A gamma-cadinene synthase glsts03, encoding gene and its application in synthesis of gamma-cadinene

Abstract

The application provides a gamma-cadinene synthase GLSTS03, a coding gene and application thereof in synthesis of gamma-cadinene. The application uses Aspergillus oryzae as a host bacterium, realizes high-efficiency biosynthesis of gamma-cadinene by heterologously expressing a gamma-cadinene synthase coding gene GLSTS03 from Ganoderma lucidum, and provides a green and economical alternative scheme for industrialized production of gamma-cadinene.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and relates to a sesquiterpene synthase and its application, more specifically to a γ-juniperene synthase GLSTS03, a gene encoding the γ-juniperene synthase, a recombinant expression vector containing the gene, a recombinant host strain containing the vector, and its application in the synthesis of γ-juniperene. Background Technology

[0002] γ-Juniperene is a natural product with a sesquiterpene chemical structure, belonging to one of the isomers of cadinene, and is widely found in the metabolites of plants and fungi. It is commonly found in the essential oils of plants from the Coniferaceae, Asteraceae, and Lamiaceae families, such as juniper, cedar, and wormwood, and is one of the important active ingredients in plant essential oils. Studies have shown that γ-juniperene has inhibitory effects on various pathogenic bacteria and also possesses certain anti-inflammatory effects, making it a potential candidate for natural antibacterial agents. γ-Juniperene has insect repellent or toxic effects, and its potential for controlling agricultural pests has been gradually explored, with the prospect of developing it into an environmentally friendly biopesticide. Due to its unique aroma and bioactivity, γ-juniperene can be used in the formulation of fragrances, cosmetics, and other daily chemical products. It also has potential activities in antitumor and antioxidant applications. As a representative product of the sesquiterpene synthesis pathway, research on the synthetic mechanism of γ-juniperene helps to reveal the regulatory rules of terpene compound biosynthesis and promotes the development of microbial synthetic biology.

[0003] Currently, the methods for obtaining γ-juniperene are mainly divided into two categories: natural extraction and biosynthesis. 1. Plant-derived extraction: Using plant roots, stems, and leaves as raw materials, crude extracts are first obtained through ultrasonic extraction or reflux extraction using organic solvents such as hexane and ethyl acetate. These crude extracts are then separated and purified using techniques such as silica gel column chromatography, high-performance liquid chromatography (HPLC), and gas chromatography (GC) to finally obtain high-purity γ-juniperene. This method is limited by factors such as raw material yield and season, resulting in higher extraction costs. 2. Microbial-derived extraction: Extracted from the fermentation products of naturally occurring γ-juniperene-producing strains such as Aspergillus and Penicillium. The fermentation broth or mycelium is first extracted with organic solvents, followed by purification using chromatography and recrystallization. This method offers relatively controllable raw material acquisition, but the yield of products from natural strains is usually low.

[0004] Therefore, at this stage, it is necessary to develop a new preparation technology that is both environmentally friendly and economical. This technology should effectively overcome the inherent drawbacks of traditional plant extraction methods, such as solvent pollution, ecological damage, and low production efficiency. At the same time, it should achieve efficient synthesis, stable mass production, and precise quality control of γ-juniperene, thereby breaking through the core technological bottlenecks in its industrialization process, promoting its green, economical, and sustainable industrial production, and fully exploring and releasing the application potential of this compound in multiple high-value fields. Summary of the Invention

[0005] To address the limitations of existing γ-juniperene sources, the low efficiency of plant extraction methods, and severe environmental pollution, this invention provides a γ-juniperene synthase GLSTS03, its encoding gene, and its application in the synthesis of γ-juniperene. This invention screened and cloned the γ-juniperene synthase encoding gene GLSTS03 from Ganoderma lucidum, and achieved efficient biosynthesis of γ-juniperene using an Aspergillus oryzae heterologous expression system.

[0006] The technical solution adopted in this invention is: a γ-juniperene synthase GLSTS03, having the amino acid sequence shown in SEQ ID NO.2. After heterologous expression of γ-juniperene synthase GLSTS03 in Aspergillus oryzae, γ-juniperene products were detected, confirming that this gene can be correctly translated into a functional protein with catalytic activity.

