Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
1999-04-23
2002-04-23
Murthy, Ponnathapuachuta (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S195000, C435S254200, C435S254220, C536S023100, C536S023200
Reexamination Certificate
active
06376222
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides for improved riboflavin production. In particular, the invention provides transformed yeast strains and processes for producing riboflavin.
BACKGROUND OF THE INVENTION
Derivatives of riboflavin (the flavocoenzymes FMN and FAD) are universally required for redox reactions in all cellular organisms. Riboflavin (vitamin B
2
) is produced by all plants and by many microorganisms (Demain A. L. Riboflavin over synthesis. Ann. Rev. Microbiol. 1972, 26, 369). The compound is not produced in vertebrates. Riboflavin is therefore an essential nutrient for man and animals.
Riboflavin may be produced by chemical synthesis and by various fermentation procedures using, for example, strains of Bacillus (e.g.,
Bacillus subtilis
), the ascomycetes
Ashbya gossypii
and
Eremothecium ashbyi
(Demain A. L. Riboflavin Over Synthesis. Ann. Rev. Microbiol. 1972, 26, 369 and Mitsuda H, Nakajima K., Effects of 8-azaguanine on Riboflavin Production and on the Nucleotide Pools in Non-Growing Cells of
Eremothecium ashbyii
. J. Nutr Sci Vitaminol (Tokyo) 1973; 19(3):215-227), various yeast strains such as
Candida guilliermondii, Candida famata
(F. W. Tanner, Jr., C. Vojnovich, J. M. Van Lanen. Riboflavin Production by Candida Species. Nature, 1945, 101 (2616):180-181) and related strains, as well as other microorganisms.
The pathway of riboflavin biosynthesis in yeast is shown in FIG.
1
. The recursors for the biosynthesis of riboflavin are guanosine triphosphate (GTP) and ribulose 5-phosphate. One mole of GTP and two moles of ribulose 5-phosphate are required to biosynthetically generate one mole of riboflavin.
In the yeast
Saccharomyces cerevisiae
, the biosynthesis of riboflavin requires at least six genes, specifically the genes RIB1, RIB2, RIB3, RIB4, RIB5 and RIB7 (Oltmanns O., Bacher A., Lingens F. and Zimmermann F. K., Biochemical and Genetic Classification of Riboflavin Deficient Mutants of
Saccharomyces cerevisiae
. Mol. Gen. Genet. 1969, 105, 306). In
C. guilliermondii
, the biosynthesis of riboflavin has also been reportedly shown to require the products of at least six genes, specifically the genes RIB1, RIB2, RIB3, RIB4, RIB5 and RIB6 (Shavlovskyy, G. M., Sibirnyy, A. A., Kshanovs'ka, B. V. Genetical classification of
C. guilliermondii
riboflavin auxotroph mutants. Genetika 15, 1561-1568, 1979). The enzymes specified by these
C. guilliermondii
genes and their roles in the biosynthetic pathway are also summarized in FIG.
1
and Table 1. In contrast to the situation in
B. subtilis
, the riboflavin biosynthetic genes are not clustered in the eucatyotes
S. cerevisiae
and
C. guilliermondii
.
TABLE 1
Enzymes and Genes of the Riboflavin Pathway
Gene
Enzyme
S. cerevisiae
C. guilliermondii
E. coli
A
GTP cyclohydrolase
RIB1
RIB1
ribA
B
bacterial deaminase
ribD
C
yeast reductase
RIB7
RIB2
D
yeast deaminase
RIB2
RIB3
E
bacterial reductase
ribD
F
unknown
phosphatase
G
lumazine synthase
RIB4
RIB5
ribE
H
riboflavin synthase
RIB5
RIB7
ribC
I
3,4-dihydroxy-2-buta
RIB3
RIB6
ribB
non 4-phosphate
synthase
The initial step in the riboflavin biosynthetic pathway is the opening of the imidazole ring of GTP catalyzed by the enzyme, GTP cyclohydrolase II. The product of this enzyme has been reported to be 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate. This intermediate is converted to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of side chain reduction, ring deamination and dephosphorylation reactions. The enzyme involved in the dephosphorylation of 5-amino-6-ribitylamino 5′-phosphate is still unknown.
