Note: Descriptions are shown in the official language in which they were submitted.
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WO 99/55829 PCT/US99/07016
Process for Preparing Doxorubicin.
Field of the Invention
The present invention concerns a process for improving daunorubicin to
s doxorubicin conversion by means of host cells transformed with recombinant
vectors
comprising DNA encoding a daunorubicin C-14 hydroxylase together with genes
conferring resistance to anthracycline antibiotics.
Background of the Invention
Anthracyclines of daunorubicin group such as doxorubicin, carminomycin and
io 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; 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,
is pp. 527-569, 1988; J. W. Lown, Pharmac. Ther. 60:185, 1993).
Anthracyclines of the daunorubicin group are naturally occurring compounds
produced by various strains of Streptomyces (S.peucetius, S.coeruleorubidus,
S.galilaeus, S.griseus, S.griseoruber, S.insignis, S.viridochromogenes,
S.bifurcus and
S.sp. strain C5) and by Acfinomyces carminata. Doxorubicin is mainly produced
by
2o strains of S. peucefius. In particular daunorubicin and doxorubicin are
synthesized in
Streptomyces peucetius ATCC 29050 and in S, peucetius subsp. caesius ATCC
27952.
The anthracycline doxorubicin is made by S.peucetius 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.
2s Aklavinone (11-deoxy-e-rhodomycinone), e-rhodomycinone, rhodomycin D,
carminomycin and daunorubicin are established intermediates in this process.
The final
step in this pathway involves the C-14 hydroxylation of daunorubicin to
doxorubicin.
Genes for daunorubicin biosynthesis have been obtained from S.peucetius
29050 and S. peucefius 27952 by cloning experiments (Stutzman-Engwall and
3o Hutchinson, Proc.NatLAcad.Sci.USA 86:3135,1988; Otten et al., J.Bacteriol.
172:3427,1990).The gene encoding the daunorubicin 14-hydroxylase, which
converts
daunorubicin to doxorubicin has been obtained from S.peucefius 29050 and its
mutants
by cloning experiments and it was overexpressed in the host cells of
Streptomyces
species and Escherichia coli as described in WO 96/27014, publication date
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2
Sept.6,1996.
Two genes of the daunorubicin biosynthetic cluster, dn-A and drr8, which
confer
doxorubicin and daunorubicin resistance to Streptomyces lividans have been
cloned
from S. peucetius ATCC 29050 strain (Guilfoile and Hutchinson,
s Proc.NatLAcad.Sci.USA 88:8553, 1991 ) (Accession Number M73758 of Genbank)
and
from the S.peucetius 7600 mutant (EP-0371,112-A and Colombo et al.,
J.Bacteriol.174:1641,1992). These genes encode two translationally coupled
proteins,
both of which are required for daunorubicin and doxorubicin resistance in this
host. The
sequence of the predicted product of one of the two genes is similar to the
products of
io other transport and resistance genes, most notably the P-glycoproteins from
mammalian tumor cells. Another gene, drrC, which confers resistance to
daunorubicin
and doxorubicin with a strong sequence similarity to the Escherichia coli and
Micrococcus luteus UvrA proteins involved in excision repair of DNA has been
cloned
from S.peucetius ATCC 29050 (Lomovskaya et al., J.Bacterio1.178:3238, 1996).
~5 Summani of the invention
The present invention provides a process for improving daunorubicin to
doxorubicin conversion in host cells by means of recombinant vectors
comprising a
DNA region or fragment containing the gene dxrA encoding daunorubicin 14-
hydroxylase together with a DNA region or fragment containing one, two or
three
2o genes, selected from the group consisting of drrA, drr8 and drrC,
conferring resistance
to daunorubicin and doxorubicin. The last three genes confer a high level of
resistance
in the host cells to doxorubicin, the product of the conversion process,
making the
process more efficient than the previous one obtained using host cells
transformed with
the recombinant vectors carrying only the DNA fragment containing the dxrA
gene,
2s described in WO 96/27014, even when a strong promoter is used.
The DNA of the invention comprises preferably all three of the drrA, drr8 and
drrC genes or only the two drrA and drr8 genes.
The DNA may be ligated to a heterologous transcriptional control sequence in
the correct fashion or cloned into a vector at the restriction site
appropriately located
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3
near a transcriptional control sequence in a vector. Typically, the vector is
a plasmid.
The recombinant vectors may be used to transform a suitable host cell. The
host may
be strains of Actinomycetes that do not or do produce anthracyclines,
preferably strains
of Streptomyces .
s Brief descrir~tion of the drav~ina~
Fig. 1 (a-c) illustrate the construction of the plasmid pIS156 described in
Example 1. This plasmid was constructed by insertion of the 2.9 kb fragment
containing
the doxA (formerly dxrA), the dnrV (formerly dnrORF10) and the C-terminal part
of the
dnrU (ddnrU, formerly dnrORF9) genes, obtained from the recombinant plasmid
pIS70
io (WO 96/27014 and A. Inventi Solari et al., GMBIM '96, P58), under the
control of the
strong promoter ermE* (Bibb et al., Molec. Microbiol. 14:533, 1994) into the
plasmid
pWHM3 (Vara et ai., J. Bacteriol. 171:5872, 1989).
