JP2014223038A - Method for screening gene coding terpene synthase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for searching for enzyme groups and variants thereof associated with terpene biosynthetic pathway, the enzyme groups and variants being used for constructing a large-scale biosynthetic system of terpene.SOLUTION: There is provided a method for screening a terpene synthase based on "substrate consuming ability" of the terpene synthase. Specifically, there is provided a method for screening genes and/or variants thereof coding an enzyme associated with terpene synthesis by introducing test genes to cells which are each introduced with a terpene synthase variant having an enhanced enzymatic activity and/or an expression thereof compared with a wild type enzyme, to culture them, and by selecting the test gene introduced to a surviving cell, among the test genes introduced into the cells.

Description

  The present invention relates to a screening method for a gene encoding terpene synthase and / or a variant thereof. More specifically, the present invention introduces a gene encoding a terpene synthase variant that encodes a terpene synthase variant that has enhanced enzyme activity and / or its expression compared to a wild-type enzyme. The test gene is introduced into the cultured cells and cultured, and the test gene introduced into the surviving cells among the test genes introduced into the cells is selected, wherein the terpene synthase mutant is The present invention relates to a screening method for a gene encoding a terpene synthase and / or a mutant thereof, characterized by causing cell death by consuming a compound involved in cell survival as a substrate or a material of the substrate.

  Terpenes are a diverse group of natural compounds produced by plants, insects, fungi, and the like, with over 55,000 structures known. Depending on the classification, a derivative having a functional group such as a carbonyl group or a hydroxy group may be called a terpenoid among terpenes. Moreover, the name isoprenoid is also used as a general term for them.

Terpenes initially pointed to hydrocarbons with the molecular formula of C ₁₀ H ₁₆ contained in essential oils, but the object called terpenes has expanded and is now derived from hydrocarbons of the composition (C ₅ H ₈ ) _n. Oxygen / nitrogen compounds as well as those with different degrees of unsaturation have come to be understood. The expanded range is often referred to as a terpenoid. In the above molecular formula, n number 2 is called monoterpene, 3 is sesquiterpene, 4 is diterpene, 6 is triterpene, 8 is tetraterpene, and many are called polyterpenes. It is said that there are over 1 million terpenes in nature, including known / unknown. All these terpene compounds are synthesized from a common precursor called isopentenyl diphosphate (hereinafter IPP) via a terpene synthesis route. IPP is synthesized via a mevalonic acid pathway starting from acetyl CoA or a non-mevalonic acid pathway starting from pyruvic acid (Non-Patent Document 1-2). IPP is converted to its structural isomer, dimethylallyl diphosphate (hereinafter abbreviated as DMAPP), by an isomerization reaction with isopentenyl diphosphate isomerase (hereinafter IDI). By the action of prenyl transferase on IPP and DMAPP synthesized through these pathways, chain alcohol diphosphate esters that are intermediates in terpene synthesis, such as geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, etc. Is generated. Terpenes are synthesized by the action of terpene synthase using these chain alcohol diphosphate esters as substrates, but these enzymes give chain terpenes, ring closure, rearrangement of hydrides and alkyl groups, addition of water and amines, Dehydrogenation, oxygen functional group introduction, and decarboxylation are performed to produce terpenes with a wide variety of skeletons.

  Terpenes are widely used as pharmaceuticals, fragrances, and synthetic raw materials for organic synthesis (Non-patent Document 3). In addition to those having such industrial value, there are those having various pharmacological activities such as antibacterial action, immunostimulatory action, tranquilizing action, and antimalarial activity. Furthermore, in recent years, terpenes having physical properties suitable for diesel and jet fuel have attracted attention as new biofuels (Non-Patent Document 4).

  Since terpenes have various physiological functions in this way, many studies have been conducted with the aim of establishing an efficient method for producing terpenes by microorganisms. Since most of the genes encoding terpene synthase are derived from plants, in production systems using hosts such as E. coli and yeast, the expression level is low, and the stability and activity of the expressed enzyme are also low. That is, in many cases, the original activity cannot be exhibited due to non-optimization of codon usage, lack of post-translational modification, absence of an association partner, and the like. Many terpene synthases do not have high enzyme activity. These causes make it difficult to construct efficient terpene microbial production.

  On the other hand, almost all cells considered to be used for industrial production hosts such as E. coli, yeast, cyanobacteria, corynebacteria, Bacillus subtilis and thermophilic bacteria synthesize direct precursors of terpenes. Yes. For this reason, it is possible to cause cells to produce terpenes by introducing only the necessary terpene synthase gene. However, the synthesis amount of these prenyl diphosphates is not necessarily high, and it is used for biosynthesis of various components necessary for cell growth such as tRNA modification, protein prenylation, and dolichol / quinone side chains. There are not always many carbon fields to be rotated.

  Various attempts have been made to improve the production route for the efficient production of terpenes. For example, overexpression of a host-derived prenyl diphosphate synthesis pathway gene (Non-patent Document 5), introduction of a heterologous prenyl diphosphate synthesis pathway gene (Non-Patent Document 6-7), and tRNA prenylating enzyme which is a competitive pathway Attempts by gene deficiency (Patent Literature 1, Non-Patent Literature 6-7), global regulator overexpression (Non-Patent Literature 7), and the like have been reported. However, the production efficiency of terpenes is overwhelmingly low (at most 1 g / L medium) compared to the production levels of bioalcohol, fatty acid esters, bioplastics, etc. (tens to hundreds of g / L medium). There is still room for improvement in the method. For the purpose of searching for genetic factors that contribute to the improvement of terpene production ability, search for complex and unexplained metabolic control networks of hosts, random gene knockout (Non-patent Documents 8-10), sigma factors and global transcription Random modification of regulators (Patent Documents 2-4 and Non-Patent Document 7), genome shuffling (Non-Patent Document 11), and the like are still vigorously performed. In addition, even in the gene (group) that exists upstream of the same terpene production pathway and enhances production, if the origin is different or the operon configuration is changed, the terpene production ability of the host increases (Non-Patent Documents 12-13). And 14-16).

  In addition, for the purpose of efficient production of terpenes, the search for synthesizing enzyme genes has been actively conducted. However, there has not been proposed a method for comprehensively acquiring all genes encoding synthases involved in terpene biosynthesis. In particular, the function of a synthase that produces an unknown terpene molecule cannot be easily identified. At present, a gene having significant sequence homology with a known terpene synthase gene is identified from a genome database, and the activity of the protein encoded by the gene is analyzed by liquid chromatography mass spectrometry (LC-MS) or gas chromatography mass spectrometry. A new terpene synthase gene is obtained by a method in which a gene encoding a protein that has been measured using a device (GC-MS) and as a result has been found to have terpene synthase activity is identified as a terpene synthase gene. (Non-patent Document 17). However, it has been difficult to obtain a gene having low sequence homology with a known terpene synthase gene by such a method.

  Screening using a carotenoid pigment synthesis pathway synthesized from prenyl diphosphate (carotenoid screening) is used for improving the terpene production pathway and searching for its synthase gene (Non-patent Documents 7 and 18). In this screening, when the amount of terpene precursor increases due to the introduction and expression of some gene, the amount of accumulated carotenoid pigment synthesized from the terpene precursor increases, and the increased amount of pigment accumulation deepens the colony color. Can be visualized.

  The present inventors have also provided a method for screening a terpene synthase gene based on the substrate consumption ability of the terpene synthase (Non-patent Document 5). That is, by constructing biosynthetic pathways of various pigments using the same substrate as the terpene synthase group as a raw material, introducing terpene synthase genes into these cells and taking the raw materials from the pigment synthesis pathway, A screening method for terpene synthase using as an index the decrease in the amount of dye synthesis per cell is provided.

  However, terpene synthase gene screening using as an index the increase or decrease in the amount of accumulated pigment has two problems. One problem is that it takes time and effort to measure the color of colonies and the screening size is limited. In such screening, in many cases, it is necessary to devise a technique for improving visibility using a nitrocellulose membrane, and there are significant limitations on the cost and throughput (Non-patent Document 19). In addition, these methods are not practical for searching with higher throughput requirements such as a metagenomic source or a random mutation library across multiple genes. Another problem is the catalytic activity of the carotenoid pigment metabolic pathway itself, and the existence of the cell host's pigment accumulation limit. Even if the upstream pathway of the terpene production pathway can be enhanced, the amount of pigment accumulated in the cell reaches a plateau due to these factors. For this reason, it is difficult to search for a terpene synthase gene having a strong activity.