[0007] SEQ ID NO.2: MSVEQLRPFPTHFRLKDLAAISGRVFEFKLNPHEKEAAKATYAWFDGRNVYHGLKKKRFLSHRFDSYAGMSFPDADVSHLETCIAFFLWAFSFDDLEGALQSKPEAHQVGVDISMEVLRNPTAPPPNFPYAAMLHDRRFRNIYSPIVRFFRAVESWMNNSQVEQARNRATDEIPSVEEFIILRRRTIGGPIVEAMVEYSLDLQIPEH VWDHPILQEMSKAVIDIMTWPNDLCSFNKEQADGDFQNLVFCIMIERDCDLQTAVDVLTEMLSQRVVDYERYRAQLPSFGPEVDAELARYNKAMEQYTQGTVVW YYHSPRYFRGQEVTGIPEIVVPVYERTTPAPEEPTAPASKSARSAPANLAPSSSKEVGLLVNAPTQHNARLSLLLISLVACLLCASFISFPSFPLNPRLVLSI.

[0008] Preferably, the recombinant γ-juniperene synthase GLSTS03 is encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.1 and catalyzes the production of γ-juniperene from farnesyl pyrophosphate.

[0009] The present invention also provides a gene encoding the γ-juniperene synthase GLSTS03 as described in claim 1.

[0010] Preferably, the encoding gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0011] SEQ ID NO.1:

[0012] The present invention also provides a recombinant expression vector containing the aforementioned coding gene.

[0013] Preferably, the recombinant expression vector is pD8-GLSTS03, which is constructed by inserting the gene fragment shown in SEQ ID NO:1 into the KpnI restriction site of the pD8 plasmid linearized by KpnI digestion.

[0014] The present invention also provides a recombinant expression strain of the recombinant expression vector.

[0015] Preferably, the recombinant expression strain is Aspergillus oryzae AO-GLSTS03, which is constructed by introducing the recombinant expression vector pD8-GLSTS03 into Aspergillus oryzae protoplasts via PEG-mediated protoplast transformation.

[0016] The present invention also provides a method for synthesizing γ-juniperene, wherein the recombinant expression strain is used for fermentation culture to synthesize γ-juniperene.

[0017] Preferably, the method for constructing the recombinant expression strain includes:

[0018] S1. Using Ganoderma lucidum genomic DNA as a template, the gene fragment encoding γ-juniperene synthase GLSTS03 was amplified by PCR; the fragment was ligated with pD8 plasmid at a molar ratio of 1:(1-6) to construct the recombinant expression vector pD8-GLSTS03.

[0019] S2. After culturing Aspergillus oryzae spores in a spore medium containing dextrin and uracil, the mycelium was collected, and the mycelium was enzymatically digested with cell wall-breaking enzyme solution to obtain protoplasts;

[0020] S3. The recombinant expression plasmid pD8-GLSTS03 obtained in step S1 was introduced into the protoplasts prepared in step S2 through PEG-mediated protoplast transformation. Positive transformants were screened, and the recombinant expression strain AO-GLSTS03 was obtained after subculture.

[0021] Preferably, in step S1, the pD8 plasmid is a pD8 plasmid fragment linearized by KpnI restriction enzyme digestion, and the molar ratio of the γ-juniperene synthase encoding gene GLSTS03 fragment to the linearized plasmid fragment is controlled at 1:(1-6), preferably 1:6, and the target gene GLSTS03 fragment is directionally inserted into the KpnI restriction enzyme site of the pD8 plasmid.

[0022] Preferably, in step S1, the amplification reaction uses a combination of forward primer pD8-GLSTS03-F and reverse primer pD8-GLSTS03-R, wherein the forward primer sequence is tccGAATTCGAGCTCGGTACCATGAGCGTCGAGCAGCTTCGCCCA and the reverse primer sequence is tactacaGATCCCCGGGTACCTCAGATCGACAGAACGAGGCGGG.

