Microorganism capable of producing monoterpenoids and use thereof

Abstract

To provide a microorganism suitable for the production of monoterpenoids, and a method for efficiently producing monoterpenoids using the microorganism.SOLUTION: A microorganism capable of producing monoterpenoids is modified to enhance activity or expression of (a) limonene synthase (LS), (b) limonene-7-hydroxylase (L7H), and (c) at least one enzyme selected from a group consisting of perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillate-O-methyltransferase (PAOMT). A method for producing monoterpenoids includes the step of exposing the microorganism to an organic raw material containing at least one selected from a group consisting of saccharides, alcohols and organic acids.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a microorganism capable of producing a monoterpenoid and a method for producing a monoterpenoid using the microorganism. [Background technology]

[0002] Monoterpenoids are compounds whose basic structure consists of two isoprene (2-methyl-1,3-butadiene) units. Monoterpenoids include linear (acyclic) and ring-containing (monocyclic, polycyclic) monoterpenoids, and some have various functional groups due to modifications such as oxidation and rearrangement reactions. Monoterpenoids are known to have various physiological activities and functions, such as limonene, perillyl alcohol, perillyl aldehyde, and perillic acid. These have attracted attention as ingredients that exhibit fragrances, food additives, antibacterial, anticancer, and antioxidant effects. In addition, methyl perillate can be derived into cumic acid, terephthalic acid, and other compounds through chemical synthesis reactions under mild conditions, and is attracting attention as an aromatic compound derivative. On the other hand, a method for producing perillyl alcohol from glucose has been reported (Non-Patent Document 1: Jorge Alonso-Gutierrez et al., Metabolic Engineering 19 (2013) 33-41). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Jorge Alonso-Gutierrez et al., Metabolic Engineering 19 (2013) 33-41 Summary of the Invention [Problem to be solved by the invention]

[0004] Aromatic compounds such as cumic acid and terephthalic acid are not easy to chemically synthesize due to their complex structures, and extracting these compounds from natural resources has been inefficient because they are only present in trace amounts in plants, etc. Therefore, there is a need to establish technology that enables the efficient production of various isoprenoids or monoterpenoids that can be derived from these aromatic compounds. [Means for solving the problem]

[0005] The present inventors have succeeded in efficiently producing monoterpenoids from organic raw materials by using microorganisms that have been modified to enhance the activity or expression of enzymes such as limonene synthase (LS), limonene-7-hydroxylase (L7H), perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillic acid-O-methyltransferase (PAOMT), thereby completing the present invention. That is, the present invention is as follows.

[0006] [1] A microorganism capable of producing monoterpenoids, (a) limonene synthase (LS), (b) limonene-7-hydroxylase (L7H), and (c) at least one enzyme selected from the group consisting of perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillic acid-O-methyltransferase (PAOMT); A microorganism modified to enhance the activity or expression of. [2] The microorganism described in [1] above, which has been modified to enhance the activity or expression of geranyl pyrophosphate synthase (GPPS) and / or neryl pyrophosphate synthase (NPPS). [3] The microorganism according to [1] or [2] above, which has been modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of acetyl-CoA acetyltransferase (AtoB), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), and isopentyl diphosphate isomerase (IDI). [4] The microorganism according to any one of the above [1] to [3], which has been modified so as to enhance the activity or expression of at least one or more enzymes selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG), and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH). [5] The microorganism according to any one of the above-mentioned [1] to [4], which has been modified so that the activity or expression of phosphoglucose isomerase (Pgi) and / or citrate synthase (GltA) is reduced. [6] The microorganism according to any one of the above [1] to [5], which is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts. [7] A method for producing a monoterpenoid, comprising a step of reacting an organic raw material with the microorganism according to any one of the above [1] to [6]. [8] The method for producing a monoterpenoid according to [7] above, wherein the organic raw material comprises one or more selected from the group consisting of sugars, alcohols and organic acids. [9] The method for producing according to [7] or [8] above, wherein the monoterpenoid is at least one selected from the group consisting of perillyl aldehyde, perillic acid and methyl perillate.

[10] The method according to [9] above, wherein the monoterpenoid is methyl perillate.

[11] (a) producing methyl perillate by the method according to

[10] above; and (b) converting the methyl perillate obtained in step (a) into aromatic compounds; A method for producing an aromatic compound, comprising:

[12] The method according to

[11] above, wherein the aromatic compound is at least one selected from the group consisting of methyl cumate, cumic acid, and terephthalic acid. Effect of the Invention

[0007] INDUSTRIAL APPLICABILITY The present invention provides a microorganism useful for producing monoterpenoids. [Brief description of the drawings]

[0008] [Figure 1] FIG. 1 is a diagram showing an example of a reaction system in the microorganism of the present invention. [Diagram 2] FIG. 2 is a graph showing the results of measuring the amount of methyl perillate produced in the microorganism of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will be described in detail below. The following embodiments are merely illustrative for explaining the present invention, and are not intended to limit the present invention to these embodiments. The present invention can be embodied in various forms without departing from the gist of the present invention.

[0010] 1. overview Monoterpenoids include limonene, perillyl alcohol, perillyl aldehyde, and perillic acid, which are attracting attention as ingredients that exhibit fragrances, food additives, antibacterial, anticancer, and antioxidant properties, etc. In addition, methyl perillate can be derived into cumic acid, terephthalic acid, etc. through chemical synthesis reactions under mild conditions, and is attracting attention as an aromatic compound derivative. However, aromatic compounds such as cumic acid and terephthalic acid are not easy to chemically synthesize due to their complex structures, and extracting these compounds from natural resources has been inefficient because the compounds are only present in trace amounts in plants, etc. Therefore, there is a need to establish technology that enables the efficient production of various isoprenoids or monoterpenoids that can be derived from these aromatic compounds. A method for producing perillyl alcohol from glucose has been reported (Jorge Alonso-Gutierrez et al., Metabolic Engineering 19 (2013) 33-41), but this report makes no mention of the production of perillyl aldehyde, perillic acid, or methyl perillate. In light of these circumstances, the present inventors have succeeded in efficiently producing monoterpenoids from organic raw materials by using microorganisms that have been modified to enhance the activity or expression of enzymes such as limonene synthase (LS), limonene-7-hydroxylase (L7H), perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillic acid-O-methyltransferase (PAOMT), thereby completing the present invention. The present invention makes it possible to provide a microorganism having a seamless production pathway from glucose to methyl perillate, and by enhancing the activity or expression of each enzyme involved in this pathway, it becomes possible to efficiently produce monoterpenoids such as methyl perillate from organic raw materials. The microorganism of the present invention and the method for producing a monoterpenoid using said microorganism are extremely useful in that they enable efficient production of a monoterpenoid from an organic raw material in a single system.

[0011] 2. Microorganisms capable of producing monoterpenoids (1) Monoterpenoids Monoterpenoids are compounds whose basic structure consists of two isoprene (2-methyl-1,3-butadiene) units. Monoterpenoids are either linear (acyclic) or contain rings (monocyclic, polycyclic), and some have various functional groups due to modifications such as oxidation and rearrangement reactions. In the present invention, examples of monoterpenoids include monoterpenoids having a limonene skeleton, and examples of monoterpenoids having a limonene skeleton include, but are not limited to, limonene, perillyl alcohol, perillyl aldehyde, perillic acid, methyl perillate, and γ-terpinene.

[0012] (2) Monoterpenoid production In the present invention, "production" of a monoterpenoid means that an organism (e.g., a microorganism) produces a monoterpenoid, and in this specification, "production" can be used interchangeably with "production" or "generation." Those skilled in the art can evaluate whether or not a monoterpenoid has been produced by known methods, for example, by separating an organic layer from a test sample (e.g., a culture solution of a microorganism) by liquid-liquid extraction and analyzing the organic layer using a gas chromatography mass spectrometer or the like. For example, if the gas chromatography analysis shows that a peak of the target monoterpenoid contained in the test sample appears at the same position as the peak of the target monoterpenoid (e.g., methyl perillate) contained in the preparation, it can be seen that the target monoterpenoid has been produced in the test sample. However, in the present invention, the method of measuring the amount of production or detecting the production of a monoterpenoid can be appropriately adopted by those skilled in the art and is not limited to the above-mentioned method.

[0013] (3) microorganisms In the present invention, a microorganism capable of producing a monoterpenoid means a microorganism that has been modified so as to efficiently produce a monoterpenoid. In the present invention, such microorganisms include, but are not limited to, the following microorganisms: (a) limonene synthase (LS), (b) limonene-7-hydroxylase (L7H), and (c) at least one enzyme selected from the group consisting of perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillic acid-O-methyltransferase (PAOMT); A microorganism modified to enhance the activity or expression of.

