CA2521075A1 - Gene involved in the biosynthesis of carotenoid and marine microorganism, paracoccus haeundaensis, producing the carotenoid - Google Patents

Abstract

The present invention relates to a gene involved in the biosynthesis of carotenoid and a marine microorganism producing the carotenoid, more particularly to a gene represented by Seq ID. No. 5, 7, 9, 11, 13 and 15 which encoding the protein needed to biosynthesis of carotenoid and a marine microorganism, Paracoccus haeundaensis, producing the carotenoid. Since the gene and the microorganism can effectively be used for, the massive-production of carotenoid.

Description

GENE INVOLVED-IN-THE BIOSYNTHESIS OF
~AROTENOID .AND MARINE MICROORGANISM
PARACOCCUS HAEUNDAENSI~, PRODUCING THE
CAROTEN~ID .
F'IEhD OF THE INVENTION
The present invention relates to genes involved in the biosynthesis of carotenoid and a marine microorganism producing the carotenoid.
BI~CKGROL1ND
Carotenoid, a C40 isoprenoid compound haring an anti-oxidant activity, means a group of pigment that is widely distributed in the nature. More than C~00 hinds c~f carotenoids have been known so far, and they are all in different forms. The color of carotenoid varies from its molecular structures that is whether it is yellow, red, scarlet or orange is decided upon the molecular structure of carotenoid. The examples of carotenoids are j3 -carotene (an orange pigment included in a carrot), licopene (a red pigment included in a tomato), fucoxanthin (a yellowish brown or a brown pigment included in marine plants), etc. As a precursor of vitamin A in human, carotenoid has activities of preventing oxidation, scavenging harmful oxygen, inhibiting the proliferation of cancer cells, and preventing the development of a cancer. It suggests that it has preventive effect on cardiovascular diseases, cancers and other adult diseases. It has been disclosed recently that carotenoid enhances immunity as being exposed on UV, so that it reduces skin damages by UV or inhibits the production of melanin. Since then, carotenoid came into a spotlight as a cosmetic material in Europe and in the U.S.A. Carotenoid is now in use as a health food ingredient (nutritional supplement), a pharmaceutical composition and a food-coloring agent, or as a pigment for animal feeds.
Among many carotenoids, astaxanthine (3, 3' dihydroxy-j3 ~ ~3 -carotene-4 , 4 ° -dione ) having the structure of below <Chemical, Formula 1> is a scarlet or light orange color pigment produced in nature.
<Chemical Formula 1>

OH
HO

Astaxanthine is mostly included in tissues of marine animals such as shrimps, red seabreams, salmons and lobsters, etc (Fujita et al., Nippon ,S'uisan Gakkaishi., 49: 1855-1869, 1983; Johnson, E..
A., Crit. Rev. Biotechnol., 11: 297-326, 1991';
Nelis et al., J. ~ppl. Bacteriol., 70: 181-191~
1991). Astaxanthine not only inhibits the reactions of active oxygen to destroy T~i\TA
proteins and lipids in cells during aerobic metabolism, to cause aging in cells and tissuesa and to induce a cancer but also suppresses the generation of hydroxy or peroxy radicals (hydroxy or peroxy radicals (Palozza et al., Aroh. Bioc~hem.
Biophys., 297: 291-295, 1992~ Shimidzu et al., Fish Sci., 62: 134-137, 1996). In addition, astaxanthine has been known to have immune modulatory activity and cardioprotective effect (Jyonuchi et al., Nutr. Cancer., 19: 269-280, 1993). In particular, an antioxidant activity of astaxanthine is 10 times as high as that of other carotenoids and 100 times as high as that of a -tocopherol. However, toxicity of astaxanthine has not been reported as of today. Astaxanthine has , been widely used for the treatment and the prevention of various diseases including neurodegenerative diseases, cancers, immune , disorders, cardiovascular diseases, etc, and studies are still going on further (Beat, H. F., The Neuroscientist, 3: 21-27, 1991; Chew et al., Anticancer Res., 19: 1849-1853, 1999° Murillo E., Arch. Latlnoam. Nutr., 42: 409-413~ 1992).
Astaxanthine is also being industrially used as a coloring agent and has been registered as a food additive in the name of 'Phaffia color' in Korea.
The consumption of astaxanthine increases over 150 every year, suggesting the importance of astaxanthine. ~ , A method for chemical synthesis of astaxanthine has been recently developed by a company (Hoffman-ZaRoche, Switzerland). However, a synthesized astaxanthine showed lower in vivo absorption and weaker stability as a food additive than a natural astaxanthine, so that the use of the synthesized astaxanthine was allowed just in some of European countries. Thus, a way to synthesize a natural astaxanthine is in strong demand and especially a way to produce astaxanthine using a microorganism producing astaxanthine becomes the focus of industria-1 interest. Phaffia rhodoxyma (Miller et al., Int.
J. Syst. Bacteriol., 48: 529-536, 1976), a kind of yeast, Haematococcus pluvialis (Bubrick, Bioresour Technol., 38: 237-239, 1991), a kind of Ch.Z~rophyta, Gram-positive Brevi.,baeterium 103 (Lizuka & Nishimura, J. Gen. Appl. Microbiol., 15:
127-134, 1969) ~ Gram-negative ~lg~r~..d~acterium aurantiacum (Yokoyama et al., Bi~sci. Bi~techn~1.
Bi~chem., 58: 184-1844~ 1994)~ Parac~ccus marcusii (Harker.et al., Int. J. Syst. Bacteriol., 48: 543-548, 1998) and Parac~e~cus car~tinifaciens (Tsubokura et al., Int. J. Syst. Bacteri~1., 49:
277-282, 1999) are the examples of the microorganisms producing astaxanthine.
Studies have been focused on a gene coding an enzyme involved in biosynthesis of carotenoid for the past 6 years . As a result, a number of genes involved in biosynthesis of carotenoid were cloned from various microorganisms and functions of them were also examined (Armstrong, G. A., J.
Bacteriol., 176: 4795-4802, 1994; Sandmann, G., 1 Eur. J. Biochem., 223: 7-24, 1994; Wieland, B., J.
Bacteriol., 176: 7719-7726, 1994). The pathway of carotenoid biosynthesis is derived from FPP
(farnesyl pyrophosphate), which is an intermediate product of general isoprenoid synthesis pathway.
As seen in FIG. 8, FPP and IPP (isopentenyl pyrophosphate) turn into GGPP (geranylgeranyl pyrophosphate) by geranylgeranyl pyrophosphate synthase encoded by crtE. Then, GGPP turns into j3 -carotene by the reactions of phytoene synthase encoded by crtB, phytoene desaturase encoded by c~rtl and lycopene cyclase encoded by crty.
carotene changes into astaxanthine finally by the reactions of j3 -carotene ketolase encoded by cr t~nT
and j3 -carotene hydroxylase encoded by ert~.
Nucleotide sequences, an organization and characteristics of crt gene (carotenogenic gene) involved in biosynthesis of carotenoid have been investigated in .Rhodo.bacter capsulatus (Armstrong et al., Mol. Gen. Genet., 216: 254-268, 1989), Erwinia herbicola (Sandimann et al., FENI,S
Micro.biol. Lett. , 71 : 77-82, 1990; Hundle et a1 .~, Photochem. Photobiol., 54: 89-93, 1991) and Ervinia uredovora (Misawa et al., J. Bacteriol., 172: 6704-6712, 1990). Besides, crt gene involved in biosynthesis of carotenoid, which is composed of crtB, crtl, crtY, crtW and crt~, has been isolated from Agrobacterium aurantiacum, a marine microorganism (Norihiko et al., J. Bacteriol., 177(22): 6575-6584, 1995). Another report has been made on three genes (crtB, crtl and crtY) coding enzymes catalyzing reactions from GGPP to ~3 -carotene and Phaffia rhodoz~rma where those three genes are inserted (WO 97/23633).
Thus, the present inventors isolated and identified a no~rel Paracoccus genus microorganism producing astaxanthine and studied further to separate a gene involved in biosynthesis of carotenoid from the microorganism. As a result, the present inventors successfully cloned crtE, crtB, crtl, crtY, crtW and crt~ genes and crt gene containing all of the above genes as well, and then the inventors examined nucleotide sequences of them, too. The present inventors completed this invention by confirming that carotenoid could be produced by using the crt gene in microorganisms that were not able to produce carotenoid.
SUMMARY OF THE INVENTION
It is an object of this invention to provide a Paracoccus haeundaensis producing astaxanthine.
It is also an object of this invention to \~
provide a protein needed in biosynthesis of carotenoid and a gene having nucleotide sequences selected from a group consisting of nucleotide sequences represented each by SEA. ID. No 5, No 7, No 9, No 11, No 13 and No 15.
It is a further object of this invention to provide a gene involved in biosynthesis of carotenoid containing the above gene. In particular, the present invention provides a ert gene having nucleotide sequences represented by SEA. ID. No 4.
It is another object of this invention to provide a recombinant vector containing the gene involved in biosynthesis of carotenoid above.
It is another object of this invention to provide a method for producing carotenoid using the above gene involved in biosynthesis of carotenoid.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
In order to achieve the above object, the present invention provides a .Paracoccus haeundaensis producing astaxanthine.
The present invention also provides a protein needed in biosynthesis of carotenoid and a gene having nucleotide sequences selected from a group consisting of nucleotide sequences each represented by SEA. ID. No 5, No 7, No 9, No 11, No 1~ and No 15.
The present invention further provides a gene involved in biosynthesis of carotenoid containing the above gene. In particular, the present invention provides crt gene having nucleotide sequences represented by SEQ. ID. No 4.
The present invention also ~ provides a recombinant vector containing the gene involved in biosynthesis of carotenoid above.
The present invention further provides a method for producing carotenoid using the above gene involved in biosynthesis of carotenoid.
Hereinafter, the present invention is described in detail.
The present invention provides a Paracoccus haeundaensis producing astaxanthine.
A microorganism was isolated from seawater sample taken from Haeundae shore in Busan, Korea as a strain having orange or red color. After investigating characteristics of the strain, it was confirmed that the strain was a rod type Gram-negative bacterium having non-motility and did not form spores (see FIG. 1) . The cell size was 0.3-0 . 7 ,um in diameter and 0 . 8-2 . 5 /gym in length . The colony had orange (scarlet) color. The optimum growth temperature of the strain was 25 C, and tYie strain was never growing under 10 C or over 40 C
( see FIG. 2 ) . The optimum NaCl concentration for the growth was 1-Co(w/w) and the strain was not growing at all with over 8o (see FIG. 4). The optimum growth pH of the strain was 8 (see FIG. 3).
The strain of the present invention used only D-arabinose and galactose as a carbon source and an energy source for the growth. Neither pentoses, hexoses, sugar alcohols, ogligosaccharides nor other amino acids were used. Starch hydrolysis, cytochrome oxidase and catalase reactions were all positive. But, urease reaction was negative. The strain could not produce indole from tryptophane, but took advantage of citric acid in deed, confirmed by citric acid test. Denitrification test was also performed. As a result, the strain reduced nitrate to nitrite, but did not reduce nitrite to NZ gas. Besides, the strain did not ferment glucose. The strain of the present invention was also confirmed to be aerobic. DNA
G+C composition.of the strain was 66.9 molo. The major non-hydroxyl fatty acid was unsaturated C18:1, and the major hydroxyl fatty acid was C10:0 (3-~H) .
In general, a Parac'~e~c~us genus microorganism is an oxidase and catalase positive, gram-negative bacterium and belongs to a -3-subclass of Pr~te~~acte.~ia phylogenetically. ~ther characteristics of the microorganism were also investigated (see Table 2). As a result, it was confirmed that the strain of the present invention belonged to Pr~teok~acteria.
In order to identify the strain of the present invention more concretely, the inventors examined sequence of 16S rDNA. As a result, the nucleotide sequence of 16S rDNA of the strain of the invention had a high homology with those of Paracoccus marcusii and Paracoccus carotinifaciens.
Nevertheless, the strain of the present invention showed different characteristics, comparing to other Paracoccus genus microorganisms including the two above (see Table 4 and Table 5).
Therefore, the present inventors confirmed that the strain of the invention was a novel Parac~ccus genus microorganism and named it 'Parac~ccus haeundaensis' .
The present inventors investigated if carotenoid was generated in the Paracoccus haeundaensis, resulting in the confirmation that the strain of the present invention produced j3 -carotene and astaxanthine (see FIG. 7). The present inventors deposited the Parac~ceus haeundaensis of the present invention at KCCM
(Korean Culture Center of Microorganisms) ~n January 24, 2003 (Accession No: KCCM - 10460).
Parac~ccus haeundaensis of the present invention can be effectively used for the production of carotenoid, especially astaxanthine.
A method to produce carotenoid using a microorganism of the present invention includes the steps of culturing Paracoccus haeundaensis in a proper medium and collecting astaxanthine from the culture solution. Particularly, the strain of the present invention was cultured in PPES-II ' medium at 25 C for 4 days, which was the primary culture, then cells were collected from the culture solution. Organic solvent was added to the cells, which were cultured at 4 C for overnight, resulting in the elution of astaxanthine. In addition to the above PPES-II
medium, ZB3 medium (L~B medium complemented with 30 lVaC1) can be used as a medium for the culture of the strain. It is also preferable to add galactose to a medium in order to induce satisfactory growth of the strain and mass-produce astaxanthine . Methanol, acetone or ethyl ether can be used as an organic solvent used for the purification of astaxanthine from the cultured cells, and methanol is more preferably used. The collection of astaxanthine from the cultured cells can be performed using HPZC (high performance liquid chromatography) or TLC (thin-layer chromatography) by the conventional method known to the people in this field.
13 °

