Valencene synthase and use thereof

Abstract

The present invention relates to the technical field of biology, and in particular to a valencene synthase and a use thereof. Pfam domains of terpene synthases PF01397 and PF03936 are used as core domains of novel valencene synthases, a novel valencene synthase having valencene synthase activity is obtained by screening, and an encoding nucleic acid molecule is designed on the basis of the amino acid sequence of the novel enzyme obtained by screening. Furthermore, a recombinant plasmid vector and an expression system for expressing the novel enzyme are prepared; the recombinant plasmid vector and the expression system can be applied to the synthesis of different citrus fragrances, thereby overcoming the time-consuming and labor-consuming problems of extracting essential oil from plants, and promoting the industrial application of the citrus fragrances.

Description

Valencene synthase and its application Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to valencene synthase and applications thereof. Background Art

[0002] Valencene, a sesquiterpenoid compound, has been reported in various plant essential oils (e.g., grape seed essential oil, citrus essential oil, etc.). It has a distinctive odor and can be used in foods and daily chemicals. However, the entire process from planting to refining plant essential oils is complex, time-consuming, and labor-intensive, requiring large amounts of plant peels, resulting in high extraction costs and considerable value. Furthermore, natural plant extracts contain very low levels of valencene, making it difficult to isolate plant essential oils containing high valencene concentrations using natural product separation methods. Therefore, finding more economical methods to obtain essential oils with high valencene concentrations and distinctive odors is an inevitable trend for achieving economically efficient and sustainable development.

[0003] Summary of the Invention

[0004] The present invention aims to provide a valencene synthase and the use of the enzyme in an expression system for preparing a composition with a special aroma containing valencene as the main component, thereby achieving the purpose of efficient and sustainable production.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a valencene synthase comprising domains with Pfam numbers PF01397 and PF03936, and having valencene synthase activity.

[0007] In alternative embodiments, the valencene synthase comprises (a) a complete protein or functional protein fragment derived from a plant; or (b) a derivative valencene synthase having at least 80% amino acid sequence homology with (a).

[0008] In an alternative embodiment, the plant is selected from a Rutaceae plant or a Ranunculaceae Juss. plant.

[0009] In an alternative embodiment, the plant is selected from the genus Cimicifuga or the genus Citrus.

[0010] In an alternative embodiment, the Cimicifuga plant includes Actaea asiatica, preferably Actaea racemosa, and the Citrus plant includes Citrus maxima.

[0011] In alternative embodiments, the valencene synthase catalyzes the synthesis of valencene in an expression system.

[0012] In an alternative embodiment, the expression system comprises a fungal expression system.

[0013] In an alternative embodiment, the fungal expression system comprises a yeast expression system.

[0014] In an alternative embodiment, the yeast expression system comprises a Saccharomyces cerevisiae expression system.

[0015] In alternative embodiments, the amino acid sequence of the valencene synthase comprises (a) or (b):

[0016] (a) any one of SEQ ID NOs: 1 to 3;

[0017] (b) a derivative valencene synthase having at least 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, or more amino acid sequence identity to (a).

[0018] In a second aspect, the present invention provides use of the valencene synthase described in any one of the aforementioned embodiments in preparing a flavor composition.

[0019] In a third aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the valencene synthase described in any one of the aforementioned embodiments or a nucleotide sequence complementary to the valencene synthase.

[0020] In an optional embodiment, the nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to any of the nucleotide sequences shown in SEQ ID No:4 to 6.

[0021] In a fourth aspect, the present invention provides a recombinant expression vector comprising the nucleic acid molecule according to any one of the aforementioned embodiments, wherein the original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

[0022] In a fifth aspect, the present invention provides a transformant, wherein the host cell of the transformant is selected from a fungal expression system, and contains the nucleic acid molecule described in any one of the preceding embodiments or the recombinant expression vector described in the preceding embodiments. Preferably, the fungal expression system is a yeast expression system, and more preferably a cerevisiae expression system.

[0023] In a sixth aspect, the present invention provides a recombinant Saccharomyces cerevisiae, which contains the nucleic acid molecule described in any one of the preceding embodiments, or contains the recombinant expression vector described in any one of the preceding embodiments; preferably, the original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

[0024] In a seventh aspect, the present invention provides a flavor composition comprising 10% to 99% valencene by mass; the valencene is synthesized by the valencene synthase described in any of the preceding embodiments.

[0025] In an eighth aspect, the present invention provides a method for preparing the flavor composition described in any of the aforementioned embodiments, the preparation method comprising fermenting and culturing the transformant or recombinant Saccharomyces cerevisiae described in the aforementioned embodiments, and recovering the flavor composition from the fermentation product.

[0026] In an alternative embodiment, the recovery method comprises extracting the fermentation product using an oil phase.

[0027] In an alternative embodiment, the oil phase comprises at least one of isopropyl myristate or n-dodecane.

[0028] In an optional embodiment, the recovery method is to add an oil phase during the fermentation process, collect the oil phase after the fermentation is completed, and separate and obtain the flavor composition enriched in the oil phase.

[0029] In an alternative embodiment, the method of collecting the oil phase comprises centrifugation.

[0030] In an alternative embodiment, the separation method comprises distillation.

[0031] In a ninth aspect, the present invention provides the use of the flavor composition described in any one of the aforementioned embodiments, or the flavor composition obtained by the preparation method described in any one of the aforementioned embodiments, in the preparation of a flavor product.

[0032] In a tenth aspect, the present invention provides a fragrance product comprising the fragrance composition described in any one of the aforementioned embodiments, or the fragrance composition prepared by the method described in any one of the aforementioned embodiments.

