Prenyltransferase

Abstract

The objective is to provide a prenyltransferase that enhances the synthetic reactivity of the target compound and suppresses isomer formation, as well as a method for producing a specified compound using this prenyltransferase. [Solution] A prenyltransferase having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, Prenyltransferase containing one mutation selected from the following mutations; (1) A230S and Y286A, (2) A230S and Y286P, (3) V47W and Y286A, (4) V47W and Y286P, (5) G284S and Y286P, (6) G284S and Y286A.

Description

Technical Field

[0001] The present invention relates to a prenyltransferase and a method for producing a compound using the prenyltransferase.

Background Art

[0002] Cannabinoids such as tetrahydrocannabinol (THC), cannabidiol (CBD), cannabigerol (CBG), and cannabimelcon (CBC) contained in Cannabis sativa L. have psychoactive effects, antibacterial effects, anticancer effects, etc., and have also been recently shown to be usable as antiemetics, antidepressant / antiepileptic drugs, and therapeutic agents for reducing intraocular pressure or treating glaucoma. Therefore, research and development for stably and low-costly supplying such cannabinoids has been underway.

[0003] The cultivation of Cannabis sativa L. in medicine requires a planned supply production and is a method carried out under strict management. In addition, the cultivated Cannabis sativa L. contains various compounds, and many processes and facilities are required to isolate and purify only the target compound. Therefore, recently, as an alternative to conventional Cannabis sativa L. cultivation, a method of biochemically synthesizing intended compounds or their precursor compounds using mutant enzymes, microorganisms, etc. has been developed.

[0004] In Patent Document 1, it has been reported that unnatural prenyltransferases having amino acid substitutions that can generate cannabigerolic acid (3-geranylolivetrate (3-GOLA)) or 5-geranyl-olivetrate (5-GOLA), which are classified as cannabinoids, from olivetolic acid and geranyl diphosphate, and enzymes that can selectively synthesize specific cannabinoids have been developed.

[0005] Furthermore, Patent Document 2 discloses the development of an amino acid substitution derivative of the prokaryotic enzyme NphB that exhibits geranyl group transfer activity, such as producing cannabigerol acid from olivetolic acid and geranyl diphosphate, and further, the development of a cell-free enzyme system using such a modified enzyme.

[0006] Furthermore, Non-Patent Document 1 reported that an amino acid substitution of NphB based on the enzyme's three-dimensional structure was produced, and the regioselectivity of the geranyl group transfer reaction was improved, resulting in a 13.6-fold increase in the yield of cannabigerol acid. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] International Publication No. 2019173770 [Patent Document 2] International Publication No. 2020028722 [Non-patent literature]

[0008] [Non-Patent Document 1] Kevin Jie Han Lim et al., ACS Catalysis. 2022, 12, 4628-4639

[0009] However, even when amino acid substitutions are designed using computational chemistry based on the three-dimensional structure of an enzyme, activity does not always increase, and achieving both the intended substrate specificity and high catalytic activity simultaneously is particularly difficult. In addition, given the recent increase in the need to synthesize only specific compounds, there is a demand for further development of various enzymes for use in diverse synthetic reactions, as well as mechanisms and methods for producing as many target compounds as possible. [Overview of the Initiative] [Problems that the invention aims to solve]

[0010] The prenyltransferase and the method for producing compounds using the same of the present invention were developed in view of these circumstances and in order to solve the above-mentioned problems. The objective is to provide a prenyltransferase that increases the yield of the target compound while suppressing by-products, and a method for producing the target compound that enables practical applications such as large-scale synthesis using this method. [Means for solving the problem]

[0011] The inventors diligently continued their research to solve the above problems and focused on the amino acid sequence of prenyltransferase. They discovered that amino acid substitutions in several specific regions significantly altered the catalytic activity for specific substrates. Furthermore, they explored regions that contributed significantly to the desired catalytic activity, and based on this knowledge, intentionally mutated specific sites simultaneously. This led to the creation of a prenyltransferase that is more specialized for the production of the target compound and suppresses by-products, thus completing the present invention.

