U.S. patent application number 10/267255 was filed with the patent office on 2003-07-03 for mitomycin biosynthetic gene cluster.
This patent application is currently assigned to Regents of the University of Minnesota. Invention is credited to He, Min, Mao, Yingqing, Sheldon, Paul, Sherman, David H., Varoglu, Mustafa.
Application Number | 20030124689 10/267255 |
Document ID | / |
Family ID | 23016731 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030124689 |
Kind Code |
A1 |
Sherman, David H. ; et
al. |
July 3, 2003 |
Mitomycin biosynthetic gene cluster
Abstract
The invention provides a biosynthetic gene cluster for
mitomycin, as well as methods of using gene(s) within the cluster
to alter biosynthesis.
Inventors: |
Sherman, David H.; (St.
Louis Park, MN) ; Mao, Yingqing; (St. Paul, MN)
; Varoglu, Mustafa; (St. Paul, MN) ; He, Min;
(St. Paul, MN) ; Sheldon, Paul; (Fitchburg,
WI) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH, P.A.
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Regents of the University of
Minnesota
|
Family ID: |
23016731 |
Appl. No.: |
10/267255 |
Filed: |
October 9, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10267255 |
Oct 9, 2002 |
|
|
|
09266965 |
Mar 12, 1999 |
|
|
|
6495348 |
|
|
|
|
Current U.S.
Class: |
435/118 ;
435/193; 435/252.3; 435/320.1; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101; C12N
9/0004 20130101; C12N 9/1007 20130101; C12Q 1/6827 20130101; C07K
14/36 20130101; C12N 9/93 20130101; C12N 9/10 20130101; C12N 9/12
20130101; C12N 15/52 20130101; C12P 17/182 20130101; C12N 9/14
20130101 |
Class at
Publication: |
435/118 ;
435/69.1; 435/193; 435/320.1; 435/252.3; 536/23.2 |
International
Class: |
C12P 017/16; C07H
021/04; C12N 009/10; C12P 021/02; C12N 001/21; C12N 015/74 |
Claims
What is claimed is:
1. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence comprising a mitomycin biosynthetic gene
cluster, a variant or a fragment thereof.
2. The isolated and purified nucleic acid molecule of claim 1 which
encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK,
MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, or any
combination thereof.
3. The isolated and purified nucleic acid molecule of claim 1 which
encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ,
MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU,
MmcV, Mct, MmcW, MmcX, MmcY, or any combination thereof.
4. The isolated and purified nucleic acid molecule of claim 1 which
is from Streptomyces spp.
5. An expression cassette comprising the nucleic acid molecule of
claim 1, 2 or 3 operably linked to a promoter functional in a host
cell.
6. A recombinant bacterial host cell in which at least a portion of
a nucleic acid molecule comprising a mitomycin biosynthetic gene
cluster is disrupted to as to result in a recombinant host cell
that produces altered levels of mitomycin relative to a
corresponding nonrecombinant bacterial host cell.
7. The recombinant host cell of claim 6 in which mitomycin levels
are increased.
8. The recombinant host cell of claim 6 in which mitomycin levels
are decreased.
9. The host cell of claim 6 wherein the nucleic acid molecule which
is disrupted encodes MitT, MitS, MitR, MitQ, MitP, MitO, MitN,
MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE, MitD, MitC,
MitB, MitA, or any combination thereof.
10. The host cell of claim 6 wherein the nucleic acid molecule
which is disrupted encodes MmcA, MmcB, MmcC, MmcD, MmcE, MmcF,
MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP, MmcQ,
MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any
combination thereof.
11. A recombinant host cell, the genome of which is augmented with
at least a portion of a nucleic acid molecule comprising a
mitomycin biosynthetic gene cluster operably linked to a promoter
functional in the host cell.
12. The recombinant host cell of claim 11 in which mitomycin levels
are increased.
13. The recombinant host cell of claim 11 in which mitomycin levels
are decreased.
14. The host cell of claim 11 wherein the genome is augmented with
a nucleic acid molecule that encodes MitT, MitS, MitR, MitQ, MitP,
MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH, MitG, MitF, MitE,
MitD, MitC, MitB, MitA, or any combination thereof.
15. The host cell of claim 11 wherein the genome is augmented with
a nucleic acid molecule that encodes MmcA, MmcB, MmcC, MmcD, MmcE,
MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO, MmcP,
MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or any
combination thereof.
