Germacradienol biosynthetase OsGMS and application thereof in prevention and control of pathogenic fungi and weeds

Abstract

The invention provides germacradienol biosynthase OsGMS and application of the germacradienol biosynthase OsGMS in prevention and control of pathogenic fungi and weeds, and belongs to the technical field of biosynthesis. The amino acid sequence of the rice germacradienol synthase OsGMS 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: 1 is a homologous sequence with at least 75% sequence similarity with the amino acid sequence as shown in SEQ ID NO: 2. 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 encoding gene of the rice germacradienol synthase OsGMS is integrated into a carrier and introduced into a host cell, so that the encoding gene is expressed in the host cell, biosynthesis of germacradienol is realized, the germacradienol has the effect of inhibiting the growth of plant pathogenic fungi and weeds, and therefore, the germacradienol synthase OsGMS can be used for preparing novel biological pesticides capable of inhibiting the fungi and the weeds, and can be used for preparing biological pesticides for inhibiting the fungi and the weeds. And a new technical path is provided for developing environment-friendly agricultural preparations.

Description

Technical Field

[0001] The present invention relates to the technical field of biosynthesis, and in particular to a germacradienol biosynthetic enzyme OsGMS and application thereof in the prevention and control of pathogenic fungi and weeds. Background Art

[0002] Germacradienol is a germarane-type sesquiterpenoid compound first isolated from Streptomyces citriodora in 1995. Streptomyces citreus ) was isolated from Streptomyces coelicolor (D. Ganßer, FC Pollak, RG Berger Asesquiterpene alcohol from Streptomyces citreus CBS 109.60 J. Nat. Prod., 58(11) (1995), pp. 1790-1793, 10.1021 / np50125a027). Streptomyces coelicolor ) was found in Streptomyces coelicolor. The N-terminal domain of the SCO6073 gene can produce Germacradienol (Jiang J, He X, Cane DE. Geosmin biosynthesis. Streptomyces coelicolor germacradienol / germacrene D synthase converts farnesyl diphosphate to geosmin. J Am Chem Soc . 2006;128(25):8128-8129). Similar to this, geosmin synthases have been identified or deduced from multiple genome sequences of Streptomyces, Frankia, Saccharopolyspora, and Myxobacterium, whose N-terminal domains can produce germacradienol (Giglio S, Jiang J, Saint CP, Cane DE, MonisPT. Isolation and characterization of the gene associated with geosminproduction in cyanobacteria. Environ Sci Technol. 2008 Nov 1;42(21):8027-32). Germacradienol is generally produced by microorganisms such as actinomycetes and Streptomyces in the soil. The present invention is the first time that it has been discovered in a rice terpene synthase.

[0003] Germacradienol has a wide range of antibacterial and anti-cytotoxic activities. Studies have shown that germacradienol has significant inhibitory activity against a variety of plant pathogens (such as Bacillus subtilis) and microorganisms, with a minimum inhibitory concentration (MIC) ranging from 12.5-25.0 μg / mL (Guiding Li, et al. The discovery of germacradienolsynthase: Construction of genetically-engineered strain, glycosylatedmodification, bioactive evaluation of germacradienol, Bioorg Chem. 2022 Jul;124:105819). In addition, germacradienol and its derivatives (such as 9β-hydroxyl germacradienol and 2-oxygermacradienol) showed strong proliferation inhibitory effects on the neuroblastoma cell line (SH-SY5Y), with a half inhibitory concentration (IC 50 ) reaches nM level (patent CN110585179A).

[0004] There are three main methods for synthesizing germacradienol: fungal culture extraction, chemical synthesis, and biosynthesis. Fungal culture extraction suffers from low efficiency and high costs, while chemical synthesis is a long route, produces numerous byproducts, and pollutes the environment. Therefore, to achieve the biosynthesis of germacradienol, it is crucial to identify sesquiterpene synthase genes that efficiently synthesize germacradienol. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention provides a germacradienol biosynthetic enzyme OsGMS and its related biomaterials for use in the biosynthesis of germacradienol or in the control of pathogenic fungi / weeds.

[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 rice germacradienol synthase OsGMS, wherein the amino acid sequence of the rice germacradienol synthase OsGMS comprises the amino acid sequence of (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 rice germacradienol synthase OsGMS comprises 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 gene for germacradienol synthase (OsGMS) from the genome of rice (Oryza sativa L.) and successfully obtained the encoded protein, which was named rice germacradienol synthase (OsGMS). By expressing the protein in recombinant Saccharomyces cerevisiae, it was found that OsGMS catalyzes the substrate farnesyl pyrophosphate (FPP) to produce the sesquiterpene product germacradienol. The rice germacradienol synthase (OsGMS) provided by the present invention provides a genetic resource for the production of germacradienol.

