JP5787341B2 - Screening method for terpene synthase gene - Google Patents

Description

  The present invention relates to a method for screening a terpene synthase gene.

Terpenes were originally hydrocarbons with the molecular formula of C ₁₀ H ₁₆ contained in essential oils, but the object called terpenes expanded, and now they are derived from hydrocarbons with the composition of (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, those having n of 2 are referred to as monoterpenes, 3 as sesquiterpenes, 4 as diterpenes, 6 as triterpenes, 8 as tetraterpenes, and many as 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 Documents 1 and 2). When prenyl transferase acts on IPP synthesized through these pathways and its structural isomer, dimethylallyl diphosphate (DMAPP), geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, etc. A chain alcohol diphosphate is produced. Terpenes are synthesized by the action of terpene synthase using these products as substrates. These enzymes give chain terpenes, ring closure, hydride and alkyl group rearrangement, addition of water and amines, dehydrogenation, oxygen Performs functional group introduction and decarboxylation to produce terpenes with various skeletons.

  Many terpenes have various pharmacological activities such as antibacterial / immunostimulatory / mental stability / antimalarial in addition to those with industrial value as perfumes, insect repellents, and aroma oils, but most of them are unknown. is there.

Since terpenes have various physiological functions, they are actively searching for their synthase genes. However, there has not been proposed a method for comprehensively obtaining all genes encoding synthases involved in terpene biosynthesis.
At present, genes that have significant homology with known terpene synthase genes are estimated from genome databases to be terpene synthase genes, and their activities are identified and certified using LC-MS and GC-MS one by one. (Non-Patent Document 3).

Furthermore, most of the terpene compounds are colorless, and high-throughput screening that directly determines the activity of the synthase based on color shading cannot be performed.
That is, in the natural world, there are an enormous amount of terpene compounds, an enormous amount of synthase genes, and an enormous number of unknown terpenes. However, it is impossible to comprehensively acquire terpene synthases, and in particular, the function of a synthase that produces an unknown terpene molecule cannot be easily identified.
In addition, a gene having a low homology with a known terpene synthase gene is not presumed to be a terpene synthase gene in the first place.

  On the other hand, since many terpene synthase genes are derived from plants, the expression level is low in hosts such as Escherichia coli and yeast, and the stability and activity of the expressed enzyme are also low. In other words, 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 have become a major impediment to the construction of biological and mass production systems for terpene synthase.

Goldstein, J.L. et al .; Nature 343,425-430 (1990) Hunter, W.N .; J Biol Chem 282, 21573-21577 (2007) Mijts, B.N. et al .; Chem Biol 12,453-460 (2005)

  In order to be able to biologically produce known and unknown terpenes in nature, it is first necessary to obtain the gene for the synthase and to develop a screening method for it. In addition, for the construction of terpene organisms and mass production systems, it is necessary to greatly improve the activity of enzymes expressed by the host, and the development of screening methods for terpene synthase mutants with high enzyme activity is necessary. It is.

  That is, an object of the present invention is to provide a general and systematic screening method for a terpene synthase gene and a general technique for obtaining a terpene synthase mutant having high enzyme activity.

  The present inventors have intensively studied to solve the above problems, and that all terpene compounds, which are said to have more than 1 million, are also synthesized from a common precursor called isopentenyl diphosphate via a terpene synthesis route. Pay attention. Then, instead of using the synthesized terpene (product) as an index, a method for screening terpene synthase based on the “substrate consumption ability” of the terpene synthase to be obtained, and a terpene synthase with high enzyme activity A method for screening mutants has been developed.

  Specifically, biosynthetic pathways of various dyes are constructed in the cell using the same substrate as the substrate of the terpene synthase group. When a terpene synthase gene is introduced into these cells, part of the substrate is taken away from the pigment synthesis pathway, and the amount of pigment synthesis per cell is reduced. As a result, the activity of the terpene synthase is visualized as cell color fading. That is, the terpene synthase can be identified by selecting a test gene introduced into a cell having a lighter color.

That is, this invention consists of the following.
1. A method for screening a terpene synthase gene and / or a mutant thereof, which comprises introducing a test gene into a cell capable of pigment synthesis and detecting a decrease in the amount of pigment synthesis.
2. The decrease in the amount of dye synthesis is a decrease in the amount of dye synthesis due to the consumption of the substrate of the enzyme involved in the dye synthesis by the terpene synthase. Screening method.
3. 3. The screening method for the terpene synthase gene and / or mutant thereof according to item 1 or 2, wherein the cell is Escherichia coli or yeast.
4). The method for screening a terpene synthase gene and / or a mutant thereof according to any one of items 1 to 3, wherein the cell capable of pigment synthesis is a cell transformed with an enzyme gene involved in pigment synthesis or a mutant thereof. .
5. Enzyme genes involved in pigment synthesis are diapophytoene synthase (CrtM) gene, dehydrosqualene desaturase (CrtN) gene, geranylgeranyl diphosphate synthase (CrtE) gene, phytoene synthase (CrtB) gene, phytoene dehydration 5. A method for screening a terpene synthase gene and / or a mutant thereof according to item 4 above, which is at least one selected from an enzyme (CrtI) gene and a geranyl diphosphate synthase (GPS) gene or a variant thereof.
6). 6. A dye-synthesizable cell transformed with a gene of an enzyme involved in dye synthesis or a mutant thereof used for the screening method according to any one of 1 to 5 above.
7). A dye-synthesizable cell transformed with a diapophytoene synthase (CrtM) gene or a variant thereof, and / or a dehydrosqualene desaturase (CrtN) gene or a variant thereof.
8). 8. The dye-synthesizable cell according to item 7, which is further transformed with a geranyl diphosphate synthase (GPS) gene or a mutant thereof.
9. A pigment-synthesizable cell transformed with a phytoene synthase (CrtB) gene or a variant thereof, and / or a phytoene dehydrogenase (CrtI) gene or a variant thereof.
10. 10. The dye-synthesizable cell according to item 9, which is further transformed with a geranylgeranyl diphosphate synthase (CrtE) gene or a mutant thereof.
11. A kit for screening for a terpene synthase gene or a mutant thereof comprising the pigment-synthesizable cell according to any one of 6 to 10 above.
12 A gene encoding a dehydrosqualene desaturase (CrtN) mutant shown in SEQ ID NO: 2.
13. A farnesyl diphosphate synthase mutant characterized in that the threonine at the 121st amino acid and / or the valine at the 157th amino acid of the FDS _Y81A mutant of farnesyl diphosphate synthase is mutated.
14. The farnesyl diphosphate synthase mutant according to item 13 above, wherein the 121st amino acid threonine is mutated to alanine or serine and / or the 157th amino acid valine is mutated to alanine.
15. 15. A gene encoding the farnesyl diphosphate synthase mutant according to item 13 or 14.

The present invention enables screening of terpene synthase genes regardless of the type of terpene that is a product, that is, the structure of the terpene, and without relying on narrowing down by sequences from a sample group (library) whose sequence is unknown, A method for comprehensively obtaining enzymes can be provided.
According to the present invention, the activity of any terpene synthase, whether known or unknown, can be visualized based on the substrate consumption ability regardless of the type of the product. Furthermore, the present invention enables color screening by using a visible dye, and enables high-throughput screening for a large number of specimens.