16. A recombinant host cell comprising a mitomycin biosynthetic
gene cluster, the genome of which is augmented by a recombinant
nucleic acid molecule, wherein the recombinant nucleic acid does
not comprise a mitomycin biosynthetic gene, and wherein the
recombinant host cell produces a biologically active agent that is
not produced by the corresponding non-recombinant host cell.
17. A product produced by the recombinant host cell of claim 6 or
11 which is not produced by the corresponding non-recombinant host
cell.
18. The product of claim 17 which comprises a biologically active
agent.
19. The product of claim 18 which is a mitomycin.
20. The product of claim 18 is not a mitomycin.
21. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence which encodes polyketide biosynthetic enzymes
or a fragment thereof, wherein the nucleic acid sequence hybridizes
under hybridizing conditions to SEQ ID NO:74.
22. An isolated and purified polypeptide comprising MitT, MitS,
MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI, MitH,
MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC, MmcD,
MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN, MmcO,
MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, Mct, MmcW, MmcX, MmcY, or
any combination thereof.
23. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence comprising sugar metabolism genes or a
fragment thereof, wherein the nucleic acid sequence hybridizes
under hybridizing conditions to a DNA comprising SEQ ID NO:75.
24. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence which encodes an aminoDAHP synthase from
Streptomyces strains that produce mitomycin.
25. A recombinant host cell in which at least a portion of a
nucleic acid sequence which encodes polyketide biosynthetic enzymes
is disrupted so as to result in a recombinant host cell that
produces altered polyketide levels or polyketides of altered
composition relative to a corresponding nonrecombinant cell,
wherein the nucleic acid sequence hybridizes under hybridizing
conditions to SEQ ID NO:74
26. A recombinant host cell in which at least a portion of a
nucleic acid sequence which encodes sugar metabolism enzymes is
disrupted so as to result in a recombinant host cell that produces
altered sugar levels or molecules with altered sugar composition
relative to a corresponding nonrecombinant cell, wherein the
nucleic acid sequence hybridizes under hybridizing conditions to a
DNA comprising SEQ ID NO:75.
27. A recombinant host cell, the genome of which is augmented with
at least a portion of a nucleic acid sequence which encodes
polyketide biosynthetic enzymes operably linked to a promoter
functional in the host cell, wherein the nucleic acid sequence
hybridizes under hybridizing conditions to SEQ ID NO:74.
28. A recombinant host cell, the genome of which is augmented with
at least a portion of a nucleic acid sequence which encodes sugar
metabolism enzymes operably linked to a promoter functional in the
host cell, wherein the nucleic acid sequence hybridizes under
hybridizing conditions to a DNA comprising SEQ ID NO:75.
29. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence that hybridizes under hybridizing conditions
to a nucleic acid segment comprising SEQ ID NO:96, or a fragment
thereof.
30. The isolated and purified nucleic acid molecule of claim 29
which is plant nucleic acid.
31. The isolated and purified nucleic acid molecule of claim 29
which is prokaryotic nucleic acid.
32. A method to introduce exogenous DNA into a refractory
Streptoinyces strain, comprising: a) contacting a bacterial donor
cell comprising a conjugative plasmid with a Streptomyces cell so
as to yield a transformed Streptomyces cell comprising at least a
portion of the plasmid; and b) identifying the transformed
Streptomyces cell.
33. The method of claim 32 wherein the Streptomyces strain produces
a mitomycin.
34. A method to identify a nucleic acid molecule that is related to
at least a portion of a nucleic acid molecule comprising a
mitomycin gene cluster, comprising: a) contacting a sample
comprising nucleic acid with an amount of a probe comprising at
least a portion of a nucleic acid molecule comprising a mitomycin
gene so as to form a complex; b) detecting the presence or absence
of the complex.
35. A method to identify a nucleic acid molecule that is related to
at least a portion of a nucleic acid molecule comprising a
mitomycin gene cluster comprising: a) contacting a sample
comprising nucleic acid with at least one oligonucleotide under
conditions effective to amplify the nucleic acid so as to yield an
amplification product, wherein the oligonucleotide specifically
hybridizes to nucleic acid comprising a mitomycin gene cluster; and
b) detecting or determining the presence or absence of the
product.
36. The method of claim 34 or 35 wherein the sample is obtained
from a plant.
37. The method of claim 34 or 35 wherein the sample is obtained
from a microorganism.
38. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence comprising a gene product selected from MitT,
MitS, MitR, MitQ, MitP, MitO, MitN, MitM, MitL, MitK, MitJ, MitI,
MitH, MitG, MitF, MitE, MitD, MitC, MitB, MitA, MmcA, MmcB, MmcC,
MmcD, MmcE, MmcF, MmcG, MmcH, MmcI, MmcJ, MmcK, MmcL, MmcM, MmcN,
MmcO, MmcP, MmcQ, MmcR, MmcS, MmcT, MmcU, MmcV, MmcW, MmcX, MmcY,
or any combination thereof.
39. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence encoding at least one gene necessary for
mitomycin biosynthesis.
40. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence encoding at least one gene for mitomycin
transport.
41. An isolated and purified nucleic acid molecule comprising a
nucleic acid sequence encoding a polypeptide that regulates
mitomycin biosynthesis or resistance.
42. A method for preparing a compound or a pharmaceutically
acceptable salt thereof from a recombinant host cell comprising
culturing the host cell of claim 6, 11 or 16 in a culture medium
containing assimilable sources of carbon, nitrogen and inorganic
salts under aerobic fermentation conditions so as to yield an
increase in the compound relative to the level of the compound
produced by the corresponding non-recombinant host cell.
43. A method for preparing a mitomycin or a pharmaceutically
acceptable salt thereof from a recombinant host cell comprising
culturing the host cell of claim 6, 11 or 16 in a culture medium
containing assimilable sources of carbon, nitrogen and inorganic
salts under aerobic fermentation conditions so as to yield an
increase in the mitomycin relative to the level of the mitomycin
produced by the corresponding non-recombinant host cell.
44. A product produced by the recombinant host cell of claim 16
which is a mitomycin.
45. A product produced by the recombinant host cell of claim 16
which is not a mitomycin.
46. An isolated and purified nucleic acid molecule comprising at
least a fragment of a nucleic acid sequence comprising a mitomycin
biosynthetic gene cluster (mit/mmc), which fragment encodes an
enzyme that during the biosyntheis of mitomycin modifies mitosane,
and which fragment has at least 80% nucleic acid sequence identity
with at least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58,
60, 62, 68, or comprising the complement of the fragment.
47. The isolated and purified nucleic acid molecule of claim 46
which encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO:
104, or MitF having SEQ ID NO: 102.
48. The isolated and purified nucleic acid molecule of claim 46
which encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO:
124, MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN
having SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.
49. The isolated and purified nucleic acid molecule of claim 46
which is from a naturally-occurring Streptomyces spp.
50. An expression cassette comprising the nucleic acid molecule of
claim 46 operably linked to a promoter functional in a host
cell.
51. A recombinant host cell comprising a recombinant nucleic acid
molecule comprising at least a fragment of a mitomycin biosynthetic
gene cluster (mit/mmc) operably linked to a promoter functional in
the host cell, which fragment encodes an enzyme that during the
biosynthesis of mitomycin modifies mitosane, and which fragment has
at least 80% nucleic acid sequence identity with at least one of
SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62, 68, or
comprising the complement of the fragment.
52. The recombinant host cell of claim 51 in which the levels of
the enzyme are increased.
53. The recombinant host cell of claim 51 in which the levels of
the enzyme are decreased.
54. The recombinant host cell of claim 51 wherein the fragment
encodes MitK having SEQ ID NO: 107, MitH having SEQ ID NO: 104, or
MitF having SEQ ID NO: 102.
55. The recombinant host cell of claim 51 wherein the fragment
encodes MmcE having SEQ ID NO: 120, MmcI having SEQ ID NO: 124,
MmcJ having SEQ ID NO: 125, MmcL having SEQ ID NO: 127, MmcN having
SEQ ID NO: 129, or MmcT having SEQ ID NO: 135.
56. The isolated and purified nucleic acid molecule of claim 46
wherein the enzyme is a hydroxylase, a reductase, a dehydrogenase,
methyltransferase, or converts a carboxyl group to a methyl
group.
57. The isolated and purified nucleic acid molecule of claim 46
wherein the fragment comprises SEQ ID NO:21, 22, 24, 38, 41, 43,
53, 57, 58, 60, 62 or 68.