[0012] In other words, the present invention is as follows. (1) A prenyltransferase having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, Prenyltransferase containing one mutation selected from the following mutations; (1) A230S and Y286A, (2) A230S and Y286P, (3) V47W and Y286A, (4) V47W and Y286P, (5) G284S and Y286P, (6) G284S and Y286A. (2) The mutations are A230S and Y286A, Furthermore, the prenyltransferase described in (1) has at least one mutation selected from the group consisting of A106G, E110G, I107V, T112V, S134A, Q159S, V143L, T161I, E148D, M163I, M160I, K166R, S162G, K167H, Q169T, S175G, L196F, S212G, T200S, V213I, T221S, L227I, D225E, S233T, V46I, G271A, I63F, T277G, S64T, E278G, S103D, A285S, M104S, H288Q, F105Y, and T273A. (3) The mutations are A230S and Y286A, Furthermore, D125G, E304G, E242Q, D6S, T42V, I289L, I292V, D6E, D39F, E242I, D39Y, D39I, D6P, S162G, D39V, E242C, T290P, E242L, D43Q, I289M, D125T, I289C, E244I, E242V, D39N, E242H, D39A, E24 The prenyltransferase described in (1), having at least one mutation selected from the group consisting of 2F, E244C, D39E, E304A, D39C, D39, D43E, E244V, D39W, D39G, Q293I, D39P, D6G, D6K, D39Q, E242W, D39M, D39R, T42A, I289F, and D39S. (4) The mutations are A230S and Y286P, Furthermore, the prenyltransferase described in (1) having the T42A and / or T290P mutation. (5) The mutations are A230S and Y286P, Furthermore, it has the T290P mutation, The prenyltransferase described in (1), which also has at least one mutation selected from the group consisting of L155V, L303I, G271F, A84D, V47I, I232H, V47L, L59M, L303V, L204T, V262D, L182K, V47F, L303F, V47A, and T290P. (6) The mutations are A230S and Y286P, Furthermore, having the mutation of T42A, and having at least one mutation selected from the group consisting of L177A, V7R, Q179M, L59M, V269I, L238M, L177I, A84T, L303I, V77Q, L238A, L303V, I232Q, A257V, L303F, A84Y, V7L, V262G, V269K and V23L, the prenyltransferase according to (1). (7) The mutation is V47W and Y286A, and further having one of the mutations of A230S and / or G284S, the prenyltransferase according to (1). (8) The mutation is V47W and Y286A, and further having the mutation of A230S, and also having the mutations of G284S and Y286A, the prenyltransferase according to (1). (9) The mutation is V47W and Y286A, and further having the mutations of A230S, G284S and Y286A, and also having the mutation of Q293F, the prenyltransferase according to (1). (10) The mutation is V47W and Y286P, and further having one of the mutations of A230S, G284S or Q293F, the prenyltransferase according to (1). (11) The mutation is V47W and Y286P, and further having the mutation of A230S, having the mutations of G284S and Y286A, the prenyltransferase according to (1). (12) The mutation is V47W and Y286P, and further having the mutations of A230S, G284S and Y286A, and also having the mutation of Q293F, the prenyltransferase according to (1). (13) The prenyltransferase has aromatic prenyltransferase activity, A prenyltransferase according to (1) which produces cannabigerolic acid and has regioselectivity of 50% or more with respect to the production of said cannabigerolic acid. (14) A genetically engineered microorganism having the prenyltransferase according to (1) to (13). (15) A method for producing a prenylated aromatic compound, comprising: a step of contacting the prenyltransferase according to (1) to (13) with a substrate, the method for producing a prenylated aromatic compound. [Advantages of the Invention]

[0013] The prenyltransferase of the present invention can efficiently synthesize a predetermined compound and greatly improve the yield or production rate of such a compound because it suppresses the production of by-products, particularly isomers. The supply of downstream compounds synthesized based on the compounds synthesized by the prenyltransferase of the present invention can be stabilized. Further, by using the prenyltransferase of the present invention, the synthetic route can be replaced by biosynthesis without relying on the production of target compounds by extraction and separation from plants or the like, making quality control easier and significantly enhancing the practicality for compound utilization. [Brief Description of the Drawings]

[0014] [Figure 1] FIG. 1 is a diagram for explaining the synthetic route from glucose to cannabidiol. [Figure 2] FIG. 2 is a diagram showing the production magnification of cannabigerolic acid by a prenyltransferase having a mutation. [Figure 3] FIG. 3 is a diagram showing the production magnification of cannabigerolic acid by a prenyltransferase having a mutation. [Figure 4] FIG. 4 is a diagram showing the production magnification of cannabigerolic acid by a prenyltransferase having a mutation. [Figure 5] FIG. 5 is a diagram showing the production magnification of cannabigerolic acid by a prenyltransferase having a mutation. [Figure 6] Figure 6 shows the cannabigerol acid production ratio of mutated prenyltransferase. [Modes for carrying out the invention]

[0015] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art. All patents, applications, and other publications (including online information) referenced herein are incorporated herein by reference in their entirety.

