant extraction requires large amounts of materials and is costly, while chemical synthesis involves complex steps, produces numerous byproducts, and has significant environmental impact. Therefore, to achieve biosynthesis of terpenoids, it is crucial to identify terpenoid synthase genes that efficiently synthesize terpenoids. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides an Astilbe truncatum eudesma-5,7-diene synthase AchTPS3 and its related biomaterials for use in the biosynthesis of eudesma-5,7-diene.

[0006] To achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a eudesma-5,7-diene synthase AchTPS3, wherein the amino acid sequence of the eudesma-5,7-diene synthase AchTPS3 includes the amino acid sequence in (i), (ii) or (iii):

[0008] (i) the amino acid sequence shown in SEQ ID NO: 1;

[0009] (ii) a functional homologous sequence having at least 75% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1;

[0010] (iii) an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1, and which has the same function as the protein composed of the amino acid sequence of SEQ ID NO: 1.

[0011] Furthermore, the Astilbe eudesma-5,7-diene synthase AchTPS3 includes a fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of the amino acid sequence shown in SEQ ID NO: 1.

[0012] The present invention discovered the eudesma-5,7-diene synthase AchTPS3 gene from the genome of Astilbe chinensis and successfully obtained the encoded protein, which was named Astilbe chinensis eudesma-5,7-diene synthase AchTPS3. Heterologous expression in Saccharomyces cerevisiae revealed that Astilbe chinensis eudesma-5,7-diene synthase AchTPS3 can catalyze the substrate farnesyl pyrophosphate (FPP) to produce the sesquiterpene product eudesma-5,7-diene. The Astilbe chinensis eudesma-5,7-diene synthase AchTPS3 provided by the present invention provides a genetic resource for the production of eudesma-5,7-diene.

[0013] The biological materials related to Astilbe truncatum eudesma-5,7-diene synthase AchTPS3 include the encoding gene of Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, a vector containing the encoding gene of Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, a genetically engineered host cell expressing Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, etc.

[0014] In a second aspect, the present invention provides a gene encoding the Astilbe truncatula eudesma-5,7-diene synthase AchTPS3, comprising the nucleotide sequence in (i) or (ii):

[0015] (i) the nucleotide sequence shown in SEQ ID NO: 2;

[0016] (ii) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO: 2.

[0017] Furthermore, the complementary sequence, degenerate sequence or homologous sequence of the nucleotide sequence shown in SEQ ID NO: 2 has more than 80% homology with the nucleotide sequence shown in SEQ ID NO: 2.

[0018] Furthermore, the gene encoding the Astilbe truncatula eudesma-5,7-diene synthase AchTPS3 also includes variants of the nucleotide sequence shown in SEQ ID NO: 2 with conservative substitutions (eg, substitutions of degenerate codons) and their complementary sequences.

[0019] Furthermore, the gene encoding the Astilbe eudesma-5,7-diene synthase AchTPS3 also includes a nucleotide sequence that is designed and artificially codon optimized using the amino acid sequence of the Astilbe eudesma-5,7-diene synthase AchTPS3 and is conducive to expression in host cells.

[0020] In a third aspect, the present invention provides a vector comprising the gene encoding the Astilbe truncatula eudesma-5,7-diene synthase AchTPS3.

[0021] A vector for expressing Astilbe truncatum eudesma-5,7-diene synthase AchTPS3 comprises a gene encoding the Astilbe truncatum eudesma-5,7-diene synthase AchTPS3. The present invention does not specifically limit the type of the vector, and a suitable vector can be selected as needed. For example, the vector includes but is not limited to pESC-Ura, pESC-Trp, pESC-Leu, pESC-His, pRS426, pET-28a, or pEAQ. Furthermore, the vector is pRS426, pET-28a, or pEAQ.

[0022] In a fourth aspect, the present invention provides a genetically engineered host cell comprising the vector.

[0023] A genetically engineered host cell for expressing Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, comprising the amino acid sequence of Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, or comprising the coding gene of Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, or having introduced therein a vector expressing Astilbe truncatum eudesma-5,7-diene synthase AchTPS3.

[0024] Furthermore, the genetically engineered host cell comprises mevalonate pathway-related genes, and can generate farnesyl pyrophosphate, which is converted into eudesma-5,7-diene under the action of the Astilbe eudesma-5,7-diene synthase AchTPS3.

[0025] Furthermore, the genetically engineered host cells include plant cells and / or microbial cells;

[0026] Furthermore, the plant cells include but are not limited to Arabidopsis cells, rice cells, tobacco cells, Artemisia annua cells, and cotton cells.