58. A method to prepare an enzyme that catalyzes a step in
mitomycin biosynthesis: expressing a recombinant DNA molecule in a
host cell so as to yield an enzyme that catalyzes a step in
mitomycin biosynthesis, wherein the recombinant DNA molecule
comprises a promoter operably linked to a DNA sequence which
encodes an enzyme that modifies mitosane, and wherein the DNA
sequence has at least 80% nucleic acid sequence identity with at
least one of SEQ ID NOs:21, 22, 24, 38, 41, 43, 53, 57, 58, 60, 62,
or 68.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/266,965, filed Mar. 12, 1999.
BACKGROUND OF THE INVENTION
[0002] Streptomyces are filamentous Gram-positive soil bacteria
with a nucleotide base composition greater than 70 mole % G+C
(Stackebrandt and Woese, 1981). They produce a wide array of
biologically active compounds including over two thirds of the
commercially important natural product metabolites (Alderson et
al., 1993; Bevax, 1998). Genetic information accumulated over the
past 15 years has demonstrated that genes encoding enzymes for
natural product assembly are clustered on the Streptomyces genome
(Martin, 1992). In addition, one or more pathway-specific
transcriptional regulatory genes, and at least one resistance gene
are typically found within the antibiotic biosynthetic gene cluster
(Chater, 1992). Heterologous hybridization with gene probes based
on highly conserved biosynthetic enzyme amino acid sequences has
been useful to clone antibiotic biosynthetic genes (Hopwood, 1997;
Seno and Baltz, 1989; Turgay and Marahiel, 1994).
[0003] The mitomycins are a group of natural products that contain
a variety of functional groups, including aminobenzoquinone and
aziridine ring systems. One representative of the family, mitomycin
C (MC), was the first recognized bioreductive alkylating agent. In
particular, since its discovery and demonstration of anticancer
activity in the 1960s, many aspects of the chemistry and biology of
MC have been investigated. This has provided detailed information
on its unprecedented molecular mechanism, unique biological and
pharmacological properties, drug resistance, and bioactive
analogues (Hata et al., 1956; Verweij, 1997). MC is regarded as the
prototype natural product alkylating agent whose activity is
dependent on the reductive activation (either chemically, such as
low pH, or enzymatically, such as DT-diaphorase, NADH cytochrome c
reductase) (Boxer, 1997; Cummings et al., 1998). Activated MC
crosslinks double-stranded DNA, which in turn induces diverse
biological effects including selective inhibition of DNA synthesis,
mutagenesis, induction of DNA repair (SOS response), sister
chromatid exchange, signal transduction, and induction of apoptosis
(Tomasz and Palem, 1997). Tumor hypoxia and the increased
expression of bioreductive enzymes in malignant cells create a
selective environment for drug activation and make MC an attractive
agent for anti-tumor therapy (Spanswick et al., 1998). MC has
become one of the most effective antitumor drugs against non-small
cell lung carcinoma and other soft tumors, as well as a clinically
important component of combination cancer chemotherapy and
radiotherapy of solid tumors (Henderson, 1993).
[0004] In addition to its biological and pharmacological
importance, MC is prominent because its molecular mechanism
represents a model for structurally related antitumor antibiotics
such as porfiromycin (Pan and Iracki, 1988), mitiromycin (Wakaki et
al., 1958), FR66979 (Paz and Hopkins, 1997), FR900482 (Williams et
al., 1997), FK973 (Hirai et al., 1994), and FK317 (Naoe et al.,
1998), as well as structurally unrelated bioreductive agents such
as EO9 (Smitskampwilms et al., 1996), and tirapazamine (Evans et
al., 1998). Numerous MC derivatives have been synthesized and
tested for enhanced activities, including the recently identified
selective protein tyrosine kinase inhibitor, 1 a-docosahexaenoyl MC
(Kasai and Arai, 1995; Shikano et al., 1998).
[0005] Streptomyces lavendulae produces MC. The molecule has an
unusual structure comprised of aziridine, pyrrolizidine,
pyrrolo-(1,2a)-indole, and amino-methylbenzoquinone rings to give
the mitosane nucleus (Webb et al., 1962). The mitosane core of MC
was shown to be derived from the junction of an
amino-methylbenzoquinone (mC.sub.7N unit) and hexosamine (C.sub.6N
unit) (Hornemann, 1981). The C.sub.6N unit consists of carbons 1,
2, 3, 9, 9a, 10, with the aziridine nitrogen derived intact from
D-glucosamine (Homemann et al., 1974).