[0016] In this specification, numerical ranges indicated using "~" represent a range that includes the numbers before and after "~" as the minimum and maximum values, respectively. In this specification, the term "process" includes not only independent processes but also any process that cannot be clearly distinguished from other processes, provided that its intended purpose is achieved.

[0017] In a first aspect, the present invention relates to a prenyltransferase having an amino acid sequence that is at least 80% identical to Sequence ID No. 1, and comprising one mutation selected from the following mutations: (1) A230S and Y286A, (2) A230S and Y286P, (3) V47W and Y286A, (4) V47W and Y286P, (5) G284S and Y286P, (6) G284S and Y286A, (7).

[0018] In some embodiments, the prenyltransferase of the present invention is A106G, E110G, I107V, T112V, S134A, Q159S, V143L, T161I, E148D, M163I, M160I, K166R, S162G, K167H, Q169T, S175G, L196F, S212G, T200S, V213I, T221S, L227I, D225E, S233T, V46I, G271A, I63F, T277G, S64T, E278G, S103D, A285S, M104S, H288Q, F105Y, T273A, D125G, E304G, E242Q, D6S, T42V, I289L, I292V, D6E, D39F, E2 42I, D39Y, D39I, D6P, S162G, D39V, E242C, T290P, E242L, D43Q, I289M, D125T, I289C, E244I, E242V, D39N, E242H, D39A, E242F, E244C, D39E, E304A, D39C, D39, D43E, E244V, D39W, D39G, Q293I, D39P, D6G, D6K, D39Q, E242W, D39M, D39R, T42A , I289F, D39S, T42A, T290P, L155V, L303I, G271F, A84D, V47I, I232H, V47L, L59M, L303V, L204T, V262D, L182K, V47F, L3 This transferase has at least one mutation selected from the group consisting of 03F, V47A, T290P, L177A, V7R, Q179M, L59M, V269I, L238M, L177I, A84T, L303I, V77Q, L238A, L303V, I232Q, A257V, L303F, A84Y, V7L, V262G, V269K, V23L, A230S, G284S, G284S, Y286A, and Q293F.

[0019] In certain embodiments, the prenyltransferase of the present invention may have at least 19, at least 18, at least 17, at least 16, at least 15, at least 14, at least 13, at least 12, at least 11, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2 mutations simultaneously or at least 1 mutation.

[0020] In some embodiments, the prenyltransferase of the present invention is sequence number 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, sequence number 29. SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO. 39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56, SEQ ID NO. 57, SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 6 2, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, A prenyltransferase may have any one amino acid sequence of SEQ ID NOs. 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, or 116 (SEQ ID NOs. 1-116).

[0021] In another embodiment, the prenyltransferase of the present invention may be a prenyltransferase that is identical (has identity) to, but is not limited to, SEQ ID NOs: 1 to 116 by at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%.

[0022] In another embodiment, the prenyltransferase of the present invention may be expressed as a recombinant protein (polypeptide), a mutant (body or type), a non-natural (type), or an isolated prenyltransferase. In one embodiment, the prenyltransferase of the present invention may also be expressed as, for example, an (aromatic)prenyl(group)transferase, though not limited to this. In another embodiment, though not limited to this, it may be a dimethylallyl(group)transferase, a geranyl(group)transferase, a farnesyl(group)transferase, a geranylgeranyl(group)transferase, or a geranylfarnesyl(group)transferase.

[0023] If the function of the prenyltransferase of the present invention is not substantially inhibited, one or more amino acids in the amino acid sequence of the prenyltransferase of the present invention may be deleted or inverted, or substituted with one or more other amino acids, or one or more amino acids may be added or inserted, and the mutation may have combinations thereof, in order to enhance or maintain its function (such as stabilization or improved heat resistance). That is, in the present invention, "mutation" includes the deletion or inversion of one or more amino acids, substitution with one or more other amino acids, addition or insertion of one or more amino acids, and combinations thereof.

