A grandisol synthetase

Abstract

The application relates to a grandisol synthetase which can catalyze the conversion of locust ketone into grandisol precursor and can have a unique bidirectional catalytic function. The enzyme and the coding gene thereof can be used for efficiently producing beta-grandisol in microorganisms or plants, and have wide application prospects in flavor improvement and the spice industry.

Description

Technical Field

[0001] This application relates to a damascene synthase, which belongs to the field of biochemistry. Background Technology

[0002] Damascene is one of the most important aroma components among C13 norisoprene compounds. It has an extremely low sensory threshold and is widely found in various fruits (such as grapes), vegetables (such as tomatoes), flowers, juices, alcoholic beverages (such as wine), tea drinks, food, daily chemicals, and fragrances. It plays a decisive role in the flavor quality and aroma characteristics of these products.

[0003] Currently, one known synthetic pathway for damascene involves the CCD-catalyzed cleavage of carotenoids to produce squalene, followed by reduction to allyl triol, and finally spontaneous conversion to damascene under acidic conditions. Existing research relies on in vitro chemical simulations and lacks direct enzymatic and genetic evidence. Detailed studies on the enzymes involved in the catalytic reduction of squalene to allyl triol are lacking, resulting in an incomplete validation of the damascene biosynthetic pathway and limiting its application in metabolic engineering for flavor enhancement and the fragrance industry. Identifying the enzyme catalyzing the conversion of squalene to allyl triol would provide a complete technological solution from gene to enzyme to application, directly applicable to transgenic breeding and microbial fermentation for the production of natural flavorings, with enormous market potential. For example, in transgenic plant breeding, the aroma quality of grapes and other fruits can be improved by introducing or overexpressing genes encoding enzymes that catalyze the conversion of damascene to allyl triols; synthetic biology platforms can also be used to construct artificial metabolic pathways in microorganisms such as yeast and bacteria to achieve the controlled synthesis of damascene; or in the production industries of food, beverages and flavorings, the efficient in vitro enzyme-catalyzed synthesis of damascene can be achieved. Summary of the Invention

[0004] To address the aforementioned problems in existing technologies, this application isolates and identifies a damascene synthase that catalyzes the conversion of grasshopper ketone to allyl triol. Furthermore, it discovers that the damascene synthase also possesses bidirectional catalytic properties, exhibiting both NADH-dependent reducing activity and NAD+-dependent reducing activity. + Dependent on oxidation activity.

[0005] This application first provides a method for producing or controlling the precursor allyl triol of damastone, comprising contacting grasshopper ketone with a damastone synthase, wherein the damastone synthase is (-)-isomenthol / (-)-carvone dehydrogenase and / or cis,trans-nepene alcohol synthase NEPS2, under conditions allowing the substrate grasshopper ketone to be converted into an allyl triol; optionally, recovering the precursor allyl triol of damastone; optionally, the method is also used in combination with other methods for producing or controlling the precursor allyl triol of damastone.

[0006] In some methods, the conditions that allow the substrate grasshopper ketone to be converted into an allyl triol include the presence of NADH.

[0007] This application also provides a method for producing or controlling damascene, comprising obtaining an allyl triol by the above method, and further converting the allyl triol into damascene under acidic conditions; optionally, recovering the damascene; optionally, the method is also used in combination with other methods for producing or controlling damascene.

[0008] This application also provides one or more applications of a biological material in the following aspects: a) preparing damascene from plants or their fruits or regulating the content of damascene in plants or their fruits; b) producing damascene in a microbial cell factory or regulating the content of damascene; c) preparing flavorings containing damascene or regulating the content of damascene in flavorings; wherein the biological material is a damascene synthase, a gene encoding a damascene synthase, a vector containing a gene encoding a damascene synthase, or a host cell; wherein the damascene synthase is (-)-isomenthol / (-)-carvone dehydrogenase, and / or cis,trans-nepene alcohol synthase.

[0009] NEPS2; Optionally, the method may also be used in conjunction with other methods for producing or controlling damascene or its precursor allyl triol.