The present invention also provides a protein , required for biosynthesis of carotenoid and a gene having nucleotide sequences selected from a group consisting of sequences each represented by SEQ.
ID. No 5, No 7, No 9, No 11, No 13 and No 15.
The gene above means a gene producing astaxanthine purified from Paracoccus haeundaensis (Accession No: KCCM-10460).
It is preferred for a gene coding a protein required for biosynthesis of carotenoid of the present invention to have a sequence selected from a group consisting of sequences each represented , by SEQ. ID. No 5, No 7, No 9, No 11, No 13 and No 15. Each protein coded by each gene above has amino acid sequence represented by SEQ. ID. No 6, No 8~ No 10, No 12, No 14 and No 16o respectively.
6 genes of the present invention and proteins coded by the same are shown in the below Table 1.
<Table 1>
Gene Gene Protein Amino name acid sequence SEQ. ID. crtW ~i -carotene ketolase SEQ. ID.

No 5 No 6 SEQ. ID. crt2 ~i -carotene hydroxylase SEQ. ID.

No 7 No 8 SEQ. ID. crtY Licopene cyclase SEQ. ID.

No 9 ~ No 10 SEQ. ID. crtl Phytoene desaturase SEQ. ID.

No 11 No 12 SEQ. ID. crtB Phytoene synthase SEQ. ID.

No 13 No 14 SEQ. ID. crtE Geranylgeranyl SEQ. ID.

No 15 pyrophosphate synthase No 16 Genes provided by the present invention can be effectively used for the production of carotenoid by being inserted in various host cells.
Those genes can be used either singly or together (more than 2, at least). For example, a gene coding licopene cyclase and represented by SEQ. ID.
No 9 can be used for the production of ~ -carotene by being inserted 111 a miCr~~rganisIrl Colltai111ng crtE, crtB and crtl only. And, genes represented by SEQ. ID. No 5 and No 7, coding j3 -carotene lsetolase and ~3 -carotene hydroxylase respectively, can be used for the production of astaxanthine by being inserted in a microorganism producing ~3 -carotene (ex: Phaffia rh~do~~rma ATCC96~15) .
r The present invention further provides a carotenoid synthesis gene containing all the above genes.
A carotenoid synthesis gene is preferred to have nucleotide sequences represented by SEQ. ID.
No 4. The carotenoid synthesis gene (referred as 'crt gene' hereinafter) of the present invention includes all the carotenoid synthesis genes involved in astaxanthine production process. An organization of ,crt gene of the present invention is presented in FIG. 15. As shown in FIG. 15, the size of crt gene of the present invention is 6,223 by and includes crtW~ crt~~ crtY~ crtl~ and crtB
in that order in 5' -~ 3' direction . Each of Kpn I , Sma I , Xma I , C1a I , HindllI and BamH I recognition sequence is located therein. Stop codon of eaoh crtW~ crt~~ crt~, crtl and crtB is overlapped to start oodon of the next gene. In particular, crtF
gene is found as a complementary strand.
The present invention also provides a recombinant vector containing the above caroteno.id biosynthesis gene.
A recombinant vector of the present invention was constructed by inserting crt gene into a basic vector. Any basic vector that was generally used for gene cloning or expression could be used for the present invention without.limitation. And a choice of a vector depended on a host cell. For example, if E. coli is used as a host cell, an E.
coli specific vector having replication origin of the E. coli is preferred. Likewise, if yeast is used as a host cell, a yeast specific vector having replication origin of yeast is preferred.
A shuttle vector that has both replication origin of E. coli and replication origin of~yeast at the same time is also available. In the preferred embodiment of the present invention, the present inventors constructed a recombinant vector containing crt gene by using pCR-XL-TOPO vector, which was named 'pCR-~L-TOPO crtfull~.
The present invention also provides a strain prepared by transformed host cells with ,a recombinant vector containing the above carotenoid biosynthesis gene.
E. coli or yeast can be used as host cells of the present invention, and in particular, E. coli is preferably selected from a group consisting of XLI-Blue, TOPO, BL21(DE3) colon plus, DH1 and DH5a , but the choice is not always limited thereto. In a preferred embodiment of the present invention, a strain was prepared by transformed BL21(DE3) codon.plus, a kind of E. coli, with a recombinant vector 'pCR-XL-TOPO crtfull' which contained crt gene represented by SEQ. ID. No 4.
The present invention further provides a method for the production of carotenoid using the carotenoid biosynthesis gene.
The carotenoid producing method of the present invention is comprised of the following steps:
1) Cloning crt gene represented by SEA. ID.
No 4;
2) Constructing a recombinant vector in which.
the gene of the above step 1) was inserted;
3) Transforming a host cell with the recombinant vector of the step 2); and 4) Recovering carotenoids from the culture solution in which a strain transformed. with the above recombinant vector was being cultured.
E. coli can be used as a host cell. At this time, any E. coli strain generally used for the transformation can be used without limitation, but it is preferred to select E. coli from a group consisting of XLI-Blue, TOPO, BL21(DE3) colon plus, DHl and DH5a . In a preferred embodiment of the present invention, BL21(DE3) colon plus was 1~

selected. The choice of a host cell for the invention is not limited to E. coli, and yeast is also available.
Particularly, a strain constructed in the above step 1) ~ step 3) was cultured in a growth medium (primary culture). Cells were recovered from the culture solution. Organic solvent was added to the cells, which was further cultured at 4 C for overnight (secondary culture). At that time, it was possible to add IPTG (isopropyl-beta-D-thiogalactopyranside), an inducer inducing the production of carotenoid, into the culture solution. Carotenoid substrates such as FPP
(farnesyl pyrophosphate), GGPP (geranylgeranyl diphosphate) or GPP (geranylpyrophosphate) could also be added. Methanol, acetone or ethyl ether could be used as an organic solvent for the _ extraction of carotenoid from the culture cells, and methanol was more preferred. Carotenoid was recovered from the culture cells by using HPLC
(high performance liquid chromatography) or TLC
(thin-layer chromatography) by following the conventional method commonly known for the people in this field.

In a preferred embodiment of the present invention, the inventors measured the amount of astaxanthine produced from a transgenic strain containing crt gene. As a result, the produced astaxanthine was 110 ~tg/g (dry weight), which was far more than that produced by an astaxanthine producing strain 'Paracoccus haeundaensis' (25 ,ug/g (dry weight)). Therefore, the method for producing astaxanthine of the present invention makes possible even for a strain which cannot produce astaxanthine itself to mass-produce astaxanthine by using carotenoid biosynthesis geue~
so that it facilitates the production of medical supplies and edible pigments as a food additive containing astaxanthine.
In an example of the present invention, a genomic DNA library of Paracoccus haeundaensis was constructed in order to clone a gene coding a protein required for carotenoid biosynthesis. The construction of a genomic DNA library was performed by the conventional method commonly , known to the people in this field. In particular, a genomic DNA library was constructed by using a cosmid vector in this invention.

In another example of the present invention, 'color complementation' was used for cloning genes, - involved in carotenoid biosynthesis, from a genomic DNA library. A microorganism not producing carotenoid (ex: E. coli) is given power to generate carotenoid by being transformed with a carotenoid biosynthesis gene that was cloned from a carotenoid producing microorganism (ex:
Parac~ccus haeundaensis of the present invention).
For example, E.coli could produce ~i -carotene and cells turned into yellow after being transformed with crtE~ crtB~ crtl and crt~. .And E. c~1i transformed with crtE~ crtB,. crtl~ crty~ crtW and crt~ could produce astaxanthine and cells turned into orange. The present inventors cultured a genomic DNA library of .Parsc~ccus haeund_aensis in a medium supplemented with FPP (farnesyl pyrophosphate), a common substrate of carotenoid.
As a result, 13 colonies having orange color were selected. Then,~a cosmid vector was isolated from each colony. Nucleotide sequence of DNA insert included in the vector was identified. As a result, it was confirmed that the ~si~e of the smallest DNA insert was 6,223 bp. The nucleotide sequence of the DNA insert was represented by SEQ.

ID. No 4.
In another example of the present invention, nucleotide sequences of 6,223 by long DNA insert assumed to contain a gene producing carotenoid was investigated, resulting in the obtainment of 6 ORFs. Those were analyzed nucleotided on NCBI
GenBank. As a result, amino acid sequences translated from each ORF had a high homology with amino acid sequences of 6 enzymes involved in the reaction inducing astaxanthine production from FPP
(see FIG. 9 - FIG. 14). From the result, the present inventors confirmed that the DNA insert isolated in this invention had crt gene coding a protein necessary for carotenoid biosynthesis (see FIG. 15) .
In another example of the present invention~
the inventors investigated if E. c'~1i not producing carotenoid could produce it by the insertion of crt gene coding a relevant protein.
First, recombinant vector 'pCR-XZ-TOPO-crtfull', in which crt gene was inserted, was constructed (see FIG. 16). Then, the prepared recombinant vector was inserted in E. coli. Zastly, E. c~1i transformant having orange color was selected. In order to confirm if the transformant could produce astaxanthine, the transformant was cultured and then cells were collected. Methanol was added to the obtained cells, which were cultured at 4 C for overnight. Then, supernatant was obtained and optical density was measured at 190-900 nm. As a result, as shown in FIG. 17, original peaks of ~iI-carotene and astaxanthine were confirmed. For more accurate analysis, HPLC assay was performed with some of the supernatant. At that time, j3 -carotene and astaxanthine purchased from Sigma were used as standard substances. As a result, it was confirmed that a transformant, in which ert gene isolated by the present inventors was inserted, could produce ~ -carotene and astaxanthine. The amount of astaxanthine produced from the transformant of the present invention was 110 ,ug/g (dry weight). The result suggested that E. c~1i in which crt gene was inserted could produce astaxanthine far more than Paracoccus haeundaensis, an astaxanthine producing strain, could do (25 ,ug/g (dry weight) ) .
BRIEF DESCRIPTION OF THE DRAWINGS
The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
FIG. 1 is a set of photographs showing the result of observation with a transmission electron microscope on the strain of the present invention in exponential stage, and each bar is 200 nm in length.
FIG. 2 is a graph showing the growth curve of the strain of the present invention depending on temperature.
FIG. 3 is a graph showing the growth curve of the strain of the present invention depending on pH.
FIG. 4 is a graph showing the growth curve of the strain of the present invention depending on culture time.
n FIG. 5 is a graph showing the growth curve of the strain of the present invention depending on NaCl concentration.
' FIG. 6 is ,a schematic diagram showing the phylogenetic dendrogram of the Paracoccus genus microorganisms of the present invention, based on 16S rDNA sequence data, and a bar represents maximum-parsimony distance (1 nucleotide substitutions per 100 nucleotides).
FIG. 7 is a set of graphs showing the results of scanning on the ranges of optical density changes at 190 ~ 090 nm (A) and at 400 ~ 550 nm (B) of a methanol extract extracted from the culture solution of the strain of the present .
invention. The peak at 450 nm is the peculiar pear of j3 -carotene, and the peak at 470 nm is the original peak of astaxanthine.
FIG. 0 is a schematic diagram showing the pathway of carotenoid biosynthesis.
FIG. 9 is a schematic diagram showing the result of comparison between an amino acid sequence of the first 0RF (open reading frame) included in a DNA insert isolated from Paracoccus ~, haeundaensis and amino acid sequences of ~i -carotene ketolase (crtW) isolated from an Alca.ligenes genus microorganism and a Bradyrhizo.bium genus microorganism.
P. haeundaensis: Paracoccus haeundaensis Alcaligenes_sp: Alcaligenes genus microorganism Bradyrhizobium sp: Bradyrhizobium genus microorganism Consensus: corresponding amino acid sequence FIG. 10 is a schematic diagram showing the result of comparison between amino acid sequences of the second ORF included in a DNA insert isolated from Paracoc~cus haeundaensis and amino acid sequences of j~ -carotene hydro~ylase (crt~) isolated from an Alcaligenes genus microorganism.
P. haeundaensis: Paracoe~cus haeundaensis Alcaligenes-sp: Aloaligenes genus microorganism Consensus: corresponding amino acid sequence FIG. 11 is a schematic diagram showing the result of comparison between amino acid sequences of the third ORF included in a DNA insert isolated from Paracoccus haeundaensis and amino acid sequences of licopene cyclase (crtY) isolated from an Flavobacterium genus microorganism.
P. haeundaensis: Paracoccus haeundaensis Flavobacterium sp: Flavobacterium genus microorganism Consensus: corresponding amino acid sequence FIG. 12 is, a schematic diagram showing the result of comparison between amino acid sequences of the fourth ORF included in a DNA insert isolated from Paracoccus haeundaensis and amino acid sequences of phytoene desaturase (ortl) isolated from an Flavol.~acterium genus microorganism.
P. haeundaensis: Paracocous haeundaensis Flavobacterium sp: Flavo.bacterium genus microorganism Consensus: corresponding amino acid sequence FIG. 13 is a schematic diagram showing the result of comparison between amino acid sequences of the fifth. ORF included in a DNA insert isolated from Paracoccus haeundaensis and amino acid sequences of phytoene synthase (crtB) isolated from an Flavobacterium genus microorganism.
P. haeundaensis: Paracoccus haeundaensis Flavobacterium sp: Flavo.bacterium genus microorganism Consensus: corresponding amino acid sequence FIG. 14 is a schematic diagram showing the result of comparison between amino acid sequences of the sixth ORF included in a DNA insert isolated from Paracoccus haeundaensis and amino acid sequences of geranylgeranyl pyrophosphate synthase ( crtE) isolated from an Flavo.baoterium genus microorganism.
P. haeundaensis: Para~oecus haeundaensis Flavobacterium-sp: Flavo.laae~teri~am genus ml~r ~orga111. sm FIG. 15 is a schematic diagram showing the organisation of crt gene isolated from Paracoccus haeundaensis.
FIG. 16 is a schematic diagram showing the cleavage map of pCR-XL-TOPO-crtfull vector of the present invention.
FIG. 17 is a set of graphs showing the results of scanning on the ranges of optical 2~