[0013] The biological materials related to rice germacradienol synthase OsGMS include a gene encoding rice germacradienol synthase OsGMS, a vector containing a gene encoding rice germacradienol synthase OsGMS, a genetically engineered host cell expressing rice germacradienol synthase OsGMS, and the like.

[0014] In a second aspect, the present invention provides a gene encoding the rice germacradienol synthase OsGMS, 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 rice germacradienol synthase OsGMS also includes conservatively substituted variants of the nucleotide sequence shown in SEQ ID NO: 2 (eg, substitutions of degenerate codons) and their complementary sequences.

[0019] Furthermore, the gene encoding the rice germacradienol synthase OsGMS also includes a nucleotide sequence that is designed and artificially codon-optimized using the amino acid sequence of the rice germacradienol synthase OsGMS and is favorable for expression in host cells.

[0020] In a third aspect, the present invention provides a vector comprising the gene encoding the rice germacradienol synthase OsGMS.

[0021] A vector for expressing rice germacradienol synthase OsGMS comprises a gene encoding the rice germacradienol synthase OsGMS. 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, or pESC-His.

[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 rice germacradienol synthase OsGMS comprises the amino acid sequence of the rice germacradienol synthase OsGMS, or comprises a gene encoding the rice germacradienol synthase OsGMS, or is introduced with a vector expressing the rice germacradienol synthase OsGMS.

[0024] Furthermore, the genetically engineered host cell comprises mevalonate pathway-related genes and can produce farnesyl pyrophosphate, which is converted into germacradienol under the action of the rice germacradienol synthase OsGMS.

[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 rice germacradienol synthase OsGMS in synthesizing germacradienol or in controlling pathogenic fungi / weeds.

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

[0032] (i) The amino acid sequence of rice germacradienol synthase OsGMS or a polypeptide comprising at least a portion of its sequence may retain biological activity or even have new biological activities after the removal or substitution of certain amino acids, or may improve the yield or optimize the protein's kinetics or other desired properties; or

[0033] (ii) involving the chemical synthesis of germacradienol; or

[0034] (iii) involved in the biosynthesis of germacradienol; or

[0035] (iiii) the fungal inhibitory activity of germacradienol, the use of germacradienol in the inhibition of fungi or in the preparation of antifungal drugs; or

[0036] (iiiii) The present invention relates to the inhibition of weed growth by germacradienol, and the use of germacradienol in inhibiting weed growth or in the preparation of a biopesticide for inhibiting weed growth.

[0037] Furthermore, the fungus includes but is not limited to Rhizoctonia solani YWK196; the weeds include but are not limited to Arabidopsis thaliana, a plant of the cruciferous family.

[0038] In a sixth aspect, the present invention provides a method for producing germacradienol, comprising: converting farnesyl pyrophosphate into germacradienol in the presence of the rice germacradienol synthase OsGMS, or using the genetically engineered host cell to produce germacradienol (including using a recombinant yeast to produce germacradienol, or heterologously expressing the rice germacradienol synthase OsGMS in a plant cell to produce germacradienol); the recombinant yeast or the plant cell contains a gene encoding a mevalonate pathway and the rice germacradienol synthase OsGMS.

[0039] Compared with the prior art, the present invention is beneficial in that:

[0040] The present invention provides a rice germacradienol synthase (OsGMS). The gene encoding the rice germacradienol synthase (OsGMS) is integrated into a vector and introduced into host cells to obtain recombinant cells or recombinant bacteria, whereupon the gene is expressed in the recombinant cells or recombinant bacteria, thereby achieving the biosynthesis of germacradienol. Biological activity experiments have shown that germacradienol inhibits the growth of plant pathogenic fungi and weeds. Therefore, the germacradienol synthase (OsGMS) of the present invention can be used to prepare novel biopesticides with fungal and weed inhibitory properties, providing a new technological path for the development of environmentally friendly agricultural formulations. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The total ion chromatogram and product structure of the fermentation extract of the recombinant strain JCR27 / OsGMS detected by GC-MS;

[0042] Figure 2 This is the mass spectrum of the fermentation extract of the recombinant strain JCR27 / OsGMS detected by GC-MS;

[0043] Figure 3 The main product of OsGMS 1 H spectrum;

[0044] Figure 4 The main product of OsGMS 13 C spectrum;

[0045] Figure 5 This is a planar display and diameter statistical graph of Rhizoctonia solani YWK196 growing on a medium containing germacradienol;

[0046] Figure 6 This is a graphic representation of Arabidopsis thaliana growing on a culture medium containing germacradienol and a statistical graph of root length. DETAILED DESCRIPTION

[0047] 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.