[0023] Preferably, in step S1, the Ganoderma lucidum is a new red Ganoderma variety, Xianzhi No. 2, bred through space mutagenesis, with the approval number: Zhe (Non) Shenjun 2014003.

[0024] Preferably, in step S1, the pD8 plasmid used is a linearized fragment digested with KpnI enzyme. Regarding the enzyme digestion system, the mass ratio of pD8 plasmid to KpnI enzyme volume is 1:(1-2), preferably 1:1; in some embodiments, this ratio is specifically 1 μg:(1-2) μL.

[0025] Preferably, in step S2, the spore culture medium comprises: NaNO3, KCl, KH2PO4, MgSO4·7H2O, FeSO4·7H2O, dextrin, (NH2)SO4, methionine, uridine, and uracil. Further, the spore culture medium comprises: 25-35 g / L NaNO3, 15-25 g / L KCl, 0.5-1.5 g / L KH2PO4, 0.1-1 g / L MgSO4·7H2O, 0.01-0.05 g / L FeSO4·7H2O, 15-25 g / L Dextrin, 5-15 g / L (NH2)SO4, 1-3 g / L Methionine, 4-6 g / L Uracil, and 1-3 g / L Uridine. The pH of the spore culture medium is 5-6, preferably 5.5.

[0026] Preferably, in step S2, the cell wall breaking enzyme solution is prepared by dissolving glycosylase in SoLI, wherein SoLI is prepared by mixing 0.8M NaCl solution and 10mM NaHPO4 solution.

[0027] Preferably, step S3 includes: washing the protoplasts with NaCl solution, adding SoLII and SoLIII solutions, then adding the recombinant expression vector pD8-GLSTS03 obtained in step S1, mixing well, and incubating on ice. Then, SoLIII solution is added to the mixture, and the mixture is incubated at room temperature for 15-20 min. Next, SoLII solution is added, mixed well, and centrifuged for 8-10 min. The supernatant is removed, and SoLII solution is added again, mixed well, and transferred to CD selection medium plates. Covering medium is then added, and the plates are incubated upside down for 2-3 days. After single colonies containing pD8-GLSTS03 transformants have grown, colony PCR is used to verify whether the target gene has been successfully transferred. After 2-3 subcultures of the obtained Aspergillus oryzae positive transformants, high-yielding strains carrying the γ-juniperene synthase gene are obtained.

[0028] Preferably, the SoLII comprises 5-30 mM CaCl2, 0.1-1 M NaCl, and 5-30 mM Tris. The pH of the SoLII is 7-8, preferably 8.0.

[0029] Preferably, the SoLIII comprises 30-50% PEG-4000, 30-60mM CaCl2 solution, and 30-60mM Tris.

[0030] Preferably, the CD screening medium comprises: 10-50 g / L NaNO3, 10-40 g / L KCl, 0.1-1.5 g / L KH2PO4, 0.1-1 g / L MgSO4·7H2O, 0.01-0.04 g / L FeSO4·7H2O, 10-30 g / L glucose, 0.5-2 g / L (NH2)SO4, 215-220 g / L sorbitol, 1-3 g / L methionine, and 10-20 g / L agar powder. The pH of the CD screening medium is 5-7, preferably 5.5.

[0031] Preferably, the covering culture medium comprises: 1-2M sorbitol and 0.2-1% agar powder.

[0032] Preferably, the fermentation culture method includes: inoculating the recombinant expression strain into a rice culture medium and culturing it at 28-30℃ for 7-10 days to produce γ-juniperene. Specifically, the cultured bacterial cells are extracted, and the extract is concentrated by rotary evaporation to obtain a fermentation broth extract, which is then purified by silica gel separation to obtain γ-juniperene.

[0033] Preferably, the rice culture medium comprises 0.1-2 g / mL rice.

[0034] The beneficial effects of this invention are:

[0035] 1. This invention uses Aspergillus oryzae as the host strain, which can efficiently synthesize and supply sufficient farnesyl pyrophosphate (FPP), laying the foundation for the biosynthesis of γ-juniperene.

