JPH07155189A - Lycopene cyclase - Google Patents

Abstract

PURPOSE: To provide a new lycopene cyclase gene encoding lycopene cyclase resistant to herbicide, expressing a biosynthesis path from geranylgeranyl pyrophosphate to β-carotene in a host cell and improving the nutrient value, etc., of organism.

CONSTITUTION: A cyanobacterium Synechococcus sp. PCC7942 is screened on a clone resistant to herbicide and a modified DNA sequence controlling the production of lycopene cyclase enzyme resistant to herbicide is purified and separated to obtain the objective new lycopene cyclase gene composed of a DNA sequence of bp 2029-3261 encoding a lycopene cyclase composed of an LCY polypeptide having an N-terminal partial amino acid sequence expressed by the formula, expressing a biosynthesis path from geranylgeranyl pyrophosphate to β-caroten in a proper host cell to improve the nutrient value, pharmacological properties and appearance of organism and useful e.g. for the construction of a transgenic plant resistant to herbicide.

COPYRIGHT: (C)1995,JPO

Description

Detailed Description of the Invention

[0001]

The present invention relates to the isolation of the gene for lycopene cyclase called lcy and its regulatory sequence, and β
To the use of said genes or said regulatory sequences in the preparation of natural or transgenic organisms in which the production of carotene and other carotenoids is regulated by the presence of said genes or to modify the expression of natural genes homologous to lcy .

[0002]

Carotenoids are the largest variety of pigments in nature. They are newly synthesized only in photosynthetic organisms and in some bacteria and fungi. Their essential function in plants is to protect against photooxidative damage sensitized by chlorophyll in photosynthetic organs ^1,2 , but they also serve various other roles. They serve as auxiliary dyes in daylighting for photosynthesis, and they are internal components of photosynthetic reaction centers.
Carotenoids are involved in the heat dissipation of light energy captured by the daylighting antenna ³ , and are substrates for the biosynthesis of plant growth-regulating abscisic acid.
^4,5 , and many flowers, fruits and animal coloring substances. Certain cyclic carotenoids, including β-carotene, are precursors of vitamin A in human and animal food and are of current interest as anti-cancer agents ^6,7 .

The generally accepted pathway of carotenoid biosynthesis in plants begins with the head-to-head condensation of two molecules of geranylgeranyl pyrophosphate, a soluble compound with 20 carbons, and the colorless, membrane-bound carotenoid phytoene. Are generated (FIG. 1). This two-step reaction in plants and blue-green bacteria is catalyzed by a single soluble enzyme, phytoene synthase ^9,10,11,12 . Sequential desaturation of phytoene produces phytofluene first and then ζ-carotene. Both of these reactions are performed by a single enzyme in plants and blue-green bacteria, phytoene desaturase ^13,14,15 . This enzyme and the enzymes that catalyze subsequent steps in the pathway are thought to be membrane bound ¹⁶ . Upon further desaturation, two symmetrical red carotenoid pigments, lycopene, are formed and then converted to yellow β-carotene by cyclization at each end of the molecule (Fig. 1). Subsequent reactions in the pathway include the addition of various oxygen functional groups to form xanthophyll or oxygenated carotenoids.

Despite many efforts, few enzymes or genes of the carotenoid biosynthetic pathway have been identified and isolated in oxygen-producing photosynthetic organisms. Maintaining catalytic activity during purification of these tightly membrane-bound enzymes has been shown to be surprisingly difficult, and the difficulty in obtaining labeled substrates for enzymatic analysis is also an obstacle. There is. The photosynthetic bacterium Rhodobacter capsulatus ¹⁷ and the non-photosynthetic bacterium Erwinia uredov
ora) ¹⁸ and Erwinia herbicola
The gene for the complete carotenoid biosynthetic pathway within ola) ¹⁹ has been cloned and sequenced. Initially, it was thought that such genes would provide molecular probes that would allow the identification of homologous genes within oxygen-producing photosynthetic organisms (blue-green bacteria, algae, and plants). However, this approach did not yield good results, as the genes of Rhodobacter and Erewinia bear little or no resemblance to the genes of the corresponding enzymes in oxygen-producing photosynthetic organisms.

Using another approach, two genes,
That gene phytoene synthase ^{10, 20} and phytoene desaturase ^14,15,21 have been cloned from the oxygen generating photosynthetic organisms. The phytoene desaturase of blue-green bacteria and the amino acid sequences of the homologous gene products of eukaryotic algae and higher plants had high conservation ^rates14,22 , but similarities with known bacterial or fungal phytoene desaturases Almost never.
In the present invention, the transformable blue-green bacterium Synechococcus sp. (Synechococcus sp.) PCC7942
Was used as a model organism to study the carotenoid biosynthetic pathway in oxygen-producing photosynthetic organisms. The present inventors have found that phytoene synthase (psy, formerly p
and phytoene desaturase (pds)
^Was cloned ^10,21 and subsequently the plant and algae genes were identified, cloned and sequenced using the blue-green bacterial gene as a probe ^14,21,22,23 . The gene pds was identified using the bleaching herbicide norflurazon, which specifically inhibits phytoene desaturation by interacting with phytoene desaturase ²⁴ . It was postulated that point mutations in the gene encoding the enzyme, which lead to amino acid substitutions in the polypeptide, may confer resistance to the herbicide. By selecting chemically induced mutants that are resistant to norflurazone and then locating these mutations by gene complementation of the resistance in wild-type strains, we found that the phytoene desaturase I found the gene.

It is useful to be able to moderate carotenoid synthesis in order to improve the nutritional value, pharmaceutical properties and appearance of plants. In doing so, it may be desirable to use the native gene when possible. Direct integration of a gene of bacterial origin into a plant may require, for example, further modification of the gene so that it can be used to regulate the photosynthetic pathway. In addition, bioengineered plants must be approved for regulation before they can be released to the outside world. If foreign genes are present, the approval process becomes more difficult. For example, β
-It is more efficient to use the natural gene to control the carotene synthetic pathway. Further, it would be useful to have a useful crop that is resistant to herbicides that targets the action of lycopene cyclase. Herbicides that target the carotenoid synthetic pathway are less toxic because this pathway does not exist in humans, animals or insects. Moreover, plants that are herbicide tolerant at two points within the carotenoid synthetic pathway allow the alternate use of two herbicides, thereby minimizing the emergence of herbicide tolerant weed species.