[0027] Furthermore, the plant cell is a tobacco cell, an Arabidopsis cell or a rice cell.

[0028] Furthermore, the microbial cells include but are not limited to Streptomyces, Pseudomonas, Bacillus, yeast cells, and Escherichia coli.

[0029] Furthermore, the microbial cells are yeast cells or Escherichia coli.

[0030] In a fifth aspect, the present invention provides the use of the Astilbe truncatula eudesma-5,7-diene synthase AchTPS3 in the synthesis of eudesma-5,7-diene.

[0031] Furthermore, the application includes the following aspects:

[0032] (i) the amino acid sequence of eudesma-5,7-diene synthase AchTPS3 or a polypeptide of at least a portion of its sequence may retain biological activity or even have new biological activity after the removal or substitution of certain amino acids, or may improve the yield or optimize the protein kinetics or other properties to be obtained; or

[0033] (ii) involving the chemical synthesis of eudesma-5,7-diene; or

[0034] (iii) Involved in the biosynthesis of eudesma-5,7-diene.

[0035] In a sixth aspect, the present invention provides a method for producing eudesma-5,7-diene, comprising: converting farnesyl pyrophosphate into eudesma-5,7-diene in the presence of the Astilbe truncatum eudesma-5,7-diene synthase AchTPS3, or using the genetically engineered host cell to produce eudesma-5,7-diene (including using a recombinant yeast bacterium to produce eudesma-5,7-diene, or heterologously expressing the Astilbe truncatum eudesma-5,7-diene synthase AchTPS3 in a plant cell to produce eudesma-5,7-diene); the recombinant yeast bacterium or the plant cell contains a gene encoding a mevalonate pathway-related gene and the Astilbe truncatum eudesma-5,7-diene synthase AchTPS3.

[0036] Compared with the prior art, the present invention is beneficial in that:

[0037] The present invention provides an Astilbe truncatum eudesma-5,7-diene synthase AchTPS3. The encoding gene of the Astilbe truncatum eudesma-5,7-diene synthase AchTPS3 is integrated into a vector and introduced into a host cell to obtain a recombinant cell or a recombinant bacterium, thereby expressing the encoding gene in the recombinant cell or the recombinant bacterium, thereby realizing the biosynthesis of eudesma-5,7-diene. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the total ion chromatogram of the fermentation extract of the recombinant strain JCR27 / AchTPS3 detected by GC-MS;

[0039] Figure 2 This is the mass spectrum of the fermentation extract of the recombinant strain JCR27 / AchTPS3 detected by GC-MS;

[0040] Figure 3 The product structure diagram of the fermentation extract of JCR27 / AchTPS3 recombinant bacteria;

[0041] Figure 4 It is the product of AchTPS3 sesquiterpenoid eudesma-5,7-diene 1 H spectrum;

[0042] Figure 5 It is the product of AchTPS3 sesquiterpenoid eudesma-5,7-diene 13 C spectrum. DETAILED DESCRIPTION

[0043] The technical solutions of the present invention are described clearly and completely below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. All other embodiments obtained by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0044] In the present invention, the term "AchTPS3" can refer to sesquiterpene synthase, or eudesma-5,7-diene synthase, or eudesma-5,7-diene synthase gene, or a nucleotide sequence encoding eudesma-5,7-diene synthase, and the specific meaning can be determined in combination with the context.

[0045] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0046] The present invention provides an Astilbe eudesma-5,7-diene synthase AchTPS3, wherein the amino acid sequence of the Astilbe eudesma-5,7-diene synthase AchTPS3 includes the amino acid sequence in (i), (ii) or (iii):

[0047] (i) the amino acid sequence shown in SEQ ID NO: 1;

[0048] (ii) a functional homologous sequence having at least 75% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1;

[0049] (iii) an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1, and which has the same function as the protein composed of the amino acid sequence of SEQ ID NO: 1.

[0050] The gene encoding the Astilbe truncatula eudesma-5,7-diene synthase AchTPS3 comprises the nucleotide sequence in (i) or (ii):

[0051] (i) the nucleotide sequence shown in SEQ ID NO: 2;

[0052] (ii) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO: 2.

[0053] In the following specific examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials, reagents, etc. used are all available from commercial sources.

