Download The dnrM gene in Streptomyces peucetius contains a

MivObiOlOgy (1996), 142,269-275
Printed in Great Britain
The dnrM gene in Streptomyces peucetius
contains a naturally occurring frameshift
mutation that is suppressed by another locus
outside of the daunorubicin-productiongene
cluster
Mark A. Gallo,l Joanne Wardlt and C. R. Hutchinson'#*
Author for correspondence: C. R. Hutchinson. Tel:
e-mail : crhutchia facstaff.wisc.edu
School of Pharmacy' and
Department of
Bacteriologyz, University of
Wisconsin, Madison, WI
53706, USA
+ 1 608 262 7582. Fax: + 1 608 262 3134.
A 2.7 kb BamHl fragment of the daunorubicin biosynthetic cluster in
Streptomyces peucetius ATCC 29050 was shown to contain two ORFs, dnrL and
dnfM#whose deduced products exhibit a high sequence similarity to a number
of glucose-l-phosphate thymidylyl transferases and TDP-D-glucose
dehydratases, respectively. Although these genes were believed to be
necessary for the synthesis of the deoxyaminosugar, daunosamine, a
constituent of daunorubicin, the dnrM gene contains a frameshift in the DNA
sequence that causes the premature termination of translation. A gene
encoding another TDP-glucose 4#6=dehydratase,
previously isolated from 5.
peucetius, was identified by PCR amplification of genomic DNA. The presence
of this gene explains why a dnrM::aphll mutation did not block daunorubicin
production.
Keywords: daunosamine, deoxyamino sugar, inactive gene, TDP-D-glucose 4,6deh ydratase, glucose-1-phosphate thymidylyl transferase
INTRODUCTION
The Streptomyes are filamentous, spore-forming soil
bacteria that produce an array of products termed
secondary metabolites. Many are useful in agriculture and
medicine, for example as antiparasitic, antimicrobial, or
anticancer agents. Relatively little is known in general
about the biochemistry, physiology and genetics of
production of secondary metabolites. The anthracyclines
are a prevalent class of such compounds isolated from
Streptomyes sp. and other actinomycetes. Two anthracyclines, daunorubicin (DNR) and doxorubicin (DXR),
have received considerable attention due to their potent
antitumour activity (Arcamone, 1981). Our laboratory has
been interested in determining the biochemical and
genetic processes involved in the synthesis of DNR and
DXR in Streptomyes pezlcetins ATCC 29050.
t Presetnt address: Sandoz Clinical DevelopmentCentre, The Quadrangle,
Frimley Business Park, Frimley, Camberley, Surrey, GU16 SSQ, UK.
Abbreviations: DNR, daunorubicin; DXR, doxorubicin; GST, glutathione
S-transferase; RHO, c-rhodomycinone; TDP, thymidylyl diphosphate.
The GenBanWEMBL accession number for the sequence reported in this
paper is L47163.
0002-0162 0 1996 SGM
Previous reports have identified a 45 kb region that
presumably contains all the genes required for the
synthesis of and resistance to DNR and DXR (Otten e t al.,
1990; Stutzman-Engwall 8c Hutchinson, 1989; Madduri
& Hutchinson, 1995a). DNR and DXR consist of two
parts, a polyketide-derived aglycone and the trideoxyamino sugar daunosamine. All of the genes required
for synthesis of the aglycone carbon skeleton have been
characterized (Guilfoile & Hutchinson, 1991; Grimm e t
al., 1994; Madduri & Hutchinson, 1995b), and most of
them share a high degree of homology with other type I1
polyketide synthases and cyclases at the level of DNA and
deduced protein sequence (Hopwood & Sherman, 1990;
Katz & Donadio, 1993; Hutchinson & Fujii, 1995).