[0007]

According to the present invention, a DNA encoding a gene for lycopene cyclase called lcy
The sequence was purified and isolated. Furthermore, the present invention includes a method of modifying the expression of a native gene, thereby not requiring the presence of the foreign gene in the organism. Furthermore, the present invention incorporates a gene encoding an enzyme containing lcy, which controls the biosynthetic pathway from geranylgeranyl pyrophosphate to β-carotene, into a suitable host cell selected from eukaryotic and prokaryotic cells. And a method of expressing. This method can be used to produce β-carotene in an organism that does not produce β-carotene, thereby improving the nutritional value, pharmaceutical properties and visible appearance value of the organism. .

The invention further provides a method of overexpressing the gene for lycopene cyclase in a transgenic organism in a constitutive or tissue-specific manner to increase the content of β-carotene or other carotenoids in the biosynthetic pathway. Including. The invention also includes transgenic organisms that introduce antisense expression of the lcy gene, either the native gene or the homologous gene, into the genomic DNA, thereby inhibiting the synthesis of lycopene cyclase and accumulating the red pigment lycopene. . This method can make the non-red creatures red, or the appearance of the actually red creatures redder. Finally, the invention also provides the construction of transgenic plants that are resistant to the action of herbicides such as the herbicide MPTA and other related compounds of trialkylamines and other herbicides that interact with the lycopene cyclase enzyme. Including. Other advantages of the present invention will be readily appreciated as the invention can be more fully understood by reference to the following detailed description in connection with the accompanying drawings.

The present invention is directed to the enzyme lycopene cyclase (LC
A gene encoding Y) and D containing the DNA flanking sequence of that gene which is best shown in SEQ ID NO: 1
It consists of NA sequence. purifying the gene called lcy,
It was isolated and cloned. Furthermore, DN modified in the promoter region of the gene for lycopene cyclase
The A sequence that confers resistance to MPTA is also
In practicing the present invention, purified, isolated and cloned (Figures 2 and 7). Herbicides generally work by disrupting selected biochemical pathways. Herbicides that interfere with the carotenoid biosynthetic pathway are particularly effective. These herbicides include several substituted triethylamine compounds that inhibit the formation of cyclic carotenoids resulting in the accumulation of lycopene in plants, algae, and blue-green bacteria ²⁶ . A particularly effective inhibitor of this type is the compound 2- (4-methylphenoxy) triethylamine hydrochloride (MPTA) ^27,28 . The target site for MPTA and related compounds is believed to be the enzyme lycopene cyclase ²⁶ .

The present inventors have found that Synechococcus sp. PC
An MPTA resistant mutant of C7942 was isolated and the gene containing one of the mutations cloned (see Figure 7). Expression of this gene in a lycopene accumulating strain of E. coli clearly showed that the MPTA resistance gene encodes lycopene cyclase. DNA hybridization analysis indicates that the sequence of this gene is conserved in other photosynthetic eukaryotic cells. The mutation conferring herbicide tolerance may be a point mutation, deletion or insertion. The mutation is such that lc is such that the resulting amino acid sequence is altered to confer herbicide resistance.
It may occur within the nucleotide sequence of y or within the flanking sequence of lcy such that regulation of lcy is altered to confer herbicide tolerance. An example of the latter mutation is the mutation of mutant 5 shown in FIG. In this mutant,
C is converted to T by a single base conversion (base number 1925 of SEQ ID NO: 1) in the promoter region of lcy, which is 104 base pairs upstream of the start codon, and highly weeded to the Cinecoccus cells containing it. It imparts drug resistance (see FIG. 2). This is probably due to a large increase in enzyme biosynthesis.

[0011] In cloning the gene for lycopene cyclase, first, Synechococcus sp. The strategy used to identify the gene for phytoene desaturase in PCC7942 was used. This approach predicts that the enzyme lycopene cyclase is the target site for the bleaching herbicide MPTA,
And the subtle changes in the main structure of lycopene cyclase are based on the speculation that it will result in an enzyme that is MPTA resistant and still retains moderate catalytic activity. The inventors were uncertain whether the resistance phenotype of the particular mutant was derived from intragenic damage of lycopene cyclase. Moreover, the phenotype selected was considered to be due to the increased rate of degradation or detoxification of the inhibitor or the result of reduced herbicide uptake. The functional expression in E. coli of a gene containing MPTA resistance mutations mutant M ^r -5, the gene called lcy what is actually demonstrated that encodes an enzyme lycopene cyclase. Several other MPTA resistance mutations were found in the lcy gene. The wild-type gene product (SEQ ID NO: 2) and the gene products of these other mutants also function well in E. coli.

Lycopene is the major substrate for cyclic carotenoid formation in plants and blue-green bacteria. The conversion of lycopene to β-carotene requires that the cyclization reaction take place at both ends of the symmetrical lycopene. The present invention demonstrates that a single blue-green bacterial enzyme efficiently catalyzes cyclization of both ends. As described below, a considerable amount of lycopene was accumulated in these cells, but accumulation of γ-carotene, which is a monocyclic species, was not observed in the culture medium.
This indicates that any lycopene molecule cyclized only at one end has a very high probability of being cyclized at the other end as well. The possibility that lycopene cyclase acts as a homodimeric complex is one explanation for these results. The novel plasmid pLCYB-M5XE was constructed and described herein for use as part of an isolation procedure and in transformation procedures. Plasmids pLCYB-M5PPF and pLCYB-M5
SP is a truncated version of pLCYB-M5XE and has been described herein. Various plasmids were constructed as described in the Materials and Methods section below.

Using the present invention, with a suitable vector carrying either the DNA sequence for lycopene cyclase or the mutant sequence of DNA for lycopene cyclase which is resistant to MPTA, It is possible to transform host cells including E. coli, blue-green bacteria, algae and plants. Such transformed cells are lc
It allows the study of the regulation of the y gene and the catalytic function of LCY. The present inventors have recognized that expression of lcy in cells of E. coli that accumulates ζ-carotene does not cause cyclization of ζ-carotene. This result means that the cyclization reaction catalyzed by the Synechococcus lcy gene product requires that the linear molecule be fully desaturated. Neurosporanes have been shown in algal fungi to be able to undergo cyclization only in half of which half is desaturated to the level of lycopene ¹⁶ . The present invention involves constructing transgenic organisms that are resistant to MPTA and other herbicides that prevent cyclization of lycopene. The agronomic benefits of using herbicide-tolerant crops are:
It has been demonstrated in Brassica species, including Brassica naous, where it has introduced atrazine resistance by genetic crossing of the crop with weeds of the same genus ²⁹ .