[0014] In the present invention, limonene synthase (LS) is an enzyme that catalyzes a reaction in which geranyl pyrophosphate (GPP) is cyclized to synthesize limonene. In the present invention, limonene-7-hydroxylase (L7H) is an enzyme that catalyzes the reaction of hydroxylating the carbon atom at position 7 of limonene to produce perillyl alcohol. As the limonene-7-hydroxylase (L7H), any enzyme that catalyzes the reaction of hydroxylating the carbon atom at position 7 of limonene to produce perillyl alcohol can be used without any particular limitation, and p-cymene monooxygenase (CymAab) can also be used. CymAab is an enzyme that catalyzes the reaction of hydroxylating the carbon atom at position 7 of p-cymene to produce 4-isopropylbenzyl alcohol, but it may also recognize limonene as a substrate and have L7H activity. Therefore, CymAab can also be used in the reaction of producing perillyl alcohol from limonene. This enzyme is composed of a hydroxylase subunit (CymAa) and a reductase subunit (CymAb). Therefore, in this specification, L7H may be referred to as "CymAab", and the subunits may be referred to as "CymAa" and "CymAb", respectively. In the present invention, perillyl alcohol dehydrogenase (POHDH) is an enzyme that catalyzes the reaction of oxidizing (dehydrogenating) perillyl alcohol to produce perillyl aldehyde. As perillyl alcohol dehydrogenase (POHDH), any enzyme that catalyzes the reaction of oxidizing (dehydrogenating) perillyl alcohol to produce perillyl aldehyde can be used without any particular limitation, and 4-isopropylbenzyl alcohol dehydrogenase (CymB) can also be used. CymB catalyzes the reaction of oxidizing (dehydrogenating) 4-isopropylbenzyl alcohol to produce 4-isopropylbenzaldehyde, while also recognizing perillyl alcohol as a substrate and may have POHDH activity. Therefore, CymB can also be used in the reaction of producing perillyl aldehyde from perillyl alcohol. In this way, CymB can have POHDH activity, and therefore POHDH may be referred to as "CymB" in this specification. In the present invention, perillyl aldehyde dehydrogenase (PAldDH) is an enzyme that catalyzes the reaction of oxidizing perillyl aldehyde to produce perillic acid. As perillyl aldehyde dehydrogenase (PAldDH), any enzyme that catalyzes the reaction of oxidizing perillyl aldehyde to produce perillic acid can be used without particular limitation, and 4-isopropylbenzaldehyde dehydrogenase (CymC) can also be used. CymC catalyzes the reaction of oxidizing 4-isopropylbenzaldehyde to produce cumic acid, while also recognizing perillyl aldehyde as a substrate and may have PAldDH activity. Therefore, CymC can also be used in the reaction of producing perillic acid from perillyl aldehyde. In this way, CymC can have PAldDH activity, and therefore, in this specification, PAldDH may be referred to as "CymC". In the present invention, perillic acid-O-methyltransferase (PAOMT) is an enzyme that catalyzes a reaction in which a methyl group is transferred (methylated) to the hydroxyl group (-OH) of perillic acid to produce methyl perillate.

[0015] In the present invention, a microorganism that has been modified to enhance the activity or expression of geranyl pyrophosphate synthase (GPPS) and / or neryl pyrophosphate synthase (NPPS) in addition to the above enzymes (a) to (c) can be used.

[0016] Furthermore, in the present invention, in addition to the enzymes (a) to (c) above, (i) GPPS and / or NPPS, and / or (ii) at least one enzyme selected from the group consisting of acetyl-CoA acetyltransferase (AtoB), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), and isopentyl diphosphate isomerase (IDI). Microorganisms that have been modified to enhance the activity or expression of can be used.

[0017] Furthermore, in the present invention, in addition to the above enzymes (a) to (c), the following enzymes (i) to (iii): (i) GPPS and / or NPPS; (ii) at least one enzyme selected from the group consisting of AtoB, HMGS, HMGR, MVK, PMK, MVD, and IDI; and (iii) at least one enzyme selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG), and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH). A microorganism that has been modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of:

[0018] Furthermore, in the present invention, in addition to the above enzymes (a) to (c), the following enzymes (i) to (iii): (i) GPPS and / or NPPS; (ii) at least one enzyme selected from the group consisting of AtoB, HMGS, HMGR, MVK, PMK, MVD, and IDI; and (iii) at least one enzyme selected from the group consisting of Dxs, Dxr, IspD, IspE, IspF, IspG and IspH and / or (iv) Microorganisms can be used that have been modified to reduce the activity or expression of phosphoglucose isomerase (Pgi) and / or citrate synthase (GltA).

[0019] Furthermore, in the present invention, a microorganism modified to enhance the activity or expression of an enzyme (MvaE) having both AtoB and HMGR activities, instead of enhancing the activity or expression of AtoB and HMGR as described in (ii) above, can also be used. 3-Hydroxy-3-methylglutaryl-CoA synthase derived from a microorganism having MvaE is sometimes called MvaS, and HMGS is sometimes referred to as "MvaS" in this specification.

[0020] As used herein, "modified to enhance activity or expression" of an enzyme means that the activity or expression of the enzyme is enhanced by, for example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more, compared to the activity or expression of the enzyme in a non-modified cell (a cell that has not been modified). Whether the activity or expression of the enzyme is enhanced can be evaluated by measuring the expression level of the enzyme in a modified cell and examining whether the expression level is increased compared to that in a non-modified cell.

[0021] As used herein, "modified to reduce activity or expression" of an enzyme means that the activity or expression of the enzyme has been modified to be reduced, for example, by 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 99% or more, or 100% compared to the activity or expression of the enzyme in a non-modified cell (a cell that has not been modified) ("100% reduction" can also be rephrased as "eliminated"). Whether the activity or expression of the enzyme is reduced can be assessed by measuring the expression level of the enzyme in the modified cells and determining whether the expression level is reduced or the expression of the enzyme is eliminated compared to unmodified cells. Furthermore, a cell in which the gene for a certain enzyme has been disrupted can be evaluated as a cell that has been "modified so that the activity or expression" of that enzyme is reduced (eliminated).

[0022] Examples of microorganisms modified to enhance the activity or expression of the above enzymes (a) to (c) include: (a) DNA encoding limonene synthase (LS) (b) DNA encoding limonene-7-hydroxylase (L7H), and (c) DNA encoding at least one enzyme selected from the group consisting of perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH), and perillic acid-O-methyltransferase (PAOMT). Microorganisms including

[0023] Examples of microorganisms that have been modified to enhance the activity or expression of GPPS, NPPS, AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, Dxs, Dxr, IspD, IspE, IspF, IspG, and IspH include microorganisms containing DNA encoding each of GPPS, NPPS, AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, Dxs, Dxr, IspD, IspE, IspF, IspG, and IspH. Examples of microorganisms that have been modified to reduce the activity or expression of Pgi and / or GltA include microorganisms that have been modified by a method of destroying the nucleic acids that encode these enzymes on the chromosome of the microorganism, a method of replacing the promoters of the nucleic acids that encode these enzymes with weaker promoters, a method of modifying expression regulatory sequences such as the promoters and ribosome binding sequences (SD sequences) of the nucleic acids that encode these enzymes so as to reduce the expression levels, a method of suppressing translation by expressing antisense RNA to inhibit ribosome binding to target mRNA, a method of suppressing transcription by CRISPR interference, and a method of reducing the activity per enzyme molecule by introducing mutations into the nucleic acids (DNA, RNA, etc.) that encode these enzymes. In the present invention, a "modified microorganism" and a microorganism containing DNA encoding each enzyme can also be referred to as a "transformant."

