Functional monoterpenoid production system with redesigned non-mevalonate metabolic pathway

Abstract

The present invention relates to a monoterpenoid production system comprising a redesigned non-mevalonic acid metabolic pathway, and more specifically, to a gene expression cassette for monoterpenoid production comprising DXS, DXR and / or LIS genes, a recombinant vector comprising the same, a plant transformed with said recombinant vector, a method for producing a transformed plant using said recombinant vector, and a method for mass production of monoterpenoids, thereby enabling the mass production of monoterpenoids with high industrial added value as synthetic raw materials for flavors and pharmaceuticals as secondary metabolites.

Description

Technology Field

[0001] The present invention relates to a monoterpenoid production system comprising a redesigned non-mevalonic acid metabolic pathway, and more specifically, to a gene expression cassette for monoterpenoid production comprising DXS, DXR and / or LIS genes and a method for monoterpenoid production using the same. Background Technology

[0003] Plants are known to produce tens of thousands of types of phytochemicals possessing various functionalities, such as anticancer, anti-inflammatory, and antioxidant properties. Among these phytochemicals, monoterpenoids, which are secondary metabolites produced by plants, are experiencing a surge in demand as raw materials for the synthesis of fragrances and pharmaceuticals.

[0004] Linalool is an important monoterpenoid for floral scents and is mainly used as a cosmetic ingredient. It is attracting attention as a material for cosmetics, perfumes, and medicines due to its ability to inhibit angiogenesis, but the supply of natural linalool is significantly insufficient compared to the demand.

[0005] Among monoterpenoids with high industrial added value, linalool is known to exist in small quantities in plants and suffers significant losses during the extraction process due to its volatility; therefore, there is a growing need for technology capable of mass-producing this functional component in small amounts by utilizing synthetic biology techniques, such as by enhancing production within the plant itself. Prior art literature

[0007] Republic of Korea Registered Patent 10-1834020 The problem to be solved

[0008] The present invention aims to solve the aforementioned problem and other related problems.

[0009] One exemplary object of the present invention is to provide a gene expression cassette for monoterpenoid production comprising: a promoter; and at least one gene selected from the group consisting of DXS, DXR, and LIS.

[0010] Another exemplary objective of the present invention is to provide a recombinant vector comprising the gene expression cassette.

[0011] Another exemplary objective of the present invention is to provide a transformant transformed with the recombinant vector.

[0012] Another exemplary objective of the present invention is to provide a transgenic plant transformed with the recombinant vector or the transgenic organism.

[0013] Another exemplary object of the present invention is to provide a method for producing a transgenic plant for monoterpenoid production, comprising the steps of: (a) preparing a recombinant vector comprising an expression cassette comprising a promoter and at least one gene selected from the group consisting of DXS, DXR, and LIS; and (b) transforming a plant with said recombinant vector.

[0014] Another exemplary object of the present invention is to provide a method for producing monoterpenoids comprising: (a) transforming a plant with a recombinant vector comprising an expression cassette comprising a promoter and at least one gene selected from the group consisting of DXS, DXR, and LIS; (b) growing and obtaining the transformed plant; and (c) isolating monoterpenoids from the obtained plant.

[0016] The technical problems to be solved according to the technical concept of the invention disclosed in this specification are not limited to those for solving the problems mentioned above, and other unmentioned problems will be clearly understood by a person skilled in the art from the description below. means of solving the problem

[0018] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application should not be considered limited by the specific descriptions provided below.

[0019] In one embodiment for achieving the above objective, the present invention provides a gene expression cassette for monoterpenoid production comprising: a promoter; and at least one gene selected from the group consisting of DXS, DXR, and LIS.

[0020] For example, at least one gene selected from the group consisting of the above DXS, DXR, and LIS may be a single gene such as the DXS gene, DXR gene, or LIS gene, or may include the DXS and DXR gene, DXS and LIS gene, or DXR and LIS gene; or may include a combination of two or more genes, i.e., multiple genes, such as the DXS, DXR, and LIS gene.

[0021] The gene expression cassette containing the above multiple genes may be such that a gene expression cassette containing two or more single genes is expressed, or a gene expression cassette containing two or more genes is expressed.