There is some debate about the target site of action of MPTA and related compounds. Previously, MPTA was shown to induce lycopene accumulation in grapefruit and to interfere with lycopene cyclization in isolated Euglena chloroplasts ²⁸ . More work was done with the structurally related compound, 2- (4-chlorophenylthio) triethylamine hydrochloride (CPTA) ²⁶ , with similar results of lycopene accumulation in vivo. However, contrary to the results obtained in vivo, C
It was reported that PTA has little or no effect on carotenoid biosynthesis in a cell-free system derived from acetone extract of tomato plastid ³⁰ , narcissus chromophore ³¹ and capsicum chromophore ³² . It has been suggested that CPTA and related compounds act at the level of gene expression rather than directly affecting enzyme function.
^30,33,34 . The blue-green bacterium Aphanocap
A more recent study using a cell-free system from
It has been shown that TA can be an effective inhibitor of lycopene cyclization in vitro, and that it inhibits the cyclization reaction in a non-competitive manner as reviewed by Sandmann et al. ²⁶ . ing. C in many other cell-free systems
The lack of inhibition by PTA suggests that physical association of the membrane and / or ordered structural association of carotenoid biosynthetic enzymes is important for effective herbicidal action. The delicate nature of carotenoid biosynthesis in cell-free systems is well known ³⁵ .

The finding of the present invention that mutations in the gene for lycopene cyclase confer resistance to MPTA is strong evidence that this enzyme is a target site for the action of MPTA and related compounds. This conclusion is further supported by the results that MPTA inhibits lycopene cyclase activity in E. coli cells presumably lacking the authentic blue-green bacterial carotene-producing organs. Accumulation of γ-carotene in MPTA-treated E. coli cells suggests that the second cyclization step performed by lycopene cyclase is more sensitive to inhibition by herbicides. These findings enable us to transform crop plants to be MPTA resistant by inserting any of our many different MPTA ^r mutant lycopene cyclase genes. More importantly, the present invention can be used to lead natural plant and algae biotechnology to confer resistance to MPTA and other herbicides. Southern hybridization analysis using the blue-green bacterial lcy sequence as a molecular probe showed that this gene was conserved in eukaryotic algae with homologous DNA sequences, and lycopene cyclase from algae and higher plants. It shows the potential of using lcy as a molecular probe for cloning. It further shows that the transgenic plants carrying the gene of the invention are functional. The fact that higher plants contain the lycopene cyclase gene, which is homologous to lcy, makes the enzymatic chemistry of lycopene cyclization in plants similar to that of blue-green bacteria, as in the case of phytoene synthase and phytoene desaturase genes. It shows that it is completely different from other microorganisms. This means that when lcy is expressed in transgenic algae or plants, lcy is functional by resembling its counterpart in plants.

In the "plant-type" pathway of carotenoid biosynthesis, we have derived from geranylgeranyl phosphate to β-
The genes were identified for all steps up to carotene, except for the two desaturation steps that convert symmetrical zeta-carotene to lycopene. Cloning of a putative gene for ζ-carotene desaturase has recently been reported ³⁶ . Desaturation of ζ-carotene in plants and algae is inhibited by several experimental bleach herbicides ²⁶ , and some of these compounds effectively inhibit ζ-carotene desaturation in Synechococcus PCC7942 ²⁶ . Our success in cloning psi, pds, and the invention, ie, lcy, using techniques known in the art is the same strategy as the induction and selection of herbicide resistance mutations derived within the scope of the invention. Mean that it makes it possible to clone ζ-carotene desaturase with a suitable inhibitor. From this,
The present invention allows the construction of a biosynthetic pathway from geranylgeranyl phosphate to β-carotene in suitable host cells by incorporating DNA sequences encoding psi, pds, zds and lcy.

The present invention also includes transgenic organisms which introduce antisense expression of the lcy gene into genomic DNA, thereby inhibiting the synthesis of lycopene cyclase and accumulating the red pigment lycopene. This method can make the non-red creatures red, or the appearance of the actually red creatures redder. The use of antisense technology to alter gene expression to alter plant phenotype has been reported. This technique has been used in tomatoes with the carotenoid biosynthetic pathway ^12,37,38 . The following examples describe the methods of isolation, purification and cloning of each lcy sequence as well as methods of probe / vector construction and their use. In addition, these examples demonstrate the transformation of suitable host cells such as bacteria, algae and plants with the vector and identification of herbicide resistant sites. Suitable host cells include carotene producing organisms where only lcy is used, or cells containing phytoene, a precursor of pds or the crtI gene product.

Raw Materials and Methods Organisms and Growth Conditions Synechococcus sp. PCC7942 (Anaquistis
Cultivation of Anacystis nidulans R2) was carried out at 35 ° C. in ³⁹ BG11 medium as previously described. For selection of MPTA resistant mutants (MPTA ^r ) and transformants, the culture was plated on solid BG11 medium containing 1.5% agar (Bacto) and 20 or 30 μM MPTA. MPTA was added to molten agar at 50 ° C. from a 20 mM methanol stock solution immediately before pouring into Petri dishes. When necessary, the BG1
Kanamycin was added to 1 agar plate at a concentration of 10 μg / ml. E. coli strain XL1-Blue with 30 μg / ml kanamycin for selection as the host for the Synechococcus genomic library in plasmid vector pBR329K ²⁵ , and plasmid pBluescript.
tIIKS ⁺ (Stratagene) and other plasmids were used with 100 μg / ml ampicillin and / or 50 μg / ml chloramphenicol as hosts.
The culture of E. coli was carried out in the LB medium at 37 ° C. in the dark ⁴⁰ .