[0010] In some embodiments, other methods for producing or regulating damastone or its precursor allyl triol include, but are not limited to, regulating (overexpressing or silencing) genes related to the synthesis of damastone or its precursor allyl triol, or other means of regulating the synthesis of damastone or its precursor allyl triol (e.g., regulating environmental factors such as light, temperature, and rainfall). These methods are conventional techniques in the art.

[0011] In some embodiments, the damascene is (Z)-β-damascene and / or (E)-β-damascene.

[0012] This application also provides the use of damascene synthase to generate oxidation and / or reduction products through bidirectional catalytic activity, wherein the bidirectional catalysis refers to NADH-dependent reduction activity and NAD+-dependent reduction activity. + Dependent oxidative activity; the damascene synthase is (-)-isomenthol / (-)-carvacrol dehydrogenase, and / or cis, trans-nepetaol synthase NEPS2.

[0013] In some embodiments, the use includes catalyzing the redox reaction of grasshopper ketone; wherein the catalytic reduction of grasshopper ketone to grasshopper alcohol under NADH conditions, and the reduction of grasshopper ketone to grasshopper alcohol under NAD conditions... +Under certain conditions, it is oxidized to 3-keto-grasshopper ketone.

[0014] This application also provides a method for producing or controlling 3-keto-grasshopper ketone, using damascene synthase in NAD+. + Damascus ketone is oxidized under certain conditions; the damascus ketone synthase is (-)-isomenthol / (-)-carvone dehydrogenase, and / or cis, trans-nepetaol synthase NEPS2.

[0015] In some embodiments, the damascene synthase is derived from berry plants and / or viticulture plants, and / or the damascene synthase is recombined in a suitable vector or host cell, and / or the damascene synthase is modified but still retains its damascene synthase activity.

[0016] In some specific embodiments, the damastone synthase is VvDMS1 (NCBI accession number: XP_002271173.1, i.e., the amino acid sequence is shown in SEQ ID NO.1), VvDMS2 (NCBI accession number: RVW91199.1, the amino acid sequence is shown in SEQ ID NO.2), or VvDMS3 (NCBI accession number: XP_002267041.1, the amino acid sequence is shown in SEQ ID NO.3).

[0017] Advantages of this application:

[0018] ① This application provides for the first time an enzyme that catalyzes the conversion of grasshopper ketone to allyl triol (a precursor of damascone), forming complete enzymatic evidence for the damascone biosynthetic pathway, making up for the non-natural defects of chemical synthesis, and is highly practical.

[0019] ②This application clarifies for the first time that (-)-isomentheptyl / (-)-carvacrol dehydrogenase and / or cis, trans-nepetaol synthase NEPS2 both catalyze the conversion of grasshopper ketone to allyl triol (a precursor of damascone).

[0020] The conversion of damascene synthase activity reveals a new use for a known enzyme.

[0021] ③ This application discovered that damastone synthase possesses a unique catalytic mechanism, exhibiting NADH-dependent reducing activity and NAD+-dependent reducing activity. + Its dependent oxidation activity, a bidirectional catalytic property rarely seen in similar enzymes, makes it possible to apply it to more complex reaction networks in synthetic biology. Attached image description:

[0022] Figure 1 Evaluation of 12 candidate damastone synthases.

[0023] Figure 2 Results of molecular docking of VvDMS3 with grasshopper ketone and NADH.

[0024] Figure 3 VvDMS3 with grasshopper ketone and NAD + The molecular docking results.

[0025] Figure 4 VvDMS3 was co-incubated with grasshopper ketone and NADH to produce the intermediate grasshopper alcohol, which then formed β-damascene under acidic conditions. (The vertical axis of the chromatogram represents the EIC selected ion abundance of the protein-free control group and the VvDMS3 experimental group, respectively, and the horizontal axis represents the retention time.)

[0026] Figure 5 VvDMS3 with grasshopper ketone and NAD + The graph shows the production of 3-keto-grasshopper ketone during co-incubation (the vertical axis of the chromatogram represents the EIC selected ion abundance of the protein-free control group and the VvDMS3 experimental group, respectively, and the horizontal axis represents the retention time).