density changes at 190 ~ 890 nm (A) and at 350 550 nm (B) of a methanol extract extracted from the culture cells of the transformant of the present invention in which crt gene was inserted.
The peak at 450 nm is the peculiar peak of ~i -carotene, and the peak at 470 nm is the original peak of astaxanthine.
EXAMPLES
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure~ may make modifications and improvements within the spirit and scope of the present invention.
Example 1- Sample collection, microoraanism .~~aaration, cultivation and maintenance of the same Seawater sample taken from Haeundae shore in Busan, Korea, was diluted by 1/1000, which was then smeared on a nutrient agar medium (Difco) and cultured at 25 C for 3 days. Among microorganisms cultured thereby, those having orange or red color were isolated. In order to isolate a microorganism producing carotenoid among strains isolated, all the candidates were cultured in PPES-II medium (tripton 1 g/.C , bacto-soyton 1 g/.C , ferric citrate 0.01 g/.~ , polypeptone 2 g/.~ and sodium chloride 3 g/.~ ). Cells were collected from the culture solution. Carotenoid was extracted by using methanol to identify a final strain producing carotenoid. The isolated microorganism was named °BC74171T strain°.
Example ~' ~ Invest ' gation of plzenot~rp~
characteristics In order to investigate morphological and physiological characteristics of the BC74171T
strain of the present invention, experiments were performed as follows. .
<2-1> Morphological characteristics BC74171T strain of the present invention was cultured in PPE.S-II medium at 25 C for 3 days.
The cultured cells were suspended in 0.1 M
phosphate buffer (pH 7.2). The cells were fixed by 2o glutaraldehyde, washed with 0.05 M
cacodylate buffer solution, and fixed again by 10 osmium tetroxide. The fixed cells were dehydrated in ethanol that was later replaced with propylene.
Those cells were put into EPON resin and sections were prepared using an ultramicrotome. The prepared sections were observed under a JEM
1200EX-II transmission electron microscope (TEM).
As a result, as shown in FIG. 1, BC74171T , strain of the present invention had a shape of rod and was 0.3-0.7 ,um in diameter and 0.0-2.5 /gym in length. The strain did not form spores. The mobility of the strain was also observed under an optical microscope by hanging-drop technique (Skerman, V. B. D. , A Guide t~ the Identificati~n of the Genera of Bacteria~ 2nd den. Batimore, 1967). As a result, BC74171T strain of the present invention was non-motile. In addition, BC74171T strain was Gram-negative, and its colonies were flat and had a light orange color.

<2-2> Phy io oa~cal characteri ics 1. Range of temperature BC74171T of the present invention was cultured in a nutrition agar medium (Difco) at different temperatures (4, 10, 20, 25, 28, 30, 37, 40 and 50 C ) for 10 days to investigate the range of growth temperature. As a result, BC74171T
strain was growing at temperature ranging 20-37 C, and the optimum growth temperature was 25 C (Talale 2 and FIG. 2).
<Ta~le 2>
Characteristics of Pa.rae~cc~us haeundaens.zs Morphological Utilization characteristics Cell 0.3-0.7 D-Glucose -diameter (,ccm) Cell length (,c~tn)~ - 8-2 Maltose -. 5 Mo~ility ~- D-Galactose *+

Spore formation - Sucrose -Optimum growth 25 Mannitol -temperature(C) Optimum growth 8 Cellobiose -pH

NaCl 7 Trehalose -resistance ( o ) Product Xylose -Indole - Dulcito -MR(Methyl red - Salicin -test) VP(Voges- - Adonitol -Proskauer test) Hydrogen sulfide - Inositol -Citrate + Arabinose +

Enzyme activity Raffinose -Catalase + Rhamnose -Urease - D-Fructose -Oxidase + D-Mannose - , Starch + Dimethylformamide -hydrolysis Denitrification Glycerol -Nitrate --' + L-Glutamic acid -Nitrite Nitrite -~ NZ - Sorbitol -gas Color +(light Lactose -orange ) Chemotaxonomic L-Asparagine -characteristics G + C content 66.9 Acetone -(mol o ) Non-hydroxyl C18:1 Major carotenoid Asta acids xant hine 3 Hydroxyl fatty 3-OH
acids Clo:o *-: Positive response, *+: Negative response 2. Range of pH

BC74171T of the present invention was cultured in PPES-II media each having different pH
(3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10Ø 10.5 and 11 . 0 ) for 10 day s to investigate growth pH range .
As a result, the optimum growth pH was 8. The growth of the strain was inhibited or retarded under pH 6 and over pH 10.5 (Table 2 and FIG. 3).
3. Growth BC74171T strain of the present invention was shaking-cultured in a PPES-II medium at 25 C for 10 days to investigate its growth by measuring turbidity. As a result6 the growth of the strain was rapidly increased from ~0 hours after the culture and slowly decreased from 50 hours after the culture (FIG. 4).
4. Resistance against NaCl BC74171T of the present invention was cultured in a trypticase soy broth medium containing 1-10 0 (w/v) NaCl for 10 days at various temperatures (4, 10, 20, ~5, 28, 30, 37, 40 and 50 C). As a result, the optimum NaCl concentration for grorath was 1-60. The growth was retarded with 7o NaCl and was stopped over 80 (Table 2 and FIG. 5).
5. Utilizing ability of carbon source In order to examine a capacity to use a carbon source, a micro-plate including 24 substrates listed in the below Table 3 was used.
The final concentration of each substrate was adjusted to 1o, which was loaded in a puple broth base (Difro), after being filtered or sterilized by moist heat. The medium was inoculated with the strain of the present invention, then was cultured at 25 C for 10 days. Yellow meant a positive reaction and purple, which was an original color of the medium~ meant a negative reaction.
<Table 3>
Substrates used in the carbon source utilizing ability test and the utilizing ability Substrate Utilizin Substrate Utilizin g g ability ability 1 D-Glucose *- 13 Arabinose +

2 Maltose - 14 Raffinose -3 D-Galactose *+ 15 Rhamnose -4 Sucrose - 16 D- -Fructose 5 Mannitol - 17 D-Mannose -6 Cellobiose - 18 Dimethylf -ormamide 7 Trehalose - 19 Glycerol 8 Xylose - 20 L- -Glutamic acid 9 Dulcitol - 21 Sorbitol . 10 Salicin - 22 Lactose -11 Adonitol - 23 L- -Asparagin a 12 Inositol - 24 Acetone -*-: Not used (positive reaction), +: Used (negative reaction) As a result~ as shown in Table 3, ~C74171T ' strain of the present invention used only D-arabinose and galactose as a Garbon source, and no other substrates were used.
6. Activity of starch hydrolysis In order t~ investigate if the strain of the present invention could hydrolyze starch, a starch agar was inoculated with the strain, then, cultured for 10 days. An activity of starch hydrolysis was measured by the method of Cowan, S.
T. & Steel, K. J. (Cowan, S. T. & Steel, K. J.~, Manual for the Identification of Medical Bacteria.
London: Cambridge University Press, 1965). As a result, the strain of the present invention was confirmed to have an activity of starch hydrolysis (Table 2).
7. Indole test (tryptophanase activity) In order to investigate if the strain of the present invention could produce indole by decomposing tryptophane, the present inventors took advantage of the method of Cappuccino et a1.
(Cappuccino J. G. and Sherman, N. In Micr~biology:
a laboratory manual (6th) 2001), resulting in the confirmation that indole was not produced from tryptophane (Table 2).
~. Acid production (fermentation) from carbohydrates Acid production was investigated by the method of Hughm et a1. (Hughm et al., J.
Bacteri~1., 66: 24-26, 1953). Methyl Red test and Voges-Proskauer test were performed and the results were all negative (Table 2).
9. Citric acid test In order to investigate if the strain of the present invention could be growing by using citric acid as a carbon source, the method of Cappuccino et a1. (Cappuccino J. G. and Sherman, N. In Microbiology: a laboratory manual (6th) 2001) was performed and the result was positive (Table 2).
10. Catalase activity The catalase activity was investigated by observing bubbles in 3o hydtogen peroxide solution by following the method of Harker et a1. (Harker et al., J. Clin. Microbi~1., 2: 463-464, 1975).
As a result, the catalse activity of the strain of the present invention was confirmed to be positive (Table 2).
11. Oxidase activity The oxidase activity was investigated by using 1o p-aminodimethylaniline oxalate as a substrate by following the method of Cappuccino et a1. (Cappuccino J. G. and Sherman, N. In Micr~.b.i~Iogy: a la.b~rat~ry manual (6th) 2001) . As a result, the cytochrome oxidase activity of the strain of the present invention was confirmed to be positive (Table 2).
12. Urease activity The urease, activity was measured by the method of Lanyi (Lanyi, B. Methods Microbiol., 19:
1-67, 1987). As a result, the urease activity of the strain of the present invention was confirmed to be negative (Table 2).

13. Denitrification In order to observe denitrification, gas production and growth were investigated through stab culture using a nutritive medium containing O.lo (w/v) agar; by following the method of Cappuccino et al,. (Cappuccino J. G. and Sherman, N.
In Microbiology: a laboratory manual (~t.h) ~001)~.
As a result, BC74171T strain of the present invention reduced nitrate to nitrite. But, the strain could not reduce nitrite to 1~T2 gas (Table 2) .