[0054] Example 1

[0055] Construction of recombinant yeast and method for producing eudesma-5,7-diene

[0056] (1) Cloning and assembly of eudesma-5,7-diene synthase AchTPS3: Based on the gene encoding eudesma-5,7-diene synthase AchTPS3 of Astilbe truncatum (nucleotide sequence shown in SEQ ID NO: 2), primers P1 and P2 were designed for assembling the gene encoding eudesma-5,7-diene synthase AchTPS3 of Astilbe truncatum with the yeast vector pZY900 (sequences of primers P1 and P2 are shown in Table 1). The yeast vector pZY900 contains the farnesyl pyrophosphate synthase gene ERG20 (for the specific construction process of the yeast vector pZY900, please refer to patent CN117187225A). The gene encoding the Astilbe eudesma-5,7-diene synthase AchTPS3 (nucleotide sequence shown in SEQ ID NO: 2) was used as a template, primers P1 and P2, and Phanta high-fidelity enzyme from Novozymes were used to amplify the Astilbe eudesma-5,7-diene synthase AchTPS3 gene fragment by PCR. The fragment was then ligated into the yeast expression vector pZY900 after BsaI digestion by homologous recombination. After sequencing confirmation, a yeast expression vector containing the Astilbe eudesma-5,7-diene synthase AchTPS3 gene was obtained.

[0057] Table 1: Sequences of primers P1 and P2

[0058]

[0059] (2) Construction of recombinant strain JCR27 / AchTPS3: The yeast expression vector containing the gene of eudesma-5,7-diene synthase AchTPS3 was introduced into the yeast strain JCR27 (the construction of yeast strain JCR27 can be found in the reference Siemon, et al. Semisynthesis of Plant-Derived Englerin A Enabled by MicrobeEngineering of Guaia-6, 10(14)-diene as Building Block) by the lithium acetate method (Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015. 2. 3. 14). Journal of the American Chemical , 2020,142(6):2760-2765) to obtain JCR27 / AchTPS3 recombinant bacteria.

[0060] (3) Strain fermentation and product identification: The JCR27 / AchTPS3 recombinant strain was inoculated into SC-URA liquid medium and cultured at 28°C and 220 rpm. Three days later, the initial OD 600 = 0.1 was transferred to 50 mL YPD liquid medium (20 g / L peptone, 10 g / L yeast powder, 10 g / L glucose, 10 g / L galactose) and cultured at 28 °C and 220 rpm in a shaking incubator for 72 h. The fermentation OD was measured. 600 , centrifuged at 5000 rpm for 20 min to collect the cells, and the final OD 600 Resuspend the cells in distilled water at 200 μL. Take 500 μL of the resuspended bacterial solution, add 1 PCR tube of glass beads and 500 μL of ethyl acetate, and grind it in a tissue grinder 15 times, each grinding intensity is 60 W for 30 seconds. Centrifuge at 12000 rpm for 5 minutes, then take the ethyl acetate layer for sample preparation and use GC-MS to detect the product. The total ion chromatogram of the extract is shown in the figure below. Figure 1 The mass spectrum of the product peak is shown in Figure 2 As shown, the product structure is as Figure 3 As shown in FIG, after comparison of nuclear magnetic resonance and mass spectrometry of reported compounds, eudesma-5,7-diene (a) was found at a retention time of 9.735 min in the extract of the fermentation broth of the recombinant bacteria JCR27 / AchTPS3.