[0022] In the present invention, the DXS, DXR, and LIS genes are genes involved in linalool biosynthesis, and the host plant is tobacco ( Nicotiana benthamiana ) closely related tobacco ( Nicotiana tabacum ) and Klarchia( Clarkia breweriSelected from ). The DXS gene is a gene (Accession number: EU650419) that encodes an enzyme involved in the reaction of pyruvate and glyceraldehyde triphosphate (G3P) to produce 1-deoxy-D-xylulose 5-phosphate (DXP), the DXR gene is a gene (Accession number: DQ839130) that encodes an enzyme involved in the production of 2-C-methylerythritol 4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP), and the LIS gene is a gene (Accession number: U58314) that encodes an enzyme involved in the production of linalool from geranyl diphosphate (GPP).

[0023] In the present invention, the DXS, DXR, and LIS genes may consist of the conventionally known DXS, DXR, and LIS gene sequences themselves or codon-optimized sequences tailored to the plant to be transformed. As an example, the DXS gene is composed of the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto if variations having biologically equivalent activity are considered. In other words, a sequence having 'substantial identity' with the nucleotide sequence of the DXS gene disclosed in the prior art (SEQ ID NO. 1) may be included within the scope of the present invention, and may mean, for example, a sequence exhibiting 80% or more sequence homology or 90% or more sequence homology.

[0024] Likewise, the DXR or LIS gene is composed of the nucleotide sequence of SEQ ID NO. 2 or SEQ ID NO. 3, but is not limited thereto if variations having biologically equivalent activity are considered, and sequences having 'substantial identity' with the nucleotide sequence of the DXR gene (SEQ ID NO. 2) or the nucleotide sequence of the LIS gene (SEQ ID NO. 3) disclosed in the prior art may be included in the scope of the present invention, and may mean, for example, sequences exhibiting 80% or more sequence homology or 90% or more sequence homology.

[0025] As an example, the DXS, DXR, and LIS genes of the present invention may be nucleotide sequences having sequence identity within 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100% of the nucleotide sequences of SEQ ID NOs 1 to 3, within the range in which they are translated into proteins and exhibit the biological activity of each enzyme.

[0026] In the present invention, 'monoterpenoid' refers to a secondary metabolite produced from plants that is widely used as a synthetic raw material for fragrances and pharmaceuticals, and is known to have high industrial added value. Examples of monoterpenoids include linalool, β-pinene, and borneol. Linalool is a natural monoterpenoid present in small amounts in flowers and spice plants, and is mainly used as a cosmetic ingredient. It is known to have effects such as inhibiting angiogenesis or inhibiting the biosynthesis of cholesterol and triglycerides in the human body through antibiotic, anti-inflammatory, and antioxidant actions. β-pinene is a natural monoterpenoid abundantly emitted by trees in forests, and is known to have effects such as skin whitening, wrinkle improvement, elasticity enhancement, and skin moisturization. Borneol is a monoterpenoid that is one of the active ingredients of mugwort, and is known to have an effect of alleviating pain and inflammation.

[0027] In the present invention, “gene expression cassette” refers to an expression structure capable of expressing a gene.

[0028] In the present invention, the gene expression cassette for monoterpenoid production may mean the enhancement of monoterpenoid production.

[0029] In the present invention, the enhancement of monoterpenoid production includes causing monoterpenoids to be produced in plants that do not produce monoterpenoids, or increasing the production of monoterpenoids in plants that produce monoterpenoids.

[0030] In this invention, 'promoter' refers to an upstream region of DNA from a structural gene and is a DNA molecule to which RNA polymerase binds to initiate transcription. A 'plant promoter' is a promoter capable of initiating transcription in plant cells. A 'constitutive promoter' is a promoter that is active under most environmental conditions, developmental states, or cell differentiation.