JP 2005-348644 A US Pat. No. 7,695,932 US Pat. No. 7,741,070 US Pat. No. 8,062,878 JP 2011-125334 A

Goldstein J. et al. L. , Et al. , Regulation of the mevalonate pathway. Nature 343, 425-430 (1990) Hunter W. N. The Non-mevalonate Pathway of Isoprenoid Precursor Biosynthesis. The Journal of Biological Chemistry 282, 21573-21571 (2007) Ajikumar P.M. K. , Et al. , Terpenoids: opportunities for biosynthesis of natural product drugs using engineering microorganisms. Molecular Pharmaceuticals 5, 2, 167-190 (2008) Lee S. K. et al. , Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels. Current Opinion in Biotechnology 19,5,556-563 (2008) Kinked Reiling K.K. et al. , Monoand diterpene production in Escherichia coli. Biotechnology and Bioengineering 87, 2, 200-212 (2004) Martin V.M. J. et al. J. et al. et al. , Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nature Biotechnology 21, 7, 796-802 (2003) Stepanopoulos G. et al. , Multi-dimentional gene target search for improving lycopene biosynthesis in Escherichia coli. Metabolic engineering 9,337-347 (2007) Oezaydin B.E. et al. , Carotenoid-based phenotypic screen of the yeast deletion collection, reviews new genes with roles in isoprenoid production. Metabolic engineering 15, 174-183 (2013) Alper H. et al. , Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli. Metabolic engineering 7,155-164 (2005) Towersamretkit I. et al. , Analysis of Metabolic Network of Synthetic Escherichia coli Producing Linear Usage Constrained-based Modeling. Procedia Computer Science 11, 24-35 (2012) Zhang Y. X. , Et al. , Genome shuffling leads to rapid phenotypic improvement in bacteria. Nature 415, 644-646 (2002) Kajiwara S. et al. , Expression of an exogenous isopentenyl diphosphite isomerase gene enhances isoprenoid biosynthesis in Escherichia coli. Biochemical Journal 324, 421-426 (1997) Tsuruta H. et al. et al. High-level production of amorphous-4, 11-diene, a preparatory of the antimalarial artemisinin, in Escherichia coli. PloS One 4, e4489 (2009) Anthony J.H. R. et al. , Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malaural drump. Metabolic Engineering 11, 13-19 (2009) Peralta-Yahya P.M. P. et al. , Identification and microbiological production of a terpene-based advanced biofuel. Nature Communications 2,483 (2011) Ajikumar P.M. K. et al. , Isoprenoid pathway optimization for Taxol pre-cursor over production in Escherichia coli. Science 330, 70-74 (2010) Mijts B.M. N. et al. , Identification of a carotenoid oxygenase synthesizing acyclic xanthoxyls: combinatorial biosynthesis and directed evolution. Chemistry and Biology 12,453-460 (2005) Prater K. J. et al. L. et al. , Proceedings of The National Academy of Sciences 107, 32, 13654-13659 (2010) Furubayashi M. et al. et al. , Directed Evolution of Carotenoid Syntheses for the Production of Unusual Carotenoids. Methods in Molecular Biology 892, 245-253 (2012)

  In order to enable biological production of known and unknown terpenes in nature, it is first necessary to acquire a gene for the synthase, and it is necessary to develop a method for acquiring such a gene. Moreover, in order to construct a terpene biological mass production system, it is necessary to improve the terpene production pathway, and it is necessary to develop a method for searching for enzymes related to the pathway and mutants thereof.

  That is, the subject of this invention is providing the search method of the enzyme group and those variants which are used in order to construct | assemble the terpene biological mass production type | system | group used in the terpene production pathway.

  The present inventors have intensively studied to solve the above problems, and cell death is caused in an E. coli strain transformed with a gene encoding a geraniol synthase (hereinafter abbreviated as GES) mutant with improved terpene production. The cell death is caused by growth inhibition caused by consumption of the substrate by GES, and the cell death can be prevented by supplying the substrate by introducing a gene encoding an enzyme that catalyzes the biosynthesis of the substrate of GES. I found. Using these findings, a test gene is introduced into a cell into which a gene encoding a terpene synthase mutant with enhanced enzyme activity and / or its expression has been introduced, and the test is introduced into the cell. A gene encoding terpene synthase and / or a variant thereof, for example, a substrate of terpene synthase or a compound serving as a material of the substrate, that is, a terpene precursor, by selecting a test gene introduced into a living cell among the genes A method for screening a gene encoding an enzyme for synthesizing a body and / or a variant thereof was constructed.

That is, this invention consists of the following.
1. A test gene is introduced into a cell into which a gene encoding a terpene synthase variant that encodes a terpene synthase variant that has an enzyme activity and / or enhanced expression compared to a wild-type enzyme. A screening method for a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell among test genes introduced, cultured, and introduced into the cell.
2. 1. The terpene synthase mutant is a terpene synthase mutant that causes cell death of cells expressing the mutant. Screening method for terpene synthase gene and / or mutant thereof.
3. 2. The cell death caused by the fact that the substrate consumed by the terpene synthase mutant or the compound serving as the material of the substrate is a compound involved in cell survival. Screening method for terpene synthase gene and / or mutant thereof.
4). 1. The terpene synthase mutant is a geraniol synthase mutant. To 3. A method for screening a gene of terpene synthase and / or a mutant thereof.
5. 1. The cell is E. coli. To 4. A method for screening a gene of terpene synthase and / or a mutant thereof.
6). A gene encoding a geraniol synthase mutant with enhanced enzyme activity, wherein the mutant has enhanced enzyme activity and / or expression compared to a wild-type enzyme and expresses the mutant In a cultured cell, a cell into which a gene encoding a mutant that causes cell death caused by a substrate consumed by the mutant or a compound serving as a material of the substrate being a compound involved in cell survival is introduced into a cell. A screening method for a terpene synthase gene and / or a variant thereof, comprising introducing a test gene and culturing, and selecting a test gene introduced into a living cell among test genes introduced into the cell.
7). 5. The gene encoding a geraniol synthase mutant with enhanced enzyme activity is the gene represented by the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. Screening method for terpene synthase gene and / or mutant thereof.
8). Of the test genes introduced into the cells, the test genes are introduced into the cells into which the gene encoding the geraniol synthase mutant represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 has been introduced. A method for screening a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell.
9. A test gene is introduced into E. coli transformed with the plasmid represented by the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 and cultured, and the test gene introduced into the surviving cells among the test genes introduced into the cells. A method for screening a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene.

  According to the present invention, a test gene is introduced into a cell into which a gene encoding a terpene synthase mutant with enhanced enzyme activity and / or expression thereof has been introduced and cultured, and the test gene introduced into the cell survived. It is possible to provide a screening method for a gene encoding terpene synthase and / or a mutant thereof including selecting a test gene introduced into a cell, and as a result, the selected gene or the mutant thereof is used. Thus, a terpene biological mass production system can be constructed.

  The method according to the present invention is a selection method based on the principle of rescue from cell death due to depletion of a substrate of a terpene synthase or a compound serving as a material of the substrate. That is, this method is a so-called selection mode in which only a cell transformed with a terpene precursor, that is, a gene encoding an enzyme that synthesizes a terpene synthase substrate or a compound that is a material of the terpene, or a mutant thereof survives. This is a gene search method. This method has an extremely high throughput and a rapid operation compared to the conventionally reported screening methods for genes that contribute to the improvement of terpene production, for example, screening methods using increase or decrease in the amount of accumulated pigment as an index. It is simple (FIG. 1). In addition, since this method is associated with cell growth (Growth-coupled), cells transformed with a gene encoding an enzyme having a stronger activity of enhancing terpene precursor synthesis or a mutant thereof are considered to have higher survival efficiency. be able to. That is, there is a possibility of further use as a technique for concentrating more preferable genes as well as selecting genes having a positive effect.