[0061] Example 2

[0062] Preparation of eudesma-5,7-diene, a terpene product of AchTPS

[0063] A small distillation column was used to separate and purify the sesquiterpene product eudesma-5,7-diene from AchTPS3. The recombinant strain JCR27 / AchTPS3 was inoculated into SC-URA liquid medium and cultured in a shaking incubator at 28°C and 220 rpm. Three days later, the initial OD 600= 0.1 was transferred to 1 L YPD liquid medium (20 g / L peptone, 10 g / L yeast powder, 10 g / L glucose, 10 g / L galactose), and 200 mL of isopropyl myristate (IPM) was added to cover it. It was fermented at 28 ° C and 220 rpm for 72 h. After centrifugation, the IPM layer was taken and the product was separated using a small distillation tower. The bottom of the distillation tower was slowly heated to 150-180 ° C. When the top temperature reached 70-80 ° C, the product was taken out. After the temperature was raised to 120 ° C, the product fraction was finished. The product fraction was passed through a 500-800 mesh silica gel column with petroleum ether as the mobile phase, 30-50 mL as a sample, spot plate confirmation, spin dryness, and GC-MS detection to confirm the product. 1 H spectrum as Figure 4 As shown ( 1 H NMR (600 MHz, CDCl3) δ 5.63 (s, 1H),5.32 (ddt, J = 6.3, 2.5, 1.4 Hz, 1H), 2.61 – 2.55 (m, 1H), 2.24 (dddd, J = 13.7,6.8, 5.3, 1.6 Hz, 1H), 2.04 – 2.00 (m, 1H), 1.87 (dd, J = 16.3, 6.5 Hz, 1H),1.79 – 1.70 (m, 1H), 1.65 – 1.60 (m, 1H), 1.56 – 1.54 (m, 1H), 1.54 – 1.50(m, 1H), 1.50 – 1.45 (m, 1H), 1.22 (td, J = 13.3, 3.9 Hz, 1H), 1.16 (d, J =7.5 Hz, 3H), 1.01 (d, J = 1.9 Hz, 3H), 1.00 (d, J = 1.8 Hz, 3H), 0.97 (s, 3H)); the product 13 C spectrum Figure 5 As shown ( 13 C NMR (151 MHz, CDCl3) δ 150.32, 142.43, 121.89, 115.15,42.41, 42.17, 36.43, 34.85, 33.70, 32.32, 24.86, 23.92, 22.18, 21.71, 18.24). After comparison with the reported literature ( Flavour Fragr. J., 15: 395-412; Phytochemistry 60, 4, 2002, 333-338), and the product was identified as eudesma-5,7-diene.

[0064] In summary, the present invention provides a eudesma-5,7-diene synthase AchTPS3 of Astilbe truncatum. The encoding gene of the eudesma-5,7-diene synthase AchTPS3 is integrated into a vector and introduced into a host cell to obtain a recombinant cell or a recombinant bacterium, so that the encoding gene is expressed in the recombinant cell or the recombinant bacterium, thereby realizing the biosynthesis of eudesma-5,7-diene, which has great economic and ecological value.

[0065] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. A eudesma-5,7-diene synthase, characterized in that The amino acid sequence of the Astilbe eudesma-5,7-diene synthase includes the amino acid sequence in (i), (ii) or (iii): (i) the amino acid sequence shown in SEQ ID NO: 1; (ii) a functional homologous sequence having at least 75% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1; (iii) an amino acid sequence in which one or more amino acids are added, deleted, or substituted in the amino acid sequence of SEQ ID NO: 1, and which has the same function as the protein composed of the amino acid sequence of SEQ ID NO:

1.

2. The eudesma-5,7-diene synthase according to claim 1, characterized in that The Astilbe eudesma-5,7-diene synthase includes a fusion protein obtained by connecting a protein tag to the N-terminus or / and the C-terminus of the amino acid sequence shown in SEQ ID NO:

1.

3. The gene encoding the eudesma-5,7-diene synthase of claim 1, characterized in that Comprising the nucleotide sequence of (i) or (ii): (i) the nucleotide sequence shown in SEQ ID NO: 2; (ii) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO:

2.

4. The gene encoding eudesma-5,7-diene synthase according to claim 3, characterized in that The complementary sequence, degenerate sequence or homologous sequence of the nucleotide sequence shown in SEQ ID NO: 2 has more than 80% homology with the nucleotide sequence shown in SEQ ID NO:

2.

5. The gene encoding eudesma-5,7-diene synthase according to claim 3, characterized in that The gene encoding the eudesma-5,7-diene synthase also includes variants of the nucleotide sequence conservatively substituted as shown in SEQ ID NO: 2 and their complementary sequences.

6. The gene encoding eudesma-5,7-diene synthase of claim 3, characterized in that The gene encoding the Astilbe eudesma-5,7-diene synthase also includes a nucleotide sequence that is advantageous for expression in a host cell and is obtained by designing and artificially codon-optimizing the amino acid sequence of the Astilbe eudesma-5,7-diene synthase.

7. A carrier, characterized in that The invention relates to a gene encoding the eudesma-5,7-diene synthase of any one of claims 3 to 6.

8. A genetically engineered host cell, characterized in that Comprising the vector according to claim 7.

9. Use of the Astilbe truncatula eudesma-5,7-diene synthase according to claim 1 or 2 in synthesizing eudesma-5,7-diene.

10. A method for producing eudesma-5,7-diene, characterized in that: include: Converting farnesyl pyrophosphate into eudesma-5,7-diene in the presence of the Astilbe nutans eudesma-5,7-diene synthase according to claim 1 or 2, or producing eudesma-5,7-diene using the genetically engineered host cell according to claim 8.

Images