[0033] In alternative embodiments, the fragrance product comprises a personal care product, a fabric care product, a cleaning product, a deodorant, a home care product, a fabric product, an air care product, a fragrance delivery system, a cosmetic formulation, a disinfectant, or a food product.

[0034] In an optional embodiment, the personal care product includes: floral water, skin care products, hair care products, antiperspirants, feminine care products, baby care products, oral hygiene products or health care.

[0035] In an alternative embodiment, the skin care product comprises toilet soap, liquid soap or body wash.

[0036] In an alternative embodiment, the oral hygiene product comprises a dental hygiene product.

[0037] In an alternative embodiment, the cosmetic formulation comprises a perfume, a fragrance liquid or an essential oil.

[0038] In an alternative embodiment, the cleaning product comprises a detergent composition.

[0039] In alternative embodiments, the detergent composition comprises a dishwashing detergent, a glass cleaner, a metal cleaner, a countertop cleaner, a floor cleaner, a carpet cleaner, a toilet cleaner, or a bleach additive.

[0040] The inventors used the Pfam domain containing the terpene synthases PF01397 and PF03936 as the core domain of valencene synthase, hoping to screen for new synthases and ultimately obtain a novel enzyme with valencene synthase activity. Based on the amino acid sequence of the screened valencene enzyme, they designed an encoding nucleic acid molecule and then prepared a recombinant plasmid vector and expression system for expressing valencene synthase. This has the potential to be applied to the synthesis of various citrus flavors, making up for the time-consuming and labor-intensive plant extraction process and promoting the industrial application of citrus flavors.

[0041] The expression system can be fermented and cultured to achieve the expression and preparation of the flavor composition, thereby obtaining a flavor composition mainly composed of valencene.

[0042] Furthermore, the present invention provides for the first time valencene synthases derived from Rutaceae and Ranunculaceae plants, which are fermented in conjunction with a yeast expression system to produce a composition with a citrus aroma. The amino acid sequences of the two valencene synthases are shown in SEQ ID NO: 1 (ArVS derived from Actaea racemosa), SEQ ID NO: 2 (ArVS-V8 derived from Actaea racemosa), and SEQ ID NO: 3 (CmVS-V4 derived from Citrus maxima), respectively.

[0043] Furthermore, using microbial fermentation to produce plant-derived fragrance products instead of extracting them from natural plant materials can mitigate the effects of climate on natural plant growth, resulting in more stable yields and quality. This also reduces the demand for plant raw materials, freeing up more planting resources for other energy crops. Therefore, the microbial fermentation method provided by the present invention for preparing compositions with unique fragrances has promising applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0045] Figure 1 is a schematic diagram of the construction of plasmid pArVS;

[0046] Figure 2 is a schematic diagram of the construction of plasmid pArVS-V8;

[0047] Figure 3 is a schematic diagram of the construction of plasmid pCmVS-V4;

[0048] Figure 4 is a schematic diagram of the construction of plasmids pVvVS, pCsVS, pShVS and pSlVS, pPvVS, pCnVS, pEgVS, and pAoVS. DETAILED DESCRIPTION

[0049] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0050] (I) Definitions or terms

[0051] The following abbreviations are used for the relevant definitions or terms used in the present invention. Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. The following terms are provided below.

[0052] 1. Protein family classification database (Pfam database)

[0053] The Pfam database is a commonly used protein family database, created based on protein data included in the UniProt database. It is currently updated to version 35.0 (November 2021, 19,632 data). In this database, you can download data such as the HMM model file, alignment sequence, species information, and HMM logo corresponding to a gene family. The HMM model file contains gene family sequence feature information and can be used with the Hmmer software to identify gene family members. The relevant website link is: https: / / www.ebi.ac.uk / interpro / entry / pfam / .

[0054] 2. Pfam domain

[0055] "Pfam domain" refers to a region within a protein sequence identified as Pfam-A or Pfam-B, and the identification process or method is designed based on multiple sequence alignments and the presence of hidden Markov motifs ("The Pfam protein families database": RDFinn, J. Mistry, J. Tate, P. Coggill, A. Heger, JE Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, ELSonnhammer, SREddy, A. Bateman Nucleic Acids Research (2010) Database Issue 38: D211-222.).

[0056] 3. NIST

[0057] The NIST referred to in this invention refers to the National Institute of Standards and Technology (NIST), which is directly under the U.S. Department of Commerce. NIST conducts basic and applied research in physics, biology, and engineering, as well as research in measurement technology and testing methods. It provides standards, standard reference data, and related services, and enjoys a high international reputation. The identification of the components in the fermented composition in this invention was based on the NIST compound name database.

[0058] 4. Novel valencene synthase

[0059] The novel valencene synthase described herein refers to a valencene synthase that had not been successfully isolated and purified prior to the present invention. This is because valencene is a terpenoid compound, and conventional terpene synthases are typically naturally occurring enzymes. Terpene-synthesizing enzymes expressed in plant cells under natural conditions are typically adapted to plant cell expression and terpene synthesis systems. The valencene synthase provided herein is a novel enzyme that can be successfully expressed in such an expression system and is capable of synthesizing valencene.

[0060] 5. Homology

[0061] The term "homology" as used herein has a meaning generally recognized in the art, and the percentage of sequence identity between two nucleic acids, polypeptides, or regions can be calculated using publicly available techniques. For example, sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Currently, there are many methods for measuring the identity between two polynucleotides or polypeptides, which can be routinely selected by those skilled in the art based on actual needs.

[0062] 6. Expression

[0063] Expression as used herein refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0064] 7. Host cells

[0065] The host cells of the present invention are cells that are used to receive, maintain, replicate, and amplify vectors. Host cells can also be used to express polypeptides encoded by the vectors. When the host cells divide, the nucleic acids contained in the vectors replicate, thereby amplifying the nucleic acids. The host cells can be eukaryotic or prokaryotic cells.