[0024] The DNAs encoding LS, L7H (CymAab), POHDH (CymB), PAldDH (CymC), PAOMT, GPPS, NPPS, AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, Dxs, Dxr, IspD, IspE, IspF, IspG, and IspH can be prepared by obtaining information on their nucleotide sequences from a known database, such as NCBI (https: / / www.ncbi.nlm.nih.gov / nucleotide / ), and using known genetic engineering techniques. In addition, if a known construct (e.g., pJBEI6409) containing a portion of the above DNA is already available, such a construct can also be used. Examples of the origin and GenBank Accession Number and / or Gene ID of the DNA encoding each enzyme that can be used in the present invention are shown below. However, the origin and GenBank Accession Number and / or Gene ID of the DNA encoding each enzyme that can be used in the present invention are not limited to those shown below. Furthermore, a person skilled in the art can appropriately optimize (codon optimize, etc.) a desired base sequence based on information on the base sequence or amino acid sequence obtained from a known database. LS from Mentha spicata: Genbank accession number L13459 L7H from Salvia dorisiana: Genbank accession number MH051318 POHDH from Salvia dorisiana: Genbank accession number MH051324 PAOMT from Salvia dorisiana: Genbank accession number MH051325 CymAb, CymB and CymC from Pseudomonas putida: Genbank accession number U24215 GPPS from Abies grandis: Genbank accession number AF513112 NPPS from Solanum lycopersicum: Genbank accession number NM_001247704 AtoB from Escherichia coli: Genbank accession number NC_000913.3 (2326109..2327293), Gene ID: 946727 HMGS and HMGR from Staphylococcus aureus: Genbank accession number AF290086 MVK from Saccharomyces cerevisiae: Genbank accession number NC_001145.3 (684467..685798), Gene ID: 855248 PMK from Saccharomyces cerevisiae: Genbank accession number NC_001145.3 (712316..713671), Gene ID: 855260 MVD from Saccharomyces cerevisiae: Genbank accession number NC_001146.8 (701895..703085), Gene ID: 855779 IDI from Escherichia coli: Genbank accession number NC_000913.3 (3033065..3033613), Gene ID: 949020 Dxs from Rhizobium radiobacter: Genbank accession number KJ619966 Dxr from Escherichia coli: Genbank accession number NC_000913.3 (193521..194717), Gene ID: 945019 IspD from Escherichia coli: Genbank accession number NC_000913.3 (2871780..2872490, complement), Gene ID: 948269 IspE from Escherichia coli: Genbank accession number NC_000913.3 (1262026..1262877, complement), Gene ID: 945774 IspF from Escherichia coli: Genbank accession number NC_000913.3 (2871301..2871780, complement), Gene ID: 945057 IspG from Escherichia coli: Genbank accession number NC_000913.3 (2640686..2641804, complement), Gene ID: 946991 IspH from Escherichia coli: Genbank accession number NC_000913.3 (26277..27227), Gene ID: 944777 Pgi from Escherichia coli: Genbank accession number NC_000913.3 (4233758..4235407), Gene ID: 948535 GltA from Escherichia coli: Genbank accession number NC_000913.3 (753185..754468, complement), Gene ID: 945323 The base sequence of DNA encoding Mentha spicata-derived LS and the amino acid sequence of Mentha spicata-derived LS are shown in SEQ ID NOs: 20 and 21, respectively. The nucleotide sequence of DNA encoding L7H derived from Salvia dorisiana and the amino acid sequence of L7H derived from Salvia dorisiana are shown in SEQ ID NOs: 22 and 23, respectively. The nucleotide sequence of DNA encoding POHDH derived from Salvia dorisiana and the amino acid sequence of POHDH derived from Salvia dorisiana are shown in SEQ ID NOs: 24 and 25, respectively. The nucleotide sequence of DNA encoding PAOMT derived from Salvia dorisiana and the amino acid sequence of PAOMT derived from Salvia dorisiana are shown in SEQ ID NOs: 26 and 27, respectively. The nucleotide sequences of DNA encoding CymAab, CymAa, CymAb, CymB and CymC derived from Pseudomonas putida are shown in SEQ ID NOs: 28, 29, 31, 33 and 35, respectively, and the amino acid sequences of CymAa, CymAb, CymB and CymC derived from Pseudomonas putida are shown in SEQ ID NOs: 30, 32, 34 and 36, respectively. However, the base sequences of the DNAs encoding the above-mentioned enzymes and the amino acid sequences of the above-mentioned enzymes in the present invention are not limited to these. As described above, a person skilled in the art can appropriately optimize (such as codon optimization) a desired base sequence based on the information of these base sequences or amino acid sequences.

[0025] In the present invention, a vector containing the DNA encoding each enzyme can be used to genetically introduce the DNA into a microorganism. Examples of such vectors include a plasmid vector and a virus vector. Those skilled in the art can appropriately select the type of vector depending on the type and purpose of the host microorganism. The vector of the present invention can contain, in addition to the DNA encoding the above enzymes, cis elements such as a promoter and an enhancer, a splicing signal, a polyA addition signal, a ribosome binding sequence (SD sequence), a selection marker gene, a reporter gene, and the like, and these can also be modified. The vector of the present invention is not limited as long as it can express the enzyme used in the present invention in a host microorganism, and for example, a vector capable of expressing two or more enzyme genes can be used. In the case of a plasmid vector, such a vector includes, but is not limited to, the pBBRA-1.1e vector, the pUCSp-2 vector, and the pUCAp-2 vector.

[0026] In the present invention, a vector can contain DNA encoding one or more enzymes. For example, one vector can contain, but is not limited to, DNA encoding CymAab (CymAa and CymAb), DNA encoding CymB and CymC, DNA encoding CymAab (CymAa and CymAb), DNA encoding AtoB, HMGS and HMGR, DNA encoding MVK, PMK, MVD and IDI, DNA encoding GPPS and LS, DNA encoding AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS and LS, etc.

[0027] In the present invention, for example, by introducing a DNA encoding LS into a microorganism, the activity or expression of LS in the microorganism can be enhanced. This can improve the production of limonene from an organic raw material such as glucose. Furthermore, by introducing a DNA encoding GPPS in addition to LS into a microorganism, the activity or expression of GPPS in the microorganism can be enhanced, and this can further improve the production of limonene from an organic raw material such as glucose. Specifically, in a microorganism, for example, acetyl CoA is supplied from glucose, and isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are produced from the supplied acetyl CoA via the methylerythritol phosphate pathway (2-C-methyl-D-erythritol 4-phosphate pathway: MEP pathway) and / or the mevalonate pathway (MVA pathway). Geranyl diphosphate (GPP) is produced from the produced IPP and DMAPP by the action of GPPS, and limonene is produced from GPP by the action of LS. That is, in the present invention, in a microorganism containing DNA encoding LS or DNA encoding LS and GPPS, it is possible to improve the production of limonene from an organic raw material such as glucose. Note that in the present invention, enhancement of the activity or expression of GPPS is selective and not essential.

[0028] Furthermore, in the present invention, by introducing a DNA encoding L7H(CymAab) into a microorganism, the activity or expression of L7H(CymAab) in the microorganism can be enhanced, thereby improving the production of perillyl alcohol from limonene. That is, in the present invention, the production of perillyl alcohol from limonene can be improved in a microorganism containing a DNA encoding L7H(CymAab).

[0029] Furthermore, in the present invention, by introducing a DNA encoding POHDH (CymB) into a microorganism, the activity or expression of POHDH (CymB) in the microorganism can be enhanced, thereby improving the production of perillyl aldehyde from perillyl alcohol. That is, in the present invention, the production of perillyl aldehyde from perillyl alcohol can be improved in a microorganism containing a DNA encoding POHDH (CymB).

[0030] Furthermore, in the present invention, by introducing DNA encoding PAldDH (CymC) into a microorganism, the activity or expression of PAldDH (CymC) in the microorganism can be enhanced, thereby improving the production of perillic acid from perillic aldehyde. That is, in the present invention, the production of perillic acid from perillic aldehyde can be improved in a microorganism containing DNA encoding PAldDH (CymC).

[0031] Furthermore, in the present invention, by introducing a DNA encoding PAOMT into a microorganism, the activity or expression of PAOMT in the microorganism can be enhanced, thereby improving the production of methyl perillate from perillic acid. That is, in the present invention, the production of methyl perillate from perillic acid can be improved in a microorganism containing a DNA encoding PAOMT.

[0032] The DNA encoding LS, the DNA encoding L7H (CymAab), the DNA encoding POHDH (CymB), the DNA encoding PAldDH (CymC), and the DNA encoding PAOMT can be combined and introduced into one type of microorganism. This allows for improved production of methyl perillate from organic raw materials such as glucose in a single system. In other words, the present invention allows for the introduction of a seamless production pathway from glucose to methyl perillate into a microorganism, and by enhancing the activity or expression of each enzyme involved in this pathway, it becomes possible to efficiently produce monoterpenoids such as methyl perillate from organic raw materials.

[0033] Thus, in the present invention, for example, by introducing DNA encoding each of the above enzymes into a microorganism, a microorganism modified to enhance the activity or expression of the enzyme can be produced. Methods for introducing DNA into a microorganism are known, and a person skilled in the art can use known gene introduction methods depending on the type of microorganism and DNA. Examples of such methods include a method of introducing a nucleic acid encoding an enzyme into a vector and transforming a microorganism with the vector system, a method of introducing an expression cassette containing a nucleic acid encoding an enzyme into the chromosome of a microorganism, a method of replacing a promoter of a nucleic acid encoding an enzyme with a stronger promoter, a method of modifying an expression regulatory sequence such as a promoter or a ribosome binding sequence (SD sequence) of a nucleic acid encoding an enzyme so as to increase the expression level, and a method of increasing the activity per molecule of an enzyme by introducing a mutation into a nucleic acid encoding an enzyme. In the present invention, a microorganism modified to reduce the activity or expression of Pgi and / or GltA can be prepared using known techniques. Examples of such techniques include a technique for destroying the nucleic acid encoding these enzymes on the chromosome of a microorganism, a technique for replacing the promoter of the nucleic acid encoding these enzymes with a weaker promoter, a technique for modifying the expression regulatory sequence such as the promoter or ribosome binding sequence (SD sequence) of the nucleic acid encoding these enzymes so as to reduce the expression level, a technique for suppressing translation by expressing antisense RNA to inhibit ribosome binding to target mRNA, a technique for suppressing transcription by CRISPR interference, and a technique for reducing the activity per enzyme molecule by introducing a mutation into the nucleic acid (DNA, RNA, etc.) encoding these enzymes.