In order to better describe the invention, we provide the SEQ.ID. No:1 of
2.867
nt consisting of the doxA, dnrV and the C-terminal part of the dnrU (ddnrU)
genes
is (complementary strand to the coding strand).
Fig. 2 (a-d) illustrate the construction of the plasmid pIS284 described in
Example 1. This plasmid contains the 2.9 kb fragment encompassing the doxA,
the
dnrV and the C-terminal part of the dnrU genes, obtained from the recombinant
plasmid
pIS70, under the control of the strong promoter ermE* together with a DNA
fragment
20 of 2.3 Kb including the drrA and drr8 resistance genes obtained from the
plasmid
pWHM603 (P. Guilfoile and C.R. Hutchinson, Proc. Natl. Acad. Sci. USA 88:8553,
1991 ) subcloned into the plasmid pWHM3.
Fig. 3 (a-c) illustrate the construction of the plasmid pIS287 described in
Example 2. Said plasmid was constructed by insertion of the 2.9 kb BamHl-
Hindlll
2s fragment containing the doxA formerly, dxrA), dnrV (formerly dnr-ORF10) and
the C
terminal part of the dnrU (ddnrU, formerly, dnr-ORF9) genes, obtained from the
recombinant plasmid pIS70 (WO 96/727014), under the control of the strong
promoter
ermE"' together with the 2.3 kb Xbal Hindlll DNA fragment containing the drrA
and drr8
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4
resistance genes and the 3.9 kb EcoRl-Hindlll fragment containing the drrC
resistance
gene into the plasmid pWHM3.
The maps shown in Figs. 1,2 and 3 do not necessarily provide an exhaustive
listing of all restriction sites present in the DNA fragments. However, the
reported sites
s are sufficient for an unambiguous recognition of the DNA segments.
Restriction sites abbreviations: Ap, apramycin;tsr, thiostrepton, amp,
ampicillin;
B, BamHl; G, Bglll; N, Notl; K, Kpnl; E, EcoRl; H, Hindlll; P, Psfl; S, Sphl;
X, Xbal, L,
Bgll ; T, Ssfl .
Detailed d rir~tion of the in~Pntion.
to The present invention provides a DNA molecule in which a DNA region or
fragment containing the gene encoding a daunorubicin C-14 hydroxylase is
joined to
a DNA region or fragment containing one, two or three different genes selected
from
the group consisting of drrA, drrB, drrC genes encoding proteins conferring to
the host
cells resistance to daunorubicin and doxorubicin.
is The DNA region containing the gene encoding a daunorubicin C-14 hydroxylase
is preferably the 2.9 kb DNA region obtained from the recombinant plasmid
pIS70
described in the patent WO 96/27014 by digestion with BamHl-Hindlll enzymes.
This
fragment contains the doxA gene, encoding the C-14 hydroxylase. Daunorubicin C-
14
hydroxylase converts daunorubicin to doxorubicin. The 2.9 kb DNA fragment also
2o comprises the dnrV gene between the Notl-Kpnl sites and a Notl-Sphl
fragment
containing the C-terminal part of the dnr(J (~dnrU ) gene.
Preferably, this 2.9 kb DNA fragment encoding a daunorubicin C-14 hydroxylase
was
ligated to both the 2.3 kb Xbal-Hindlll DNA fragment containing the drrA and
drrB
resistance genes obtained from the plasmid pWHM603 and the 3.9 kb EcoRl-
HindlIl
2s fragment containing the drrC gene obtained from the plasmid pWHM264; in
another
preferred embodiment, the 2.9 kb DNA fragment is ligated to the 2.3 kb Xbal -
Hindlll
DNA fragment only.
All the DNA molecules encoding a daunorubicin C-14 hydroxylase described in
WO 96/27014 may be employed in the present invention.
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s
in particular the DNA molecule of the present invention may comprise all of
the
2.9 kb DNA fragment or only a part of the fragment, at least 1.2 kb in length
corresponding to the Kpnl-BamHl fragment containing the DNA molecule of doxA,
encoding a daunorubicin C-14 hydroxylase, which converts daunorubicin to
doxorubicin.
s This DNA molecule consists essentially of the sequence reported in the
patent
application WO 96/27014, which sequence is referred to as the "dxrA" sequence.
Also,
the deduced amino acid sequence of the daunorubicin C-14 hydroxylase is shown
in
that patent application.
The DNA molecule of the present invention may comprise at least 2247 nt of the
io 2.3 kb Xbal-Hindlll DNA fragment containing the drrA and drrB genes
encoding proteins
conferring to host cells resistance to daunorubicin and doxorubicin.
The DNA molecule of the invention may comprise all or part of the 3.9 kb EcoRl-
Hindlll fragment containing the drrC resistance gene, at least 2.5 kb in
length
corresponding to the Sstl-Sphl fragment containing the DNA molecule of drrC,
encoding
is a protein conferring to host cells resistance to daunorubicin and
doxorubicin.
The present invention also includes DNA comprising genes conferring resistance
to doxorubicin and daunorubicin having a sequence at least 80% identical to
the
sequences of the drrA and drrB genes (Guilfoile and Hutchinson,
Proc.NatLAcad.Sci.USA 88:8553, 1991 ) and or drrC gene (Lomovskaya et al.,
2o J.Bacteriol.178:3238, 1996).