It is a figure explaining the screening method of the enzyme gene in connection with terpene synthesis. Panel A shows a screening method that has been reported previously and that is carried out using carotenoid pigments with color as an indicator. Panel B shows a screening method which is a method according to the present invention and which is carried out using avoidance of cell death due to substrate depletion as an index. It is a figure which shows the schematic diagram of the expression construct of the gene which codes a geraniol synthetase variant, and its plasmid map. Panel A shows the expression construct pLac− of a gene encoding a mutant (GESM53) in which the N-terminal 52 amino acids of geraniol synthase (GES) are deleted and a 6 × histidine tag (6 × His) is added to the C-terminus. It is a schematic diagram of gesM53. Panel C shows the plasmid map of pLac-gesM53. Panel B shows a mutant (GESM53) in which glycine, which is the amino acid corresponding to the 54th GES in the amino acid sequence of GESM53, is replaced with valine in the amino acid sequence of GESM53, and 6 × His is added to the C-terminus. It is a schematic diagram of the expression construct pBAD-gesM53 _G54V of the gene encoding _G54V ). Panel D shows the plasmid map of pBAD-gesM53 _G54V . (Example 1) It is a figure which shows the result of having measured the geraniol production in colon_bacillus | E._coli strain XL-1 blue which introduce | transduced the mutant of the geraniol synthetase gene by the gas chromatography / hydrogen ion detector. A. XL-1 blue transformed with pLac-gesM53, B.I. Shows the analysis result of geraniol in XL-1 blue transformed with pLac-ges _D323A . C. Indicates the analysis result of the geraniol preparation. The horizontal axis represents the holding time (RT [min]). (Example 1) In Escherichia coli in which the carotenoid pigment synthase genes diapophytoene synthase (CrtM) gene and dehydrosqualene desaturase (CrtN) gene are expressed, when the geraniol synthase gene is co-expressed, the substrate of the pigment synthase is It is a figure explaining that the reduction | decrease of the accumulation amount of the amount of carotenoid pigment | dye was recognized because it was deprived by geraniol synthase. Panel A shows that in E. coli expressing CrtM and CrtN genes, geranylgeranyl diphosphate (GPP) is synthesized through several pathways using pyruvate as a raw material, and geraniol (GES) is further synthesized from GPP. It will be described that yellow pigment, a kind of carotenoid, is synthesized from GPP by relaying farnesyl diphosphate (FPP) while Geraniol is produced. Panel B shows that in E. coli XL-1 Blue in which CrtM gene and CrtN gene are expressed, when wild-type GES (indicated as WT in the figure) or GESM53 (indicated as M53 in the figure) is co-expressed, an empty vector ( It shows that the amount of carotenoid synthesis was significantly reduced compared to when transforming Void (indicated in the figure) or expressing a GES-inactivating mutant (indicated as WT _{D323A in the} figure). The amount of carotenoid synthesis is shown as the amount synthesized (μg / g DCM) per gram of dry cell weight (DCW). The results are shown as an average value of N = 3, and error bars indicate a standard deviation of N = 3. (Example 1) Colony formation was observed in Escherichia coli JW3686 expressing wild type GES and Escherichia coli JW3686 expressing GES inactivating mutant (indicated as D323A in the figure), but colony formation was observed in Escherichia coli JW3686 expressing GESM53. It is a figure explaining that almost was not observed. In the figure, wild type GES is indicated as WT, GES inactivating mutant is indicated as D323A, and GESM53 is indicated as M53. The vertical axis in the figure represents the colony forming unit (CFU / TF) per transformation. Example 3 It is a figure explaining colony formation was observed when the colony was not formed in colon_bacillus | E._coli which expressed only GESM53, but isopentenyl diphosphate isomerase (IDI) was co-expressed in this colon_bacillus | E._coli. When E. coli JW3686 was cotransformed with pLac-gesM53 and pAC-void, an empty vector, or pAC-crtMN, a chromogenic enzyme gene expression vector, no colonies were formed, but pLac-gesM53 and pAC-idi Were cotransformed to form colonies. The left vertical axis of the figure shows colony forming units (CFU / TF) per transformation. In the figure, ◯ indicates a colony forming unit (CFU _{GES (+)} ) by co-transformation of pLac-gesM53 and pAC-void, pAC-idi, or pAC-crtMN. ● indicates a colony forming unit (CFU _{GES (-)} ) by cotransformation of pLac plasmid and pAC-void, pAC-idi, or pAC-crtMN. The right vertical axis of the figure shows the survival rate, which is the ratio of CFU _{GES (+)} to CFU _{GES (-)} . The results are shown as an average value of N = 3, and error bars indicate a standard deviation of N = 3. Example 3 It is a figure which shows the result of having investigated whether the kind of host influences the cell death by GES mutant expression by transforming various GES mutant expression plasmids and a green fluorescent protein expression plasmid into three types of E. coli strains. Three types of E. coli strains, JW3686 strain (described as JW / Δtna in the figure), BW25113 strain, and XL-1 Blue were used. In the figure, D323A and G285S represent GES inactivating mutants, wt represents wild type GES, and M53 represents GESM53. The vertical axis represents the number ratio of fluorescent colonies and non-fluorescent colonies to colony forming units (cfu / plate) per plate, and the numbers in the figure indicate the number of colonies actually counted. Example 4 By cotransforming pLac-gesM53 and a mixed solution of pAC-idi and pAC-gfp (equivalent mixture of pAC-gfp / pAC-idi) into E. coli, and culturing the resulting transformant, It is a figure which shows that IDI gene was able to be selectively concentrated from the mixed system with a GFP gene. The left panel shows the results of counting the formed colonies after co-transforming pLac-gesM53 or pLac-ges _D323A and the pAC-gfp / pAC-idi mixed solution into the JW3686 strain. In the bar graph, dark gray indicates a green fluorescent colony (fluorescence (+)), and light gray indicates a non-fluorescent colony (fluorescence (-)). The numbers in the figure indicate the number of colonies actually observed. The right panel shows the plasmid portion of 3 fluorescent colonies and 3 of 30 non-fluorescent colonies (see left panel) generated by co-transformation of pLac-gesM53 and pAC-gfp / pAC-idi mixture. The results of amplification by polymerase chain reaction (PCR) and analysis by agarose gel electrophoresis are shown. In the figure, M is a molecular weight marker. (Example 5) Using arabinose inducible system of GESM53 _G54V, it illustrates switching and the examination results on the rescue effects of the cells death by IDI by arabinose _{GESM53 G54V} expression and it by cell death. The left vertical axis shows the number of colonies formed on an agar medium containing arabinose (○ CFU / TF-arabinose (+)) and colony formation on an agar medium containing no arabinose (● CFU / TF-arabinose (−)). Indicates a number. The number of colonies formed was expressed as colony forming units (CFU / TF) per transformation. The right vertical axis shows the viable cell rate (bar graph). The viable cell rate was calculated as the ratio of the number of colonies on the agar medium containing arabinose and the number of colonies on the agar medium not containing arabinose. (Example 6) An Escherichia coli (JW3686 / pBAD-gesM53 _G54V ) into which an arabinose induction system of GESM53 _G54V was introduced was _transformed with an equiconcentration mixture (pAC-gfp / pAC-idi mixture) of pAC-idi and pAC-gfp. It is a figure explaining that the IDI gene was able to be selectively concentrated from the mixed system with a GFP gene by culturing the transformant which was prepared. The left panel (A) shows JW3686 / pBAD-gesM53 G54V _transformed with a pAC-gfp / pAC-idi mixed solution and an agar medium (upper) containing 0.2% arabinose and an agar medium containing no arabinose (upper). The lower part) shows the state of colonies formed by seeding. White colonies are fluorescent colonies, that is, transformants of pAC-gfp. The right panel (B) shows colony forming units (CFU / TF) per transformation determined from the number of non-fluorescent colonies formed and the number of fluorescent colonies. The numbers in the figure indicate the number of colonies actually counted. (Example 7)

  The present invention relates to a screening method for a gene encoding terpene synthase and / or a variant thereof. More specifically, the present invention relates to a screening method for a gene encoding an enzyme that synthesizes a terpene precursor and / or a variant thereof.

  The screening method according to the present invention has found that cell death is caused by growth inhibition resulting from substrate consumption by the enzyme in a cell transformed with a gene encoding a terpene synthase mutant with enhanced enzyme activity. The test gene is introduced into a cell into which a gene encoding a terpene synthase mutant with enhanced enzyme activity and / or expression thereof has been introduced and cultured, and the target introduced into the cell is cultured. Among the test genes, a test gene introduced into a living cell is selected.

  “Terpenes” are hydrocarbons having isoprene as a structural unit, and are biological substances produced by plants, insects, fungi, and the like. It is the name given to a group of 10 carbon compounds originally found in large quantities in essential oils, and is therefore organized on the basis of 10 carbons. Depending on the classification, a derivative having a functional group such as a carbonyl group or a hydroxy group may be called a terpenoid among terpenes. Moreover, the name isoprenoid is also used as a general term for them.

  Terpenes are biosynthesized from mevalonic acid or pyruvic acid in vivo through several steps of reaction. The biosynthesis pathway of terpenes differs depending on the species.

  The first step in terpene biosynthesis is the generation of isoprene units, from the mevalonate pathway or the non-mevalonate pathway (also referred to as the NEP / DOXP pathway) to the C5 molecule (C5PP) that is the source of the isoprene unit, ie isopentenyl diphosphorus. Acid (IPP) is synthesized. In addition, IPP produces its isomer, dimethylallyl diphosphate (DMAPP).

  The isomerization reaction from IPP to DMAPP is catalyzed by isopentenyl pyrophosphate isomerase (IDI).

  The second stage of terpene biosynthesis includes the coupling process of isoprene units in which isopentenyl diphosphate (IPP) is successively added to dimethylallyl diphosphate (DMAPP). As a result, the number of carbons increases by 5 and various chain alcohol diphosphates such as those having 10 carbon atoms (C10PP), those having 15 carbon atoms (C15PP), those having 20 carbon atoms (C20PP) Synthesized. These chain alcohol diphosphates are used as terpenoid precursors in the final stage of the terpenoid biosynthetic pathway.

  C10PP, such as geranyl diphosphate (GPP), is synthesized by a head-to-tail type condensation reaction that occurs between IPP and DMAPP. This reaction is catalyzed by dimethylallyltransferase (EC 2.5.1.1). C10PP is a precursor for monoterpenes such as geraniol and linalool.

  C15PP is synthesized by a condensation reaction of IPP and C10PP. For example, farnesyl diphosphate (FPP) is synthesized by a condensation reaction of IPP and GPP. This reaction is catalyzed by farnesyl diphosphate synthase (farnesyl diphosphate synthase). This is a precursor of sesquiterpenes such as farnesol. In addition, when two molecules of C15PP are bound by the catalytic action of squalene synthase, squalene is generated and becomes a precursor of triterpene such as cholesterol and phytosterol.

  C20PP is synthesized by a condensation reaction of IPP and C15PP. For example, geranylgeranyl diphosphate (GGPP) is synthesized by a condensation reaction of IPP and FPP. This reaction is catalyzed by geranylgeranyl diphosphate synthase. C20PP is the basic skeleton of diterpenes such as geranylgeraniol, phytol, and taxol. Since many diterpenes have physiological activity, the development of mass production methods is extremely important. Furthermore, when two molecules of C20PP are bound, phytoene is produced and becomes a tetraterpene precursor such as carotenoid.