[0006] The mC.sub.7N unit in MC and the ansamycins is derived from
3-amino-5-hydroxybenzoic acid (AHBA) (Becker et al., 1983; Kibby
and Richards, 1981). AHBA was first shown to be incorporated into
the ansamycin antibiotic actamycin (Kibby et al., 1980).
Subsequently, it was confirmed as an efficient precursor for
rifamycin (Becker et al., 1983; Kibby and Rickards, 1981; Ghilsalba
and Neuesch, 1981), geldanamycin (Potgieter, 1983), ansamitocin
(Hatano et al., 1982), ansatrienin (Wu et al., 1987),
streptovaricin (Staley and Rinehart, 1991) and naphthomycin A (Lee
et al., 1994). Anderson et al. (1980) demonstrated that
[carboxy-.sup.13C] AHBA could be efficiently and specifically
incorporated into the C-6 methyl group of porfiromycin, which
contains the same mitosane core as MC. Incorporation experiments
with radiolabeled precursors have demonstrated that the mitosane
core of MC was derived from the junction of AHBA and D-glucosamine
(Anderson et al., 1980; Homemann, 1981).
[0007] Meanwhile the O- and N- (but not C-) methyl groups were
shown to be derived from L-methionine, while the C-10 carbamoyl
group came from L-arginine or L-citrulline (Bezanson and Vining,
1971; Homemann and Eggert, 1975; Homemann et al., 1974).
[.sup.14C]-labeled precursor feeding studies with D-glucose,
pyruvate and D-erythrose indicated that de novo biosynthesis of
AHBA resulted directly from the shikimate pathway. However, no
incorporation into the mC.sub.7N unit of either MC (Homemann, 1981)
or the ansamycin antibiotics (Chiao et al., 1998) was found from
labeling studies with shikimic acid, the shikimate precursor
3-dehydroquinic acid, or the shikimate derived amino acids. These
results led to the hypothesis of a modified shikimate pathway, in
which a 3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP)
synthase-like enzyme catalyzes the conversion to
3,4-dideoxy-4-amino-D-arabino-heptulos- onic acid-7-phosphate
(amino-DAHP), to give the ammoniated shikimate pathway (Kim et al.,
1992). Floss (1997) provided strong support for this new variant of
the shikimate pathway by showing that aminoDAHP,
5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and
5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) could be
efficiently converted into AHBA by a cell-free extract of
Amycolatopsis mediterranei (rifamycin producer), in contrast to the
normal shikimate pathway intermediate DAHP which was not converted
(Kim et al., 1992; Kim et al., 1996). Recently, the AHBA synthase
(rifk) gene from A. mediterranei has been cloned, sequenced and
functionally characterized (Kim et al., 1998).
[0008] Little is known regarding the details of the convergent
assembly of MC from AHBA and D-glucosamine in S. lavendulae, i.e.,
whether its de novo biosynthesis is related to the primary
metabolic shikimate pathway, an important route in microorganisms
and plants for aromatic amino acid biosynthesis (Floss, 1997). In
addition, it is unclear how S. lavendulae resists the activity of
MC since the preferred MC alkylation sites in DNA are guanine and
cytosine, and MC-induced cell death can result from a single
crosslink per genome (Tomasz, 1995).
[0009] Thus, there is a continuing need for the identification and
isolation of antibiotic biosynthetic genes, including genes which
confer resistance to antibiotics or result in enhanced production
of antibiotics.
SUMMARY OF THE INVENTION
[0010] The present invention provides an isolated and purified
nucleic acid molecule, e.g., DNA, comprising a gene cluster for
mitomycin, a variant or a fragment thereof (the mit/mmc gene
cluster). As described hereinbelow, the S. lavendulae mitomycin
gene cluster includes the mitomycin biosynthetic gene cluster
comprising 47 mitomycin biosynthetic genes spanning 55 kb of
contiguous DNA. The biosynthetic portion of the gene cluster
includes genes that encode polypeptides involved in the generation
of biosynthetic precursors, mitosane ring system assembly and
functionalization (e.g., methylation, hydroxylation, aminotransfer,
carbamoylation, and carbonyl reduction), a mitomycin resistance
gene which is different than mrd and the unlinked mcr, as well as
several regulatory genes. Gene disruption was employed to further
characterize some of the genes. Fourteen of 22 gene disruption
mutants affected mitomycin biosynthesis, resulting in abrogation or
overexpression of drug production, e.g., targeted genetic
disruption of a mitomycin pathway regulator (e.g., mmcW) led to a
substantial increase in drug production. It is preferred that the
isolated and purified nucleic acid molecule of the invention is
nucleic acid from Streptomyces spp., such as Streptomyces
lavendulae (e.g., B19/ATCC 27422, NRRL 2564, KY681, ATCC 27423, or
PB1000), Streptomyces caespitosus, Streptomyces verticillatus, and
Streptomyces sandaensis (FERM-P7654), although isolated and
purified nucleic acid molecules from other organisms which produce
mitomycin or biological or functional equivalents thereof are also
within the scope of the invention. The nucleic acid molecules of
the invention are double-stranded or single-stranded.