The present invention also enables selection of the type of dye synthesis pathway to be co-introduced by 1) searching for sesquiterpene / triterpene synthase using farnesyl diphosphate (hereinafter C15PP) as a raw material, 2) geranylgeranyl diphosphate ( In the following, search for diterpene / tetraterpene synthase using C20PP as a raw material, 3) search for monoterpene synthase using geranyl diphosphate (hereinafter C10PP) as a raw material, Enables more detailed screening support, such as enzyme optimization and screening of mutants with mutations.
In addition, unlike conventional approaches (for example, Non-Patent Document 1 and Non-Patent Document 3) based on similarity in structure (array), the method is based on a functional approach. That is, since the screening method according to the present invention is a technique based on enzyme activity, terpene synthase genes with low sequence homology can be obtained without leakage.

In the screening method according to the present invention, a gene encoding a more active terpene synthase in a host cell such as E. coli shows a high score. Therefore, a gene encoding a more active enzyme, that is, a valuable terpene synthase can be obtained by the screening method according to the present invention.
For example, a large number of mutants of known terpene synthase genes are prepared, and a host into which the mutants are transfected is used to produce mutants with high enzyme activity, high enzyme stability, or high expression levels. Can be screened.

FIG. 1 is a diagram showing a terpene synthesis route. FIG. 2 is a diagram showing the visualization mechanism of three types of terpene synthesis activity. When E. coli (B) expressing CrtM / CrtN is co-expressed with a terpene synthase gene, the substrate (C15PP) is deprived from the yellow pigment synthesis pathway, and the cell color becomes white and the terpene synthesis activity can be visualized. Similarly, the expression of the diterpene synthesis gene that consumes C20PP whitens the color of Escherichia coli (C) expressing CrtE / CrtB / CrtI from pink so that the terpene synthesis activity can be visualized. Since the expression of the monoterpene synthesis gene that consumes C10PP changes the color of the GPS / CrtM / CrtN expression strain (A) from yellow to white, the terpene synthesis activity can be visualized. FIG. 3 shows the results of measuring the activity of squalene synthase using pigment synthesizing cells. Squalene synthase (shown as Sqs _{wt in the} figure) and its inactive mutant (shown as Sqs _{Y171S in the} figure), E. coli (b) expressing CrtM and CrtN or CrtM, CrtN and isopentenyl diphosphate isomerase It was introduced into Escherichia coli (c) expressing (idi), and the amount of dye synthesis was analyzed. In the figure, “null” means a control in which Sqs _wt and Sqs _Y171S are not introduced. The E. coli pellet is shown in (a) (Example 1). FIG. 4 shows changes in cell color of C15PP screening cells using the CrtN mutant (Example 2). FIG. 5 shows the results of measuring the activity of geraniol synthase using pigment synthesizing cells. Geraniol synthase (shown as GES _{Full in the} figure) and its inactive mutation in E. coli expressing CrtM and CrtN (b) or E. coli expressing CrtM, CrtN and isopentenyl diphosphate isomerase (idi) (c) The body (indicated as GES _c-end in the figure) was introduced, and the amount of dye synthesis was analyzed. In the figure, “null” means a control in which GES _Full and GES _c-end are not introduced. These E. coli pellets are shown in (a) (Example 3). FIG. 6 is a diagram showing colonies formed by introducing and co-expressing a mutant library gene prepared using FDS _Y81A as a parent into Escherichia coli into which pAC-EBI-idi has been introduced, and then _distributing the genes on an agar medium. (Example 5). Since the parent FDS _Y81A itself has weak geranyl farnesyl diphosphate synthase (CP25PP) activity, it forms a light pink colony. White colonies are clones expressing mutants with high geranyl farnesyl diphosphate synthase activity. FIG. 7 shows the C25PP synthesizing ability of FDS _Y81A and its amino acid substitution product: specifically, when a mutant of carotenoid synthase (CrtM) with reduced substrate selectivity is co-expressed in E. coli. (Example 5) which represents the skeleton spectrum of the carotenoid accumulated in. The left figure of FIG. 8 shows the crystal structure of a complex of E. coli-derived farnesyl diphosphate synthase (FDS: ispA) and an artificial inhibitor, and a mutant with enhanced geranyl farnesyl diphosphate (C25PP) synthesis activity. The amino acid mutation found in is shown on the structure. The right figure of FIG. 8 shows C20PP consumption activity of FDS, FDS _Y81A, and its variant (Example 6). FIG. 9 shows a 5-epiallystroken synthase (TEAS) gene-containing vector (Ptrc-TEAS). (Example 7) FIG. 10 shows C15PP consumption activity of TEAS. pUC18Nm-TEAS was coexpressed with pAC-MNwt (left) or pAC-EBI (right), and the cell color and the amount of dye synthesis were tested (Example 7). FIG. 11 shows C15PP consumption activity of TEAS and its inactive form. pUC18Nm-TEAS was co-expressed with pAC-MNwt and assayed for cell color and dye synthesis (Example 7). FIG. 12 shows C15PP consumption activity of TEAS and its inactive form. pUC18Nm-TEAS was coexpressed with pAC-EBI and assayed for cell color and pigment synthesis (Example 7). FIG. 13 shows C15PP consumption activity of taxadiene synthase (TS). pUC18Nm-TS was co-expressed with pAC-MNwt and assayed for cell color and dye synthesis (Example 8). FIG. 14 shows C20PP consumption activity of taxadiene synthase (TS). pUC18Nm-TS was co-expressed with pAC-EBI and assayed for cell color and pigment synthesis (Example 8). FIG. 15 shows (a) C15PP and (b) C20PP carotenoid consumption functions of three DNAs selected from the KafTom library. (a) is pAC-MN +, (b) is pAC-EBI +, the bar graph is the average value of n = 4, and the error bar is the standard deviation of n = 4 (Example 9).

Since the present invention searches for terpene synthases (for example, monoterpenes, sesquiterpenes, diterpenes, sesterterpenes, triterpenes, tetraterpene synthases, etc.), the same substrate (for example, geranyl diphosphate (hereinafter referred to as C10PP)) is used. Introducing a terpene synthase gene into cells capable of synthesizing pigments from farnesyl diphosphate (hereinafter C15PP), geranylgeranyl diphosphate (hereinafter C20PP, etc.), competing for substrate consumption, The present invention relates to a screening method for a terpene synthase gene by detecting a decrease in the amount of dye synthesis.
Furthermore, the present invention relates to a method for screening a highly active terpene synthase gene or a variant thereof by introducing a terpene synthase gene into a cell capable of dye synthesis and detecting a decrease in the amount of dye synthesis.

  “Terpenes” are also called terpenoids and isoprenoids. The “terpene” of the present invention is a terpene used in a broad sense. It is a hydrocarbon with isoprene as a structural unit and is a biological material 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 based on 10 carbons. Terpenes are biosynthesized from the mevalonate and non-mevalonate pathways. These pathways differ depending on the species.

A “terpene synthase” is an enzyme involved in terpene synthesis.
Terpenes are biosynthesized from mevalonic acid or pyruvate in vivo. The first step in the biosynthesis of terpenes is the generation of isoprene units, and isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are synthesized from the mevalonate or non-mevalonate (MEP) pathway. Two are the two C5 molecules (C5PP) from which the isoprene unit originates.

The second step in the biosynthesis of terpenes is the coupling process of isoprene units in which isopentenyl diphosphate (IPP) is added to dimethylallyl diphosphate (DMAPP) one after the other, which results in five carbon atoms. It will increase gradually.
The first step is the synthesis of C10PP from IPP and DMAPP with dimethylallyltransferase (EC 2.5.1.1), which is the starting material for monoterpene biosynthesis.
Farnesyl diphosphate synthase (farnesyl diphosphate synthase) combines IPP and C10PP to synthesize C15PP. This is used for sesquiterpene biosynthesis. When two molecules of C15PP are bound, squalene is produced (squalene synthase) and converted to triterpenes such as cholesterol and phytosterols.
Geranylgeranyl diphosphate synthase synthesizes C20PP, the basic skeleton of diterpenes, from C15PP and IPP. Furthermore, when two molecules of C20PP are bound, phytoene is produced, which becomes a precursor of a carotenoid that is a tetraterpene.

