JPH10327865A - Carotenoid glycoside and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce a new carotenoid glycoside, comprising astaxanthin diglucoside and adonixanthin 3'-glucoside, having a high polarity and a high solubility in water and useful as a color tone improving agent for cultured fishes and shellfishes, a food additive, etc. SOLUTION: This new carotenoid glycoside comprises astaxanthin diglucoside represented by a chemical structural formula of formula I and adonixanthin 3'-glucoside represented by a chemical structural formula of formula II, has a high polarity and a high solubility in water and is useful as a color tone improving agent for cultured fishes and shellfishes, a food additive, etc. The compounds are obtained by transferring all or a part of carotenoid biosynthesis genes crtE, crtB, crtI, crtY, crtZ, crtX and crtW into a microorganism, making the seven genes present therein so as to enable the expression, culturing the resultant transformed microorganism in a culture medium and collecting the prepared product from the resultant cultured product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[Background of the Invention]

The present invention relates to novel carotenoid glycosides such as novel astaxanthin diglucoside and adonixanthin-3'-glucoside, and a process for producing these novel carotenoid glycosides and known astaxanthin monoglucoside. It is about their use. These pigments have high solubility in water and are useful as color improvers for cultured fish and shellfish, food additives and the like.

[0002]

[0002] Carotenoids (carotenoids) such as β-carotene (beta-carotene) are widely distributed in various animals including microorganisms and plants. Also,
Astaxanthin, a typical carotenoid contained in animals, is known as a red carotenoid distributed on the surface of shrimp, crab, red sea bream, and muscle of salmon, and is used as a color improver and deepener for cultured fish and shellfish. ing. In addition, astaxanthin has recently been reported to have an antioxidant action (Nobuto Shimizu, Wataru Watari, Carotenoids of marine organisms, Wataru Watari, Koseisha Koseikaku, 1993), and is expected to be applied to food additives, etc. ing. However, astaxanthin is a fat-soluble substance like other carotenoid pigments, and thus causes problems in solubility such as solubility in a water-soluble solvent and addition to a water-soluble compound.

[Summary of the Invention]

Therefore, it is desired to develop a novel high-polar carotenoid, particularly a high-polar carotenoid having the same basic skeleton as astaxanthin. In view of the above circumstances, an object of the present invention is to search for a novel highly polar carotenoid, and to provide a novel substance thereof, a production method thereof, and a use thereof.

[0004]

Therefore, the present inventors focused on carotenoid glycosides, combined carotenoid biosynthesis genes, introduced them into microorganisms such as bacteria, and searched for cultures. A novel carotenoid glycoside was found, and the present invention was completed based on this finding. That is, the carotenoid glycosides according to the present invention are astaxanthin diglucoside and adonixanthin-3'-glucoside, which are novel compounds represented by the following chemical structural formulas (1) and (2), respectively.

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The present invention also provides a method for producing the above-mentioned novel carotenoid glycoside and a method for producing astaxanthin monoglucoside [chemical formula (3) shown below] which is a known substance. That is, the method for producing a carotenoid glycoside according to the present invention comprises a carotenoid biosynthesis gene crtE , crtB , crt
All or part of I , crtY , crtZ , crtX , and crtW are introduced into a microorganism to allow these seven genes to be expressed, and the transformed microorganism is cultured in a medium, and astaxanthin diglucoside is cultured from the culture. Adonixanthin-3 '
-At least one of glucoside and astaxanthin monoglucoside is collected.

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[0006] The present invention also relates to a food use of the carotenoid and a method for producing a plant producing the carotenoid. That is, the present invention relating to food applications comprises a food improving agent comprising at least one of astaxanthin diglucoside, adonixanthin-3'-glucoside and astaxanthin monoglucoside (excluding the case of astaxanthin monoglucoside alone). Or food.

Further, the method for producing a plant having a carotenoid glycoside according to the present invention provides a method for producing a carotenoid biosynthesis gene crt.
X , crtW , crtZ , crtE , crtB , crtI , crtY are partially or entirely introduced into a plant so that these seven genes can be expressed so that the transformed plant is cultivated, and the astaxanthin diglucoside is cultivated in the cultivation. , Adonixanthin-3'-glucoside and astaxanthin monoglucoside.

[Specific description of the invention]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below in detail. Carotenoid glycoside As described above, the novel carotenoid glycoside according to the present invention is astaxanthin diglucoside (chemical structural formula (1)) and adonixanthin-3'-glucoside (chemical structural formula (2)). And have the following physicochemical properties.

<Physicochemical properties of carotenoid glycoside> Physicochemical properties of astaxanthin diglucoside Astaxanthin diglucoside [Chemical Structural Formula (1)
Of the compound]. (1) Color of substance: red powder (2) Molecular weight: 920 (3) Molecular formula: C ₅₂ H ₇₂ O ₁₄ (4) Mass spectrometry: high resolution FABMS Theoretical value: 921.5000 (M + H) ⁺ actual value: 921.4992 ( 5) Visible absorption spectrum (measured in chloroform: methanol = 2: 1): λmax 492 nm (6) ¹ H-NMR (measured in heavy chloroform: methanol = 2: 1, 400 MHz) δppm (hydrogen number) , Multiplicity, coupling constant): 1.19 (6H, s),
1.31 (6H, s), 1.86 (6H, s), 1.94 (2H, m), 1.95 (6
H, s), 1.97 (6H, s), 2.18 (2H, dd, 5,13), 3.27 (2
H, m), 3.36 (2H, t, 9), 3.42 (2H, t, 9), 3.43 (2H,
t, 9), 3.70 (2H, dd, 5,12), 3.83 (2H, dd, 3,12),
4.49 (2H, d, 8), 6.18-6.67 (14H, m) (7) Solubility: Easily soluble in aqueous methanol, methanol and ethanol. Poorly soluble in chloroform, acetone, ethyl acetate and hexane.

[0010] 2. Physicochemical properties of adonixanthin-3'-glucoside The physicochemical properties of adonixanthin-3'-glucoside [compound of the chemical structural formula (2)] are shown below. (1) Color of substance: red powder (2) Molecular weight: 744 (3) Molecular formula: C ₄₆ H ₆₄ O ₈ (4) Mass spectrometry: high resolution FABMS Theoretical value: 745.4679 (M + H) + Actual value: 745.4677 ( 5) Visible absorption spectrum (measured in chloroform: methanol = 2: 1): λmax 487 nm (6) ¹ H-NMR (measured in heavy chloroform: methanol = 2: 1, 400 MHz) δppm (hydrogen number) , Multiplicity, coupling constant): 1.03 (6H, s),
1.18 (3H, s), 1.29 (3H, s), 1.52 (1H, t, 12), 1.6
9 (3H, s), 1.79 (1H, m), 1.83 (1H, m), 1.87 (3H,
s), 1.92 (3H, s), 1.94 (6H, s), 1.96 (3H, s), 2.06
(1H, m), 2.08 (1H, m), 2.42 (1H, dd, 4,17), 3.19
(1H, m), 3.27 (1H, m), 3.38 (2H, m), 3.72 (1H, dd,
5,12), 3.83 (1H, dd, 3,12), 4.05 (1H, m), 4.28 (1
H, dd, 6,14), 4.42 (1H, d, 8), 6.05-6.70 (14H, m) (7) Solubility: Easily soluble in aqueous methanol, methanol and ethanol. Poorly soluble in chloroform, acetone, ethyl acetate and hexane.

The carotenoid glycoside according to the present invention typically means each isolate of the above-mentioned novel carotenoid glycosides, and the two carotenoid glycosides and the known astaxanthin monoglucoside (chemical structure Equation (3) 2
Also included are species or mixtures of three.

Process for producing carotenoid glycoside and method for producing the same
Method for Producing Producing Plant The carotenoid glycoside according to the present invention can be produced chemically or genetically by any suitable method, but appropriately combines genes involved in carotenoid biosynthesis. Thus, it can be mass-produced by genetic engineering.

The method for producing a carotenoid glycoside according to the present invention comprises the steps of carotenoid biosynthesis genes crtE , crtB , crtI , crt
All or part of Y , crtZ , crtX , and crtW are introduced into a microorganism so that these seven genes can be expressed, and the transformed microorganism is cultured in a medium, and astaxanthin diglucoside, Nixanthin-3'-
As described above, at least one of glucoside and astaxanthin monoglucoside is collected.

Further, in the present invention, the carotenoid biosynthesis genes crtX , crtW , crtZ , crtE , crtB , crtI , crtY
Is introduced into a plant so that these seven genes can be expressed, and the transformed plant is cultivated. Astaxanthin diglucoside, adonixanthin-3'-glucoside and astaxanthin monoglucoside are cultivated in the cultivation. By producing at least one kind of the above, a plant having the carotenoid glycoside can be produced. Here, "all or part ... 7
The expression of a gene of a species is referred to as "if the host already has some of the above seven genes (or genes having the same functions as those described above), as described below. This means that seven types of genes, including the retained gene and the supplementary transgene, are present in an expressible state.

It was unexpectedly possible to obtain the carotenoid glycoside of the present invention with high polarity by using the above-mentioned gene combination.

<Genes Required for the Synthesis of Carotenoid Glycosides and Methods for Producing the Same> Astaxanthin diglucoside, adonixanthin-3'-glucoside,
And a group of genes required for the synthesis of astaxanthin monoglucoside, and examples of their production methods. (1) Astaxanthin diglucoside (and astaxanthin monoglucoside) A gene group necessary for producing astaxanthin diglucoside (and astaxanthin monoglucoside) and preferable examples of the production method will be described below. Plant resident (ep
iphytic) crtE , crtB , crtI , cr , carotenoid biosynthesis genes of bacteria Erwinia (e.g., Erwinia uredovora )
tY, crtZ, crtX gene, and, carotenoid biosynthesis genes in which crtW gene appropriate vector marine bacteria which produce astaxanthin (e.g. Agrobacterium aurantiacum) (specific examples are described below), bacteria such as E. coli, Sacc
By introducing and expressing in a microorganism such as yeast such as haromyces cerevisiae or Candida utilis , preferably a bacterium such as Escherichia coli, astaxanthin diglucoside and astaxanthin monoglucoside can be mainly obtained. The target carotenoid glycoside can be isolated and purified by a usual method for separating a microbial metabolite from a culture as described below. However, depending on the host microorganism, for example, in yeast and the like, marine bacteria may be used instead of Erwinia 's crtZ due to differences in expression levels.
crtZ may be used. In addition, cr tY may be derived from marine bacteria.

[0017] In addition, the marine bacteria crtB, crtI, crtY, crt
Since they have the Z and crtW genes, these five genes derived from marine bacteria can be used together with the crtE and crtX genes of Erwinia . In the present invention, the above seven genes i.e. crtE, cr tB, crtI, crtY , crtZ,
It is sufficient that crtX and crtW are expressed in combination. Therefore, the origin of each gene is Flavobacterium sp.
1534 (Luis Pasamontes et al., Isolation and Charact
erization of the carotenoid biosynthesis genes of
Flavobacterium sp.strain R1534 Gene, 185, 35-41, 1
997), and if necessary, the above 7
Species genes can also be distributed and introduced and expressed in multiple hosts (homogeneous or heterologous).