[0048] In the present invention, the term "OsGMS" may refer to sesquiterpene synthase, or germacradienol synthase, or germacradienol synthase gene, or a nucleotide sequence encoding germacradienol synthase, and the specific meaning can be determined in combination with the context.

[0049] 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.

[0050] The present invention provides a rice germacradienol synthase OsGMS, wherein the amino acid sequence of the rice germacradienol synthase OsGMS includes the amino acid sequence in (i), (ii) or (iii):

[0051] (i) the amino acid sequence shown in SEQ ID NO: 1;

[0052] (ii) a functional homologous sequence having at least 75% sequence similarity to the amino acid sequence shown in SEQ ID NO: 1;

[0053] (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.

[0054] The gene encoding rice germacradienol synthase OsGMS comprises the nucleotide sequence in (i) or (ii):

[0055] (i) the nucleotide sequence shown in SEQ ID NO: 2;

[0056] (ii) a complementary sequence, a degenerate sequence or a homologous sequence of the nucleotide sequence shown in SEQ ID NO: 2.

[0057] 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.

[0058] Example 1

[0059] Construction of recombinant yeast and method for producing germacradienol

[0060] (1) Cloning and assembly of rice germacradienol synthase OsGMS: Based on the coding gene of rice germacradienol synthase OsGMS (nucleotide sequence shown in SEQ ID NO: 2), primers P1 and P2 were designed for assembling the coding gene of rice germacradienol synthase OsGMS with 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 yeast vector pZY900, please refer to patent CN117187225A). Using the rice germacradienol synthase OsGMS gene (nucleotide sequence shown in SEQ ID NO: 2) as a template, primers P1 and P2, and Novozymes' Phanta high-fidelity enzyme were used to amplify the rice germacradienol synthase OsGMS 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 rice germacradienol synthase OsGMS gene was obtained.

[0061] Table 1: Sequences of primers P1 and P2

[0062]

[0063] (2) Construction of JCR27 / OsGMS recombinant bacteria: The yeast expression vector containing the rice germacradienol synthase OsGMS gene 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 Microbe Engineering 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 / OsGMS recombinant bacteria.

[0064] (3) Strain fermentation and product identification: The JCR27 / OsGMS 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 for 72 h. The fermentation broth 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 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 peak was subjected to nuclear magnetic resonance analysis, and its nuclear magnetic resonance hydrogen spectrum 1 H NMR Figure 3 As shown ( 1 H NMR (600 MHz, Chloroform-d) δ 5.67(d, J = 3.5 Hz, 1H), 5.64 (d, J = 3.5 Hz, 1H), 5.06 –4.93 (m, 5H), 3.48 (s, 1H), 2.49 – 2.36 (m, 5H), 2.33 – 2.16 (m, 8H), 1.90 (dddt, J = 12.9, 4.9,3.2, 1.7Hz, 3H), 1.79 – 1.40 (m, 22H), 1.37 – 0.85 (m, 34H)), C NMR 13 C NMR Figure 4 As shown ( 13C NMR (151 MHz, CDCl3) δ 143.24, 131.27, 130.74, 123.91, 71.92, 59.07,41.46, 34.02, 32.95, 26.99, 26.46, 23.89, 22.20, 16.87, 14.88, which are consistent with the data of germacradienol reported in the literature (Xu H, Rinkel J, Chen X, Köllner TG, Chen F, DickschatJS. Mechanistic divergence between (4S,7R)-germacra-(1(10)E,5E)-dien-11-ol synthses from Dictyostelium purpureum and Streptomyces coelicolor. OrgBiomol Chem. 2021;19(2):370-374), the product was identified as germacradienol with a retention time of 12.475 min and a CAS number of 172927-59-2.