[0031] In the present invention, the promoter refers to an upstream region of a gene to which RNA polymerase binds to initiate the transcription of the gene, and is not limited to any type of promoter as long as it can efficiently express proteins encoded by the DXS, DXR, and LIS genes. The type of promoter can be a conventional promoter, such as the CaMV 35S promoter, SlRbcsl promoter, ProG10 promoter, AtUBQ promoter, AtRPS5a promoter, RPS5A (ribosomal protein subunit 5a) promoter, Agrobacterium tumefaciens Nopaline synthase (NOS) promoter, Agrobacterium tumefaciens octopine synthase gene (OCS) promoter, small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) promoter, and preferably the CaMV 35S promoter, SlRbcs1 promoter, and AtRPS5 promoter, but is not limited thereto.

[0033] Another aspect of the present invention for achieving the above objective provides a recombinant vector for monoterpenoid production comprising the gene expression cassette.

[0034] In the present invention, the recombinant vector may specifically include a DXS, DXR, or LIS gene, or include at least two genes selected from the group consisting of DXS, DXR, and LIS.

[0035] As an example, the recombinant vector may include one of the DXS, DXR, and LIS genes, or two or more genes such as the DXS and LIS genes, the DXR and LIS genes, or a combination of the DXS, DXR, and LIS genes.

[0036] The above 'DXS, DXR, and LIS genes', 'gene expression cassette', and 'monoterpenoid' are as described above.

[0037] In the present invention, the recombinant vector is referred to as an 'expression vector,' which is a vector capable of expressing a target protein or nucleic acid (RNA) in a suitable host cell, and refers to a genetic construct comprising essential regulatory elements operably linked to enable the expression of a gene insert. It refers to a bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector, and generally, any plasmid or vector may be used if it can replicate and stabilize within the host.

[0038] In the present invention, the recombinant vector may be one in which the DXS, DXR and / or LIS genes are operably linked to a promoter.

[0039] In the present invention, the term “operably linked” refers to a state in which a nucleic acid expression control sequence and a sequence encoding a target protein are functionally linked to each other to perform a general function. The term “expression control sequence” refers to a DNA sequence that regulates the expression of an operantly linked polynucleotide sequence in a specific host cell. Such a control sequence includes a promoter for carrying out transcription, any operator sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer, etc.

[0040] In the present invention, 'recombinant' can be used interchangeably with 'genetic manipulation' and refers to the production of a form of gene that does not exist in nature by using molecular cloning experimental techniques such as modifying, cutting, and linking genes.

[0041] The expression vector comprising the DXS, DXR, and LIS gene sequences of the present invention and a suitable transcription / translation regulatory signal can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be effectively linked to a suitable promoter within the expression vector to lead mRNA synthesis. Additionally, the expression vector may include a ribosome binding site and a transcription terminator as a translation initiation site.

[0042] In the present invention, as an example, the pICH41308 vector may be used as the host vector for the preparation of the recombinant vector, and any Agrobacterium commonly used for plant transformation may be used as the Agrobacterium used in the present invention, and specifically, Agrobacterium tumefaciens GV3101 may be used.

[0043] The expression vector may include one or more selectable markers to distinguish transformed cells, said markers being nucleic acid sequences having characteristics that can typically be selected by chemical methods, and any gene capable of distinguishing transformed cells from non-transformed cells is included. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, and aadA genes.

[0044] In the recombinant vector of the present invention, a conventional terminator may be used, for example, Cauliflower Mosaic Virus 35S (CaMV 35S) terminator, AtuNos terminator, Ocs (A. tumefaciens octopine synthase gene) terminator, Mas (A. tumefaciens mannopine synthase) terminator, Atug7 (7th gene isolated from Agrobacterium tumefaciens L.) terminator, actin2 terminator, tomato ATPase terminator, potato Histone H4 terminator, etc., and preferably, CaMV 35S terminator and AtuNos terminator may be used, but are not limited thereto.

[0046] Another aspect of the present invention for achieving the above objective provides a transformant transformed with the recombinant vector.

[0047] Any host cell known in the art that can stably clone and express the recombinant vector may be used as the transformant of the present invention, and may be, for example, Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, Staphylococcus, and Agrobacterium tumefaciens, preferably Agrobacterium tumefaciens, and as an example, Agrobacterium tumefaciens GV3101.

[0049] Another aspect of the present invention for achieving the above objective provides a transgenic plant for producing transgenic monoterpenoids using a recombinant vector comprising DXS, DXR and / or LIS genes or a transgenic plant transformed with such a vector.