[0011] As described hereinbelow, a 3.8 kb BamHI fragment from the
S. lavendulae genome was isolated which comprises three open
reading frames (ORFs). One of the ORFs (mitA) showed high
similarity to previously identified AHBA synthase genes (Kim et
al., 1998), while another (mitB) showed sequence similarity to
several prokaryotic and eukaryotic glycosyltransferases. Nucleotide
sequence analysis showed that mitA encodes a 388 amino acid protein
that has 71% identity (80% similarity) with the rifamycin AHBA
synthase from Amycolatopsis mediterranei, as well as with two
additional AHBA synthases from related ansamycin
antibiotic-producing microorganisms. Gene disruption and
site-directed mutagenesis of the S. lavendulae chromosomal copy of
mitA completely blocked the production of MC. The function of mitA
was confirmed by complementation of a S. lavendulae strain
containing a K191A mutation in MitA with 3-amino-5-hydroxybenzoic
acid, i.e., MC production was restored when the mitA mutant strain
was cultured in the presence of exogenous 3-amino-5-hydroxybenzoic
acid. mitB encodes a 272 amino acid protein.
[0012] Seven gene products (aminoDHQ synthase (MitP), aminoquinate
dehydrogenase (MitT), aminoDHQ dehydratase (MmcF), AHBA synthase
(MitA), oxidoreductase (MitG), phosphatase (MitJ), and kinase
(MitS)) are likely responsible for assembly of the intermediate
3-amino-5-hydroxybenzoic acid (AHBA) through a variant of the
shikimate pathway. However, the gene encoding aminoDAHP synthase,
the first presumed enzyme involved in AHBA biosynthesis from
phosphoenol pyruvate (PEP) and erythrose 4-phosphate (E4P), is not
linked within the mitomycin biosynthetic gene cluster.
[0013] A mitomycin resistance determinant (mct) encodes a
membrane-associated protein involved in excretion of mitomycin from
cells. Disruption of met by insertional inactivation resulted in a
S. lavendulae mutant strain that was considerably more sensitive to
MC. Expression of mct in E. coli conferred a 5-fold increase in
cellular resistance to MC, led to the synthesis of a membrane
associated protein, and correlated with reduced intracellular
accumulation of drug. Co-expression of mct and mrd in E. coli
resulted in a 150-fold increase in resistance, as well as reduced
intracellular accumulation of MC. The results establish that MRD
maintains a high affinity for MC and may serve as the primary
receptor (participating as an accessory component in a drug export
system) for subsequent transport by MCT.
[0014] The cloned mitomycin biosynthetic genes are useful to
elucidate the molecular basis for the biosynthesis of the mitosane
ring system, as well as to engineer the biosynthesis of novel
natural products. Moreover, genetic engineering or overexpression
of the transport, resistance and regulatory proteins may lead to
higher titers of mitomycin compounds from production cultures.
[0015] Preferably, the isolated nucleic acid molecule comprising
the gene cluster includes a nucleic acid sequence comprising SEQ ID
NO:96 or SEQ ID NO:76, a variant or a fragment thereof, e.g., a
nucleic acid molecule that hybridizes under moderate, or more
preferably stringent, hybridization conditions to SEQ ID NO:96, SEQ
ID NO:76 or a fragment thereof. Moderate and stringent
hybridization conditions are well known to the art, see, for
example sections 9.47-9.51 of Sambrook et al. (Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989). For example, stringent conditions are those
that (1) employ low ionic strength and high temperature for
washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC);
0.1% sodium lauryl sulfate (SDS) at 50.degree. C., or (2) employ a
denaturing agent such as formamide during hybridization, e.g., 50%
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM NaCl, 75 mM sodium citrate at 42.degree. C. Another example
is use of 50% formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium
citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium
pyrophosphate, 5.times.Denhardt's solution, sonicated salmon sperm
DNA (50 .mu.g/ml), 0.1% sodium dodecylsulfate (SDS), and 10%
dextran sulfate at 42.degree. C., with washes at 42.degree. C. in
0.2.times.SSC and 0.1% SDS.