Selection of MPTA-Resistant Mutants Synechococcus cultures were prepared as previously described.
³⁹ ethane methylsulfonate a chemical mutagen treated (EMS, Sigma Chemical Co., St. Louis, MO), the after grown for 24 hours in a liquid medium, containing MPTA of 20 or 30 [mu] M BG11 Selected on agar plates. Herbicide tolerance in selected mutants was tested by spotting 3 μl of diluted culture onto BG11 agar master petri dishes. After growing for 2 weeks, different concentrations of M
Replicas were made on plates containing PTA and grown for an additional 2 weeks. Mutants were maintained on BG11 agar plates containing 4 μM MPTA. This MP
The TA was a gift from Dr. Henry Yokoyama of the Fruit and Vegetable Chemistry Laboratory within the US Department of Agriculture's Agricultural Research Department in Pasadena, Calif. (91106).

[0020] Molecular Cloning MPTA resistant Synechococcus mutant 5 (M ^r -5)
Genomic DNA was extracted from ⁴¹ , digested to completion with EcoRI, and a genomic library was constructed within the EcoRI site of pBR329K ²⁵ . The parental wild type strain was ^{designated as} Mr-5
The DNA of the library was transformed ⁴¹ and the transformants were selected on BG11 agar plates containing both MPTA (20 or 30 μM) and kanamycin (10 μM). Was recovered the plasmid vector with flanking genomic DNA using the strategy described in Kamobittsu (Chamovitz) al ^25. 2.1 Collected in this way
The kb EcoRI-SalI genomic DNA fragment was used as a molecular probe for Southern DNA analysis. ³² P labeling was performed by the random priming method ⁴² . An 8.5 kb genomic clone identified by colony hybridization ⁴⁰ using this probe was transformed into the plasmid pBlue.
It was subcloned into escriptIIKS ⁺ (Stratagene) and designated as pM5EE. Various fragments of this clone were cloned into the vector pBluescriptIIKS ^+.
Subcloned into Synechococcus sp. PCC
Wild-type strains of 7942 were tested for their ability to transform MPTA resistance. Operation by Del Sal et al. ⁴³
Was used to prepare a miniprep of plasmid DNA.

Construction of lycopene-accumulating strain of Escherichia coli To confirm the activity of lycopene cyclase (LCY),
It was essential to modify E. coli cells by genetic engineering to produce lycopene, a substrate for the enzyme LCY. Several such techniques performed in E. coli using genes from Erewinia species have been published in the past
^{18,19,44,45,46,47} . Briefly, a vector was constructed from a cluster of genes encoding a carotenoid biosynthetic enzyme cloned from Erewinia uredbora ¹⁸ . The 2.26 kb BstEII-SnaBI fragment was deleted from plasmid pCAR (Misawa et al., 19
90), crtE, crtB, and crtI 3.
The 75 kb Asp718-EcoRI fragment was subcloned into the EcoRV site of the plasmid vector pACYC184. A lycopene accumulating strain of E. coli was then constructed using the resulting vector, which has the same insert as the recombinant plasmid designated pCAR-ADE ¹⁸ .

Functional expression of lycopene cyclase in E. coli 7.2 kb XbalI-E containing MPTA resistance mutation
The coRI fragment into the vector pBluescriptIIK
It was cloned at multiple cloning sites of S ⁺ (Stratagen) and then excised as a SacI-EcoRI fragment. This SacI-EcoRI fragment is then
PTG (isopropylthio-β-D-galactoside) inducible expression vector pTrcHisB (Invitrogen)
X containing the pCAR-ADE cloned in and using the resulting plasmid designated pLCYB-M5XE
Competent cells of the L1-Blue E. coli strain were transformed. Transformed cells were plated on LB agar plates containing ampicillin (100 μg / ml) and chloramphenicol (50 μg / ml), and 100 mM aqueous solution of IPTG 10 h before plating the cells.
μl was applied.

Carotenoid pigment analysis 18 at 37 ° C. in LB medium containing 1 mM IPTG.
Culturing of E. coli was carried out for an hour. Bacterial cells were harvested from 50 ml of the suspension culture by centrifugation, and the carotenoid pigment was extracted by dissolving the precipitate in 90% acetone. As described previously ¹⁰ , Spherisolve
(Spherisorb) ODS1 HP on 25 cm reverse phase column
Carotenoids were analyzed by LC. Acetonitrile /
L6200 using an isocratic solvent system of methanol / isopropanol (85: 10: 5).
A Merck / Hitachi HPLC system consisting of a pump, L300 multichannel photodetector with D6000 mesophase was used. Hitachi DAD Manager software allowed simultaneous detection of phytoene and colored carotenoids. Individual carotenoids were identified based on their on-line absorption spectra and typical retention times compared to lycopene and β-carotene controls.

[0024]

【Example】

1. Selection of MPTA-resistant mutants and confirmation of gene presence The blue-green bacterium Synechococcus sp. PCC7942 wild-type cells did not grow on BG11 agar plates containing> 2 μM bleaching herbicide MPTA. EMS
After mutagenesis of the wild-type strain and growth on agar plates containing 30 μM MPTA at
We have selected some herbicide resistant mutants. Mutant 5 ( ^Mr- 5) showed the highest resistance, 5
Grown on agar plates containing 0 μM MPTA (FIG. 2). This mutant was used in all subsequent experiments described herein. The genetic basis of MPTA resistance in M ^r -5, were obtained by transformation experiments. Genomic DNA was extracted from the M ^r -5, endonuclease EcoRI
Digested with Synechococcus sp. PCC7942 wild type cells were transfected. The high frequency of appearance of colonies on agar plates containing 30 μM MPTA (approximately 10 ⁴ times that of the non-transformed control) demonstrated a genetic trait of resistance to MPTA, and single lesions or intimate contact. It has been shown that any of the multiple mutations involved are responsible for the resistance trait.