The DNA molecule of the invention may be ligated to a heterologous
transcriptional control sequence in the correct fashion or cloned into a
vector at a
restriction site appropriately located near a transcriptional control sequence
in the
vector. Preferably the transcription of the different genes may be coordinated
by a
2s common strong promoter such as ermE'"'(Bibb et al., Molec. Microbiol.
14:533, 1994).
The DNA molecule of the invention may be ligated into any autonomously
replicating and/or integrating agent comprising a DNA molecule to which one or
more
additional DNA segments can be added. Typically, however, the vector is a
plasmid. A
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6
preferred plasmid is the high-copy number plasmid pWHM3 or pIJ702 (Katz et
al., J.
Gen. Microbiol. 129:2703, 1983). Other suitable plasmids are pIJ680 (Hopwood
et al.,
Genetic Manipulation of Streptomyces. A laboratory Manual, John Innes
Foundation,
Norwich, UK,1985) and pWHM601 (Guilfoile and Hutchinson, Proc. Natl. Acad.
Sci.
s USA 88:8553, 1991 ).
Any suitable technique may be used to insert the DNA into the vector.
Insertion
can be achieved by ligating the DNA into a linearized vector at an appropriate
restriction
site. For this, direct combination of sticky or blunt ends, homopolymer
tailing, or the use
of a Pinker or adapter molecule may be employed.
io The recombinant vector may be used to transform a suitable host cells that
do
not or do produce anthracyclines.
The host cells may be ones that are daunorubicin or doxorubicin sensitive,
i.e.,
cannot grow in the presence of a certain amount of daunorubicin or
doxorubicin, or that
are daunorubicin or doxorubicin resistant. In any case the resulting
recombinant clones
is obtained by transformation with the new recombinant vectors of the
invention show
higher level of resistance to daunorubicin and doxorubicin than the parental
host. The
level of doxorubicin resistance in recombinant S. lividans is much higher than
the level
observed in anthracycline producing strains S. peucetius ATCC 29050 and ATCC
27952.
2o The host may be a microorganism such as a bacterium. Strains of
Actinomycetes, in particular strains of S. lividans and other strains of
Strepfomyces
species that do not produce anthracyclines may be transformed. S. lividans TK
23 is a
more suitable host in comparison to the S. peucetius dnrN mutant transformed
with the
recombinant plasmid pIS70 containing the dxrA gene used for daunorubicin to
2s doxorubicin bioconversion (WO 96/27014).
The recombinant vectors of the invention may also be used to transform a
suitable host cell which produces daunorubicin, in order to enhance the
conversion of
daunorubicin to doxorubicin.
S. peucetius ATCC 29050 and ATCC27952 strains including their mutants that
produce
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7
anthracyclines may therefore be transformed. )n particular S. peucetius strain
WMH1654, a mutant strain obtained from S.peucetius ATCC 29050 and deposited at
the American Type Culture Collection, 10801 University Boulevard, Manassas,
Virginia
20110-2209, USA, under the accession number ATCC55936 may be used.
s Transformants of Streptomyces strains are typically obtained by protoplast
transformation.
The invention includes processes for improving doxorubicin production by
conversion of daunorubicin, which processes comprise a bioconversion process
of
added daunorubicin into doxorubicin in hosts which do not produce
anthracyclines and
io a fermentation process for producing doxorubicin in hosts which directly
produce
daunorubicin.
Bioconversion procPS~ of da ~nnmbi in to doxo »ir9n
This process comprises:
1 ) culturing the recombinant host cells not producing daunorubicin
transformed with the
is vectors of the invention to which daunorubicin is added and
2) isolating doxorubicin from the culture.
In this process the recombinant strain may be cultured at temperatures from
20°C to 40°C, for example from 24°C to 37°C. The
daunorubicin is added to the culture
medium from 24 to 96 hours of the growth phase. The culture is preferably
carried out
2o with shaking. The duration of the culture in the presence of daunorubicin
may be from
12 to 72 hours. The concentration of daunorubicin in the culture may be from
20 to
1000 mcg/ml; for example from 100 to 400 mcg/ml.
Doxorubicin production by ferment~~: ;;,
This process comprises:
2s 1 ) culturing recombinant daunorubicin-producing host cells transformed
with the vectors
of the invention and
2) isolating doxorubicin from the culture.
In this process the recombinant strain may be cultured at temperature from
20°C
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8
to 40°C; for example from 26°C to 34°C. The culture is
carried out with shaking. The
duration of the culture may be from 72 to 168 hours.
Materials and Methods
s Bacterial strains and r~lasmidw E, coli strain DHSa, which is sensitive to
ampicillin and
apramycin is used for subcloning DNA fragments. The host S. lividans TK23 was
obtained from D. A. Hopwood (John lnnes Institute, Norwich,United Kingdom) and
the
host S. peucetius WMH1654 is a mutant strain obtained from S.peucetius ATCC
29050
and has been deposited at the American Type Culture Collection, 10801
University
io Boulevard, Manassas, Virginia 20110-2209, USA, under the accession number
ATCC55936.
The plasmid cloning vectors are pGem-7Zf(+) and related plasmids (Promega,
Madison,
WI), pIJ4070 (D. A. Hopwood) and the E.coli-Streptomyces shuttle vector pWHM3
(Vara et al., J. Bacteriol. 171:5872, 1989).
is
Media and bcff .r~ E. coli strain DHSa is maintained on LB agar (Sambrook et
al.,
Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold
Spring Harbor, NY, 1989). When selecting for transformants, ampicillin or
apramycin
are added at concentrations of 100 micrograms/ml.