[0066] 8. Carrier

[0067] The vectors of the present invention are replicable nucleic acids that can express one or more heterologous proteins when transformed into appropriate host cells. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors generally remain episomal, but can be designed to integrate genes or parts thereof into genomic chromosomes. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vectors are well known to those skilled in the art.

[0068] (II) Detailed technical plan

[0069] In a specific embodiment, in a first aspect, the present invention provides a valencene synthase comprising domains with Pfam numbers PF01397 and PF03936, and having valencene synthase activity.

[0070] Among them, PF01397 is the N-terminal conserved domain of terpene synthase, and PF03936 is the C-terminal metal ion binding domain of terpene synthase. Screening genes containing both PF01397 and PF03936 Pfam domains can obtain complete plant terpene synthase.

[0071] The Pfam database is a collection of protein families, each of which is represented in the form of multiple sequence alignments and hidden Markov models.

[0072] 1. Download the Pfam database information (http: / / ftp.ebi.ac.uk / pub / databases / Pfam / releases / Pfam35.0 / ).

[0073] 2. Use hmmpress software to create an index for the local database.

[0074] 3. Use hmmscan software (E<0.01) to align the input protein sequence with the HMM domain library and return the domains contained in each protein.

[0075] 4. Filter out results containing both PF03936 and PF01397 domains.

[0076] In alternative embodiments, the valencene synthase comprises (a) a complete protein or functional protein fragment derived from a plant; or (b) a derivative valencene synthase having at least 80% amino acid sequence homology with (a).

[0077] Taking into account the cyclical growth and yield characteristics of plants, developing microbial alternative production methods for plant essential oils or plant-derived spice products has greater economic value and social significance than developing microbial alternative production methods for spices from other sources.

[0078] In some preferred embodiments, the above-mentioned derivatization process can be to replace the amino acid site with another amino acid of the same type (having similar chemical properties or functions). As an example, amino acids can be divided into the following categories according to their side chain properties: (1) non-polar amino acids: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar amino acids: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic amino acids: Asp (D), Glu (E); (4) basic amino acids: Lys (K), Arg (R), His (H). Alternatively, amino acids can be classified based on common side chain properties: (1) hydrophobic amino acids: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic amino acids: Cys, Ser, Thr, Asn, Gln; (3) acidic amino acids: Asp, Glu; (4) basic amino acids: His, Lys, Arg; (5) amino acids that affect chain orientation: Gly, Pro; (6) aromatic amino acids: Trp, Tyr, Phe. Conservative substitutions between amino acids in the above homologous property categories have little or no effect on the function, activity, or other biological properties of the polypeptide.

[0079] In an alternative embodiment, the plant is selected from a Rutaceae plant or a Ranunculaceae Juss. plant.

[0080] In an alternative embodiment, the plant is selected from the genus Cimicifuga or the genus Citrus.

[0081] In an alternative embodiment, the Cimicifuga plant includes Actaea asiatica, preferably Actaea racemosa, and the Citrus plant includes Citrus maxima.

[0082] Hemp plants are widely distributed and have long been used as medicinal plants. In existing phytochemical research reports, the most widely studied ones include Actaea asiatica from China and Actaea racemosa which is widely distributed in Europe and the United States.

[0083] In alternative embodiments, the valencene synthase catalyzes the synthesis of valencene in an expression system.

[0084] In an alternative embodiment, the expression system comprises a fungal expression system.

[0085] In an alternative embodiment, the fungal expression system comprises a yeast expression system.

[0086] In an alternative embodiment, the yeast expression system comprises a Saccharomyces cerevisiae expression system.

[0087] In alternative embodiments, the amino acid sequence of the valencene synthase comprises (a) or (b):

[0088] (a) any one of SEQ ID NOs: 1 to 3;

[0089] (b) a derivative valencene synthase having at least 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, or more amino acid sequence identity to (a).

[0090] In a second aspect, the present invention provides use of the valencene synthase described in any one of the aforementioned embodiments in preparing a flavor composition.

[0091] In a third aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the valencene synthase described in any one of the aforementioned embodiments or a nucleotide sequence complementary to the valencene synthase.

[0092] In an optional embodiment, the nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology to any of the nucleotide sequences shown in SEQ ID No:4 to 6.

[0093] In a fourth aspect, the present invention provides a recombinant expression vector comprising the nucleic acid molecule according to any one of the aforementioned embodiments, wherein the original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

[0094] In a fifth aspect, the present invention provides a transformant, wherein the host cell of the transformant is selected from a fungal expression system, and contains the nucleic acid molecule described in any one of the preceding embodiments or the recombinant expression vector described in the preceding embodiments. Preferably, the fungal expression system is a yeast expression system, and more preferably a cerevisiae expression system.

[0095] In a sixth aspect, the present invention provides a flavor composition comprising 10% to 99% valencene by mass, including but not limited to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 99%; wherein the valencene is synthesized by the valencene synthase described in any of the preceding embodiments.

[0096] In a seventh aspect, the present invention provides a recombinant Saccharomyces cerevisiae, which contains the nucleic acid molecule described in any one of the preceding embodiments, or contains the recombinant expression vector described in any one of the preceding embodiments; preferably, the original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

[0097] In an eighth aspect, the present invention provides a method for preparing the flavor composition described in any of the aforementioned embodiments, the preparation method comprising fermenting and culturing the transformant or recombinant Saccharomyces cerevisiae described in the aforementioned embodiments, and recovering the flavor composition from the fermentation product.

[0098] In an alternative embodiment, the recovery method comprises extracting the fermentation product using an oil phase.

[0099] In an alternative embodiment, the oil phase comprises at least one of isopropyl myristate or n-dodecane.