2. The genomic library of cloned mutant M ^r -5 of MPTA resistance gene was constructed into the EcoRI site of the plasmid vector pBR329K ^25. The DNA of this library was transfected as a closed circular plasmid into cells of the wild type strain and transformants were selected for growth on BG11 agar plates containing both MPTA and kanamycin. Synechococcus sp.
Stable transformation in PCC7942 occurs by the integration of foreign DNA into the bacterial chromosome after homologous recombination ^48,49 . Double resistant transformant is MP
It occurred as a result of one crossover phenomenon between a plasmid containing genomic DNA carrying a mutation for TA resistance (MPTA ^r ) and its homologous sequence in the chromosome. After all,
The pBR329K plasmid DNA was integrated into the blue-green bacterial chromosome flanking the MPTA resistance gene (19
This is explained in detail in FIG. 2 of the 1990 reference to Kamovitz et al. ²⁵ ). The genomic DNA of the double resistant transformant was transformed into S
A portion of the pBR329K vector was flanked by digestion with either alI or BamHI endonucleases, followed by DNA ligation, transfection of E. coli cells, and selection for kanamycin resistance (Kan ^r ). Collected with genomic DNA fragments. From such one Kan ^r -MPTA ^r transformant, we recovered a 2.1 kb EcoRI-SalI fragment of blue-green bacterial genomic DNA. This fragment was screened original M ^r -5 genomic DNA library using as a molecular probe to identify a plasmid containing the EcoRI genomic insert of 8.5 kb. Synechococcus sp. Into the cell of the wild-type strain of PCC7942
Transfection of the 8.5 kb DNA fragment resulted in a high frequency of herbicide-resistant colonies and thus this fragment
It was confirmed to contain a mutation that confers A ^r . In a similar test, the mutation resulted in a 4.6 kb Eco
Has been shown to be located within the RI-BamHI fragment,
It was subsequently assigned to a 0.2 kb region delimited by PstI and SalI restriction sites (FIG. 3).

3. MPTA resistance gene from the MPTA encoding lycopene cyclase to inhibit cyclization of lycopene, the present inventors have, MPTA resistance in strains M ^r -5 is a due changes in lycopene cyclase, the said mutation We thought that we would arrive at the gene of this enzyme by examining the position. Therefore, we tested the presence of this gene by expressing a blue-green bacterial genomic fragment containing the mutation in E. coli that produces lycopene. The ¹⁸ constructed plasmid was used as described previously. This is due to the enzymes GGPP synthase (crtE), phytoene synthase (crtB) and phytoene desaturase (c
rtI) containing the gene from the bacterium Elewinia uredbora. E. coli cells carrying this plasmid accumulate lycopene (Fig. 6) and produce pink colonies on agar plates.

The plasmid pLCYB-M5XE (FIG. 5) containing the 7.2 kb genomic insert was introduced into E. coli cells already containing pCAR-ADE. Colonies and cultures of cells containing both plasmids in the presence or absence of IPTG were yellow and contained β-carotene in addition to lycopene as evidenced by HPLC analysis (FIG. 6). The smallest is the vector pTrcHis
1.5 kb in B (plasmid pLCYB-M5PPF)
Several other genomic fragments containing the mutation, PstI-PstI fragment of E. coli, gave results similar to those shown in Figure 6 (see also Figure 4). pTrcHis
Cloned in all three frames of the vector 1.
Enzyme activity was observed with the 5 kb PstI fragment, which is I
It was not dependent on PTG induction. No activity was observed when the fragment was cloned in the reverse orientation. It was concluded that the binding of E. coli DNA polymerase to the promoter of the vector promotes the recognition and utilization of the blue-green bacterial promoter and the authentic blue-green bacterial enzyme rather than the fusion protein. The truncated form of pLCYB-M5PPF lacking only the 0.2 kb PstI-SalI portion containing the MPTA resistance mutation did not maintain lycopene cyclase activity in E. coli cells. DN
A sequence analysis revealed a single open reading frame that perfectly matched the assigned position of lycopene cyclase activity (FIG. 7). The present inventors
It was concluded that a putative gene, designated cy, encodes the enzyme lycopene cyclase, and that this single gene product is sufficient to catalyze the two-end cyclization step necessary to produce β-carotene from lycopene.

4. Lycopene cyclase is a target site for MPTA inhibition. To test the interaction between MPTA and lycopene cyclase activity, 10 suspension cultures of transformed E. coli cells were tested.
Grow in the presence of 0 μM MPTA. The HPLC analysis of carotenoids shown in FIG. 6 shows that these cells accumulate considerable amounts of monocyclic γ-carotene in addition to lycopene and β-carotene, but γ-carotene is not detected in the absence of MPTA. It was We conclude that MPTA acts directly as an inhibitor of lycopene cyclase.

5. Identification of mutations for MPTA resistance (Fig. 7) DNA in the region conferring lycopene cyclase activity
Sequence analysis (Fig. 4) revealed a single open reading frame, lcy. This open reading frame has a predicted molecular weight of 46,093g
The size encodes a protein of 411 amino acids with / mol (SEQ ID NO: 2). Mutant 5 is M
104 from the position considered to be the start codon of the gene in the promoter region of lcy that confers resistance to PTA
It differs from its wild type at one site upstream of base pair (position 1925 of SEQ ID NO: 1). The mutation does not change the amino acid sequence of the protein encoded by open reading frame 2 (ORF2). Conversion to T in M ^r -5 of C in the wild-type results in a consensus sequence or Pribnow box (TATAAT) by 6 base sequences approximating about -10 E. coli promoter region (TACAAT). This T residue at position 6 of the E. coli consensus promoter is highly conserved (96%), while the residue at position 3 is the least conserved base (44).
%) ⁵⁰ . Mutations to T at ^Mr- 5 are believed to promote DNA strand separation, thereby enhancing mRNA production. This is the most relevant explanation for the significant increase in MPTA resistance of mutant 5 due to the overproduction of this message, and thus of the enzyme itself. This mutant is considered to be resistant to all other herbicides that act by interfering with lycopene cyclase activity.

A search of the Genbank and Swissprot DNA and protein sequence data banks found no genes or proteins with obvious homology to lcy. The only similarity between the deduced amino acid sequence of the protein encoded by lcy and other known or reported proteins lies at the amino terminus of LCY. The nucleotide binding motif (SEQ ID NO: 3) is present at this location in LCY, indicating that this enzyme binds to and utilizes the cofactor FAD (FIG. 8). By using the blue-green bacterium Synechococcus PCC7942 as a model organism for the study of carotenoid biosynthetic pathways, from this genetically simple and convenient organism all oxygen-producing photosynthetic organisms (plants, algae and blue-green bacteria)
The findings have been widely applied to That is, once the carotenoid biosynthesis gene is identified in Synechococcus, it is possible to isolate the homologous gene from any plant or algae. This was evidenced by the inventors using the phytoene desaturase Synecococcus gene to clone homologous genes from plants and algae. If the plant or algae gene has not been identified, the initial identification of our blue-green bacterial gene allows the necessary and sufficient means to allow identification and cloning of the plant and algae gene of phytoene desaturase. Was provided. Also in the present invention, identification of the lycopene cyclase gene from Synechococcus PCC7942 provided a necessary and sufficient means for identifying and cloning homologous algae and plant genes. Like the earlier pds and psy genes, the Synechococcus lycopene cyclase gene has two genes from the genus Eleunia.
It has little similarity to known bacterial genes of the species and to any other gene in the major databases (Genbank and Swissprot).