[0027] Figure 6 MS of grasshopper ketone (top left), grasshopper alcohol (top right), 3-keto-grasshopper ketone (bottom left) and β-damascene (bottom right) 1 (Level 1 mass spectrometry, with ion abundance on the vertical axis and mass-to-charge ratio on the horizontal axis).

[0028] Figure 7 MS analysis of grasshopper ketone (top), grasshopper alcohol (middle), and 3-keto-grasshopper ketone (bottom) 2 (Secondary mass spectrometry, with ion abundance on the vertical axis and mass-to-charge ratio on the horizontal axis).

[0029] Figure 8 Proposed MS of grasshopper ketone standard at a collision energy of 20 eV 2 Fragmentation pattern diagram.

[0030] Figure 9 The proposed MS analysis of grasshopper ketone reduction products after in-source cleavage at a collision energy of 20 eV. 2 Fragmentation pattern diagram.

[0031] Figure 10 Proposed MS of grasshopper ketone oxidation products at a collision energy of 20 eV 2 Fragmentation pattern diagram.

[0032] Figure 11 Multiple sequence alignment diagram of VvDMS3 and its four neighboring tandem genes.

[0033] Figure 12 SDS-PAGE results of the MBP fusion protein of VvDMS3 and its four neighboring tandem gene products.

[0034] Figure 13 A comparison of the ability of VvDMS3 and its four neighboring tandem gene products to catalyze the production of β-damascene (vertical axis represents the relative abundance of the sum of the three β-damascene characteristic ions, and horizontal axis represents retention time).

[0035] Figure 14 Schematic diagram of the damascene synthesis pathway. Detailed implementation method:

[0036] The present application will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present application, but do not limit the present application in any way.

[0037] The damascenone in this application is also known as β-damascenone ((E / Z)-β-damascenone), CAS Registry Numbers: 23726-93-4 and 59739-63-8, including trans-β-damascenone ((E)-β-damascenone or trans-β-damascenone) and cis-β-damascenone ((Z)-β-damascenone or cis-β-damascenone).

[0038] The allenic triol in this application refers to grasshopper alcohol, which is a trihydroxy compound generated by the reduction of grasshopper ketone and is a direct precursor of β-damascone.

[0039] The damascene synthase (VvDMS) of this application refers to an enzyme with the activity of catalyzing the reduction of grasshopper ketone to allyl triol (a precursor of damascene). Some specific examples are: (-)-isopiperitenol / (-)-carveol dehydrogenase, whose amino acid sequence in Vitaceae (Vitis vinifera) is, for example, NCBI accession number: XP_002266902.2, RVW91199.1 (SEQ ID NO.2), RVW91200.1, RVW35501.1, XP_034673826.1, XP_034673749.1, RVW35564.1, XP_002267041.1 (SEQ ID NO.3), XP_034673797.1, RVW35183.1, etc. Specific examples include cis,trans-nepetalactol synthase NEPS2 ((+)-cis,trans-nepetalactol synthase), whose amino acid sequence in Vitaceae plants is, for example, NCBI accession number: XP_002271173.1 (SEQ ID NO.1), XP_057951916.1, etc. In some embodiments, the damascene synthase is derived from plant leaves, fruits, and callus tissue. In some embodiments, the plant is a dicotyledonous or monocotyledonous plant, such as rice, Arabidopsis thaliana, tomato, coffee tree, cocoa tree, lavender, grape, etc. In some preferred embodiments, the plant is a plant of the Vitaceae family or the genus Vitis, or a berry-bearing plant. In some preferred embodiments, the damascene synthase is derived from tomato, grape, or their fruit.

[0040] The methods or uses of this application can also be combined with other genes or methods to prepare or regulate damascene synthesis. Prior art has reported that increased expression levels of the VviWRKY24 gene can significantly enhance the metabolic flux to the downstream compound β-damascene synthesis; damascene synthesis requires the catalysis of neoflavin by carotenoid cleavage dioxygenases (CCDs) to form damascene, such as VvCCD1, VvCCD4a, and VvCCD4b; increasing the activity of 1-deoxyxylulose 5-phosphate synthase (DXS) and / or isopentenyl pyrophosphatase isomerase (IPP isomerase) can increase damascene content, etc. Therefore, other genes or methods related to the synthesis of damascene or its precursor allyl triols can be, for example, genes such as VviWRKY24, VvCCD1, VvCCD4a, and VvCCD4b (overexpressed or silenced), or methods such as increasing or inhibiting DXS activity and / or IPP isomerase activity. This is something that can be achieved by those skilled in the art through routine experiments. Therefore, regulation can refer to either inhibitory or promoting effects.

