Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification
Reexamination Certificate
2001-05-24
2004-02-24
Nashed, Nashaat T. (Department: 1652)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
C435S471000, C435S254110, C435S320100, C536S023100, C536S023200, C536S023700
Reexamination Certificate
active
06696293
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the manufacture of carotenoids and biological materials useful therefor.
BACKGROUND OF THE INVENTION
Astaxanthin is known to be distributed in a wide variety of organisms, such as, for example, animals (e.g., birds, such as, for example, flamingos and scarlet ibis, and fish, such as, for example, rainbow trout and salmon), algae, and microorganisms. It is also recognized that astaxanthin possesses a strong antioxidation property against reactive oxygen species, which suggests a pharmaceutical applicability in protecting living cells against some diseases, such as, for example, cancer. Moreover, from a viewpoint of industrial application, demand for astaxanthin as a coloring reagent is increasing, especially in the industry of farmed fish, such as, for example, salmon, because astaxanthin imparts a distinctive orange-red coloration to the animals that contributes to consumer appeal in the marketplace.
Phaffia rhodozyma
is known as a carotenogenic yeast strain that produces astaxanthin. In contrast to other carotenogenic yeast, such as, for example, Rhodotorula,
Phaffia rhodozyma
(
P. rhodozyma
) can ferment some sugars such as, for example, D-glucose. This is an important feature from the viewpoint of industrial application. In a recent taxonomic study, the 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).
In order to obtain hyper-producers of astaxanthin from
P. rhodozyma
, some strain improvement studies have been conducted. However, in this decade, such efforts have been restricted to employing conventional mutagenesis and protoplast fusion techniques. Recently, Wery et al. developed a host vector system using
P. rhodozyma
in which multicopies of a non-replicable plasmid were integrated into the genome of
P. rhodozyma
at the ribosomal DNA locus (Wery et al., Gene, 184, 89-97, 1997). Verdoes et al., International Patent Publication No. WO97/23633, reported the use of improved vectors to transform
P. rhodozyma
with copies of the three carotenogenic genes that encode enzymes that catalyze the reaction from geranylgeranyl pyrophosphate to beta-carotene.
Many researchers have speculated that astaxanthin might function as an antioxidant in
Phaffia rhodozyma
because its production is stimulated during the respiration phase of growth rather than during the fermentation phase. In general, reactive oxygen species tend to be generated during the respiration phase as a result of electron overflow in the respiratory chain. Electron overflow in the respiratory chain is caused by an imbalance of electron transfer during reduction of the ubiquinone pool and electron transfer occurring downstream in the respiratory chain. It is speculated that astaxanthin might quench such reactive oxygen species in a manner analogous to superoxide dismutase.
Schroeder and Johnson reported that the respiratory chain of
Phaffia rhodozyma
shifted from KCN-sensitive respiration to KCN-resistant respiration during the late phase of growth when astaxanthin production was stimulated (J. Biol. Chem., 270, 18374-18379, 1995). The KCN-sensitive respiratory chain, in which an electron from the ubiquinone pool is transferred to complex IV via complex III, is a common electron transfer chain that is found in a wide variety of organisms. It is known that this respiratory chain is inhibited by KCN or antimycin A.
The KCN-resistant respiratory chain is found in both plants and fungi. In this respiratory chain, a mitochondrial membrane protein, alternative oxidase (AOX), plays a substantial role in transferring an electron from the ubiquinone pool to an H
2
O molecule by using an oxygen molecule as an acceptor. AOX activity is known to be inhibited by n-propyl gallate (n-PG) or salicylhydroxamic acid (SHAM).
In their characterization study of antimycin-sensitive hyper-producers of astaxanthin derived from
Phaffia rhodozyma
, An et al. speculated that such mutants produced increased amounts of astaxanthin to quench reactive oxygen species, which might be produced by electron overflow from the electron transfer chain (Appl. Env. Microbiol, 55, 116-124, 1989).
SUMMARY OF THE INVENTION
This invention was conceived based on the presumption that the biosynthesis of astaxanthin might be upregulated under conditions in which the electron transfer chain is in the reduced state. The reduced state might be induced by addition of a specific inhibitor such as antimycin A, KCN, n-PG or SHAM. The reduced state might also be induced by a mutation that would result in an imbalance in electron transfer.
In accordance with this invention, mutants were obtained that displayed resistance to SHAM. Surprisingly, these mutants displayed 50% higher productivity of astaxanthin than their parent strain.
In the present invention, the cloning of a gene that codes for an alternative oxidase from
Phaffia rhodozyma
is disclosed. In the present invention, the enzymatic characterization of the expression of the gene in suitable host organisms such as
E. coli
or
Saccharomyces cerevisiae
is also disclosed. The cloned gene may be used for the reduction of AOX activity using methods such as, for example, site-directed mutagenesis of promoter sequences or anti-sense methods in a suitable host, such as
P. rhodozyma
. The effects of gene expression on carotenogenesis can be studied by cultivating transformants in an appropriate medium under appropriate cultivation conditions.
An object of the present invention is a process for producing a carotenoid involving:
(a) culturing a mutant microorganism in culture medium containing an alternative oxidase (AOX) inhibitor, wherein the mutant microorganism produces at least 10% more of the carotenoid compared to the parental stain of the mutant microorganism; and
(b) recovering the carotenoid produced by the mutant microorganism from the culture media.
Another object of the present invention is a process for producing a carotenoid involving:
(a) culturing a microorganism containing a polynucleotide sequence encoding an alternative oxidase (AOX), which polynucleotide sequence has been altered to form a mutant microorganism compared to a parental microorganism containing an unaltered polynucleotide sequence encoding AOX, which mutant microorganism has a reduced level of AOX expression and produces at least 10% more carotenoid as compared to the unaltered parental microorganism; and
(b) recovering the carotenoid produced by the mutant microorganism.
A further object of the present invention is a process for engineering a carotenoid-producing microorganism involving:
(a) selecting a parental microorganism that produces a carotenoid;
(b) culturing the parental microorganism in a culture medium containing an alternative oxidase (AOX) inhibitor; and
(c) selecting a mutant microorganism that grows in the culture medium containing the AOX inhibitor and which microorganism produces at least 10% more of the carotenoid compared to the parental microorganism.
A further object of the present invention is a process for engineering an enhanced carotenoid-producing microorganism involving:
(a) selecting a parental microorganism that produces a carotenoid;
(b) altering a polynucleotide sequence encoding an alternative oxidase (AOX) in the parental microorganism to form a mutant microorganism, which mutant has a reduced level of AOX expression compared to the parental microorganism; and
(c) selecting a mutant microorganism that produces at least 10% more of the carotenoid compared to the parental microorganism.
Another object of the present invention is a recombinantly-produced mutant microorganism produced from a parental carotenoid-producing microorganism having a gene that encodes alternative oxidase (AOX), wherein the gene expression of the AOX in the parental microorganism is altered to produce the mutant microorganism, whereby the efficiency of expression of the AOX in the mutant is reduced compared to the parental microorganism and t
Hoshino Tatsuo
Ojima Kazuyuki
Setoguchi Yutaka
Bryan Cave LLP
Nashed Nashaat T.
Roche Vitamins Inc.
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