[0034] In the present invention, a tag can be fused to an enzyme protein. That is, in the present invention, a DNA encoding a fusion protein of a tag and an enzyme can be used. Examples of tags that can be used in the present invention include, but are not limited to, tags that promote solubilization to improve the soluble expression of an enzyme, and tags for purification (e.g., His tags). Examples of tags that promote solubilization of an enzyme include, but are not limited to, Sumo, Fh8, GST, and the like. By fusing a tag that promotes solubilization of an enzyme to an enzyme (e.g., PAOMT), the soluble expression of the enzyme is promoted, and as a result, the production amount of monoterpenoids such as methyl perillate can be improved.

[0035] The microorganisms used in the present invention are not limited as long as they can express the enzymes used in the present invention, and examples thereof include Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, yeast, etc. Those skilled in the art can appropriately select expression vectors so that the enzymes of the present invention can be expressed in these microorganisms, and can also optimize the codons of DNA encoding the enzymes according to the types of these microorganisms.

[0036] Furthermore, those skilled in the art can appropriately set the culture conditions (temperature, time, medium composition, etc.) of the microorganisms used in the present invention and the reaction conditions (temperature, reaction time, pH, etc.) of the enzymes used in the present invention depending on the type of microorganism.

[0037] 3. Method for producing monoterpenoids (1) Monoterpenoids As stated in “2(1)” above. (2) Method for producing monoterpenoids In the present invention, the method for producing monoterpenoids includes a step of reacting an organic raw material with the microorganism described above in "2.(3) Microorganisms." In the present invention, "acting an organic raw material with a microorganism" means culturing the microorganism in the presence of the organic raw material. In the present invention, the term "organic raw material" refers to a carbon source or nitrogen source that can be assimilated and grown by microorganisms. Examples of carbon sources include, but are not limited to, sugars, alcohols, and organic acids, and examples of nitrogen sources include, but are not limited to, organic nitrogen compounds. In the present invention, examples of "sugars" include, but are not limited to, glucose, fructose, sucrose, xylose, arabinose, cellulose, and the like. In the present invention, examples of "alcohols" include, but are not limited to, methanol, ethanol, propanol, butanol, glycerol, mannitol, inositol, and the like. In the present invention, examples of "organic acids" include, but are not limited to, formic acid, acetic acid, benzoic acid, citric acid, lactic acid, oxalic acid, succinic acid, and the like. In the present invention, examples of "organic nitrogen compounds" include, but are not limited to, urea, soybean hydrolysates, casein hydrolysates, peptone, yeast extract, meat extract, corn steep liquor, and the like.

[0038] Furthermore, the method for producing a monoterpenoid of the present invention may include, for example, the following steps. (a) preparing and culturing a microorganism comprising at least one DNA selected from (i) a DNA encoding LS, (ii) a DNA encoding L7H, and (iii) a DNA encoding at least one enzyme selected from the group consisting of POHDH, PAldDH, and PAOMT; (b) adding an organic raw material to the culture solution of the microorganism obtained in the step (a) and further culturing the mixture;

[0039] The microorganism in the above step (a) may further contain DNA encoding GPPS and / or NPPS. The microorganism in the above step (a) may further contain DNA encoding at least one enzyme selected from the group consisting of AtoB, HMGS, HMGR, MVK, PMK, MVD and IDI. The microorganism in step (a) may further contain DNA encoding at least one enzyme selected from the group consisting of Dxs, Dxr, IspD, IspE, IspF, IspG and IspH. The microorganism in step (a) may further include a sequence modification on the chromosome that reduces the activity or expression of Pgi and / or GltA, for example, a deletion or mutation in the Pgi and / or GltA gene or its promoter sequence on the chromosome. The microorganism in step (a) may further include an RNA or protein that suppresses the expression of Pgi and / or GltA, for example, an antisense RNA complementary to a ribosome binding sequence, Cas9 (dCas9) lacking nuclease activity, a guide RNA targeting the Pgi and / or GltA gene, etc.

[0040] Furthermore, the production method of the present invention may further include a step of recovering the produced monoterpenoid. Furthermore, the production method of the present invention may further include a step of purifying the produced monoterpenoid. Those skilled in the art can recover and purify the produced monoterpenoid by known methods suited to the physical properties of the monoterpenoid, such as distillation, membrane dehydration, desalting using ion exchange resins, crystallization, column chromatography, and the like.

[0041] In the present invention, when Escherichia coli is used as the microorganism, the method for producing monoterpenoids can be carried out, for example, as follows, but is not limited thereto. First, a transformant is prepared by introducing DNA encoding each enzyme into the host Escherichia coli, and the transformant is cultured overnight at about 37° C. in a known culture medium (e.g., LB medium). Next, when the monoterpenoid to be produced is perillic acid, the grown transformant is suspended in a minimal medium (e.g., M9 medium) containing an organic source (e.g., glucose) and incubated at about 37°C until an appropriate OD 600The transformants are cultured until the enzyme activity or expression reaches a certain value, and then expression of the enzyme genes is induced using IPTG or the like. The transformants in which the activity or expression of each enzyme has been enhanced are contacted with the organic raw material, and then cultured at about 37° C. for an appropriate period of time (e.g., 24 hours). The minimal medium may contain known antibiotics. On the other hand, when the monoterpenoid to be produced is methyl perillate, the grown transformant is suspended in Terrific Broth (TB medium) containing an organic source (e.g., glucose) and incubated at about 37°C until an appropriate OD 600 The transformants are cultured until the enzyme activity or expression reaches a certain value, and then expression of the enzyme genes is induced using IPTG or the like. The transformants in which the activity or expression of each enzyme has been enhanced are contacted with the organic raw material, and then cultured at about 20° C. for an appropriate period of time (e.g., 48 hours). The TB medium may contain known antibiotics. Next, the obtained culture liquid is mixed with an organic solvent (e.g., ethyl acetate) to perform liquid-liquid extraction, and the organic layer containing the extract is analyzed by a known method, for example, gas chromatography. As a result of the gas chromatography analysis, the presence or absence of a peak exhibited by the target monoterpenoid is confirmed, and if a peak is present, it indicates that the target monoterpenoid has been produced. The production amount of the monoterpenoid can be calculated by creating a calibration curve using a commercially available monoterpenoid.

[0042] 4. Method for producing aromatic compounds In the present invention, aromatic compounds can be produced using monoterpenoids (e.g., methyl perillate) produced by the method for producing monoterpenoids of the present invention. That is, the present invention provides a method for producing aromatic compounds, comprising: (a) a step of producing a monoterpenoid by the method for producing monoterpenoids of the present invention; and (b) a step of converting the monoterpenoid obtained in step (a) into an aromatic compound. In the present invention, the aromatic compound means an aromatic compound produced via an intermediate from a monoterpenoid (for example, methyl perillate). Those skilled in the art can produce aromatic compounds by using monoterpenoids as intermediates according to known methods. In addition, those skilled in the art can appropriately set the production conditions (Esmer Jongedijk, et al., ChemistryOpen 2018, 7, 201-203). Aromatic compounds that can be produced via monoterpenoids as intermediates include, but are not limited to, for example, methyl cumate, cumic acid, terephthalic acid, cumic acid methyl ester, and the like. The methyl perillate produced by the monoterpenoid production method of the present invention can then be converted into cumic acid and further into terephthalic acid by known chemical synthesis reactions (Esmer Jongedijk, et al., ChemistryOpen. 2018 Feb; 7(2): 201-203.).

[0043] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0044] [Comparative Example 1] Regarding E. coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_pp gene, and CymAb_pp gene was enhanced as follows.

[0045] (A) Obtaining pJBEI6409 Plasmid pJBEI6409 (Metab Eng. 2013 Sep;19:33-41) containing the AtoB gene from Escherichia coli, the HMGS gene and HMGR gene from Staphylococcus aureus, the MVK gene, PMK gene, and MVD gene from Saccharomyces cerevisiae, the IDI gene from Escherichia coli, the GPPS gene from Abies grandis, and the LS gene from Mentha spicata was obtained from Addgene (plasmid #47048). The full-length nucleotide sequence of pJBEI6409 is shown in SEQ ID NO:18.

