Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Fungi
Reexamination Certificate
2001-03-01
2003-01-07
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Fungi
C435S052000, C435S069900, C435S193000, C435S255100, C435S255200, C435S254110, C435S254210, C435S320100, C435S483000, C536S023200, C536S023740
Reexamination Certificate
active
06503749
ABSTRACT:
The formation of 3-oxo-delta
4
-steroids from 3&bgr;-hydroxy-delta
5
precursors in the biosynthesis of all classes of steroid hormones in mammals is catalysed by the enzyme system 3&bgr;-hydroxy-delta
5
-steroid dehydrogenase (EC 1.1.1.145) and delta
5
-delta
4
-steroid isomerase (EC 5.3.3.1), designated 3&bgr;-HSD. For example, 3&bgr;-HSD catalyses the transformation of pregnenolone to progesterone, of 17&agr;-hydroxypregnenolone to 17&agr;-hydroxyprogesterone, of dehydroepiandrosterone to delta
4
-androstenedione or of 5-androstene-3&bgr;-17&bgr;-diol to testosterone (Simard et al., 1996).
Thus, 3&bgr;-HSD is one of the key enzymes in the route for biosynthesis of hydrocortisone starting from cholesterol in the adrenal cortex of mammals (FIG.
1
).
The use of recombinant microorganisms, especially modified yeasts, permitting heterologous expression of one or more of the mammalian enzymes of this biosynthetic route for producing hydrocortisone or intermediates of this biosynthesis was described for example in European patent application EP 340878, U.S. Pat. No. 5,137,822, Dumas et al., 1994 and Cauet et al., 1994.
When functional 3&bgr;-HSD is expressed in yeast, the transformed yeast cells do not completely convert 3&bgr;-hydroxysteroids to the corresponding 3-oxosteroids, for example pregnenolone to progesterone, but accumulate a compound which is also observed in the case of cells of untransformed yeast. Identification of the compound accumulated as the 3&bgr;-acetate ester of the starting steroid and characterization of the enzyme possessing acyltransferase activity which is responsible for this esterification (designated hereinafter as APAT for “acetyl-coenzyme A pregnenolone acetyltransferase”) are described in the present application. Furthermore, accumulation of pregnenolone acetate by a pregnenolone-producing transformed yeast strain was described in European patent application EP 727489. It can be considered on the basis of these observations that esterification of the 3&bgr;-hydroxysteroids produced by the yeast is undesirable as it is responsible for secondary reactions and by-products leading to a decrease in the yield of accumulated 3&bgr;-hydroxysteroids, for example pregnenolone, or to a decrease in the yield of bioconversion of 3&bgr;-hydroxy-delta
5
-steroids to 3-oxo-delta
4
-steroids, particularly in the production of progesterone or of 17&agr;-hydroxy-progesterone leading to a decrease in subsequent production of hydrocortisone by the biosynthetic route already mentioned.
On the basis of the results obtained, mentioned above, the present invention describes the construction of yeast strains that have lost the undesirable APAT activity, by alteration of the gene coding for this activity, resulting in stabilization of the 3&bgr;-hydroxysteroids in the presence of the latter. These strains can therefore be used as starting strains for constructing recombinant strains that are capable of converting 3&bgr;-hydroxysteroids to further products with improved yields.
The invention also describes the construction of yeast strains that have lost APAT activity by alteration of the gene coding for this activity and either expressing 3&bgr;-HSD or the cytochrome P
450
17&agr;, or co-expressing 3&bgr;-HSD and the cytochrome P
450
17&agr; of the route of biosynthesis of hydrocortisone from cholesterol. The strains expressing for example 3&bgr;-HSD make it possible to improve the yields in bioconversion of 3&bgr;-hydroxy-delta
5
-steroids to 3-oxo-delta
4
-steroids and can therefore be used in processes of improved production of hydrocortisone or of its intermediates in yeast.
A subject of the present invention is therefore a modified yeast strain in which the acetyl-CoA pregnenolone acetyltransferase (APAT) activity is eliminated by altering the gene coding for this activity, resulting in stabilization of the 3&bgr;-hydroxysteroids.