14. Hydrogen sulfide production test In order to investigate if the strain of the present invention could produce hydrogen sulfide from amino acid containing sulfur like cysteine or a substrate like inorganic sulfur compounds, , hydrogen sulfide production test was performed using TSI (Triple Sugar Iron Agar) medium (Cappuccino J. G. and Sherman, N. In Microbiology:
a laboratory manual (6th) 2001). The result was negative (Table 2).
Example 3: Fatty acid analysis BC74171T strain of the present invention was cultured on trypticase soy agar medium (pH 8.0) supplemented with 2% NaCl at 25 C for 2 days.
Then, harvesting, saponification, methylation and extraction of FAMEs (fatty acid methyl esters) were performed with the cells cultured by the method of Sasser (Sasser, M. In Methods in Phytobaeteriology, 199-204~ 1990). GC-MS was performed following the method of hipske et a1.
(Lipski et a1. ~ Syst. ~l~a~tal . Mio-robiol. , 20 : 448-457~ 1997), leading to the investigation of FAMEs.
As a result, according to the confirmed fatty acid profile (C18:1, 84 . 32 a ~ Cls:o~ 7 . 79 0 ~ Cio:o (5-~H) 2.06 of Cl~:lcis5 2.0 0; Ci4:o (3-OH) , 1.47 0~ Cl7:o, 0.80 0~ Cl6:o, 0.78 0; and unknown peak, 0.78 a) the strain of the present invention was°confirmed to belong to a -subclass of Proteobacteria. And a major hydroxyl fatty acid was Clo:o (3-CH) (Table 2) .

Example 4- Base composition of DNA
The base composition of genomic DNA of BC74171T strain of the present invention was identified by the method of Tamaoka et a1.
(Tamaoka, J. & Komagata, K. FEMS Microbiol. .Lett.~, 25: 125-128, 1984). Particularly, the genomic DNA
was extracted from BC74171T strain of the present invention by following the conventional method known to the people in this field. The extracted genomic DNA was hydrolysed. The obtained nucleotides were analy~,ed by HPLC (reverse-phase HPLC). As a result, G+C content of DNA of BC74171T strain was 66.9 molo (Table 2) .
Example 5' 16S rDNA sec~uencinc~ and phylogenetic dendroc~ram analysis Genomic DNA was extracted from BC74171T
strain of the present invention by the method of Rainey et a1. (Rainey et al., Syst. Appl.
Micro.,taiol., 15: 197-202, 1992). 16S rDNA was PCR
amplified by using primers represented by SEA. ID.
No 1 and no 2. PCR was performed as follows. 20 pmol each of two primers, 10 ng of genomic DNA, 1 unit of Taq polymerase and 10x buffer solution0(with MgCl2) were mixed to make the reacting solution. The prepared reacting solution was pre-denatured at 94 C for 5 minutes, followed by 25 cycles of denaturation at 94'C for 1 minute, annealing at 56C for 30 seconds, polymerization at 72 C for 90 seconds, and final extension at 72 C for 10 minutes. The PCR product was cloned into pGEM-T vector (Promega), and isolated DNA
clones were reacted with ABI PRISMTM staining reagent (Perkin Elmer, LISA). Nucleotide sequence was determined by using ABI 377 genetic analyzer ( Perkin Elmer~ LTSA) .
As a result, 16S rDNA of BC74171T strain of the present invention was 1451 by long and had nucleotide sequences represented by SEQ. ID. No 3.
The nucleotide sequence of 16S rDNA of BC74171T
strain was analyzed by using BLASTN and BLASTX of NCBI GenBank database. As a result, the nucleotide sequence of 16S rDNA of BC74171T strain showed 99.80 and 99.60 homology each with those of Paracoccus marcusii and Paracoccus carotinifaciens which have been known to produce astaxanthine among various Paracoccus genus microorganisms.

Thus, BC74171T strain of the present invention was confirmed to be a Paracoccus genus strain. And the characteristics of the strain of the present invention were compared with those of various Paracoccus genus microorganisms. The characteristics of other Paracoccus genus microorganism listed in the below Table 4 (2-15) were the results of investigations by Harker et al.
(Harker et al., Int. J. Syst. Bacteriol., 48: 543-548, 1998), Lipski et a1. (Lipski et al., Syst.
Appl. Microbiol.., 20: 448-457, 1997), Tsubokura et a1. (Tsubokura et al., Int. J. Syst. Bacteri~1.', 49: 277-282, 1999)~ Kelly et a1. (Kelly et al., The genus Parac~ccus. In Th.e Pr~karyotes, http://www.prokaryotes.com., Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer ~ E.
Stackebrandt. ~Tew York: Springer., 2000) and Doronina et al. (Doronina et al., Int. J. Syst.
.uV~1. Bacteri~1.~ 52: 679-6, 2002). As shown in Table 4, BC74171T strain of the present invention did not share any characteristics with any other Parac~ccus genus~microorganism. , <Table 4>
Comparison of the characteristics of the strain of the present invention and those of other Paracoccus genus microorganisms . 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mobilit - - - - - - - - - - - - +

y - +

Growth + - NR - - NR - - - ~n1NR NR - NR NR

in NaCl Gluco - + + + + + + + + + + +

se , Arabi + + + + +

s nose a f Glyce - + + + NR + + + + + +

a rol 1 Sucro - NR + + + + +

n se a - Manni - + + + + + + + + + +

s tol s Fruct - + + NR + + + + + + +

ose Denitri - - + - - - + + + - - + + + +

ficatio n Llrease - + NR - - - + - + NR + - NR - -activit y Yellow + - NR - - + - - - + - - - - -color G 66 64 NR 63 67 67 64 71.62 66 67 66 68 66 67 +
C

content .9 - - .5 .5 .5 -(mol 66 67 - - 68 o ) .6 *1: The strain of the present invention 2: P. alcaliphibus (JCM 7364T) 3: P. alkenifer (DSM 11593T) 4: P. aminophilus (JCM 7686T) 5: P. aminovorans (JCM 7685T) 6: P. carotinifaciens (IFO 16121T) 7: P. denitrificans (ATCC 17741T) 8: P. kocurii (JCM 7684T) 9: P. kondratievae (VKM B-2222T) 10: P. marcusii (DSM 11574T) 11: P. methylutens (VKM B-2164T) . 10 12: P. pantotrophus (ATCC 35512T) 13: P. solventivorans (DSM 6637T) 14: P. thiocyanatus (IAM 12816T) 15, P. versutus (ATCC 25364T) '~+: Positi~ae reaction . Negative reaction W: Weak reaction) NR: Not reported The characteristics of BC74171T strain of the present invention were different in many ways even with Paracoccus marcusii and Paracoccus carotinifaciens showing the highest 16S rDNA
Sequence homology. More precisely, as shown in the below Table 5, Paracoccus marcusii (Hacker et al., Int. J. Syst. Bacteriol.,. 48: 543-548, 1998) was a coccus or a short rod type forming a short chain. On the contrary, BC74171T strain of the present invention was a rod type not forming a chain. Paracoccus carotinifaciens (Tsubokura et al., Int. J. Syst. Bacteriol., 49: 277-282, 1999) was also a rod type not forming a chain but had flagella. However, BC74171T strain of the present invention had not flagella. The strain of the present invention did not use any of glucose, mannitol, maltose or mannose, but the other two strains did use them. Besides, BC74171T strain of the present invention decomposed starch. But, the two other strains did not.
<Table 5>
Comparison of the characteristics of the strain of the present invention and those of other Parac~c~cus genus microorganisms Characteris BC74171T Parac~ccus Paracoccus tics marcusii carotinifacien s Cell Rod type Coccus or Rod type morphology short rod type Mobility -~ - +~

U Glucose - + +

s Maltose - + +

f Mannitol - +

a 1 Arabinose + + -n a s 46 s Citric + + -acid Mannose - NR* +

Starch + -hydrolysis Color + + +

*+: Positive reaction, . Negative reaction, NR: Not reported Considering all the above results, BC74171T
strain of the present invention was confirmed to be a novel microorganism belonging to Paracoccus genus. The present inventors named the BC74171T
strain as 'Parac~occus haeundaensis~ and deposited it at KCCM (Korean Culture Center of Microorganisms), on January 24, 2008 (accession No: KCCM-10460). Systematic position of the Parac~ccus .haeuno_'aensis of the present invention was shown in FIG. 6. Phylogenetic dendrogram was made out by using Treeview program. Bootstrap analysis (1000 replications) was performed using a method measuring distance and parsimony (Agnes Groisillier and Aline Lonvaud-Funel, International Journal of Systematic Bacteriology, 49: 1417-1428, 1999 ) .

Examx~le 6: Astaxanthine production in the strain Qf the present invention The strain of the present invention was t cultured in 50 m.~ of medium (yeast extract 1%, tryptone 0.50, NaCl 30) at 25'C for 6 days. The cells were collected by centrifugation (13,000 rpm). The collected cells were suspended in 20 m.~
of methanol, which. were then cultured overnight at 4 C. Centrifugation was performed again with 13,000 rpm to obtain supernatant. ~ptical density was measured at 190-900 nm.
As a result, as shown in FIG. 7, peaks were observed both. at 450 nm and at 470 nm, and those peaks were confirmed to be original peaks of j3,.-carotene and asta_xanthine .
For the accuracy of analysis~ 1 m.~ of the supernatant was taken and filtered by a 0.45 ,um filter, followed by HPLC analysis (column: 4.6~
250 mm, uBondapak C10, ~nlaters, Milford, MA~ mobile phase: acetonitrile-methanol-water(49:44:7 v/v), Flow: 10 m.~/min, Detector: 470 nm) . ~3 -carotene and astaxanthine purchased from Sigma were used as a standard substance. As a result, the strain of the present invention did produce astaxanthine and j3 -carotene. The amount of astaxanthine produced , from the strain was 25 ug/g (dry weight).
Therefore, it was confirmed that bright orange color of the strain was caused by carotenoid biosynthesis in a cell and a major pigment accumulated in the cell was astaxanthine.
Example 7: Preparation of aenomic DNA for the cloning of a carotenoid biosynthesis gene .Pa.racoccus haeunclaensis (KCCM-10460) was cultured in a PPES-II medium (tripton 1 g/~;, bacto-soyton 1 g/.~ , ferric citrate 0.01 g/~ , polypepton 2 g/.~ and NaCl 3 g/.C ) at 25 C for 10 days. Then, the culture solution was centrifuged at 13,000 rpm to collect cells. In order to isolate genomic DNA from cells, the cells were suspended in STE buffer solution (10 mM Tris, 1 mM
EDTA, 100 mM NaCl, pH 8.0), which was further reacted at 68 C for 15 minutes. The cells obtained from centrifugation were re-suspended in solution I (50 mM glucose, 25 mM Tris, 10 mM EDTA, pH 8 . 0 ) . 5 mg/m.~ of lysozyme and 100 ,ug/m.~ of RNase A were added, followed by reaction at 37 C for 1 hour. Then, 250 ,ug/m.~ of proteinase K was added, followed by reaction at 37 Cfor 3 hours. N-lauroylsarcosine was added by to of total volume, followed by reaction at 37 C. Genomic DNA was purified by phenol-chloroform extraction method.
Extraction was performed by adding same volume of phenol-chloroform and genomic DNA was precipitated by adding 1000 ethanol twice as much as the whole volume. The precipitated DNA was washed with 70~
ethanol. TE buffer solution was added to dissolve the precipitated DNA completely at 65 C, which was then ready to be used.
Example 8- Construction of genomic DNA library <8-1> Preparation of a cosmid vector ~laa I (9 U/,~g) restriction enzyme was added to 25 ,fig of a cosmid vector (SuperCos 1 Cosmid Vector, Stratagene), making the total volume of reacting solution 200 ,cry, and the solution was reacted at 37 C for 1 hour, followed by digestion.
Vector DNA was purified. by phenol-chloroform v.
extraction method, which was precipitated by 1000 ethanol. For dephosphorylation of the vector digested with X.ba I, CIAP enzyme (.Promega) was added, followed by reaction at 37°C for 30 minutes.