By contrast, little is known about the genes involved in
biosynthesis of daunosamine (Krugel etal., 1993;Thorson
etal., 1993; Otten etal., 1995b). In this paper we report the
sequence of a 2682 bp DNA segment containing two
putative ORFs, dnrL and dnrM (Fig. l), located within the
known boundaries of the 45 kb dnr gene cluster. We show
that dnrL likely encodes a glucose-1-phosphate thymidylyl
transferase and dnrM encodes a thymidylyldiphospho(TDP)-D-glucose 4,6-dehydratase7on the basis of
sequence comparisons to other known transferases and
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269
M. A. GALLO, J. W A R D a n d C. R. H U T C H I N S O N
I
B
B
I
1
dnrR2
deoxysugar
B B
I I
regulatory
Bg
B
// I
1
oxidation
dehydratases involved in deoxysugar metabolism. However, the dnrM sequence was unexpectedly found to
contain a frameshift that results in the formation of a
truncated protein. Thus, inactivation of dnrM in S.
pezlcetim 29050 did not prevent DNR o r DXR production,
suggesting that another dehydratase-encoding gene
existed outside the 45 k b dnr region. Southern analysis of
chromosomal DNA using dnrM as a probe showed, in
addition to dnrM, several weakly hybridizing bands ; one
of these appeared t o be the gene for another dehydratase
that, from the results of initial PCR amplification and
D N A sequence analysis, encodes a dehydratase previously
purified from S. pet/cetit/s (Thompson e t a/., 1992).
METHODS
Bacterial strains and plasmids. S. peucetius ATCC 29050 was
obtained from the American Type Culture Collection. Escber i c h coli strains DH5aMCR (Life Technologies) and JM105
(Yanisch-Perron e t al., 1985) were used for routine subcloning
and preparation of ssDNA. The glutathione S-transferase (GST)
vector pGEX-4 was obtained from Pharmacia. The high-copy
number E. coli-Streptomyces shuttle vector pWHM3 was from
Vara e t al. (1989). M13 phage-derived mp18 and mp19 vectors
(Yanisch-Perron e t al., 1985) were used for sequencing by
previously published methods (Summers e t a/., 1992). The
pUC4-KIXX plasmid containing the neomycinlkanamycin
resistance gene (aphll) from Tn5 was obtained from Pharmacia.
The plasmid pDH5 (Hillemann et al., 1991) was used to generate
ssDNA for transformation of Streptomyces sp. and gene disruption ; pWHM3 was used to clone the wild-type dnrLM
genes.
Media and growth conditions. S. peucetius was grown at 30 OC
on R2YE (Hopwood etal., 1985) for propagation of cells, DNA
isolation, and protoplast preparation. ISP4 medium (Difco) was
used to produce spores. Transformants of S. peucetius were
selected on 10 pg thiostrepton ml-', 25 pg apramycin ml-' or
50 pg neomycin ml-'. E. coii transformants were selected with
100 pg ampicillin ml-', 100 pg apramycin ml-' or 50 pg neomycin ml-'. S. peucetius strains were grown in 5 ml seed medium
(Guilfoile & Hutchinson, 1991) for 2 d, then transferred to GPS
production medium (Dekleva e t al., 1985) and grown in 50 ml
broth in a 250 ml baffle-bottom flask for an additional 3 d at
30 "C with shaking at 300 r.p.m. Cultures were acidified with
oxalic acid, heated at 60 OC for 45 min, adjusted to pH 8.5, and
extracted with chloroform (Otten e t al., 1990). The extracted
anthracycline metabolites were analysed by TLC or HPLC
(Otten e t al., 1990).
DNA isolation and manipulation. DNA isolation, restriction
270
2kb
dnrR1
I
B
1
resktanc
..................................... ......... .............................. ................................
Fig. I . Partial restriction map of a selected
region of the dnr cluster of S. peucetius
29050. dnrN, 0 (Otten et a/., 1995a); dnrF, A,
B (Guilfoile & Hutchinson, 1991); dnrl, I
(Madduri & Hutchinson, 199513; StutzmanEngwall et a/., 1992). Restriction sites: B,
BamHI; Bg, Bglll.
endonuclease digestions, and ligations were performed according to standard techniques (Sambrook e t al., 1989).
Hybridization analysis was carried out using the Genius nonradioactive kit (Boehringer Mannheim) according to the manufacturer's instructions. DNA was electrophoresed on 0.7 YO
agarose gels, and alkali transfer to Hybond-N (Amersham) was
performed for Southern analysis.
DNA sequencing. DNA fragments were subcloned into
M13mp18 and M13mp19, and single-stranded templates were
sequenced by the dideoxy chain-termination method using (a35S)dCTP and Sequenase version 2.0 (USB) according to the
manufacturer's instructions. 7-Deaza-dGTP was used instead of
dGTP to avoid compressions.