[0041] This application provides a method for generating grasshopper alcohol, which involves reducing grasshopper ketone with damascone synthase under NADH conditions. This application also provides a method for generating damascone, where grasshopper alcohol is obtained using this method, and then damascone is formed under acidic conditions. Additionally, this application provides a method for generating 3-keto-grasshopper ketone, which involves using damascone synthase under NAD... + It is formed by the oxidation of grasshopper ketone under certain conditions.

[0042] In some embodiments, the damascene synthase is (-)-isomenthol / (-)-carvacrol dehydrogenase, and / or cis,trans-nepene alcohol synthase NEPS2; in other embodiments, the damascene synthase may be recombined in a suitable vector or host cell or modified but still retain damascene synthase activity.

[0043] This application provides a method for producing or controlling β-damascene or its precursor allyl triol, comprising: contacting grasshopper ketone with the damascene synthase under conditions allowing the conversion of the substrate grasshopper ketone to allyl triol (e.g., in the presence of NADH); wherein the damascene synthase is (-)-isomenthol / (-)-carvone dehydrogenase, and / or cis,trans-nepetalol synthase NEPS2. Optionally, the generated allyl triol is then treated under acidic conditions to convert it to β-damascene. Optionally, the β-damascene or its precursor allyl triol is recovered.

[0044] This application provides an application of a biomaterial in the following aspects: a) preparing β-damascene from plants or their fruits or regulating the content of β-damascene in plants or their fruits; b) producing β-damascene or regulating the content of β-damascene in a microbial cell factory; c) preparing fragrances and flavorings containing β-damascene or regulating the content of β-damascene in fragrances and flavorings; wherein the biomaterial is a damascene synthase, a gene encoding a damascene synthase, a vector containing a gene encoding a damascene synthase, or a host cell.

[0045] This application also provides a bidirectional catalytic use of damastone synthase, wherein the bidirectional catalysis refers to NADH-dependent reducing activity and NAD+-dependent reducing activity. + Dependent oxidation activity. In some embodiments, the damascene synthase is VvDMS1 (NCBI accession number: XP_002271173.1, i.e., amino acid sequence as shown in SEQ ID NO.1), VvDMS2 (NCBI accession number: RVW91199.1, amino acid sequence as shown in SEQ ID NO.2) and / or VvDMS3 (NCBI accession number: XP_002267041.1, amino acid sequence as shown in SEQ ID NO.3). In some embodiments, the use is for catalyzing the redox reaction of damascene.

[0046] The methods or uses of this application can be carried out in vivo or in vitro, for example in an in vitro enzyme reaction system or in microorganisms expressing the VvDMS.

[0047] Example 1: Genome alignment and candidate gene screening

[0048] This application uses grapes (as the test material) as an example to identify the damascone synthase responsible for catalyzing the conversion of damascone to allyl triol in the damascone biosynthesis pathway. The specific steps are as follows:

[0049] A genome-wide search and candidate gene screening were performed on the grape (Vitis vinifera PN40024) genome. First, the genome sequence and gene structure annotation files of Vitis vinifera PN40024.v4 were downloaded from the Grape Genome Browser database (https: / / grapedia.org / files-download / ), and a grape genome-wide protein database was constructed locally. Subsequently, a Hidden Markov Model (HMM) method was used to perform genome-wide alignment on this protein database. The seed sequences for the HMM model were derived from conserved reductase domains in the InterPro database, including the ADH domain (PF08240), AKR domain (PF00248), and SDR domain (PF00106). The search was conducted using TBtools software, with an e-value threshold of less than 0.05 to ensure high reliability of candidate sequences.