[0024] Furthermore, the mutations that are substituted, added, or inserted may not be limited to amino acids, but may also be compounds such as pseudo-amino acids or artificial amino acids.

[0025] In one embodiment, one or more linkers (spacers) or tags may be added to the prenyltransferase of the present invention to enhance or maintain its function (such as stabilization or improved heat resistance), provided that this does not substantially inhibit the function of the prenyltransferase of the present invention. Such linkers may include, but are not limited to, keyhole limpet hemocyanin (KLH), carrier proteins such as bovine serum albumin (BSA), peptides or amino acids, aminocaproic acid (Ahx), polyethylene glycol (PEG), fluorescent dyes, or biotin.

[0026] The number of amino acids of the present invention that can be mutated by deletion, inversion, substitution, addition, or insertion is not particularly limited as long as the resulting prenyltransferase activity of the present invention is present. In one embodiment, the prenyltransferase of the present invention is a prenyltransferase having one sequence number selected from the group consisting of sequence numbers 1 to 116, and may have, but is not limited to, 1 to 200, 1 to 150, 1 to 120, 1 to 100, 1 to 80, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, or at least 19, at least 18, at least 17, at least 16, at least 15, at least 14, at least 13, at least 12, at least 11, at least 10, at least 9, at least 8, at least 7, at least 6, at least 5, at least 4, at least 3, at least 2, or at least 1 further mutations.

[0027] In another aspect, the present invention may be a prenyltransferase that promotes or catalyzes a reaction between a hydrophobic substrate and an aromatic substrate.

[0028] In some embodiments of the present invention, the hydrophobic substrate may be, but is not limited to, an isoprenoid moiety, a geranyl moiety, a farnesyl moiety, and a group that may contain one or more phosphate groups. In one embodiment, it may be isopentenyl pyrophosphate, dimethylallyl pyrophosphate, farnesyl pyrophosphate, geranyl pyrophosphate (GPP), or geranylgeranyl diphosphate.

[0029] In some embodiments of the present invention, the aromatic substrate may be 2,4-dihydroxy-6-pentylbenzoic acid, (E)-2,4-dihydroxy-6-styrylbenzoic acid, olivetol, olivetolic acid, divalinol, divalinolic acid (DVA), orcinol, or orceric acid (OSA).

[0030] In certain embodiments, the substrate of the prenyltransferase of the present invention is not limited, but may include, for example, dihydroxynaphthalene (DHN), flavonoids, polyketides, resveratrol, or pyrophosphate, and in one embodiment, may include 1,6-DHN, 2,7-DHN, naringenin, apigenin, genistein, or daidzein.

[0031] In some embodiments of the present invention, the substance produced by the prenyltransferase of the present invention is not limited to but may be cannabinoids, such as 5-geranyl olivelate (5-GOLA), cannabigerol acid (3-geranyl olivelate (3-GOLA)), cannabigerol (CBG) (2-GOL), or cannabigerovalic acid (3-GDVA), cannabigerolsinic acid (3-GOSA and 5-GOSA), or 2-O-geranyl olivetolic acid (2-O-GOA).

[0032] In certain embodiments, the by-products may be isomers, and the isomers are not limited to 2-O-geranylolibetolic acid (2-O-GOA), 4-O-geranylolibetolic acid (4-O-GOA), or 5-geranyl-olibetrate (5-GOLA).

[0033] In another aspect, the present invention is a regioselective prenyltransferase that catalyzes or promotes the regioselective or stereoselective synthesis (reaction) of a desired (target) compound. The present invention may also be a prenyltransferase having high specificity to a desired compound. In certain embodiments, but not limited to, for example, in the production (manufacturing) reaction of a desired canbinide, it may have regioselectivity for cannabigerol acid in the total geranyl olivetolate (3-GOLA+5-GOLA) produced. In one embodiment, the prenyltransferase of the present invention may be a prenyltransferase having at least 50% regioselectivity for cannabigerol acid from, for example, geranyl pyrophosphate and olivetolate.