Disruption of dnrM. A 725 bp BglII fragment internal to dnrM
was removed from the 2.7 kb BamHI fragment containing dnrL
and dnrM, and the DNA was blunt-ended by treatment with the
Klenow fragment and ligated to a 1.1 kb SalI fragment
containing apbll. The resulting 3.1 kb fragment was subcloned
into pDH5 (Hillemann e t al., 1991) to give pWHM209, and
ssDNA was produced and used to transform S. peucetitls 29050
protoplasts. Transformants were selected on R2YE plates
containing thiostrepton or neomycin to verify integration into
the 5'.peucetius genome by homologous recombination. Several
of the transformants were subjected to two rounds of sporulation, and single colonies were picked and scored for the
expected neomycin-resistant thiostrepton-sensitive phenotype
of a double crossover recombination event. Four mutants were
analysed by Southern analysis to verify gene disruption and
replacement by digesting total chromosomal DNA with BamHI
and probing with either the 725 bp BglII or the 2.7 kb BamHI
fragment. All neomycin-resistant, thiostrepton-sensitive mutants lacked the 725 bp BglII fragment and contained a BamHI
fragment of 3.1 kb, properties one would expect of gene
replacement mutants (data not shown). One dnrM: :aphll clone
was chosen and named WMH1590.
PCR amplification. PCR was used to amplify an additional
putative dehydratase from S. peucetius 29050 chromosomal
DNA. The PCR mixture contained 1 x PCR buffer [SO mM
KC1, 10mM Tris/HCl, pH 9.0 (at 25 "C), 0.1 YOTriton X-100,
and 2-5 mM MgCl,] supplied by Promega. Each primer was
present at 0.3 pM, and each dNTP was present at 0.1 mM. The
codon bias of Streptomyces genes (Wright & Bibb, 1992) was
taken in to account in designing the following two primers:
GTCAACGTCACCGTCACCGGCGCCGCCGGCCAGATC-
GG(G/C) TACGC(G/C)CT, which corresponds to the Nterminal peptide sequence reported previously (Thompson e t
al., 1992); and TCA(G/C)AG(G/C)GGCTCCCACCAG(G/C)(A/T)(G/C)CG, which corresponds to the complementary
sequence of a conserved peptide in the C-termini of TDP-Dglucose dehydratases. Taq polymerase (1 U, Promega) and
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Daunorubicin synthesis genes
approximately 100 ng target DNA was added in a final reaction
volume of 100 pl. Amplification was performed in a thermal
cycler (model 480, Perkin-Elmer Cetus) by denaturing the
samples at 100 "C for 5 min and then subjecting them to 25
cycles of denaturing (97 "C, 30 s), annealing (55 "C, 45 s) and
elongation (70 "C, 90 s). Amplification products were bluntended with Klenow and 0.1 mM dNTPs, separated by agarose
gel electrophoresis, and purified with Qiaex resin (Qiagen).
The resulting DNA fragment was ligated into M13mp18 and 19 for sequencing.
Expression of dnrM in E. coli. The GST gene fusion system
(Pharmacia)was used to express dnrM in E. coli. Plasmid pGEX4T-1 was cut with XhoI, and linkers were added to introduce an
SstI site. This modified plasmid was cut with EcoRI, bluntended by treatment with the Klenow fragment, then the DNA
was cut with JstI and ligated to a 1 kb BspHIlblunt-JstI
fragment containing dnrM to give pWHM204. Expression of
GST:DnrM in E. coli(pWHM204) strains was induced with
1 mM IPTG. The formation of a novel, unique protein was
demonstrated by SDS-PAGE analysis.
RESULTS
Sequence analysis of dnrL and dnrM
Sequence analysis by CODONPREFERENCE (Devereux e t al. ,
1984) of a 2.7 kb BamHI fragment from within the dnr
gene cluster (Fig. 1) revealed two ORFs, dnrL and dnrM.
The region encompassing nt 1-655 appeared to contain
no long ORFs with any recognizable similarity to known
proteins by TFASTA analysis (Devereux e t al., 1984). The
most likely translational start site for dnrL is the ATG
beginning at nt 655 and ending with TGA at nt
1708-1710, producing a polypetide of 351 aa.