[0046] (B) Construction of CymAab_pp enhancing plasmid A DNA fragment containing the base sequences of the CymAa gene and CymAb gene derived from Pseudomonas putida F1 was synthesized (base sequence of synthetic DNA: SEQ ID NO: 1). The base sequences of the CymAa gene and CymAb gene were designed to optimize the codons for efficient expression in E. coli. The synthesized DNA was inserted into the BsaI site of the pBBRA-1.1e vector (SEQ ID NO: 2) by Type IIS cloning. The constructed plasmid was named pBBRA-CymAab_pp.

[0047] (C) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_pp-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1 (A) and the plasmid pBBRA-CymAab_pp constructed in Comparative Example 1 (B), and the resulting strain was named DH5α / pJBEI6409 / pBBRA-CymAab_pp.

[0048] [Comparative Example 2] Regarding E. coli DH5α, the expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_px gene, and CymAb_px gene was enhanced as follows.

[0049] (A) Construction of CymAab_px enhancing plasmid A DNA fragment containing the base sequences of the CymAa gene and CymAb gene derived from Paraburkholderia xenovorans was synthesized (base sequence of synthetic DNA: SEQ ID NO: 3). The base sequences of the CymAa gene and CymAb gene were designed to optimize the codons in order to efficiently express them in E. coli. The synthesized DNA was inserted into the BsaI site of the pBBRA-1.1e vector (SEQ ID NO: 2) by Type IIS cloning. The constructed plasmid was named pBBRA-CymAab_px.

[0050] (B) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_px-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1 (A) and the plasmid pBBRA-CymAab_px constructed in Comparative Example 2 (A), and the resulting strain was named DH5α / pJBEI6409 / pBBRA-CymAab_px.

[0051] [Comparative Example 3] Regarding E. coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_F1g gene, and CymAb_F1g gene was enhanced as follows.

[0052] (A) Construction of CymAab_F1g enhancing plasmid The CymAa gene and CymAb gene regions were amplified by PCR using the Pseudomonas putida F1 genome (Genbank Accession No. CP000712) as a template and the p_CymAab_F primer (SEQ ID NO: 4) and the p_CymAab_R primer (SEQ ID NO: 5). This PCR fragment (amplified product) was inserted into the BsaI site of the pBBRA-1.1e vector (SEQ ID NO: 2) by Type IIS cloning. The constructed plasmid was named pBBRA-CymAab_F1g.

[0053] (B) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_F1g-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1 (A) and the plasmid pBBRA-CymAab_F1g constructed in Comparative Example 3 (A), and the resulting strain was named DH5α / pJBEI6409 / pBBRA-CymAab_F1g.

[0054] [Example 1] For Escherichia coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_F1g gene, CymAb_F1g gene, CymB_F1g gene and CymC_F1g gene was enhanced as follows.

[0055] (A) Construction of CymBCAab_F1g enhancing plasmid Using the Pseudomonas putida F1 genome (Genbank Accession No. CP000712) as a template and the p_CymB_F primer (SEQ ID NO: 6) and the p_CymAab_R primer (SEQ ID NO: 5), the CymB gene, CymC gene, CymAa gene, and CymAb gene regions were amplified by PCR. This PCR fragment was inserted into the BsaI site of the pBBRA-1.1e vector (SEQ ID NO: 2) by Type IIS cloning. The constructed plasmid was named pBBRA-CymBCAab_F1g. The full-length nucleotide sequence of pBBRA-CymBCAab_F1g is shown in SEQ ID NO: 19.

[0056] (B) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymBCAab_F1g-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1(A) and the plasmid pBBRA-CymBCAab_F1g constructed in Example 1(A), and the resulting strain was named DH5α / pJBEI6409 / pBBRA-CymBCAab_F1g.

[0057] [Comparative Example 4] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_pp enhanced strains prepared in Comparative Example 1 (C) were evaluated for perillic acid production as follows.

[0058] DH5α / pJBEI6409 / pBBRA-CymAab_pp prepared in Comparative Example 1(C) was cultured overnight at 37° C. with shaking at 200 rpm in LB medium (16 g / L Bacto-tryptone, 10 g / L Yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin and 34 μg / mL chloramphenicol. Add 10 mL of M9 medium (6 g / L Na2HPO4, 3 g / L KH2PO4, 0.5 g / L NaCl, 1 g / L NH4Cl, 2 mM MgSO4·7H2O, 0.1 mM CaCl2·2H2O, 10 mg / L thiamine hydrochloride, 8.3 mg / L FeSO4·7H2O) containing 1% glucose, 50 μg / mL carbenicillin, and 34 μg / mL chloramphenicol to a 50 mL tube (with a filter cap), and add the culture solution in the LB medium to the M9 medium at OD. 600 = 0.2, and cultured with shaking at 200 rpm at 37°C. When the OD600 reached 0.6 to 1.0, IPTG solution was added to a final concentration of 0.1 mM, and cultured with shaking for an additional 24 hours.

[0059] 1 mL of the resulting culture solution was mixed with 1 mL of ethyl acetate using a vortex mixer, and liquid-liquid extraction was performed. 1 μL of the ethyl acetate layer was used for analysis using a gas chromatography mass spectrometer (Shimadzu Corporation GCMS-TQ8040 NX, autosampler AOC-20i Plus). The carrier gas was helium, the column was Agilent HP-5MS (inner diameter 0.25 mm, length 30 m, film thickness 0.5 μm), and the column oven temperature was set to 50 ° C. The analysis conditions were: temperature increase from 50 ° C to 200 ° C at 10 ° C / min, held at 200 ° C for 1 minute, vaporizer temperature was set to 200 ° C, and splitless injection mode was used. Gas chromatography analysis identified the peaks of perillyl aldehyde (11.1 min), perillyl alcohol (11.4 min), and perillic acid (13.4 min). The standard solutions used were purchased perillyl aldehyde, perillyl alcohol, and perillic acid. Calibration curves were created to calculate the production amounts, which were 0.36 mg / L for perillyl aldehyde, 0.87 mg / L for perillic alcohol, and 98.3 mg / L for perillic acid.

[0060] [Comparative Example 5] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_px enhanced strains prepared in Comparative Example 2 (B) were evaluated for perillic acid production as follows.

[0061] In this comparative example, the evaluation of perillic acid production was performed in the same manner as in Comparative Example 4, except that DH5α / pJBEI6409 / pBBRA-CymAab_px prepared in Comparative Example 2(B) was used. As a result of gas chromatography analysis, perillic aldehyde was 0.61 mg / L, perillic alcohol was 3.48 mg / L, and perillic acid was 112.0 mg / L.

[0062] The results of Comparative Examples 4 and 5 are shown in Table 1.

[0063] [Table 1]

[0064] [Comparative Example 6] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymAab_F1g enhanced strains prepared in Comparative Example 3 (B) were evaluated for perillic acid production as follows.

[0065] In this comparative example, the evaluation of perillic acid production was performed in the same manner as in Comparative Example 4, except that DH5α / pJBEI6409 / pBBRA-CymAab_F1g prepared in Comparative Example 3(B) was used. As a result of gas chromatography analysis, perillic aldehyde was below the detection limit, perillic alcohol was 0.03 mg / L, and perillic acid was 46.3 mg / L.

[0066] [Example 2] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, and CymBCAab_F1g enhanced strains prepared in Example 1(B) were evaluated for perillic acid production as follows.

[0067] In this example, the evaluation of perillic acid production was performed in the same manner as in Comparative Example 4, except that DH5α / pJBEI6409 / pBBRA-CymBCAab_F1g prepared in Example 1(B) was used. As a result of gas chromatography analysis, perillic aldehyde and perillic alcohol were below the detection limit, and perillic acid was 82.0 mg / L.

[0068] The results of Comparative Example 6 and Example 2 are summarized in Table 2. As shown in Table 2, the amount of perillic acid produced in Example 2 was increased by 1.8 times compared to Comparative Example 6. The amount of perillic acid produced in Example 2 was unexpectedly high, considering that the CymB gene, CymC gene, CymAa gene, and CymAb gene used in Example 2 were not codon-optimized. These results indicate that the efficiency of perillic acid production can be improved by enhancing CymB and CymC.