[0034] Furthermore, the regioselectivity of the prenyltransferase of the present invention is not limited to, but applies to the desired compound, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, and at least 72%. It may have at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or more.

[0035] In another embodiment, the prenyltransferase of the present invention may be a prenyltransferase that reduces the rate of byproduct formation of a given compound (a product other than the target compound, or an isomer) to at least 50% compared to wild-type prenyltransferase. In one embodiment, but not limited to, such a rate of formation may be at least 49%, at least 48%, at least 47%, at least 46%, at least 45%, at least 44%, at least 43%, at least 42%, at least 41%, at least 40%, at least 39%, at least 38%, at least 37%, at least 36%, at least 35%, at least 34%, at least 33%, at least 32%, at least 31%, at least 30%, at least 29%, at least 28%, at least 27%, and less It may be at least 26%, at least 25%, at least 24%, at least 23%, at least 22%, at least 21%, at least 20%, at least 19%, at least 18%, at least 17%, at least 16%, at least 15%, at least 14%, at least 13%, at least 12%, at least 11%, at least 10%, at least 9%, at least 8%, at least 7%, at least 6%, at least 5%, at least 4%, at least 3%, at least 2%, or at least 1%.

[0036] In a second aspect, the present invention may be a vector expressing the above-mentioned prenyltransferase. In one embodiment, the present invention may also be an expression vector comprising SEQ ID NOs: 1 to 116.

[0037] In a third aspect, the present invention may be a transformant into which a vector (plasmid) expressing the above-mentioned prenyltransferase has been introduced. The present invention is, but is not limited to, a recombinant microorganism into which the above-mentioned prenyltransferase has been incorporated, or a genetically modified cell expressing the above-mentioned prenyltransferase. In some embodiments, such (host) microorganism or cell may be a microorganism in which the microalgae are selected from the group consisting of diatoms or green algae, yeast and actinomycetes. In another embodiment of the present invention, such yeast may be, but is not limited to, the genera Yarrowia or Saccharomyces, and the microalgae may be Phaeodactylum tricornutum or Chlamydomonas reinhardtii, typically Komagataella (pichia), Escherichia, Bacillus or Aspergillus. In another embodiment, the actinomycetes may be, but are not limited to, Streptomyces.

[0038] Figure 1 shows the synthesis pathway of cannabinoid compounds from glucose, which is the starting material. In the cannabinoid biosynthesis pathway, olivetolic acid is produced from trioxododecanoyl-CoA generated via the malonyl-CoA production pathway. Furthermore, geranyl pyrophosphate is biosynthesized by the condensation reaction of farnesyl pyrophosphate and isopentenyl pyrophosphate catalyzed by geranylgeranyl pyrophosphate synthase. Cannabigerolic acid is synthesized from the resulting olivetolic acid and geranyl pyrophosphate, and then cannabidiol is produced.

[0039] In a fourth aspect, the present invention can be used as an alternative biosynthetic or metabolic pathway in the synthesis pathway of cannabinoid compounds. In some embodiments of the present invention, but not limited to, a method for producing cannabinoids or prenylated (aromatic) compounds, comprising the step of contacting (reacting) a prenyltransferase with a hydrophobic substrate and an aromatic substrate. In a particular embodiment of the present invention, but not limited to, the aromatic substrate may be olivetol, olivetolic acid, divalinol, divalinolic acid, orsinol, or orceric acid. Also, but not limited to, the hydrophobic substrate may be an isoprenoid moiety, a geranyl moiety, a farnesyl moiety, or a substrate having one or more phosphate groups. Also in one embodiment, but not limited to, geranyl diphosphate or geranyl pyrophosphate, etc. Furthermore, in a particular aspect of the present invention, but not limited to, a method for producing cannabigerolic acid from olivetolic acid and geranyl pyrophosphate in the cannabinoid synthesis pathway.

[0040] In certain embodiments of the present invention, cofactors or coenzymes may be added, but are not limited to, magnesium ions, platinum (PT), and the like.

[0041] In another aspect, certain embodiments of the present invention may include a culturing (or fermentation) step using cells (microorganisms) into which the gene expressing the prenyltransferase described above has been introduced. In a particular embodiment, such culturing may occur before the contact step, and may be aerobic or anaerobic, and may be continuous or batch. Furthermore, the culturing period may be 1, 2, 3, 4, 5, 6, or 7 days or more, and may be 1, 2, 3, or 4 weeks or more. The culturing temperature may be 23 to 44 degrees Celsius, and the pH may be 4 to 9. In yet another embodiment, an aromatic substrate may be added to such culturing step.