The structures and functions of the genes of Erwinia and marine bacteria, the biosynthetic pathway, and the like are described in detail in the following four documents, for example, and also in Example 1. In brief the function of each crt gene used in the present invention, crtE gene is one that encodes a protein having geranylgeranyl pyrophosphate synthase activity, cr
The tB gene encodes a protein having phytoene desaturase activity, the crtI gene encodes a protein having phytoene desaturase activity, the crtY gene encodes a protein having lycopene cyclase activity, and crtZ Is 3,
(3 ') - are those that encode a protein having a β- ionone ring hydroxylase activity, crtX is one that encodes a protein having a peptidase Aki Kang Chin glucosyltransferase activity, cr tW is 4, (4')
It encodes -β-ionone ring oxygenase. In the present invention, by using the above crt gene in combination, astaxanthin monoglucoside and astaxanthin diglucoside through astaxanthin or the like as an intermediate, and further adonixanthin-3′-glucoside using adonixanthin or the like as an intermediate Is converted to・ Norihiko Misawa, Carotenoid biosynthetic pathway elucidated at the gene level, Protein nucleic acid enzyme, 41, 337-346, 1996 ( Er
(Carotenoid biosynthesis) genes of winia and marine bacteria) ・ Norihiko Misawa et al., Elucidation of the Erwin
ia uredovora carotenoid biosynthetic pathway by fu
nctional analysis of gene products expressed in Esc
herichia coli.J. Bacteriol., 172, 6704-6712, 1990
( Erwinia gene) ・ Norihiko Misawa et al., Structure and functional
analysis of a marinebacterial carotenoid biosynth
esis gene cluster and astaxanthin biosynthetic pat
hway proposed at the gene level.J. Bacteriol., 17
7, 6575-6584, 1995 (marine bacterial genes) ・ Paul D. Fraser, Y. Miura, N. Misawa, In vitro ch
aracterization of astaxanthin biosynthetic enzyme
s. J. Biol. Chem., 272, 6128-6135, 1997 (Erwi nia
Of crtZ and of marine bacteria crtZ, crtW gene)

On the other hand, when it is desired to produce carotenoid glycosides such as astaxanthin diglucoside in organisms (microorganisms, plants, etc.) that originally produce carotenoids,
Needless to say, it is not necessary to introduce all seven genes starting from the crtE gene (it is only necessary to supplement the missing gene in the host). For example, the yeast Phaffia rhod
In the case of ozyma , since up to astaxanthin is produced, introduction and expression of only crtX will be sufficient. A mutant strain of Phaffia rhodozyma that accumulates β-carotene has also been isolated (P. Girard et al., Β-carotene produc).
ing mutants of Phaffia rhodozyma.Appl.Microbiol.
Biorechnol, 41, 183-191, 1994). When it is desired to produce a carotenoid glycoside such as astaxanthin diglucoside in this mutant strain, three genes of crtZ , crtX and crtW may be introduced.

Plants can also naturally synthesize carotenoids. For example, tomato fruits produce large amounts of lycopene and small amounts of β-carotene, carrots produce large amounts of β-carotene, and corn seeds produce large amounts of zeaxanthin. Therefore, when it is desired to produce carotenoid glycosides such as astaxanthin diglucoside in these tissues, at least two genes, crtX and crtW , are required, but in addition to these, a lacking crt gene is introduced and expressed. It should be done.

The genes used in the present invention are those having different origins.
In addition to the crt gene, it may be a gene having the same function (encoding a mutant enzyme having the same activity). Representative examples of the sequences of these crt genes are described in the above-mentioned documents. To be specific, crtE is amino acid numbers 1 to 302 of SEQ ID NO: 7 of the present specification, and crtB is amino acid number 1 of SEQ ID NO: 6. -296, crtI is the amino acid number 1-4 of SEQ ID NO: 8
92, crtY is amino acid numbers 1 to 382 of SEQ ID NO: 10 or 1 to 386 of SEQ ID NO: 3, crtZ is amino acid numbers 1 to 162 of SEQ ID NO: 2 or 5, or amino acid numbers 1 to 175 of SEQ ID NO: 11, and crtX is SEQ ID NO: 9, amino acid numbers 1 to 431, and crtW are amino acid numbers 1 to 21 of SEQ ID NO: 1.
2 or a protein having an amino acid sequence substantially identical to amino acid Nos. 1-242 of SEQ ID NO: 4 (for example, an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted or added) And a gene encoding a protein having the same enzymatic activity).

The above-mentioned specific crt gene has been deposited in the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology as described below.

(2) Adonixanthin-3'-glucoside Next, the genes required for producing adonixanthin-3'-glucoside and preferred examples of the production method will be described. Plant-resident bacteria Erwinia (for example, Erwinia uredovor
a ) the carotenoid biosynthesis genes crtE , crtB , crt
The I , crtY , crtX genes and crtZ , crtW genes, which are carotenoid biosynthesis genes of marine bacteria (for example, Agrobacterium aurantiacum ) that produce astaxanthin, are transformed into bacteria such as Escherichia coli and Saccharomyces cerevisiae using appropriate vectors (specific examples are described later). Alternatively, adonixanthin- 3'-glucoside can be mainly obtained by introducing into a microorganism such as yeast such as Candida utilis or the like, preferably a bacterium such as Escherichia coli and expressing it. The target carotenoid glycoside can be isolated and purified by a usual method for separating a microbial metabolite from a culture as described below. However, depending on the host microorganism, for example, yeast, etc.
It may be used crtZ of Erwinia instead of crtZ.

Marine bacteria are crtB , crtI , crtY , crt
Since they have the Z and crtW genes, these five genes derived from marine bacteria can be used together with the crtE and crtX genes of Erwinia . In the present invention, the above seven genes i.e. crtE, cr tB, crtI, crtY , crtZ,
It is sufficient that crtX and crtW are expressed in combination. Therefore, the origin of each gene is Flavobacterium sp.
1534 (Luis Pasamontes et al., Isolation and Charact
erization of the carotenoid biosynthesis genes of
Flavobacterium sp.strain R1534 Gene, 185, 35-41, 1
997), and if necessary, the above 7
Species genes can also be distributed and introduced and expressed in multiple hosts (homogeneous or heterologous).

The crt gene used in the present invention is as defined above, and it may be mentioned that each of the crt genes having different origins may be a gene having the same function (encoding a mutant enzyme having the same activity). It is as follows.

<Methods for preparing plasmids containing various crt genes and methods for introducing and expressing them in various microorganisms> Next, methods for preparing plasmids containing various crt genes described above, and introducing and introducing these plasmids into various organisms are described. Examples of the expression method will be described in more detail. 1) Acquisition of DNA chain One means of acquiring the above-mentioned various crt genes is to chemically synthesize at least a part of the chain length according to the method of nucleic acid synthesis. Given this, Erwinia
A chromosomal DNA library such as uredovora or Agrobacterium aurantiacum is prepared in Escherichia coli, and a method commonly used in the field of genetic engineering from this library, such as a hybridization method using an appropriate probe (eg, a chemically synthesized DNA probe), It can be said that it is faster to obtain this.

2) Transformation of organism and gene expression A plasmid containing the crt gene group as described above was prepared,
By introducing this into a suitable host microorganism such as Escherichia coli and expressing it, a target carotenoid glycoside can be obtained.

The following is an outline of a preferred method for introducing and expressing a foreign gene into a microorganism. Procedures and methods for preparing a plasmid containing a foreign gene and introducing and expressing the plasmid into a microorganism such as Escherichia coli are not limited to those described below in the present invention, and may be those commonly used in the field of genetic engineering. And its methods or methods (eg, "Vectors for clo
ning genes ", Methods in Enzymology, 216, p. 469-63
1, 1992, Academic Press and "Other bacterial
systems ", Methods in Enzymology, 204, p.305-636,
1991, Academic Press).

(1) Escherichia coli There are several well-established methods for introducing a foreign gene ( crt gene group) into Escherichia coli, such as the Hanahan's method and the rubidium method. For example, Sambrook, J., Fritsch, EF, Maniatis, T.,
"Molecular cloning -A laboratory manual." Cold Sp
ring Harbor Laboratory Press, 1989). Expression of a foreign gene in Escherichia coli may be performed according to a conventional method (for example, as described in "Molecular cloning -A laboratory manu
al. "), for example, pUC and pBluescript
c) using a vector for E. coli having the promoter and the like. The present inventors have, for E. coli vector pBluescript II SK having promoter of the lac etc. ^- with, in the direction to receive a read-through transcription of the promoter of the lac, the Agrobacterium aurantiacum crtW or c,
The rtW and crtZ genes were inserted and these genes were expressed in E. coli. In addition, Erwinia uredovor a crtE ,
crtB , crtI , crtY , crtZ (when crt Z of Agrobacterium was not used) and crtX were expressed using an E. coli vector pACYC184 that can coexist with pBluescript II in one cell.

[0030] (2) Zymomonas mobilis ethanol producing bacterium Zymomonas mobilis of foreign genes into (seven crt genes) transfer method can be carried out by common conjugal transfer method Gram-negative bacteria, Zymomona s mobili
expression of foreign genes in s, for example Zymomonas mobil
can be carried out using a is a vector for pZA22 (Katsumi Nakamura, "molecular breeding of Zymomonas bacteria", Japan Agricultural Chemistry Journal, 63, p.1016-1018, 1989, and, N. Misawa, S.
Yamano, H. Ikenaga, Production of β-carotene in Zy
momonas mobilis and Agrobacteri um tumefaciens by i
ntroduction of the biosynthesis genes from Erwinia
ur edovora .Appl.Environ.Microbiol., 57, 1847-184
9, 1991).

(3) Yeast A foreign gene to the yeast Saccharomyces cerevisiae (7
There are already established methods such as the lithium method for introducing the crt gene), and the method may be used (for example,
Edited by the Bioindustry Association, supervised by Yuichi Akiyama, see "Yeast New Biotechnology", published by Medical Publishing Center). Expression of foreign genes in yeast is controlled by PGK or GPD (GAP)
Using such a promoter and terminator, an expression cassette is constructed in which a foreign gene is inserted between the promoter and the terminator so as to receive read-through of transcription, and this expression cassette is used as a vector of S. cerevisiae , for example, a YRp system. (Multicopy vector for yeast using the ARS sequence of the yeast chromosome as an origin of replication), YEp system (multicopy vector for yeast having a replication origin of 2 μm DNA of yeast), YIp system (yeast chromosome having no yeast replication origin) This can be performed by inserting the vector into a vector such as an integration vector ("Yeast New Biotechnology" mentioned above, published by the Medical Publishing Center, ABC Series of Japanese Society for Agricultural Chemistry, "Genetic Engineering for Substance Production," published by Asakura Shoten, And S. Yamano, T. Ishii, M. Nakagawa, H. Ikenag
a, N. Misawa, Metabolic engineering for production
ofβ-carotene and lycopene in Saccharomyces cerev
isiae. Biosci. Biotech. Biochem., 58, 1112-1114, 19
94). The method for introducing a foreign gene into the yeast Candida utilis can be carried out according to the method already disclosed by the present inventors (Kondo, Misawa, Kajiwara, JP-A-8-173170). Specifically, a cycloheximide resistance gene,
After linearizing a plasmid containing a drug resistance marker gene such as a G418 resistance gene or a hygromycin resistance gene, the plasmid can be integrated on a chromosome by an electric pulse method or a lithium method. GAP is the expression of foreign genes described in the patent application publication, P GK, PMA
Such promoters can be used. Yeast Phaf
The method of introducing a foreign gene (seven crt genes) into fia rhodozyma is disclosed by Van Ooyen et al. (Van Ooyen et al., Transformation of Phaffia rh).
odozyma, WO94 / 06918, 1994), a plasmid containing a selectable marker gene such as a G418 resistance gene can be integrated on a chromosome by a lithium method or the like.

The introduction of foreign genes (seven kinds of crt genes) into the yeast Candida tropicalis was carried out by the method disclosed in Japanese Patent Application Laid-Open No. 4505557 and by the method disclosed in Yarrowia lipolytica.
For example, Japanese Patent Laid-Open Nos. 60199386 and 61
21087 and JP-A-63164889, respectively.

<Culture Method of Microorganism and Purification Method of Carotenoid Glycoside> In the method of producing a carotenoid glycoside according to the present invention, the above transformed microorganism into which seven kinds of crt genes have been introduced is generally cultured with a host microorganism. The astaxanthin diglucoside, astaxanthin monoglucoside, and adonixanthin-3'-glucoside produced by culturing in the medium used for the above are collected from the culture by a conventional method.