The translational start site for dnrM is believed to be the
ATG beginning at nt 1707. This start site overlaps the
TGA stop of dnrL, implying a possible translational
coupling of these two proteins. A T G A stop codon
beginning at nt 1890 allowed for production of a 61 aa
polypeptide (6572 Da). CODONPREFERENCE analysis
revealed a switch in reading frames approximately 97 nt
beyond the ATG, at nt 1707. If the D N A sequence data
are interpreted in a manner that allows introduction of a
frameshift, a TGA stop codon beginning at nt 2680 is
found in this new frame, implying production of a
polypeptide of 324 aa (35 177 Da) for the combined ORFs
from nt 1707 to 2679. Codon usage analysis of this frame
showed very few rare Streptomyes codons (Wright &
Bibb, 1992).
Characteristics of the deduced gene products of dnrL
and dnrM
The deduced product of dnrL has a high sequence
similarity to several glucose-1 -phosphate thymidylyl transferases (Table l),enzymes that are involved in transferring
a TDP moiety to glucose. This irreversible reaction is an
important first step in the activation of sugars for eventual
transfer to other molecules (Liu & Thorson, 1994).
Consequently, dnrL most likely provides this enzyme for
daunosamine biosynthesis.
The deduced product of the truncated dnrM gene shares a
significant sequence similarity to the N-terminal portion
of several TDP-glucose 4,6-dehydratasesmTranslation of
the second reading frame identified by CODONPREFERENCE
analysis reveals a remarkable sequence similarity to the
remaining amino acid residues of this family of dehydratases (Table 1). Therefore, we believe that these two
frames should be linked (Fig. 2). A simple sequencing
error was not responsible for the shift in translational
sequence revealed in Fig. 2 because the region was
sequenced several times in both directions from several
Table I . Comparisons between the amino acid sequences of DnrL and DnrM with other
deoxysugar biosynthesis enzymes having similar functions
(Devereux et al., 1984) analysis was used for comparisons. DnrM in this table refers to the
combined reading frames as noted in Fig. 2 and the text.
BESTFIT
Protein
Number
of amino
acids
Similarity
Identity
Reference
(%)
DnrL
StrD
TylAl
RfbA
355
304
76.3
59.3
293
55.6
59.7
36.9
31-5
Pissowotzki et al. (1991)
Merson-Davies & Cundliffe (1994)
Stevenson e t al. (1994);
Yao & Valvano (1994)
61.6
46.7
58.1
Pissowotzki et al. (1991)
Stevenson e t af. (1994)
Linton e t al. (1995)
DnrM
StrE
RfbB
Gdh
328
361
329
76.2
63.0
71.4
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M. A. GALLO, J. W A R D a n d C. R. H U T C H I N S O N
1
121
241
361
481
601
721
841
961
1081
1201
1321
1441
1561
1681 G A C C A ~ T C T
D D S R V E I S S .
C
C
7
C
A
l
Q
A
C
C
A
l
G
M
G
A
X
C
T
W
l
C
k
~
~
C
T
A
& H - > f M T Y K I L V T G G A G P I G S H Y V R A L L G P R G D G S V G
T
1801 GGGTCACKiCPCGACAGCCKiACCTATOCAOaClUTCCOO
AC
C
V T A G Q P D L C R Q S G Q P R S R P R R R A V R L R P R *
O
C
T
G
A
C
A
~
G
~
G
A
~
* L L D S L T Y A G N P A N L D P V R G D E R F A , F V H G D I C D A P L V D R
1921
m
--
C
A
mCCCAA-lUGGTACGCAM-
m
L V A E H D Q I V H F A A E S H V D R S V R S G , P R . S S C A P N V M G T Q T L L
2041 G
A
C
O
C
O
O
C
C
C
r
O
C
O
C
C
A
D A A L R ~ A P R I P V H I S ~ D E V Y O S V O V O A S T E T D P L H P S S A Y
~
m
~
~
m
~
A
P
~
~
2161 G C G Q G C A ~ ~ A C C A C C b C G C A C ~ C W X C G G A ~ C A A C T A C G G C M X C A
A G T K A S S D L L A L S Y H R T H G L D V R I T R C S N N Y G T H Q H P E K V
T
G
h
W
W
A
c
G
G
T
G
h
~
~
~
G
G
n
~
2281 A
I P R P V T T L L S G G R V P L Y G D G G N L R N W L H V E D H V D A U E L V R
~
C
A
A
C
A
A
~
~
~
2401 GAGGGA-TCTACAACAE G G S P G E I Y N I G G G T E L S N K E L T A L L L D A V G A G W D S V E L U
~~~
--AGCcG
2521
Ac
- - - CAC l
A D R K G H D R R Y A V D H G K I T A L L G Y R P R R D F R D S L A E T V A W Y
2641
~~~~~
R D R R S W W A P L T G S .