[0042] In some embodiments of the present invention, the process may include a step of separating, isolating, or purifying the product obtained in the culture step (or a step of recovering the product). In certain embodiments, however, the process may include, but is not limited to, partition extraction, liquid extraction, permeation vaporization, evaporation, filtration, membrane filtration, dialysis, reverse osmosis, distillation, crystallization and recrystallization, centrifugation, extractive filtration, ion exchange chromatography, size exclusion chromatography, or adsorption chromatography, or a combination thereof. In one embodiment, however, the product may be a cannabinoid or a prenylated (aromatic) compound, and may be cannabigerol acid. [Examples]

[0043] The present invention will be described in more detail below based on specific examples, but these examples are illustrative and not intended to limit the present invention.

[0044] Test Example 1: Mutant Expression The NphB gene (GenScript Japan) was synthesized using a gene synthesis service. Furthermore, mutant genes (GenScript Japan) were synthesized by site-directed mutation using a site-directed mutant generation service. To express them as His-tag fusion proteins, the NphB gene or the mutant gene was introduced into a pET28(+) vector, and plasmids were created.

[0045] Test Example 2: Protein Expression and Purification Each of the above plasmids was used to transform E. coli strain BL21(DE3). The transformants were transferred to LB medium containing 50 μg / ml kanamycin. When the OD600 of each culture was between 0.4 and 0.6, 100 μM IPTG was added, and the cultures were incubated at 18°C ​​for approximately 16 hours. Subsequently, the cultures were centrifuged (6000xg, 4°C, 30 minutes) to obtain wet cells. The wet cells were suspended in a 5% v / v cell lysate of the culture (10% BugBuster 10X (Merck), 50 mM Tris-HCl (pH 7.2), 500 mM sodium chloride, 20 mM imidazole, 20% glycerol), and left to stand at room temperature for 10 minutes. Each lysate was centrifuged (10000xg, 4°C, 30 minutes), and the supernatant was collected. Next, the recovered supernatant was washed with three times the column volume of buffer A (50 mM Tris-HCl (pH 7.2), 500 mM sodium chloride, 20 mM imidazole, 20% glycerol) using a Ni-NTA column (Cytiva). Then, the produced prenyltransferase was eluted with twice the column volume of elution buffer (buffer A, but used as 250 mM imidazole). Prenyltransferase was quantified from the obtained samples using the Bradford method to obtain prenyltransferases of sequence numbers 1 to 116.

[0046] Test Example 3: Enzyme Assay Condition A: The test was performed on prenyltransferase with 2 mg / ml NphB or amino acid substitutions using 5 mM geranyl diphosphate, 5 mM olivetolic acid, 5 mM magnesium chloride, and 50 mM Tris-HCl (pH 7.2) buffer in a total volume of 50 μl.

[0047] Condition B: A total volume of 50 μl was used with 5 mM geranyl diphosphate, 1 mM olivetolic acid, 5 mM magnesium chloride, and 50 mM Tris (pH 7.2) buffer, and the test was performed on prenyltransferase with 0.4 mg / ml NphB or amino acid substitutions.

[0048] Each reaction product was incubated at 30°C for 40 minutes, and then extracted twice with 100 μl of ethyl acetate. The extracted samples were air-dried. The samples were redissolved in methanol, passed through a PVDF filter, and analyzed by LC-MS.

[0049] Test Example 4: Quantitative Determination of Products The reaction products were fractionated by reverse-phase chromatography (using an ACQUITY BEH C18 1.7 μm column (2.1 × 100 mm)) using an LC-MS2050 (Shimadzu). The column compartment temperature was set to 55°C, and analysis was performed at a flow rate of 0.8 ml / min. Compounds were separated using gradient elution of solvent A (water + 0.1% formic acid) and solvent B (acetonitrile + 0.1% formic acid) as the mobile phase. For the first 2 minutes, 1.8 ml of 35% solvent B was initially eluted by isocratic elution, then the concentration of solvent B was increased to 60% over a 0.4 ml flow, and 3.2 ml of 60% solvent B was eluted again by isocratic elution. Subsequently, the concentration of solvent B was increased to 100% over a 0.4 ml flow, and 1.8 ml of 100% solvent B was eluted by isocratic elution. The chromatographic area of ​​the purchased analytical standards was measured, and each sample was quantified using an interpolation calibration curve.