[0050] Using the methods described above, a total of 267 potential reductase candidate genes were obtained, including 45 alcohol dehydrogenases (ADHs), 179 short-chain dehydrogenases / reductases (SDRs), and 42 aldehyde-ketone reductases (AKRs). These candidate genes are considered to be involved in the damascene synthesis pathway, providing a gene resource library for subsequent functional verification.

[0051] Example 2: Candidate gene selection

[0052] To further screen for the genes most relevant to damascene synthesis from candidate reductases, this application combined gene expression correlation analysis and molecular docking simulation for optimization. The specific steps are as follows:

[0053] First, using the accumulation pattern of damascene at different fruit development stages as a reference, the Pearson correlation coefficient between the expression levels of candidate reductase genes and damascene content was calculated to obtain candidate genes significantly co-expressed with damascene. Using this method, a total of 43 candidate reductase genes were screened, suggesting their potential involvement in the biosynthesis of damascene. Subsequently, at the structural level, active sites of these candidate genes were predicted, identifying their typical catalytic motifs: Ser-Thr (ADHs), Ser / Tyr-Lys (SDRs), and Tyr-His-Lys (AKRs). The substrate damascene was docked with each candidate protein using the molecular docking tool (CB-Dock2), with a focus on analyzing its binding mode with catalytic residues. The results showed that some candidate enzymes could form stable hydrogen bonds, with the hydrogen bond distance between the substrate carbonyl group and key residues (such as Tyr hydroxyl, Ser hydroxyl, Thr hydroxyl, and His amino groups) concentrated in... This suggests that it has potential substrate recognition and catalytic capabilities.

[0054] To comprehensively evaluate the priority of candidate genes, this application established a scoring formula: Score = R + 0.5 × HB_number. Where R is the correlation coefficient between the candidate gene and the damascene content, and HB_number is the number of hydrogen bonds formed between the substrate and the active residues. Based on this standard, 12 candidate genes were ultimately selected as key research targets for subsequent functional validation experiments.

[0055] Example 3: Gene Cloning and Protein Expression

[0056] The 12 candidate genes selected in Example 2 were cloned and expressed in *E. coli*. After protein purification, their catalytic activity against damascone was tested. The activity was based on the correlation between damascone content and the predicted hydrogen bond formation between damascone and key catalytic residues. Specific experiments are as follows:

[0057] The open reading frames (ORFs) of 12 candidate reductase genes were amplified from a cDNA library obtained by reverse transcription of total RNA from grape 'Marselan' green fruit (E-L33 stage). After the PCR products were confirmed by sequencing, they were directionally cloned into the pMal-c5x expression vector (New England Biolabs) and inserted at the NdeI and BamHI sites to obtain a recombinant expression construct with an MBP fusion tag.

[0058] The resulting recombinant plasmid was transformed into *E. coli* Transetta (DE3) competent cells and cultured in LB medium containing 100 μg / mL ampicillin and 34 μg / mL chloramphenicol. When the cell density reached OD600 = 0.7, 0.5 mM IPTG was added for induction, and the cells were cultured at 16℃ and 100 rpm for 16 h to promote the expression of the soluble fusion protein. After cell collection, the cells were suspended in PBS buffer (containing 1 mM PMSF and 1 mM DTT), lysed by sonication, and centrifuged to obtain the soluble protein supernatant.

[0059] In the initial screening stage, crude protein extract (900 μL) was taken and mixed with substrate grasshopper ketone (400 μM) and cofactor (500 μM NADH / NAD). + The reaction was carried out overnight at 30°C. The reaction products were analyzed by UHPLC-QTOF-MS and GC-MS, and the formation of damascene was detected under acidic conditions (0.3M citrate-phosphate buffer, pH 3.0, 4h).

[0060] The scores of candidate damastone synthases were based on their correlation with β-damastone content and the predicted hydrogen bond formation between damastone and key catalytic residues. Figure 1 The results included gene number, gene family, Pearson correlation coefficient between expression level and β-damascene content, whether the inferred enzyme active site could form hydrogen bonds with the substrate carbonyl group, total score, and whether β-damascene was detected in the initial screening enzyme activity reaction. It can be seen that only the product of the Vitvi17g00537 gene (i.e., (-)-isomenthol / (-)-carvyl alcohol dehydrogenase) can catalyze the conversion of grasshopper ketone to the intermediate grasshopper alcohol, and generate (Z)- and (E)-β-damascene under acidic conditions. Therefore, (-)-isomenthol / (-)-carvyl alcohol dehydrogenase was identified as a type of damascene synthase (VvDMS).