..,..........,......,....................................................................,...,,......................................................................,....,.....................,........,........................,..................................................................................................
Fig. 2. Nucleotide sequence of the 2682 bp BamHl fragment containing dnrL and dnrM. Putative RBSs are underlined.
The translated amino acid sequences are shown below the DNA sequence. The presumed site of the frameshift in dnrM is
indicated by a ' 0 and asterisks indicate stop codons.
M13 plaques derived from independent primary clones.
Additionally, the region was cloned by PCR from S.
pezicetizls 29050 chromosomal DNA and again shown to
contain the same frameshift by DNA sequencing.
Expression of dnrM in E. co/i
A GST: DnrM fusion protein (Methods) appeared as a
32.6 kDa band (Fig. 3), which is 6.6 kDa larger than the
band corresponding to GST alone. This indicates that a
polypeptide of 61 aa is present at the C-terminal end of
GST, in agreement with the predicted size if the truncated
272
DnrM protein is fused to GST. A fusion protein of
60 kDa [equal to GST (26 kDa) plus a full-length DnrM
(34 kDa)] was not detected (Fig. 3). This result is
consistent with the sequence analysis and corroborates the
conclusion that dnrM contains a naturally occurring
frameshift mutation.
Disruption of dnrM
The chromosomal copy of dnrM in S. pezrcetitls 29050 was
inactivated by insertion of apb11 to produce the
WMH1590 strain (Methods). Wild-type S.pezlcetizls 29050
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~
~
Daunorubicin synthesis genes
kDa
94
67 -
43
1
2
3
Isolation of another TDP-D-glucose 4,6-dehydratase
gene
-
-
GST: DnrM
fusion
-
30
GST-
21.5
formation of TDP-D-glucose is not a rate-limiting step in
the conversion of RHO to DNR.
4
-
Fig. 3. SDS-PAGE of E. coli cell extracts containing the
GST: DnrM fusion vector, pWHM204. Lanes: 1, molecular
mass markers; 2, E. coli DH5aMCR(pGEX4), induced; 3,
DH5aMCR(pWHM204), uninduced; 4, DH5aMCR(pWHM204),
induced.
Table 2. Effects on secondary metabolite production of
the introduction of dnrM into 5. peucetius strains 29050
and WMH1590
............................................................................ ..............................
Values are means of three samples for RHO ( & 417 ) and DNR
(& 1.02) and are in pg ml-'.
1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
Strainlplasmid
RHO DNR Ratio
29050/pWHM3
29050/pWHM205
WMH1590/pWHM3
WMH1590/pWHM205
27-7
34.5
35-5
21.2
2.9
3.6
2.4
1.7
9.6
9.6
14.8
12-5
and WMH1590 strains were transformed with the
pWHM3 vector DNA (Vara e t al., 1989) or pWHM205, a
plasmid containing the wild-type copy of the 2.7 kb
BamHI fragment with the dnrLM genes that had been
cloned in pWHM3. The levels of erhodomycinone
(RHO) or DNR produced by these strains (Table 2) were
the same within experimental error.
These results indicate the following : first, an uninterrupted copy of dnrM is not required for the synthesis of
DNR; second, a gene encoding a functional TDP-Dglucose 4,6-dehydratase must be present in S. pezlcetitls
29050 since DNR was produced in both wild-type and
WMHl590 strains. Finally, since additional copies of
dnrL do not enhance the conversion of RHO to DNR, the
Thompson et al. (1992) have partially purified a TDP-Dglucose 4,6-dehydratase from S.petleetins. The N-terminal
sequence of their protein does not agree with that
predicted for DnrM, although it does show some
similarity to this family of dehydratases (Liu & Thorson,
1994). We synthesized degenerate oligodeoxynucleotides
based on highly conserved regions at the N-terminus of
the reported amino acid sequence (Thompson e t al., 1992)
and the reverse complement of the C-termini of such
dehydratases (Liu & Thorson, 1994), then amplified by
PCR the targeted region in the DNA from S. pezlcetias
29050. The DNA sequence of a small portion of the 1.1 kb
fragment amplified was determined. Its translated sequence is : VNVTVTGAAGQIGYALVTVIGAAGLIG
YALQRT; residues in bold are highly conserved in
TDP-D-glucose 4,6-dehydratases (Liu & Thorson, 1994).