  The third step in terpene biosynthesis is the generation of various skeletons. Each terpene having each carbon number synthesized by the isoprene unit bond is biosynthesized through various skeleton formation processes. Various enzymes are also involved in this biosynthetic pathway.

The terpene synthase that can be screened in the present invention is an enzyme that is involved in the synthetic pathway from isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP) to each terpene, and is a pigment that cells used in the screening have The enzyme and substrate involved in the synthesis pathway are shared. It may be a natural enzyme possessed in various organisms or a mutant.
Examples of the common substrate include chain alcohol diphosphates such as C15PP, C20PP, and C10PP.

  Examples of the enzyme using C15PP as a substrate include γ-humulene synthase, farnesol synthase, aristoloken synthase, β-farnesene synthase, notekaton synthase, squalene synthase and the like. These desorb diphosphate from C15PP and cause the resulting carbocation to attack the substrate itself to produce various hydrocarbon compounds (or hydroxide compounds). Squalene synthase synthesizes hydrocarbon squalene with C30 skeleton by condensing two C15PP head-to-head. In any enzyme reaction, since the substrate, the intermediate, and the product are colorless and transparent, it is difficult to analyze the substrate itself at high throughput. However, if the method of the present invention is used, squalene synthesis is performed. Enzyme activity can be measured non-destructively in cells.

  Examples of the enzyme using C20PP as a substrate include taxadiene synthase, coparyl diphosphate synthase, and kasbene synthase. Many diterpenes have physiological activity, and gene search methods for their synthases are extremely important. In these enzyme reactions, the substrate and the product are both colorless and transparent.

  Enzymes using C10PP as a substrate include geraniol synthase, limonene synthase, menthol synthase, pinene synthase, farnesyl diphosphate synthase (FDS), and the like. In the enzyme reaction catalyzed by these substances, both the substrate and the product are colorless and transparent.

The “terpene synthase gene” in the present invention is a gene encoding terpene synthase. The “variant thereof” is a concept of a gene in which the DNA of a terpene synthase gene is mutated by substitution, deletion, addition or the like of a part of its nucleotides. It may be a naturally occurring mutation or an artificially made mutation.
The mutation is preferably a mutation that increases the enzyme activity, expression level or stability of the expressed enzyme. Also preferred are mutations that increase substrate specificity. In particular, mutations that increase enzyme activity are preferred.
A library of mutants is prepared, and mutants having high enzyme activity, expression level or stability in the host can be screened from the library by the method of the present invention. For example, BstFDS _Y81A (geranyl farnesyl diphosphate synthesizing activity) is a mutant of farnesyl diphosphate synthase (BstFDS) derived from Bacillus stearothermophilus. (Hereinafter _referred to as FDS _Y81A ) (Ohnuma, S. et al .; J Biol Chem 271,30748-30754 (1996)). In addition, mutants of FDS _Y81A having higher enzyme activity can be mentioned.

  The test gene introduced into the cell capable of dye synthesis in the present invention is a gene or a gene group considered to contain a terpene synthase gene or a variant thereof. It does not matter if it is derived from a natural cell, a gene from a library produced, a chemically synthesized gene, a PCR amplified gene, or the like. Since the present invention uses the substrate consumption ability of the expressed enzyme as an index, an unidentified gene can be screened.

  Introduction of a test gene or a terpene synthase gene or a mutant thereof into a cell capable of pigment synthesis can be performed by a conventional method used in gene recombination techniques. For example, a gene to be screened is incorporated into a vector and cells are transformed.

  The dye used in the present invention is a dye that is biosynthesized via isopentenyl diphosphate (IPP) and its derivatives, and the substrate of the enzyme involved in the dye synthesis is the same as the substrate of the terpene synthase. If there is no limit. A dye having good visibility of color change due to a decrease in the amount of dye synthesized is preferred. For example, a red pigment is preferable to yellow. Also preferred are dyes produced by mutants of enzymes involved in dye synthesis.

For dyes that share a substrate with terpenes, this method can be used to make more than 700 other carotenoids known in nature or artificial carotenoids with unnatural skeletons (Umeno, D. et al .; J Bacteriol 186, 1531 -1536 (2004)). In addition, proteins that exhibit a blue color by binding to specific carotenoid pigments, such as cluster cyanine (Cianci, M. et al .; Proc Natl Acad Sci USA 99, 9795-9800 (2002)) are also promising. Desirably, a carotenoid pigment having good visibility (red to reddish purple to bluish purple) is preferable.
In the present invention, the carotenoid is a yellow, red, or purple water-insoluble polyene pigment, and includes those having 40 (C40), C30, C45, and C50 carbon atoms. Examples of the carotenoid of C40 include lycopene, β-carotene, astaxanthin, lutein, 3,4,3 ′, 4′-tetradehydrolycopene, tolylene and the like. Examples of C30 (diapocarotenoid) include diaponenurosporene, diapolicopene, diapolicopene dial, staphyloxanthine, and the like.

  Enzymes involved in dye synthesis are enzymes involved in the synthesis route from isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP) to each dye, and include enzymes whose direct products are not dyes. In addition, enzymes involved in dye synthesis share the same substrate as the enzyme involved in the terpene synthesis pathway. It may be a natural enzyme possessed in various organisms or a mutant. By mutating an enzyme involved in dye synthesis, a dye with good visibility can be obtained.

An enzyme gene involved in pigment synthesis is a gene that encodes an enzyme involved in pigment synthesis, and its “mutant” refers to substitution or deletion of a part of the nucleotide of the DNA of the enzyme gene involved in pigment synthesis. This is a concept of a gene mutated by addition or the like. It may be a naturally occurring mutation or an artificially made mutation.
The mutation is preferably a mutation that increases the enzyme activity, expression level or stability of the expressed enzyme. Moreover, the variation | mutation which makes the pigment | dye which is a product of an enzyme more vivid color is preferable. By making the color vivid, it is possible to easily detect and measure a decrease in the amount of dye synthesis.

Suitable enzyme genes involved in pigment synthesis include diapophytoene synthase (CrtM) gene, diapophytoene dehydrogenase (CrtN) gene, geranylgeranyl diphosphate synthase (CrtE) gene, phytoene synthase (CrtB) gene Phytoene dehydrogenase (CrtI) gene and geranyl diphosphate synthase (GPS) gene, and variants thereof. The cell is transformed with at least one gene selected from these enzyme genes and variants thereof.
Diapophytoene synthase (CrtM) synthesizes diapophytoene using C15PP as a substrate. Diapophytoene dehydrogenase (CrtN) synthesizes diaponeurosporene using diapophytoene as a substrate. Geranylgeranyl diphosphate synthase (CrtE) synthesizes C20PP using C15PP as a substrate. Phytoene synthase (CrtB) synthesizes phytoene using C20PP as a substrate. Phytoene dehydrogenase (CrtI) synthesizes lycopene using phytoene as a substrate. Geranyl diphosphate synthase (GPS) synthesizes C10PP using C5PP as a substrate.