[0100] In an optional embodiment, the recovery method is to add an oil phase during the fermentation process, collect the oil phase after the fermentation is completed, and separate and obtain the flavor composition enriched in the oil phase.

[0101] In an alternative embodiment, the method of collecting the oil phase comprises centrifugation.

[0102] In an alternative embodiment, the separation method comprises distillation.

[0103] In a ninth aspect, the present invention provides the use of the flavor composition described in any one of the aforementioned embodiments, or the flavor composition obtained by the preparation method described in any one of the aforementioned embodiments, in the preparation of a flavor product.

[0104] In a tenth aspect, the present invention provides a fragrance product comprising the fragrance composition described in any one of the aforementioned embodiments, or the fragrance composition prepared by the method described in any one of the aforementioned embodiments.

[0105] In alternative embodiments, the fragrance product comprises a personal care product, a fabric care product, a cleaning product, a deodorant, a home care product, a fabric product, an air care product, a fragrance delivery system, a cosmetic formulation, a disinfectant, or a food product.

[0106] In an optional embodiment, the personal care product includes: floral water, skin care products, hair care products, antiperspirants, feminine care products, baby care products, oral hygiene products or health care.

[0107] In an alternative embodiment, the skin care product comprises toilet soap, liquid soap or body wash.

[0108] In an alternative embodiment, the oral hygiene product comprises a dental hygiene product.

[0109] In an alternative embodiment, the cosmetic formulation comprises a perfume, a fragrance liquid or an essential oil.

[0110] In an alternative embodiment, the cleaning product comprises a detergent composition.

[0111] In alternative embodiments, the detergent composition comprises a dishwashing detergent, a glass cleaner, a metal cleaner, a countertop cleaner, a floor cleaner, a carpet cleaner, a toilet cleaner, or a bleach additive.

[0112] The following embodiments of the present invention are described in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0113] Example 1 Preparation and Application of Valencene Synthase from Actaea racemosa

[0114] 1.1 Valencene synthase gene mining in Actaea racemosa

[0115] With reference to the published protein sequence information of Actaea racemosa (https: / / ftp.cngb.org / pub / gigadb / pub / 10.5524 / 100001_101000 / 100627 / assemblies / CYVA-Cimicifuga_racemosa / ), we searched for protein sequences containing both the Pfam domains of terpenoid synthase PF01397 and PF03936. A total of 13 potential terpenoid synthase protein sequences were obtained.

[0116] 1.2 Identification of citrus aroma synthases and genes in Actaea racemosa

[0117] 1.2.1 Construction of yeast expression vector

[0118] Primers were designed to construct the gene to be verified on the universal vector pZY900 (using the same method as patent CN202210473488.4).

[0119] The validation process of ArVS and ArVS-V8 from Actaea racemosa is demonstrated here. The amino acid sequences of ArVS and ArVS-V8 are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively:

[0120] The genes ArVS and ArVS-V8 from Actaea racemosa were directly synthesized according to Saccharomyces cerevisiae codon optimization, and the nucleotide sequences are shown in SEQ ID NOs: 4 and 5, respectively.

[0121] Specific gene primer pairs ArVS-1F / R and ArVS-V8-1F / R were designed. The synthesized genes were used as templates and the ArVS and ArVS-V8 gene fragments were obtained by PCR amplification using GXL high-fidelity enzyme from Novozymes. After gel recovery using Tiangen Gum Recovery Kit, the fragments were ligated into the yeast expression vector pZY900 after BsaI cutting using the homologous recombination kit from Yishen Company. After sequencing confirmation, yeast expression vectors containing the ArVS and ArVS-V8 gene fragments were obtained and named pArVS and pArVS-V8. The plasmid maps are shown in Figures 1 and 2, respectively. The lacZ gene in pZY900 was replaced by the ArVS and ArVS-V8 genes, respectively.

[0122] The ArVS-1F / R primer sequences are shown in Table 1 below:

[0123] Table 1

[0124] The ArVS-V8-1F / R primer sequences are shown in Table 1 below:

[0125] Table 2

[0126] 1.2.2 Strain construction

[0127] Plasmids pArVS, pArVS-V8 and pZY900 were respectively introduced into strain JCR27 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 LiAc transformation of Saccharomyces cerevisiae) (the construction of yeast strain JCR27 is shown in the literature Siemon, Thomas et al. "Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block." Journal of the American Chemical Society vol.142,6(2020):2760-2765.doi:10.1021 / jacs.9b12940), and the transformed strains were named JArVS, JArVS-V8 and S900.

[0128] 1.2.3 Strain fermentation and product identification

[0129] Strains JArVS, JArVS-V8, and S900 were inoculated into SC-Ura liquid medium (Li Xiaowei. Engineering the acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015, uracil-deficient medium), and cultured overnight at 30°C and 200 rpm in a shaking incubator. The next day, the inoculation volume was 1% and the inoculation volume was transferred to 45 ml of YPDHG liquid medium (20 g / L peptone, 10 g / L yeast powder, 10 g / L glucose, 10 g / L galactose), 5 ml of isopropyl myristate was added, and the culture was shaken at 30°C and 200 rpm for 72 h. The oil layer was collected and diluted to an appropriate concentration with n-hexane. The product was detected by GC-MS.

[0130] GCMS detection conditions were a Thermo Fisher Scientific TRACE GCμLTRA gas chromatograph equipped with an AS 3000 autosampler and a split / splitless injector, and a TSQ QUANTμm XLS MS with a triple quadrupole detector. The column was a TR-5MS colμmn (30 m × 0.25 mm × 0.25 μm). The carrier gas was high-purity helium at a flow rate of 1 mL / min. Acetone was used as the needle wash. The injection volume was 1 μL, with a split ratio of 50. The inlet temperature was 240°C, and the ion transfer tube temperature was 270°C. The detection procedure was as follows: the column temperature was initially set at 50°C and held for 1 min; the temperature was increased at 15°C / min to 280°C; the temperature was increased at 20°C / min to 300°C and held for 2 min.