  The third stage of terpene biosynthesis is the process of converting chain alcohol diphosphates into terpenes with various skeletons. Various enzymes are also involved in this biosynthetic pathway.

  Examples of the enzyme using C10PP as a substrate include geraniol synthase, limonene synthase, menthol synthase, and pinene synthase.

  Examples of the enzyme using C15PP as a substrate include γ-humulene synthase, farnesol synthase, aristoloken synthase, β-farnesene synthase, and notekatone synthase-squalene synthase. Squalene synthase catalyzes the reaction of condensing two molecules of C15PP through a head-to-tail bond to synthesize hydrocarbon squalene having a C30 skeleton.

  Examples of the enzyme using C20PP as a substrate include taxadiene synthase, coparyl diphosphate synthase, and kasbene synthase.

  A “terpene precursor” refers to a compound that is in a stage prior to a certain product in the terpene biosynthetic pathway and can be converted into the product by one to several steps of reaction. In other words, a terpene precursor means a substrate of an enzyme that synthesizes a product or a compound that becomes a material of the substrate in a terpene biosynthesis pathway. Terpene precursors include linear alcohol diphosphates exemplified by C5PP such as IPP and DMAPP, and C10PP such as GPP, C15PP such as FPP, and C20PP such as GGPP.

  “Terpene synthase” means an enzyme that catalyzes the biosynthesis of terpene. In the present invention, the term “terpene synthase” includes any enzyme involved in each process of terpene biosynthesis. In addition, an enzyme is obtained by adding another protein such as a histidine tag to the N-terminal side or C-terminal side of its amino acid sequence, as long as its basic properties such as enzyme activity are not inhibited. There may be. The enzyme may be a natural enzyme possessed in various organisms or a mutant. The “terpene synthase mutant” is a concept of a protein in which a terpene synthase is mutated by substitution, deletion, addition or the like of a part of amino acid residues of the protein. Some mutants cause changes in properties such as enzyme activity, substrate specificity, and stability due to such mutations. In contrast, an enzyme that has not been mutated is referred to as a wild-type enzyme.

  In the present invention, a gene encoding terpene synthase is referred to as “terpene synthase gene”. The “variant thereof” is a concept of a gene in which DNA of a terpene synthase gene is mutated by substitution, deletion, addition or the like of a part of the nucleotides. It may be a naturally occurring mutation or an artificially made mutation. In addition, a terpene synthase gene encodes another protein on the 5 ′ end side or 3 ′ end side of its DNA as long as its function, for example, the expression of the encoded protein or the function of the expressed protein is not inhibited. For example, a gene encoding a tag peptide such as a histidine tag may be added.

  Mutants can be obtained by creating a library of mutants and selecting mutants having high enzyme activity, expression level or stability in the host. Such selection can be carried out by a conventionally reported terpene synthase gene screening method using the increase or decrease in the amount of accumulated pigment as an index, or the screening method according to the present invention. Moreover, a variant and its library can be created using a known genetic engineering technique.

  The terpene synthase encoded by the terpene synthase gene to be introduced into the cell or a variant thereof and the variant thereof may be any as long as they have sufficient substrate consumption ability. Preferably, it is a terpene synthase variant whose enzyme activity and / or its expression is enhanced compared to the wild type enzyme, more preferably a terpene synthase variant whose enzyme activity is enhanced compared to the wild type enzyme . “Enzyme activity and / or its expression is enhanced compared to the wild-type enzyme” means that the enzyme activity per cell and / or its expression level is higher than that of the wild-type enzyme. A terpene synthase mutant whose enzyme activity and / or its expression is enhanced compared to the wild-type enzyme has a high substrate consumption capacity, resulting in depletion of the substrate and the compound that is the material of the substrate. For this reason, growth inhibition and cell death are caused in cells expressing a terpene synthase or a mutant thereof, because the substrate and the compound serving as the material of the substrate are compounds involved in cell survival. The degree of enhancement of the enzyme activity and / or expression of the terpene synthase mutant may be enhanced to the extent that cell death is induced by the increased consumption of the substrate and the compound serving as the material of the substrate.

  The terpene synthase encoded by a terpene synthase gene to be introduced into a cell or a mutant thereof, and the mutant thereof preferably have a substrate or a compound that is a material of the substrate that is involved in cell survival. It is the characteristic terpene synthase or its variant. More preferably, it is a terpene synthase or a variant thereof characterized by causing cell death of cells expressing the terpene synthase or a variant thereof. More preferably, in a cell expressing a terpene synthase or a mutant thereof, cell death is caused by depletion of a substrate of the enzyme encoded by the enzyme or the mutant or a compound serving as a material of the substrate. Synthetic enzyme or its variant. Even more preferably, the enzyme activity and / or expression thereof is enhanced compared to the wild-type enzyme and is consumed by the enzyme or variant thereof in a cell expressing the terpene synthase or variant thereof. It is a terpene synthase mutant characterized by causing cell death caused by a substrate or a compound serving as a material of the substrate being a compound involved in cell survival. Specific examples of the terpene synthase or a variant thereof include a terpene synthase or a variant thereof that consumes a chain alcohol diphosphate ester such as GPP or FPP with high efficiency.

As a terpene synthase encoded by a terpene synthase gene to be introduced into a cell or a mutant thereof and a mutant thereof, for example, a mutant of geraniol synthase (GES) can be preferably exemplified. Specifically, as a GES variant, a variant of GES derived from sweet basil, which was prepared by excising 52 amino acids at the N-terminal from the amino acid sequence of full-length GES (567 amino acids) (GESM53 and Can be mentioned). GESM53 has enhanced enzyme activity compared to the wild type, and caused cell death in cells expressing GESM53. Another specific example is a mutant (referred to as GESM53 _G54V ) in which glycine, which is the amino acid corresponding to the 54th amino acid of GES, is substituted with valine in the amino acid sequence of GESM53. Since GESM53 _G54 has a higher translation efficiency of its gene than GESM53, higher activity can be expected. The nucleotide sequence of the gene encoding GESM53 is shown in SEQ ID NO: 1, the amino acid sequence of GESM53 is shown in SEQ ID NO: 2, the nucleotide sequence of the gene encoding GESM53 _G54V is shown in SEQ ID NO: 3, and the amino acid sequence of GESM53 _G54V is shown in SEQ ID NO: 4. .

  As a gene to be introduced into cells by the method according to the present invention, a gene encoding an enzyme having the ability to consume a terpene synthase gene or a compound serving as a material of the substrate, instead of a terpene synthase gene or a variant thereof, Such variants can be used. For example, by introducing into a cell a gene encoding an enzyme having an activity of consuming IPP, which is a compound serving as a material for a substrate of terpene synthase, preferably an enzyme having a high activity of consuming IPP, Depletion is caused, resulting in cell death. An example of such an enzyme is pyrophosphatase that hydrolyzes and removes pyrophosphate.

  The cell may be any one as long as it introduces a terpene synthase gene or a variant thereof and consumes a compound involved in cell survival, resulting in cell death. . Preferably, cells that synthesize terpene precursors, such as IPP or DMAPP, or cells that have a terpene biosynthesis pathway are used. Considering the growth rate, ease of cloning, ease of genetic manipulation, and ease of use of terpenes in the construction of a mass production system for terpenes, the cells can be transformed into Escherichia coli, cyanobacteria, corynebacterium, Bacillus subtilis, thermophilic bacteria. Such bacteria and yeasts are preferred. It is also possible to use cells artificially damaged in the terpene biosynthesis pathway or part of its control system. Suitable cells include Escherichia coli, for example, JW3686 strain and BW25113 strain. The JW3686 strain is a strain obtained by adding the gene modification of ΔtnaA :: kanR to the BW25113 strain (Baba T. et al., Construction of Escherichia coli K-12 in-frame, single-gene knockout mutcntulc. Systems Biology 2, Article No. 0008 (2006)), since tryptophan metabolizing enzyme TnaA necessary for indole synthesis is missing, indole synthesis is not performed. Therefore, there is an advantage that the terpene product can be analyzed without suffering from the indole peak in the gas chromatogram. The BW25113 strain is an Escherichia coli strain derived from the Escherichia coli K-12 strain, and is widely used as a parent strain for preparing various genetically modified Escherichia coli.

Introduction of a gene or a mutant thereof into a cell can be performed using a known genetic engineering technique. For example, by inserting a terpene synthase gene or a mutant thereof into an appropriate vector DNA, creating a recombinant vector containing the gene or the mutant, and transforming the resulting recombinant vector into a cell Introducing a gene or a variant thereof into a cell can be performed. As a vector DNA into which a terpene synthase gene or a variant thereof has been inserted, a plasmid vector in which a gene encoding a protein having a 6 × histidine tag added to the C-terminus of GESM53 represented by the amino acid sequence shown in SEQ ID NO: 2 is inserted (FIG. 2). Panel A and C). The nucleotide sequence of this plasmid vector is shown in SEQ ID NO: 5. As another example, a plasmid vector (see panels B and D in FIG. 2) in which a gene encoding a protein having a 6 × histidine tag added to the C-terminus of GESM53 G54V _represented by the amino acid sequence shown in SEQ ID NO: 4 is inserted. It can be illustrated. The nucleotide sequence of this plasmid vector is shown in SEQ ID NO: 6.