First, a general culture method will be described. For culturing a transformed microorganism capable of producing astaxanthin diglucoside, astaxanthin monoglucoside, and adonixanthin-3'-glucoside, a usual culturing method can be used for the host microorganism to be used (for example, see Molecular Cloning described above). ). In general, peptones, yeast extracts, sugars (such as glucose) and inorganic substances (such as NaCl (sodium chloride)) are used as culture media.
Can be used (for example, the molecular clonin described above).
g and LIST OF CULTURES issued by Fermentation Research Institute).
In addition, metabolic maps considered to be precursors for carotenoid biosynthesis (edited by the Biochemical Society of Japan, published by Tokyo Chemical Dojin, 1980
), Pages 123-125 (e.g., pyruvate and mevalonic acid) and trace components that promote the production of astaxanthin, adonixanthin, etc. (e.g., Fe2 ⁺ ,
α-Ketoglutaric acid (2-oxoglutaric acid) etc.)
(PDFraser et al., J. Biol. Chem. 272, 6128-6135, 19
97, ref.) Can be added as needed.

The most suitable culture method is a liquid culture method. The optimal culture conditions vary depending on the type of host microorganism, but usually the culture temperature is 16 to 40 ° C, particularly 20 to 30 ° C, and the pH of the medium during the culture is maintained at 4 to 10, particularly 6 to 8 It is desirable. When liquid culture is performed for half to four days, astaxanthin diglucoside, astaxanthin monoglucoside, and adonixanthin-3'-glucoside are produced and accumulated in the microorganism. It is desirable to stop the culture when the production in the culture reaches a maximum. For general culture methods related to the present invention, for example, the above-mentioned Molecular Cloning and the like can be referred to.

The isolation and purification of astaxanthin diglucoside, astaxanthin monoglucoside and adonixanthin-3'-glucoside from the culture are carried out according to a method commonly used for isolating and purifying microbial metabolites from the culture. (For example, by Noboru Otake,
Isolation and purification of substances, see The University of Tokyo Press, 1981).
For example, the culture is separated into a culture filtrate and cells by filtration or centrifugation, and the cells are extracted with an organic solvent (eg, methanol, chloroform, acetone or a mixture of two or more of these). Next, after the extract is concentrated, and if necessary, the culture filtrate can be purified by liquid chromatography using, for example, silica gel, chemically bonded silica gel, a gel filtration agent, or the like. The trends of astaxanthin diglucoside, astaxanthin monoglucoside and adonixanthin-3'-glucoside are tracked, for example, using the red-orange color characteristic of each carotenoid glycoside by thin-layer chromatography as a guide. It can be separated and purified.

The carotenoid glycosides described above may be obtained as a single glycoside or as a mixture of two or three types, and in any form, as a highly water-soluble carotenoid, a food additive or the like. Can be used. This means, as mentioned above, that the carotenoid glycosides according to the invention are typically isolates of the novel substances astaxanthin diglucoside and adonixanthin-3'-glucoside, respectively. It is meant to include mixtures of two or three of the novel carotenoid glycosides and known astaxanthin monoglucosides.

[0038] 3) As Transformation and Gene Expression aforementioned plants, seven plants the crt gene missing all or plant cells crt genes to produce the carotenoid glycosides by introducing into the cell Can be produced. As a preferred example, for example, a plasmid containing the above-mentioned crtX and crtW genes and five other crtZ genes is prepared, introduced into an appropriate plant such as tomato, and expressed, whereby the desired carotenoid glycoside is obtained. A plant that produces a body can be obtained.

The following is an outline of a method for introducing and expressing a foreign gene into a plant. The method for preparing a plasmid containing a foreign gene and the procedure or method for introducing and expressing the plasmid into a plant (cells such as leaves, stems and roots) are not limited to those described below in the present invention. The method may be performed according to the method or method (for example, Isao Ishida and Norihiko Misawa, Introduction to Cell Engineering Experiment Operation, Kodansha, 1992), including those commonly used in the field of engineering.

As a method for introducing a foreign gene into a plant, a method involving a plant pathogenic bacterium Agrobacterium tumefaciens , an electroporation method, a method using a particle gun, and the like are known. You can use these methods depending on the type of plant you want to introduce, but currently Agroba
The method via cterium tumefaciens is most frequently used. As the promoter, a 35S promoter of cauliflower mosaic virus (CaMV), which is a high expression promoter for whole body, and other promoters that are expressed in various organs can be used. Binary vector pBI121 containing the CaMV 35S promoter can be obtained from Clontech, Inc., as a vector to through Agrobacterium tumefaci ens, those widely used. Er
A crt gene such as winia crtI can be introduced into plants such as tobacco and tomato by using pBI121 as a vector, and it has been shown that the crt gene is expressed and functions in tobacco leaves and tomato fruits. Norihiko Misawa, Production of Plants Resistant to Carotenoid Biosynthesis Inhibitors, Chemical Regulation of Plants, 31, 143-149, 1996). At this time, the transit peptide sequence (for example, Rubisc) was used to transfer the Crt protein synthesized in the plant cytoplasm to plastids such as chloroplasts and plastids, which are places for carotenoid production.
cr the transit peptide sequence) of the small subunit of o
It must be added immediately before the start codon of the t gene (Norihiko Misawa, creation of carotenoid biosynthesis inhibitor-resistant plants,
Chemical regulation of plants, 31, 143-149, 1996).

4) Deposit of Microorganism The Escherichia coli incorporating the isolated gene, which has become the source of the DNA strand of the present invention, has been deposited with the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology as follows. (1) Escherichia coli JM109 (pCAR1) Accession number: FERM BP-2377 Accession date: April 11, 1989 (2) Escherichia coli JM101 (pAccrt-EIB, pAK92) Accession number: FERM BP-4505 Date: December 20, 1993 Strain (1) was obtained from crtE , crtB , crt of Erwinia uredovora
Contains the I , crtY , crtZ , and crtX genes, and is a strain (2)
Contains the crtZ and crtW genes of Agrobacterium aurantiacum .

Use of carotenoid glycoside The carotenoid glycoside according to the present invention, which can be obtained as described above, can be used in the food field. That is, the present invention relates to a method of optionally adding one or more of ataxanthin diculcoside, adonixanthin-3'-glucoside and astaxanthin monoglucoside (excluding the use of astaxanthin monoglucoside alone) to food oil, glycerin, There is also provided a food improving agent or food which is contained in an appropriate amount together with an additive such as ethanol. Here, the food improving agent includes a food color improving agent, a color enhancer, a food additive, an antioxidant, and the like, and the food includes a solid (such as kamaboko) and a semi-fluid (such as stew). ,
It includes various foods and drinks such as liquids (food oil, soft drinks, etc.).

[0043]

The present invention will be described more specifically with reference to the following examples.
However, the present invention is not limited thereby.
Needless to say. [Example 1] Preparation of plasmid Plant-resident (epiphytic) bacteriaErwinia Can be obtained from
TocrtEThe gene gels farnesyl pyrophosphate (FPP)
An enzyme that converts to nilgeranyl pyrophosphate (GGPP)
Encodes GGPP synthase.Erwinia Get from
Can becrtBGene converts GGPP to phytoene
It encodes the enzyme phytoene synthase. Sa
In addition,Erwinia Can be obtained fromcrtIGenes are
Phytoende, an enzyme that converts phytoene to lycopene
Encodes a saturase,Erwinia Gaining from
CancrtYGene converts lycopene to β-carotene
Encoding lycopene cyclase,
(The above, Norihiko Misawa, Carote clarified at the genetic level
Noid biosynthetic pathway, protein, nucleic acid, enzyme, 41, 337-346,
 1996, Norihiko Misawa et al., Elucidation of the
 Erwinia uredovoracarotenoid biosynthetic pathway
by functional analysis of gene productsexpressed i
n Escherichia coli. J. Bacteriol., 172, 6704-6712,
 1990, and Gerhard Sandmann, N. Misawa, New fun
ctional aasignment of the carotenogenic genes crtB
 and crtE with constructs of these genes from Erwi
niaspecies.FEMS Microbiol. Lett. 90, 253-258, 1992
 See). Therefore, the above four crt genes were
When introduced and expressed in microorganisms such as
Begins to synthesize β-carotene. Astaxanthi
Marine bacteriaAgrobacterium aurantiacum OrAlca
ligene s sp. can be obtained from strain PC-1crtW
Is the carotenoid 4, (4 ')-β-ionone ring oxygenator
CodeA. aurantiacumOrAlcaligene
s sp. strain PC-1 orErwiniaCan get from
WearcrtZIs a carotenoid 3, (3 ′)-β-ionone ring
Encodes idroxylase (Paul D. Fraser,
Y. Miura, N. Misawa, In vitro characterization of
astaxanthin biosynthetic enzymes. J. Biol. Chem.,
272, 6128-6135, 1997). Large intestine producing beta-carotene
For microorganisms such as bacteriacrtW WhencrtZIntroduce and express both
To produce astaxanthin as a final product
Become. Microorganisms such as E. coli that produce β-carotene
TocrtWWhen introduced and expressed, canthaxanthin,c
rtZZeaxanthin is produced when introduced and expressed
(Norihiko Misawa, Caroteno revealed at the genetic level
Id biosynthetic pathway, protein nucleic acid enzyme, 41, 337-346,19
96, Norihiko Misawa et al., Structure and functio
nal analysis of a marine bacterial carotenoid bios
ynthesis gene cluster and astaxanthin biosynthetic
 pathway proposed at the gene level.J. Bacterio
l., 177, 6575-6584, 1995, and Paul D. Fraser,
Y. Miura, N. Misawa, In vitro characterization of
astaxanthin biosynthetic enzymes. J. Biol. Chem.,
272, 6128-6135, 1997).Erwinia Can get from
it cancrtXIs zeaxanthin glucosyltransfer
Enzyme that produces zeaxanthin
For microorganisms such as enterobacteriacrtX Zeaxa is introduced and expressed
To synthesize tintin-β-D-diglucoside (No
rihiko Misawa et al., Elucidation of the Erwiniaur
edovora carotenoid biosynthetic pathway by functio
nal analysis of gene products expressed in Escheri
See chia coli. J. Bacteriol., 172, 6704-6712, 1990.
See).A. aurantiacum ofcrtW0.91 kb including BamHI-Hin
Insert the cII fragment into the BamHI-HincII site of pBluescript II SK-
Then, the plasmid pAK916 was prepared. further,A. aur
antiacumofcrtWWhencrtZ1.88 kb Ba containing two genes
Insert the mHI-KpnI fragment into the BamHI-KpnI site of pBluescript II SK-
The resulting plasmid was inserted into the plasmid pAK96K. These plastic
Sumid is described in N. Misawa et al., J. Bacteriol., 177, 65.
75-6584, 1995.Erwinia uredovora
Of all sixcrt 6.50 kb Asp718 (KpnI)-including gene
The EcoRI fragment was converted to plasmid pCAR25 (N. Misawa et al.,
J. Bacteriol., 172, 6704-6712, 1990)
After Klenow fragment treatment, the vector pACYC184 (R.E.Ro
se, The nucleotide sequence of pACYC184, Nucleic A
cids Res., 16, 355, 1988).
Smid pACCAR25 (FIG. 1) was prepared. next,E. uredovo
raofcrtZOther than 5crt 6.01 kb Asp718 including gene
 The (KpnI) -EcoRI fragment was ligated with plasmid pCAR16 (N. Misawa
et al., J. Bacteriol., 172, 6704-6712, 1990).
After processing the Klenow fragment, the vector pACYC184
And the plasmid pACCAR16 (Figure 1) was inserted into the EcoRV site of
Produced. [Example 2] Method for obtaining astaxanthin diglucoside and the like Escherichia coli JM101 (pACCAR25, pAK916) was used as the inoculum.
For culture, use LB medium (10 g Tryptone (Bacto), 5 g Yeast E
xtract (Bacto), dissolve 10g NaCl in 1 liter of distilled water
After adjusting to pH 7.2, sterilize in an autoclave).
Syrin 150 mg, chloramphenicol 30 mg, isopro
Pill β-D-thiogalactopyranoside 50 mg (1 medium per medium)
(In the bottle) was used. 1000 ml volume triangle
The bacteria were added to a total of 12 liters of 40 300 ml culture media in a flask.
Inoculate the strains one platinum loop at a time and shake at 30 ° C for 48 hours (100 rp
m) Cultured. Centrifuge the culture solution (5,000 rpm) and centrifuge the cells.
After obtaining the fraction, add 500 ml of acetone, stir,
The precipitate is filtered off and the precipitate is acetone: methanol (7: 3)
Extracted again with 500 ml. The extracts are combined, concentrated,
Partition with 300 ml of xanth and 300 ml of 80% aqueous methanol.
Was. After concentrating the 80% aqueous methanol fraction,
(2x20 cm, Silica gel 60 manufactured by Merck)
Lum: fractionation using methanol (8: 2) as the elution solvent
Was. In addition, TSK gel ODS-80Ts (7.8x300 mm, Tosoh Corporation)
Liquid) and distilled water: high-speed liquid crystal using methanol (1: 9).
Astaxanthin diglucoside by chromatography
 1 mg was obtained. In addition, an asterisk
Fractions that elute earlier than xanthine diglucoside are
gel ODS-80Ts (7.8x300 mm, manufactured by Tosoh Corporation) and distilled water:
High Performance Liquid Chromatography Using Tanol (5:95)
As a result, 3 mg of astaxanthin monoglucoside was obtained.
Visible absorption spectrum of astaxanthin monoglucoside
, Mass spectrometry and 1H-NMR spectra have already been reported
Astaxanthin monoglucoside
 (Akihiro Yokoyama, Kyouko Adachi, and Yoshikazu S
hizuri, Journal of Natural Products, Vol. 58, pp.
1929-1933).

