Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for...
Reexamination Certificate
2000-03-03
2002-04-02
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
C435S006120, C435S252300, C435S320100, C536S023200
Reexamination Certificate
active
06365386
ABSTRACT:
The present invention relates to recombinant production of carotenoids and biological materials useful therefor.
Phaffia rhodozyma
(
P. rhodozyma
) is a carotenogenic yeast strain which produces astaxanthin. Astaxanthin is distributed in a wide variety of organisms such as animals (birds such as flamingo and scarlet ibis, and fish such as rainbow trout and salmon), algae and microorganisms. It is also recognized that astaxanthin has a strong antioxidation property against oxygen radicals, and is expected to be useful pharmaceutically for protecting living cells against certain diseases, such as a cancer. Moreover, industrial need for astaxanthin as a coloring reagent is increasing, especially in the industry of farmed fish like salmon, because astaxanthin imparts a distinctive orange-red coloration to the animals and contributes to consumer appeal in the marketplace.
P. rhodozyma
is known as a carotenogenic yeast strain which produces astaxanthin. Different from the other carotenogenic yeast, Rhodotorula species,
P. rhodozyma
can ferment some sugars such as D-glucose. This is an important feature from a viewpoint of industrial application. In a recent taxonomic study, a sexual cycle of
P. rhodozyma
was revealed and its telemorphic state was designated under the name of
Xanthophyllomyces dendrorhous
(W. I. Golubev; Yeast 11, 101-110, 1995). Some strain improvement studies to obtain hyper producers of astaxanthin from
P. rhodozyma
have been conducted, but such efforts have been restricted to employ the method of conventional mutagenesis and protoplast fusion in this decade. Recently, Wery et al. developed a host vector system using
P. rhodozyma
in which a non-replicable plasmid was integrated in multiple copies into the genome of the ribosomal DNA of
P. rhodozyma
(Wery et al., Gene, 184, 89-97, 1997). Verdoes et al. reported more improved vectors to obtain a transformant of
P. rhodozyma
as well as its three carotenogenic genes which code for the enzymes that catalyze the reactions from geranylgeranyl pyrophosphate to beta-carotene (WO 97/23633).
A specific biosynthetic pathway for carotenogenesis branches from the general isoprenoid pathway at the point of an important intermediate, farnesyl pyrophosphate (FPP) (FIG.
1
). FPP and IPP are condensed by geranylgeranyl pyrophosphate (GGPP) synthase which is encoded by crtE in
P. rhodozyma
to produce GGPP. GGPP is then converted to beta-carotene by the sequential reaction of an enzyme functioning doubly as phytoene synthase and lycopene cyclase which is encoded by crtBY and phytoene desaturase encoded by crtI.
In bacteria, enzymes and genes which are involved in xanthophyll formation have been isolated and characterized in detail. Beta-carotene hydroxylase which is coded by crtZ is involved in the two steps of hydroxylation for the beta-ionone-ring of beta-carotene at both of the ends. The crtZ gene has been cloned from a wide variety of organisms such as
Erwinia uredovora
(Misawa et al., J. Bacteriol., 172, 6704-6712, 1990), Flavobactor species (
L. Pasamontes
et al., 185 (1), 35-41, 1997) and
Agrobacterium aurantiacum
(Misawa et al., J. Bacteriol., 177 (22), 6575-6584, 1995). Beta-carotene ketolase which is encoded by crtW catalyzes the two steps of introduction of an oxo-group into the beta-ionone -ring of beta-carotene at both of the ends. Kajiwara et al. cloned and sequenced the bkt gene corresponding to crtW in eubacteria from
Haematococcus bluvialis
(Kajiwara et al.,
P. Mol. Biol.,
29, 343-352, 1995). Harker et al. also cloned and sequenced the crtO gene corresponding to crtW in eubacteria from Synechococcus PCC7942 (Harker et al., FEBS Letters, 404, 129-134, 1997). Both enzymes, i.e., the hydroxylase and the ketolase, have wide substrate specificity and this ensures the formation of a wide variety of xanthophylls in case both of the enzymes react at the same time, depending on the reaction condition. (
FIG. 1
)
As described above, all the genes which were involved in the formation of beta-carotene from FPP have been isolated but the enzymes and genes which would be involved in the last step of xanthophyll formation from beta-carotene have not been identified on the protein and DNA level in
P.rhodozyma
. Although Johnson et al. (Crit. Rev. Biotechnol, 11 (4), 297-326, 191) proposed the existence of two independent pathways for astaxanthin formation by assuming that some of the xanthophyll compounds isolated by them would be intermediates of astaxanthin biosynthesis, these two independent pathways could not be proven because enzymes and genes which are involved in such pathways could not be isolated. Furthermore, it can not be excluded that these xanthophyll compounds could have resulted from an experimental artifact in the isolation step of these compounds. Failure to isolate a mutant from
P. rhodozyma
which accumulates intermediates in the biosynthetic pathway from beta-carotene to astaxanthin made it difficult to clarify the biosynthetic pathway from beta-carotene to astaxanthin.