  As the vector, an expression vector is preferably used. The vector DNA is not particularly limited as long as it can replicate in the host, and is appropriately selected depending on the type of host and intended use. The vector DNA may be one in which a part of the DNA other than the part necessary for replication is missing, in addition to one extracted from a naturally occurring one. Representative examples include plasmid, bacteriophage and virus-derived vector DNA. Examples of plasmid DNA include E. coli-derived plasmids, Bacillus subtilis-derived plasmids, yeast-derived plasmids, and the like. Specific examples include pUC18 and pACYC184 in E. coli, pYB110, pE194, pC194, pHY300PLK DNA and the like in Bacillus subtilis, and pRS303, YEp213, and TOp2609 in yeast. It is necessary to incorporate the gene into the vector so that the function of the target gene is exhibited, and at least the target gene sequence and the promoter are constituent elements. In addition to these elements, a gene sequence carrying information on replication and control, if desired, for example, a ribosome binding sequence, a terminator, a signal sequence, a cis element such as an enhancer, a splicing signal, a selection marker, and the like. Alternatively, a plurality of gene sequences can be combined into a vector DNA by a method known per se. Examples of selection markers include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like. As a method for incorporating the target gene sequence into the vector DNA, a method known per se can be applied. For example, a method is used in which a target gene sequence is treated with an appropriate restriction enzyme, cleaved at a specific site, then mixed with a similarly treated vector DNA, and religated by ligase. Alternatively, a desired recombinant vector can also be obtained by ligating a suitable linker to the gene sequence of interest and inserting it into a multicloning site of a vector suitable for the purpose.

  For introduction of the vector into the host cell, means known per se can be applied. For example, the standard method described in the book (Sambrook et al., “Molecular Cloning, Laboratory Manual Second Edition”, 1989, Cold Spring Harbor Laboratory). Specific methods include calcium phosphate transfection, DEAE-dextran mediated transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape loading, ballistic introduction and infection. Etc.

  When a bacterium is used as a host, any promoter can be used as long as it can be expressed in a host such as Escherichia coli. For example, promoters derived from Escherichia coli or phage, such as trp promoter, lac promoter, PL promoter, and PR promoter can be exemplified. An artificially designed and modified promoter such as a tac promoter may be used. The method for introducing a recombinant vector into bacteria is not particularly limited as long as it is a method for introducing DNA into bacteria. Preferable examples include a method using calcium ions and an electroporation method.

  When yeast is used as a host, the promoter is not particularly limited as long as it can be expressed in yeast, and any promoter may be used. For example, gal1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like can be exemplified. The method for introducing a recombinant vector into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and preferred examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

  When an animal cell is used as a host, it is preferable that the recombinant vector is capable of autonomous replication in the cell and at the same time comprises a promoter, an RNA splice site, a target gene, a polyadenylation site, and a transcription termination sequence. Moreover, the replication origin may be included if desired. As the promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter or the like can be used, and a cytomegalovirus early gene promoter or the like may be used. Preferred examples of the method for introducing a recombinant vector into animal cells include the electroporation method, the calcium phosphate method, and the lipofection method.

  When a prokaryotic organism is used as a host, it is preferable that the recombinant vector is capable of autonomous replication in the bacterium, and at the same time is composed of a promoter, a ribosome binding sequence, a target gene and a transcription termination sequence. Moreover, the gene which controls a promoter may be contained.

As the expression vector, it is preferable to use an expression induction system (gene expression induction system) capable of controlling the expression of a desired gene. The gene expression induction system is a cell into which the system is introduced, and the expression of a desired gene is induced or suppressed in the presence of a specific drug or the like. A cell transformed with a gene encoding a terpene synthase or a variant thereof used in the screening method according to the present invention is maintained because cell death is caused by growth inhibition due to substrate consumption by the expressed enzyme. When culturing is desired, the expression of the gene needs to be suppressed. By using an expression induction system that can control the expression of genes, in normal maintenance culture, the cells are made to survive by suppressing the expression of the genes. Can be triggered. Any gene expression induction system can be used as long as it can control the expression of a gene encoding a terpene synthase or a mutant thereof. Preferably, one with a low level of leakage before induction of expression, more preferably one with little leakage before expression induction is used. Various systems have been reported as gene expression induction systems, and some are commercially available, so that an appropriate one can be selected and used. As a preferred gene expression induction system, a pBAD protein expression system can be exemplified. The pBAD protein expression system is a plasmid vector incorporating the pBAD promoter, and can control the expression level of the target protein in a dose-dependent manner with L-arabinose. An example of an expression vector for a terpene synthase mutant gene using the pBAD protein expression system is a plasmid represented by the nucleotide sequence set forth in SEQ ID NO: 6 (see panels B and D in FIG. 2). This plasmid is an expression construct in which a gene encoding a protein having a 6 × histidine tag added to the C-terminus of GESM53 G54V _represented by the amino acid sequence shown in SEQ ID NO: 4 is introduced under the _pEI of a ColEI type vector.

  “Test gene” means a gene to be tested by the screening method of the present invention. The test gene is not particularly limited and may be a naturally occurring gene or an artificially created mutant. For example, metagenome, mutants of known terpene synthases involved in the terpene biosynthetic pathway, genes prepared from cells and organisms where terpene production is observed, known genomic libraries and cDNA libraries, artificial terpene biosynthetic pathway operons Libraries and libraries obtained by randomization of enzyme genes that contribute to the improvement of the pathway upstream of the terpene biosynthesis pathway can be mentioned. The metagenome means a group of microbial genomes extracted from an environmental sample such as soil without culturing and separating microorganisms.

  Introduction of a test gene into a cell can be performed by a conventional method used in gene recombination techniques. For example, the transformation method using the vector which integrated the test gene can be mentioned. As a method for introducing a vector and a vector into a host cell, the above-described method for introducing a vector and a vector into a host cell can be used.

  The introduction of the terpene synthase gene or a variant thereof and the test gene into the cell may be performed simultaneously, or the former may be introduced into the cell and the latter may be introduced into the cell later.

  After introducing a test gene into a cell into which a terpene synthase gene or a mutant thereof has been introduced, the cell is cultured under a known medium or condition suitable for the cell. For example, in the case of bacteria such as Escherichia coli, the culture is performed under conditions that form colonies.

  The determination that the test gene is a terpene synthase gene and / or a mutant thereof is performed using the survival of the cell into which the test gene is introduced as an index. That is, among the test genes introduced into the cells, the test gene introduced into the cells that survived the culture is selected as a terpene synthase gene and / or a mutant thereof. As described above, in cells transformed with a gene encoding terpene synthase or a mutant thereof, cell death is caused by growth inhibition due to substrate consumption by the expressed enzyme. When a gene encoding an enzyme capable of increasing the synthesis amount of a terpene synthase substrate or a compound serving as a material of the substrate, that is, a terpene precursor, or a mutant thereof is present in the test gene, Only the cells into which the gene has been introduced can avoid growth inhibition due to substrate consumption, and can survive and proliferate against cell death. Therefore, it can be determined that, in the cells that were able to survive by the introduction of the test gene, the amount of terpene precursor synthesis increased by the introduction of the test gene, and as a result, survival was possible. That is, it can be determined that a test gene introduced into a surviving cell into which a test gene has been introduced is a gene encoding an enzyme that synthesizes a terpene precursor and / or a variant thereof. A test gene is collected from the surviving cells, and the test gene is identified by a known method.

  By using the method according to the present invention, a gene encoding terpene synthase and / or a variant thereof can be obtained without destroying cells. Specifically, in the case of bacteria such as Escherichia coli, colonies are formed on a plate, and the formed colonies may be picked up. The entire screening can be semi-automated using a colony picker. The method according to the present invention enables efficient and high-throughput selection of genes that improve the raw material supply route in terpene production.

  A preferred embodiment is a gene encoding a geraniol synthase mutant with enhanced enzyme activity, wherein the mutant has enhanced enzyme activity and / or expression compared to the wild-type enzyme, and A mutant characterized in that, in a cell expressing the mutant, cell death caused by a substrate consumed by the mutant or a compound serving as a material of the substrate being a compound involved in cell survival A terpene synthase gene comprising introducing a test gene into a cell into which a gene encoding the gene has been introduced, culturing, and selecting a test gene introduced into a living cell among the test genes introduced into the cell And / or a mutant screening method thereof.

  In another preferred embodiment, a test gene is introduced into a cell into which a geraniol synthase gene variant represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 has been introduced, and then introduced into the cell. A screening method for a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell among test genes.

  In another preferred embodiment, a test gene is introduced into E. coli transformed with a plasmid represented by the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 and cultured, and the test gene introduced into the cell is transformed. A screening method for a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell.

  In order to deepen the understanding of the present invention, the present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

[Production of geraniol synthase mutant with improved terpene production]
In developing a method for improving terpene production, first, a geraniol synthase mutant with improved terpene production was prepared.

  The catalytic activity of terpene synthase is generally very low, and the kcat value is about 0.01 to 0.1 / sec. Among them, monoterpene synthases such as geraniol synthase (GES) show the fastest catalytic activity. Therefore, geraniol synthase derived from sweet basil was selected as an object of study and an attempt was made to produce a mutant thereof with enhanced terpene production activity in Escherichia coli. Specifically, a geraniol synthase derived from sweet basil is used as a geraniol synthase (Ijima Y. et al., Charactorization of Gentanol Synthet from The Pellets of Sandy 3). ), A geraniol synthase mutant was prepared by the following method.