Example 3 Method for Obtaining Adonixanthin-3'-Glucoside Escherichia coli JM101 (pACCAR16, pAK96K) was used as the inoculum.
For cultivation, LB medium supplemented with 150 mg of ampicillin, 30 mg of chloramphenicol, and 50 mg of isopropyl β-D-thiogalactopyranoside (in 1 liter of each medium) was used. 300 ml of medium 40 in a 1000 ml Erlenmeyer flask
A total of 12 liters of this strain was inoculated with one platinum loop at a time.
The culture was carried out with shaking (100 rpm) at 48 ° C for 48 hours. After centrifuging the culture solution (5,000 rpm) to obtain a cell fraction, acetone 500
After adding and stirring the precipitate, the precipitate was separated by filtration and the precipitate was extracted again with 500 ml of acetone: methanol (7: 3). The extracts were combined, concentrated, and partitioned with 300 ml of hexane and 300 ml of 80% aqueous methanol. After concentrating the 80% aqueous methanol fraction, a silica gel column (2x20 cm,
Ca gel 60) was fractionated using chloroform: methanol (8: 2) as an elution solvent. Furthermore, TSK gel ODS-80Ts
(7.8x300 mm, manufactured by Tosoh Corporation) and distilled water: methanol (1:
9 mg of adonixanthin-3'-glucoside was obtained by high performance liquid chromatography using 9).

Example 4 Example of Formulation of Food Improving Agent and Food After 1 mg of carotenoid glycoside containing astaxanthin diglucoside was uniformly dissolved in 25 ml of ethanol,
When 1 ml of the mixture was added to 350 ml of clear soft drink (for example, Kirin Lemon [Select]), the mixture was uniformly colored red (reddish orange).

[0046]

Industrial Applicability According to the present invention, novel astaxanthin diglucoside and adonixanthin-3'-glucoside, which are highly polar carotenoid glycosides, as well as these novel carotenoid glycosides and astaxanthin monosaccharide which is a known highly polar carotenoid A method for producing a glucoside is provided. The carotenoid glycoside obtained in the present invention is useful as a water-soluble food additive, a color improver for cultured fish and shellfish, and the like, and can be produced in large quantities by a genetic engineering technique. In addition, by expressing the carotenoid glycoside obtained in the present invention (in fruits, leaves, flowers, stems, roots, etc.) as a plant for edible or ornamental use by expressing in a plant using the above crt gene. Useful plants can be obtained. In the present invention, it is understood that the combination of the crt gene and the carotenoid glycoside with high polarity was unexpectedly obtained.

[0047]

[Sequence list]

SEQ ID NO: 1 Sequence length: 639 Sequence type: number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Agrobacterium aurantiacum Sequence characteristics: Other information: crtW (gene Name) Sequence GTG CAT GCG CTG TGG TTT CTG GAC GCA GCG GCG CAT CCC ATC CTG GCG 48 Met His Ala Leu Trp Phe Leu Asp Ala Ala Ala His Pro Ile Leu Ala 1 5 10 15 ATC GCA AAT TTC CTG GGG CTG ACC TGG CTG TCG GTC GGA TTG TTC ATC 96 Ile Ala Asn Phe Leu Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile 20 25 30 ATC GCG CAT GAC GCG ATG CAC GGG TCG GTG GTG CCG GGG CGT CCCGCGC 144 Ile Ala His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg 35 40 45 GCC AAT GCG GCG ATG GGC CAG CTT GTC CTG TGG CTG TAT GCC GGA TTT 192 Ala Asn Ala Ala Met Gly Gln Leu Val Leu Trp Leu Tyr Ala Gly Phe 50 55 60 TCG TGG CGC AAG ATG ATC GTC AAG CAC ATG GCC CAT CAC CGC CAT GCC 240 Ser Trp Arg Lys Met Ile Val Lys His Met Ala His His Arg His Ala 65 70 75 80 GGA ACC GAC GAC GAC CCC GAT TTC GAC C AT GGC GGC CCG GTC CGC TGG 288 Gly Thr Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp 85 90 95 TAC GCC CGC TTC ATC GGC ACC TAT TTC GGC TGG CGC GAG GGG CTG CTG 336 Tyr Ala Arg Phe Ile Gly Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu 100 105 110 CTG CCC GTC ATC GTG ACG GTC TAT GCG CTG ATC CTT GGG GAT CGC TGG 384 Leu Pro Val Ile Val Thr Val Tyr Ala Leu Ile Leu Gly Asp Arg Trp 115 120 125 ATG TAC GTG GTC TTC TGG CCG CTG CCG TCG ATC CTG GCG TCG ATC CAG 432 Met Tyr Val Val Phe Trp Pro Leu Pro Ser Ile Leu Ala Ser Ile Gln 130 135 140 CTG TTC GTG TTC GGC ACC TGG CTG CCG CAC CGC CCC GGC CAC GAC GCG 480 Leu Phe Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Ala 145 150 155 160 TTC CCG GAC CGC CAC AAT GCG CGG TCG TCG CGG ATC AGC GAC CCC GTG 528 Phe Pro Asp Arg His Asn Ala Arg Ser Ser Arg Ile Ser Asp Pro Val 165 170 175 TCG CTG CTG ACC TGC TTT CAC TTT GGC GGT TAT CAT CAC GAA CAC CAC 576 Ser Leu Leu Thr Cys Phe His Phe Gly Gly Tly His His Glu His His 180 185 190 CTG CAC CCG ACG GTG CCG TGG TGG CGC CTG CCC AGC ACC CGC ACC AAG 624 Leu His Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys 195 200 205 GGG GAC ACC GCA TGA 639 Gly Asp Thr Ala *** 210

SEQ ID NO: 2 Sequence length: 489 Sequence type: number of strands: double stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Agrobacterium aurantiacum Sequence characteristics: Other information: crtZ (Gene name) Sequence ATG ACC AAT TTC CTG ATC GTC GTC GCC ACC GTG CTG GTG ATG GAG TTG 48 Met Thr Asn Phe Leu Ile Val Val Ala Thr Val Leu Val Met Glu Leu 1 5 10 15 ACG GCC TAT TCC GTC CAC CGC TGG ATC ATG CAC GGC CCC CTG GGC TGG 96 Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro Leu Gly Trp 20 25 30 GGC TGG CAC AAG TCC CAC CAC GAG GAA CAC GAC CAC GCG CTG GAA AAG 144 Gly Trp His Lys Ser His His Glu Glu His Asp His Ala Leu Glu Lys 35 40 45 AAC GAC CTG TAC GGC CTG GTC TTT GCG GTG ATC GCC ACG GTG CTG TTC 192 Asn Asp Leu Tyr Gly Leu Val Phe Ala Val Ile Ala Thr Val Leu Phe 50 55 60 ACG GTG GGC TGG ATC TGG GCG CCG GTC CTG TGG TGG ATC GCC TTG GGC 240 Thr Val Gly Trp Ile Trp Ala Pro Val Leu Trp Trp Ile Ala Leu Gly 65 70 75 80 ATG ACT GTC TAT GGG CTG ATC TAT TTC GTC CTG CAT GAC GGG CTG GTG 288 Met Thr Val Tyr Gly Leu Ile Tyr Phe Val Leu His Asp Gly Leu Val 85 90 95 CAT CAG CGC TGG CCG TTC CGT TAT ATC CCG CGC AAG GGC TAT GCC AGA 336 His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Lys Gly Tyr Ala Arg 100 105 110 CGC CTG TAT CAG GCC CAC CGC CTG CAC CAT GCG GTC GAG GGG CGC GAC 384 Arg Leu Tyr Gln Ala His Arg Leu His His Ala Val Glu Gly Arg Asp 115 120 125 CAT TGC GTC AGC TTC GGC TTC ATC TAT GCG CCC CCG GTC GAC AAG CTG 432 His Cys Val Ser Phe Gly Phe Ile Tyr Ala Pro Pro Val Asp Lys Leu 130 135 140 AAG CAG GAC CTG AAG ATG TCG GGC GTG CTG CGG GCC GAG GCG CAG GAG 480 Lys Gln Asp Leu Lys Met Ser Gly Val Leu Arg Ala Glu Ala Gln Glu 145 150 155 160 CGC ACG TGA 489 Arg Thr --- ***