[0050] The plant in the present invention may preferably be a seed plant that reproduces by producing and spreading seeds, and may include both gymnosperms and angiosperms; angiosperms may include both dicotyledons and monocotyledons. Seed plants are broadly composed of vegetative organs such as roots, stems, and leaves, and reproductive organs such as flowers, and include seeds, which are plants in an undeveloped embryonic state. The plant in the present invention may be a whole plant having a complete structure, or a part of a plant composed of plant organs, tissues such as roots, stems, leaves, flowers, and seeds, or a number of plant cells, and may be a culture of plant cells or tissues. The part of the plant may be connected to the whole plant or may be separated.

[0051] In addition, the type of cells or tissues of the transformed plant according to the present invention is not limited as long as they are derived from a plant. Examples of plant tissues include epidermal tissue composed of epidermal cells constituting the surface of a plant, stomatal tissue composed of two guard cells in the epidermis, hair formed from epidermal cells, root hair formed from epidermal cells of a root, parenchyma tissue including parts excluding the vascular bundles of a stem or root and photosynthetic mesophyll tissue (palm tissue, spongy tissue), hypertrophied posterior tissue from the parenchyma tissue of a stem or petiole, thickened membrane tissue with a hypertrophied cell wall, xylem or tracheids involved in the movement of water within the plant body, xylem including xylem parenchyma tissue and xylem fibers, phloem parenchyma tissue responsible for the transport of organic matter, phloem fibers, phloem tissue including companion cells, etc.

[0052] The plants of the present invention may be used without limitation as long as they are plants capable of mass-producing monoterpenoids by DXS, DXR and / or LIS gene expression, for example, tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, sunflower, alfalfa, sorghum, wheat, cotton, peanut, tomato, potato, lettuce, or chili pepper, and more specifically, tobacco.

[0053] In the present invention, tobacco is a plant of the genus Nicotiana, and as long as it can overexpress the DXS, DXR, and / or LIS genes, its type is not particularly limited; the present invention can be implemented by selecting an appropriate variety suitable for the purpose of the transformation method and the mass production of monoterpenoids. For example, closely related tobacco ( Nicotiana tabacum ) and Klarchia( Clarkia breweri Varieties such as ) can be used.

[0054] Plants transformed using a recombinant vector containing DXS, DXR, and / or LIS genes according to the present invention or a transformant transformed with such a vector may be produced using plant transformation methods known in the art without limitation. Examples of plant transformation methods may include a method of fusing a liposome containing a recombinant vector according to the present invention with a plant protoplast, a method of injecting a recombinant vector into a plant protoplast using PEG, a method of direct injection of said recombinant vector into a plant cell, a microparticle impaction method, a gene gun, an electroporation method, a transformation method using a virus, a vacuum infiltration method, a floral meristem dipping method, etc., and preferably, a transformation method using Agrobacterium may be used.

[0056] Another aspect of the present invention for achieving the above objective provides a method for producing a transgenic plant comprising: (a) a recombinant vector comprising a gene expression cassette for monoterpenoid production, comprising a promoter; and at least one gene selected from the group consisting of DXS, DXR, and LIS; and (b) a step of transgenicizing a plant with said recombinant vector.

[0057] The above 'DXS, DXR and LIS genes', 'gene expression cassette', 'recombinant vector', 'monoterpenoid', 'plant and transformation' are as described above.

[0058] The method for transforming the above-mentioned plant may use any plant transformation method known in the art without limitation. A person skilled in the art may select and implement a known transformation method suitable for a specific plant by considering the characteristics of the plant selected as a host. Examples of plant transformation methods may include a method of fusing a liposome containing a recombinant vector with a plant protoplast, a method of injecting a recombinant vector into a plant protoplast using PEG, a method of direct injection of the recombinant vector into a plant cell, a microparticle impaction method, a gene gun, electroporation, a transformation method using a virus, a vacuum infiltration method, a floral meristem dipping method, etc., and preferably, a transformation method using Agrobacterium may be used.