2o S. lividans TK23 and S. peucetius WMH1654 are maintained on R2YE (Hopwood
et
al., Genetic Manipulation of Streptomyces. A Laboratory Manual, John Innes
Foundation, Norwich, UK, 1985) and ISP4 (Difco, Detroit, MI} agar media,
respectively.
When selecting for transformants, the plates are overlayed with soft agar
containing
thiostrepton at a concentration of 50 microgramslml.
2s
Subcloning D A fragm n : DNA samples are digested with appropriate restriction
enzymes and separated on agarose gels by standard methods (Sambrook et al.,
Molecular Cloning. A Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold
Spring Harbor, NY, 1989). Agarose slices containing DNA fragments of interest
are
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9
excised from a gel and the DNA is isolated from these slices using the
GENECLEAN
device (Bio101, La Jolla, CA) or an equivalent. The isolated DNA fragments are
subcloned using standard techniques (Sambrook et al., Molecular Cloning. A
Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY,
1989)
s into E. coli for routine manipulations, and E, coli-Streptomyces shuttle
vectors or
Streptomyces vectors for expression experiments.
Transformation of tr ,otom Pt Sp~GI~S and E coli: Competent cells of E. coli
are
prepared by the calcium chloride method (Sambrook et al., Molecular Cloning. A
i~ Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor,
NY, 1989)
and transformed by standard techniques (Sambrook et al., Molecular Cloning. A
Laboratory Manual, 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor, NY,
1989).
S. lividans TK23 is grown in liquid R2YE medium (Hopwood et al., Genetic
Manipulation
of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK,
1985)
is and harvested after 48 hr. The mycelial pellet is washed twice with 10.3%
(wt/vol)
sucrose solution and used to prepare protoplasts according to the method
outlined in
the Hopwood manual (Hopwood et al., Genetic Manipulation of Streptomyces. A
Laboratory Manual, John Innes Foundation, Norwich, UK, 1985). The protoplast
pellet
is suspended in about 300 microlitres of P buffer (Hopwood et al., Genetic
Manipulation
20 of Streptomyces. A Laboratory Manual, John Innes Foundation, Norwich, UK,
1985)
and 50 microlitres aliquot of this suspension is used for each transformation.
Protoplasts are transformed with plasmid DNA according to the small scale
transformation method of Hopwood et al. (Genetic Manipulation of Streptomyces.
A
LaboraforyManual, John Innes Foundation, Norwich, UK, 1985), Stutzman-Engwall
and
2s Hutchinson (Proc. Natl. Acad. Sci. USA. 86:3135, 1988) or Otten et al. (J.
Bacteriol.
172: 3427, 1990). After 17 hr of regeneration on R2YE medium at 30°C,
the plates are
overlayed with 200 micrograms/ml of thiostrepton and allowed to grow at
30°C until
sporulated.
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F_val~ation of daunor~bicin and doxorubicin resistan hPVel: The level of
resistance is
expressed as Minimal Inhibitory Concentration (MIC) and is determined by the
standard
two-fold dilution method using R2YE medium. The strains are cultured in slants
of
s R2YE medium and incubated at 28°C for 8-10 days. Recombinant strains
are grown
in the same medium added with 20 micrograms/ml of thiostrepton. Bacterial
cultures
containing approximately 106-10'viable cells/ml are prepared from cultures
grown at
28°C at 280 rpm for 48 hours in Tryptic Soy Broth (Difco). The cultures
are
homogenized by glass beads. One loopful of the homogenized cultures is
inoculated
to on the agar plates containing different concentrations of daunorubicin and
doxorubicin
from 0.39 to 800 micrograms/ml. The agar plates are incubated at 30°C
for 7 days and
the MICs are determined as the lowest_concentrations that prevent visible
growth.
I5 Daunorubicin to Doxorubicin bioconversion: S. lividans TK23 transformants
harboring
a plasmid of the invention are inoculated into 25 ml of liquid R2YE medium
with 40
micrograms/ml of thiostrepton. Cultures are grown in 300 ml Erlenmeyer flasks
and
incubated on a rotary shaker at 280 rpm at 30 C°. After 2 days of
growth, 2.5 ml of this
culture are transferred to 25 ml of APM production medium: ((g/I) glucose
(60), yeast
2o extract (8), malt extract (20), NaCI (2), 3-(morpholino)propanesulfonic
acid (MOPS
sodium salt) (15), MgS04 .7H20 (0.2), FeS04 .7H20 (0.01 ), ZnS04.7H20 (0.01 ),
supplemented with 20 micrograms/ml of thiostrepton. 400 micrograms/ml of
daunorubicin are added at 48 hr.of the growth phase. Cultures are grown in 300
ml
Erlenmeyer flasks and incubated on a rotary shaker at 280 rpm at 30 C°
for 72 hr.
zs Each culture is acidified with 25 milligrams/ml of oxalic acid and after
incubation at 30°C
on a rotary shaker at 280 rpm for 30 min. is extracted with an equal volume of
acetonitrile:methanol (1:1 ) at 30°C and 300 rpm for 2 hr. The extract
is filtered and the
filtrate is analyzed by reversed-phase high pressure liquid chromatography (RP-
HPLC).