Then, the purified vector was reacted with BamH I
(5 U/,ctg) at 37 C for 1 hour. Extraction was performed by phenol-chloroform extraction method, and precipitation was induced by using ethanol.
The precipitate was dissolved in TE buffer solution, making the concentration 1 ,ug/,ue .
<8-2> Construction of aenomic DNA library 100 ,ttg of Paracoccus haeundaensis genomic DNA
obtained in the above Example 7 was treated with Sau3A I (10 U), inducing a partial enzyme reaction.
Upon completing the enzyme reaction, 0.5 1 EDTA
was added. Then, genomic DNA was separated by phenol-chloroform extraction method, which. was precipitated by 1000 ethanol. Genomic DNA, a product of a partial enzyme reaction, was dissolved in TE buffer solution, and treated with CIAP enzyme by the same method as used in the above Example 7, leading to dephosphorylation.
DNA was separated again by phenol-chloroform extraction method. For the ligation with a cosmid vector prepared in the above Example <8-1>, T4 ligase (Promega) and 10x ligase buffer (Promega) were added to the isolated DNA, followed by reaction at 12 C for 18 hours. After completing the reaction, an E. coli strain XZ1-Blue (Stratagene) was transformed with the ligation mixture, resulting in the construction of a genomic DNA library.
Example 9- Examination and analysis of a transaenic strain containing a color-producing sane FPP (Sigma), one of common substrates included in carotenoids, was added to a ZB agar medium by to out of total volume. A genomic library constructed in the above Example 0 was smeared on the plate, which was cultured at 37 C .
Among cultured colonies, the ones having orange color were selected (13 out of about X000 ' colonies). Cosmid vectors were isolated from those 13 colonies selected above. Then, primer working sequencing was performed ~to identify nucleotide sequences of DNA fragments inserted in each cosmid vector. Identification of the nucleotide sequences was committed to GenoTech Corp., Korea.