[0050] The wild-type prenyltransferase obtained as described above and prenyltransferases with amino acid substitutions as one form of mutation (SEQ ID NOs. 2-5) were used in an enzyme assay under test condition B described above. Furthermore, the products were analyzed, and the amount of cannabigerol acid produced was measured. Table 1 shows the relative production ratios of cannabigerol acid for prenyltransferases with amino acid substitutions (SEQ ID NOs. 2-5), with the amount of cannabigerol acid produced by wild-type prenyltransferase set to 1. For prenyltransferases with amino acid substitutions, the ratios were approximately 5, 16, and 148 times, respectively. In contrast, the production ratio of cannabigerol acid by prenyltransferase SEQ ID NO. 5 was approximately 430 times higher than that of wild-type prenyltransferase.

[0051] [Table 1]

[0052] Furthermore, using wild-type prenyltransferase and prenyltransferases with amino acid substitutions (SEQ ID NOs. 2-5), the amount of isomers produced other than cannabigerol acid, which is generated by the prenyltransferase-catalyzed reaction of olivetolic acid and geranyl diphosphate under test condition B, was measured in the same manner as described above. Table 2 shows the ratio of the isomer amount of prenyltransferases with amino acid substitutions (SEQ ID NOs. 2-5) to the isomer amount of wild-type prenyltransferase. For prenyltransferases with amino acid substitutions (SEQ ID NOs. 2-5), the ratios were 0.38, 0.41, 0.00, and 0.00, respectively.

[0053] [Table 2]

[0054] Enzyme assays were performed using prenyltransferases with amino acid substitutions (SEQ ID NOs. 5 and 12-14) under test condition B, as described above. The amount of cannabigerol acid produced was measured, and the relative production ratio is shown in Figure 2, with the amount of cannabigerol acid produced by the prenyltransferase with amino acid substitution (SEQ ID NO. 5) set as 1. The highest value was 1.94 times for the prenyltransferase of SEQ ID NO. 12.

[0055] Furthermore, prenyltransferases containing amino acid substitutions (SEQ ID NOs. 5-11) were used, and the enzyme assay was performed under test condition A, as described above. The amount of cannabigerol acid produced was measured, and the relative production ratio is shown in Figure 3, with the amount of cannabigerol acid produced by prenyltransferase containing amino acid substitutions (SEQ ID NO. 5) set as 1. The highest value was achieved with prenyltransferase SEQ ID NO. 8, at 1.32 times.

[0056] Furthermore, the homology between wild-type prenyltransferase (SEQ ID NO: 1) and prenyltransferases with amino acid substitutions as one form of mutation (SEQ ID NOs: 6-11) is shown below.

[0057] [Table 3]

[0058] As one form of mutation, prenyltransferases with amino acid substitutions (SEQ ID NOs. 5 and 17-46) were used, and enzyme assays were performed under test condition A, as described above. The relative production of cannabigerol acid was measured with the amount of cannabigerol acid produced by the prenyltransferase with amino acid substitution (SEQ ID NOs. 5) set to 1, and the results are shown in Figure 4. The highest value was achieved by prenyltransferases SEQ ID NOs. 22 and 24, at 1.51 times.

[0059] Furthermore, prenyltransferases with amino acid substitutions (SEQ ID NO: 5, and 47-81) were used, and the enzyme assay was performed under test condition B, as described above. The amount of cannabigerol acid produced was measured, and the relative production ratio is shown in Figure 5, with the amount of cannabigerol acid produced by prenyltransferase with amino acid substitutions (SEQ ID NO: 5) set to 1. The highest value was achieved with prenyltransferase SEQ ID NO: 49, at 1.61 times.

[0060] Furthermore, prenyltransferases with amino acid substitutions as one form of mutation (SEQ ID NO: 5, and 82-116) were used, and enzyme assays were performed under test condition B, as described above. The amount of cannabigerol acid produced was measured, and the relative production ratio is shown in Figure 6, with the amount of cannabigerol acid produced by the prenyltransferase with amino acid substitution (SEQ ID NO: 5) set to 1. The highest value was 2.72 times for the prenyltransferase of SEQ ID NO: 107.