RP-HPLC is performed by using a Vydac C,e column (4.6 x 250 millimeters; 5
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11
micrometers particle size) at a flow rate of 0.385 ml/min. Mobiie phase A is
0.2%
trifluoroacetic acid (TFA, from Pierce Chemical Co.) in H20 and mobile phase B
is
0.078% TFA in acetonitrile (from J.T.Baker Chemical Co.). Elution is performed
with a
linear gradient from 20 to 60% phase B in phase A in 33 minutes and monitored
with
s a diode array detector set at 488 nm (bandwidth 12 micrometers).
Daunorubicin and
doxorubicin (10 micrograms/ml in methanol) are used as external standards to
quantitate the amount of these metabolites isolated from the cultures.
I~xer~bis'ttion: The S. peucetius WMH1654 mutant is transformed with a
plasmid of the invention. Transformant~ arP i~~~~~m+o.~ ir,f~, ~~ .",i ..r
onvr .r_~:____
supplemented with 20 micrograms/ml thiostrepton. Cultures are grown in 300 ml
Erlenmeyer flasks on a rotary shaker at 280 rpm at 30°C. After 2 days
of growth, 2.5
ml of this culture are transferred to 25 ml of APM medium supplemented with 20
micrograms/ml thiostrepton. Cultures are grown in 300 ml Erlenmeyer flasks on
a rotary
is shaker at 280 rpm at 28°C for 96 - 120 hours. Each culture is
acidified with 25
milligrams/ml of oxalic acid and, after 45 min. incubation at 30°C on a
rotary shaker at
280 rpm, is extracted with an equal volume of acetonitrile:methanol (1:1 ) at
30°C and
300 rpm for 2 hr. The extract is filtered and the filtrate is analyzed by RP-
HPLC
following the same method used to analyze the bioconversion products.
Example 1
Ex~mpl~1 (Fig. 1 (a-c) and Fig. 2 (a-d).
In order to remove a non-essential region, the plasmid pIS70 (W096/27014) is
before
2s digested EcoRl-Hindlll and the 3.5 kb fragment is subcloned into the same
sites of the
multiple cloning site sequence of the plasmid pGEM-7Zf (+) (Promega, Madison-
WI
USA) to obtain another BamHl restriction site. The new plasmid pGendoxAUV was
BamHl digested and the fragment, now reduced to 2.9 kb, was transferred into
the
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12
plasmid pIJ4070 (from the John Innes Institute, Norwich, UK) under the control
of strong
promoter ermE*. This new plasmid, named p7doxAUV, was digested Bglll and the
fragment inserted into the plasmid pWHM3 (J.Vara et al., J. Bacteriol.
171:5872-5881,
1989) to obtain the plasmid pIS156 (fig. 1c).
s The 2.3 kb Bgll fragment containing the drrA and drrB resistance genes is
transferred
after blunt ending from the plasmid pWHM603 into the Smal site of the plasmid
pBluescript II SK + (Stratagene) to obtain the plasmid pdrrAB and an Xbal-
Hindlll
fragment is transferred from pdrrAB into the vector pIJ4070 to obtain pIS278.
Afterwards, pIS278 is digested with EcoRl Xbal and inserted into the EcoRl
Xbal
to plasmid pWHM3 to obtain the plasmid pIS281. This plasmid is digested with
Xbal and
the Xbal fragment of plasmid pIS156 is inserted to obtain the plasmid pIS284.
Example 2
is
Con tr ~ ion of th _ la mid I 87 tFig~~~; The drrC resistance gene contained
in the plasmid pWHM264 is excised by EcoRl-Hindlll digestion and inserted into
the
plasmid pIJ4070 to obtain the plasmid pIS282. From this plasmid, the drrC
resistance
gene is transferred as a Bglll fragment to pIS252 (this plasmid is a modified
form of
2o pWHM3 containing an extra Bglll site close to the EcoRl site) to obtain the
plasmid
pIS285. pIS285 is EcoRl digested and ligated with the 5.5 kb DNA fragment
excised
from plasmid pIS284 to obtain the plasmid pIS287.
Example 3
2s R istanc~ of th abov r ombinant olas~~~ids t~ 'IOXOtLhlr'~n~ The level of
resistance
to daunorubicin and doxorubicin of S. lividans TK23 transformed with the
recombinant
plasmids pIS70, pIS284 or pIS287 in comparison with S. lividans TK23, S.
lividans
TK23 transformed with the vector pWHM3 and the anthracycline producing S.
peucetius ATCC 29050 and ATCC 27952 strains is determined as MICs on R2YE
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13
medium following the procedure described in Materials and Methods. The maximum
level of daunorubicin and doxorubicin resistance is obtained with the plasmid
pIS287
containing the drrA, drr8 and drrC resistance genes. The level of doxorubicin
resistance
was increased 64 times also with the plasmid containing only the drrA and
s drrB.resistance genes (Table 1 ).
Table 1. Resistance of recombinant strains to doxorubicin.