This sequence is not the same as that proposed for DnrM
(Fig. 2), but the first 14 aa are identical to those reported
by Thompson e t al. (1992). It thus appears that we have
located the gene for a functional TDP-D-glucose 4,6dehydratase of S. pezmtizls. The DNA sequence was not
identical to any part of the 45 kb region of the dnr gene
cluster sequenced, indicating that this other dehydratase is
not encoded by a gene inside the defined dnr cluster. We
were also unable to amplify this other dehydratase gene
from pWHM341 (Otten e t al., 1990), a cosmid containing
a 27 kb DNA segment to the left of the defined dnr cluster
(Madduri & Hutchinson, 1995a), as in Fig. 1.
DISCUSSION
The genes for the biosynthesis, regulation, and resistance
to secondary metabolites are normally clustered in Streptomzces species, perhaps because this arrangement confers a
selective advantage in maintenance and regulation. Coordinate expression of such genes has often been found
(Chater & Bibb, 1995). In this regard, we have shown that
a transcript containing dnrL and dnrM is present in S.
petlcetz'zjs 29050, and is regulated by the dnrI gene encoding
a transcription factor (Madduri & Hutchinson, 1995a).
On the basis of the above, we were surprised to find an
inactive dnrM gene in the dnr cluster. The lack of a
functional gene encoding a TDP-D-glucose 4,6dehydratase in this gene cluster implies the presence of
another functional gene elsewhere in the organism, as
suggested by the results of our exploratory studies and the
work of Thompson e t al. (1992). Moreover, the existence
of a dnrL homologue at another locus that provides a
functional glucose-1-phosphate thymidylyl transferase is
not excluded by our data. Since there are many other
glycosylated anthracyclines produced by Streptomtees species, it would be interesting to investigate whether other
DNR-producing species, or descendants of strain 29050,
such as S.peucetitls subsp. caesizls ATCC 27952 (Arcamone
e t al., 1969), also contain a mutant dnrM homologue. (It
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M. A. G A L L O , J. W A R D a n d C. R. H U T C H I N S O N
would be surprising to find that dnrM was mutated only in
the isolate of the 29050 strain in our collection, but we
have not tested this possibility.)
Although it is unusual to find nonfunctional genes in
Streptomyes operons, nonfunctional ketoreductase domains in the products of polyketide synthase genes have
been reported in the erythromycin-producing Saccharopobspora eytbraea (Donadio & Katz, 1992) and the
avermectin-producing Streptomyes avermitilis (MacNeil e t
al., 1994). DNR biosynthesis is unique in this regard since
a nonfunctional gene is present, but another gene product
from outside the biosynthetic gene cluster complements
the missing activity. In erythromycin biosynthesis, the
cluster of production genes, as currently defined, lacks
genes encoding glucose-1-phosphate thymidylyl transferase and TDP-D-glucose 4,6-dehydratase, but genes for
these enzymes have been found elsewhere in S. eytbraea
(Linton e t al., 1995; S. Zotchev & C. R. Hutchinson,
unpublished results).
The unglycosylated intermediate RHO is approximately
10-fold more prevalent than DNR when S. pegcetizls is
grown in GPS medium (Dekleva et al., 1985). Therefore,
it appears quite likely that one of the steps of daunosamine
biosynthesis or attachment is rate-limiting. It is also
possible that the TDP-D-glucose 4,6-dehydratase that
actually catalyses the second step of daunosamine biosynthesis in S.pezlcetitls is not well-adapted for this function
and hence is forming a metabolic bottleneck. Consequently, it would be interesting to determine the effect on
DNR production in S. petlcetitls 29050 of repairing the
frameshift in dnrM or placing the other TDP-D-glucose
4,6-dehydratase gene under control of dnrl (Madduri &
Hutchinson, 1995a), or introducing additional copies of a
known TDP-D-glucose 4,6-dehydratase.
ACKNOWLEDGEMENTS
We are grateful to K. Linton and B. Jarvis for sharing sequence
data prior to publication. The authors would also like to thank
B. Jarvis and S. L. Otten for helpful discussions. This work was
supported by grants from the National Institutes of Health (GM
25799 and 31925) and Pharmacia Spa, Milan, Italy.