[0036] 2. The expression plasmid pD8-GLSTS03 used contains the pAmyB amylase promoter. When rice is used as the culture medium, this promoter can strongly drive the efficient transcriptional expression of the γ-juniperene synthase gene GLSTS03.

[0037] 3. The high-yield strain AO-GLSTS03 constructed uses rice as the fermentation medium, which has the advantages of inexpensive raw materials, simple operation, short culture cycle, and easy product separation and purification.

[0038] 4. This invention is suitable for large-scale industrial fermentation. Using the culture medium and conditions of this invention, and through separation, purification, and multi-dimensional spectroscopic identification, the heterologous expression strain of *Aspergillus oryzae* can efficiently synthesize and accumulate γ-juniperene, solving the problems of high extraction cost and difficult separation and purification of plant-derived γ-juniperene. This method can provide stable raw materials for the green synthesis of natural products and pharmaceutical intermediates containing the γ-juniperene skeleton, possessing both significant industrial application value and academic significance. Attached Figure Description

[0039] Figure 1 This is an electrophoresis image of the fragment of the γ-juniperene synthase encoding gene GLSTS03 obtained by amplifying Ganoderma lucidum cDNA as a template in an embodiment of the present invention.

[0040] Figure 2 This is an electrophoresis image of colony PCR verification of the obtained pD8-GLSTS03 single clone in an embodiment of the present invention.

[0041] Figure 3 This is an electrophoresis image of the obtained pD8-GLSTS03 plasmid verified by enzyme digestion in an embodiment of the present invention.

[0042] Figure 4 This is the GC detection spectrum of γ-juniperene in an embodiment of the present invention;

[0043] Figure 5 This is the MS detection spectrum of γ-juniperene in an embodiment of the present invention. Detailed Implementation

[0044] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0045] Example 1

[0046] A method for preparing γ-juniperene using Aspergillus oryzae is carried out according to the following steps.

[0047] Step S1: Construction of expression plasmid pD8-GLSTS03

[0048] Using the genomic DNA sequence of *Xianzhi No. 2* as a template, and pD8-GLSTS03-F as the forward primer and pD8-GLSTS03-R as the reverse primer, the GLSTS03 fragment encoding the γ-juniperene synthase gene was amplified. Figure 1 Using a pD8 plasmid fragment linearized by KpnI digestion as a vector, the GLSTS03 gene fragment encoding γ-juniperene synthase and the pD8 plasmid fragment linearized by KpnI digestion were mixed at a molar ratio of 1:6. Using a non-ligase-dependent single-fragment one-step cloning kit, the GLSTS03 gene fragment was inserted into the KpnI restriction site of the pD8 plasmid. The mass ratio of pD8 plasmid to KpnI was 1 μg: 1 μL. After ligation, the ligation system was transformed into *E. coli* DH5α, and positive clones were obtained after overnight culture. Finally, PCR verification was performed. Figure 2 ) and enzyme digestion verification ( Figure 3 After that, the expression plasmid pD8-GLSTS03 was obtained.

[0049] The sequence of the forward primer pD8-GLSTS03-F is tccGAATTCGAGCTCGGTACCATGAGCGTCGAGCAGCTTCGCCCA, and the sequence of the reverse primer pD8-GLSTS03-R is tactacaGATCCCCGGGTACCTCAGATCGACAGAACGAGGCGGG.

[0050] Step S2: Obtaining high-yield γ-juniperene strains

[0051] Aspergillus oryzae spores were evenly spread on potato dextrose agar (PDA) plates containing uridine-uracil (UU) and incubated at 30°C for 7 days to harvest a sufficient quantity of Aspergillus oryzae spores. The Aspergillus oryzae spore preservation solution was inoculated into 100 mL of spore culture medium and incubated at 30°C and 200 rpm for 2 days with constant temperature shaking to obtain uniformly growing Aspergillus oryzae mycelia.