SEQ ID NO: 3 Sequence length: 1161 Sequence type: Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Agrobacterium aurantiacum Sequence characteristics: Other information: crtY (Gene name) Sequence GTG ACC CAT GAC GTG CTG CTG GCA GGG GCG GGC CTT GCC AAC GGG CTG 48 Met Thr His Asp Val Leu Leu Ala Gly Ala Gly Leu Ala Asn Gly Leu 1 5 10 15 ATC GCC CTG GCG CTG CGC GCG GCG CGG CCC GAC CTG CGC GTG CTG CTG 96 Ile Ala Leu Ala Leu Arg Ala Ala Arg Pro Asp Leu Arg Val Leu Leu 20 25 30 CTG GAC CAT GCC GCA GGA CCG TCA GAC GGC CAC ACC TGG TCC TGC CAC 144 Leu Asp His Ala Ala Gly Pro Ser Asp Gly His Thr Trp Ser Cys His 35 40 45 GAC CCC GAC CTG TCG CCG GAC TGG CTG GCG CGG CTG AAG CCC CTG CGC 192 Asp Pro Asp Leu Ser Pro Asp Trp Leu Ala Arg Leu Lys Pro Leu Arg 50 55 60 CGC GCC AAC TGG CCC GAC CAG GAG GTG CGC TTT CCC CGC CAT GCC CGG 240 Arg Ala Asn Trp Pro Asp Gln Glu Val Arg Phe Pro Arg His Ala Arg 65 70 75 80 CGG CTG GCC ACC GGT TAC GGG TCG CTG GAC GGG GCG GCG CTG GCG GAT 288 Arg Leu Ala Thr Gly Tyr Gly Ser Leu Asp Gly Ala Ala Leu Ala Asp 85 90 95 GCG GTG GTC CGG TCG GGC GCC GAG ATC CGC TGG GAC AGC GAC ATC GCC 336 Ala Val Val Arg Ser Gly Ala Glu Ile Arg Trp Asp Ser Asp Ile Ala 100 105 110 CTG CTG GAT GCG CAG GGG GCG ACG CTG TCC TGC GGC ACC CGG ATC GAG 384 Leu Leu Asp Ala Gln Gly Ala Thr Leu Ser Cys Gly Thr Arg Ile Glu 115 120 125 GCG GGC GCG GTC CTG GAC GGG CGG GGC GCG CAG CCG TCG CGG CAT CTG 432 Ala Gly Ala Val Leu Asp Gly Arg Gly Ala Gln Pro Ser Arg His Leu 130 135 140 ACC GTG GGT TTC CAG AAA TTC GTG GGT GTC GAG ATC GAG ACC GAC CGC 480 Thr Val Gly Phe Gln Lys Phe Val Gly Val Glu Ile Glu Thr Asp Arg 145 150 155 160 CCC CAC GGC GTG CCC CGC CCG ATG ATC ATG GAC GCG ACC GTC ACC CAG 528 Pro His Gly Val Pro Arg Pro Met Ile Met Asp Ala Thr Val Thr Gln 165 170 175 CAG GAC GGG TAC CGC TTC ATC TAT CTG CTG CCC TTC TCT CCG ACG CGC 576 Gln Asp Gly Tyr Arg Phe Ile Tyr Leu Leu Pro Phe Ser Pro Thr Arg 180 185 190 ATC CTG ATC GAG GAC ACG CGC TAT TCC GAT GGC GGC GAT CTG GAC GAC 624 Ile Leu Ile Glu Asp Thr Arg Tyr Ser Asp Gly Gly Asp Leu Asp Asp 195 200 205 GAC GCG CTG GCG GCG GCG TCC CAC GAC TAT GCC CGC CAG CAG GGC TGG 672 Asp Ala Leu Ala Ala Ala Ser His Asp Tyr Ala Arg Gln Gln Gly Trp 210 215 220 ACC GGG GCC GAG GTC CGG CGC GAA CGC GGC ATC CTT CCC ATC GCG CTG 720 Thr Gly Ala Glu Val Arg Arg Glu Arg Gly Ile Leu Pro Ile Ala Leu 225 230 235 240 GCC CAT GAT GCG GCG GGC TTC TGG GCC GAT CAC GCG GCG GGG CCT GTT 768 Ala His Asp Ala Ala Gly Phe Trp Ala Asp His Ala Ala Gly Pro Val 245 250 255 CCC GTG GGA CTG CGC GCG GGG TTC TTT CAT CCG GTC ACC GGC TAT TCG 816 Pro Val Gly Leu Arg Ala Gly Phe Phe His Pro Val Thr Gly Tyr Ser 260 265 270 CTG CCC TAT GCG GCA CAG GTG GCG GAC GTG GTG GCG GGT CTG TCC GGG 864 Leu Pro Tyr Ala Ala Gln Val Ala Asp Val Val Ala Gly Leu Ser Gly 275 280 285 CCG CCC GGC ACC GAC GCG CTG CGC GGC GCC ATC CGC GAT TAC GCG ATC 912 Pro Pro Gly Thr Asp Ala Leu Arg Gly Ala Ile Arg Asp Tyr Ala Ile 290 295 300 GAC CGG GCG CGC CGC GAC CGC TTT CTG CGC CTT TTG AAC CGG ATG CTG 960 Asp Arg Ala Arg Arg Asp Arg Phe Leu Arg Leu Leu Asn Arg Met Leu 305 310 315 320 TTC CGC GGC TGC GCG CCC GAC CGG CGC TAT ACC CTG CTG CAG CGG TTC 1008 Phe Arg Gly Cys Ala Pro Asp Arg Arg Tyr Thr Leu Leu Gln Arg Phe 325 330 335 TAC CGC ATG CCG CAT GGA CTG ATC GAA CGG TTC TAT GCC GGC CGG CTG 1056 Tyr Arg Met Pro His Gly Leu Ile Glu Arg Phe Tyr Ala Gly Arg Leu 340 345 350 AGC GTG GCG GAT CAG CTG CGC ATC GTG ACC GGC AAG CCT CCC ATT CCC 1104 Ser Val Ala Asp Gln Leu Arg Ile Val Thr Gly Lys Pro Pro Ile Pro 355 360 365 CTT GGC ACG GCC ATC CGC TGC CTG CCC GAA CGT CCC CTG CTG AAG GAA 1152 Leu Gly Thr Ala Ile Arg Cys Leu Pro Glu Arg Pro Leu Leu Lys Glu 370 375 380 AAC GCA TGA 1161 Asn Ala *** 385

SEQ ID NO: 4 Sequence length: 729 Sequence type: Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Alcaligenes strain: sp. PC-1 sequence Features: Other information: crtW (gene name) Sequence ATG TCC GGA CGG AAG CCT GGC ACA ACT GGC GAC ACG ATC GTC AAT CTC 48 Met Ser Gly Arg Lys Pro Gly Thr Thr Gly Asp Thr Ile Val Asn Leu 1 5 10 15 GGT CTG ACC GCC GCG ATC CTG CTG TGC TGG CTG GTC CTG CAC GCC TTT 96 Gly Leu Thr Ala Ala Ile Leu Leu Cys Trp Leu Val Leu His Ala Phe 20 25 30 ACG CTA TGG TTG CTA GAT GCG GCC GCG CAT CCG CTG CTT GCC GTG CTG 144 Thr Leu Trp Leu Leu Asp Ala Ala Ala His Pro Leu Leu Ala Val Leu 35 40 45 TGC CTG GCT GGG CTG ACC TGG CTG TCG GTC GGG CTG TTC ATC ATC GCG 192 Cys Leu Ala Gly Leu Thr Trp Leu Ser Val Gly Leu Phe Ile Ile Ala 50 55 60 CAT GAC GCA ATG CAC GGG TCC GTG GTG CCG GGG CGG CCG CGC GCC AAT 240 His Asp Ala Met His Gly Ser Val Val Pro Gly Arg Pro Arg Ala Asn 65 70 75 80 GCG GCG ATC GGG CAA CTG GCG CTG TGG CTC TAT GCG GGG TTC TCG TGG 288 Ala Ala Ile Gly Gln Leu Ala Leu Trp Leu Tyr Ala Gly Phe Ser Trp 85 90 95 CCC AAG CTG ATC GCC AAG CAC ATG ACG CAT CAC CGG CAC GCC GGC ACC 336 Pro Lys Leu Ile Ala Lys His Met Thr His His Arg His Ala Gly Thr 100 105 110 GAC AAC GAT CCC GAT TTC GGT CAC GGA GGG CCC GTG CGC TGG TAC GGC 384 Asp Asn Asp Pro Asp Phe Gly His Gly Gly Pro Val Arg Trp Tyr Gly 115 120 125 AGC TTC GTC TCC ACC TAT TTC GGC TGG CGA GAG GGA CTG CTG CTA CCG 432 Ser Phe Val Ser Thr Tyr Phe Gly Trp Arg Glu Gly Leu Leu Leu Pro 130 135 140 GTG ATC GTC ACC ACC TAT GCG CTG ATC CTG GGC GAT CGC TGG ATG TAT 480 Val Ile Val Thr Thr Tyr Ala Leu Ile Leu Gly Asp Arg Trp Met Tyr 145 150 155 160 GTC ATC TTC TGG CCG GTC CCG GCC GTT CTG GCG TCG ATC CAG ATT TTC 528 Val Ile Phe Trp Pro Val Pro Ala Val Leu Ala Ser Ile Gln Ile Phe 165 170 175 GTC TTC GGA ACT TGG CTG CCC CAC CGC CCG GGA CAT GAC GAT TTT CCC 576 Val Phe Gly Thr Trp Leu Pro His Arg Pro Gly His Asp Asp Phe Pro 180 185 190 GAC CG G CAC AAC GCG AGG TCG ACC GGC ATC GGC GAC CCG TTG TCA CTA 624 Asp Arg His Asn Ala Arg Ser Thr Gly Ile Gly Asp Pro Leu Ser Leu 195 200 205 CTG ACC TGC TTC CAT TTC GGC GGC TAT CAC CAC GAA CAT CAC CTG CAT 672 Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His 210 215 220 CCG CAT GTG CCG TGG TGG CGC CTG CCT CGT ACA CGC AAG ACC GGA GGC 720 Pro His Val Pro Trp Trp Arg Leu Pro Arg Thr Arg Lys Thr Gly Gly 225 230 235 240 CGC GCA TGA 729 Arg Ala ***

SEQ ID NO: 5 Sequence length: 489 Sequence type: Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Alcaligenes strain: sp. PC-1 sequence Features: Other information: crtZ (Gene name) Sequence ATG ACG CAA TTC CTC ATT GTC GTG GCG ACA GTC CTC GTG ATG GAG CTG 48 Met Thr Gln Phe Leu Ile Val Val Ala Thr Val Leu Val Met Glu Leu 5 10 15 ACC GCC TAT TCC GTC CAC CGC TGG ATT ATG CAC GGC CCC CTA GGC TGG 96 Thr Ala Tyr Ser Val His Arg Trp Ile Met His Gly Pro Leu Gly Trp 20 25 30 GGC TGG CAC AAG TCC CAT CAC GAA GAG CAC GAC CAC GCG TTG GAG AAG 144 Gly Trp His Lys Ser His His Glu Glu His Asp His Ala Leu Glu Lys 35 40 45 AAC GAC CTC TAC GGC GTC GTC TTC GCG GTG CTG GCG ACG ATC CTC TTC 192 Asn Asp Leu Tyr Gly Val Val Phe Ala Val Leu Ala Thr Ile Leu Phe 50 55 60 ACC GTG GGC GCC TAT TGG TGG CCG GTG CTG TGG TGG ATC GCC CTG GGC 240 Thr Val Gly Ala Tyr Trp Trp Pro Val Leu Trp Trp Ile Ala Leu Gly 65 70 75 80 ATG ACG GTC TAT G GG TTG ATC TAT TTC ATC CTG CAC GAC GGG CTT GTG 288 Met Thr Val Tyr Gly Leu Ile Tyr Phe Ile Leu His Asp Gly Leu Val 85 90 95 CAT CAA CGC TGG CCG TTT CGG TAT ATT CCG CGG CGG GGC TAT TTC CGC 336 His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Arg Gly Tyr Phe Arg 100 105 110 AGG CTC TAC CAA GCT CAT CGC CTG CAC CAC GCG GTC GAG GGG CGG GAC 384 Arg Leu Tyr Gln Ala His Arg Leu His His Ala Val Glu Gly Arg Asp 115 120 125 CAC TGC GTC AGC TTC GGC TTC ATC TAT GCC CCA CCC GTG GAC AAG CTG 432 His Cys Val Ser Phe Gly Phe Ile Tyr Ala Pro Pro Val Asp Lys Leu 130 135 140 AAG CAG GAT CTG AAG CGG TCG GGT GTC CTG CGC CCC CAG GAC GAG CGT 480 Lys Gln Asp Leu Lys Arg Ser Gly Val Leu Arg Pro Gln Asp Glu Arg 145 150 155 160 CCG TCG TGA 489 Pro Ser ***