[0065] Example 2

[0066] Antibacterial activity assay of germacradienol, the main product of OsGMS

[0067] In the clean bench, a sterilized toothpick was used to pick up the hyphae of R. solani AG1-IA strainYWK196 and spot it on the center of the surface of PDA culture medium (potato dextrose agar, 20% potato, 2% glucose, 2% agar, w / v), and cultured upside down in a 28°C incubator for 2-3 days. A bacterial disc was punched out at the edge of the colony with a 6 mm punch and inoculated in the center of the PDA culture medium containing germacradienol. Germacradienol was prepared into a 100,000 ppm stock solution with DMSO and diluted to 250 ppm and 500 ppm with culture medium, respectively. The low concentration solution was supplemented with DMSO according to the amount of DMSO added to the high concentration solution, and the control group was added with the corresponding amount of DMSO (CK). After static culture in a 28°C incubator for 18-24 hours, the growth diameter of the hyphae was measured by cross hair. The results are shown in the figure. Figure 5 As shown in the results, germacradienol at a concentration of 500 ppm almost completely inhibited the growth of Rhizoctonia solani.

[0068] Example 3

[0069] Detection of the Arabidopsis thaliana growth inhibitory activity of germacradienol, the main product of OsGMS

[0070] Prepare a 100,000 ppm stock solution of germacradienol with DMSO for later use.

[0071] Soak wild-type Arabidopsis seeds in 1 mL of sterile distilled water for 1 minute, sterilize with 80% ethanol for 2 minutes, and then rinse with sterile distilled water 5-6 times to remove the ethanol from the surface of the seeds. Sow the sterilized Arabidopsis seeds evenly on 1 / 2 MS culture medium. After sowing, vernalize in a refrigerator at 4°C for 2 days and then incubate in a light-controlled culture room (16 h light, 8 h dark, 22°C) for 5-6 days.

[0072] Heat and melt 1 / 2 MS culture medium, take 4 mL of culture medium into a shaking tube, add 4 μL of germacradienol stock solution to make the final concentration of germacradienol in the culture medium 500 ppm, add 2 μL of stock solution to make the final concentration 250 ppm, add the corresponding amount of DMSO (CK) to the control group, and divide 4 mL of culture medium into two six-well plates. After the culture medium cools and solidifies, use a sterilized toothpick to transplant the seedlings onto the surface of 1 / 2 MS culture medium containing germacradienol. Transplant 10 seedlings of the same growth in each group and culture them vertically in a light culture room. After culturing for 3-4 days, take pictures to record the growth of Arabidopsis, measure the root length and leaf spacing with a ruler, and calculate the statistics. The results are as follows Figure 6 As shown in Figure 3, 250 ppm of germacradienol had a significant inhibitory effect on root growth and leaf development in Arabidopsis.

[0073] In summary, the present invention provides a rice germacradienol synthase (OsGMS) that produces germacradienol. The gene encoding the rice germacradienol synthase (OsGMS) is integrated into a vector and introduced into host cells to obtain recombinant cells or recombinant bacteria, whereupon the gene is expressed in the recombinant cells or recombinant bacteria, thereby achieving the biosynthesis of germacradienol. Biological activity experiments have shown that germacradienol inhibits the growth of plant pathogenic fungi and weeds. Therefore, the germacradienol synthase (OsGMS) of the present invention can be used to prepare novel biopesticides that inhibit fungi and weeds, providing a new technological path for the development of environmentally friendly agricultural formulations.

[0074] 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 rice germacradienol synthase, characterized in that The amino acid sequence of the rice germacradienol 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 rice germacradienol synthase according to claim 1, characterized in that The rice germacradienol synthase comprises 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 rice germacradienol synthase encoding gene according to 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 rice germacradienol synthase encoding gene 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 rice germacradienol synthase according to claim 3, characterized in that The rice germacradienol synthase encoding gene also includes conservatively substituted variants of the nucleotide sequence shown in SEQ ID NO: 2 and their complementary sequences.

6. The gene encoding rice germacradienol synthase according to claim 3, characterized in that The rice germacradienol synthase encoding gene 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 rice germacradienol synthase.

7. A carrier, characterized in that A gene encoding the rice germacradienol synthase according to 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 rice germacradienol synthase according to claim 1 or 2 in synthesizing germacradienol or in controlling pathogenic fungi / weeds.

10. A method for producing germacradienol, characterized in that: include: Farnesyl pyrophosphate is converted into germacradienol in the presence of the rice germacradienol synthase according to claim 1 or 2, or germacradienol is produced using the genetically engineered host cell according to claim 8.

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