(1) Preparation of geraniol synthase mutant First, 52 amino acids at the N-terminal were excised from the amino acid sequence of geraniol synthase derived from sweet basil (567 amino acids) in order to remove the transport sequence to the plastid. For affinity purification, a 6 × histidine tag (6 × His) was introduced immediately before the stop codon. The mutant thus prepared is hereinafter referred to as GESM53. As a control, an inactivated mutant (referred to as GES _D323A ) in which the 323rd aspartic acid (referred to as D323) essential for the enzyme activity in the full-length amino acid sequence of geraniol synthase was substituted with alanine was used. Produced.

  A gene encoding GESM53 (referred to as gesM53) was introduced into the ColEI type vector under pLac to prepare an expression construct of GESM53 (referred to as pLac-gesM53) (Panel A in FIG. 2). The plasmid map is shown in panel C of FIG. The nucleotide sequence of plasmid pLac-gesM53 is shown in SEQ ID NO: 5.

In addition, in order to introduce gesM53 under the pEI of the ColEI type vector and further improve the translation efficiency, in the amino acid sequence of GESM53, a mutation that replaces glycine, the amino acid corresponding to the 54th amino acid of geraniol synthase, with valine To construct an expression construct (referred to as pBAD-gesM53 _G54V ) (panel B in FIG. 2). The plasmid map is shown in panel D of FIG. The nucleotide sequence of plasmid pBAD-gesM53 _G54V is shown in SEQ ID NO: 6.

A gene encoding GES _D323A (referred to as ges _D323A ) was introduced under the pLac of the ColEI type vector to _prepare an expression construct for GES _D323A (referred to as pLac-ges _D323A ).

(2) Production of geraniol by geraniol synthase and its mutant The geraniol production by the above geraniol synthase and its mutant was confirmed. Specifically, XL-1 Blue was used as a host, and pLac-gesM53 was introduced. As a control, pLac-gesM53 _D323A was introduced into XL-1 Blue. XL-1 Blue is an Escherichia coli strain transformed with pBBRSOE6 (Non-patent Document 5) in order to increase terpene production. PLac-gesM53 _D323A is an GESM53 gene encoding an inactivating mutant (GESM53 _D323A ) in which aspartic acid, which is the amino acid corresponding to the 323rd amino acid necessary for the enzyme activity of geraniol synthase, is substituted with alanine. Expression construct.

After the gene introduction, 1 mM isopropyl-β-thiogalactopyranoside (IPTG) and 0.02% arabinose were added, and the gene expression was induced by shaking culture at 30 ° C. and 200 rpm for 8 hours. The culture was extracted with ethyl acetate (including limonene internal standard). The extract was analyzed using a gas chromatograph / hydrogen ion detector (GC-FID) under the following conditions. Column: DB-5 ms, inlet temperature: 250 ° C., column temperature: 60 ° C. (3 min), 60-150 ° C. (6 ° C./min), 150-230 ° C. (15 ° C./min), detector temperature: 250 ° C. . Carrier _{gas: N} 2.

As a result, as shown in FIG. 3, production of geraniol was observed in XL-1 Blue transformed with pLac-gesM53, but production of geraniol was not observed in XL-1 Blue transformed with pLac-gesM53 _D323A. It was.

(3) Intracellular geraniol synthase activity of cells into which geraniol synthase and its mutants were introduced The intracellular geraniol synthase activity of cells into which the geraniol synthase or its mutants were introduced was measured. Intracellular geraniol synthase activity was measured by constructing biosynthetic pathways for various dyes using the same substrate as the terpene enzyme group as the raw material, and introducing the terpene synthase genes into these cells. This was carried out by a method (Patent Document 5) for measuring terpene enzyme activity using as an index the decrease in the amount of dye synthesis per cell by taking the raw material from As shown in panel A of FIG. 4, geranylgeranyl diphosphate (GPP) is expressed in E. coli expressing the diapophytoene synthase (CrtM) gene and dehydrosqualene desaturase (CrtN) gene derived from Staphylococcus aureus. From the above, farnesyl diphosphate (FPP) is relayed to synthesize a yellow pigment 4,4′-diaponeurosporene, which is a kind of carotenoid. Geraniol synthase (GES) is an enzyme that converts GPP, which is an intermediate thereof, into geraniol. Therefore, when co-expressed in Escherichia coli in which CrtM and CrtN are expressed, the substrate is deprived of the pigment synthesis system. As a result, the amount of accumulated carotenoid pigment decreases. That is, when a decrease in the amount of accumulated carotenoid pigment is observed, it can be determined that the intracellular geraniol synthase is high.

  Specifically, the CrtM gene and CrtN gene derived from Staphylococcus aureus are introduced into Escherichia coli, the geraniol synthase or a mutant thereof is introduced into the obtained Escherichia coli and cultured, and then the dye synthesized in the Escherichia coli Using the amount as an indicator, intracellular geraniol synthase activity was measured.

As shown in panel B of FIG. 4, a CrtM-CrtN gene expression construct pAC-CrtM-CrtN transformed with E. coli transformed with an empty vector (Void) is a yellow pigment 4,4′-diaponeurospo Len accumulated and the cell color became yellow. In contrast, when E. coli transformed with pAC-CrtM-CrtN is further transformed with the wild-type GES gene expression construct pLac-ges or the GESM53 gene expression construct pLac-gesM53, the amount of accumulated yellow pigment is significantly reduced. And the cells turned white. Moreover, the substrate consumption by GESM53 is clearly higher than that of the wild type. This is because the enzyme activity in the cell is sufficiently improved by introducing a deletion mutation of the N-terminal amino acid and a mutation that improves the translation efficiency. Such cell whitening, ie, reduction in pigment production, was not observed in E. coli transformed with pAC-CrtM-CrtN and the inactivated GES mutant gene expression construct pLac-ges _D323A .

  The above results reflect that GES has a high substrate consumption activity in the cells and that the amount of geranyl diphosphate (GPP), which is a precursor for carotenoid pigment production, is significantly reduced.

[Confirmation of improved terpene production by geraniol synthase mutant]
The gene encoding the geraniol synthase mutant with improved terpene production produced in Example 1 was introduced into an Escherichia coli strain to confirm improvement in terpene production.

Specifically, pLac-gesM53 was transformed into E. coli strain JW3686 (BW25113ΔtnaA) and cultured. Inactivation GES mutant (GES _D323A ) expression plasmid pLac-ges _D323A and empty vector pLac-void were similarly transformed and cultured as controls.

As a result, no colony was formed in the JW3686 strain transformed with pLac-gesM53, and the transformation efficiency was zero. On the other hand, when pLac-ges _D323A or pLac-void was transformed into the JW3686 strain, colonies were formed. This result means that forced expression of the GES gene is lethal to cells.

[Investigation of cell death mechanism by forced expression of geraniol synthase mutant]
We investigated the mechanism by which forced expression of the gene encoding geraniol synthase mutant is lethal to cells. There are two possible mechanisms by which forced expression of GES mutant genes is lethal to cells: (1) GES or its products have host toxicity; (2) cells for geraniol synthesis The quinone, dolichol, and tRNA modification sources essential for growth are depleted, resulting in cell death.

(1) Examination of possibility of GES or its product having host toxicity Geraniol produced by geraniol synthase is a monoterpene, and monoterpene is known to exhibit antibacterial activity. Therefore, it may have been toxic to E. coli. However, there is a report that the minimum inhibitory concentration (MIC) of E. coli for geraniol is about 800 mg / L (Dunrop MJ et al., Engineering Microbiological Tolerance and Exploited Efflux Pump. (2011)). The amount of geraniol accumulated in the medium in this example is about 10-20 mg / L, which is sufficiently lower than the reported MIC. From this, it is considered that the product toxicity by geraniol synthase is not the reason for lethality due to the forced expression of the GES gene observed in this Example.

(2) Examination of possibility of cell death due to depletion of substances essential for cell growth by geraniol synthesis GPP which is a substrate of GES is consumed by geraniol synthesis by GES mutant. The possibility that this consumption of GPP is the cause of cell death was examined. That is, whether or not cell death can be prevented by introducing a gene encoding an enzyme involved in GPP synthesis together with a gene encoding a geraniol synthase mutant into the cell to compensate for the consumption of GPP by the GES mutant. It was investigated. As an enzyme involved in the synthesis of GPP, isopentenyl diphosphate isomerase (IDI; GenBank ID: 949020) derived from E. coli was used. IDI is an enzyme that catalyzes the isomerization reaction between isopentenyl diphosphate (IPP) and its isomer, dimethylallyl diphosphate (DMAPP). A head-to-tail type condensation reaction occurs between the produced DMAPP and IPP to produce GPP. A plasmid pAC-idi that expresses a gene encoding IDI was prepared by inserting the gene into a pAC plasmid.

First, one of the following plasmids was introduced into E. coli strain JW3686, cultured for 1 hour, and sprayed on LB agar medium to form a colony: expressing an inactivated mutant in which D323A mutation was introduced into full-length GES Plasmid pLac-ges _D323A , plasmid pLac-gesM53 expressing mutant GESM53 in which 52 amino acid residues on the N-terminal side of GES are deleted and 6 × His is added to C-terminal, and full-length wild-type GES are expressed Plasmid pLac-ges.