[0061] Example 4: Enzyme Activity Verification

[0062] To further verify the enzyme's function in catalyzing the conversion of grasshopper ketone to the intermediate grasshopper alcohol (an allyl triol), a VvDMS–MBP fusion protein was constructed, purified by affinity chromatography (Amylose resin), and quantified using the Bradford method after concentration of the eluent. The purified candidate VvDMS protein was then systematically validated for its substrate specificity and catalytic function under different cofactor conditions. The Vitvi17g00537 protein was combined with grasshopper ketone and NADH or NAD. + Molecular docking was performed, and the results are shown in [the table]. Figures 2-3 .

[0063] Using grasshopper ketone as a substrate, NADH and NAD were added respectively. + NADPH and NADP + As a cofactor, the reaction system was carried out at 30℃, and the product was detected by UHPLC-QTOF-MS. For example, using 10 μg of purified protein as the core component of the reaction system, it was reacted with 20 mM grasshopper ketone and the cofactor at 30℃ for 1 h, terminated with methanol, and the precipitate was removed by centrifugation. The final product was identified by UHPLC-QTOF-MS and GC-MS. Experimental results show that this VvDMS has specific catalytic function (see...). Figures 4-7 ):

[0064] ① In the presence of NADH, the protein encoded by Vitvi17g00537 can efficiently catalyze the reduction of grasshopper ketone to generate the intermediate grasshopper alcohol. This intermediate can spontaneously rearrange and dehydrate under acidic conditions, ultimately generating (Z)- and (E)-β-damascone.

[0065] ②In NAD + In its presence, Vitvi17g00537 can also oxidize grasshopper ketone to 3-keto-grasshopper ketone, demonstrating its unique bidirectional redox catalytic properties.

[0066] ③ In NADPH and NADP + Under the specified conditions, no obvious product was detected, indicating that the enzyme has strict selectivity for cofactors.

[0067] Mass spectrometry evidence further supports the above conclusions: the reduction product shows a +2 Da mass shift, consistent with the structural characteristics of carbonyl reduction to hydroxyl; the oxidation product shows a -2 Da shift, and fragment peaks confirm the structural characteristics of the C3 hydroxyl group being oxidized to a carbonyl group. Acidification experiments show that only the reduction product generated by NADH can be converted into β-damascene, while the oxidation product does not release volatile damascene, revealing the crucial role of cofactor specificity in aroma formation.

[0068] When identifying the product, the MS / MS fragmentation diagram of the grasshopper ketone standard at a collision energy of 20 eV was used as a reference for the structural analysis of its redox-related derivatives (see [link to documentation]). Figure 8 ); MS analysis of grasshopper ketone reduction products 1 The mass spectrum revealed intrasource cleavage, with the product undergoing three dehydration processes (–3H₂O), indicating the presence of three hydroxyl groups, consistent with the structure of allyl triols (see [link to mass spectrometry]). Figure 6 The proposed MS analysis of grasshopper ketone reduction products after in-source cleavage at a collision energy of 20 eV. 2 See Shatter mode Figure 9 Proposed MS / MS fragmentation mode data of the grasshopper ketone oxidation derivative at a collision energy of 20 eV indicate that the 3-hydroxyl group is oxidized to a 3-keto group, forming 3-keto-grasshopper ketone (see...). Figure 10 ).

[0069] Example 5: Identification of other damascene synthases (VvDMS)

[0070] To determine whether other (-)-isomenthol / (-)-carvacrol dehydrogenases also possess damascene synthase activity, this embodiment isolates another (-)-isomenthol / (-)-carvacrol dehydrogenase from grapes, namely the gene product of Vitvi17g00535, and verifies its damascene synthase activity, while comparing it with the gene product of Vitvi17g00537.

[0071] To discover more possible types of damascene synthases, this application analyzed a gene cluster consisting of Vitvi17g00537 and its four neighboring genes in a tandem distribution (see...). Figures 11-12 The study was conducted.