SEQ ID NO: 6 Sequence length: 891 Sequence type: number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Erwinia uredovora Sequence characteristics: Other information: crtB (gene name) Sequence ATG GCA GTT GGC TCG AAA AGT TTT GCG ACA GCC TCA AAG TTA TTT GAT 48 Met Ala Val Gly Ser Lys Ser Phe Ala Thr Ala Ser Lys Leu Phe Asp 1 5 10 15 GCA AAA ACC CGG CGC AGC GTA CTG ATG CTC TAC GCC TGG TGC CGC CAT 96 Ala Lys Thr Arg Arg Ser Val Leu Met Leu Tyr Ala Trp Cys Arg His 20 25 30 TGT GAC GAT GTT ATT GAC GAT CAG ACG CTG GGC TTT CAG GCC CGG CAG 144 Cys Asp Asp Val Ile Asp Asp Gln Thr Leu Gly Phe Gln Ala Arg Gln 35 40 45 CCT GCC TTA CAA ACG CCC GAA CAA CGT CTG ATG CAA CTT GAG ATG AAA 192 Pro Ala Leu Gln Thr Pro Glu Gln Arg Leu Met Gln Leu Glu Met Lys 50 55 60 ACG CGC CAG GCC TAT GCA GGA TCG CAG ATG CAC GAA CCG GCG TTT GCG 240 Thr Arg Gln Ala Tyr Ala Gly Ser Gln Met His Glu Pro Ala Phe Ala 65 70 75 80 GCT TTT CAG GAA GTG GCT ATG GCT CAT GAT ATC GCC CCG GCT TAC GCG 288 Ala Phe Gln Glu Val Ala Met Ala His Asp Ile Ala Pro Ala Tyr Ala 85 90 95 TTT GAT CAT CTG GAA GGC TTC GCC ATG GAT GTA CGC GAA GCG CAA TAC 336 Phe Asp His Leu Glu Gly Phe Ala Met Asp Val Arg Glu Ala Gln Tyr 100 105 110 AGC CAA CTG GAT GAT ACG CTG CGC TAT TGC TAT CAC GTT GCA GGC GTT 384 Ser Gln Leu Asp Asp Thr Leu Arg Tyr Cys Tyr His Val Ala Gly Val 115 120 125 GTC GGC TTG ATG ATG GCG CAA ATC ATG GGC GTG CGG GAT AAC GCC ACG 432 Val Gly Leu Met Met Ala Gln Ile Met Gly Val Arg Asp Asn Ala Thr 130 135 140 CTG GAC CGC GCC TGT GAC CTT GGG CTG GCA TTT CAG TTG ACC AAT ATT 480 Leu Asp Arg Ala Cys Asp Leu Gly Leu Ala Phe Gln Leu Thr Asn Ile 145 150 155 160 GCT CGC GAT ATT GTG GAC GAT GCG CAT GCG GGC CGC TGT TAT CTG CCG 528 Ala Arg Asp Ile Val Asp Asp Ala His Ala Gly Arg Cys Tyr Leu Pro 165 170 175 GCA AGC TGG CTG GAG CAT GAA GGT CTG AAC AAA GAG AAT TAT GCG GCA 576 Ala Ser Trp Leu Glu His Glu Gly Leu Asn Lys Glu Asn Tyr Ala Ala 180 185 190 CCT GAA AAC CGT CA G GCG CTG AGC CGT ATC GCC CGT CGT TTG GTG CAG 624 Pro Glu Asn Arg Gln Ala Leu Ser Arg Ile Ala Arg Arg Leu Val Gln 195 200 205 GAA GCA GAA CCT TAC TAT TTG TCT GCC ACA GCC GGC CTG GCA GGG TTG 672 Glu Ala Glu Pro Tyr Tyr Leu Ser Ala Thr Ala Gly Leu Ala Gly Leu 210 215 220 CCC CTG CGT TCC GCC TGG GCA ATC GCT ACG GCG AAG CAG GTT TAC CGG 720 Pro Leu Arg Ser Ala Trp Ala Ile Ala Thr Ala Lys Gln Val Tyr Arg 225 230 235 240 AAA ATA GGT GTC AAA GTT GAA CAG GCC GGT CAG CAA GCC TGG GAT CAG 768 Lys Ile Gly Val Lys Val Glu Gln Ala Gly Gln Gln Ala Trp Asp Gln 245 250 255 CGG CAG TCA ACG ACC ACG CCC GAA AAA TTA ACG CTG CTG CTG GCC GCC 816 Arg Gln Ser Thr Thr Thr Pro Glu Lys Leu Thr Leu Leu Leu Ala Ala 260 265 270 TCT GGT CAG GCC CTT ACT TCC CGG ATG CGG GCT CAT CCT CCC CGC CCT 864 Ser Gly Gln Ala Leu Thr Ser Arg Met Arg Ala His Pro Pro Arg Pro 275 280 285 GCG CAT CTC TGG CAG CGC CCG CTC TAG 891 Ala His Leu Trp Gln Arg Pro Leu * 290 295

SEQ ID NO: 7 Sequence length: 909 Sequence type: Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin: Organism: Erwinia uredovora Sequence characteristics: Other information: crtE (gene name) Sequence ATG ACG GTC TGC GCA AAA AAA CAC GTT CAT CTC ACT CGC GAT GCT GCG 48 Met Thr Val Cys Ala Lys Lys His Val His Leu Thr Arg Asp Ala Ala 1 5 10 15 GAG CAG TTA CTG GCT GAT ATT GAT CGA CGC CTT GAT CAG TTA TTG CCC 96 Glu Gln Leu Leu Ala Asp Ile Asp Arg Arg Leu Asp Gln Leu Leu Pro 20 25 30 GTG GAG GGA GAA CGG GAT GTT GTG GGT GCC GCG ATG CGT GAA GGT GCG 144 Val Glu Gly Glu Arg Asp Val Val Gly Ala Ala Met Arg Glu Gly Ala 35 40 45 CTG GCA CCG GGA AAA CGT ATT CGC CCC ATG TTG CTG TTG CTG ACC GCC 192 Leu Ala Pro Gly Lys Arg Ile Arg Pro Met Leu Leu Leu Leu Thr Ala 50 55 60 CGC GAT CTG GGT TGC GCT GTC AGC CAT GAC GGA TTA CTG GAT TTG GCC 240 Arg Asp Leu Gly Cys Ala Val Ser His Asp Gly Leu Leu Asp Leu Ala 65 70 75 80 TGT GCG GTG GAA ATG GTC CAC GCG GCT TCG CTG ATC CTT GAC GAT ATG 288 Cys Ala Val Glu Met Val His Ala Ala Ser Leu Ile Leu Asp Asp Met 85 90 95 CCC TGC ATG GAC GAT GCG AAG CTG CGG CGC GGA CGC CCT ACC ATT CAT 336 Pro Cys Met Asp Asp Ala Lys Leu Arg Arg Gly Arg Pro Thr Ile His 100 105 110 TCT CAT TAC GGA GAG CAT GTG GCA ATA CTG GCG GCG GTT GCC TTG CTG 384 Ser His Tyr Gly Glu His Val Ala Ile Leu Ala Ala Val Ala Leu Leu 115 120 125 AGT AAA GCC TTT GGC GTA ATT GCC GAT GCA GAT GGC CTC ACG CCG CTG 432 Ser Lys Ala Phe Gly Val Ile Ala Asp Ala Asp Gly Leu Thr Pro Leu 130 135 140 GCA AAA AAT CGG GCG GTT TCT GAA CTG TCA AAC GCC ATC GGC ATG CAA 480 Ala Lys Asn Arg Ala Val Ser Glu Leu Ser Asn Ala Ile Gly Met Gln 145 150 155 160 GGA TTG GTT CAG GGT CAG TTC AAG GAT CTG TCT GAA GGG GAT AAG CCG 528 Gly Leu Val Gln Gly Gln Phe Lys Asp Leu Ser Glu Gly Asp Lys Pro 165 170 175 CGC AGC GCT GAA GCT ATT TTG ATG ACG AAT CAC TTT AAA ACC AGC ACG 576 Arg Ser Ala Glu Ala Ile Leu Met Thr Asn His Phe Lys Thr Ser Thr 180 185 190 CTG TTT TGT GCC TC C ATG CAG ATG GCC TCG ATT GTT GCG AAT GCC TCC 624 Leu Phe Cys Ala Ser Met Gln Met Ala Ser Ile Val Ala Asn Ala Ser 195 200 205 AGC GAA GCG CGT GAT TGC CTG CAT CGT TTT TCA CTT GAT CTT GGT CAG 672 Ser Glu Ala Arg Asp Cys Leu His Arg Phe Ser Leu Asp Leu Gly Gln 210 215 220 GCA TTT CAA CTG CTG GAC GAT TTG ACC GAT GGC ATG ACC GAC ACC GGT 720 Ala Phe Gln Leu Leu Asp Asp Leu Thr Asp Gly Met Thr Asp Thr Gly 225 230 235 240 AAG GAT AGC AAT CAG GAC GCC GGT AAA TCG ACG CTG GTC AAT CTG TTA 768 Lys Asp Ser Asn Gln Asp Ala Gly Lys Ser Thr Leu Val Asn Leu Leu 245 250 255 GGC CCG AGG GCG GTT GAA GAA CGT CTG AGA CAA CAT CTT CAG CTT GCC 816 Gly Pro Arg Ala Val Glu Glu Arg Leu Arg Gln His Leu Gln Leu Ala 260 265 270 AGT GAG CAT CTC TCT GCG GCC TGC CAA CAC GGG CAC GCC ACT CAA CAT 864 Ser Glu His Leu Ser Ala Ala Cys Gln His Gly His Ala Thr Gln His 275 280 285 TTT ATT CAG GCC TGG TTT GAC AAA AAA CTC GCT GCC GTC AGT TAA 909 Phe Ile Gln Ala Trp Phe Asp Lys Lys Leu Ala Ala Val Ser * 290 295 300