[0059] The above 'transformation method using Agrobacterium' is a method of delivering foreign genes into plant cells using Agrobacterium, a Gram-negative bacterium found in soil that causes tumors in the roots and stems of plants. It is a method that utilizes the phenomenon in which T-DNA (transfer DNA) of a tumor-inducing plasmid (Ti plasmid) found in Agrobacterium species such as Agrobacterium tumefaciens and Agrobacterium rhizogenes is inserted into the plant genome.

[0060] Using the transformation method using Agrobacterium in the present invention, DXS, DXR, and / or LIS genes may be transiently expressed or stably expressed.

[0061] In addition, using the transformation method using Agrobacterium in the present invention, CMK, IDI, and / or GPPS genes may be transiently expressed and stablely expressed in addition to DXS, DXR, or LIS genes.

[0062] For the aforementioned transient expression, a part of a plant, for example, a plant leaf, is transformed by infecting it with Agrobacterium containing a recombinant vector, and the infected part can be obtained from the plant after a sufficient amount of time has passed for the target gene to be expressed. For stable expression, plant cells or tissues are cultured, infected with Agrobacterium, and transformed; then, through further culture, suitable transformants are selected and subjected to a regeneration process to be cultured into transformants with a complete structure. By obtaining seeds from the transformed plants and germinating them, stable transformants can be obtained in the next generation as well.

[0063] In the present invention, in the step of infecting a plant with the Agrobacterium to transform it, Agrobacterium containing the p19 gene may be co-transfected to increase the expression stability of the foreign gene by suppressing RNA silencing.

[0065] Another aspect of the present invention for achieving the above objective provides a method for producing monoterpenoids comprising: (a) transforming a plant with a recombinant vector comprising a promoter and at least one gene selected from the group consisting of DXS, DXR, and LIS; (b) culturing and obtaining the transformed plant; and (c) isolating monoterpenoids from the obtained plant.

[0066] The above 'DXS, DXR and LIS genes', 'gene expression cassette', 'recombinant vector', 'monoterpenoid', 'method for transforming plants', and 'range and types of plants to be transformed' are as described above.

[0067] (b) The step of cultivating the above-mentioned plant means providing environmental conditions such as light, temperature, and humidity necessary for plant growth, as well as elements necessary for plant growth such as water, inorganic salts, nutrients, and hormones, during the time required to express a desired amount of gene after the plant has been transformed. When cells, tissues, or cultures thereof isolated from a plant are transformed, the elements necessary for plant tissue culture, such as water, nutrients, inorganic salts, and growth regulators, may be delivered through a culture medium. Additionally, when the DXS, DXR, and / or LIS genes according to the present invention are expressed in the plant using an inducible promoter, or when the CMK, IDI, and / or GPPS genes are additionally expressed, the plant may be cultivated while applying the corresponding stimulus necessary to activate the inducible promoter, such as light, heat, or hormones.

[0068] The step of obtaining the above-mentioned plant means obtaining the whole or part of a plant that is transformed and overexpresses the desired gene. For example, it may involve obtaining a transformed part such as a root, stem, or leaf that overexpresses the DXS, DXR, and / or LIS genes of the present invention, or seeds of a transformed plant body, and it may also involve obtaining a culture of plant cells or tissues, such as a callus or protoplast transformed with the DXS, DXR, and / or LIS genes.

[0069] (c) The step of isolating monoterpenoids from the obtained plants of step (c) involves, for example, crushing the transgenic plants and filtering them to extract monoterpenoids. The extracted monoterpenoids can be further isolated with high purity by performing known filtration methods such as chromatography. Additionally, pretreatment such as freezing and drying the plants can be performed to extract monoterpenoids. Monoterpenoids can be mass-produced by rapidly multiplying transgenic plants expressing DXS, DXR, and / or LIS genes in large quantities using the production method of the present invention. Effects of the invention

[0071] The present invention provides a functional monoterpenoid production system comprising a redesigned non-mevalonic acid metabolic pathway, in particular a gene expression cassette for monoterpenoid production comprising DXS, DXR and / or LIS genes, a recombinant vector comprising the same, a plant transformed with said recombinant vector, a method for producing a transformed plant using said recombinant vector, and a method for producing monoterpenoids.