[0072] The results showed that both Vitvi17g00534 (a cis,trans-nepetaol synthase NEPS2) and Vitvi17g00535 (a (-)-isomenthyl / (-)-carvacrol dehydrogenase) could catalyze the conversion of grasshopper ketone to β-damastone under NADH conditions, but their catalytic efficiency was significantly lower than that of Vitvi17g00537. Notably, only Vitvi17g00537 exhibited NAD... + The dependent oxidation activity indicates that it is the most functionally complete core enzyme in this gene cluster. Specifically, the protein encoded by Vitvi17g00534 is named VvDMS1 (NCBI accession number: XP_002271173.1, amino acid sequence as shown in SEQ ID NO.1), the protein encoded by Vitvi17g00535 is named VvDMS2 (NCBI accession number: RVW91199.1, amino acid sequence as shown in SEQ ID NO.2), and the protein encoded by Vitvi17g00537 is named VvDMS3 (NCBI accession number: XP_002267041.1, amino acid sequence as shown in SEQ ID NO.3). VvDMS1 is cis,trans-nepenicillin synthase NEPS2, and both VvDMS2 and VvDMS3 are (-)-isomenthyl / (-)-carvyl dehydrogenases. Therefore, cis,trans-nepetaol synthase NEPS2 and (-)-isomenthol / (-)-carvacrol dehydrogenase are both damastone synthases.

[0073] The specific experiment is as follows:

[0074] All five genes were constructed in the same pMal-c5x expression vector and induced to express in *E. coli* Transetta (DE3). Bacterial cells were lysed by sonication in an ice bath, and cell debris was removed by centrifugation. The supernatant containing the target protein was purified by starch affinity chromatography, and the eluent was then concentrated by ultrafiltration and quantified using the Bradford method. All recombinases were obtained under identical conditions to ensure comparability in subsequent activity comparisons.

[0075] In in vitro enzymatic experiments, each reaction system contained 10 μg of purified recombinant protein, 400 μM NADH, and 20 mM damascene. The reaction buffer was uniformly 1 M Tris-HCl (pH 7.4). The reaction was carried out at 30 °C for 1 h, followed by termination with methanol and centrifugation to remove the precipitate. The supernatant was further treated under acidic conditions by adding 0.3 M citrate-phosphate buffer (pH 3.0) and incubating at room temperature for 4 h to allow the reaction intermediate to spontaneously dehydrate and rearrange, releasing β-damascene. All samples underwent the same acidification procedure to ensure that differences in products stemmed solely from varying enzymatic reaction efficiencies.

[0076] The enzymatic reaction products were analyzed using a unified headspace solid-phase microextraction-gas chromatography-mass spectrometry (SPME–GC–MS) method. The specific procedure was as follows: 5 mL of acidified reaction solution was taken, and 1.00 ± 0.01 g NaCl and 10 μL of internal standard (4-methyl-2-pentanol, 1.0018 g / L) were added, and the solution was sealed in a 15 mL headspace vial. The fibers were extracted using DVB / CAR / PDMS, equilibrated at 40 °C for 30 min, followed by 30 min of volatile extraction. The fibers were then thermally desorbed at 250 °C for 8 min at the injection port. Chromatographic separation was performed using an HP-INNOWAX capillary column (60 m × 0.25 mm × 0.25 μm) with helium as the carrier gas (1 mL / min). The oven temperature program was set as follows: 50 °C for 1 min, increased to 220 °C at 3 °C / min and held for 5 min. Mass spectrometry was performed in EI ionization mode (70 eV) with a scan range of m / z 30–350. The interface, ion source, and quadrupole temperatures were set to 280 °C, 250 °C, and 150 °C, respectively. The obtained products were qualitatively analyzed by retention time, retention index, and NIST11 library, and quantitatively analyzed by external standard calibration curves.