The present invention also relates to a mutant of diapophytoene desaturase (CrtN) derived from Staphylococcus aureus and a gene thereof. Wild-type diapophytoene desaturase (CrtN) mainly synthesizes the yellow pigment diaponeurosporene (Raisig, A. et al .; Biochim Biophys Acta1533, 164-170 (2001)), but CrtN The mutant synthesizes a red diapolicopene. Such improvements improve visibility and dramatically improve all of the S / N ratio, quantitativeness, and reliability.
Specifically, it is a CrtN mutant (SEQ ID NO: 2) characterized by mutating glutamic acid at the 209th amino acid of CrtN to glycine, 242st asparagine to serine, and 286th methionine to valine. This CrtN mutant enzyme synthesizes red diapolicopene (FIG. 4).
Examples of the gene encoding the CrtN mutant include the sequence of SEQ ID NO: 1. The diapophytoene desaturase (CrtN) gene from Staphylococcus aureus can be obtained from the S. aureus (ATCC 35556) genome and can be replaced by various site-specific mutation methods it can.

  The cells capable of dye synthesis used in the present invention are not limited as long as the cells can biosynthesize pigments with the enzymes involved in the dye synthesis. Natural cells or cells transformed by genetic recombination may be used. Examples of the genetically modified cells include Escherichia coli, Bacillus subtilis, and yeast, which are simple cells for genetic engineering.

  A preferable cell capable of dye synthesis used in the present invention is a cell transformed with an enzyme gene involved in pigment synthesis or a mutant thereof. Further, cells in which Idi is introduced into cells by an isopentenyl diphosphate isomerase (idi) gene to enhance the isoprenoid pathway are preferred.

  Preferably, the dye-synthesizable cell used in the present invention is transformed with a diapophytoene synthase (CrtM) gene or a variant thereof, and / or a dehydrosqualene desaturase (CrtN) gene or a variant thereof. Cells. The pigment-synthesizable cell produces diaponeurosporene (yellow) diapolicopene (red) or the like using C15PP as a substrate, and the cells become yellow or red (FIG. 2). The CrtM gene and the CrtN gene or their mutants may be incorporated into the same vector or may be incorporated into different vectors.

  Another preferred embodiment is a cell transformed with a CrtM gene or variant thereof, and / or a CrtN gene or variant thereof, in addition to a geranyl diphosphate synthase (GPS) gene or variant thereof. The cells capable of dye synthesis produce diaponeurosporene (yellow) diapolicopene (red) or the like using C10PP as a substrate, and the cells become yellow or red. The CrtM gene, CrtN gene and GPS gene or their variants may be incorporated into the same vector, or may be incorporated into different vectors.

  Another preferred embodiment is a cell transformed with a phytoene synthase (CrtB) gene or variant thereof, and / or a phytoene dehydrogenase (CrtI) gene or variant thereof. The pigment-synthesizable cell produces lycopene (red) or the like using C20PP as a substrate, and the cell becomes pink or the like. The CrtB gene and the CrtI gene or variants thereof may be incorporated into the same vector or may be incorporated into different vectors.

  Another preferred embodiment is a cell transformed with the CrtB gene or variant thereof, and / or the CrtI gene or variant thereof, in addition to a geranylgeranyl diphosphate synthase (CrtE) gene or variant thereof. The pigment-synthesizable cell produces lycopene (red) or the like using C20PP as a substrate, and the cell becomes pink or the like. In particular, introduction of the CrtE gene is necessary for hosts that do not synthesize C20PP (for example, E. coli). The CrtB gene, the CrtI gene and the CrtE gene or their mutants may be incorporated into the same vector or into different vectors.

  In the present invention, “decrease in the amount of dye synthesis” means that the amount of dye biosynthesis in the cells decreases and the amount of dye in the cells decreases. The terpene synthase gene is present in the sample, and the substrate of the enzyme involved in pigment biosynthesis is deprived by the terpene synthase, thereby reducing the amount of pigment synthesis. That is, in the method according to the present invention, the amount of dye synthesis in a cell capable of synthesizing a dye into which a test gene has been introduced is compared with the amount of dye synthesis in a cell capable of synthesizing the dye not into which the test gene has been introduced. Is selected, and the test gene introduced into the cell is determined to be a terpene synthase gene. In other words, in the method according to the present invention, a terpene synthase gene can be obtained by selecting a test gene that causes a decrease in the amount of dye synthesis in the pigment cells.

The method for detecting the decrease in the amount of dye synthesis or the method for measuring the decrease in the amount of dye synthesis can be carried out by visual recognition. Moreover, it can detect with various measuring instruments, such as various measuring instruments, a microplate reader, a spectrophotometer, and a colorimetric photometer.
By using the method of the present invention, the activity of terpene synthase can be detected or measured without destroying cells. Specifically, when visual recognition is performed, a colony of cells is created on a plate and the colony color is observed. If it is by visual inspection, colonies with a diameter of 0.5-1.0 mm are formed and searched by visual inspection.
When it is necessary to increase the throughput, it is possible to use a technique of densely gathering smaller colonies using a low-magnification magnifier or the like. Further, by using image analysis software, the intensity of colony color can be quantified, or the entire screening can be semi-automated using a colony picker.

  The present invention relates to a dye-synthesizable cell transformed with a gene of an enzyme involved in dye synthesis or a mutant thereof used in the screening method of the present invention.

Examples of the pigment-synthesizable cell include pigment-synthesizable cells transformed with a diapophytoene synthase (CrtM) gene and a diapophytoene desaturase (CrtN) gene or mutants thereof. Using the cell capable of dye synthesis, a terpene synthase gene using C15PP, which is a substrate of CrtM, as a substrate can be detected, and the enzyme activity can be measured. In addition, a terpene synthase gene using diapophytoene, which is a substrate of CrtN, as a substrate can be detected, and the enzyme activity can be measured.
The dye-synthesizable cell produces diaponeurosporene (yellow) diapolicopene (red) or the like by CrtM and CrtN or a mutant thereof, and becomes colored such as yellow or red. If an enzyme gene having a common substrate is present in the subject, the amount of dye synthesis in the host decreases due to the enzyme competing for the substrate, and decolorization or lightness is reduced.

  Pigment transformed with diapophytoene synthase (CrtM) gene, diapophytoene desaturase (CrtN) gene and geranyl diphosphate synthase (GPS) gene or a variant thereof Synthesizeable cells can be exemplified. Using the dye-synthesizable cell, a terpene synthesis gene using C10PP as a substrate can be detected, and its enzyme activity can be measured. In cells where C10PP does not accumulate in cells (for example, E. coli), even if a terpene synthesis gene with C10PP as a substrate is present in the sample, it may not be able to compete for the substrate, so there may be no color change Is expensive. Therefore, a terpene synthase gene using C10PP as a substrate can be detected by introducing a GPS gene into the cell, accumulating C10PP in the cell, and competing with it. Due to the presence of the terpene synthase gene using C10PP as a substrate, the cell color is decolorized or lightened.

Further, examples of cells capable of dye synthesis include cells capable of pigment synthesis transformed by a phytoene synthase (CrtB) gene and a phytoene dehydrogenase (CrtI) gene or a mutant thereof.
Using the cell capable of dye synthesis, a terpene synthesis gene using C20PP as a substrate of CrtB as a substrate can be detected, and the enzyme activity can be measured. Further, a terpene synthesis gene using phytoene as a substrate for CrtI can be detected, and the enzyme activity can be measured.
The dye-synthesizable cell produces lycopene (red) or the like and becomes red or the like by CrtB and CrtI or a mutant thereof. If an enzyme gene having a common substrate is present in the subject, the amount of dye synthesis in the host decreases due to the enzyme competing for the substrate, and decolorization or lightness is reduced.