As a result, the smallest DNA insert of all fragments inserted in the cosmid vectors was 6,223 by long, and had nucleotide sequences represented by SEQ . ID . No 4 . A cosmid vector containing the j nucleotide sequence represented by SEQ. ID. No ~4 was named 'COSCRT'.
Example 10: See~uence anal~rsis of a DNA insert containing a carotenoid biosynthesis related aene Sequence analysis of a DNA insert obtained in the above Example 9 was performed using NCEI ORF
Finder program (http://www.ncbi,.nlm.nih.gov/gorf/gorf.html) to analyze ORF.
As a result~ 6 ORFs were included in a DNA
insert obtained in the above Example 3. Each. ORF
showed a relevant homology with nucleotide . sequences of crtW coding ~3 -carotene ketolase, crt2 coding ~3 -carotene hydroxlylase, crtY coding licopene cyclase, crtl coding phytoene desaturase, crtB coding phytoene synthase and crtE coding geranylgeranyl pyrophosphate synthase. All of those enzymes were confirmed to be involved in carotenoid biosynthesis. , 53 , The comparison of homology between amino acid sequences translated from each ORF and amino acid sequences of each carotenoid biosynthesis enzyme isolated from Alcaligenes sp. (Misawa et al., Biochem. Biophys. Res. Commun., 209(3): 867-876, 1995), Bradyrhizobium sp. (Hannibal et al., J.
Bacteriol., 182(13): 3850-3853, 2000) and Flavolaacterium sp. (Pasamotes et a1. , Gene, 185.( 1 ) : 35-41, 1997 ) was shown in FIG. 9 and FIG.
14. The nucleotide sequences of 6 ORF genes cloned by the present inventors and amino acid sequences translated therefrom were each represented by ~SEQ. ID. No 5 ~ No 16. In , particular, or tG~~ e~r t~, c~r t~, crtl and ortB genes were each represented by SEA. ID. No 5, No 7, No 9, No 11, No 13 and No 15 and amino acid sequences of each gene abo~re were represented by SEA. ID. No 6, No 8, No 9, No 12, No 13 and No 16, respectively.
It was confirmed from the above results that crt gene involved in biosynthesis of carotenoid was included in a DNA fragment inserted in the cosmid vector above.
The composition of crt gene of the present invention was shown in FIG. 15. As shown in FIG. ~, 15, termination codon and start codon were overlapped in each crtW, crt2, crtY, and crtl. ~~
Especially, crtE gene seemed to have a directionality of complementary strands, and had each of recognition sequences of KpnI, XmaI, SmaI, ClaI, HindILI and BamH in its sequences.
xample 11' EXTJrP~g ~ On pf rrt rrPnc~ -j n F' C07 ~
In order to investigate if carotenoid could be produced by a protein expressed by ert gene of Paraooccus .haeundaensis isolated in the above Example 9, crt gene was first amplified by PCR
using HL premix (Bioneer). At that time~
oligoneucleotide primers represented by SEQ. ID.
No 17 and No 18 were used. All the PCRs were performed as followso pre-denaturation at 94 C for 5 minutes, denaturation at 94°C for 50 seconds~
annealing at 66C for 30 seconds, polymerization at 72 C for 6 minute, 25 cycles from denaturation to polymerization, and final extension at 72 C for minutes. The PCR product was inserted in Topo-XL-vector (Invitrogen), which was used for transduction of E. coli. At that time, XL1-Blue ' (Stratagene), TOPO (Invitrogen), BL21(DE3) codon plus (Stratagene) , DH1 (Takara) and DHSa (Takara) were used as E. coli of the present invention. As a result, all transformants of BL21(DE3), XLl-Blue, BL2l(DE3) codon plus were confirmed to have orange color. Thereafter, each transformant of E. coli was selected for culture. As a result, transformed BL21(DE3) codon plus produced astaxanthine most.
The present inventors inserted crt gene having nucleotide sequences represented by SEQ. ID.
No 4, isolated in the above Example 9, into pCR-XL-TOPO vector (Invitrogen), a gene expression vector, which was then named 'pCR-XL-TOPO-crtfull°
(FIG. 1~). BL21(DE3) codon plus cells were transfected with the vector 'pCR-XL-TOPO-crtfull, followed by selection of strains having orange color. The selected strains were cultured in 50 m.@ of LB medium at 37 °C for 8 hours . The culture solution was centrifuged at 13~000 rpm, then supernatant was discarded and cells were collected.
20 m.~ of methanol was added to the collected cells. , After vortexing, the cells were cultured at 4 C
for overnight. Centrifugation was performed (13,000 rpm) to obtain supernatant. In order to confirm whether carotenoid was generated, optical density was measured at 190-900 nm and 400-550 nm.
As a result, peaks were observed at 450 nm and 470 nm, which were confirmed to be original peaks of ~i -carotene and astaxanthine (FIG. 17).
For the accuracy of analysis, 1 m.~ of the above supernatant was obtained and filtered with a 0.45 ,ccm (pore sire) filter, followed by HPLC v analysis (column: 4.6x250mm, uBondapak C18, Waters, Milford, MA~ mobile phase: acetonitrile-methanol-water(49:44:7 v/v), Flow: l0ml/min, Detector:
470nm). At that time, ~3 -carotene and astaxanthine, purchased from Sigma, were used as a standard substance. As a result, the substances produced from the strain of the present invention were confirmed to be astaxanthine and j3 -carotene.
Astaxanthine produced from the strain of the present invention was also quantified, resulting in the production of 110 ,c.~g/g (dry weight) . For that result, neither inducer nor carotenoid substrate was added, suggesting that ~i -carotene and astaxanthine could be produced in~E. c~1i only by nucleotide sequences having 6,223 by isolated in the present invention. The amount of astaxanthine quantified above was far more than that produced (25 ,ug/g) by Paracoccus .haeundaensis (,Accession No: KCCM-10460).
INDUSTRIAL APPLICABILITY
As explained hereinbefore, the present inventors have isolated and identified a novel Paracoccus genus microorganism mostly producing astaxanthine among many carotenoids, and also have cloned 6 genes coding proteins involved in carotenoid biosynthesis and crt gene containing the same from the above microorganism. The present inventors also have confirmed that carotenoid can be produced even in E. col.i not producing carotenoid, by using crt gene.
Therefore, a gene of the present invention and ~a microorganism producing the gene can be effectively used for the production of carotenoids such as ~i -carotene and astaxanthine which. are available for making food, medicines and beauty stuffs.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE LISTING
<110> KIM, Young Tae LEE, Jae Hyung ALGENETECH
<120> Gene involved in the biosynthesis of carotenoid and marine microorganism, paracoccus haeundaesis, producing the carotenoid <130> 4fpo-02-06 <150> KR2003-20222 <151> 2003-03-31 <150> KR2003-20023 <151> 2003-03-31 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer for Paracoccus haeundaesis 16S rDNA
<400> 1 cataagtaat tatggttttg t 21 <210> 2 ' <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> reverse primer for Paracoccus haeundaesis 16S rDNA
<400> 2 cgcttcctta gaaaggag 18 <210> 3 <211> 1454 <212> DNA
<213> Paracoccus haeundaesis <400> 3 caacttgaga gtttgatcct ggctcagaac gaacgctggc ggcaggctta acacatgcaa 60 gtcgagcgag accttcgggt ctagcggcgg acgggtgagt aacgcgtggg aacgtgccct 120 tctctacgga atagccccgg gaaactggga gtaataccgt atacgccctt tgggggaaag 180 atttatcgga gaaggatcgg cccgcgttgg attaggtagt tggtggggta atggcccacc 240 aagccgacga tccatagctg gtttgagagg atgatcagcc acactgggac tgagacacgg 300 cccagactcc tacgggaggc agcagtgggg aatcttagac aatgggggca accctgatct 360 agccatgccg cgtgagtgat gaaggcctta gggttgtaaa gctctttcag ctgggaagat 420 aatgacggta ccagcagaag aagccccggc taactccgtg ccagcagccg cggtaatacg 480 gagggggcta gcgttgttcg gaattactgg gcgtaaagcg cacgtgggcg gactggaaag 540 tcagaggtga aatcccaggg ctcaaccttg gaactgcctt tgaaactatc agtctggagt 600 tcgagagagg tgagtggaat tccgagtgta gaggtgaaat tcgtagatat tcggaggaac 660 accagtggcg aaggcggctc actggctcga tactgacgct gaggtgcgaa agcgtgggga 720 gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgaatgcc agacgtcggc 780 aagcatgctt gtcggtgtca cacctaacgg attaagcatt ccgcctgggg agtacggtcg 840 caagattaaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 900 attcgaagca acgcgcagaa ccttaccaac ccttgacatg gcaggaccgc tggagagatt 960 cagctttctc gtaagagacc tgcacacagg tgctgcatgg ctgtcgtcag ctcgtgtcgt 1020 gagatgttcg gttaagtccg gcaacgagcg caacccacgt ccctagttgc cagcattcag 1080 ttgggcactc tatggaaact gccgatgata agtcggagga aggtgtggat gacgtcaagt 1140 tctcatggcc cttacgggtt gggctacaca cgtgctacaa tggtggtgac' agtgggttaa 1200 tccccaaaag ccatctcagt tcggattgtc ctctgcaact cgagggcatg aagttggaat 1260 cgctagtaat cgcggaacag catgccgcgg tgaatacgtt cccgggcctt gtacacaccg 1320 cccgtcacac catgggagtt ggttctaccc gacgacgctg cgctaacctt cggggggcag 1380 gcggccacgg taggatcagc gactggggtg aagtcgtaac aaggtagccg taggggaacc 1440 tgcggctgga tcac 1454 <210> 4 <211> 6223 <212> DNA
<213> crt gene <400> 4 gttccacgac tggggcatcc ccacgaccgc gtcgctgcgc gccatcgcgc cgatgatggg 60 gccggaccgg gttctggtcg ggtcgggcgg ggtgcgtcac gggctggacg ccgcgcgggc 120 catccgcctc ggcgcggacc tcgtggggca ggcggcccgc gcgctgcccg ccgcgcgcca 180 cagcgccgag gccctgtccg atcacctgtc cgacgtcgtg acccagctgc gcatcgcgat 240 gttctgcacc ggatcgggcg accttgcagc gctgcgctgc gcgcctctgc tggtgccggg 300 gccgggtggc caatggtcgc aagcaacggg gatggaaacc ggcgatgcgg gactgtagtc 360 tgcgcggatc gccggtccgg gggacaagat gagcgcacat gccctgccca aggcagatct 420 gaccgccacc agcctgatcg tctcgggcgg catcatcgcc gcgtggctgg ccctgcatgt 480 gcatgcgctg tggtttctgg acgcggcggc gcatcccatc ctggcgatcg cgaatttcct 540 ggggctgacc tggctgtcgg tcggtctgtt cttcatcgcg catgacgcga tgcacgggtc 600 ggtcgtgccg gggcgtccgc gcggcaatgc ggcgatgggc cagctggtcc tgtggctgta 660 tgccggattt tcgtggcgca agatgatcgt caagcacatg gcccatcacc gccataccgg 720 aaccgacgac gaccccgatt tcgaccatgg cggcccggtc cgctggtacg cgcgcttcat 780 cggcacctat ttcggctggc gcgaggggct gctgctgccc gtcatcgtga cggtctatgc 840 gctgatcctg ggggatcgct ggatgtacgt ggtcttctgg ccgctgccgt cgatcctggc 900 gtcgatccag ctgttcgtgt tcggcacctg gctgccgcac cgccccggcc acgacgcgtt 960 cccggaccgc cataatgcgc ggtcgtcgcg gatcagcgac cccgtgtcgc tgctgacctg 1020 ctttcacttt ggtggttatc atcacgaaca ccacctgcac ccgacggtgc cttggtggcg 1080 cctgcccagc acccgcacca agggggacac cgcatgacca atttcctgat cgtcgtcgcc 1140 accgtgctgg tgatggagtt gacggcctat tccgtccacc gttggatcat gcacggcccc 1200 ctgggctggg gctggcacaa gtcccaccac gaggaacacg accacgcgct ggaaaagaac 1260 gacctgtacg gcctggtctt tgcggtgatc gecacggtgc tgttcacggt gggctggatc 1320 tgggcgccgg tcctgtggtg gatcgctttg ggcatgaccg tctatgggct gatctatttc 1380 gtcctgcatg acgggctggt tcatcagcgc tggccgttcc gctatatccc gcgcaagggc 1440 tatgcccgcc gcctgtatca ggcccaccgc ctgcaccacg cggtcgaggg, acgcgaccat 1500 tgcgtcagct tcggcttcat ctatgcgccg ccggtcgaca agctgaagca ggacctgaag 1560 acgtcgggcg tgctgcgggc cgaggcgcag gagcgcacgt gacccatgac gtgctgctgg 1620 caggggcggg ccttgcgaac gggctgatcg ccctggcgct gcgcgcggcg cggcccgacc 1680 tgcgggtgct gctgctggat catgcggcgg gaccgtcaga cggccatacc tggtcctgcc 1740 acgaccccga tctgtcgccg cactggctgg cgcggctgaa gcccctgcgc cgcgccaact 1800 ggcccgacca ggaggtgcgc tttccccgcc atgcccggcg gctggccacc ggttacgggt 1860 cgctggacgg ggcggcgctg gcggatgcgg tggcccggtc gggcgccgag atccgctgga 1920 acagcgacat cgccctgctg gatgaacagg gggcgacgct gtcctgcggc acccggatcg 1980 aggcgggcgc ggtcctggac gggcgcggcg cgcagccgtc gcggcatctg accgtgggtt 2040 tccagaaatt cgtgggcgtc gagatcgaga ccgactgccc ccacggcgtg ccccgcccga 2100 tgatcatgga cgcgaccgtc acccagcagg acgggtaccg attcatctat ctgctgccct 2160 tctctccgac gcgcatcctg atcgaggaca ctcgctattc cgatggcggc aatctggacg 2220 acgacgcgct ggcggcggcg tcccacgact atgcccgcca gcagggctgg accggggccg 2280 aggtccggcg cgaacgcggc atcctgccca ttgcgctggc ccatgacgcg gcgggcttct 2340 gggccgatca cgcggagggg cctgttcccg tgggactgcg cgcggggttc tttcacccgg 2400 tcaccggcta ttcgctgccc tatgcggcgc aggtggcgga cgtggtggcg ggcctgtccg 2460 ggccgcccgg caccgacgcg ctgcgcggcg ccatccgcga ttacgcgatc gaccgggcac 2520 gccgtgaccg ctttctgcgc ctgctgaacc ggatgctgtt ccgcggctgc gcgcccgacc 2580 ggcgctatac cctgctgcag cggttctacc gcatgccgca tggactgatc gaacggttct 2640 atgccggccg gctgagcgtg gcggatcagc tgcgcatcgt gaccggcaag cctcccattc 2700 cccttggcac ggccatccgc tgcctgcccg aacgtcccct gctgaaggaa aacgcatgaa 2760 cgcccattcg cccgcggcca agaccgccat cgtgatcggc gcaggctttg, gcgggctggc 2820 cctggccatc cgcctgcagt ccgcgggcat cgccaccacc ctggtcgagg cccgggacaa 2880 gcccggcggg cgcgcctatg tctggcacga tcagggccat gtcttcgacg cgggcccgac 2940 cgtcatcacc gaccccgatg cgctcaagga gctgtgggcg ctgaccgggc aggacatggc 3000 gcgcgacgtg acgctgatgc cggtgtcgcc cttctatcga ctgatgtggc cgggcgggaa 3060 ggtcttcgat tacgtgaacg aggccgatca gctggagcgc cagatcgccc agttcaaccc 3120 ggacgacctg gaaggatacc gccgcttccg tgattacgcg gaggaggtgt atcaggaggg 3180 ctacgtcaag ctgggcaccg tgcccttcct caagctgggc cagatgctca aggccgcgcc 3240 cgcgctgatg aagctggagg cctataagtc cgtccatgcc aaggtcgcga ccttcatcaa 3300 ggacccctat ctgcggcagg cgttttcgta tcacacgctg ctggtgggcg ggaatccctt 3360 ctcgaccagc tcgatctatg cgctgatcca cgcgctggag cggcgcggcg gggtctggtt 3420 cgccaagggc ggcaccaacc agctggtcgc gggcatggtc gcgctgttcg aacggcttgg 3480 cggccagatg atgctgaacg ccaaggtcgc ccggatcgag accgagggcg cgcggaccac 3540 gggcgtcacc ctggcggacg ggcggtcttt aagggccgac atggtcgcca gcaacggcga 3600 cgtcatgcac aactatcgcg acctgctggg ccacacggcc cgcgggcaga gccgcgcgaa 3660 atcgctggac cgcaagcgct ggtccatgtc gttgttcgtg ctgcatttcg gtctgcgcga 3720 ggcgcccaag gacatcgcgc atcacaccat cctgttcggc ccccgctaca gggagctggt 3780 caacgagatc ttcaagggcc cgaagctggc cgaggatttc tcgctgtacc tgcattcgcc 3840 ctgcacgacc gatccggaca tggcgcctcc gggcatgtcc acgcattacg tgctggcccc 3900 cgtgccgcat ctgggccgcg ccgagatcga ttgggcggtc gaggggccgc gctatgccga 3960 ccgcatcctg gcgtccctgg aggagcggct gatcccgaac ctgcgcgcca acctgaccac 4020 gacgcgcatc ttcacgcccg ccgatttcgc cagcgaactg aacgcccatc acggcagcgc 4080 cttctcggtc gagccgatcc tgacgcaatc cgcgtggttc cggccgcaca accgcgacaa 4140 gacgatccgc aacttctatc tggtcggcgc gggcacccat ccgggcgcgg gcattccggg 4200 cgtcgtgggc tcggccaagg ccacggecca ggtgatgctg tccgacctgg cgggcgcatg 4260 agcgatctgg tcctgacctc gaccgaggcg atcacccaag ggtcgcaaag ctttgccacg 4320 gcggccaagc tgatgccgcc gggcatccgc gacgacacgg tgatgctcta tgcctggtgc 4380 cgccacgcgg atgacgtgat cgacggtcag gccctgggca gccgccccga ggcggtgaac 4440 gacccgcagg cgcggctgga cggcctgcgc gtcgacacgc tggcggccct gcagggcgac 4500 ggtccggtga ccccgccctt tgccgcgctg cgcgcggtgg cgcggcggca tgatttcccg 4560 caggcctggc ccatggacct gatcgaaggc ttcgcgatgg atgtcgaggc gcgcgactat 4620 cgcacgctgg atgacgtgct ggaatattcc tatcacgtcg caggcatcgt cggcgtgatg 4680 atggcccgcg tgatgggcgt gcgcgacgat cctgtcctgg accgcgcctg cgacctgggg 4740 ctggcgttcc agctgaccaa catcgcgcgc gacgtgatcg acgatgcgcg catcgggcgg 4800 tgctatctgc cgggggactg gctggaccag gcgggcgcgc ggatcgacgg gccggtgccg 4860 tcgccggagc tgtacacagt gatcctccgg ctgttggatg aggcggaacc ctattacgcg 4920 tcggcgcggg tgggtctggc ggatctgcca ccgcgctgcg cctggtccat cgccgccgcg 4980 ctacggatct atcgcgccat cgggctgcgc atccgcaaga gcgggccgca ggcctatcgc 5040 cagcggatca gcacgtccaa ggctgccaag atcggcctgc tgggcgtcgg, gggctgggat 5100 gtcgcgcgat cacgcctgcc gggggcgggc gtgtcgcggc agggcctctg gacccggccg 5160 catcacgtct aggcgcgcgc ggcgtagggc agaacccgtt ccagcagggc cgcgatttcc 5220 ggagcctgaa ggcgcttgct gcgcagcatc gcgtccagtt gggcgcggct ggcctcgtaa 5280 tgacgggaca cgttctgcag gtctgacacg gccagaaggc cgcgccgcgg gccgggggcc 5340 gcggcatcgc gaccggtatc cttgccaagc gccgcctggt cgcccacgac gtccagcagg 5400 tcgtcatagg actggaacac gcggcccagc tgacggccaa agtcgatcat ctgggtctgc 5460 tcctcggcgt cgaactcctt gatcacggcc agcatctcca gcccggcgat gaacagcacg 5520 ccggtcttca ggtcctgttc ctgttcgacc cccgcgccgt tcttggccgc gtgcaggtcc 5580 aggtcctggc cggcgcacag gccctgcggc cccagggacc gcgacaggat ccgcaccagc 5640 tgcgcccgca ccgtgcccga cgcgccgcgc gcaccggcca gcagggccat tgcctcggtg 5700 atcagggcga tgccgcccag cacggcacgg ctttcgccat gcgccacatg ggtcgcgggc 5760 r cggccgcggc gcagcccggc atcgtccatg cagggcaggt cgtcgaagat cagcgatgcg 5820 gcatgcacca tctcgaccgc gcaggcggcg tcgacgatcg tgtcgcagac cccgcccgag 5880 gcctctgccg caagcagcat cagcatgccg cggaaccgcc tgcccgacga cagcgcgcca 5940 tggctcatgg ccgcgccgag cggctgcgac acggcaccga atccctgggc gatctcctca 6000 agtctggtct gcagaagggt ggcgtggatc gggttgacgt ctcgtctcat cagtgccttc 6060 gcgcttgggt tctgacctgg cgggaaggtc aggccggggc ggcaccccgt gacccgtcat 6120 ccaccgtcaa cagtccccat gttggaacgg ttcacgcccg attgcgagcc ttttcgacgg 6180 cgacgcgggg tcgcgcggca atttgtccaa caaggtcagt gga 6223 <210> 5 <211> 729 <212> DNA
<213> crtW gene <400> 5 atgagcgcac atgccctgcc caaggcagat ctgaccgcca ccagcctgat cgtctcgggc 60 ggcatcatcg ccgcgtggct ggccctgcat gtgcatgcgc tgtggtttct ggacgcggcg 120 gcgcatceca tcctggcgat cgcgaatttc ctggggctga cctggctgtc ggtcggtctg 180 ttcttcatcg cgcatgacgc gatgcacggg tcggtcgtgc cggggcgtcc~ gcgcggcaat 240 gcggcgatgg gccagctggt cctgtggctg tatgccggat tttcgtggcg caagatgatc 300 gtcaagcaca tggcccatca ccgccatacc ggaaccgacg acgaccccga tttcgaccat 360 ggcggcccgg tccgctggta cgcgcgcttc atcggcacct atttcggctg gcgcgagggg 420 ctgctgctgc ccgtcatcgt gacggtctat gcgctgatcc tgggggatcg ctggatgtac 480 gtggtcttct ggccgctgcc gtcgatcctg gcgtcgatcc agctgttcgt gttcggcacc 540 tggctgccgc accgccccgg ccacgacgcg ttcccggacc gccataatgc gcggtcgtcg 600 cggatcagcg accccgtgtc gctgctgacc tgctttcact ttggtggtta tcatcacgaa 660 caccacctgc acccgacggt gccttggtgg cgcctgccca gcacccgcac caagggggac 720 accgcatga 729 <210> 6 <211> 242 <212> PRT , <213> crtW inoacid am <400> 6 Met Ala HisAlaLeu ProLysAla AspLeuThr AlaThrSer Leu Ser Ile Ser GlyGlyIle IleAlaAla TrpLeuAla LeuHisVal His Val Ala Trp PheLeuAsp AlaAlaAla HisProIle LeuAlaIle Ala Leu Asn Leu GlyLeuThr TrpLeuSer ValGlyLeu PhePheIle Ala Phe His Ala MetHisGly SerValVal ProGlyArg ProArgGly Asn Asp Ala Met GlyGlnLeu ValLeuTrp LeuTyrAla GlyPheSer Trp Ala Arg Met IleValLys HisMetAla HisHisArg HisThrGly Thr Lys Asp Asp Asp Pro Asp Phe Asp His Gly Gly Pro Val Arg Trp Tyr Ala ArgPheIle GlyThrTyr PheGlyTrp ArgGluGly LeuLeuLeu Pro ValIleVal ThrValTyr AlaLeuIle LeuGlyAsp ArgTrpMet Tyr ValValPhe TrpProLeu ProSerIle LeuAlaSer IleGlnLeu Phe ValPheGly ThrTrpLeu ProHisArg ProGlyHis AspAlaPhe Pro AspArgHis AsnAlaArg SerSerArg IleSerAsp ProValSer Leu f Leu Thr Cys Phe His Phe Gly Gly Tyr His His Glu His His Leu His Pro Thr Val Pro Trp Trp Arg Leu Pro Ser Thr Arg Thr Lys Gly Asp Thr Ala <210> 7 <211> 489 <212> DNA
<213> crt2 gene <400> 7 atgaccaatt tcctgatcgt cgtcgccacc gtgctggtga tggagttgac ggcctattcc 60 gtccaccgtt ggatcatgca cggccccctg ggctggggct ggcacaagtc ccaccacgag 120 gaacacgacc acgcgctgga aaagaacgac ctgtacggcc tggtctttgc ggtgatcgcc l80 acggtgctgt tcacggtggg ctggatctgg gcgccggtcc tgtggtggat cgctttgggc 240 atgaccgtct atgggctgat ctatttcgtc ctgcatgacg ggctggttca tcagcgctgg 300 ccgttccgct atatcccgcg caagggctat gcccgccgcc tgtatcaggc ccaccgcctg 360 caccacgcgg tcgagggaog cgaccattgc gtcagcttcg gcttcatcta tgcgccgccg 420 gtcgacaagc tgaagcagga cctgaagacg tcgggcgtgc tgcgggccga ggcgcaggag 480 cgcacgtga a8~
<210> 8 <211> 162 <212a PRT
<213> crtZ amino acid <400> 8 MetThr AsnPheLeu IleValVal AlaThrVal LeuValMet GluLeu ThrAla TyrSerVal HisArgTrp IleMetHis GlyProLeu GlyTrp GlyTrp HisLysSer HisHisGlu GluHisAsp HisAlaLeu GluLys AsnAsp LeuTyrGly LeuValPhe AlaValIle AlaThrVal LeuPhe ThrVal GlyTrpIle TrpAlaPro ValLeuTrp TrpIleAla LeuGly MetThr ValTyrGly LeuIleTyr PheValLeu HisAspGly LeuVal His Gln Arg Trp Pro Phe Arg Tyr Ile Pro Arg Lys Gly Tyr Ala Arg ArgLeuTyr GlnAla ArgLeuHis His Val GluGlyArg Asp His Ala HisCysVal SerPhe PheIleTyr Ala Pro ValAspLys Leu Gly Pro LysGlnAsp LeuLys SerGlyVal Leu Ala GluAlaGln Glu Thr Arg ArgThr <210> 9 <211> 1161 <212> DNA
<213> crtY gene <400> 9 gtgacccatg acgtgctgct ggcaggggcg ggccttgcga acgggctgat cgccctggcg 60 ctgcgcgcgg cgcggcccga cctgcgggtg ctgctgctgg atcatgcggc gggaccgtca 120 gacggccata cctggtcctg ccacgacccc gatctgtcgc cgcactggct ggcgcggctg 180 aagcccctgc gccgcgccaa ctggcccgac caggaggtgc gctttccccg ccatgcccgg 240 cggctggcca ccggttacgg gtcgctggac ggggcggcgc tggcggatgc~ ggtggcccgg 300 tcgggcgccg agatccgctg gaacagcgac atcgccctgc tggatgaaca gggggcgacg 360 ctgtcctgcg gcacccggat cgaggcgggc gcggtcctgg acgggcgcgg cgcgcagccg 420 tcgcggcatc tgaccgtggg tttccagaaa ttcgtgggcg tcgagatcga gaccgactgc 480 ccccacggcg tgccccgccc gatgatcatg gacgcgaccg tcacccagca ggacgggtac 540 cgattcatct atctgctgcc cttctctccg acgcgcatcc tgatcgagga cactcgctat 600 tccgatggcg gcaatctgga cgacgacgcg ctggcggcgg cgtcccacga ctatgcccgc 660 cagcagggct ggacc,ggggc cgaggtccgg cgcgaacgcg gcatcctgcc cattgcgctg 720 gcccatgacg cggcgggctt ctgggccgat cacgcggagg ggcctgttcc cgtgggactg 780 cgcgcggggt tctttcaccc ggtcaccggc tattcgctgc cctatgcggc gcaggtggcg 840 gacgtggtgg cgggcctgtc cgggccgccc ggcaccgacg cgctgcgcgg cgccatccgc 900 gattacgcga tcgaccgggc acgccgtgac cgctttctgc gcctgctgaa ccggatgctg 960 ttccgcggct gcgcgcccga ccggcgctat accctgctgc agcggttcta ccgcatgccg 1020 catggactga tcgaacggtt ctatgccggc cggctgagcg tggcggatca gctgcgcatc 1080 gtgaccggca agcctcccat tccccttggc acggccatcc gctgcctgcc cgaacgtccc 1140 ctgctgaagg aaaacgcatg a 1161 <210> 10 <211> 386 <212> PRT