[0061] Industrial applicability The cannabinoids produced using this invention can be further synthesized into CBD, THC, CBG, or CBC, and used as active ingredients in pharmaceuticals, health foods, and cosmetics. Furthermore, due to the high production efficiency, yields increase significantly, contributing to industrialization by maintaining a stable supply and price.

Claims

1. A prenyltransferase having an amino acid sequence that is at least 80% identical to SEQ ID NO: 1, Prenyltransferase containing one mutation selected from the following mutations; (1) A230S and Y286A, (2) A230S and Y286P, (3) V47W and Y286A, (4) V47W and Y286P, (5) G284S and Y286P, (6) G284S and Y286A.

2. The aforementioned mutations are A230S and Y286A, Furthermore, the prenyltransferase according to claim 1, having at least one mutation selected from the group consisting of A106G, E110G, I107V, T112V, S134A, V143L, T161I, E148D, M163I, M160I, K166R, S162G, K167H, Q169T, S175G, L196F, S212G, T200S, V213I, T221S, L227I, D225E, S233T, V46I, G271A, I63F, T277G, S64T, E278G, S103D, A285S, M104S, H288Q, F105Y, and T273A.

3. The aforementioned mutations are A230S and Y286A, Furthermore, D125G, E304G, E242Q, D6S, T42V, I289L, I292V, D6E, D39F, E242I, D39Y, D39I, D6P, S162G, D39V, E242C, T290P, E242L, D43Q, I289M, D125T, I289C, E244I, E242V, D39N, E242H, D39A, E24 The prenyltransferase according to claim 1, having at least one mutation selected from the group consisting of 2F, E244C, D39E, E304A, D39C, D39, D43E, E244V, D39W, D39G, Q293I, D39P, D6G, D6K, D39Q, E242W, D39M, D39R, T42A, I289F, and D39S.

4. The mutations are A230S and Y286P, Furthermore, the prenyltransferase according to claim 1, having the T42A and / or T290P mutation.

5. The mutations are A230S and Y286P, Furthermore, it has the T290P mutation, The prenyltransferase according to claim 1, further comprising at least one mutation selected from the group consisting of L155V, L303I, G271F, A84D, V47I, V47L, L59M, L303V, L204T, V262D, L182K, V47F, L303F, V47A, and T290P.

6. The mutations are A230S and Y286P, Furthermore, it has the T42A mutation, The prenyltransferase according to claim 1, further comprising at least one mutation selected from the group consisting of L177A, V7R, Q179M, L59M, V269I, L238M, L177I, A84T, L303I, V77Q, L238A, L303V, I232Q, A257V, L303F, A84Y, V7L, V262G, V269K, and V23L.

7. The mutations are V47W and Y286A, Furthermore, the prenyltransferase according to claim 1, having either the A230S mutation or the G284S mutation.

8. The mutations are V47W and Y286A, Furthermore, it has the A230S mutation, Furthermore, the prenyltransferase according to claim 1, having the G284S and Y286A mutations.

9. The mutations are V47W and Y286A, Furthermore, it has the A230S, G284S, and Y286A mutations, Furthermore, the prenyltransferase according to claim 1, having the Q293F mutation.

10. The mutations are V47W and Y286P, Furthermore, the prenyltransferase according to claim 1, having one of the mutations A230S, G284S, or Q293F.

11. The mutations are V47W and Y286P, Furthermore, it has the A230S mutation, The prenyltransferase according to claim 1, having the G284S and Y286A mutations.

12. The mutations are V47W and Y286P, Furthermore, it has the A230S, G284S, and Y286A mutations, Furthermore, the prenyltransferase according to claim 1, having the Q293F mutation.

13. The aforementioned prenyltransferase has aromatic prenyltransferase activity, The prenyltransferase according to claim 1, which produces cannabigerol acid and has regioselectivity of 50% or more for the production of said cannabigerol acid.

14. A genetically modified microorganism having the prenyltransferase described in claims 1 to 13.

15. A method for producing prenylated aromatic compounds, A method for producing a prenylated aromatic compound, comprising the step of contacting a prenyltransferase according to claims 1 to 13 with a substrate.

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