[0072] Specifically, the present invention enables the mass production of monoterpenoids, which are high-value industrial and medical materials, without the need for a separate substrate by expressing DXS, DXR, and LIS genes as a single or multiple expression in crops such as tobacco. Brief explanation of the drawing

[0074] Figure 1 shows the linalool biosynthetic pathway. Figure 2a is a schematic diagram showing the configuration of a single gene expression cassette (module), and Figure 2b is the result of confirming the gene expression level by qRT-PCR in tobacco leaves in which the single gene expression cassette (level 1 module) of Figure 2a was transiently expressed. Figure 3 shows the results of confirming the linalool production level during transient expression of a single gene expression cassette. Figure 4 shows the results of analyzing the linalool content in the case where single gene expression cassettes were combined (DXS+LIS; DXR+LIS; DXS+DXR+LIS) and co-transfected into tobacco leaves. Figure 5 shows the results of analyzing the beta-pinene content when single gene expression cassettes (DXS, DXR, CMK, IDI, GPPS, and LIS) were co-transfected into tobacco leaves, either individually or in combination. Figure 6 shows the results of analyzing the borneol content when single gene expression cassettes (DXS, DXR, CMK, IDI, GPPS, and LIS) were co-transfected into tobacco leaves, either individually or in combination. Specific details for implementing the invention

[0075] The present invention will be explained in more detail below through the following examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0077] Example 1. Selection of Linalool Biosynthetic Gene

[0078] To redesign the metabolic pathway of linalool among monoterpenoids, six genes previously reported to be involved in linalool biosynthesis were used in the host plant tobacco ( Nicotiana benthamiana ) closely related tobacco ( Nicotiana tabacum ) and Klarchia( Clarkia breweri Selected from ) (Fig. 1 and Table 1).

[0079] No. name channel Access number CDS size (bp) Sequence number base amino acids 1 DXS Pyruvate + G3P → DXP EU650419 2154 1 7 2 DXR DXP → MEP DQ839130 1422 2 8 3 LIS GPP → Linalool U58314 2613 3 9 4 CMK CDP-ME → CDP-MEP KJ159923 1230 4 10 5 IDI DMAPP ↔ IPP AB049815 873 5 11 6 GPPS DMAPP + IPP → GPP KF977582 1248 6 12

[0081] Example 2. Construction of recombinant vector and tobacco leaf transformation

[0082] To simplify the metabolic pathway, recombinant vectors were constructed by introducing six previously selected genes into a level 0 vector (pICH41308) provided by the MoClo system (modular cloning system, Addgene). The level 0 module containing the nucleotide sequence of each gene was used to construct a level 1 module that could be introduced into and expressed in plants using different promoter and terminator modules (level 0, MoClo system, and Table 2) (Fig. 2a).

[0083] division name Sequence number Promoter CaMV35S-D 13 SlRbcs1 14 ProG10-90 15 CaMV35S-L 16 AtUBQ-10 17 AtRPS5a 18 Terminator CaMV35S-ter 19 AtuNos-ter 20

[0084] Level 1 plasmid DNAs of each gene were transformed into Agrobacterium cells (GV3101) using the freeze-thaw method. Agrobacterium cells containing each module were diluted to an OD600 of 0.8 in a solution (infiltration buffer, IB) containing 10 mM MES, 10 mM MgCl2, and 200 μM Acetosyringone, and then transfected onto the back of tobacco leaves using a needleless syringe. At this time, Agrobacterium cells (p19_GV3101) containing the p19 gene, which helps increase the expression stability of foreign genes by inhibiting RNA silencing, were mixed in a 1:1 ratio and transfected. After 5 days, the tobacco leaves injected with Agrobacterium cells were sampled to investigate gene expression (Fig. 2b) and the production of substances. After confirming the expression of the level 1 module for each gene (Fig. 2b), key genes were selected through the single transient expression results of the level 1 module to select the optimal production combination.