  Furthermore, as a cell capable of pigment synthesis, a cell capable of pigment synthesis transformed by a phytoene synthase (CrtB) gene, a phytoene dehydrogenase (CrtI) gene and a geranylgeranyl diphosphate synthase (CrtE) gene or a variant thereof. Can be illustrated. Using the dye-synthesizable cell, a terpene synthesis gene using C20PP as a substrate can be detected, and its enzyme activity can be measured. When cells that do not synthesize C20PP (such as E. coli) are used as hosts, it is possible to detect terpene synthase genes using C20PP as a substrate by further introducing a geranylgeranyl diphosphate synthase (CrtE) gene. . Due to the presence of the terpene synthase gene using C20PP as a substrate, the cell color is decolorized or lightened.

The dye-synthesizable cell can be produced by a conventional method. For example, a pigment synthase gene is purchased or prepared by PCR or the like in a conventional manner, the gene is incorporated into an expression vector suitable for the host by a conventional method, a target vector is selected, and a host cell is transformed with the vector by a conventional method. Obtained by conversion. When two or more types of pigment synthesizing genes are incorporated, they are used for transformation into the same expression vector, or are incorporated into different vectors and cotransformed.
The host cell to be used is not limited, but Escherichia coli, Bacillus subtilis, yeast and the like are preferable in view of shortening of the culture time and ease of cloning. In particular, Escherichia coli and yeast are preferable, and suitable Escherichia coli includes cloning strains such as Escherichia coli XL1-Blue, expression strains such as HB101 and BL21, and gene knockout strains with abundant terpene precursor synthesis, such as JW1750 ΔgdhA (glutamate dehydrogenase deficiency), JW0110 ΔaceE (pyruvate dehydrogenase deficiency) (Baba, T. et al .; Mol Syst Biol 2, 2006 0008 (2006)) and the like. INVSc1 (invitrogen), YPH499 (stratagene), etc. are mentioned.
A suitable vector is not particularly limited, and a commonly used vector may be used. For example, when the host is E. coli, examples include those derived from pUC18, pACYC184, etc., when the host is Bacillus subtilis, pUB110, pE194, pC194, pHY300PLK DNA, etc., when the host is yeast, pRS303, YEp213, TOp2609 and the like.

The present invention also relates to a screening kit for a terpene synthase gene or a variant thereof.
The kit according to the present invention may contain cells capable of pigment synthesis. The dye-synthesizable cells are preferably those stored frozen and enclosed in a vial. Alternatively, the kit according to the present invention may contain a pigment synthase gene expression vector for transforming desired cells into pigment-synthesizable cells as a component.
Furthermore, the kit according to the present invention includes a (candidate) gene library, microplate, isoprenoid pathway (terpene upstream pathway) prepared based on an expression vector for a test gene, a cloning enzyme set, a metagenome source, or a sequence database. ) -Enhanced strains, substances and instruments useful for screening such as culture media and additives (image scanners, colorimetric photometers, color samples, colony pickers), etc. can be added as components of the kit.

The present invention further relates to a variant of farnesyl diphosphate synthase (FDS) obtained by the screening method according to the present invention. FDS is a C15PP synthase, but the mutant is an enzyme having an activity of synthesizing geranyl farnesyl diphosphate (C25PP).
A preferred variant is a further variant of FDS _Y81A in which amino acid 81 tyrosine (Y) of BstFDS from Bacillus Stearothermophilus is replaced with alanine (A). _{Examples thereof} include an enzyme in which threonine (T) at position 121 of FDS _Y81A is mutated or an enzyme in which valine (V) at position 157 is mutated. A preferred mutation of threonine (T) at No. 121 is a mutation to alanine (A) or serine (S), and a preferred mutation of valine (V) at No. 157 is a mutation to alanine (A).

Terpenoids are classified by skeletons such as mono (C10), sesqui (C15), di (C20), sesta (C25), tri (C30), tetra (C40). Sesterterpenes are increasingly reported to fungi and marine organisms, but their types are less than others, and their genes are unknown. Furthermore, there is no example of establishing a supply route for C25PP, and there is no example of biological production yet.
The obtained FDS mutant derived from Bacillus Stearothermophilus gives the most efficient C25PP supply ability to E. coli and is useful for providing a cell production system for sesterterpenes. The linear C25 isoprenoid membrane is used as the main component of membranes such as archaea, but its synthetic gene is unknown. C25PP has been reported by Tachibana et al. (Tachibana, A et al; Eur J Biochem 267, 321-328 (2000)), but its activity in Escherichia coli was very weak. These mutants were the first to provide an efficient supply route to C25PP.

  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.

<Reduction of CrtM / CrtN dye synthesis due to C15PP consumption of squalene synthase>
Squalene synthase is an enzyme that synthesizes hydrocarbon squalene having a C30 skeleton by condensing two C15PPs head-to-head. Since the substrate, the intermediate, and the product are both colorless and transparent, their analysis is difficult to perform at high throughput.
However, the activity of squalene synthase could be measured non-destructively in E. coli by using E. coli that acts on the same substrate as that of squalene synthase to synthesize pigment.

(1) Preparation of cells capable of dye synthesis
The E. coli expression plasmid pAC-MNwt containing the CrtM gene and the CrtN gene was introduced into Escherichia coli XL1-Blue by a conventional method to obtain cells capable of synthesizing carotenoids (diaponeurosporen). pAC-MNwt is obtained by PCR amplification of CrtM and CrtN genes (GEN Bank ID: 1125277 and 1125276) from the genome of Staphylococcus aureus, and a plasmid for expression of E. coli (pAmod (Schmidt-Dannert, C. et al.) downstream of lacP. al .; Nat Biotechnol 18, 750-753 (2000))).
In addition, E. coli expression plasmid pAC-MNwt-idi containing CrtM gene, CrtN gene and isopentenyl diphosphate isomerase (idi) gene (GEN Bank ID: 949020) was transferred to Escherichia coli XL1-Blue by a conventional method. The cells were synthesized to synthesize more carotenoids (diaponeurosporene). pAC-MNwt-idi was incorporated into pAC-MNwt in the form of PCR amplified idi from the E. coli genome placed under the control of lacP / O.

(2) Preparation of human squalene synthase gene In order to express squalene synthase, which is originally a membrane protein, in E. coli without creating inclusion bodies (to increase solubility), the membrane domain of this enzyme must be excised. is important. Therefore, a gene encoding squalene synthase hSqS _wt consisting of 340 amino acids lacking the N-terminal 30 amino acids and the C-terminal 47 amino acids of human-derived squalene synthase (total length 417 amino acids) was obtained (Thompson, JFet al. Arch Biochem Biophys 350, 283-290 (1998)).
Furthermore, a gene encoding an amino acid substitution product hSqS _{Y171S in} which the 171st tyrosine of squalene synthase hSqS _wt was substituted with serine was obtained. Specifically, using hSqS _WT as a template, a gene encoding hSqS _{Y171S in} which the codon TAC (tyrosine: Y) of Y171 was replaced with AGC (serine: S) by site-directed mutagenesis was obtained. hSqS _Y171S is an inactive mutant with a broken catalytic site (Gu, P. et al .; J Biol Chem 273, 12515-12525 (1998)). These were inserted into Escherichia coli expression plasmid pUC18Nm (Umeno, D. et al .; J Bacteriol 184, 6690-6699 (2002)) to obtain pUC-hSqS _wt and pUC-hSqS _Y171S .

(3) Observation of cell color
pAC-MN _wt, or pAC-MN _wt -idi Escherichia coli XL1-Blue with, was transformed with pUC-hSqS _wt or pUC-hSqS _Y171S, it was compared pellet cells color and liquid culture at 30 ° C.. As a control, pUC18Nm was used.
The cell color of cells transformed with pUC-hSqS _wt of Escherichia coli XL1-Blue having pAC-MN _wt was significantly more decolorized and lighter than that of the control (pUC18Nm). The cell color of the cells transformed with pUC-hSqS _Y171S of E. coli XL1-Blue having pAC-MN _wt was not changed compared to the cells into which the control (pUC18Nm) was introduced (FIG. 3 (a)).