[0069] [Table 2]

[0070] [Example 3] For Escherichia coli DH5α, expression of the PAOMT gene with a His tag at the N-terminus was enhanced as follows. (A) Construction of His-PAOMT enhancing plasmid A DNA fragment containing a His tag and the base sequence of the PAOMT gene derived from Salvia dorisiana was synthesized (base sequence of synthetic DNA: SEQ ID NO: 7). The base sequence of the PAOMT gene was designed to optimize the codons for efficient expression in E. coli. This DNA fragment was treated with restriction enzymes XhoI and HindIII and inserted into the same restriction enzyme sites of the pUCSp-2 vector (SEQ ID NO: 8). The constructed plasmid was named pUCSp-His-SdPAOMT.

[0071] (B) Generation of His-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pUCSp-His-SdPAOMT constructed in Example 3(A), and the resulting strain was designated DH5α / pUCSp-His-SdPAOMT.

[0072] [Example 4] For Escherichia coli DH5α, expression of the PAOMT gene with an Fh8 solubility tag at the N-terminus was enhanced as follows. (A) Construction of Fh8-PAOMT enhancing plasmid A DNA fragment containing the Fh8 tag and the base sequence of the PAOMT gene derived from Salvia dorisiana was synthesized (base sequence of the synthetic DNA: SEQ ID NO: 9). The base sequence of the PAOMT gene was designed to optimize the codons in order to efficiently express it in E. coli. This DNA fragment was treated with restriction enzymes XhoI and HindIII and inserted into the same restriction enzyme sites of the pUCSp-2 vector (SEQ ID NO: 8). The constructed plasmid was named pUCSp-Fh8-SdPAOMT.

[0073] (B) Generation of Fh8-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pUCSp-FH8-SdPAOMT constructed in Example 4(A), and the resulting strain was designated DH5α / pUCSp-FH8-SdPAOMT.

[0074] [Example 5] For Escherichia coli DH5α, expression of the PAOMT gene with a SUMO solubility tag at the N-terminus was enhanced as follows. (A) Construction of SUMO-PAOMT enhancing plasmid A DNA fragment containing a SUMO tag and the base sequence of the PAOMT gene derived from Salvia dorisiana was synthesized (base sequence of synthetic DNA: SEQ ID NO: 10). The base sequence of the PAOMT gene was designed to optimize the codons for efficient expression in E. coli. This DNA fragment was treated with restriction enzymes XhoI and HindIII and inserted into the same restriction enzyme site of the pUCSp-2 vector (SEQ ID NO: 8). The constructed plasmid was named pUCSp-SUMO-SdPAOMT.

[0075] (B) Generation of SUMO-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pUCSp-SUMO-SdPAOMT constructed in Example 5(A), and the resulting strain was designated DH5α / pUCSp-SUMO-SdPAOMT.

[0076] [Example 6] For Escherichia coli DH5α, expression of the PAOMT gene with a GST soluble tag at the N-terminus was enhanced as follows. (A) Construction of GST-PAOMT enhancing plasmid A DNA fragment containing a GST tag and the base sequence of the PAOMT gene derived from Salvia dorisiana was synthesized (base sequence of synthetic DNA: SEQ ID NO: 11). The base sequence of the PAOMT gene was designed to optimize the codons for efficient expression in E. coli. This DNA fragment was treated with restriction enzymes XhoI and HindIII and inserted into the same restriction enzyme sites of the pUCSp-2 vector (SEQ ID NO: 8). The constructed plasmid was named pUCSp-GST-SdPAOMT.

[0077] (B) Generation of GST-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pUCSp-GST-SdPAOMT constructed in Example 6(A), and the resulting strain was designated DH5α / pUCSp-GST-SdPAOMT.

[0078] [Example 7] The His-PAOMT-enhanced strain prepared in Example 3(B) was subjected to evaluation of perillic acid methylation activity as follows.

[0079] In this example, the methylation activity of perillic acid was evaluated by culturing DH5α / pUCSp-His-SdPAOMT prepared in Example 3(B) in LB medium (16 g / L Bacto-tryptone, 10 g / L Yeast extract, 5 g / L NaCl) containing 10 μg / mL streptomycin overnight at 37°C with shaking at 200 rpm. 10 mL of Terrific Broth (TB) medium containing 1% glucose and 10 μg / mL streptomycin was added to a 50 mL tube (with filter cap), and the OD of the culture in the LB medium was measured. 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 37°C with shaking at 200 rpm. 600 When the pH reached 0.8 to 1.0, IPTG solution was added to a final concentration of 0.1 mM, and the mixture was cultured with shaking at 200 rpm at 20° C. After 16 hours, perillic acid was added to a final concentration of 30 mg / L, and the mixture was cultured with shaking for an additional 24 hours.

[0080] 500 μL of the obtained culture solution was mixed with 500 μL of ethyl acetate using a vortex mixer to perform liquid-liquid extraction. 1 μL of the ethyl acetate layer was used for analysis using a gas chromatography mass spectrometer (Shimadzu Corporation GCMS-TQ8040 NX, autosampler AOC-20i Plus) in the same manner as in Comparative Example 4. Gas chromatographic analysis identified a peak for methyl perillate (13.3 min) using organically synthesized methyl perillate as the standard solution. A calibration curve was created to calculate the amount of methyl perillate produced, which was 0.059 mg / L.

[0081] [Example 8] The Fh8-PAOMT-enhanced strain prepared in Example 4(B) was subjected to evaluation of perillic acid methylation activity as follows.

[0082] In this example, the evaluation of perillic acid methylation activity was performed in the same manner as in Example 7, except that DH5α / pUCSp-Fh8-SdPAOMT prepared in Example 4(B) was used. As a result of gas chromatography analysis, the amount of methyl perillate produced was 0.070 mg / L.

[0083] [Example 9] The SUMO-PAOMT-enhanced strain prepared in Example 5(B) was evaluated for perillic acid methylation activity as follows.

[0084] In this example, the evaluation of perillic acid methylation activity was performed in the same manner as in Example 7, except that DH5α / pUCSp-SUMO-SdPAOMT prepared in Example 5(B) was used. As a result of gas chromatography analysis, the amount of methyl perillate produced was 0.068 mg / L.

[0085] [Example 10] The GST-PAOMT-enhanced strain prepared in Example 6(B) was evaluated for perillic acid methylation activity as follows.

[0086] In this example, the evaluation of perillic acid methylation activity was performed in the same manner as in Example 7, except that DH5α / pUCSp-GST-SdPAOMT prepared in Example 6(B) was used. As a result of gas chromatography analysis, the amount of methyl perillate produced was 0.089 mg / L.

[0087] The results of Examples 7 to 10 are summarized in Figure 2. In Examples 7 to 10, methyl perillate was detected in all of them, and PAOMT activity was confirmed in all of the expression constructs. Moreover, the production amounts of methyl perillate in Examples 8 to 10 increased by 19%, 15%, and 51%, respectively, compared to Example 7. The above results demonstrated that PAOMT activity can be improved by adding a soluble tag.

[0088] [Example 11] For Escherichia coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_pp gene, CymAb_pp gene, and PAOMT gene was enhanced using three plasmids as follows.

[0089] DH5α / pJBEI6409 / pBBRA-CymAab_pp prepared in Comparative Example 1(C) was transformed with the plasmid pUCSp-SUMO-SdPAOMT constructed in Example 5(A), and the resulting strain was named DH5α / pJBEI6409 / pBBRA-CymAab_pp / pUCSp-SUMO-SdPAOMT.

[0090] [Example 12] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, CymAab_pp, and PAOMT enhanced strains prepared in Example 11 were evaluated for methyl perillate production as follows.

[0091] DH5α / pJBEI6409 / pBBRA-CymAab_pp / pUCSp-SUMO-SdPAOMT prepared in Example 11 was cultured overnight at 37°C with shaking at 200 rpm in LB medium (16 g / L Bacto-tryptone, 10 g / L Yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, and 50 μg / mL spectinomycin. 100 mL of Terrific Broth (TB) medium containing 1% glucose, 50 μg / mL carbenicillin, 34 μg / mL chloramphenicol, and 50 μg / mL spectinomycin was added to a 500 mL Erlenmeyer flask, and the culture solution in the LB medium was measured at OD. 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 37°C with shaking at 200 rpm. 600 When the pH reached 0.8 to 1.0, IPTG solution was added to a final concentration of 0.1 mM, and the mixture was cultured at 20° C. with shaking at 200 rpm for 48 hours.

[0092] From 100 mL of the obtained culture solution, liquid-liquid extraction with ethyl acetate was performed using a separatory funnel. Extraction with 50 mL of ethyl acetate was repeated three times, and 150 mL of the collected ethyl acetate layer was concentrated with a rotary evaporator to finally obtain 2 mL of ethyl acetate solution. 1 μL of this concentrated solution was used and analyzed with a gas chromatography mass spectrometer (Shimadzu Corporation GCMS-TQ8040 NX, autosampler AOC-20i Plus) in the same manner as in Example 7. A calibration curve was created to calculate the production amount, and the amount of methyl perillate was 1.27 mg / L.