[0016] A preferred nucleic molecule of the invention comprises a
nucleic acid sequence encoding a polypeptide including, but not
limited to, MitA (e.g., SEQ ID NO:10 encoded by SEQ ID NO:97), MitB
(e.g., SEQ ID NO:11 encoded by SEQ ID NO:98), MitC (e.g., SEQ ID
NO:12 encoded by SEQ ID NO:99), MitD (e.g., SEQ ID NO:100 encoded
by SEQ ID NO:45), MitE (e.g., SEQ ID NO:101 encoded by SEQ ID
NO:44), MitF (e.g., SEQ ID NO:102 encoded by SEQ ID NO:43), MitG
(e.g., SEQ ID NO:103 encoded by SEQ ID NO:42), MitH (e.g., SEQ ID
NO:104 encoded by SEQ ID NO:41), MitI (e.g., SEQ ID NO:105 encoded
by SEQ ID NO:40), MitJ (e.g., SEQ ID NO:106 encoded by SEQ ID
NO:39), MitK (e.g., SEQ ID NO:107 encoded by SEQ ID NO:38), MitL
(e.g., SEQ ID NO:108 encoded by SEQ ID NO:37), MitM (e.g., SEQ ID
NO:109 encoded by SEQ ID NO:36), MitN (e.g., SEQ ID NO:108 encoded
by SEQ ID NO:35), MitO (e.g., SEQ ID NO:111 encoded by SEQ ID
NO:34), MitP (e.g., SEQ ID NO:112 encoded by SEQ ID NO:33), MitQ
(e.g., SEQ ID NO:113 encoded by SEQ ID NO:32), MitR (e.g., SEQ ID
NO:114 encoded by SEQ ID NO:31), MitS (e.g., SEQ ID NO:115 encoded
by SEQ ID NO:30), MitT (e.g., SEQ ID NO:140 encoded by SEQ ID
NO:29), MmcA (SEQ ID NO:116 encoded by SEQ ID NO:49), MmcB (SEQ ID
NO:117 encoded by SEQ ID NO:50), MmcC (SEQ ID NO:118 encoded by SEQ
ID NO:51), MmcD (SEQ ID NO:119 encoded by SEQ ID NO:52), MmcE (SEQ
ID NO:120 encoded by SEQ ID NO:53), MmcF (SEQ ID NO:121 encoded by
SEQ ID NO:54), MmcG (SEQ ID NO:122 encoded by SEQ ID NO:55), MmcH
(SEQ ID NO:123 encoded by SEQ ID NO:56), Mmcl (SEQ ID NO:124
encoded by SEQ ID NO:57), MmcJ (SEQ ID NO:125 encoded by SEQ ID
NO:58), MmcK (SEQ ID NO:126 encoded by SEQ ID NO:59), MmcL (SEQ ID
NO:127 encoded by SEQ ID NO:60), MmcM (SEQ ID NO:128 encoded by SEQ
ID NO:61), MmcN (SEQ ID NO:129 encoded by SEQ ID NO:62), MmcO (SEQ
ID NO:130 encoded by SEQ ID NO:63), MmcP (SEQ ID NO:131 encoded by
SEQ ID NO:64), MmcQ (SEQ ID NO:132 encoded by SEQ ID NO:65), MmcR
(SEQ ID NO:133 encoded by SEQ ID NO:66), MmcS (SEQ ID NO:134
encoded by SEQ ID NO:67), MmcT (SEQ ID NO:135 encoded by SEQ ID
NO:68), MmcU (SEQ ID NO:136 encoded by SEQ ID NO:69), MmcV (SEQ ID
NO:137 encoded by SEQ ID NO:70), MmcW (SEQ ID NO:138 encoded by SEQ
ID NO:71), MmcX (SEQ ID NO:139 encoded by SEQ ID NO:72), MmcY (SEQ
ID NO:141 encoded by SEQ ID NO:73), Mct (SEQ ID NO:117 encoded by
SEQ ID NO:16), a variant or a fragment thereof, e.g., a nucleic
acid molecule that hybridizes under moderate, or more preferably
stringent, hybridization conditions to at least one of the nucleic
acid sequences identified hereinabove.