Strain MIC for doxorubicin (micrograms/ml)
S. peucetius ATCC 29050 12.5
to S. peucetius ATCC 27952 12.5
S. lividans TK23 12.5
S. lividans TK23(pWHM3) 12.5
S. lividans TK23(pIS284) 800
S. lividans TK23(pIS287) >800
is
Example 4
2o different r i tan P n~PrnPs: The pIS70, pIS284 or pIS287 plasmids are
introduced into
S. l ividans TK23 by transformation with selection for thiostrepton
resistance, according
to the procedures described in the Materials and Methods section. The
resulting S.
lividans TK23(pIS70), S. lividans TK23(pIS284) and S, lividans TK23(pIS287)
transformants are tested for the ability to bioconvert a high level (400
micrograms/ml)
2s of daunorubicin to doxorubicin using the APM medium as described above. S.
lividans
TK23(pIS70) transformants can convert up to 11.5% of added daunorubicin to
doxorubicin (Table 2). S. lividans TK23(pIS284) and S. lividans TK23(pIS287)
transformants can convert up to 73.5% of added daunorubicin to doxorubicin
(Table 2).
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Table 2. Bioconversion of daunorubicin to doxorubicin by S. lividans strains.
Strain Anthracycline (micrograms/ml)
DOX DNR 13-dihydroDNR
s S. lividans TK23(pIS70) (control) 46 250 70
S. lividans TK23(pIS284) 294 33 21
S. iividans TK23(pIS287) 288 24 35
to Example 5
different resistan~r~P a ne : The pIS284 and pIS287 plasmids are introduced
into S.
is peucetius WMH1654 dnrX mutant strain by protoplasts transformation with
selection for
thiostrepton resistance, according to the procedures described in the
Materials and
Methods section. The resulting S. peucetius transformants are fermented and
the
fermentation broths analyzed according to the method previously described. S.
peucefius WMH1654{pIS284) produced up to 81 micrograms/ml of doxorubicin and
up
2o to 18 micrograms/ml of daunorubicin after a 120 hr fermentation (Table 3).
S.peucefius
WMH1654(pIS287) produced up to 92 micrograms/ml of doxorubicin and no
detectable
amount of daunorubicin (Table 3). .
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TabIP ~. Doxorubicin production by S. peucefius WMH1654 dnrX strains.
Strain Anthracycline (micrograms/ml)
DOX DNR 13-dihydroDNR
S. peucetius WMH1654 41 35 18
s S. peucetius WMH1654(pIS284) 81 18 g
S. peucetius WMH1654(pIS287) g2 p
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SEQ ID.1
1 GGATCCGCAC CGGGTACACG GCACGGGACC GCCCACCGCG CGGTGCGCGG
51 TGGGCGGTCC CGTGCCGGTC GCGGCCGGCG GATCAGCGCA GCCAGACGGG
101 CAGTTCGGTG AGCCGCGCCG TCTGGGCCCC CTTCCGGCAC CACCGCAACT
151 CGTCGTACGG CACGGCCAGT CGGGCCTCGG GGAACCTGCT GCGCAGTACG
201 CCGATCATCG TGCGCGACTC CAGCTGGGCG AGCTGCTCCC CGATGCAGTA
251 GTGCGGCCCG TCGCCGAAGG TGAGCCGCCG CCACGAGGGA CGGTCCGGGT
301 GGAAGGCGTG CGGGGCGTCG TGATGGCGGC CGTCGGTGTT GGTGCCCTCG
351 ATGTCCACCA GCACCGGCGC TCCGCGGGGC AGCCGGACGC CGCCGATGGT
401 CACCTCCGTG GCAGCGAACC TCCACAACGT GTAGGGCACC GGCGGGTGGT
451 AGCGCAGCGC CTCCTCCACG AACCGGGAGA CGGCGTCCTC GTCGGCATCC
501 GCCGCGAGGC GGCCCGCCAG GACCTCCGCG AGCAGGAAGC CCAGGAAGGA