SEQ ID NO: 8 Sequence length: 1479 Sequence type: Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Erwinia uredovora Sequence characteristics: Other information: crtI (Gene name) Sequence ATG AAA CCA ACT ACG GTA ATT GGT GCA GGC TTC GGT GGC CTG GCA CTG 48 Met Lys Pro Thr Thr Val Ile Gly Ala Gly Phe Gly Gly Leu Ala Leu 1 5 10 15 GCA ATT CGT CTA CAA GCT GCG GGG ATC CCC GTC TTA CTG CTT GAA CAA 96 Ala Ile Arg Leu Gln Ala Ala Gly Ile Pro Val Leu Leu Leu Glu Gln 20 25 30 CGT GAT AAA CCC GGC GGT CGG GCT TAT GTC TAC GAG GAT CAG GGG TTT 144 Arg Asp Lys Pro Gly Gly Arg Ala Tyr Val Tyr Glu Asp Gln Gly Phe 35 40 45 ACC TTT GAT GCA GGC CCG ACG GTT ATC ACC GAT CCC AGT GCC ATT GAA 192 Thr Phe Asp Ala Gly Pro Thr Val Ile Thr Asp Pro Ser Ala Ile Glu 50 55 60 GAA CTG TTT GCA CTG GCA GGA AAA CAG TTA AAA GAG TAT GTC GAA CTG 240 Glu Leu Phe Ala Leu Ala Gly Lys Gln Leu Lys Glu Tyr Val Glu Leu 65 70 75 80 CTG CCG GTT ACG CCG TTT TAC CGC CTG TGT TGG GAG TCA GGG AAG GTC 288 Leu Pro Val Thr Pro Phe Tyr Arg Leu Cys Trp Glu Ser Gly Lys Val 85 90 95 TTT AAT TAC GAT AAC GAT CAA ACC CGG CTC GAA GCG CAG ATT CAG CAG 336 Phe Asn Tyr Asp Asn Asp Gln Thr Arg Leu Glu Ala Gln Ile Gln Gln 100 105 110 TTT AAT CCC CGC GAT GTC GAA GGT TAT CGT CAG TTT CTG GAC TAT TCA 384 Phe Asn Pro Arg Asp Val Glu Gly Tyr Arg Gln Phe Leu Asp Tyr Ser 115 120 125 CGC GCG GTG TTT AAA GAA GGC TAT CTA AAG CTC GGT ACT GTC CCT TTT 432 Arg Ala Val Phe Lys Glu Gly Tyr Leu Lys Leu Gly Thr Val Pro Phe 130 135 140 TTA TCG TTC AGA GAC ATG CTT CGC GCC GCA CCT CAA CTG GCG AAA CTG 480 Leu Ser Phe Arg Asp Met Leu Arg Ala Ala Pro Gln Leu Ala Lys Leu 145 150 155 160 CAG GCA TGG AGA AGC GTT TAC AGT AAG GTT GCC AGT TAC ATC GAA GAT 528 Gln Ala Trp Arg Ser Val Tyr Ser Lys Val Ala Ser Tyr Ile Glu Asp 165 170 175 GAA CAT CTG CGC CAG GCG TTT TCT TTC CAC TCG CTG TTG GTG GGC GGC 576 Glu His Leu Arg Gln Ala Phe Ser Phe His Ser Leu Leu Val Gly Gly 180 185 190 AAT CCC TTC GCC ACC TCA TCC ATT TAT ACG TTG ATA CAC GCG CTG GAG 624 Asn Pro Phe Ala Thr Ser Ser Ile Tyr Thr Leu Ile His Ala Leu Glu 195 200 205 CGT GAG TGG GGC GTC TGG TTT CCG CGT GGC GGC ACC GGC GCA TTA GTT 672 Arg Glu Trp Gly Val Trp Phe Pro Arg Gly Gly Thr Gly Ala Leu Val 210 215 220 CAG GGG ATG ATA AAG CTG TTT CAG GAT CTG GGT GGC GAA GTC GTG TTA 720 Gln Gly Met Ile Lys Leu Phe Gln Asp Leu Gly Gly Glu Val Val Leu 225 230 235 240 AAC GCC AGA GTC AGC CAT ATG GAA ACG ACA GGA AAC AAG ATT GAA GCC 768 Asn Ala Arg Val Ser His Met Glu Thr Thr Gly Asn Lys Ile Glu Ala 245 250 255 GTG CAT TTA GAG GAC GGT CGC AGG TTC CTG ACG CAA GCC GTC GCG TCA 816 Val His Leu Glu Asp Gly Arg Arg Phe Leu Thr Gln Ala Val Ala Ser 260 265 270 AAT GCA GAT GTG GTT CAT ACC ACC TAT CGC GAC CTG TTA AGC CAG CAC CCT 864 Asn Ala Asp Val Val His Thr Tyr Arg Asp Leu Leu Ser Gln His Pro 275 280 285 GCC GCG GTT AAG CAG TCC AAC AAA CTG CAG ACT AAG CGC ATG AGT AAC 912 Ala Ala Val Lys Gln Ser Asn Lys Leu Gln Thr Lys Arg Met Ser Asn 290 295 300 TCT CTG TTT GTG CTC TAT TTT GGT TTG AAT CAC CAT CAT GAT CAG CTC 960 Ser Leu Phe Val Leu Tyr Phe Gly Leu Asn His His Asp Gln Leu 305 310 315 320 GCG CAT CAC ACG GTT TGT TTC GGC CCG CGT TAC CGC GAG CTG ATT GAC 1008 Ala His His Thr Val Cys Phe Gly Pro Arg Tyr Arg Glu Leu Ile Asp 325 330 335 GAA ATT TTT AAT CAT GAT GGC CTC GCA GAG GAC TTC TCA CTT TAT CTG 1056 Glu Ile Phe Asn His Asp Gly Leu Ala Glu Asp Phe Ser Leu Tyr Leu 340 345 350 CAC GCG CCC TGT GTC ACG GAT TCG TCA CTG GCG CCT GAA GGT TGC GGC 1104 His Ala Pro Cys Val Thr Asp Ser Ser Leu Ala Pro Glu Gly Cys Gly 355 360 365 AGT TAC TAT GTG TTG GCG CCG GTG CCG CAT TTA GGC ACC GCG AAC CTC 1152 Ser Tyr Tyr Val Leu Ala Pro Val Pro His Leu Gly Thr Ala Asn Leu 370 375 380 GAC TGG ACG GTT GAG GGG CCA AAA CTA CGC GAC CGT ATT TTT GCG TAC 1200 Asp Trp Thr Val Glu Gly Pro Lys Leu Arg Asp Arg Ile Phe Ala Tyr 385 390 395 400 CTT GAG CAG CAT TAC ATG CCT GGC TTA CGG AGT CAG CTG GTC ACG CAC 1248 Leu Glu Gln His Tyr Met Pro Gly Leu Arg Ser Gln Leu Val T hr His 405 410 415 CGG ATG TTT ACG CCG TTT GAT TTT CGC GAC CAG CTT AAT GCC TAT CAT 1296 Arg Met Phe Thr Pro Phe Asp Phe Arg Asp Gln Leu Asn Ala Tyr His 420 425 430 GGC TCA GCC TTT TCT GTG GAG CCC GTT CTT ACC CAG AGC GCC TGG TTT 1344 Gly Ser Ala Phe Ser Val Glu Pro Val Leu Thr Gln Ser Ala Trp Phe 435 440 445 CGG CCG CAT AAC CGC GAT AAA ACC ATT ACT AAT CTC TAC CTG GTC GGC 1392 Arg Pro His Asn Arg Asp Lys Thr Ile Thr Asn Leu Tyr Leu Val Gly 450 455 460 GCA GGC ACG CAT CCC GGC GCA GGC ATT CCT GGC GTC ATC GGC TCG GCA 1440 Ala Gly Thr His Pro Gly Ala Gly Ile Pro Gly Val Ile Gly Ser Ala 465 470 475 475 480 AAA GCG ACA GCA GGT TTG ATG CTG GAG GAT CTG ATT TGA 1479 Lys Ala Thr Ala Gly Leu Met Leu Glu Asp Leu Ile * 485 490

SEQ ID NO: 9 Sequence length: 1296 Sequence type: number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism name: Erwinia uredovora Sequence characteristics: Other information: crtX (gene name) Sequence ATG AGC CAT TTC GCG GCG ATC GCA CCG CCT TTT TAC AGC CAT GTT CGC 48 Met Ser His Phe Ala Ala Ile Ala Pro Pro Phe Tyr Ser His Val Arg 1 5 10 15 GCA TTA CAG AAT CTC GCT CAG GAA CTG GTC GCG CGC GGT CAT CGG GTG 96 Ala Leu Gln Asn Leu Ala Gln Glu Leu Val Ala Arg Gly His Arg Val 20 25 30 ACC TTT ATT CAG CAA TAC GAT ATT AAA CAC TTG ATC GAT AGC GAA ACC 144 Thr Phe Ile Gln Gln Tyr Asp Ile Lys His Leu Ile Asp Ser Glu Thr 35 40 45 ATT GGA TTT CAT TCC GTC GGG ACA GAC AGC CAT CCC CCC GGC GCG TTA 192 Ile Gly Phe His Ser Val Gly Thr Asp Ser His Pro Pro Gly Ala Leu 50 55 60 ACG CGC GTG CTA CAC CTG GCG GCT CAT CCT CTG GGG CCG TCA ATG CTG 240 Thr Arg Val Leu His Leu Ala Ala His Pro Leu Gly Pro Ser Met Leu 65 70 75 80 AAG CTC ATC AAT GAA ATG GCG CGC ACC ACC GAT ATG CTG TGC CGC GAA 288 Lys Leu Ile Asn Glu Met Ala Arg Thr Thr Asp Met Leu Cys Arg Glu 85 90 95 CTC CCC CAG GCA TTT AAC GAT CTG GCC GTC GAT GGC GTC ATT GTT GAT 336 Leu Pro Gln Ala Phe Asn Asp Leu Ala Val Asp Gly Val Ile Val Asp 100 105 110 CAA ATG GAA CCG GCA GGC GCG CTC GTT GCT GAA GCA CTG GGA CTG CCG 384 Gln Met Glu Pro Ala Gly Ala Leu Val Ala Glu Ala Leu Gly Leu Pro 115 120 125 TTT ATC TCT GTC GCC TGC GCG CTG CCT CTC AAT CGT GAA CCG GAT ATG 432 Phe Ile Ser Val Ala Cys Ala Leu Pro Leu Asn Arg Glu Pro Asp Met 130 135 140 CCC CTG GCG GTT ATG CCT TTC GAA TAC GGG ACC AGC GAC GCG GCT CGC 480 Pro Leu Ala Val Met Pro Phe Glu Tyr Gly Thr Ser Asp Ala Ala Arg 145 150 155 160 GAA CGT TAT GCC GCC AGT GAA AAA ATT TAT GAC TGG CTA ATG CGT CGT 528 Glu Arg Tyr Ala Ala Ser Glu Lys Ile Tyr Asp Trp Leu Met Arg Arg 165 170 175 CAT GAC CGT GTC ATT GCC GAA CAC AGC CAC AGA ATG GGC TTA GCC CCC 576 His Asp Arg Val Ile Ala Glu His Ser His Arg Met Gly Leu Ala Pro 180 185 190 CGG CAA AAG CTT CAC CAG TGT TTT TCG CCA CTG GCG CAA ATC AGC CAG 624 Arg Gln Lys Leu His Gln Cys Phe Ser Pro Leu Ala Gln Ile Ser Gln 195 200 205 CTT GTT CCT GAA CTG GAT TTT CCC CGC AAA GCG TTA CCG GCT TGT TTT 672 Leu Val Pro Glu Leu Asp Phe Pro Arg Lys Ala Leu Pro Ala Cys Phe 210 215 220 CAT GCC GTC GGG CCT CTG CGC GAA ACG CAC GCA CCG TCA ACG TCT TCA 720 His Ala Val Gly Pro Leu Arg Glu Thr His Ala Pro Ser Thr Ser Ser 225 230 235 240 TCC CGT TAT TTT ACA TCC TCA GAA AAA CCC CGG ATT TTC GCC TCG CTG 768 Ser Arg Tyr Phe Thr Ser Ser Glu Lys Pro Arg Ile Phe Ala Ser Leu 245 250 255 GGC ACG CTT CAG GGA CAC CGT TAT GGG CTG TTT AAA ACG ATA GTG AAA 816 Gly Thr Leu Gln Gly His Arg Tyr Gly Leu Phe Lys Thr Ile Val Lys 260 265 270 GCC TGT GAA GAA ATT GAC GGT CAG CTC CTG TTA GCC CAC TGT GGT CGT 864 Ala Cys Glu Glu Ile Asp Gly Gln Leu Leu Leu Ala His Cys Gly Arg 275 280 285 CTT ACG GAC TCT CAG TGT GAA GAG CTG GCG CGA AGC CGT CAT ACA CAG 912 Leu Thr Asp Ser Gln Cys Glu Glu Leu Ala Arg Ser Arg His Thr Gln 290 295 300 GTG GTG GAT TTT GCC GAT CAG TCA GCC GCG CTG TCT CAG GCG CAG CTG 960 Val Val Asp Phe Ala Asp Gln Ser Ala Ala Leu Ser Gln Ala Gln Leu 305 310 315 320 GCG ATC ACC CAC GGC GGC ATG AAT ACG GTA CTG GAC GCG ATT AAT TAC 1008 Ala Ile Thr His Gly Gly Met Asn Thr Val Leu Asp Ala Ile Asn Tyr 325 330 335 CGG ACG CCC CTT TTA GCG CTT CCG CTG GCC TTT GAT CAG CCC GGC GTC 1056 Arg Thr Pro Leu Leu Ala Leu Pro Leu Ala Phe Asp Gln Pro Gly Val 340 345 350 GCG TCA CGC ATC GTT TAT CAC GGC ATC GGC AAG CGT GCT TCC CGC TTT 1104 Ala Ser Arg Ile Val Tyr His Gly Ile Gly Lys Arg Ala Ser Arg Phe 355 360 365 ACC ACC AGC CAT GCT TTG GCT CGT CAG ATG CGT TCA TTG CTG ACC AAC 1152 Thr Thr Ser His Ala Leu Ala Arg Gln Met Arg Ser Leu Leu Thr Asn 370 375 380 GTC GAC TTT CAG CAG CGC ATG GCG AAA ATC CAG ACA GCC CTT CGT TTG 1200 Val Asp Phe Gln Gln Arg Met Ala Lys Ile Gln Thr Ala Leu Arg Leu 385 390 395 400 GCA GGG GGC ACC ATG GCC GCT GCC GAT ATC ATT GAG CAG GTT ATG TGC 1248 Ala Gly Gly Thr Met Ala Ala Ala Asp Ile Ile Glu Gln Val M et Cys 405 410 415 ACC GGT CAG CCT GTC TTA AGT GGG AGC GGC TAT GCA ACC GCA TTA TGA 1296 Thr Gly Gln Pro Val Leu Ser Gly Ser Gly Tyr Ala Thr Ala Leu * 420 425 430