[0077] from Figure 13It can be seen that under NADH conditions, the enzymes expressed by the three genes Vitvi17g00534, Vitvi17g00535, and Vitvi17g00537 can catalyze the conversion of grasshopper ketone to β-damascenone, but the efficiencies of the three are extremely different: VvDMS1 only detected (E)-β-damascenone, 0.73 μg / L, indicating that its catalytic efficiency is extremely low. VvDMS2 simultaneously detected the generation of (Z)- and (E)-β-damascenone, with the (Z)- isomer at 0.58 μg / L and the (E)- isomer at 6.51 μg / L, and the total product at approximately 7.09 μg / L. In comparison, VvDMS3 exhibits significantly higher catalytic efficiency than the other two, with its (Z)-isomer at 54.47 μg / L, its (E)-isomer reaching a high of 720.99 μg / L, and the total product reaching 775.46 μg / L, resulting in an E / Z ratio of 13.24. This demonstrates that VvDMS3 produces approximately 100 times more β-damascene than VvDMS2 and 1000 times more than VvDMS1.

[0078] In summary, among various types of damascene synthases, cis,trans-nepetalone synthase NEPS2 exhibits relatively low catalytic efficiency, while (-)-isomentheptyl / (-)-carvacrol dehydrogenases demonstrate higher catalytic efficiency. In particular, some (-)-isomentheptyl / (-)-carvacrol dehydrogenases, such as Vitvi17g00537 (VvDMS3), are the most functionally complete core enzymes, with catalytic efficiency and product abundance far exceeding other damascene synthases. This application provides for the first time the pathway of "carotenoid cleavage product neoxanthin → damascene → β-damascene" (see...). Figure 14 The damascene synthase in the study not only verified its function in catalyzing the reduction of grasshopper ketone to allyl triol, but also discovered that the damascene synthase has a unique cofactor-dependent bidirectional catalytic mechanism.

Claims

1. A method for producing or regulating the precursor allyl triol of damastone, comprising contacting grasshopper ketone with a damastone synthase under conditions allowing the substrate grasshopper ketone to be converted into an allyl triol, wherein the damastone synthase is (-)-isomenthol / (-)-carvone dehydrogenase, and / or cis,trans-nepene alcohol synthase NEPS2; wherein: The amino acid sequence of the cis, trans-nepeta alcohol synthase NEPS2 is shown in SEQ ID NO.1; The amino acid sequence of the (-)-isomentheptyl / (-)-carvacrol dehydrogenase is shown in SEQ ID NO.2 or SEQ ID NO.3; The conditions that allow the substrate grasshopper ketone to be converted into an allyl triol include the presence of NADH.

2. The method as described in claim 1, characterized in that, The method further includes recovering the damascene precursor allyl triol or using it in combination with other methods for producing or controlling the damascene precursor allyl triol.

3. The method as described in claim 1 or 2, characterized in that, The damascene is (Z)-β-damascene and / or (E)-β-damascene.

4. A method for producing or controlling damascone, comprising obtaining an allyl triol by the method of any one of claims 1-3, and further converting the allyl triol into damascone under acidic conditions.

5. The method as described in claim 4, characterized in that, The damascene may be recovered or used in combination with other methods for producing or controlling damascene.

6. The method as described in claim 4 or 5, characterized in that, The production or regulation of damascone in a microbial cell factory using biological materials, wherein the biological materials are selected from damascone synthase, genes encoding damascone synthase, or vectors or host cells containing genes encoding damascone synthase.

7. Use of a damastone synthase for bidirectional catalysis of grasshopper ketone to 3-keto-grasshopper ketone and / or grasshopper alcohol, wherein the bidirectional catalysis refers to NADH-dependent reducing activity and NAD+-dependent reducing activity. + Dependent oxidation activity; the damascene synthase is (-)-isomenthyl / (-)-carvacrol dehydrogenase, the amino acid sequence of which is shown in SEQ ID NO.3; wherein, The damascone synthase catalyzes the reduction of grasshopper ketone to grasshopper alcohol under NADH conditions, and its oxidation to 3-keto-grasshopper ketone under NAD+ conditions.

8. A method for producing or regulating 3-keto-grasshopper ketone, using damascene synthase in NAD... + Damascus ketone is oxidized under certain conditions; the damascus ketone synthase is (-)-isomenthol / (-)-carvone dehydrogenase, and its amino acid sequence is shown in SEQ ID NO.3.

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