(4) Comparison of carotenoid (diaponeurosporene) synthesis amount Simultaneously with observation of pellet cell color, the carotenoid synthesis amount (TB medium, 2 mL) in liquid culture was measured (pAC-MNwt and pAC-MNwt-idi). The results are shown in FIGS. 3 (b) and 3 (c).

When wild-type squalene synthase hSqS _wt was co-expressed with CrtM and CrtN, the amount of dye synthesis was clearly reduced compared to the control (FIGS. 3 (b) and (c)). This is because C15PP, which should be used for dye synthesis, was used for squalene synthesis.
The amino acid substitution product hSqS _Y171S is an inactive mutant with a broken catalytic site. The expression of such mutant genes had almost no effect on pigment synthesis and was at the same level as the control.
From these, the dye created by CrtM-CrtN was reduced by the activity of the C15PP-consuming enzyme, which could be visualized as a decrease in cell color.
When the isoprenoid pathway was enhanced by the introduction of the idi gene (Fig. 3 (c)), the substantial concentration of C15PP in the cells increased and the amount of dye synthesis also increased. For the search for stronger terpene synthase activity, it is preferable to use cells into which the CrtM gene, CrtN gene and idi gene have been introduced.

<CrtN gene mutant; improvement of synthetic pigment>
C20PP consumption activity measures lycopene color fading, that is, a clear color change from red to white, allowing accurate and rapid screening. On the other hand, the consumption activity of C15PP is detected and measured based on the color fading of diaponenurosporene, that is, the degree of fading from yellow to white, so that there is a problem that visibility is poor and false positives frequently occur. Therefore, protein engineering was applied to CrtN (dehydrosqualene desaturase) to obtain a route to convert C15PP into a bright red pigment instead of yellow.
Specifically, random gene mutations were introduced into the CrtN gene by mutation PCR (manganese method) to create a library. Incorporated into the expression plasmid for Escherichia coli (pUC) together with the CrtM gene, a cell with red cell color was obtained. The nucleotide sequence of the obtained gene variant was the sequence shown in SEQ ID NO: 1 (amino acid sequence is SEQ ID NO: 2).

The obtained gene mutant was incorporated into the E. coli expression plasmid pAmod together with the CrtM gene to obtain a pAC-MN _{red mutant} . When this plasmid was introduced into Escherichia coli XL1-Blue by a conventional method, cells having a pathway (FIG. 4) for converting C15PP into a bright red pigment instead of yellow were obtained.
This improvement has dramatically improved the S / N ratio, quantification, and reliability of terpene synthase gene screening technology.

<Reduction of CrtM / CrtN dye synthesis by geraniol synthase>
Geraniol synthase (GES) is a kind of monoterpene synthase, which synthesizes the fragrance geraniol from geranyl diphosphate. Both the substrate and the product are clear and colorless.
GES gene derived from sweet basil (Iijima, Y. et al .; Plant Physiol134, 370-379 (2004)) and the carboxyl terminal domain (287-567) or N-terminal part (total 286 amino acids) of the same gene The (inactive) gene (only the C-terminal part of the above GES gene was PCR amplified and incorporated into a pUC vector) was incorporated into an E. coli expression plasmid (pUC18Nm) by a conventional method, and pUC-GES _Full and pUC- Got GES _C-end .
pUC-GES _Full or pUC-GES _C-end was introduced into E. coli XL1-Blue carrying pAC-MNwt, pAC-MNwt-idi or pAC-MN _{red mutant} .
When colonies were formed, colonies into which pUC-GES _Full had been introduced were observed as lighter colonies than those into which the control (pUC18Nm) was introduced.
Further, when the plasmid pAC-MN _{red mutant} containing the CrtN mutant gene was used, the visibility of the presence or absence of the activity was significantly improved.
At the same time, after liquid culture (TB 2 mL, 30 ° C., 48 h), the carotenoid component was extracted with 1 mL of acetone, and the amount of carotenoid synthesis per cell was calculated from the absorption spectrum. A significant decrease in the amount of dye synthesis was observed (FIG. 5).

<Reduction of GPS / CrtM / CrtN dye synthesis due to C10PP consumption of monoterpene synthase>
E. coli farnesyl diphosphate synthase (FDS) may convert C5PP to C15PP with little release of C10PP. At this time, even if there is a monoterpene synthesis activity (C10PP consumption activity), the C10PP that it should deprive is not accumulated in the cells, so there may be a problem that it cannot be detected as a color change.
To solve this problem, the S82F mutant of BstFDS was created separately from this endogenous FDS. This enzyme mutant has a function as a geranyl diphosphate synthase (GPS) in which the 82st serine of the BstFDS is substituted with phenylalanine (Japanese Patent Laid-Open No. 11-169178). The same thing was tried about FDS derived from E. coli, and it has been reported that this increased the monoterpene activity introduced into E. coli (Reiling, KK et al .; Biotechnol Bioeng 87, 200-212 (2004)).

In cells into which BstFDS _S82F has been introduced, C10PP is synthesized using C5PP (dimethylallyl diphosphate) as a substrate, and C10PP accumulates in a large amount in the cell. This C10PP is converted to C15PP by endogenous FPS (ispA), and then the dye accumulates by CrtM followed by CrtN. At this time, if there is a monoterpene synthase that consumes C10PP, part of the precursor (C15PP) via C10PP will not be used for dye synthesis, and the cell color will fade.

<Screening of C25PP (geranyl farnesyl diphosphate) synthase / CrtE / CrtB / CrtI dye synthesis reduction by C20PP consumption>
FDS (farnesyl diphosphate synthase) is an enzyme that synthesizes C15PP by combining dimethylallyl diphosphate and geranyl diphosphate. On the other hand, FDS _Y81A in which amino acid tyrosine at position 81 of FDS is substituted with alanine gives a mixture of C20PP / C25PP, not C15PP. In order to obtain mutants with higher product specificity for C25PP, a library with FDS _Y81A as a parent was created and screened for function for 20 consumption activity (C25PP synthesis ability).

(1) Preparation of CrtE / CrtB / CrtI-expressing Escherichia coli (cells capable of dye synthesis) that consumes C20PP
A plasmid pAC-EBI-idi for expression of E. coli containing CrtE gene, CrtB gene, CrtI gene and idi gene was prepared. Specifically, CrtE, CrtB and CrtI genes were PCR amplified from the genome of soil bacteria (Pantoea ananatis, DSM30080), and incorporated into an expression plasmid for E. coli (pAmod) downstream of lacP / O to obtain pAC-EBI. In this pAC-EBI, idi amplified by PCR from the E. coli genome was incorporated downstream of lacP / O to obtain pAC-EBI-idi.
PAC-EBI-idi was introduced into E. coli XL1-Blue by a conventional method to obtain a carotenoid (lycopene) -synthesizeable cell. Since Escherichia coli does not synthesize C20PP, it is necessary to introduce CrtE that catalyzes the synthesis of C20PP from C15PP.

