Process for preparing doxorubicin

Details Process for preparing doxorubicin Process for preparing doxorubicin

1999-01-14

2001-04-03

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S189000, C435S252350, C435S320100, C435S471000, C536S023200

Reexamination Certificate

active

06210930

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns a process for improving doxorubicin production by means of a recombinant strain bearing a mutation in a gene of daunorubicin metabolism.
BACKGROUND OF THE INVENTION
Anthracyclines of the daunorubicin group such as doxorubicin, carminomycin and aclacinomycin and their synthetic analogs are among the most widely employed agents in antitumoral therapy (F. Arcamone, Doxorubicin, Academic Press New York, 1981, pp. 12-25; A. Grein, Process Biochem., 16:34, 1981; T. Kaneko, Chimicaoggi May 11, 1988; C. E. Myers et al., “Biochemical mechanism of tumor cell kill” in Anthracycline and Anthracenedione-Based Anti-cancer Agents (Lown, J. W., ed.) Elsevier Amsterdam, pp. 527-569, 1988; J. W. Lown, Pharmac. Ther. 60:185-214, 1993). Anthracyclines of the daunorubicin group are naturally occurring compounds produced by various Streptomyces species and by
Actinomyces carminata
. Doxorubicin is mainly produced by strains of
Streptomyces peucetius
while daunorubicin is produced by many other Actinomycetes. In particular daunorubicin and doxorubicin are synthesized in
S. peucetius
ATCC 29050 and 27952 from malonic acid, propionic acid and glucose by the pathway summarized in Grein (Advan. Applied Microbiol. 32:203, 1987) and in Eckart and Wagner (J. Basic Microbiol. 28:137, 1988). Aklavinone (11-deoxy-&egr;-rhodomycinone), &egr;-rhodomycinone and carminomycin are established intermediates in this process. The final step in this pathway involves the hydroxylation of daunorubicin to doxorubicin by the DoxA enzyme ({U.S. Ser. No. 08/396,218, WO96/27014}; M. L. Dickens and W. R. Strohl, J. Bacteriol. 178:3389 (1996)), which is reported to occur only in
S. peucetius.
13-Dihydrodaunorubicin may be an intermediate in the conversion of &egr;-rhodomycinone to daunorubicin via rhodomycin D (
FIG. 1
) according to Dickens et al. (J. Bacteriol. 179:2641 (1997)). Daunorubicin is bioconverted to (13S)-13-dihydrodaunorubicin when added to cultures of
S. peucetius
and some other Streptomycetes (N. Crespi-Perellino et al., Experientia, 38:1455, 1982; T. Oki et al., J. Antibiotics, 34:1229, 1981; G. Cassinelli et al., Gazz. Chim. Ital. 114:185,1984). It is not known whether the 13-dihydrodaunorubicin that may be an intermediate of daunorubicin and doxorubicin production in
S. peucetius
is identical to the (13S)-13-dihydrodaunorubicin formed by this bioconversion. Since these two compounds can differ in their C-13 stereochemistry, one diastereomer of 13-dihydrodaunorubicin might be a substrate for DoxA and the other one would not. In the latter case, C-13 reduction of daunorubicin would block its further oxidation to doxorubicin.
Several genes for daunorubicin and doxorubicin biosynthesis and resistance have been isolated from
S. peucetius
29050 and 27952 by cloning experiments. The
S. peucetius
dnrU gene identified herein is a homolog of the Streptomyces sp. strain C5 gene ORF1 (syn. dauU) described by Dickens and Strohl (J. Bacteriol. 178:3389 (1996)). Since the predicted protein products of the dnrU and dauU genes resemble enzymes known to reduce ketone groups, the dnrU and dauU proteins may catalyze the reduction of daunorubicin, formed in vivo or added to cultures exogenously, to 13-dihydrodaunorubicin.
Daunorubicin is known to be converted to 4′-O-glycosides called baumycins in Streptomyces species (Y. Takahashi, H. Naganawa. T. Takeuchi, H. Umezawa, T. Komiyama, T. Oki and T. Inui. J. Antibiot. 30:622, 1977) thus decreasing also the amount of doxorubicin potentially obtainable through oxidation of daunorubicin. For recovering daunorubicin at the end of fermentation, baumycins are converted to daunorubicin by acid hydrolysis. However this process presents certain drawbacks in that the amount of doxorubicin thus produced is low and the process is complicated by the acid hydrolysis step. The present invention solves this problem by providing a mutated Streptomyces strain in which one of the genes responsible for the conversion of daunorubicin to baumycins has been insertionally inactivated.
SUMMARY OF THE INVENTION
The present invention concerns a process for preparing doxorubicin by means of a bacteria recombinant strain bearing at least one mutation and preferably two mutations blocking the function of at least one gene of daunorubicin metabolism. The inactivating mutations increase daunorubicin and doxorubicin production levels and cause the disappearance of baumycin-like products resulting in daunorubicin and doxorubicin secretion directly into the culture medium. Consequently, there is no need to acidify the cultures at the end of fermentation. The relative amounts of 6-rhodomycinone and 13-dihydrodaunorubicin may also be altered, as an incidental consequence of the mutation. Preferably the bacterium is a strain of Streptomyces sp. producing daunorubicin and doxorubicin, having at least one mutation blocking the function of at least one gene of daunorubicin metabolism.
One of the inactivated genes is preferably in the DNA fragment having the configuration of restriction sites shown in
FIG. 5
or in a fragment derived therefrom containing a gene, dnrX, encoding a protein involved in the metabolism of daunorubicin to acid-sensitive, baumycin-like compounds.
The second inactivated gene is preferably comprised in the DNA fragment having the configuration of restriction sites shown in
FIG. 3
or in a fragment derived therefrom containing a gene, dnrU, coding for a protein involved in the metabolism of daunorubicin.
The present invention provides a mutant strain of
S. peucetius
, obtained from
S. peucetius
ATCC 29050, having a mutation blocking the function of the dnrU gene. This mutation greatly increases the doxorubicin production level relative to the amount of daunorubicin, and by coincidence may also increase the amount of &egr;-rhodomycinone.
The present invention also provides a mutant strain of
S. peucetius
, obtained from
S. peucetius
ATCC 29050, having a mutation inactivating the function of the dnrX gene.
The present invention also provides a mutant strain of
S. Peucetius
, obtained from
S. peucetius
ATCC 29050, having a mutation inactivating the function of both the dnrX and dnrU genes.
Genes for daunorubicin and doxorubicin biosynthesis and resistance have been obtained from
S. peucetius
29050 and
S. peucetius
27952 by cloning experiments as described in Stutzman-Engwall and Hutchinson (Proc. Natl. Acad. Sci. USA, 86: 3135 (1988)) and Otten et al., (J. Bacteriol. 172: 3427 (1990)).
The dnrU mutant can be obtained by disrupting the dnrU gene, obtained from the
S. peucetius
29050 anthracycline production genes described by Stutzman-Engwall and Hutchinson (Proc. Natl. Acad. Sci. USA, 86: 3135 (1988)) and Otten et al. (J. Bacteriol. 172: 3427 (1990)), by insertion of the neomycin/kanamycin resistance gene (aphII) into the BaII restriction site located at the beginning of dnrU. This disrupted dnrU::aphII gene is used to replace the normal dnrU gene in the 29050 strain.
The dnrX mutant was obtained by disrupting the dnrX gene of the anthracycline biosynthetic gene cluster by insertion of the neomycin/kanamycin resistance gene (aphII) in the NcoI restriction site of dnrX.


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