REFERENCES
Arcamone, F. (1981). Doxorubicin. Med Chem Ser Monogr 17,25-31.
Arcamone, F., Cassinelli, G., Fantini, G., Grein, A,, Orezzi, P., Pol,
C. & Spalla, C. (1969). 14-hydroxydaunomycin, a new antitumor
antibiotic from Streptomycespeucetiusvar. caesius. Biotechnol Bioeng 11,
1101-1 110.
Chater, K. F. & Bibb, M. J. (1995). Regulation of bacterial antibiotic
production. In Products of Secondav Metabolism. Biotechnology, vol. 7.
Weinheim : VCH.
Dekleva, M. L., Titus, J. A. & Strohl, W. R. (1985). Nutrient effects
on anthracycline production by Streptomyces peucetius in a defined
medium. Can J Microbiol 31, 287-294.
Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive
set of sequence analysis programs for the VAX. Nucleic Acids Res
12, 387-395.
Donadio, 5. & Katz, L. (1992). Organization of the enzymatic
domains in the multifunctional polyketide synthase involved in
274
erythromycin formation in SaccbaropoEyspora erythraea. Gene 110,
5140.
Grimm, A., Madduri, K., Ali, A. & Hutchinson, C. R. (1994).
Characterization of the Streptomyces peucetius ATCC 29050 genes
encoding doxorubicin polyketide synthase. Gene 151, 1-10.
Guilfoile, P. G. & Hutchinson, C. R. (1991). A bacterial analog of
the mdr gene of mammalian tumor cells is present in Streptomyces
peucetius, the producer of daunorubicin and doxorubicin. Proc Natl
Acad Sci U S A 88, 8553-8557.
Hillemann, D., Puhler, A. & Wohlleben, W. (1991). Gene disruption
and gene replacement in Streptomyces via single stranded DNA
transformation of integration vectors. Nucleic Acids Res 19,
727-73 1.
Hopwood, D. A. & Sherman, D. H. (1990). Molecular genetics of
polyketides and its comparison to fatty acid biosynthesis. Annu Rev
Genet 24, 37-66.
Hopwood, D. A., Bibb, M. J., Chater, K. F., Kieser, T., Bruton,
C. J., Kieser, H. M., Lydiate, D. J., Smith, C. P., Ward, J. M. &
Schrempf, H. (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich : The John Innes Foundation.
Hutchinson, C. R. & Fujii, 1. (1995). Polyketide synthase gene
manipulation : a structure-function approach in engineering novel
antibiotics. Annu Rev Microbiol49, 201-38.
Katz, L. & Donadio, 5. (1993). Polyketide synthesis: prospects for
hybrid antibiotics. Annu Rev Microbiol47, 875-912.
Krugel, H., Schumann, G., Hanel, F. & Fielder, G. (1993). Nucleotide
sequence analysis of five putative Streptomyces griseus genes, one of
which complements an early function in daunorubicin biosynthesis
that is linked to a putative gene cluster involved in TDPdaunosamine formation. Mol b Gen Genet 241, 326-333.
Linton, K. J., Jarvis, B. W. & Hutchinson, C. R. (1995). Cloning of
the genes encoding thymidine diphosphoglucose 4,6-dehydratase
and thymidine diphospho-4-keto-6-deoxyglucose3,5-epimerase
from the erythromycin-producing SaccharopoEyspora evtbraea. Gene
153, 33-40.
Liu, H. W. &Thorson, J. 5. (1994). Pathways and mechanisms in the
biogenesis of novel deoxy sugars by bacteria. Annu Rev Micrubiol48,
223-256.
MacNeil, D. J., Occi, J. L., Gewain, K. M. & MacNeil, T. (1994).
Correlation of the avermectin polyketide synthase genes to the
avermectin structure : implications for designing novel avermectins.
Ann NY Acad Sci 721, 123-132.
Madduri, K. & Hutchinson, C. R. (1995a). Functional characterization and transcriptional analysis of the dnrR 1 locus, which
controls daunorubicin biosynthesis in Streptomyces peucetius. J
Bacterioll77, 1208-1215.
Madduri, K. & Hutchinson, C. R. (1995b). Functional characterization and transcriptional analysis of a gene cluster governing
early and late steps in daunorubicin biosynthesis in Streptomyces
peucetius. J Bacterioll77, 3879-3884.