(2) Preparation of mutant FDS gene library derived from moderately thermophilic bacterium (Bacillus genus) A mutant library was prepared for the FDS gene derived from moderately thermophilic bacterium (genus Bacillus). Specifically, the FDS _Y81A gene (PCR-amplified from the purified genomic DNA (ATCC 12980D-5) obtained from ATCC. GENE BankID: 13293, literature: Koyama T, et al., J. Biochem., 133 (3), 355-63 (1993)), introduced mutation into the mutant PCR (manganese method) (Cadwell, RC et al .; PCR Methods Appl 2, 28-33 (1992)) An integrated mutant library (hereinafter referred to as EP-PCR library) was obtained in (pUC18Nm).

(3) Introduction and screening of CrtE / CrtB / CrtI-expressing E. coli library genes
XL1-Blue with pAC-EBI-idi was transfected with plasmid pUC18Nm (described above) containing the genes of these libraries and colonies were formed at 37 ° C, then allowed to stand at room temperature and colony color Was observed (FIG. 6). At the same time, the amount of carotenoid synthesis in liquid culture (TB medium, 2 mL) was also measured.
By selecting white colonies, three C25PP synthase mutants having activity for synthesizing geranyl farnesyl diphosphate, ie, synthesizing C25PP from C20PP, were obtained. The sequences of these enzyme variants were as follows:

In addition to the substitution of amino acid 81, the two mutants are those in which threonine (T) at 121 is mutated to alanine (A) or serine (S), and the other is the substitution of amino acid 81, It was FDS in which 157 valine (V) was mutated to alanine (A).

(4) Gene analysis before and after screening
1. The above-mentioned EP-PCR library was introduced into Escherichia coli, colonized on LB plate medium, and 7 randomly selected sequences were determined.

2. The same library was introduced into an E. coli strain carrying pAC-EBI. Mutant DNA was extracted from the white colonies on the plate and the sequence was read.

The number of gene mutations at the nucleotide sequence level was 6.9 nucleotide mutations / gene before screening, but the average number of mutations after screening dropped to 3.0 mutations / gene. In addition, mutants before screening have many non-synonymous mutations and non-trivial (substitution with amino acids with greatly different chemical properties) inside the structure, while mutants after screening have most mutations. Concentrated on the surface.
Therefore, it is strongly suggested that screening (picking white colonies) selects for an enzyme that retains the C20PP consumption function and removes an enzyme that has a mutation that is detrimental to the function / structure retention. All four mutations found in the core part of the protein after screening were Trivial mutations.

(5) C25PP synthesis ability of novel C25PP synthase mutants
The three FDS81 mutants that showed an increase in C20 consumption activity score had T121A or V157A mutations in addition to Y81A.
Thus, co-expression of the obtained three FDS81 mutant genes 10-1, 50-1, _50-2 and the CrtM _26/38 gene was attempted.
CrtM is an enzyme that synthesizes the C15PP → C30 carotenoid skeleton. Mutant M _26/38 introduced with the F26A / W38A mutation uses prenyl diphosphates with various skeletons up to C15, C20, and C25 as substrates. , C30, C35, C40, C45, C50 and various skeleton carotenoids are produced. If FDS's ability to synthesize C25PP increases (if the intracellular C25 / C20 ratio increases), it is expected that the peak ratio of C50 carotenoids will also increase. In fact, 50-2 (mutated to residue 157) showed a particularly high C50 carotenoid synthesis function. The amount of carotenoid synthesis per dry cell weight (DCW) of the enzyme mutant is shown in FIG.
The 50-2 obtained here gives E. coli an efficient C25PP supply capacity, and is thought to be important for the construction of a cell production system for sesterterpenes. These mutants were the first to provide an efficient supply route to C25PP.

<Creation of new C25PP synthase mutant, screening and C20PP consumption ability>
Seven types of gene variants were created by combining three amino acid substitutions (Y81A, V157A, T121A) alone or in combination, and their C20PP consumption activity was compared.

1. Using site-directed mutagenesis (SDM), mutants were prepared with all combinations of Y81A, V157A, and T121A and incorporated into the pUC18Nm vector.
2. E. coli (XL1 Blue) was cotransformed with pAC-EBI-idi to form colonies.
3. The colony was inoculated into LB (Carb / Cm) 500 μL and cultured (37 ° C., 16 h, n = 4).
4). 10 μL of this culture solution was inoculated into 2 mL of TB (Carb / Cm) and cultured (30 ° C., 48 h).
5. E. coli was collected by centrifugation, and the intracellular fat-soluble fraction was extracted with 1 mL of acetone.
6). The absorption spectrum of 300 μL of the extract was measured, and the amount of dye was calculated from the absorbance at the maximum absorption wavelength.

When wild-type FDS was added, the synthesis amount of lycopene (C40 carotenoid) was improved by 50% from 400 to 600μg / gDCW. This is because a large amount of C15PP, which is a precursor of C20PP, can be supplied. On the other hand, when the Y81A single mutant was added, the synthesis amount became about 1/4. Y81A single mutant is a mutant that has been adopted as a C25PP supply channel.
The mutation of T121A is an amino acid mutation newly found in this study. The V157A mutation was previously reported as an amino acid mutation in the 1996 Ohnuma paper. These alone increased the amount of pigment synthesis (C40 carotenoid synthesis ability) rather. The same was true for the T121A-V157A mutant.
On the other hand, when the V157A mutation or the T121A mutation was combined with the Y81A mutation, the dye (C40 carotenoid) did not accumulate at all in the cells and showed very strong C20PP consumption activity (FIG. 8).

<Screening of sesquiterpene synthase TEAS>
This example shows that this screening system can be used for visualization of sesquiterpene synthase activity using TEAS which is a sesquiterpene synthase.
As the sesquiterpene synthase, 5-epiallystroken synthase (TEAS) derived from tobacco (Nicotiana tabacum) was used. The main product of this enzyme, (+)-5-epiaristroken, is known as the precursor of capsidiol, a tobacco phytoalexin. TEAS is the first plant-derived sesquiterpene for which genes have been identified, and various biochemical and structural studies have been conducted for its ease of use (Back K and Chappel J, J Biol Chem. 270 (1995) 7375-7381, Back K et al. Arch Biochem Biophys 315 (1994) 527-532, Mathis JR et al. Biochemistry 36 (1997) 8340-8348).

(1) Synthesis of TEAS gene and integration into vector
We asked Mr. Gene (GENEART) to codon-optimize the Tobacco 5-epi-Aristolokene synthase (TEAS) gene (1,653 bps) derived from Nicotiana tabacum in Escherichia coli and added a Trc promoter upstream of it. Synthesized (Ptrc-TEAS) (FIG. 9). Ptrc-TEAS was treated with Xba I and Xho I restriction enzymes and incorporated into a pUC18Nm vector (pUC18Nm-TEAS).

(2) Decrease in the amount of dye synthesized by TEAS E. coli (XL1 Blue) was cotransformed with Ptrc-TEAS and pAC-MNwt or pAC-EBI to form colonies.
2. The colony was inoculated into LB (Carb / Cm) 500 μL and cultured (37 ° C., 16 h, n = 4).
3. 10 μL of this culture solution was inoculated into 2 mL of TB (Carb / Cm / IPTG 0.1 mM) and cultured (30 ° C., pAC-MN: 24 h culture, pAC-EBI: 48 h culture).
4). E. coli was collected by centrifugation, and the intracellular fat-soluble fraction was extracted with 1 mL of acetone.
5. The absorption spectrum of 300 μL of the extract was measured, and the amount of dye was calculated from the absorbance at the maximum absorption wavelength.

  TEAS, a sesquiterpene synthase, should exhibit C15PP consumption activity. There was a report that its E. coli activity was very low (0.24μg / L; Martin VJJ et al. Biotechnol Bioeng. 75 (2001) 497-503). When actually expressed in the cell together with the C30 carotenoid pathway, Dye synthesis was almost completely suppressed, and the activity was easily visible (FIG. 10).