[0093] [Example 13] For Escherichia coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_pp gene, CymAb_pp gene, and PAOMT gene was enhanced using two plasmids as follows. (A) Construction of CymAab_pp and SUMO-PAOMT enhancing plasmids DNA fragments containing the base sequences of an arabinose-inducible promoter (SEQ ID NO: 12), a SUMO-tagged PAOMT gene derived from Salvia dorisiana (SEQ ID NO: 13), a CymAa gene derived from Pseudomonas putida F1 (CymAb gene) (SEQ ID NO: 14), and a terminator (SEQ ID NO: 15) were synthesized. The PAOMT gene, CymAa gene, and CymAb gene were designed to have codon-optimized sequences for efficient expression in E. coli. These synthesized DNAs were inserted into the BsmBI site of the pUCAp-2 vector (SEQ ID NO: 16) by Type IIS cloning. The constructed plasmid was named pUCAp-Para-SUMOSdPAOMT-CymAab_pp.

[0094] (B) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, CymAab_pp, and SUMO-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1 (A) and the plasmid pUCAp-Para-SUMOSdPAOMT-CymAab_pp constructed in Example 13 (A), and the resulting strain was named DH5α / pJBEI6409 / pUCAp-Para-SUMOSdPAOMT-CymAab_pp.

[0095] [Example 14] For Escherichia coli DH5α, expression of the AtoB gene, HMGS gene, HMGR gene, MVK gene, PMK gene, MVD gene, IDI gene, GPPS gene, LS gene, CymAa_px gene, CymAb_px gene, and PAOMT gene was enhanced using two plasmids as follows. (A) Construction of CymAab_px and SUMO-PAOMT enhancing plasmids DNA fragments containing the base sequences of an arabinose-inducible promoter (SEQ ID NO: 12), a SUMO-tagged PAOMT gene derived from Salvia dorisiana (SEQ ID NO: 13), a CymAa gene derived from Paraburkholderia xenovorans (CymAa gene), a CymAb gene (SEQ ID NO: 17), and a terminator (SEQ ID NO: 15) were synthesized. The PAOMT gene, CymAa gene, and CymAb gene were designed to have codon-optimized sequences for efficient expression in E. coli. These synthesized DNAs were inserted into the BsmBI site of the pUCAp-2 vector (SEQ ID NO: 16) by Type IIS cloning. The constructed plasmid was named pUCAp-Para-SUMOSdPAOMT-Cymab_px.

[0096] (B) Generation of AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, CymAab_px, and SUMO-PAOMT-enhanced strains Escherichia coli DH5α was transformed with the plasmid pJBEI6409 obtained in Comparative Example 1 (A) and the plasmid pUCAp-Para-SUMOSdPAOMT-CymAab_px constructed in Example 14 (A), and the resulting strain was designated DH5α / pJBEI6409 / pUCAp-Para-SUMOSdPAOMT-CymAab_px.

[0097] [Example 15] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, CymAab_pp, and PAOMT enhanced strains prepared in Example 13(B) were evaluated for methyl perillate production as follows.

[0098] DH5α / pJBEI6409 / pUCAp-Para-SUMOSdPAOMT-CymAab_pp prepared in Example 13(B) was cultured overnight at 37°C with shaking at 200 rpm in LB medium (16 g / L Bacto-tryptone, 10 g / L Yeast extract, 5 g / L NaCl) containing 50 μg / mL carbenicillin and 34 μg / mL chloramphenicol. 100 mL of Terrific Broth (TB) medium containing 1% glucose, 50 μg / mL carbenicillin, and 34 μg / mL chloramphenicol was added to a 500 mL Erlenmeyer flask, and the culture solution in the LB medium was measured at OD. 600 The bacteria were inoculated so that the OD was 0.3, and cultured at 37°C with shaking at 200 rpm. 600 When the pH reached 0.8 to 1.0, IPTG solution was added to a final concentration of 0.1 mM, and L-arabinose was added to a final concentration of 0.2%, followed by shaking culture at 20° C. and 200 rpm for 48 hours.

[0099] From 100 mL of the obtained culture solution, liquid-liquid extraction with ethyl acetate was performed using a separatory funnel. Extraction with 50 mL of ethyl acetate was repeated three times, and 150 mL of the collected ethyl acetate layer was concentrated with a rotary evaporator to finally obtain 2 mL of ethyl acetate solution. 1 μL of this concentrated solution was used and analyzed with a gas chromatography mass spectrometer (Shimadzu Corporation GCMS-TQ8040 NX, autosampler AOC-20i Plus) in the same manner as in Example 7. A calibration curve was created to calculate the production amount, and the results were 84.6 mg / L for perillic acid and 2.32 mg / L for methyl perillate.

[0100] [Example 16] The AtoB, HMGS, HMGR, MVK, PMK, MVD, IDI, GPPS, LS, CymAab_px, and PAOMT enhanced strains prepared in Example 14(B) were evaluated for methyl perillate production as follows.

[0101] In this example, the evaluation of methyl perillate production was performed in the same manner as in Example 13, except that DH5α / pJBEI6409 / pUCAp-Para-SUMOSdPAOMT-CymAab_px prepared in Example 14(B) was used. As a result of gas chromatography analysis, perillic acid was 27.0 mg / L and methyl perillate was 2.06 mg / L.

[0102] The results of Examples 12, 15, and 16 are summarized in Table 3. Methyl perillate was detected in all of Examples 12, 15, and 16, demonstrating that methyl perillate can be produced using glucose as a raw material. That is, it was demonstrated that the present invention enables production of microorganisms capable of producing monoterpenoids. It was also demonstrated that the use of the modified microorganisms of the present invention enables efficient production of monoterpenoids such as methyl perillate from organic raw materials such as glucose.

[0103] [Table 3]

[0104] [Example 17] For Escherichia coli HMS174(DE3), expression of the Dxs gene, Dxr gene, IDI gene, GPPS gene, LS gene, CymAa_F1g gene, CymAb_F1g gene, CymB_F1g gene, and CymC_F1g gene was enhanced as follows.

[0105] (A) Construction of Dxs, Dxr, IDI, GPPS, and LS augmentation plasmids The GPPS gene derived from Abies grandis and the LS gene derived from Mentha spicata were synthesized by adding the recognition sequence of the restriction enzyme ScaI at the 5' end and the recognition sequence of the restriction enzyme XhoI at the 3' end (SEQ ID NO: 37). The GPPS gene and the LS gene were designed to have codon-optimized sequences for efficient expression in E. coli. The synthesized DNA was transformed into pCOLA_P T7 -dxs-dxr_P T7 The plasmid was inserted into the same restriction enzyme site of -idi-ispS (JP Patent Publication No. 2022-150327, Example 1(D)) and linked downstream of the T7 promoter. T7 -dxs-dxr_P T7 The plasmid was named -idi-GPPS-LS, and contains the Dxs gene from Rhizobium radiobacter, the Dxr gene from Escherichia coli, the IDI gene, the GPPS gene from Abies grandis, and the LS gene from Mentha spicata.

[0106] (B) Generation of Dxs, Dxr, IDI, GPPS, LS, and CymBCAab_F1g-enhanced strains E. coli HMS174(DE3) (Novagen) was transformed with the plasmid pCOLA_P obtained in Example 17(A). T7 -dxs-dxr_P T7 The strain was transformed with -idi-GPPS-LS and the plasmid pBBRA-CymBCAab_F1g constructed in Example 1(A), and the resulting strain was T7 -dxs-dxr_P T7 The plasmid was named -idi-GPPS-LS / pBBRA-CymBCAab_F1g.

[0107] [Example 18] For Escherichia coli HMS174(DE3), expression of the Dxs gene, Dxr gene, IDI gene, NPPS gene, LS gene, CymAa_F1g gene, CymAb_F1g gene, CymB_F1g gene and CymC_F1g gene was enhanced as follows.

[0108] (A) Construction of Dxs, Dxr, IDI, NPPS, and LS augmentation plasmids The NPPS gene derived from Solanum lycopersicum and the LS gene derived from Mentha spicata were synthesized by adding the recognition sequence of the restriction enzyme ScaI at the 5' end and the recognition sequence of the restriction enzyme XhoI at the 3' end (SEQ ID NO: 38). The NPPS gene and the LS gene were designed to have codon-optimized sequences for efficient expression in E. coli. The synthesized DNA was transformed into pCOLA_P T7 -dxs-dxr_P T7 The plasmid was inserted into the same restriction enzyme site of -idi-ispS (JP Patent Publication No. 2022-150327, Example 1(D)) and linked downstream of the T7 promoter. T7 -dxs-dxr_P T7 The plasmid was named -idi-NPPS-LS, and contains the Dxs gene from Rhizobium radiobacter, the Dxr gene from Escherichia coli, the IDI gene, the NPPS gene from Solanum lycopersicum, and the LS gene from Mentha spicata.