<213> crtY inoacid am <400> 10 Val Thr His AspValLeu LeuAlaGly AlaGlyLeu AlaAsn GlyLeu Ile Ala Leu AlaLeuArg AlaAlaArg ProAspLeu ArgVal LeuLeu Leu Asp His AlaAlaGly ProSerAsp GlyHisThr TrpSer CysHis Asp Pro Asp LeuSerPro HisTrpLeu AlaArgLeu LysPro LeuArg Arg Ala Asn TrpProAsp GlnGluVal ArgPhePro ArgHis AlaArg Arg Leu Ala ThrGlyTyr GlySerLeu AspGlyAla AlaLeu AlaAsp Ala Val Ala Arg Ser Gly Ala Glu Ile Arg Trp Asn Ser Asp Ile Ala LeuLeuAspGlu GlnGlyAla ThrLeuSer CysGlyThr ArgIleGlu AlaGlyAlaVal LeuAspGly ArgGlyAla GlnProSer ArgHisLeu ThrValGlyPhe GlnLysPhe ValGlyVal GluIleGlu ThrAspCys ProHisGlyVal ProArgPro MetIleMet AspAlaThr ValThrGln GlnAspGlyTyr ArgPheIle TyrLeuLeu ProPheSer ProThrArg IleLeuIleGlu AspThrArg TyrSerAsp GlyGlyAsn LeuAspAsp AspAlaLeuAla AlaAlaSer HisAspTyr AlaArgGln GlnGlyTrp ThrGlyAlaGlu ValArgArg GluArgGly IleLeuPro IleAlaLeu AlaHisAspAla AlaGlyPhe TrpAlaAsp HisAlaGlu GlyProVal ProValGlyLeu ArgAlaGly PhePheHis ProValThr GlyTyrSer LeuProTyrAla AlaGlnVal AlaAspVal ValAlaGly LeuSerGly ProProGlyThr AspAlaLeu ArgGlyAla IleArgAsp TyrAlaIle AspArgAlaArg ArgAspArg PheLeuArg LeuLeuAsn ArgMetLeu ' PheArgGlyCys AlaProAsp ArgArgTyr ThrLeuLeu GlnArgPhe TyrArgMetPro HisGlyLeu IleGluArg PheTyrAla GlyArgLeu SerValAlaAsp GlnLeuArg IleValThr GlyLysPr~ ProIlePro LeuGlyThrAla IleArgCys LeuProGlu ArgProLeu LeuLysGlu AsnAla <210> 11 <211> 1506 <212> DNA

<213> crtI ne ge <400> 11 atgaaCgCCC attCC~CCCC~C ggCCaagaCC gCCatCgtga tCggCC~Cagg CtttggCggg 60 ctggccctgg ccatccgcct gcagtccgcg ggcatcgcca ccaccctggt cgaggcccgg 120 gacaagcccg gcgggcgcgc ctatgtctgg cacgatcagg gccatgtctt'cgacgcgggc 180 ccgaccgtca tcaccgaccc cgatgcgctc aaggagctgt gggcgctgac cgggcaggac 240 atggcgcgcg acgtgacgct gatgccggtg tcgcccttct atcgactgat gtggccgggc 300 gggaaggtct tcgattacgt gaacgaggcc gatcagctgg agcgccagat cgcccagttc 360 aacccggacg acctggaagg ataccgccgc ttccgtgatt acgcggagga ggtgtatcag 420 gagggctacg tcaagctggg caccgtgccc ttcctcaagc tgggccagat gctcaaggcc 480 gcgcccgcgc tgatgaagct ggaggcctat aagtccgtcc atgccaaggt cgcgaccttc 540 atcaaggacc cctatctgcg gcaggcgttt tcgtatcaca cgctgctggt gggcgggaat 600 cccttctcga ccagctcgat ctatgcgctg atccacgcgc tggagcggcg cggcggggtc 660 tggttcgcca agggcggcac caaccagctg gtcgcgggca tggtcgcgct gttcgaacgg 720 cttggcggcc agatgatgct gaacgccaag gtcgcccgga tcgagaccga gggcgcgcgg 780 accacgggcg tcaccctggc ggacgggcgg tctttaaggg ccgacatggt cgccagcaac 840 ggcgacgtca tgcacaacta tcgcgacctg ctgggccaca cggcccgcgg gcagagccgc 900 gcgaaatcgc tggaccgcaa gcgctggtcc atgtcgttgt tcgtgctgca, tttcggtctg 960 cgcgaggcgc ccaaggacat cgcgcatcac accatcctgt tcggcccccg ctacagggag 1020 ctggtcaacg agatcttcaa gggcccgaag ctggccgagg atttctcgct gtacctgcat 1080 tcgccctgca cgaccgatcc ggacatggcg cctccgggca tgtccacgca ttacgtgctg 1140 gcccccgtgc cgcatctggg ccgcgccgag atcgattggg cggtcgaggg gccgcgctat 1200 gccgaccgca tcctggcgtc cctggaggag cggctgatcc cgaacctgcg cgccaacctg 1260 accacgacgc gcatcttcac gcccgccgat ttcgccagcg aactgaacgc ccatcacggc 1320 agcgccttct cggtcgagcc gatcctgacg caatccgcgt ggttccggcc gcacaaccgc 1380 gacaagacga tccgcaactt ctatctggtc ggcgcgggca cccatccggg cgcgggcatt 1440 ccgggcgtcg tgggctcggc caaggccacg gcccaggtga tgctgtccga cctggcgggc 1500 gcatga 1506 <210> 12 <211> 501 <212> PRT