[0131] The results showed that no valencene was detected in the oil phase corresponding to the blank control strain S900, while valencene was detected in the oil phase corresponding to the strains JArVS and JArVS-V8, with the valencene production levels being 110 mg / L and 230 mg / L, respectively.

[0132] Example 2 Functional Identification of Valencene Synthase from Citrus maxima (Pomelo, pummelo)

[0133] 2.1 Gene mining for citrus aroma synthesis in Citrus maxima (Pomelo, pummelo)

[0134] With reference to the published protein sequence information of Citrus maxima (http: / / citrus.hzau.edu.cn / data / Genome_info / HWB.v1.0 / HWB.v1.0.protein.fa), we searched for protein sequences containing both the Pfam domains of terpenoid synthase PF01397 and PF03936, and obtained a total of 91 potential terpenoid synthase protein sequences.

[0135] 2.2 Identification of citrus aroma synthases and genes in Citrus maxima (Pomelo, pummelo)

[0136] 2.2.1 Construction of yeast expression vector

[0137] Primers were designed to construct the gene to be verified on the universal vector pZY900 (see patent CN202210473488.4).

[0138] The verification process of CmVS-V4 from Citrus maxima (Pomelo, pummelo) is shown here. The amino acid sequence of CmVS-V4 is:

[0139] The gene CmVS-V4 from Citrus maxima (Pomelo, pummelo) was directly synthesized according to the codon optimization of Saccharomyces cerevisiae, and the sequence is shown in SEQ ID NO: 6:

[0140] A specific gene primer pair, CmVS-V4-1F / R, was designed. Using the synthesized gene as a template, the CmVS-V4 gene fragment was obtained by PCR amplification using Novozymes' GXL high-fidelity enzyme. After gel recovery using the Tiangen Gum Recovery Kit, the fragment was ligated into the yeast expression vector pZY900 after BsaI cutting using the homologous recombination kit from Yishen Company. After sequencing confirmation, a yeast expression vector containing CmVS-V4 was obtained and named pCmVS-V4. The schematic diagram of the construction of plasmid pCmVS-V4 is shown in Figure 3, which replaces the lacZ gene in pZY900 with the gene CmVS-V4. The primer sequences of CmVS-V4-1F / R are shown in Table 3 below.

[0141] Table 3

[0142] 2.2.2 Strain construction

[0143] The plasmid pCmVS-V4 was introduced into strain JCR27 by the lithium acetate method, and the transformed strain was named JCmVS-V4.

[0144] 2.2.3 Strain fermentation and product identification

[0145] The strain JCmVS-V4 was fermented and the product extracted according to Example 1.2.3.

[0146] The results showed that the valencene production of strain JCmVS-V4 was 215 mg / L.

[0147] Comparative Example Expression of Existing Valencene Synthase Gene

[0148] 1.1 Construction of plasmids containing valencene synthase genes from Vitis vinifera (Grape), Citrus sinensis (Sweet orange) (Citrus aurantium var. sinensis), Solanum habrochaites (Wild tomato) (Lycopersicon hirsutum), Solanum lycopersicum (Tomato) (Lycopersicon esculentum), Switchgrass (Panicum virgatum L.), Callitropsis nootkatensis, Eryngium glaciale, and Alpinia oxyphylla Miq.

[0149] The valencene synthases from different sources used in this comparative example are the most extensively studied in the art and are generally recognized to have high yields.

[0150] Primers were designed to construct the codon-optimized valencene synthase encoding gene on the universal vector pZY900.

[0151] The primer sequences designed for each gene are shown in the table below. Using the synthesized genes described above as templates, yeast expression vectors containing the VvVS, CsVS, ShVS, SlVS, PvVS, CnVS, EgVS, and AoVS genes were constructed using the same method as in 2.1 of Example 1. These vectors were designated pVvVS, pCsVS, pShVS, pSlVS, pPvVS, pCnVS, pEgVS, and pAoVS, respectively. A schematic diagram of the construction of the plasmids pVvVS, pCsVS, pShVS, pSlVS, pPvVS, pCnVS, pEgVS, and pAoVS is shown in Figure 4 . These vectors replace the lacZ gene in pZY900 with the VvVS, CsVS, ShVS, SlVS, PvVS, CnVS, EgVS, and AoVS genes, respectively.

[0152] Table 4

[0153] The amino acid sequence of valencene synthase from Vitis vinifera (Grape) is:

[0154] The nucleotide sequence VvVS after codon optimization in Saccharomyces cerevisiae is:

[0155] The nucleotide sequence CsVS after codon optimization of Saccharomyces cerevisiae is:

[0156] The amino acid sequence of valencene synthase from wild tomato (Lycopersicon hirsutum) is:

[0157] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0158] The amino acid sequence of valencene synthase from Solanum lycopersicum (Tomato) (Lycopersicon esculentum) is:

[0159] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0160] The amino acid sequence of valencene synthase from switchgrass (Panicum virgatum L.) is:

[0161] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0162] The amino acid sequence of valencene synthase from Callitropsis nootkatensis is:

[0163] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0164] The amino acid sequence of valencene synthase from Eryngium glaciale is:

[0165] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0166] Plasmid Construction of the Valencene Synthase Gene from Alpinia oxyphylla Miq. The amino acid sequence of the valencene synthase derived from the gene is:

[0167] The nucleotide sequence of Saccharomyces cerevisiae after codon optimization is:

[0168] 1.2 Construction and shake flask fermentation of strains containing valencene synthase genes from Vitis vinifera (Grape), Citrus sinensis (Sweet orange) (Citrus aurantium var. sinensis), Solanum habrochaites (Wild tomato) (Lycopersicon hirsutum), Solanum lycopersicum (Tomato) (Lycopersicon esculentum), Switchgrass (Panicum virgatum L.), Callitropsis nootkatensis, Eryngium glaciale, and Alpinia oxyphylla Miq.