551 GCCGGTGGTG TCGTGGCCGG CGAAGATGAG CCCGGTGATC ATGTAGACGA
601 GCTGGTCGTC GGAGACCGAG CCGAACTCGG CCTGCGCGCG CTCGTACAGC
651 ACGCGGGTCA TGGTCGGGGT GTCGTTCCGC CGGGCTGAGT GCACGGCTTC
701 GAGGAGCAGG CTCTCCAGGG CCGAGGTGTC CGGCACGCCC CCGGCAGGGT
751 CCGTGCCGTC ACCCCCGCCG CTCTGCGGGC CGCCGAGGCC GAGTGCCTTG
801 AGAACGCTGA CGGCCTCGCG GGCCATCGCC GGATCGGTGA CCGGCACACC
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851 GAGCAGCTCG CAGATGACCA ACAGCGGGAA GTGGTACGCG AAGCCGCCGA
901 TCAGCTCGGC CGGTTTGCCC GACCGGCCGG AGGCGTCGGC GAGTTCGGTG
951 AGCAGCCGGC CGGCGATCGC GGCGATGCGA TCCGTCCGCT CGGCCAGCCG
1001 GCGCGGGTTG AACGCAGGTG CGTGGATGCG GCGCAGGCGC CGGTGGGCCT
1051 CGCCGTCCAC GGCGATGAGC GTGAACGGAC GCAGCTCCGG AACGGGGATG
1101 TCGAGACCGT CGTCCACCCC CCGCCAGGCG GCGGGGGCGA GGTCGGGGTC
1151 CTTCACGAAC CGGGGATCGG CCAGCACCTC GCGGGCGAGG GCGTCATCGG
1201 TGATGACCCA GGCGGGTCCG CCCGCGGGGG CGTTCACCTC GACGACCGGG
1251 CCCGCCTCCC GGAAGGCGTC GTGCACCTCG GGCTTGCGCT GCATGGTCAT
1301 CATGGGACAC GCGAACGGGT CGACGGCCAC CCGGGGCGCC TCGCCGCTCA
9351 CGAGGCACCG CCCGCCGCCG CGGGGTACCC CTCCCGCAGT TCGACCACCG
1401 AGAAGCCGGC CCCGTGCGGG TCGAGCAGGT CCGCCCGCCG CCCCCTGGGC
1451 GTGTCGGCGG GCTCGTTCTC GACGGAGCCG CCGAGTTCAA CGGCGCGCCG
1501 GACCGTCGCG TCGCAGTCGT GCACGGCGAA CAGCACGGCC CAGTGCGGCC
1551 GTACCGCGCC GGTGACGCCC AGCTCCTGGG TGCCGGCGAC CGGTGTGTCA
1601 CCGATGTGCC AGACCGGGTC GGTGACGCCC TTCAGTCCGG TGTCGGCCGG
1651 AGCCAGGCCG AGGGTCGCCG GGTAGAAGTC CCGGGCGGCC CCGATGCCGT
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1701 CGGTCACCAG CTCGACCCAG CCGACCGAGC CGGGCACGCC CGTCACCTCC
1751 GCGCCCTCCA TGACTCCCTT GCGCCAGACC GCGAACGCGG CCCCGGCGGG
1801 GTCGGCGAAG ACCGCCATCC GGCCGAGGCC GAGGACGTCC ATCGGAGTCA
1851 TGATGACCTC GCCGCCCGCC GTCTCGACCC GCTTGGTCAG TGCGTCGGCG
1901 TCGTCGGTGG CGAAGTACAC GGTCCAGATG GCCGGCATGC CGTGCTGGTC
1951 GTTCCCGGGC CCGTACGGCC GGTGGTAGGG GGTGTCGATC TGGTGGCGGG
2001 CGACCGCGGC GACCAGCTTC CCGTCGGAGC TGAACGTCGT GTATCCCCCG
2051 GCGCCCGGGT CGCTGACCAC GGTGGCGGTC CAGCCGAACA GGCCGGTGTA
2101 GAAGTCGGCC GAGGCGGCGA CATCGGGCGA ACCGAGGTCG AACCATGCGG
2151 GGGCGCCGGG CGCGAACCTG GTCACGAATC GTTCCTTTCG ATGGATCGGC
2201 ACACGAGCGT CTGCGCTCGC GGATGAGACG GACATCTCGC GGATGAGACG
2251 GACATGCGGG CGGGGCGGGC CGCCGCCGTC AGTGCGCGGT GTCGCCGACG
2301 GCGGCCGCGC CGGCCTCCCA GAGCTTCGCC GCGAGGCCGG CGTCGGCGGT
2351 CGGGCCGCTC ACCGGGGACA GCCGCCGGTC GCTGTAGTAG CCGCCCGTGG
2401 TCAACTCCTC GGCCGGCGCG GACGCCAGCC ACACGAGGGT GTCGGCGCCC
2451 TTCGCCGCGG AGCGCAGGAA GGGGTTGAAC CGGAAGTAGG ACGAGGCGAC
2501 CGTGCCCCGT CCGATGCGGG TGCGGACCTC ACCGGGGTGA TAGCTGACCG
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2551 CCAGCACGTC CGGCCAGCGC CTGGCGGCCT CCGCCGCGGT CATGATGTTG
2601 GCCTGTTTGG ACGTGCCGTA CGCCTGGCCG GCGCTGTAGC GGTGACGGTC
2651 GCCGTTGAGG TCGTCCGGGT CGATCCGGCC CTGGGTGTAC GCGTCGGACG
2701 AGGTGAGGAT CAGCCGCCCG CCCGCGAGCC GCTCCCGCAG CAGCCGTGCC
2751 AGCAGGAAGC CTGCGAGGTG ATTGACCTGG ATGGTGGCCT CGAACCCGTC
2801 CTGGGTCGTG GTGCGCGACC AGAACATGCC GCCGGCGTTG CTGGCCATGA
2851 CATCGATGCG CGGGTACCGG
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SEQUENCE LISTING
<110> PHARMACIA & UPJOHN S.P.A.
