Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Patent
1996-04-23
1999-06-08
Patterson, Jr., Charles L.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
435 67, 435189, 4352523, 43525233, 4353201, 435946, 536 232, 536 243, C12P 726, C12N 902, C12N 112, C07H 2104
Patent
active
059104333
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a keto group-introducing enzyme necessary for synthesizing ketocarotenoids, such as astaxanthin, which are useful for a red-color enhancing treatment of cultured fishes and shellfishes (such as sea bream, salmon and shrimp) and are also applied to foods as a coloring agent or an antioxidant; a DNA coding for the above enzyme; a recombinant vector comprising the DNA; a microorganism into which the DNA has been introduced; and a method for producing ketocarotenoids using the above microorganism.
BACKGROUND ART
"Ketocarotenoid" is a general term for keto group-containing carotenoid pigments. Carotenoids are synthesized from mevalonic acid as a starting substance via isoprenoid basic biosynthesis pathway which shares an initial part with the synthesis pathway for steroids and other isoprenoids (see FIG. 6). Isopentenyl pyrophosphate (IPP) with 5 carbon atoms, which is a basic unit, generated from the isoprenoid basic biosynthesis pathway condenses with its isomer dimethylallyl pyrophosphate (DMAPP) to produce geranyl pyrophosphate (GPP) with 10 carbon atoms and, in addition, IPP condenses to produce farnesyl pyrophosphate (FPP) with 15 carbon atoms. FPP produces geranylgeranyl pyrophosphate (GGPP) with 20 carbon atoms by condensing with IPP again. Then, GGPPs condense with each other to produce colorless phytoene which is the initial carotenoid. Through a series of unsaturated reactions, phytoene is converted to phytofluene, .zeta.-carotene, neurosporene and finally to lycopene. Subsequently, lycopene is converted by a cyclization reaction to a .beta.-carotene containing two .beta.-ionone rings. Finally, it is believed that a keto-groups, a hydroxyl group, etc. are introduced into the .beta.-carotene to thereby synthesize astaxanthin, zeaxanthin and the like (see Britton, G., "Biosynthesis of Carotenoids", Plant Pigments, Goodwin, T. W (ed.), London, Academic Press, 1988, pp. 133-182).
Recently, the present inventors have cloned a group of carotenoid biosynthesis genes of the non-photosynthetic bacterium Erwinia uredovora present in plant from the genomic DNA library in E. coli using its yellow color formation as an indicator. Further, by expressing a various combinations of these genes in microorganisms such as E. coli, the inventors has made it possible to produce in microorganisms such as E. coli phytoene, lycopene, .beta.-carotene and zeaxanthin which is a yellow carotenoid pigment wherein a hydroxyl group has been introduced into .beta.-carotene (see FIG. 7) (Misawa, N., Nakagawa, M., Kobayashi, K., Yamano, S., Izawa, Y., Nakamura, K., and Harashima, K., "Elucidation of the Erwinia uredovora Carotenoid Biosynthetic Pathway by Functional Analysis of Gene Products Expressed in Escherichia coli", J. Bacteriol., 172, pp. 6704-6712, 1990; Misawa, N., Yamano, S, Ikenaga, H., "Production of .beta.carotene in Zymomonas mobilis and Agrobacterium tumefaciens by Introduction of the Biosynthesis Genes from Erwinia uredovora", Appl. Environ. Microbiol., 57, pp. 1847-1849, 1991; and Japanese Unexamined Patent Publication No. 3-58786).
On the other hand, astaxanthin which is a red ketokarotenoid is a representative animal carotenoid widely present in marine organisms, e.g. red fishes such as sea bream and salmon, and crustaceans such as crab and shrimp. Since animals generally cannot biosynthesize carotenoids, they have to take in from outside those catotenoids synthesized by microorganisms or plants. For this reason, astaxanthin has been widely used for the purpose of red color enhancing for cultured fishes and shellfishes such as sea bream, salmon and shrimp.
Astaxanthin is also used as a coloring agent for foods. Furthermore, astaxanthin is attracting attention as an antioxidant to remove activated oxygen generated in a body which is causative of a cancer (see Takao Matuno and Wataru Inui, "Physiological Functions and Biological Activities of Carotenoids in Animals", KAGAKU TO SEIBUTU (Chemistry and Organisms), 28, pp. 219-227, 1990).
As sources of astaxanth
REFERENCES:
Yamano, et al. "Metabolic Engineering for Production of .beta.-Carotene and Lycopene in Saccharomyces cerevisiae," Biosci. Biotech. Biochem. 58(6): 1112-14 (1994).
Anderson, et al. "Isopentenyl Diphosphate:Dimethylallyl Diphosphate Isomerase: An Improved Purification of The Enzyme and Isolation of the Gene from Saccharomyces cerevisiae," J. of Biochem. 264(32):19169-19175 (1989).
Misawa, et al. "Production of .beta.-Carotene in Zymomonas mobilis and Agrobacterium tumefaciens by Introduction of the Biosysnthesis Genes from Erwinia uredovora," Appl. Envirom, Microbiol. 57(6):1847-49 (1991).
Misawa, et al. "Symposium Papers of 36th Symposium on the Chemistry of Natural Products," pp. 175-180 (1994).
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"Production of .beta.-Carotene in Zymomonas Mobilis and Agrobacterium Tumefaciens by Introduction of the Biosynthesis Genes from Erwinia Uredovora", Applied and Environmental Microbiology, (Jun. 1991), vol. 57, pp. 1847-1849.
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"Cloning and Expression in Escherichia Coli of the Gene Encoding .beta.-C-4-Oxygenase, that Converts .beta.-Carotene to the Ketocarotenoid Canthaxanthin in Haematococcus Pluvialis", Lotan et al., FEBS Letters (1995) vol. 364, pp. 125-128.
Kajiwara Susumu
Kondo Keiji
Misawa Norihiko
Kirin Beer Kabushiki Kaisha
Patterson Jr. Charles L.
Slobodyansky Elizabeth
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