[0169] Plasmids pVvVS, pCsVS, pShVS, pSlVS, pPvVS, pCnVS, pEgVS, and pAoVS were introduced into strain JCR27 using the lithium acetate method, and the transformed strains were named JVvVS, JCsVS, JShVS, JSlVS, JPvVS, JCnVS, JEgVS, and JAoVS, respectively. Fermentation was performed using the same shake flask fermentation conditions and method as in sections 1.2.3 and 1.3 of Example 1. The shake flask fermentation yields of strains JVvVS, JCsVS, JShVS, JSlVS, JPvVS, JCnVS, JEgVS, and JAoVS were 0.35 mg / L, 15 mg / L, 0.06 mg / L, 0.07 mg / L, 1.5 mg / L, 40 mg / L, 80 mg / L, and 65 mg / L, respectively.

[0170] Comparison of Examples 1 and 2 with the comparative example shows that the valencene synthase derived from Actaea racemosa and Citrus maxima (Pomelo, pummelo) provided by the present invention has higher synthesis activity than existing valencene synthases.

[0171] Example 3 Construction of Valencene High-yielding Strain

[0172] 3.1 Recombinant plasmid construction

[0173] 3.1.1 Construction of high-yield plasmid corresponding to ArVS

[0174] Primers ZL-ArVS-1F / R and ZL-ArVS-2F / R were used, respectively, with pYR017 (see Patent 202210473488.4) and pArVS as templates to amplify the long fragment (containing the Ura3 left homology arm, histidine selection marker, CYC1 terminator, tHMG1 gene, GAL1-GAL10 promoter, PGK1 terminator, Ura3 right homology arm, and plasmid backbone) and ArVS. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pArVS-P2.

[0175] The primer sequences of ZL-ArVS-1F / R and ZL-ArVS-2F / R are shown in the following table:

[0176] Primers ZL-ArVS-1F / R and ZL-ArVS-2F / R were used, respectively, with pYR018 (see Patent 202210473488.4) and pArVS as templates to amplify the long fragment (containing the yprcdelta15 left homology arm, tryptophan selection marker, GPM1 terminator, GAL1-GAL10 promoter, PGK1 terminator, yprcdelta15 right homology arm, and plasmid backbone) and ArVS. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pArVS-P3.

[0177] 3.1.2 Construction of high-yield plasmid corresponding to ArVS-V8

[0178] Primers ZL-ArVS-V8-1F / R and ZL-ArVS-V8-2F / R were used to amplify pYR017 (see Patent 202210473488.4) and pArVS-V8 as templates, respectively, to obtain a long fragment (containing the Ura3 left homology arm, histidine selection marker, CYC1 terminator, tHMG1 gene, GAL1-GAL10 promoter, PGK1 terminator, Ura3 right homology arm, and plasmid backbone) and ArVS-V8. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pArVS-V8-P2.

[0179] The primer sequences of ZL-ArVS-V8-1F / R and ZL-ArVS-V8-2F / R are shown in the following table:

[0180] Primers ZL-ArVS-V8-1F / R and ZL-ArVS-V8-2F / R were used to amplify pYR018 (see Patent 202210473488.4) and pArVS-V8 as templates, respectively, to obtain a long fragment (containing the yprcdelta15 left homology arm, tryptophan selection marker, GPM1 terminator, GAL1-GAL10 promoter, PGK1 terminator, yprcdelta15 right homology arm, and plasmid backbone) and ArVS-V8. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pArVS-V8-P3.

[0181] 3.1.3 Construction of high-yield plasmid corresponding to CmVS-V4

[0182] Primers ZL-CmVS-V4-1F / R and ZL-CmVS-V4-2F / R were used, respectively, with pYR017 (see Patent 202210473488.4) and pCmVS-V4 as templates to amplify the long fragment (containing the Ura3 left homology arm, histidine selection marker, CYC1 terminator, tHMG1 gene, GAL1-GAL10 promoter, PGK1 terminator, Ura3 right homology arm, and plasmid backbone) and CmVS-V4. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pCmVS-V4-P2.

[0183] The primer sequences of ZL-ArVS-1F / R and ZL-ArVS-2F / R are shown in the following table:

[0184] Primers ZL-CmVS-V4-1F / R and ZL-CmVS-V4-2F / R were used to amplify pYR018 (see Patent 202210473488.4) and pCmVS-V4 as templates, respectively, to obtain a long fragment (containing the yprcdelta15 left homology arm, tryptophan selection marker, GPM1 terminator, GAL1-GAL10 promoter, PGK1 terminator, yprcdelta15 right homology arm, and plasmid backbone) and CmVS-V4. These fragments were then recombined in Saccharomyces cerevisiae using yeast assembly methods to construct pCmVS-V4-P3.

[0185] 3.1.4 Construction of gene editing plasmids

[0186] The fragment was amplified using primers GHX07-1F / R and pKlURA100 (see patent 202210473488.4) as a template. The fragment was then assembled with pCas (see patent 202210473488.4) using the Goldengate method to construct plasmid pGHX07 targeting the ACH-TPS07 gene in strain LXF-5 (see patent 202210473488.4).