<120> .PROCESS FOR PREPARING DOXORUBICIN
<130> 1615-9003
<140> PCT/UNKNOWN
<141> 1999-04-22
<150> 09/065,606
<151> 1998-09-24
<160> 1
<170> PatentIn Ver. 2.0
<210> 1
<211> 2870
<212> DNA
<213> Streptomyces peucetius
<220>
<221> misc feature
<222> Complement((1)..(2870))
<223> Complementary strand to the coding strand
<400> 1
ggatccgcac cgggtacacg gcacgggacc gcccaccgcg cggtgcgcgg tgggcggtcc 60
cgtgccggtc gcggccggcg gatcagcgca gccagacggg cagttcggtg agccgcgccg 120
tctgggcccc cttccggcac caccgcaact cgtcgtacgg cacggccagt cgggcctcgg 180
ggaacctgct gcgcagtacg ccgatcatcg tgcgcgactc cagctgggcg agctgctccc 240
cgatgcagta gtgcggcccg tcgccgaagg tgagccgccg ccacgaggga cggtccgggt 300
ggaaggcgtg cggggcgtcg tgatggcggc cgtcggtgtt ggtgccctcg atgtccacca 360
gcaccggcgc tccgcggggc agccggacgc cgccgatggt cacctccgtg gcagcgaacc 420
tccacaacgt gtagggcacc ggcgggtggt agcgcagcgc ctcctccacg aaccgggaga 480
cggcgtcctc gtcggcatcc gccgcgaggc ggcccgccag gacctccgcg agcaggaagc 540
ccaggaagga gccggtggtg tcgtggccgg cgaagatgag cccggtgatc atgtagacga 600
gctggtcgtc ggagaccgag ccgaactcgg cctgcgcgcg ctcgtacagc acgcgggtca 660
tggtcggggt gtcgttccgc cgggctgagt gcacggcttc gaggagcagg ctctccaggg 720
ccgaggtgtc cggcacgccc ccggcagggt ccgtgccgtc acccccgccg ctctgcgggc 780
cgccgaggcc gagtgccttg agaacgctga cggcctcgcg ggccatcgcc ggatcggtga 840
ccggcacacc gagcagctcg cagatgacca acagcgggaa gtggtacgcg aagccgccga 900
tcagctcggc cggtttgccc gaccggccgg aggcgtcggc gagttcggtg agcagccggc 960
cggcgatcgc ggcgatgcga tccgtccgct cggccagccg gcgcgggttg aacgcaggtg 1020
cgtggatgcg gcgcaggcgc cggtgggcct cgccgtccac ggcgatgagc gtgaacggac 1080
gcagctccgg aacggggatg tcgagaccgt cgtccacccc ccgccaggcg gcgggggcga 1140
ggtcggggtc cttcacgaac cggggatcgg ccagcacctc gcgggcgagg gcgtcatcgg 1200
tgatgaccca ggcgggtccg cccgcggggg cgttcacctc gacgaccggg cccgcctccc 1260
ggaaggcgtc gtgcacctcg ggcttgcgct gcatggtcat catgggacac gcgaacgggt 1320
cgacggccac ccggggcgcc tcgccgctca cgaggcaccg cccgccgccg cggggtaccc 1380
ctcccgcagt tcgaccaccg agaagccggc cccgtgcggg tcgagcaggt ccgcccgccg 1490
ccccctgggc gtgtcggcgg gctcgttctc gacggagccg ccgagttcaa cggcgcgccg 1500
gaccgtcgcg tcgcagtcgt gcacggcgaa cagcacggcc cagtgcggcc gtaccgcgcc 1560
ggtgacgccc agctcctggg tgccggcgac cggtgtgtca ccgatgtgcc agaccgggtc 1620
ggtgacgccc ttcagtccgg tgtcggccgg agccaggccg agggtcgccg ggtagaagtc 1680
ccgggcggcc ccgatgccgt cggtcaccag ctcgacccag ccgaccgagc cgggcacgcc 1740
cgtcacctcc gcgccctcca tgactccctt gcgccagacc gcgaacgcgg ccccggcggg 1800
gtcggcgaag accgccatcc ggccgaggcc gaggacgtcc atcggagtca tgatgacctc 1860
gccgcccgcc gtctcgaccc gcttggtcag tgcgtcggcg tcgtcggtgg cgaagtacac 1920
ggtccagatg gccggcatgc cgtgctggtc gttcccgggc ccgtacggcc ggtggtaggg 1980
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ggtgtcgatc tggtggcggg cgaccgcggc gaccagcttc ccgtcggagc tgaacgtcgt 2040
gtatcccccg gcgcccgggt cgctgaccac ggtggcggtc cagccgaaca ggccggtgta 2100
gaagtcggcc gaggcggcga catcgggcga accgaggtcg aaccatgcgg gggcgccggg 2160
cgcgaacctg gtcacgaatc gttcctttcg atggatcggc acacgagcgt ctgcgctcgc 2220
ggatgagacg gacatctcgc ggatgagacg gacatgcggg cggggcgggc cgccgccgtc 2280
agtgcgcggt gtcgccgacg gcggccgcgc cggcctccca gagcttcgcc gcgaggcc 2340
gg
cgtcggcggt cgggccgctc accggggaca gccgccggtc gctgtagtag ccgcccgtgg 2900
tcaactcctc ggccggcgcg gacgccagcc acacgagggt gtcggcgccc ttcgccgcgg 2460
agcgcaggaa ggggttgaac cggaagtagg acgaggcgac cgtgccccgt ccgatgcggg 2520
tgcggacctc accggggtga tagctgaccg ccagcacgtc cggccagcgc ctggc
ccgccgcggt catgatgttg gcctgtttgg acgtgccgta cgcctggcc
g gcgctgtagc 2690
ggtgacggtc cc tt a tc tcc t c atcc 9gcct 2580
g g g gg g ggg g ggcc ctgggtgtac gcgtcggacg 2700
aggtgaggat cagccgcccg cccgcgagcc gctcccgcag cagccgtgcc agcaggaagc 2760
ctgcgaggtg attgacctgg atggtggcct cgaacccgtc ctgggtcgtg gtgcgcgacc 2820
agaacatgcc gccggcgttg ctggccatga catcgatgcg cgggtaccgg 2870