  As shown in FIG. 5, no colonies were formed in E. coli strain JW3686 in which only pLac-gesM53 was transformed to express GESM53. On the other hand, colony formation was observed in the E. coli strain expressing full-length wild-type GES and the JW3686 strain expressing GES-inactivating mutant.

Next, pLac-gesM53 was cotransformed into JW3686 strain together with any of pAC-idi, plasmid pAC-crtMN for expression of E. coli containing CrtM gene and CrtN gene, and pAC-void. Specifically, any of pAC-idi, pAC-crtMN, and pAC-void was introduced into the JW3686 strain together with pLac-gesM53 and cultured for 1 hour, and then sprayed on an LB agar medium to form colonies. The number of colonies formed was counted, and the viable cell rate was determined using the following formula 1. In the following formula, CFU _{GES (+)} means the number of colonies formed of E. coli strain co-expressed with pLac-gesM53, and CFU _{GES (-)} represents the number of colonies formed of E. coli strain not co-expressed with pLac-gesM53. means. Here, the number of colonies formed is expressed in colony forming units (abbreviated as CFU or cfu).

(Equation 1)

CFU _{GES (+)}
Viable bacteria rate = ───────────
CFU _{GES (-)}

  As shown in FIG. 6, when pLac-gesM53 and the empty vector pAC-void were co-introduced, no colonies were formed, and the transformation efficiency was almost zero. No colony formation was observed when pLac-gesM53 and pAC-crtMN were co-introduced. When Crt-M and / or CrtN is expressed, it competes with GES, consumes the substrate of GES, and synthesizes carotenoid pigment. As a result, it can be considered that geraniol production is reduced as compared with the case where only GES is expressed. If the toxicity of the GES product geraniol indicates cell lethality, cell death may be prevented by expressing Crt-M and / or CrtN, but the transformation efficiency is completely restored. From the absence, the assumption that the toxicity of the GES product is related to cell lethality can be denied.

  On the other hand, by cotransforming the isopentenyl diphosphate isomerase (IDI) gene derived from Escherichia coli with pLac-gesM53, colonies were formed and the transformation efficiency was remarkably recovered (FIG. 6). From this result, cell death due to forced expression of the GESM53 gene is not due to the toxicity of the GESM53 product geraniol, but due to its high enzymatic activity, consumption of isoprenyl diphosphate essential for cell growth is depleted. Was strongly suggested. It has been conventionally reported that the production amount of carotenoids, terpenes and the like is increased by overexpressing IDI (Non-patent Document 12). It can be considered that the overexpression of IDI increases the terpene raw material in the host, that is, isoprenyl diphosphate, and as a result, the cells can grow even under the expression of GES.

  From the above results, the cause of cell death due to forced expression of the gene encoding geraniol synthase mutant is the consumption of the substrate by the geraniol synthase mutant, and the supply of the substrate, that is, the enzyme involved in the production of the substrate It became clear that the cell death can be prevented by expression.

  Utilizing the above phenomenon, by co-expressing a gene encoding a terpene synthase having high enzyme activity and a gene encoding an enzyme involved in production of the substrate of the enzyme, survival of cells transformed with these genes The terpene production can be improved by using such cells.

  Furthermore, by using a cell transformed with a gene encoding a terpene synthase having high enzyme activity, introducing the test gene, culturing the cell, and selecting the gene introduced into the living cell, the substrate of the enzyme Alternatively, a gene encoding an enzyme involved in the production of the substrate material can be selected.

[Examination of host used in gene selection method using cell death by forced expression of geraniol synthase mutant]
Whether cell death by forced expression of a gene encoding a geraniol synthase mutant and prevention of cell death by supplying a substrate for the enzyme is a phenomenon peculiar to the JW3686 strain, or regardless of the type of E. coli strain It was examined whether it was also observed in E. coli strains.

  E. coli strains examined are BW25113 strain, JW3686 strain, and XL-1 Blue. The BW25113 strain is an Escherichia coli strain that is widely used for the production of genetically modified strains. The JW3686 strain is a strain obtained by adding ΔtnaA :: kanR gene modification to the BW25113 strain. XL-1 Blue is a strain frequently used for cloning.

Each of these three strains was transformed with a mixed solution containing two types of plasmid DNA, namely, green fluorescent protein expression plasmid pLac-sfgfp and expression plasmids of various GES mutants at a concentration ratio of 1: 5. pLac-sfgfp is a plasmid that constantly expresses superfolder green fluorescent protein (Super Folder GFP). The following GES mutant expression plasmids were used: plasmid pLac-ges _D323A expressing an inactivated mutant in which a D323A mutation was introduced into a full-length GES, and inactivation in which a G285S mutation was introduced into a full-length GES. Plasmid pLac-ges _G285S expressing mutant, plasmid pLac-gesM53 expressing mutant GESM53 in which 52 amino acid residues on the N-terminal side of GES are deleted and 6 × His is added to the C-terminal. Moreover, the plasmid pLac-ges which expresses a full-length wild type GES was used as a GES expression plasmid.

  After transforming the expression plasmid into E. coli, each transformant was seeded on an agar medium to form colonies. Transformants with pLac-sfgfp give colonies with green fluorescence, and transformants with various GES expression plasmids give nonfluorescent colonies.

As shown in FIG. 7, the GES-inactivating mutants (GES _D323A and GES _G285S ) are naturally transformed, and the wild-type GES-transformed mutant has a fluorescent colony / non-fluorescent colony number ratio regardless of the host. The concentration ratio of pLac-sfgfp to pLac-ges in the mixed solution used during the transformation operation, that is, 1: 5 was not substantially changed. From this, it is clear that the GES inactivating mutant and the wild type GES are not cytotoxic to any E. coli strain.

  On the other hand, when GESM53 was introduced into the JW3686 strain and the BW25113 strain, all of the generated colonies were fluorescent, and no fluorescent colonies were observed. This result indicates that GESM53 is lethal to the two cells. Interestingly, this lethality was not observed when XL-1 Blue was the host. That is, the number ratio of non-fluorescent colony / fluorescent colony was the same as that of the GES inactive mutant. GESM53 expression does not appear to cause lethal substrate depletion in XL-1 Blue.

[Gene selection using cell death by forced expression of geraniol synthase mutant]
As shown in Example 4, cell death due to forced expression of a gene encoding a geraniol synthase mutant can be prevented by supplying a substrate for the enzyme, that is, expressing an enzyme involved in substrate production. Using this principle, cells transformed with a gene encoding a terpene synthase mutant with high enzyme activity are introduced, the cells are cultured by introducing the test gene, and the genes introduced into the living cells are selected. Thus, a gene encoding an enzyme involved in the production of the enzyme substrate can be selected. This selection method was actually examined.

  Specifically, an attempt was made to select the former from a mixed system of a gene having an effect of enhancing the terpene production pathway and a gene having no such effect. PAC-idi was used as an expression plasmid for a gene having an effect of enhancing the terpene production pathway, and a green fluorescent protein expression plasmid pAC-gfp was used as an expression plasmid for a gene having no such effect.

First, plasmid solutions containing equal amounts (5 ng / μL) of pAC-idi and pAC-gfp were prepared. JW3686 strain was transformed with 1 μL of this plasmid solution and 1 μL of GES mutant expression plasmid pLac-gesM53 or GES inactivating mutant expression plasmid pLac-gesM53 _D323A . After adding SOC culture medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ , 20 mM glucose) for 1 hour, agar containing 1 mM IPTG It was sprayed on the medium to form colonies. The number of fluorescent colonies and non-fluorescent colonies per plate was counted.

As shown in the left panel of FIG. 8, in the experiment in which the plasmid solution and pLac-gesM53 _D323A were cotransformed, 101 colonies were formed, and the numbers of fluorescent colonies and non-fluorescent colonies were 41 and 60, respectively. Met. The number ratio of fluorescent colonies to non-fluorescent colonies was almost the same as the 1: 1 mixture ratio of pAC-idi and pAC-gfp in the plasmid solution before transformation. That is, it can be said that almost no selection has occurred between the IDI gene and the GFP gene.

  On the other hand, in the experiment in which the plasmid solution and pLac-gesM53 were cotransformed, 33 colonies were formed, and the numbers of fluorescent colonies and non-fluorescent colonies were 3 and 30, respectively. That is, the non-fluorescent colonies expressing IDI were more concentrated than the fluorescent colonies expressing GFP.

The ratio of the fluorescent colony to the non-fluorescent colony is 0.68 in the experimental system using pLac-gesM53 _D323A and 0.1 in pLac-gesM53. That is, the apparent efficiency of “selection of gene encoding IDI” is only 6.8 times.

  On the other hand, considering that the transformation efficiency ratio between pAC-idi and pAC-gfp is about 1000 times as seen from FIG. 6, this selection efficiency is not high at all. Therefore, a plasmid was amplified by polymerase chain reaction from three fluorescent colonies generated by transformation of pLac-gesM53 and the plasmid mixture, and gel electrophoresis analysis was performed. In addition, the same analysis was performed by randomly selecting 3 from 30 non-fluorescent colonies.