[0187] The primer sequences for GHX07-1F / R are shown in the following table:

[0188] 3.2 Construction of high-yield strains

[0189] 3.2.1 Construction of high-yield strains corresponding to ArVS

[0190] Plasmids pArVS and pArVS-P2 were linearized using MssI, and plasmid pArVS-P3 was linearized using NotI, and the fragment containing ArVS was recovered from the gel.

[0191] Plasmid pGHX07 (transfer plasmid) and the linearized fragment of pArVS, the linearized fragment of pArVS-P2, and the linearized fragment of pArVS-P3 were co-transferred into the strain LXF-5 via the lithium acetate method (see Patent 202210473488.4). The colonies were verified for the correctness of the strain by PCR, and then cultured with YPD (tryptone, yeast extract, glucose) and washed with water. The bacteria were streaked onto 5-FOA plates, and colonies were picked from the plates to continue colony PCR verification for the correctness of the knockout. The correct strain was named JArVS-P1; the pZY521 knockout cassette (see Patent 202210473488.4) was transferred into the strain JArVS-P1 to knock out the transcription repressor GAL80, obtaining the strain JArVS-P2.

[0192] 3.2.2 Construction of high-yield strain corresponding to ArVS-V8

[0193] Plasmids pArVS-V8 and pArVS-V8-P2 were linearized using MssI, and plasmid pArVS-V8-P3 was linearized using NotI, and the fragment containing ArVS-V8 was recovered from the gel.

[0194] Plasmid pGHX07 (transfer plasmid) and the linearized fragment of pArVS-V8, the linearized fragment of pArVS-V8-P2, and the linearized fragment of pArVS-V8-P3 were co-transferred into the strain LXF-5 by the lithium acetate method (see Patent 202210473488.4). The colonies were verified for the correctness of the strain by PCR, and then cultured with YPD (tryptone, yeast extract, glucose) and washed with water. The bacteria were streaked onto 5-FOA plates, and bacteria were picked from the plates to continue colony PCR to verify the correctness of the knockout. The correct strain was named JArVS-V8-P1; the pZY521 knockout cassette (see Patent 202210473488.4) was transferred into the strain JArVS-V8-P1 to knock out the transcription repressor GAL80, obtaining the strain JArVS-V8-P2.

[0195] 3.2.3 Construction of high-yield strain corresponding to CmVS-V4

[0196] Plasmids pCmVS-V4 and pCmVS-V4-P2 were linearized using MssI, and plasmid pCmVS-V4-P3 was linearized using NotI. The fragment containing CmVS-V4 was recovered from the gel.

[0197] Plasmid pGHX07 (transfer plasmid) and the linearized fragment of pCmVS-V4, the linearized fragment of pCmVS-V4-P2, and the linearized fragment of pCmVS-V4-P3 were jointly transferred into the strain LXF-5 by the lithium acetate method (see Patent 202210473488.4). The correctness of the strain was verified by PCR, and then the colonies were cultured with YPD (tryptone, yeast extract, glucose) and washed with water. The bacteria were streaked on 5-FOA plates, and the bacteria were picked from the plates and continued to be verified by colony PCR for the correctness of the knockout. The correct strain was named JCmVS-V4-P1; the pZY521 knockout cassette (see Patent 202210473488.4) was transferred into the strain JCmVS-V4-P1 to knock out the transcription repressor GAL80, obtaining the strain JCmVS-V4-P2.

[0198] 3.3 Strain shake flask fermentation and fermentation tank evaluation

[0199] Strains JArVS-P1, JArVS-P2, JArVS-V8-P1, JArVS-V8-P2, JCmVS-V4-P1, and JCmVS-V4-P2 were inoculated into YPD liquid medium, respectively, and cultured overnight at 30°C and 200 rpm. The next day, the strains were transferred to 45 ml of YPDHG liquid medium (JArVS-P1, JArVS-V8-P1, JCmVS-V4-P1) or YPD liquid medium (JArVS-P2, JArVS-V8-P2, JCmVS-V4-P2) according to the initial OD600 = 0.1, 5 ml of isopropyl myristate was added, and the cultures were cultured at 30°C and 200 rpm for 72 h, and the oil layer was collected. Among them, the valencene production of JArVS-P1, JArVS-P2, JArVS-V8-P1, JArVS-V8-P2, JCmVS-V4-P1, and JCmVS-V4-P2 were 186 mg / L, 203 mg / L, 414 mg / L, 603 mg / L, 344 mg / L, and 468 mg / L, respectively.

[0200] Referring to the fermentation tank culture medium and fermentation method described in the literature (Ye Z, Huang Y, Shi B, et al. Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of (+)-valencene and its chemical conversion to (+)-nootkatone [published online ahead of print, 2022 Mar 13]. Metab Eng. 2022; 72: 107-115. doi: 10.1016 / j.ymben.2022.03.005), the constructed strains JArVS-P2, JArVS-V8-P2, and JCmVS-V4-P2 were subjected to fed-batch fermentation. During the fermentation process, a covering agent (isopropyl myristate) was added to enable in situ extraction. During the fermentation process, the dissolved oxygen content was controlled to be above 20%, the pH was 5, the glucose concentration was 1-2 g / L, and the ethanol concentration was below 5 g / L. Finally, in a 1L fermentation tank, the valencene production of the strains reached 6.1g / L, 17.5g / L and 13.1g / L respectively.

[0201] Example 4 Fermentation of Valencene Synthase with Highly Homological Amino Acid Sequences

[0202] Based on the amino acid sequences of ArVS and ArVS-V8 provided in Example 1, and CmVS-V4 provided in Example 2, amino acid sequences with 80%, 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97%, and 99% homology, respectively, were designed. The corresponding nucleotide sequences were optimized according to the codons of Saccharomyces cerevisiae, and the corresponding upstream and downstream primers were then constructed. Then, strains expressing valencene synthase with the homologous amino acid sequences were constructed according to the method of Example 1, and fermentation was carried out. The results showed that the valencene production obtained by fermentation of strains corresponding to ArVS, ArVS-V8, and CmVS-V4 with 83% or greater homology was comparable to that of Examples 1 and 2, respectively. However, the valencene production obtained by fermentation of strains corresponding to ArVS, ArVS-V8, and CmVS-V4 with 80% homology decreased by more than 93% compared to the production of strains corresponding to 83% homology.