<213> crtI inoacid am <400> 12 Met Ala HisSerPro AlaAla LysThrAla IleValIle GlyAla Asn Gly Gly GlyLeuAla LeuAla I12ArgLeu GlnSerAla GlyIle Phe Ala Thr LeuValGlu AlaArg AspLysPro GlyGlyArg AlaTyr Thr Val His AspGlnGly HisVal PheAspAla GlyProThr ValIle Trp Thr Pro AspAlaLeu LysGlu LeuTrpAla LeuThrGly GlnAsp Asp Met Ala Arg Asp Val Thr Leu Met Pro Val Ser Pro Phe Tyr Arg Leu 85 90 95 ' MetTrpPro GlyGlyLys ValPheAsp TyrValAsn GluAlaAsp Gln LeuGluArg GlnIleAla GlnPheAsn ProAspAsp LeuGluGly Tyr ArgArgPhe ArgAspTyr AlaGluGlu ValTyrGln GluGlyTyr Val LysLeuGly ThrValPro PheLeuLys LeuGlyGln MetLeuLys Ala AlaProAla LeuMetLys LeuGluAla TyrLysSer ValHisAla Lys ValAlaThr PheIleLys AspProTyr LeuArgGln AlaPheSer Tyr HisThrLeu LeuValGly GlyAsnPro PheSerThr SerSerIle Tyr AlaLeuIle HisAlaLeu GluArgArg GlyGlyVal TrpPheAla Lys GlyGlyThr AsnGlnLeu ValAlaGly MetValAla LeuPheGlu Arg LeuGlyGly GlnMetMet LeuAsnAla LysValAla ArgIleGlu Thr 245 250 . 255 GluGlyAla ArgThrThr GlyValThr LeuAlaAsp GlyArgSer Leu ArgAlaAsp MetValAla SerAsnGly AspValMet HisAsnTyr Arg AspLeuLeu GlyHisThr AlaArgGly GlnSerArg AlaLysSer Leu AspArgLys ArgTrpSer MetSerLeu PheValLeu HisPheGly Leu ArgGluAla ProLysAsp IleAlaHis HisThrIle LeuPheGly Pro ArgTyrArg GluLeuVal AsnGluIle PheLysGly ProLysLeu Ala GluAspPhe SerLeuTyr LeuHisSer ProCysThr ThrAspPro Asp MetAlaPro ProGlyMet SerThrHis TyrValLeu AlaProVal Pro HisLeuGly ArgAlaGlu IleAspTrp AlaValGlu GlyProArg Tyr 385 390 395 . 400 Ala Asp Arg Ile Leu Ala Ser Leu Glu Glu Arg Leu Ile Pro Asn Leu Arg Ala Asn Leu Thr Thr Thr Arg Ile Phe Thr Pro Ala Asp Phe Ala Ser Glu Leu Asn Ala His His Gly Ser Ala Phe Ser Val Glu Pro Ile Leu Thr Gln Ser Ala Trp Phe Arg Pro His Asn Arg Asp Lys Thr Ile Arg Asn Phe Tyr Leu Val Gly Ala Gly Thr His Pro Gly Ala Gly Ile Pro Gly Val Val Gly Ser Ala Lys Ala Thr Ala Gln Val Met Leu Ser Asp Leu Ala Gly Ala <210> 13 <211> 915 <212> DNA
<213> crtB gene <400>

atgagcgatctggtcctgacctcgaccgaggcgatcacccaagggtcgcaaagctttgcc60 acggcggccaagctgatgccgccgggcatccgcgacgacacggtgatgctctatgcctgg120 tgccgccacgcggatgacgtgatcgacggtcaggccctgggcagccgccccgaggcggtg180 aaCgaCCCgCaggCgCggCtggaCggCCtgCC~CgtCgaCaCgCtggCggCCCtgCagggC24O

gacggtccggtgaccccgccctttgccgcgctgcgcgcggtggcgcggcggcatgatttc300 ccgcaggcctggcccatggacctgatcgaaggcttcgcgatggatgtcgaggcgcgcgac360 tatcgcacgctggatgacgtgctggaatattcctatcacgtcgcaggcatcgtcggcgtg420 atgatggcccgcgtgatgggcgtgcgcgacgatcctgtcctggaccgcgcctgcgacctg480 gggctggcgttccagctgaccaacatcgcgcgcgacgtgatcgacgatgcgcgcatcggg540 cggtgctatctgccgggggactggctggaccaggcgggcgcgcggatcga.cgggccggtg600 ccgtcgccggagctgtacacagtgatcctccggctgttggatgaggcggaaccctattac660 gcgtcggcgcgggtgggtctggcggatctgccaccgcgctgcgcctggtccatcgccgcc720 gcgctacggatctatcgcgccatcgggctgcgcatccgcaagagcgggccgcaggcctat780 cgccagcggatcagcacgtccaaggctgccaagatcggcctgctgggcgtcgggggctgg840 gatgtcgcgc gatcacgcct gccgggggcg ggcgtgtcgc ggcagggcct ctggacccgg 900 ccgcatcacg tctag 915 <210> 14 <211> 304 <212> PRT

<213> crtB inoacid am <400> 14 Met Ser Asp LeuValLeu ThrSerThr GluAlaIle ThrGln GlySer Gln Ser Phe AlaThrAla AlaLysLeu MetProPro GlyIle ArgAsp Asp Thr Val MetLeuTyr AlaTrpCys ArgHisAla AspAsp ValIle Asp Gly Gln AlaLeuGly SerArgPro GluAlaVal AsnAsp ProGln Ala Arg Leu AspGlyLeu ArgValAsp ThrLeuAla AlaLeu GlnGly ~5 70 75 80 Asp Gly Pro ValThrPro ProPheAla AlaLeuArg AlaVal AlaArg Arg His Asp PheProGln AlaTrpPro MetAspLeu IleGlu GlyPhe Ala Met Asp ValGluAla t~rgAspTyr ArgThrLeu AspAsp ValLeu Glu Tyr Ser TyrHisVal AlaGlyIle ValGlyVal MetMet AlaArg Val Met Gly Val Arg Asp Asp Pro Val Leu Asp Arg Ala Cys Asp Leu Gly Leu Ala Phe Gln Leu Thr Asn Ile Ala Arg Asp Val Ile Asp Asp AlaArgIleGly ArgCysTyr LeuProGly AspTrpLeuAsp GlnAla GlyAlaArgIle AspGlyPro ValProSer ProGluLeuTyr ThrVal IleLeuArgLeu LeuAspGlu AlaGluPro TyrTyrAlaSer AlaArg ValGlyLeuAla AspLeuPro ProArgCys AlaTrpSerIle AlaAla Ala Leu Arg Ile Tyr Arg Ala Ile Gly Leu Arg Ile Arg Lys Ser Gly Pro Gln Ala Tyr Arg Gln Arg Ile Ser Thr Ser Lys Ala Ala Lys Ile 260 ~ 265 270 Gly Leu Leu Gly Val Gly Gly Trp Asp Val Ala Arg Ser Arg Leu Pro Gly Ala Gly Val Ser Arg Gln Gly Leu Trp Thr Arg Pro His His Val <210>

<211>

<212>
DNA

<213>
crtE
gene <400>

atgagacgagacgtcaacccgatccacgccacccttctgcagaccagacttgaggagatc60 gcccagggattcggtgccgtgtcgcagccgctcggcgcggccatgagccatggcgcgctg120 tcgtcgggcaggcggttccgcggcatgctgatgctgcttgcggcagaggcctcgggcggg180 gtctgcgacacgatcgtcgacgccgcctgcgcggtcgagatggtgcatgccgcatcgctg240 atcttcgacgacctgccctgcatggacgatgccgggctgcgccgcggccggcccgcgacc300 catgtggcgcatggcgaaagccgtgccgtgctgggcggcatcgccctgatcaccgaggca360 atggccctgctggccggtgcgcgcggcgcgtcgggcacggtgcgggcgcagctggtgcgg420 atcctgtcgcggtccctggggccgcagggcctgtgcgccggccaggacctggacctgcac480 gcggccaagaacggcgcgggggtcgaacaggaacaggacctgaagaccggcgtgctgttc540 atcgccgggctggagatgctggccgtgatcaaggagttcgacgccgaggagcagacccag600 atgatcgactttggccgtcagctgggccgcgtgttccagtcctatgacgacctgctggac660 gtcgtgggcgaccaggcggcgcttggcaaggataccggtcgcgatgccgcggcccccggc720 ccgcggcgcggccttctggccgtgtcagacctgcagaacgtgtcccgtcattacgaggcc780 agccgcgcccaactggacgcgatgctgcgcagcaagcgccttcaggctccggaaatcgcg840 gccctgctggaacgggttctgccctacgccgcgcgcgcctag 882 <210> 16 ' <211> 293 <212> PRT
<213> crtE amino acid <400> 16 Met Arg Arg Asp Val Asn Pro Ile His Ala Thr Leu Leu Gln Thr Arg Leu Glu Glu Ile Ala Gln Gly Phe Gly Ala Val Ser Gln Pro Leu Gly Ala Ala Met Ser His Gly Ala Leu Ser Ser Gly Arg Arg Phe Arg Gly Met Leu Met Leu Leu Ala Ala Glu Ala Ser Gly Gly Val Cys Asp Thr Ile Val Asp Ala Ala Cys Ala Val Glu Met Val His Ala Ala Ser Leu Ile Phe Asp Asp Leu Pro Cys Met Asp Asp Ala Gly Leu Arg Arg Gly Arg Pro Ala Thr His Val Ala His Gly Glu Ser Arg Ala Val Leu Gly Gly Ile Ala Leu Ile Thr Glu Ala Met Ala Leu Leu Ala Gly Ala Arg Gly Ala Ser Gly Thr Val Arg Ala Gln Leu Val Arg Ile Leu Ser Arg Ser Leu Gly Pro Gln Gly Leu Cys Ala Glg~ Gln Asp Leu Asp Leu His 145 150 155 ~ 160 Ala Ala Lys Asn Gly Ala Gly Val Glu Gln Glu Gln Asp Leu Lys Thr Gly Val Leu Phe Ile Ala Gly Leu Glu Met Leu Ala Val Ile Lys Glu Phe Asp Ala Glu Glu Gln Thr Gln Met Ile Asp Phe Gly Arg Gln Leu Gly Arg Val Phe Gln Ser Tyr Asp Asp Leu Leu Asp Val Val Gly Asp Gln Ala Ala Leu Gly Lys Asp Thr Gly Arg Asp Ala Ala Ala Pro Gly Pro Arg Arg Gly Leu Leu Ala Val Ser Asp Leu Gln Asn Val Ser Arg 245 250 255 ' His Tyr Glu Ala Ser Arg Ala Gln Leu Asp Ala Met Leu Arg Ser Lys Arg Leu Gln Ala Pro Glu Ile Ala Ala Leu Leu Glu Arg Val Leu Pro Tyr Ala Ala Arg Ala <210> 17 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> forward primer for crt gene <400> 17 gttccacgac tggggcatc lg <210> 18 <211> 28 <212> DNA ' <213> Artificial Sequence <220>
<223> reverse primer for crt gene <400> 18 tccactgacc ttgttggaca aattgccg 2g

Claims (19)

1. A gene coding a protein involved in carotenoid biosynthesis, which has nucleotide sequences selected from a group consisting of nucleotide sequences represented by SEQ. ID. No 5, No 7, No 9, No 11, No 13 and No 15.

2. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtW coding .beta.-carotene ketolase and represented by SEQ.
ID. No 5.

3. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtZ coding .beta.-carotene hydroxylase and represented by SEQ.
ID. No 7.

4. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtY coding licopene cyclase and represented by SEQ. ID.
No 9.

5. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtI coding phytoene desaturase and represented by SEQ. ID.
No 11.

6. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtB coding phytoene synthase and represented by SEQ. ID.
No 13.

7. The gene as set forth in claim 1, wherein the gene has nucleotide sequences of crtE coding geranylgeranyl pyrophosphate synthase and represented by SEQ. ID. No 15.

8. A crt gene containing all the genes of claim 2 ~ claim 7 and represented by SEQ. ID. No 4.

9. A protein encoded by the gene of claim 1, which has nucleotide sequences selected from a group consisting of nucleotide sequences represented by SEQ. ID. No 6, No 8, No 10, No 12, No 14 and No 16.

10. A recombinant vector containing the crt gene of claim 8.

11. The recombinant vector as set forth in claim 10, wherein the vector is pCR-XL-TOPO-crtfull having a cleavage map represented in FIG. 16.

12. An E. coli transformant transformed with the recombinant vector of claim 11.

13. A method for producing carotenoid comprising the following steps:

1) Cloning the crt gene of claim 8;
2) Constructing a recombinant vector in which the crt gene of the above step 1) was inserted;
3) Transfecting a host cell with the recombinant vector of the step 2); and 4) Recovering carotenoids from the culture cells in which a strain transformed with the above recombinant vector was being cultured.

14. The method as set forth in claim 13, wherein the recombinant vector is that of claim 11.

15. The method as set forth in claim 13, wherein the host cell is E. coli or yeast.

16. The method as set forth in claim 13, wherein the recovery of carotenoids is performed from the culture cells in which the E. coli was being cultured.

17. The method as set forth in claim 13, wherein the cartenoid is .beta.-carotene or astaxanthine.

18. A Paracoccus haeundaensis producing astaxanthine, which has a 16S rDNA nucleotide sequence represented by SEQ. ID. No 3.

19. The Paracoccus haeundaensis as set forth in claim 18, wherein the strain is represented by accession No: KCCM-10460.