SUMMARY OF THE INVENTION
This invention relates to a gene and an enzyme which is involved in the last step of astaxanthin biosynthesis (i.e., from beta-carotene to astaxanthin).
The present invention provides an isolated DNA, for example, a cDNA including a nucleotide sequence coding for astaxanthin synthase which is involved in the reaction from beta-carotene to astaxanthin in
P. rhodozyma
, like the AST gene.
In a preferred embodiment, the cloned DNA fragment can be characterized in that
(a) the nucleotide sequence encodes an enzyme having the amino acid sequence described in SEQ ID NO: 1, or
(b) the nucleotide sequence encodes a variant of the enzyme selected in (a), which nucleotide sequence is either (i) an allelic variant or (ii) an enzyme having one or more amino acid insertions, deletions, and/or substitutions and having the stated enzyme activity.
In another preferred embodiment, the isolated cDNA fragment can be derived from a gene of
Phaffia rhodozyma
and is selected from:
(i) a cDNA sequence represented by SEQ ID NO: 2;
(ii) an isocoding or an allelic variant for the cDNA sequence represented by SEQ ID NO: 2; and
(iii) a derivative of a cDNA sequence represented by SEQ ID NO: 2 with insertions, deletions, and/or substitutions of one or more nucleotide(s), and encoding a polypeptide having the enzyme activity.
In another preferred embodiment, the present invention includes the isolated cDNA as described above, which is characterized in that the nucleotide sequence is:
(i) a nucleotide sequence represented in SEQ ID NO: 2;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes an astaxanthin synthase having the same amino acid sequence as that encoded by the nucleotide sequence in (i); and
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from i) or ii) under standard hybridizing conditions.
In still another preferred embodiment, an isolated genomic DNA fragment can be derived from a gene of
Phaffia rhodozyma
and is selected from:
(i) a genomic DNA sequence represented by SEQ ID NO: 3;
(ii) an isocoding or an allelic variant for the genomic DNA sequence represented by SEQ ID NO: 3; and
(iii) a derivative of a genomic DNA sequence represented by SEQ ID NO: 3 with insertions, deletions, and/or substitutions of one or more nucleotide(s), and coding for a polypeptide having the enzyme activity.
In another preferred embodiment the present invention includes the isolated genomic DNA as described above, which is characterized in that the nucleotide sequence is:
(i) a nucleotide sequence represented in SEQ ID NO: 3;
(ii) a nucleotide sequence which, because of the degeneracy of the genetic code, encodes an astaxanthin synthase having the same amino acid sequence as that encoded by the nucleotide sequence in (i); and
(iii) a nucleotide sequence which hybridizes to the complement of the nucleotide sequence from i) or ii) under standard hybridizing conditions.
Another aspect of the present invention is a recombinant polypeptide having astaxa
Hoshino Tatsuo
Ojima Kazuyuki
Setoguchi Yutaka
Bryan Cave LLP
Monshipouri Maryam
Prouty Rebecca E.
Roche Vitamins Inc.
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