Alteration of the gene coding for APAT activity can be effected for example by insertion, deletion or substitution of a DNA sequence in the functional elements of the gene, for example the promoter or the sequence coding for the protein possessing APAT activity. Integration of the DNA sequence altered in this way in a host strain of yeast can then be effected for example by the technique of homologous recombination and leads to the generation of chromosomal mutants of yeast corresponding to the modified strains of the invention in which the disappearance of APAT activity and the stabilization of 3&bgr;-hydroxysteroids are demonstrated, for example by cell culture in the presence of pregnenolone and by measuring the pregnenolone content as a function of time, following the operating conditions described later in the experimental section.
The following may be mentioned in particular as host yeast strains used for the invention: strains of Saccharomyces such as
S. cerevisiae
, strains of Candida such as
C. maltosa
, strains of Kluyveromyces such as
K. lactis
or strains of Pichia such as
P. pastoris.
A particular subject of the invention is a yeast strain modified as above in which the gene altered is the ATF2 gene of
S. cerevisiae
or a homologue of the latter.
By gene ATF2 we mean the gene of
S. cerevisiae
identified in the yeast genome at locus ATF2 or YGR177c, of “Saccharomyces Genome Database” (SGD); (Cherry et al. http://genome-www.stanford.edu/Saccharomyces/) of which the open reading frame (ORF) designated YGR177c is translated into an amino acid sequence in the Mips database, accessible under accession number S64491 (Hebling U., Hofmann B. and Delius H. (May 1996)) and whose sequence is shown in FIG.
4
. This gene codes for a protein possessing APAT activity, as is shown later in the experimental section.
By gene that is a homologue of the ATF2 gene, we mean a gene that codes for a protein possessing APAT activity and possessing sequence identity of about 60% or more with the sequence of protein YGR177C.
A more particular subject of the invention is a modified yeast strain as above in which the altered gene is the ATF2 gene of
S. cerevisiae
, designated hereinafter as atf2 mutant strain.
A quite particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of a DNA sequence that has at least one nucleotide.
The DNA sequence that is inserted in the ATF2 gene so as to lose all APAT activity can be, for example, an auxotrophic selection gene supplying a nutritional requirement of the host strain such as the gene URA3, the gene LEU2, the gene TRP1, the gene HIS3 or the gene ADE2, for example a dominant selection gene such as a gene for resistance to an antibiotic such as G418, phleomycin or hygromycin B or for example a reporter gene such as the &bgr;GAL gene.
The DNA sequence that is inserted in the ATF2 gene can also be a yeast expression block made up of a promoter and a transcription terminator, for example a yeast promoter such as PGK, TDH3, CYC1 or TEF1, for example a yeast terminator such as CYC1, TDH3, TEF1 or PGK. The expression block can be a combination of the elements mentioned above, for example the block TEF1
prom
/PGK
term
.
A more quite particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the URA3 selection gene or of the expression block TEF1
prom
/GK
term
.
A particular subject of the invention is a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the URA3 selection gene.
The atf2 mutant strains of the invention, devoid of APAT activity and in which the URA3 gene has been inserted, designated hereinafter as atf2-&Dgr;::URA3, could thus be selected by prototrophy with uracil.
A quite particular subject of the invention is modified strains of
S. cerevisiae
designated as TGY156 and TGY158, the detailed constructions of which are given later in the experimental section.
A particular subject of the invention is also a modified yeast strain as above, in which the ATF2 gene is altered by insertion of the expression block TEF1
prom
/PGK
term
, The atf2 mutant
Achstetter Tilman
Cauet Gilles
Degryse Eric
Aventis Pharma S.A.
Bierman, Muserlian and Lucas
Myers Carla J.
LandOfFree
Yeast strains possessing the interrupted ATF2 gene and their... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Yeast strains possessing the interrupted ATF2 gene and their..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Yeast strains possessing the interrupted ATF2 gene and their... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3065100