[0052] Under aseptic conditions, mycelia were collected by filtration using a 5 mm pore size sterile filter. The mycelia were washed three times with sterile water to thoroughly remove any residual culture medium components from the cell surface, and then repeatedly squeezed in the filter to fully drain the water. The pretreated mycelia were transferred to 50 mL sterile centrifuge tubes, 20 mL of cell wall lysis buffer was added, and the tubes were incubated at 30°C with gentle shaking for 2.5 h. During the enzymatic digestion process, the dissociation efficiency and morphological integrity of the protoplasts were dynamically monitored under a microscope.

[0053] The enzymatic hydrolysate was filtered using sterile Miracloth filter cloth (Merck, Germany), and the filtrate was collected in 50 mL sterile centrifuge tubes. The collected filtrate was centrifuged at 800 rpm for 6 min at 4°C. The supernatant was then gently poured off and discarded in a laminar flow hood; the precipitate at the bottom of the tube was the Aspergillus oryzae protoplasts. The protoplast precipitate was washed twice with sterile 0.8 M NaCl solution, and a SoLII / SoLIII mixture was prepared at a volume ratio of 4:1. The protoplast concentration was precisely adjusted to 2 × 10⁻⁶ using microscopic examination and counting. 8 cells / mL. Transfer 200 mL of the prepared protoplast solution to a new 50 mL centrifuge tube, add 2 μg of expression plasmid pD8-GLSTS03, gently pipette to mix thoroughly, and incubate on ice for 20 min. Add 1 mL of SoLIII to the mixture and incubate at room temperature for 20 min. Then add 10 mL of SoLII, mix well, and centrifuge at 800 rpm for 10 min at 4 °C. Remove the supernatant, add 1 mL of SoLII, and gently pipette to mix. Transfer 200 mL of the mixture to a CD selection medium plate, then add 5-10 mL of 30-40 °C cover medium. After adding the cover medium, gently shake to mix thoroughly with the 200 mL mixture, and evenly cover the surface of the CD selection medium plate. After the cover medium solidifies, seal the plate and incubate upside down for 3 days.

[0054] After the Aspergillus oryzae transformants containing pD8-GLSTS03 grew hyphae on the surface of CD selection medium plates, they continued to grow and form colonies. A suitable amount of hyphae was picked and added to 30 μL of TE SoL (1M Tris, 0.5M EDTA, pH 8.0), vortexed, and 1 μL was used as a template. PCR verification was performed using pD8-GLSTS03-F and pD8-GLSTS03-R primers. Positive transformants were subcultured three times on the same CD selection medium plates to obtain a high-yielding strain (AO-GLSTS03) carrying a genetically stable γ-juniperene synthase gene.

[0055] The spore culture medium was prepared according to the following steps: 3.0g NaNO3, 2.0g KCl, 0.1g KH2PO4, 0.05g MgSO4. 4▪ 7H2O, 0.002g FeSO4 4▪ 7H2O, 2g dextrin, 900mg ammonium sulfate, 150mg methionine, 500mg uridine and 200mg uracil, pH 5.5, add water to a final volume of 100mL.

[0056] The cell wall-breaking enzyme solution was prepared by dissolving 1.0% takara shuzo enzyme in SoLI; the SoLIII consisted of 0.8 M sodium chloride solution and 10 mM sodium dihydrogen phosphate solution.

[0057] The SoLII is composed of 1.2M sorbitol, 50mM calcium chloride solution, 50mM sodium chloride solution and 10mM glycerol (Tris), and the pH is adjusted to 7.5.

[0058] The SoLIII is composed of 40% polyethylene glycol-4000 (PEG-4000), 50 mM calcium chloride solution and 50 mM aminobutanetriol (Tris).

[0059] The CD screening medium was prepared according to the following steps: each 200 mL CD screening medium contained 6.0 g NaNO3, 4.0 g KCl, 0.2 g KH2PO4, 0.1 g MgSO4▪7H2O, 0.004 g FeSO4▪7H2O, 4 g Glucose, 0.2 g (NH4)2SO4, 43.72 g sorbitol, 300 mg methionine and 3 g agar powder, with a pH of 5.5.

[0060] The covering medium consists of 1.2 M sorbitol and 0.5% agar powder.