SEQ ID NO: 10 Sequence length: 1149 Sequence type: Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism: Erwinia uredovora Sequence characteristics: Other information: crtY (gene name) Sequence ATG CAA CCG CAT TAT GAT CTG ATT CTC GTG GGG GCT GGA CTC GCG AAT 48 Met Gln Pro His Tyr Asp Leu Ile Leu Val Gly Ala Gly Leu Ala Asn 1 5 10 15 GGC CTT ATC GCC CTG CGT CTT CAG CAG CAG CAA CCT GAT ATG CGT ATT 96 Gly Leu Ile Ala Leu Arg Leu Gln Gln Gln Gln Pro Asp Met Arg Ile 20 25 30 TTG CTT ATC GAC GCC GCA CCC CAG GCG GGC GGG AAT CAT ACG TGG TCA 144 Leu Leu Ile Asp Ala Ala Pro Gln Ala Gly Gly Asn His Thr Trp Ser 35 40 45 TTT CAC CAC GAT GAT TTG ACT GAG AGC CAA CAT CGT TGG ATA GCT CCG 192 Phe His His Asp Asp Leu Thr Glu Ser Gln His Arg Trp Ile Ala Pro 50 55 60 CTG GTG GTT CAT CAC TGG CCC GAC TAT CAG GTA CGC TTT CCC ACA CGC 240 Leu Val Val His His Trp Pro Asp Tyr Gln Val Arg Phe Pro Thr Arg 65 70 75 80 CGT CGT AAG CTG AAC A GC GGC TAC TTT TGT ATT ACT TCT CAG CGT TTC 288 Arg Arg Lys Leu Asn Ser Gly Tyr Phe Cys Ile Thr Ser Gln Arg Phe 85 90 95 GCT GAG GTT TTA CAG CGA CAG TTT GGC CCG CAC TTG TGG ATG GAT ACC 336 Ala Glu Val Leu Gln Arg Gln Phe Gly Pro His Leu Trp Met Asp Thr 100 105 110 GCG GTC GCA GAG GTT AAT GCG GAA TCT GTT CGG TTG AAA AAG GGT CAG 384 Ala Val Ala Glu Val Asn Ala Glu Ser Val Arg Leu Lys Lys Gly Gln 115 120 125 GTT ATC GGT GCC CGC GCG GTG ATT GAC GGG CGG GGT TAT GCG GCA AAT 432 Val Ile Gly Ala Arg Ala Val Ile Asp Gly Arg Gly Tyr Ala Ala Asn 130 135 140 TCA GCA CTG AGC GTG GGC TTC CAG GCG TTT ATT GGC CAG GAA TGG CGA 480 Ser Ala Leu Ser Val Gly Phe Gln Ala Phe Ile Gly Gln Glu Trp Arg 145 150 155 160 TTG AGC CAC CCG CAT GGT TTA TCG TCT CCC ATT ATC ATG GAT GCC ACG 528 Leu Ser His Pro His Gly Leu Ser Ser Pro Ile Ile Met Asp Ala Thr 165 170 175 GTC GAT CAG CAA AAT GGT TAT CGC TTC GTG TAC AGC CTG CCG CTC TCG 576 Val Asp Gln Gln Asn Gly Tyr Arg Phe Val Tyr Ser Leu Pro Leu Ser 180 185 190 CCG ACC AGA TTG TTA ATT GAA GAC ACG CAC TAT ATT GAT AAT GCG ACA 624 Pro Thr Arg Leu Leu Ile Glu Asp Thr His Tyr Ile Asp Asn Ala Thr 195 200 205 TTA GAT CCT GAA TGC GCG CGG CAA AAT ATT TGC GAC TAT GCC GCG CAA 672 Leu Asp Pro Glu Cys Ala Arg Gln Asn Ile Cys Asp Tyr Ala Ala Gln 210 215 220 CAG GGT TGG CAG CTT CAG ACA CTG CTG CGA GAA GAA CAG GGC GCC TTA 720 Gln Gly Trp Gln Leu Gln Thr Leu Leu Arg Glu Glu Gln Gly Ala Leu 225 230 235 240 CCC ATT ACT CTG TCG GGC AAT GCC GAC GCA TTC TGG CAG CAG CGC CCC 768 Pro Ile Thr Leu Ser Gly Asn Ala Asp Ala Phe Trp Gln Gln Arg Pro 245 250 255 CTG GCC TGT AGT GGA TTA CGT GCC GGT CTG TTC CAT CCT ACC ACC GGC 816 Leu Ala Cys Ser Gly Leu Arg Ala Gly Leu Phe His Pro Thr Thr Gly 260 265 270 TAT TCA CTG CCG CTG GCG GTT GCC GTG GCC GAC CGC CTG AGT GCA CTT 864 Tyr Ser Leu Pro Leu Ala Val Ala Val Ala Asp Arg Leu Ser Ala Leu 275 280 285 GAT GTC TTT ACG TCG GCC TCA ATT CAC CAT GCC ATT ACG CAT TTT GCC 912 Asp Val Phe Thr Ser Ala Ser Ile His His Ala Ile Thr His Phe Ala 290 295 300 CGC GAG CGC TGG CAG CAG CAG GGC TTT TTC CGC ATG CTG AAT CGC ATG 960 Arg Glu Arg Trp Gln Gln Gln Gly Phe Phe Arg Met Leu Asn Arg Met 305 310 315 320 CTG TTT TTA GCC GGA CCC GCC GAT TCA CGC TGG CGG GGG ATG CAG CGT 1008 Leu Phe Leu Ala Gly Pro Ala Asp Ser Arg Trp Arg Val Met Gln Arg 325 330 335 TTT TAT GGT TTA CCT GAA GAT TTA ATT GCC CGT TTT TAT GCG GGA AAA 1056 Phe Tyr Gly Leu Pro Glu Asp Leu Ile Ala Arg Phe Tyr Ala Gly Lys 340 345 350 CTC ACG CTG ACC GAT CGG CTA CGT ATT CTG AGC GGC AAG CCG CCT GTT 1104 Leu Thr Leu Thr Asp Arg Leu Arg Ile Leu Ser Gly Lys Pro Pro Val 355 360 365 CCG GTA TTA GCA GCA TTG CAA GCC ATT ATG ACG ACT CAT CGT TAA 1149 Pro Val Leu Ala Ala Leu Gln Ala Ile Met Thr Thr His Arg * 370 375 380

SEQ ID NO: 11 Sequence length: 528 Sequence type: number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Organism name: Erwinia uredovora Sequence characteristics: Other information: crtZ (gene name) Sequence ATG TTG TGG ATT TGG AAT GCC CTG ATC GTT TTC GTT ACC GTG ATT GGC 48 Met Leu Trp Ile Trp Asn Ala Leu Ile Val Phe Val Thr Val Ile Gly 1 5 10 15 ATG GAA GTG ATT GCT GCA CTG GCA CAC AAA TAC ATC ATG CAC GGC TGG 96 Met Glu Val Ile Ala Ala Leu Ala His Lys Tyr Ile Met His Gly Trp 20 25 30 GGT TGG GGA TGG CAT CTT TCA CAT CAT GAA CCG CGT AAA GGT GCG TTT 144 Gly Trp Gly Trp His Leu Ser His His Glu Pro Arg Lys Gly Ala Phe 35 40 45 GAA GTT AAC GAT CTT TAT GCC GTG GTT TTT GCT GCA TTA TCG ATC CTG 192 Glu Val Asn Asp Leu Tyr Ala Val Val Phe Ala Ala Leu Ser Ile Leu 50 55 60 CTG ATT TAT CTG GGC AGT ACA GGA ATG TGG CCG CTC CAG TGG ATT GGC 240 Leu Ile Tyr Leu Gly Ser Thr Gly Met Trp Pro Leu Gln Trp Ile Gly 65 70 75 80 GCA GGT ATG ACG GCG TAT GGA TTA CTC TAT TTT ATG GTG CAC GAC GGG 288 Ala Gly Met Thr Ala Tyr Gly Leu Leu Tyr Phe Met Val His Asp Gly 85 90 95 CTG GTG CAT CAA CGT TGG CCA TTC CGC TAT ATT CCA CGC AAG GGC TAC 336 Leu Val His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Lys Gly Tyr 100 105 110 CTC AAA CGG TTG TAT ATG GCG CAC CGT ATG CAT CAC GCC GTC AGG GGC 384 Leu Lys Arg Leu Tyr Met Ala His Arg Met His His Ala Val Arg Gly 115 120 125 AAA GAA GGT TGT GTT TCT TTT GGC TTC CTC TAT GCG CCG CCC CTG TCA 432 Lys Glu Gly Cys Val Ser Phe Gly Phe Leu Tyr Ala Pro Pro Leu Ser 130 135 140 AAA CTT CAG GCG ACG CTC CGG GAA AGA CAT GGC GCT AGA GCG GGC GCT 480 Lys Leu Gln Ala Thr Leu Arg Glu Arg His Gly Ala Arg Ala Gly Ala 145 150 155 160 GCC AGA GAT GCG CAG GGC GGG GAG GAT GAG CCC GCA TCC GGG AAG TAA 528 Ala Arg Asp Ala Gln Gly Glu Asp Glu Pro Ala Ser Gly Lys * 165 170 175

[Brief description of the drawings]

FIG. 1. Plasmid pACCA containing six crt genes.
Plasmid pAC containing R25 and 5 crt genes
Explanatory drawing showing CAR16.

────────────────────────────────────────────────── ─── Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 23/00 C12P 23/00 // A23L 2/58 C09B 61/00 A C09B 61/00 A23L 2/00 M (C12N 15 / 09 ZNA C12R 1:01) (C12N 15/09 ZNA C12R 1:05) (C12N 15/09 ZNA C12R 1:18) (C12N 1/21 C12R 1:19) (C12P 23/00 C12R 1:19)

Claims (9)

[Claims] 1. An astaxanthin diglucoside represented by the following chemical structural formula (1). Embedded image 2. Adonixanthin-3'-glucoside represented by the following chemical structural formula (2). Embedded image 3. A mixture of two or three of astaxanthin diglucoside, adonixanthin-3'-glucoside and astaxanthin monoglucoside. 4. A carotenoid biosynthesis gene crtE , crtB , cr
All or a part of tI , crtY , crtZ , crtX , and crtW are introduced into a microorganism to allow these seven genes to be expressed, and the transformed microorganism is cultured in a medium, and astaxanthin diglucoside is cultured from the culture. Adonixanthin-3 '
-A method for producing carotenoid glycosides, comprising collecting at least one of glucoside and astaxanthin monoglucoside. 5. A carotenoid biosynthesis gene crtE , crtB , cr
tI , crtY , crtX are genes from Erwinia , and crtZ is
The method for producing a carotenoid glycoside according to claim 4, wherein the gene is derived from Erwinia or astaxanthin-producing marine bacteria, and crtW is a gene derived from astaxanthin-producing marine bacteria. 6. The method according to claim 4, wherein the microorganism is a bacterium or a yeast.
Or the method for producing a carotenoid glycoside according to 5. 7. The method for producing a carotenoid glycoside according to claim 6, wherein the bacterium is Escherichia coli. 8. A food improving agent or food comprising at least one of astaxanthin diglucoside, adonixanthin-3'-glucoside and astaxanthin monoglucoside (excluding the case of astaxanthin monoglucoside alone). 9. The carotenoid biosynthesis genes crtX , crtW , cr
Some or all of tZ , crtE , crtB , crtI , and crtY are introduced into plants so that these seven genes can be expressed, and the transformed plants are cultivated. Astaxanthin diglucoside, adonixanthin A method for producing a plant having a carotenoid glycoside, comprising producing at least one of -3'-glucoside and astaxanthin monoglucoside.