[0086] Example 3. Confirmation of monoterpenoid production according to gene expression cassettes (modules) and combinations thereof

[0087] We investigated whether the production of linalool was enhanced using single gene expression cassettes (Fig. 2a) introduced into tobacco leaves and combinations thereof. First, linalool, beta-pinene, or borneol were extracted from tobacco leaves in which single gene expression modules were transiently expressed, and GC-MS or HPLC analysis was performed to determine whether the production of linalool was enhanced for each gene cassette.

[0088] As a result, unlike the case where CMK, GPPS, and IDI genes were expressed, it was confirmed that linalool production increased compared to the wild type (WT) when DXS, DXR, and LIS genes were expressed individually (Fig. 2c). Specifically, when level 1 modules were expressed individually, the linalool production of the DXS, DXR, and LIS modules was 70–77 ng·mg -1 It was confirmed that this resulted in an approximately 1.4 to 2-fold increase compared to the control group (WT) (Fig. 3). Based on these results, when each level module was combined and co-transplanted into tobacco leaves, 94 ng.mg was obtained when all DXS, DXR, and LIS genes were expressed. -1 Compared to the control group (WT, 38 ng.mg -1 It was confirmed that linalool production increased 2.5 times compared to (Fig. 4).

[0089] In addition, HPLC content analysis of other major monoterpenoids, β-pinene and borneol, showed that when the DXR, GPPS, and LIS genes were expressed, the β-pinene content was 135 ng·mg -1 As wild type (75ng.mg -1 A 1.8-fold increase compared to ) was confirmed (Fig. 5), and when DXS, GPPS, and LIS genes were expressed, the borneol content was 13 mg·g -1 As wild type (6 mg.g -1 A 2.2-fold improvement compared to ) was confirmed (Fig. 6).

[0090] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 A gene expression cassette for monoterpenoid production comprising: a promoter comprising the nucleotide sequence of SEQ ID NO. 18 and LIS (S-linalool synthase); a gene expression cassette comprising a promoter comprising the nucleotide sequence of SEQ ID NO. 17 and GPPS (geranyl diphosphate synthase); and a gene expression cassette comprising a promoter comprising the nucleotide sequence of SEQ ID NO. 14 and DXR (1-deoxy-D-xylulose-5-phosphate reductoisomerase); wherein the monoterpenoid is beta-pinene. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A gene expression cassette for monoterpenoid production according to claim 1, wherein the DXR gene is composed of the nucleotide sequence of SEQ ID NO.

2. Claim 6 A gene expression cassette for monoterpenoid production according to claim 1, wherein the LIS gene is composed of the nucleotide sequence of SEQ ID NO.

3. Claim 7 A recombinant vector comprising a gene expression cassette according to claim 1. Claim 8 Transformed bodies, excluding human bodies transformed with a recombinant vector according to Paragraph 7. Claim 9 In paragraph 8, the transformant is a transformant that is Agrobacterium. Claim 10 A transgenic plant transformed by the recombinant vector of claim 7 or the transgenic organism of claim 8. Claim 11 A transgenic plant according to claim 10, characterized in that the plant is selected from the group consisting of tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce, and chili pepper. Claim 12 A method for producing a transgenic plant for monoterpenoid production, comprising: (a) a step of preparing a recombinant vector of claim 7; and (b) a step of transforming a plant with said recombinant vector. Claim 13 A method for producing a transgenic plant according to claim 12, wherein the transformation is performed by an Agrobacterium that has been introduced with the recombinant vector. Claim 14 A method for producing a transgenic plant according to claim 12, characterized in that the plant is selected from the group consisting of tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce, and chili pepper. Claim 15 A method for producing monoterpenoids comprising: (a) transforming a plant with a recombinant vector of claim 7; (b) culturing and obtaining the transformed plant; and (c) extracting monoterpenoids from the obtained plant. Claim 16 A method for producing monoterpenoids according to claim 15, wherein the transformation is performed by Agrobacterium that has been introduced with the recombinant vector. Claim 17 A method for producing monoterpenoids according to claim 15, characterized in that the above-mentioned plant is selected from the group consisting of tobacco, Arabidopsis thaliana, corn, rice, soybean, canola, alfalfa, sunflower, sorghum, wheat, cotton, peanut, tomato, potato, lettuce, and chili pepper.

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