Methods of producing carotenoids using DNA molecules...

Details Methods of producing carotenoids using DNA molecules... Methods of producing carotenoids using DNA molecules...

1996-11-12

2004-11-23

Kerr, Kathleen (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing a carotene nucleus

C536S023100, C536S023200, C536S023600, C536S023740, C435S233000

Reexamination Certificate

active

06821749

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a DNA chain which provides higher carotenoid content during biosynthesis of carotenoid and a method for producing carotenoids characterized by introducing said DNA chain into carotenoid producing microorganism to express said chain and to obtain higher carotenoid content.
BACKGROUND OF THE INVENTION
Carotenoid is a general name of a kind of natural pigments. Generally, carotenoids have 40 carbon atoms and consists of isoprene skeletons, and Carotenoids are abundant in the natural world. Approximately 600 kinds of carotenoids have been isolated and identified up to the present [(see Key to carotenoids. Basel-Boston, Birkhauser, 1987(Pfander, H. ed.)]. Carotenoids are synthesized through the isoprenoid biosynthetic pathway, a part of which is common to the pathways for steroids and other terpenoids. Passing through the isoprene common biosynthetic pathway, hydroxymethylglutaryl-CoA(HMG-CoA) is converted to isopentenyl pyrophosphate(IPP), which has 5 carbon atoms, via mevalonate. Then IPP is converted to dimethylallyl pyrophosphate(DMAPP) by isomerization. Then, by polycondensation with IPP which has 5 carbon atoms, DMAPP is converted sequentially to geranyl pyrophosphate(GPP which has 10 carbon atoms), farnesyl pyrophosphate(FPP which has 15 carbon atoms), geranylgeranyl pyrophosphate(GGPP which has 20 carbon atoms) and so forth (FIG.
1
).
The carotenoid biosynthetic pathway is branched from the isoprene common pathway at the point of GGPP is formed. At the point, two molecules of GGPP are condensed to synthesize phytoene which is the first carotenoid and colorless. Then, phytoene is converted to lycopene by desaturation reaction. Then, lycopene is converted to &bgr;-carotene by cyclization. Various xanthophylls such as zeaxanthin and astaxanthin are synthesized by introducing hydroxyl groups or keto groups to &bgr;-carotene.
Recently, the inventors of the present invention cloned the carotenoid biosynthesis genes derived from
Erwinia uredovora,
which is a non-photosynthetic epiphytic bacterium in
Escherichia coli
by using yellowish color of
Er. uredovora
as markers and elucidated the functions of the genes. Then, various combinations of these genes are introduced to express, and it made possible that microorganisms such as
E. coli
and yeast produce phytoene, lycopene, &bgr;-carotene, zeaxanthin and so forth(See FIG.
2
): [See 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: 6704-6712 (1990); Misawa, N., Yamano, S., and Ikenaga, H., “Production of &bgr;-carotene in
Zymomonas mobilis
and
Agrobacterium tumefaciens
by introduction of the biosynthesis genes from
Erwinia uredovora”,
Appl. Environ. Microbiol., 57: 1847-1849 (1991); Yamano, S., Ishii, T., Nakagawa, M., Ikenaga, H., and Misawa, N., “Metabolic engineering for production of &bgr;-carotene and lycopene in
Saccharomyces cerevisiae”,
Biosci. Biotech. Biochem., 58: 1112-1114 (1994) and Japanese Patent Application laid-open No. HEI 3-58786(Japanese Patent Application filing No. HEI 2-53255):“A DNA chain useful for synthesis of carotenoids” by the inventors of the present invention]. With the carotenoid biosynthesis genes from
Er. uredovora,
carotenoids can be synthesized from FPP. Since FPP is the common substrate not only for carotenoids but also for steroids and other terpenoids, bacteria incapable of synthesizing carotenoids also have FPP. Accordingly, for example, when four crt genes, crtE, crtB, crtI and crtY, which are necessary for biosynthesis of &bgr;-carotene from FPP are introduced in microorganisms, the microorganism becomes capable of producing &bgr;-carotene (See FIG.
2
). Furthermore, by the same procedures as mentioned above, the inventors cloned the carotenoid biosynthesis genes derived from a marine bacterium,
Agrobacterium aurantiacum
in
E. coli.
By expressing various combinations of the genes from the bacterium and those from the above-mentioned
Er. uredovora,
it made possible that the microorganisms such as
E. coli
produce astaxanthin, canthaxanthin and so forth (See FIG.
3
): (Norihiko Misawa et al., “Elucidation of an astaxanthin biosynthetic pathway at the level of the biosynthesis genes”, Abstract of the 36th Symposium on the chemistry of natural products: 175-180 (1994)). Among the above carotenoids, astaxanthin, zeaxanthin and &bgr;-carotene are already in practical use and are regarded as promising substances. They are used for food or feed additives as red or yellow natural coloring agents or as nutritional aid having cancer prophylactic activity, immunopotentiating activity or provitamin A activity. Accordingly, when the carotenoid biosynthesis genes obtained by the inventors is used as exogenous genes for transforming microorganisms such as
E. coli
to express, it gave microorganisms such as
E. coli
the capability of biosynthesis for producing useful carotenoids. Up to now, it is the only way to improve production of useful carotenoids was to find out microorganism which can synthesize sufficient amount of a targeted carotenoid, and to try to increase its production by investigating culture conditions or mutation treatment. Owing to the studies done by the inventors, it became possible to choose host microorganism which is cultured easily and proliferates rapidly, and is guaranteed to be safe for food regardless of its carotenoid producing capability. As a matter of course, it is also possible to use microorganisms which can synthesize sufficient amount of useful carotenoids originally. In such a case, by transforming the microorganisms with carotenoid biosynthesis genes, it became possible to obtain higher carotenoid production or to alter final carotenoid products. For example, when both crtW and crtZ genes from
Ag. aurantiacum
were introduced into a microorganism capable of producing &bgr;-carotene as a final product to express them, the microorganism was transformed to another one which produce astaxanthin as a final product.
On the other hand, both astaxanthin and &bgr;-carotene can also be synthesized by organic synthesis methods. In these cases, considering these carotenoids are used for feed or food additives, there is problems that by-products are also produced and such synthetic products are not preferred by consumers because they prefer natural products. However, carotenoids produced by the conventional fermentation methods could not compete with those by the organic synthesis methods in price. As mentioned earlier, when the above mentioned carotenoid biosynthesis genes are used, it improves the fermentation methods, thereby it is considered that the carotenoid produced by the fermentation methods will be able to compete with those by the organic synthesis methods in price. If the microorganism can accumulate enough amount of carotenoid in itself, the carotenoid produced by the microorganisms will succeed in such price competition. Therefore, a technology to obtain higher carotenoid content by using microorganisms has been longed for.
Until now, in order to obtain higher carotenoid production in its biosynthesis; the traditional random mutation method is only employed to select mutant strains having higher carotenoid content with mutagenic agent such as NTG. However, this method requires huge amount of time and labor of technicians. In addition, even if enhancement of carotenoid synthesis is successfully achieved, the method requires both huge amount of time and effort to inhibit decreasing of carotenoid content caused by frequent reverse mutations naturally happens because the method lacks its theoretical basis.
SUMMARY OF THE INVENTION
The object of the present invention is to increase amount of carotenoids biosynthetically produced by microorganisms.
To solve the above problem, the inventors have investigated the problem thoroughly and developed a novel technology

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