Merson-Davies, L. A. & Cundliffe, E. (1994). Analysis of five tylosin
biosynthetic genes from the t$lBA region of the Streptomycesfradiae
genome. Mol Microbioll3, 349-355.
Otten, 5. L., Stutzman-Engwall, K. J. & Hutchinson, C. R. (1990).
Cloning and expression of daunorubicin biosynthesis genes from
Streptomyces peucetius and S. peucetius subsp. caesius. J Bacteriof 172,
3427-3434.
Otten, 5. L., Ferguson, J. & Hutchinson, C. R. (1995a). Regulation
of daunorubicin production in Streptomyces peucetius by the dnrR2
locus. J Bacterioll77, 12161224.
Otten, 5. L., Liu, X., Ferguson, J. & Hutchinson, C. R. (1995b).
Cloning and characterization of the Streptomyces peucetius dnrQS
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 16:16:23
Daunorubicin synthesis genes
Thompson, M. W., Strohl, W. R. & Floss, H. G. (1992). Purification
genes encoding a daunosamine biosynthesis enzyme and a glycosyl
transferase involved in daunorubicin biosynthesis. J Bacterioll77,
6688-6692.
Pissowotzki, K., Mansouri, K. & Piepersberg, W. (1991). Genetics
of streptomycin production in Streptomyces griseus : molecular
structure and putative function of genes strELMBZN. M o l 6 Gen
Genet 231, 113-123.
Sambrook,J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
a Laborato? Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Spring Harbor Laboratory.
Thorson, J. S., Lo, S. F., Liu, H.-W. & Hutchinson, C. R. (1993).
Stevenson, G., Neal, B., Liu, D., Hobbs, M., Packer, N. H., Batley,
M., Redmond, J. W., Lindquist, L. & Reeves, P. (1994). Structure of
the 0 antigen of Escbericbia coli K-12 and the sequence of its r- gene
oxysugar portion of the erythromycin biosynthesis pathway in
Saccbaropo&ora elytbraea (Streptomyces etytbreus). J Bacteriol 171,
5872-5881.
cluster. J Bacterioll76, 4144-4156.
Stutzman-Engwall, K. J. & Hutchinson, C. R. (1989). Multigene
families for anthracycline antibiotic production in Streptomyces
peucetius. Proc Natl Acad Sci U S A 86, 3135-3139.
Stutzman-Engwall, K. J., Otten, 5. L. & Hutchinson, C. R. (1992).
Regulation of secondary metabolism in Streptomyces: its use for the
overproduction of daunorubicin in Streptomycespeucetius.J Bacteriol
174, 144-154.
Summers, R. G., Wendt-Pienkowski, E., Motamedi, H. &
Hutchinson, C. R. (1992). Nucleotide sequence of the tcmll-tcmlV
region of the tetracenomycin C biosynthetic gene cluster of
Streptomyces glawescens and evidence that the tcmN gene encodes a
multifunctional cyclase-dehydratase-O-methyl transferase. J
Bacterioll74, 1810-1820.
and characterization of TDP-D-glucose 4,6-dehydratase from
anthracycline-producing streptomycetes. J Gen Microbiol 138,
779-786.
Biosynthesis of 3,6-dideoxy-hexoses : new mechanistic reflections
upon 2,6-dideoxy, 4,6-dideoxy and amino sugar construction. J A m
Cbem Soc 115, 6993-6994.
Vara, J., Lewandowska-Skarbek, M., Wang, Y.-G., Donadio, 5.81
Hutchinson, C. R. (1989). Cloning of genes governing the de-
Wright, F. & Bibb, M. J. (1992). Codon usage in the G+C-rich
Streptomyces genome. Gene 113, 55-65.
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13
phage cloning vectors and host strains: nucleotide sequences of the
M13mp18 and pUC19 vectors. Gene 33, 103-119.
Yao, 2.81Valvano, M. A. (1994). Genetic analysis of the O-specific
lipopolysaccharide biosynthesis region ( r - )of Escbericbia coli K-12
W31100: identification of genes that confer group 6 specificity to
Sbigella jexneri serotypes Y and 4a. J Bacterioll76, 41334143.
Received 27 June 1995; revised 14 September 1995; accepted 28
September 1995.
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