[0109] (B) Generation of Dxs, Dxr, IDI, NPPS, LS, and CymBCAab_F1g-enhanced strains E. coli HMS174(DE3) (Novagen) was transformed with the plasmid pCOLA_P obtained in Example 18(A). T7 -dxs-dxr_P T7 The resulting strain was transformed with -idi-NPPS-LS and the plasmid pBBRA-CymBCAab_F1g constructed in Example 1(A), and the resulting strain was T7 -dxs-dxr_PT7 The plasmid was named -idi-NPPS-LS / pBBRA-CymBCAab_F1g.

[0110] [Example 19] For Escherichia coli HMS174(DE3), the Pgi gene was disrupted and expression of the Dxs gene, Dxr gene, IDI gene, GPPS gene, LS gene, CymAa_F1g gene, CymAb_F1g gene, CymB_F1g gene and CymC_F1g gene was enhanced as described below.

[0111] The E. coli Pgi-disrupted strain HMS174(DE3) / Δpgi (JP Patent Publication No. 2022-150327, Example 1(C)) was transformed into the plasmid pCOLA_P obtained in Example 17(A). T7 -dxs-dxr_P T7 The strain was transformed with -idi-GPPS-LS and the plasmid pBBRA-CymBCAab_F1g constructed in Example 1(A), and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7 -dxs-dxr_P T7 The plasmid was named -idi-GPPS-LS / pBBRA-CymBCAab_F1g.

[0112] [Example 20] For Escherichia coli HMS174(DE3), the Pgi gene was disrupted and expression of the Dxs gene, Dxr gene, IDI gene, NPPS gene, LS gene, CymAa_F1g gene, CymAb_F1g gene, CymB_F1g gene and CymC_F1g gene was enhanced as described below.

[0113] The E. coli Pgi-disrupted strain HMS174(DE3) / Δpgi (JP Patent Publication No. 2022-150327, Example 1(C)) was transformed into the plasmid pCOLA_P obtained in Example 18(A). T7 -dxs-dxr_P T7 The strain was transformed with -idi-NPPS-LS and the plasmid pBBRA-CymBCAab_F1g constructed in Example 1(A), and the resulting strain was named HMS174(DE3) / Δpgi / pCOLA_P T7-dxs-dxr_P T7 The plasmid was named -idi-NPPS-LS / pBBRA-CymBCAab_F1g.

[0114] [Example 21] The Dxs, Dxr, IDI, GPPS, LS, and CymBCAab_F1g enhanced strains prepared in Example 17(B) were evaluated for perillic acid production as follows.

[0115] In this example, the perillic acid production evaluation was performed using HMS174(DE3) / pCOLA_P prepared in Example 17(B). T7 -dxs-dxr_P T7 Except for using -idi-GPPS-LS / pBBRA-CymBCAab_F1g, the same procedure as in Comparative Example 4 was repeated. As a result of gas chromatography analysis, the perillic acid concentration was 0.03 mg / L.

[0116] [Example 22] The Dxs, Dxr, IDI, NPPS, LS, and CymBCAab_F1g enhanced strains prepared in Example 18(B) were evaluated for perillic acid production as follows.

[0117] In this example, the perillic acid production evaluation was performed using HMS174(DE3) / pCOLA_P prepared in Example 18(B). T7 -dxs-dxr_P T7 Except for using -idi-NPPS-LS / pBBRA-CymBCAab_F1g, the same procedure as in Comparative Example 4 was repeated. As a result of gas chromatography analysis, the perillic acid concentration was 4.89 mg / L.

[0118] [Example 23] The Pgi-disrupted, Dxs, Dxr, IDI, GPPS, LS, and CymBCAab_F1g-enhanced strains prepared in Example 19 were evaluated for perillic acid production as follows.

[0119] In this example, the perillic acid production evaluation was performed using HMS174(DE3) / Δpgi / pCOLA_P prepared in Example 19. T7 -dxs-dxr_PT7 Except for using -idi-GPPS-LS / pBBRA-CymBCAab_F1g, the same procedure as in Comparative Example 4 was repeated. As a result of gas chromatography analysis, the perillic acid concentration was 0.07 mg / L.

[0120] [Example 24] The Pgi-disrupted, Dxs, Dxr, IDI, NPPS, LS, and CymBCAab_F1g-enhanced strains prepared in Example 20 were evaluated for perillic acid production as follows.

[0121] In this example, the perillic acid production evaluation was performed using HMS174(DE3) / Δpgi / pCOLA_P prepared in Example 20. T7 -dxs-dxr_P T7 Except for using -idi-NPPS-LS / pBBRA-CymBCAab_F1g, the same procedure as in Comparative Example 4 was repeated. As a result of gas chromatography analysis, the perillic acid content was 7.52 mg / L.

[0122] The results of Examples 21 to 24 are shown in Table 4. In Examples 21 to 24, perillic acid was detected in all cases, demonstrating that perillic acid can be produced from glucose as a raw material by utilizing the MEP pathway instead of the MVA pathway. As shown in Table 4, the perillic acid production in Example 22 was increased by 163 times compared to Example 21, and the perillic acid production in Example 24 was increased by 107 times compared to Example 23. From the above results, it was shown that the efficiency of perillic acid production can be improved by increasing NPPS instead of GPPS. Furthermore, as shown in Table 4, the amount of perillic acid produced in Example 23 was increased by 2.3 times compared to Example 21, and the amount of perillic acid produced in Example 24 was increased by 1.5 times compared to Example 22. These results demonstrate that the efficiency of perillic acid production can be improved by disrupting the Pgi gene. That is, it was demonstrated that the present invention enables production of microorganisms capable of producing monoterpenoids. It was also demonstrated that the use of the modified microorganisms of the present invention enables efficient production of monoterpenoids such as perillic acid from organic raw materials such as glucose.

[0123] [Table 4] [Industrial Applicability]

[0124] The present invention can provide a microorganism useful for producing monoterpenoids. [Sequence List Free Text]

[0125] SEQ ID NOs: 1 to 19, 37 and 38: synthetic DNA

Claims

1. A microorganism capable of producing monoterpenoids, (a) limonene synthase (LS), (b) limonene-7-hydroxylase (L7H), and (c) at least one enzyme selected from the group consisting of perillyl alcohol dehydrogenase (POHDH), perillyl aldehyde dehydrogenase (PAldDH) and perillic acid-O-methyltransferase (PAOMT); A microorganism modified to enhance the activity or expression of.

2. The microorganism according to claim 1 , which has been modified to enhance the activity or expression of geranyl pyrophosphate synthase (GPPS) and / or neryl pyrophosphate synthase (NPPS).

3. The microorganism according to claim 1, which has been modified to enhance the activity or expression of at least one enzyme selected from the group consisting of acetyl-CoA acetyltransferase (AtoB), 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), and isopentyl diphosphate isomerase (IDI).

4. The microorganism according to claim 1, which has been modified so as to enhance the activity or expression of at least one enzyme selected from the group consisting of 1-deoxy-D-xylulose 5-phosphate synthase (Dxs), 1-deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr), 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (IspD), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (IspE), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF), 1-hydroxy-2-methyl-2-butenyl 4-diphosphate synthase (IspG), and 1-hydroxy-2-methyl-2-butenyl 4-diphosphate reductase (IspH).

5. The microorganism according to claim 1 , which has been modified to reduce the activity or expression of phosphoglucose isomerase (Pgi) and / or citrate synthase (GltA).

6. The microorganism according to claim 1, which is at least one selected from the group consisting of Escherichia coli, coryneform bacteria, Bacillus bacteria, Pantoea bacteria, Enterobacter bacteria, Pseudomonas bacteria, Streptomyces bacteria, Actinomyces bacteria, filamentous fungi, and yeasts.

7. A method for producing a monoterpenoid, comprising a step of reacting an organic raw material with the microorganism according to claim 1.

8. The method for producing a monoterpenoid according to claim 7, wherein the organic raw material comprises one or more selected from the group consisting of sugars, alcohols and organic acids.

9. The method according to claim 7, wherein the monoterpenoid is at least one selected from the group consisting of perillyl aldehyde, perillic acid, and methyl perillate.

10. The method of claim 9, wherein the monoterpenoid is methyl perillate.

11. (a) producing methyl perillate by the method of claim 10; and (b) converting the methyl perillate obtained in step (a) into aromatic compounds; A method for producing an aromatic compound, comprising:

12. The method according to claim 11, wherein the aromatic compound is at least one selected from the group consisting of methyl cumate, cumic acid, and terephthalic acid.

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