[0203] The different homologous amino acid sequences, nucleotide sequences and corresponding primers are shown in the following table:

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

[0205] Partial nucleotide and amino acid sequences are as follows:

Claims

1. Valencene synthase, characterized in that The valencene synthase comprises domains with Pfam numbers PF01397 and PF03936 and has valencene synthase activity.

2. The valencene synthase according to claim 1, characterized in that The valencene synthase includes (a) a complete protein or a functional protein fragment derived from a plant; or (b) a derived valencene synthase having at least 80% amino acid sequence homology with (a).

3. The valencene synthase according to claim 2, characterized in that The plant is selected from the Rutaceae plant or the Ranunculaceae Juss. plant.

4. The valencene synthase according to claim 2 or 3, characterized in that The plant is selected from the group consisting of Cimicifuga plants or Citrus L. plants.

5. The valencene synthase according to claim 4, characterized in that The Cimicifuga plant includes Actaea asiatica, preferably Actaea racemosa, and the Citrus plant includes Citrus maxima.

6. The valencene synthase according to any one of claims 1 to 5, characterized in that The valencene catalyzes the synthesis of valencene in the expression system.

7. The valencene synthase according to claim 6, characterized in that The expression system includes a fungal expression system.

8. The valencene synthase according to claim 7, characterized in that The fungal expression system includes a yeast expression system.

9. The valencene synthase according to claim 8, characterized in that The yeast expression system includes a Saccharomyces cerevisiae expression system.

10. The valencene synthase according to any one of claims 1 to 9, characterized in that The amino acid sequence of the valencene synthase comprises (a) or (b): (a) any one of SEQ ID NOs: 1 to 3; (b) a derivative valencene synthase having at least 83%, 85%, 87%, 89%, 91%, 93%, 95%, 97% or more amino acid sequence identity to (a).

11. Use of the valencene synthase according to any one of claims 1 to 10 in the preparation of a flavor composition.

12. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the valencene synthase according to any one of claims 1 to 10 or a nucleotide sequence complementary to the valencene synthase.

13. The nucleic acid molecule according to claim 12, characterized in that The nucleic acid molecule comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology with any of the nucleotide sequences shown in SEQ ID No: 4 to 6.

14. A recombinant expression vector containing the nucleic acid molecule according to claim 12 or 13, characterized in that: The original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

15. A transformant containing the nucleic acid molecule according to claim 12 or 13, or containing the recombinant expression vector according to claim 14, wherein the host cell of the transformant is selected from a fungal expression system, preferably a yeast expression system, and more preferably a cerevisiae expression system.

16. Recombinant Saccharomyces cerevisiae, characterized in that The recombinant Saccharomyces cerevisiae contains the nucleic acid molecule according to claim 12 or 13, or contains the recombinant expression vector according to claim 14; preferably, the original vector of the recombinant expression vector is pZY900, pYR017 or pYR018.

17. A fragrance composition, characterized in that The flavor composition comprises 10% to 99% valencene by mass; the valencene is synthesized by catalysis of the valencene synthase according to any one of claims 1 to 10.

18. The method for preparing the flavor composition according to claim 17, characterized in that: The preparation method comprises fermenting and culturing the transformant according to claim 15 or the recombinant Saccharomyces cerevisiae according to claim 16, and recovering the flavor composition from the fermentation product.

19. The preparation method according to claim 18, characterized in that: The recovery method includes extracting the fermentation product using an oil phase.

20. The preparation method according to claim 19, characterized in that: The oil phase includes at least one of isopropyl myristate or n-dodecane.

21. The preparation method according to claim 19, characterized in that: The recovery method is to add the oil phase during the fermentation process, collect the oil phase after the fermentation is completed, and separate and obtain the flavor composition enriched in the oil phase.

22. The preparation method according to claim 21, characterized in that: The method of collecting the oil phase includes centrifugation.

23. The preparation method according to claim 21, characterized in that: The separation method includes distillation.

24. Use of the flavor composition according to claim 17, or the flavor composition obtained by the preparation method according to any one of claims 18 to 23, in the preparation of flavor products.

25. A flavor product comprising the flavor composition of claim 17, or a flavor composition obtained by the preparation method of any one of claims 18 to 23.

26. The scented product according to claim 25, characterized in that The fragrance product includes: a personal care product, a fabric care product, a cleaning product, a deodorant, a home care product, a fabric product, an air care product, a fragrance delivery system, a cosmetic preparation, a disinfectant or a food.

27. The scented product according to claim 26, characterized in that The personal care products include: floral water, skin care products, hair care products, antiperspirants, feminine care products, baby care products, oral hygiene products or health care.

28. The fragrance product according to claim 27, characterized in that The skin care product includes soap, liquid soap or shower gel.

29. The scented product of claim 27, wherein: The oral hygiene products include dental hygiene products.

30. The scented product of claim 26, wherein: The cosmetic preparations include perfumes, fragrance liquids or essential oils.

31. The scented product of claim 26, wherein: The cleaning products include detergent compositions.

32. The scented product according to claim 31, characterized in that The decontamination composition includes a dishwashing detergent, a glass cleaner, a metal cleaner, a countertop cleaner, a floor cleaner, a carpet cleaner, a toilet cleaner or a bleach additive.

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