(3) Inactive form of TEAS and amount of dye synthesis PUC18Nm-TEAS _D303A and pUC18Nm-TEAS _R443A were prepared by substituting A (GCG) for D303 (GAT) or R443 (CGT) of TEAS using site-directed mutagenesis using PCR.
2. E. coli (XL1 Blue) was co-transformed with various TEAS plasmids and pAC-MNwt or pAC-EBI to form colonies.
3. The colony was inoculated into 500 μL of LB (Carb / Cm) and cultured (37 ° C., 16 h, n = 4).
4). 10 μL of this culture solution was inoculated into 2 mL of TB (Carb / Cm / IPTG 0.1 mM) and cultured (30 ° C., pAC-MN: 24 h culture, pAC-EBI: 48 h culture).
5. E. coli was collected by centrifugation, and the intracellular fat-soluble fraction was extracted with 1 mL of acetone.
6). The absorption spectrum of 300 μL of the extract was measured, and the amount of dye was calculated from the absorbance at the maximum absorption wavelength.

The TEAS mutant was inactive (FIGS. 11 and 12). The first step of the TEAS reaction is the elimination of the phosphate group of FPP to form a carbocation. This phosphate group elimination reaction occurs when Mg ²⁺ binds to a phosphate group. TEAS D303 retains Mg ²⁺ in the vicinity of FPP. R443 is an amino acid that plays a role of 1) directly binding to the phosphate group of FPP and 2) retaining Mg ²⁺ via water. When these were substituted with A, Mg ²⁺ and phosphate groups could not be bound, and the TEAS mutant was considered to be inactive.

<Screening of taxadiene synthase (TS)>
This example shows that this screening system can be used for visualization of diterpene synthase activity using taxadiene synthase (TS), which is a diterpene synthase.
Taxadiene synthase (TS), a diterpene cyclase from yew (Taxus brevifolia), was used to confirm C20PP substrate consumption activity. Taxadiene synthase is an enzyme that catalyzes the initial reaction of the taxol (paclitaxel) biosynthetic pathway. Taxol is known to have an antitumor action, and in recent years, it has been reported that it is useful as a treatment for neurodegenerative diseases such as Alzheimer's disease. The chemical synthesis of taxol has a very poor yield (up to 0.4%; Walji AM and MacMillan EWC. SYNLETT.10 (2007) 1477-1489). Furthermore, the amount obtained from plants is very small and difficult to scale up. Biosynthesis using metabolic engineering of microorganisms has attracted attention (Ajikumar PK et al. Science 330 (2010) 70-75, Dejong JM et al. Biotechnol Bioeng. 93 (2006) 212-24).

We commissioned Mr. Gene (GENEART) to perform total synthesis of Taxus brevifolia-derived taxadiene synthase (TS) gene with 59 residues at the N-terminus by codon optimization in E. coli (Ptrc-TS).
Ptrc-TS was treated with Xba I and Xho I restriction enzymes and incorporated into a pUC18Nm vector (pUC18Nm-TS).
Taxadiene synthase has a transport peptide sequence to plastid at the N-terminus. By cutting the sequence, the solubility of taxadiene synthase in E. coli is increased.

1. E. coli (XL1 Blue) was co-transformed with pUC18Nm-TS and pAC-MNwt or pAC-EBI to form colonies.
2. The colony was inoculated into LB (Carb / Cm) 500 μL and cultured (37 ° C., 16 h, n = 4).
3. 10 μL of this culture solution was inoculated into 2 mL of TB (Carb / Cm / IPTG 0.1 mM) and cultured (30 ° C., pAC-MN: 24 h culture, pAC-EBI: 48 h culture).
4). E. coli was collected by centrifugation, and the intracellular fat-soluble fraction was extracted with 1 mL of acetone.
5. The absorption spectrum of 300 μL of the extract was measured, and the amount of dye was calculated from the absorbance at the maximum absorption wavelength.

  When the liquid culture pellet of the cells co-transformed with pAC-MNwt was observed with the naked eye, the yellow color was slightly light (C15 consumption ability, FIG. 13). The liquid culture pellets of cells cotransformed with pAC-EBI clearly showed a color difference between the naked eye and the photograph (C20 consumption ability, FIG. 14).

<Screening of terpene synthase gene from tomato cDNA>
The method of the present invention was applied to unidentified DNA.
1. Blast search in Kazusa DNA Research Institute's tomato cDNA library: KaFTom (http://www.pgb.kazusa.or.jp/kaftom/) with sesquiterpene synthase (kaurene synthase, taxadiene synthase, copalyl synathase) as Quety As a result, AK327297, AK324962, and AK321354 were hit.
2. From the top 3 of the search, the sequence giving the longest ORF was PCR amplified and incorporated into pUC18Nm. The numbers in parentheses indicate the amino acid length of the polypeptide estimated from the ORF.
LEFL2026E14 (773 aa) AK327297
LEFL1089AH03 (551 aa) AK324962
LEFL1023CC09 (381 aa) AK321354
3. These plasmids were cotransformed with pAC-MN or pAC-EBI in E. coli (XL1 Blue) to form colonies.
4). The colony was inoculated into LB (Carb / Cm) 500 μL and cultured (37 ° C., 16 h, n = 6).
5. 10 μL of this culture solution was inoculated into 2 mL of TB (Carb / Cm / IPTG 0.1 mM) and cultured (30 ° C., 48 h).
6). E. coli was collected by centrifugation, and the intracellular fat-soluble fraction was extracted with 1 mL of acetone.
7). The absorption spectrum of 300 μL of the extract was measured, and the amount of dye accumulated was calculated from the absorbance at the maximum absorption wavelength.

In the C15P screening system, only E14 showed strong activity (decrease in the amount of accumulated carotenoid pigment), but there was almost no change in activity for other than that. On the other hand, in the C20PP consumption screening, the amount of synthesis decreased slightly. This strongly suggests that this E14 gene is a sesquiterpene synthase (FIG. 15).
A Blast search using E14 as a query yielded the results shown in the table. Thus, the effectiveness of this method as a gene search method was suggested.

  The present invention provides a general and systematic search for terpene synthase genes. Enables acquisition of highly active terpene synthase mutants.

Claims (5)

A test gene is introduced into a cell capable of dye synthesis, and a decrease in the amount of dye synthesis is detected using the color fading as an index , wherein the dye passes through isopentenyl diphosphate (IPP). A method for screening a terpene synthase gene and / or a mutant thereof , which is a biosynthesized pigment .   The terpene synthase gene and / or variant thereof according to claim 1, wherein the decrease in the amount of dye synthesis is a decrease in the amount of dye synthesis due to consumption of a substrate of an enzyme involved in dye synthesis by the terpene synthase. Screening method.   The method for screening a terpene synthase gene and / or a mutant thereof according to claim 1 or 2, wherein the cell is Escherichia coli or yeast.   The terpene synthase gene and / or its mutant screening according to any one of claims 1 to 3, wherein the cell capable of pigment synthesis is a cell transformed with an enzyme gene involved in pigment synthesis or a mutant thereof. Method.   Enzyme genes involved in pigment synthesis are diapophytoene synthase (CrtM) gene, dehydrosqualene desaturase (CrtN) gene, geranylgeranyl diphosphate synthase (CrtE) gene, phytoene synthase (CrtB) gene, phytoene dehydration 5. The method for screening a terpene synthase gene and / or a variant thereof according to claim 4, wherein the terpene synthase gene and / or a variant thereof are at least one selected from an enzyme (CrtI) gene and a geranyl diphosphate synthase (GPS) gene.