[0061] Step S3: Structural identification of the γ-juniperene skeleton

[0062] Mycelia of a high-yielding γ-juniperene synthase strain (AO-GLSTS03) carrying the gene for γ-juniperene synthase were inoculated into sterilized culture medium containing rice (each test tube contained 1 gram of rice and 0.7 mL of water, the rice was soaked for six hours and then sterilized), and incubated statically at 30°C for one week. Once the mycelia covered the surface of the culture medium and produced a large number of spores, the γ-juniperene synthesized by the mycelium was secreted into the culture medium. GC-MS analysis of the crude ethyl acetate extract of the metabolites of the host strain *Aspergillus oryzae* and the high-yielding γ-juniperene strain (AO-GLSTS03) is as follows: Figure 4 As shown. The GC characteristics of γ-juniperene are as follows. Figure 4 As shown, to further verify the structure of this new compound, we performed GC-MS analysis. The mass spectrometric characteristics of γ-juniperene are shown below. Figure 5 As shown, the result displays: [M+H] + The value was 204.30, consistent with the previously reported GC-MS values ​​for γ-juniperene.

[0063] GC-MS Detection Method: This experiment used a Shimadzu GCMS-TQ8050 NX gas chromatograph-triple quadrupole mass spectrometer, equipped with an Rtx-5MS column (inner diameter 0.25 mm, film thickness 0.25 μm). The operating parameters of the detection system were as follows: the column oven temperature program was 80℃ for 5 min, then increased to 100℃ at a rate of 5℃ / min, then increased to 300℃ at a rate of 14℃ / min, and finally increased to 320℃ at a rate of 15℃ / min and held for 5 min; the mass spectrometry acquisition range was 33~300 m / z, and the acquisition rate was set to 2 spectra / second; the ionization method was electron impact ionization, the ionization energy was 70 eV, and the ion source temperature was 230℃.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A γ-juniperene synthase GLSTS03, characterized in that, It has the amino acid sequence shown in SEQ ID NO.

2.

2. The γ-juniperene synthase GLSTS03 as described in claim 1, characterized in that, The recombinant γ-juniperene synthase GLSTS03 is encoded by a gene with the nucleotide sequence shown in SEQ ID NO.1 and catalyzes the production of γ-juniperene from farnesyl pyrophosphate.

3. The gene encoding the γ-juniperene synthase GLSTS03 as described in claim 1.

4. The encoding gene as described in claim 3, characterized in that, It has a nucleotide sequence as shown in SEQ ID NO.

1.

5. A recombinant expression vector containing the coding gene as described in claim 3 or 4.

6. A recombinant expression strain containing the recombinant expression vector of claim 5.

7. A method for synthesizing γ-juniperene, characterized in that, γ-juniperene was synthesized by fermentation culture using the recombinant expression strain described in claim 6.

8. The method as described in claim 7, characterized in that, The method for constructing the recombinant expression strain includes: S1. Using Ganoderma lucidum genomic DNA as a template, the gene fragment encoding γ-juniperene synthase GLSTS03 was amplified by PCR; the fragment was ligated with pD8 plasmid at a molar ratio of 1:(1-6) to construct the recombinant expression vector pD8-GLSTS03. S2. After culturing Aspergillus oryzae spores in a spore medium containing dextrin and uracil, the mycelium was collected and then enzymatically hydrolyzed to obtain protoplasts; S3. The recombinant expression plasmid pD8-GLSTS03 obtained in step S1 was introduced into the protoplasts prepared in step S2 through PEG-mediated protoplast transformation. Positive transformants were screened, and the recombinant expression strain AO-GLSTS03 was obtained after subculture.

9. The method as described in claim 7 or 8, characterized in that, The fermentation culture method includes: inoculating the recombinant expression strain into a rice culture medium and culturing it at 28-30℃ for 7-10 days to ferment and produce γ-juniperene; the rice culture medium contains 0.1-2 g / mL rice.

10. The use of the γ-juniperene synthase GLSTS03 according to claim 1 or 2, the encoding gene according to claim 3 or 4, the recombinant expression vector according to claim 5, or the recombinant expression strain according to claim 6 in the synthesis of γ-juniperene.

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