  As shown in the right panel of FIG. 8, three fluorescent colonies were transformed with pAC-gfp as well as pAC-idi. As a result, these three fluorescent colonies are proliferated while preventing cell death under the expression of the GES mutant by the effect of the enzyme encoded by the introduced IDI gene, and the GFP gene introduced together is encoded. It shows that it was fluorescing by the protein. That is, it was revealed that all 33 colonies formed by culturing Escherichia coli co-transformed with a plasmid solution and pLac-gesM53 were transformed with pAC-idi.

  In this way, by culturing cells transformed with a mixed plasmid solution containing pAC-idi and pLac-gesM53, a gene encoding IDI is removed from a mixed system with a gene having no effect of enhancing the terpene production pathway. It could be selectively concentrated.

  Therefore, by using a cell transformed with a gene encoding a terpene synthase having high enzyme activity, introducing the test gene, culturing the cell, and selecting the gene introduced into the living cell, the substrate of the enzyme The gene encoding the enzyme involved in the production of can be selected.

[Construction of an arabinose induction system for improving the efficiency of gene selection methods using cell death by forced expression of geraniol synthase mutants]
Production of a substrate of the enzyme by using a cell transformed with a gene encoding a terpene synthase having high enzyme activity, introducing the test gene, culturing the cell, and selecting the gene introduced into the living cell It is possible to select genes that encode enzymes involved in. However, when co-transforming E. coli with two plasmids, the transformation efficiency is never high. In order to efficiently perform such a selection method, a gene encoding a terpene synthase having high enzyme activity is previously transformed into a host such as E. coli strain JW3686, and the obtained transformant is made into a competent cell. On the other hand, it is preferable to transform a test gene, for example, a gene library. However, since terpene synthase having high enzyme activity causes cell death by depleting its substrate in the cell, transformation of the terpene synthase gene having high enzyme activity in the cell supplies the substrate of the enzyme. It is difficult to carry out unless there is a gene encoding an enzyme having an effect of enhancing terpene production such as IDI. Therefore, the genes encoding terpene synthases having high enzymatic activity were prepared inserted constructs P _BAD downstream. In this type of construct, since the terpene synthase gene is in an expression-repressed state before being induced by arabinose, transformation of the gene into E. coli does not cause cell death, and transformation should be possible. is there.

First, GESM53 _G54V was used as a terpene synthase having high enzyme activity, and pBAD-gesM53 _G54V (Panel B and Panel D in FIG. 2 and SEQ ID NO: 6) containing the GESM53 _G54V gene was introduced into the JW3686 strain. Unlike pLac-gesM53, pBAD-gesM53 _G54V was not toxic in the absence of arabinose even when transformed into E. coli (FIG. 9). Moreover, a competent cell was able to be produced without any problem from a transformant obtained by transforming pBAD-gesM53 _G54V into the JW3686 strain (hereinafter referred to as JW3686 / pBAD-gesM53 _G54V ).

_Any of the three expression plasmids of pAC-idi, pAC-gfp, and pAC-void was introduced into the prepared JW3686 / pBAD-gesM53 _G54V , and an agar medium containing or not containing 0.2% arabinose Were inoculated to form colonies. The ratio of the number of colonies on the agar medium containing arabinose and the number of colonies on the agar medium not containing arabinose was calculated and defined as the viable cell ratio of cells expressing the geraniol synthase mutant.

As shown in FIG. 9, the viable cell rate of E. coli expressing only GESM53 _G54V was about 10 ⁻⁴ −10 ⁻⁵ . The viable cell rate of Escherichia coli co-expressed with GESM53 _G54V and GFP decreased to about 10 ^{−3 to} 10 ⁻⁴ . On the other hand, the viable cell rate of Escherichia coli co-expressed with GESM53 and IDI was about 1. Further, a similar experiment was performed using the plasmid _{pBAD-gesM53 D323A} expressing _{GESM53 D323A} unactivated. The viable cell rate of E. coli was almost 1 both when only GESM53 _D323A was expressed and when co-expressed with GFP or IDI. That is, if the expression of GESM53 _G54V is not induced, such a difference in survival rate does not occur.

These results indicate _{that GESM53} the cells by depletion of its substrate by expression of _G54V is subjected to growth inhibition, and capable of restoring the growth of cells by increasing the precursor supply amount of substrate _{GESM53 G54V} by IDI . That is, even when the GESM53 _G54V gene is introduced into the cells, the gene can be kept in an expression-suppressed state by not adding arabinose, and the transformant can be maintained in a alive state.

  In this way, by using an expression vector containing an arabinose-inducible promoter, a gene encoding a terpene synthase having high enzymatic activity is transformed into a host in advance, and the resulting transformant is made into a competent cell. be able to. Using the obtained competent cells, a gene selection method using cell death by forced expression of a geraniol synthase mutant can be efficiently carried out.

[Study of mock selection in arabinose induction system]
Using the arabinose induction system constructed in Example 6, an experiment was conducted in which only pAC-idi was selected from the plasmid mixture containing pAC-gfp and pAC-idi.

Specifically, the JW3686 / pBAD-gesM53 _G54V prepared in Example 6 was _transformed with a pAC-idi and pAC-gfp equal concentration plasmid mixed solution (pAC-gfp / pAC-idi mixed solution). The agar medium containing 2% arabinose and the agar medium not containing arabinose were seeded. Preparation and transformation of the plasmid mixture were performed in the same manner as described in Example 5. The formed fluorescent colonies and non-fluorescent colonies were counted, and the colony forming unit (CFU / TF) per transformation was calculated.

In the agar medium containing no arabinose, both fluorescent colonies and non-fluorescent colonies were observed (FIG. 10). On the other hand, in the medium containing arabinose, the number of non-fluorescent colonies, that is, pAC-idi transformants was the same as that in the case of no induction, but no fluorescent colonies, that is, pAC-gfp transformants were observed at all. (FIG. 10). This is because GESM53 _G54V expression is induced in a medium containing arabinose, and thus, in Escherichia coli into which pAC-idi has not been introduced, the GESM53 _G54V substrate is depleted and cell death occurs.

Thus, it became clear that a gene having the ability to supply a precursor of the terpene synthesis pathway can be selected using the arabinose induction system of the GESM53 _G54V gene.

  The present invention can provide an efficient search method for a terpene synthase gene used in a method for improving terpene production. Terpenes are useful compounds as biofuels, pharmaceuticals, agricultural chemicals, fragrances, and synthetic raw materials for organic synthesis. Therefore, the present invention is useful for efficient production of terpenes and has extremely high industrial applicability.

SEQ ID NO: 1: A gene encoding a sweet basil geraniol synthase mutant lacking the first to 52nd amino acid residues.
SEQ ID NO: 3: A gene encoding a sweet basil geraniol synthase mutant in which the first to 52nd amino acid residues are deleted and the 53rd glycine is replaced with valine.
SEQ ID NO: 5: An expression plasmid for a gene encoding a sweet basil geraniol synthase mutant in which the first to 52nd amino acid residues are deleted and a 6 × histidine tag is added to the C-terminus thereof.
SEQ ID NO: 6: Sweet basil geraniol synthase mutant in which the 1st to 52nd amino acid residues are deleted, the 53rd glycine is substituted with valine, and the 6 × histidine tag is added to the C terminus An expression plasmid for a gene encoding

Claims (9)

  A test gene is introduced into a cell into which a gene encoding a terpene synthase variant that encodes a terpene synthase variant that has an enzyme activity and / or enhanced expression compared to a wild-type enzyme. A screening method for a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell among test genes introduced, cultured, and introduced into the cell.   The method for screening a terpene synthase gene and / or a mutant thereof according to claim 1, wherein the terpene synthase mutant is a terpene synthase mutant that causes cell death in cells expressing the mutant.   The terpene synthase according to claim 2, wherein the cell death is cell death caused by a substrate consumed by the terpene synthase mutant or a compound serving as a material of the substrate being a compound involved in cell survival. A screening method for genes and / or mutants thereof.   The method for screening a terpene synthase gene and / or a variant thereof according to any one of claims 1 to 3, wherein the terpene synthase mutant is a geraniol synthase mutant.   The method for screening a terpene synthase gene and / or variant thereof according to any one of claims 1 to 4, wherein the cell is Escherichia coli.   A gene encoding a geraniol synthase mutant with enhanced enzyme activity, wherein the mutant has enhanced enzyme activity and / or expression compared to a wild-type enzyme and expresses the mutant In a cultured cell, a cell into which a gene encoding a mutant that causes cell death caused by a substrate consumed by the mutant or a compound serving as a material of the substrate being a compound involved in cell survival is introduced into a cell. A screening method for a terpene synthase gene and / or a variant thereof, comprising introducing a test gene and culturing, and selecting a test gene introduced into a living cell among test genes introduced into the cell.   The gene of the terpene synthase according to claim 6 and / or the gene encoding the geraniol synthase mutant with enhanced enzyme activity is a gene represented by the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Or a screening method for the mutant.   Of the test genes introduced into the cells, the test genes are introduced into the cells into which the gene encoding the geraniol synthase mutant represented by the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 has been introduced. A method for screening a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene introduced into a living cell.   A test gene is introduced into E. coli transformed with the plasmid represented by the nucleotide sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6 and cultured, and the test gene introduced into the surviving cells among the test genes introduced into the cells. A method for screening a terpene synthase gene and / or a mutant thereof, comprising selecting a test gene.