The conversion of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione to 6,7-(1dimethyl-8-ribityllumazine by the enzyme, 6,7-dimethyl-8-ribityllumazine synthase, requires a second substrate, 3,4-dihydroxy-2-butanone 4-phosphate, which is obtained from ribulose 5-phosphate by the catalytic action of 3,4-dihydroxy-2-butanone-4-phosphate synthase. Finally, 6,7-dimethyl-8-ribityllumazine is converted to riboflavin by a dismutation reaction catalyzed by riboflavin synthase. The sequence of the RIB1 gene directing the synthesis of GTP cyclohydrolase II, the initial enzyme of the riboflavin pathway, reportedly has been established in the yeast,
C. guilliermondii
(Liauta-Teglivets, O., Hasslacher, M., Boretskyy, Y., Kohlwein, S. D., Shavlovskii, G. M. Molecular cloning of the GTP cyclohydrolase. Structural gene RIB1 of
Pichiia guilliermondii
involved in riboflavin biosynthesis. Yeast 11, 945-952, 1995).
Recombinant strains of
Bacillus subtilis
used in the production of riboflavin by fermentation have been reportedly described, e.g. in EP 405 370. These strains carry the riboflavin operon under the control of a strong promoter directing the production of the cognate enzymes. The gene constructs of the riboflavin operon under the control of a strong; promoter may be present at one or several different locations on the
B. subtilis
chromosome. The incorporation of an additional gene of the riboflavin pathway under the control of a strong promoter at a separate locus on the
B. subtilis
chromosome reportedly has also been shown to increase the yield of riboflavin obtained by fermentation. See, EP 821 063.
Whereas the production of riboflavin by native strains of yeasts such as
C. guilliermondii
have been reported, these reports suffer from the drawback that relatively small amounts of riboflavin are produced therefrom. Heretofore, recombinant DNA technology has not been applied to over-express riboflavin biosynthetic genes in
C. guillietmondii
or in related flavinogenic yeasts.
SUMMARY OF THE INVENTION
The invention is a recombinant DNA construct used to transform yeast strains which over-produce riboflavin.
More specifically, the present invention provides a yeast strain transformed by a recombinant DNA sequence such as a DNA sequence which, upon expression in a suitable host cell, encodes at least one polypeptide with riboflavin biosynthetic activity and which DNA sequence is transcriptionally linked to a promoter that is functional in such a yeast strain.
Another embodiment of the invention is a transformed yeast strain which belongs to the group of flavinogenic yeasts which over-produce riboflavin under conditions of iron starvation.
A further embodiment of the present invention is a transformed yeast strain wherein the polypeptide encoding DNA sequence is isolated or derived from yeast, preferably a flavinogenic yeast which over-produces riboflavin under conditions of iron starvation, such as Candida, e.g.
Candida guilliermondii
or
Candida famata.
Another embodiment of the present invention is a yeast strain wherein the polypeptide encoding DNA sequence encodes a protein with GTP cyclohydrolase II activity and is selected from the following DNA sequences:
a) the DNA sequence as shown in
FIG. 4
or its complementary strand;
b) DNA sequences which hybridize under standard conditions to the protein coding regions of the DNA sequences defined in (a) or fragments thereof; and
c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).
Another embodiment is a yeast strain transformed with a DNA construct as set forth above, wherein the promoter is the TEF
S. cerevisiae
promoter.
Another embodiment is a process for producing riboflavin by culturing a yeast strain as set forth above under suitable culture conditions to express riboflavin and then isolating the riboflavin from the medium or the yeast strain.
Another embodiment of the present invention is a process for producing riboflavin by mixing the isolated riboflavin with one or more suitable food or feed ingredients to form a food or feed composition.
Another embodiment is an isolated and purified DNA molecule which encodes a polypeptide with riboflavin biosynthetic activity isolated from a yeast strain and which encodes a polypeptide with riboflavin biosynthetic activity that is encoded by a nucleotide sequence including the DNA sequence in FIG.
4
and functionally equival
Babyak Lyubov Ya.
Bacher Adelbert
Boretskyy Yuriy R.
Demchyshyn Vasyl V.
Eberhardt Sabine
Bryan Cave LLP
Kerr Kathleen
Murthy Ponnathapuachuta
Roche Vitamins Inc.
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