It is now possible to modify the natural genes of plants and algae. For example, the results obtained with the blue-green bacterial gene can locate the site of a herbicide resistance mutation, which can be used to determine where to alter the natural gene to make it herbicide resistant. For any newly emerged herbicide, resistant mutants are selected to determine what modification of the enzyme's amino acid sequence or regulatory DNA sequence can confer resistance. Similarly, the native enzyme or regulatory sequence of any plant can be altered to create similar mutants. in addition,
The expression of the gene can be adjusted and manipulated in a tissue-specific manner, and it is also possible to use antisense technology with natural genes. While the invention has been described with examples,
The terminology used is for the purpose of description rather than limitation of the invention. Obviously, it is possible to modify and change the present invention based on the above technique.
Therefore, within the scope of each claim described in the claims,
It should be understood that the invention may be practiced otherwise than as specifically described.

References 1. Koyama, Y., (1991) Photochem. Photobi
ol B 9: 265-280 2. Siefermann-Harms,
D.), (1987) Physiol. Plant., 69: 265-280 3. Demmig-Adams, B. et al., (199
2) Ann. Rev. Plant Physiol. Plant Mol. Biol., 43:
599-626 4. Zeevaart, J. et al., (1988) Ann. Re
v. Plant Physiol. Plant Mol. Biol., 39: 439-473 5. Rock (Rock, CD) et al., (1991) Proc. Natl. Aca
d. Sci. USA, 88: 7496-7499 6. Mathews-Roth, M. et al., (1991) O
ncology, 48: 177-179 7. Parozza, P. et al., (1992) Methods Enz
ymol., 213: 403-420 8. Britton, G. (1988) Plant Pigments
(T. Goodwin), Academic Press, N
Y) pp. 133-180 9. Dogbo, O. et al., (1988) Proc. Natl. Acad.
Sci. USA, 85: 7054-7058 10. Chamovitz, D. et al., (1992) FEBS Let.
t., 296: 305-310 11. Bartley (Bartley, GE) et al., (1992) J. Bio.
L. Chem., 267: 5036-5039 12. Bramley, P. et al., (1992) Plant J.,
2: 343-349 13. Linden, H. et al., (1991) Z. Naturforsc
h., 46c: 1045-1051 14. Pecker, I. et al., (1992) Proc. Natl. A
cad. Sci. USA, 89: 4962-4966 15. Hugueney, P. et al., (1992) Eur. J.
Biochem., 209: 399-407 16. Bramley, PM, (1985) Adv. Lipid
Res., 21: 243-279 17. Armstrong (GA) et al., (1989).
Mol. Gen. Genet., 216: 254: 268 18. Misawa, N. et al., (1990) J. Bacteriol.,
172: 6704-6712 19. Hundle, BS et al., (1993) FEBS Lett.,
315: 329-334 20. Ray (JA) et al., (1987) Nucleic Acids Re
s., 15: 10587-10588 21. Kamovitz et al., (1991) Plant Mol. Biol., 16: 967.
-974 22. Pecker et al., (1993) Research in Photosynthesis,
Vol III (N. Murata), Kluwer Academic Pu
blishers, Dordrectht) pp. 11-18 23. Bartley et al., (1991) Proc. Natl. Acad. Sci. US
A, 88: 6532-6536 24. Sandmann, G. et al., (1989) Z. Naturf.
orsch., 44c: 787-790 25. Kamovitz et al., (1990) Z. Naturforsch., 45c: 482.
-486 26. Sandman et al., (1989) Target sites of herbicide.
action (Boger and Sandman) CRC Press,
Florida, pp. 25-44 27. Yokoyama, H. et al., (1982) Carotenoid
Chem. And Biochem. (Britton and Goodwin)
Pergamon Press, Oxford, pp. 371-385 28. Cunningham, Jr. (Cunningham, FX, J
r.), (1985) "Carotenoid of Euglena gracilis Klebs v
ar bacillaris Cori: photoisomerization, biosynthes
is localization and assoc. with cholorphyllis ... ",
(Ph. D. Thesis, Brandeis Univ.) Chap. 4, pp. 5-17 29. Beaversdorf, W. D. et al., (1980).
Crop Science, 20: 289 30. Bucholtz, ML et al., (1977) Chem. Bio.
l. Interactions, 17: 359-362 31. Beyer, P. et al., (1980) Planta, 150: 43.
5-438 32. Camara, B. et al., (1982) Physiol. Veg. 2
0: 757-773 33. Fosket, DE, et al., (1983) Plant S
ci. Lett., 30: 165-175 34. Shu (Hsu, WJ) et al., (1972) Phytochemistry, 1
1: 2985-2990 35. Bayer, (1989) Physiology, Biochemistry and
Genetics of Nongreen Plastids (Boyer,
Shannon and Hardison)
American Society of Plant Physiology, pp. 157-170 36. Linden et al., (1993) FEMS Microbiol. Lett., 106:
99-104 37. Bird, CR et al., (1991) Biotechnology,
9: 635-639.37.22 38. Hali, LN, et al., (1993) Plant J., 3: 121-
129 39. Hirschberg, J. et al., (1987) Z.
Naturforsch., 42c: 102-112 40. Sambrook, JF et al., (1989) Molec.
ular Cloning: A Lab. Manual, 2nd, Cold Spring Lab.
Press, Cold Spring Harbor, pp. 316-317, 326-328 41. Williams (J.), (1988) Methods En
zymol., 167: 766-778 42. Feinberg, AP, et al., (1983) Ana.
L. Biochem., 132: 6:13 43. Del Sal, G. et al., (1988) Nucleic Aci.
ds Res., 16: 9878 44. Hundle et al., (1991) Photochem. Photobiol 54: 89.
093 45. Misawa et al., (1991) Appl. Environ. Microbiol., 5
7: 1847-1849 46. Sandman et al., (1990) FEMS Microbiol. Lett., 7
1: 77-82 47. Schnurr G. et al., (1991) FEMS Microb.
iol. Lett., 78: 157-162 48. Williams, (1988) Methods Enzymol., 167: 76
6-778 49. Golden, SS, (1988) Methods Enz
ymol., 167: 714-7277 50. Hawley et al., (1983) Nucleic Acids Re
s., 11: 2244

[Sequence list]

SEQ ID NO: 1 Sequence length: 4928 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Genomic DNA Sequence features Characteristic symbols: CDS Location: 2029. ． 3261 array

[0034]

[Table 1]

[0035]

[Table 2]

[0036]

[Table 3]

[0037]

[Table 4]

SEQ ID NO: 2 Sequence length: 411 Sequence type: Amino acid Topology: Linear Sequence type: Protein sequence

[0039]

[Table 5]

[0040]

[Table 6]

SEQ ID NO: 3 Sequence length: 29 Sequence type: Amino acid Number of chains: Single strand Topology: Linear sequence

[0042]

[Table 7]

[0043]

[Brief description of drawings]

FIG. 1 is a schematic diagram of the carotenoid biosynthetic pathway in blue-green bacteria and plants. IPP = isopentenyl pyrophosphate; GPP = geranyl pyrophosphate; FPP = farnesyl pyrophosphate; GGPP = geranylgeranyl pyrophosphate; PPPP = prephytoene pyrophosphate.

FIG. 2: Blue-green bacterium Synechococcus sp. PCC794
Photo of Petri dishes showing MPTA resistance in two mutants (numbered) and two wild type strains (C and S). The wild type strain designated by C is the parent strain of the mutant. MP in micromolar on the upper right of each Petri dish
The TA concentration is shown. Each strain was spotted in several places on each petri dish to minimize positional effects on survival.

FIG. 3 is a schematic diagram showing a gene map showing mutation sites of MPTA ^r- 5 (mutant 5). 4.6 kb E
Various small fragments of the coRI-BamHI genomic fragment (shown in the restriction map below) were transformed into the vector pBluescr.
cloned into iptKS ⁺ and cloned into Synechococcus sp.
Their ability to confer herbicide resistance by transfecting cells of the wild-type strain of PCC7942 and plating the transformants on MPTA-containing medium (indicated by +)
It was confirmed. B = BamHI; C = ClaI; E = Ec
oRI; K = KpnI; H = HindIII; P = Pst
I; S = SalI; X = XbaI.

FIG. 4 is a schematic diagram showing a gene map showing that lycopene cyclase activity is in a region containing MPTA resistance damage. Each symbol is the same as in FIG.

FIG. 5: Plasmids pLCYB-M5XE and pLCY
FIG. 3 is a schematic diagram of the B-M5PPF structure. The putative position of the lcy gene within mutant 5 was identified based on expression experiments.

FIG. 6: Lycopene-accumulating strain of E. coli (panel A), lycopene-accumulating strain of E. coli and pLCYB-M5XE (panel B), lycopene-accumulating strain of E. coli in the presence of MPTA and p.
Graph showing HPLC analysis of carotenoid dye extracted from cells of LCYB-M5XE (panel C). 100m
3 shows the effect of treatment of E. coli cells with M MPTA. The lower panels show the absorption spectra of peaks 1, 3 and 5. (─) Peak 1 is lycopene; (・ ・ ・ ・)
Peak 3 is γ-carotene; (-) peak is β-carotene.

FIG. 7: Schematic showing the identification of mutations in the lycopene cyclase gene for MPTA resistance.

FIG. 8 is a schematic diagram showing a dinucleotide binding motif at the amino terminus of LCY showing that FAD is utilized by lycopene cyclase. a is an acidic amino acid, ie D or E, p is a polar or charged amino acid, ie D, E, K, R, H, S, T, Q, N, s is a small or hydrophobic amino acid, That is, A,
I, L, V, M, C, and the asterisk is any amino acid.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C12N 5/10 // C12N 9/04 9359-4B (C12N 15/09 ZNA C12R 1:01) ( (C12N 1/21 C12R 1:19) (C12N 1/21 C12R 1:01) (C12N 9/04 C12R 1:01) (C12N 9/04 C12R 1:19) (C12N 15/00 ZNA A C12R 1:01 ) (72) Inventor Joseph Hirschberg Israel Jerusalem 93714 Burla Street 26 (72) Inventor Francis Xavier Cunningham Zhenia United States Maryland 20815 Chevi Chess Washington Avenue 2727 (72) Inventor Elizabeth Bant United States Maryland 20814 Bethesda Bellevueー 9603

Claims (67)

[Claims] 1. A DNA sequence (SEQ ID NO: 1, bp20) encoding a gene for lycopene cyclase called lcy.
29-3261), a purified, isolated and cloned DNA sequence consisting essentially of 2. A purified, isolated and cloned DNA sequence which comprises a modified DNA sequence encoding the gene for lycopene cyclase which is resistant to herbicides. 3. A modified DNA sequence according to claim 2, characterized in that said resistance comprises resistance to bleaching herbicides of the trialkylamine family including CPTA and MPTA. 4. A transformed Escherichia coli carrying a DNA sequence for lycopene cyclase. 5. A transformed Escherichia coli carrying a DNA sequence for lycopene cyclase which is resistant to herbicides. 6. The transformed E. coli according to claim 5, wherein the resistance comprises resistance to a bleaching herbicide of the trialkylamine family including CPTA and MPTA. 7. A vector containing the DNA according to claim 1. 8. A host cell transformed with the vector of claim 7, wherein the host cell is selected from the group of suitable eukaryotic cells and prokaryotic cells. 9. The host cell according to claim 8, which is Escherichia coli. 10. The host cell according to claim 8, which is a blue-green bacterium. 11. The host cell of claim 10, which is selected from the group comprising Synechococcus PCC7942 and Synechocystis PCC6803. 12. The host cell according to claim 8, which is an alga. 13. The host cell according to claim 8, which is a plant cell. 14. A vector containing the DNA according to claim 2. 15. A host cell transformed with the vector of claim 14 which is selected from the group of suitable eukaryotic cells and prokaryotic cells. 16. The host cell according to claim 15, which is Escherichia coli. 17. The host cell according to claim 15, which is a blue-green bacterium. 18. The host cell of claim 17, which is selected from the group comprising Synechococcus PCC7942 and Synechocystis PCC6803. 19. The host cell according to claim 15, which is an alga. 20. The host cell according to claim 15, which is a plant cell. 21. A vector containing the DNA according to claim 3. 22. A host cell transformed with the vector of claim 21, wherein the host cell is selected from the group of suitable eukaryotic cells and prokaryotic cells. 23. The host cell according to claim 22, which is Escherichia coli. 24. The host cell according to claim 22, which is a blue-green bacterium. 25. The host cell of claim 24, which is selected from the group comprising Synechococcus PCC7942 and Synechocystis PCC6803. 26. The host cell of claim 22, which is an alga. 27. The host cell according to claim 22, which is a plant cell. 28. Geranyl pyrophosphate derived from geranylgeranyl pyrophosphate
A method for expressing a carotene pathway by incorporating a gene encoding an enzyme that controls the biosynthetic pathway into a suitable host cell. 29. Incorporating a DNA sequence encoding a gene for phytoene synthase designated psi; pd
the step of incorporating a DNA sequence encoding the gene for phytoene desaturase, designated s; the step of incorporating the DNA sequence encoding the gene for zeta-carotene desaturase, designated zds; and the DNA encoding the gene for lycopene cyclase, designated lcy. 29. The method of claim 28, including the step of incorporating the sequence. 30. Incorporating a DNA sequence encoding a gene for phytoene synthase designated psi; cr
incorporating a DNA sequence encoding a gene for phytoene desaturase in Erewinia, designated tI;
29. The method according to claim 28, characterized in that it comprises the step of incorporating a DNA sequence coding for the gene for lycopene cyclase designated as 1 and lcy. 31. The method of claim 28, wherein the suitable host cell is E. coli. 32. An lc encoding LCY polypeptide.
The purified, isolated and cloned DNA sequence of SEQ ID NO: 1, including the nucleotide sequence of y and its flanking DNA sequence. 33. The DNA sequence according to claim 32, characterized in that the resulting sequence is point-mutated, deleted or inserted so as to impart herbicide resistance. 34. A point mutation, a deletion or an insertion is made in the nucleotide sequence of lcy so that the resulting sequence can confer herbicide resistance.
DNA sequence according to 3. 35. A point mutation, deletion or insertion is made in the flanking sequence of lcy so that the regulation of lcy can confer herbicide resistance.
DNA sequence according to 3. 36. The DNA sequence according to claim 35, wherein C is converted to T at base number 1925 of SEQ ID NO: 1 in the sequence. 37. DN according to claim 32, characterized in that it is a homologous sequence encoding an LCY polypeptide.
A array. 38. The DNA sequence according to claim 37, characterized in that the obtained sequence is point-mutated, deleted or inserted so as to impart herbicide resistance. 39. An effective fragment of a sequence including the point mutation, deletion or insertion, 33.
The described DNA sequence. 40. A vector containing the DNA according to claim 32. 41. A host cell transformed with the vector of claim 40, which is selected from the group of suitable eukaryotic cells and prokaryotic cells. 42. The host cell of claim 41, which is E. coli. 43. The host cell of claim 41, which is a blue-green bacterium. 44. The host cell of claim 41, selected from the group comprising Synechococcus PCC7942 and Synechocystis PCC6803. 45. The host cell of claim 41, which is an alga. 46. The host cell of claim 41, which is a plant cell. 47. A vector containing the DNA according to claim 33. 48. A host cell transformed with the vector of claim 47, which host cell is selected from the group of suitable eukaryotic cells and prokaryotic cells. 49. The host cell of claim 48, which is E. coli. 50. The host cell of claim 48, which is a blue-green bacterium. 51. The host cell of claim 48, which is selected from the group comprising Synechococcus PCC7942 and Synechocystis PCC6803. 52. The host cell of claim 48, which is an alga. 53. The host cell according to claim 48, which is a plant cell. 54. A transgenic organism in which antisense expression of the lcy gene is introduced into genomic DNA, thereby inhibiting the synthesis of lycopene cyclase and accumulating the red pigment lycopene. 55. A transgenic organism in which the lcy gene is introduced into genomic DNA, whereby lycopene cyclase is overproduced and β-carotene is excess. 56. The DNA sequence according to claim 2 is Genome D.
A transgenic organism introduced into NA and thereby becoming a herbicide-resistant organism. 57. A transgenic organism in which the DNA sequence according to claim 33 is introduced into genomic DNA, whereby a herbicide-resistant organism is obtained. 58. The DNA sequence according to claim 3 is genome D
Introduced in NA, thereby CPTA and MPTA
A transgenic organism that has become resistant to a bleaching herbicide of the trialkylamine family containing. 59. A transgenic organism in which the DNA sequence according to claim 35 is introduced into genomic DNA, whereby a herbicide-resistant organism is obtained. 60. A transgenic organism into which the DNA sequence according to claim 36 has been introduced into genomic DNA, thereby making the herbicide resistant organism. 61. An lc encoding LCY polypeptide.
preparing a suitable vector of the DNA sequence set forth in SEQ ID NO: 1, including the nucleotide sequence of y and its flanking DNA sequence; and inserting said vector into bacterial, algae and plant cells, thereby transgenic A method of altering carotenoid biosynthesis, content or composition within bacteria, algae, and plants by forming an organism. 62. The method according to claim 61, characterized in that a point mutation, a deletion or an insertion is made in the DNA sequence so that the obtained sequence can confer herbicide resistance. 63. The DNA sequence consisting of a modified DNA sequence for the vector encoding a gene for lycopene cyclase that is resistant to herbicides.
The method described in 1. 64. The method of claim 61, wherein said vector comprises antisense expression of the lcy gene. 65. The method according to claim 61, wherein the vector comprises the lcy gene and its promoter or a modified promoter. 66. A plasmid designated as pLYCB-M5XE. 67. Screening blue-green bacteria for herbicide resistant clones; purifying and isolating denatured DNA sequences that control the production of herbicide resistant lycopene cyclase enzyme; required for the production of said herbicide resistant enzyme. The DNA
Determining sequence modifications; and natural plant or algae DN
A method of constructing a plant or algae that is resistant to the action of a herbicide that interacts with the lycopene cyclase enzyme by denaturing the plant or algae to produce a similar modification in the A sequence.