[0017] The invention further provides an isolated and purified
nucleic acid molecule which is linked to a mitomycin biosynthetic
gene cluster and which encodes polyketide biosynthetic enzymes, a
variant or a fragment thereof. Preferably, the nucleic acid
molecule of this embodiment of the invention comprises at least
one, preferably at least five, and more preferably at least nine,
open reading frames. More preferably, the nucleic acid molecule
hybridizes under moderate, or more preferably stringent,
hybridization conditions to SEQ ID NO:74, or a portion thereof.
[0018] The invention also provides an isolated and purified nucleic
acid molecule which is linked to a mitomycin biosynthetic gene
cluster and which encodes sugar biosynthetic enzymes, a variant or
a fragment thereof. Preferably, the nucleic acid molecule of this
embodiment of the invention comprises at least one, preferably at
least five, more preferably at least nine, and even more preferably
at least twelve, open reading frames. Preferably, the nucleic acid
molecule of this embodiment of the invention hybridizes under
moderate, or more preferably stringent, hybridization conditions to
SEQ ID NO:75, or a portion thereof.
[0019] The invention also provides a variant polypeptide having at
least about 80%, more preferably at least about 90%, and even more
preferably at least about 95%, but less than 100%, contiguous amino
acid sequence identity to a polypeptide having an amino acid
sequence encoded by SEQ ID NO:76, or a fragment thereof. A
preferred variant polypeptide includes a variant polypeptide or
fragment thereof having at least about 1%, more preferably at least
about 10%, and even more preferably at least about 50%, the
activity of the polypeptide having the amino acid sequence
comprising SEQ ID NO: 10-12, 17 or 100-141. Thus, for example, the
activity of a polypeptide having SEQ ID NO:98 can be compared to a
variant of SEQ ID NO:98 having at least one amino acid
substitution, insertion, or deletion relative to SEQ ID NO:98.
[0020] A variant nucleic acid sequence of the invention has at
least about 80%, more preferably at least about 90%, and even more
preferably at least about 95%, but less than 100%, contiguous
nucleic acid sequence identity to a nucleic acid sequence
comprising SEQ ID NO:76, or a fragment thereof. The amino acid
and/or nucleic acid similarity (or homology) of two sequences may
be determined manually or using algorithms well known to the
art.
[0021] The invention also provides probes and primers comprising at
least a portion of the nucleic acid molecules of the invention. The
probes or primers of the invention are preferably detectably
labeled or have a binding site for a detectable label. Preferably,
the probes or primers of the invention are at least about 7, more
preferably at least about 15, contiguous nucleotides bases having
at least about 80% identity, more preferably at least about 90%
identity, to the isolated nucleic acid molecules of the invention.
Such probes or primers are useful to detect, quantify, isolate
and/or amplify DNA strands with complementary to sequences related
to the mitomycin biosynthetic gene cluster, sequences related to
those encoding the polyketide biosynthetic enzymes linked to the
mitomycin biosynthetic gene cluster, sequences related to those
encoding sugar biosynthetic enzymes linked to the mitomycin
biosynthetic gene cluster, a variant or a fragment thereof.
[0022] Also provided is an expression cassette comprising a nucleic
acid molecule comprising at least a portion of a mitomycin
biosynthetic gene cluster, a nucleic acid molecule which is linked
to a mitomycin biosynthetic gene cluster and which encodes
polyketide biosynthetic enzymes, a nucleic acid molecule which is
linked to a mitomycin biosynthetic gene cluster and which encodes
sugar biosynthetic enzymes, a variant or fragment thereof, operably
linked to a promoter functional in a host cell. Host cells that
have been modified genetically, i.e., recombinant host cells,
include host cells comprising an expression cassette, e.g., an
expression cassette of the invention, or host cells in which the
genome has been genetically manipulated, e.g., by deletion of a
portion of, replacement of a portion of, or by disruption of, the
host chromosome, so as to reduce or eliminate the expression of a
particular mitomycin biosynthetic gene, polyketide biosynthetic
gene or a sugar biosynthetic gene of the invention.
[0023] One embodiment of the invention is a recombinant host cell,
e.g., a bacterial cell, in which a portion of a nucleic acid
sequence comprising the mitomycin gene cluster, i.e., the
endogenous or native genomic sequence, is disrupted or replaced,
for example, by an insertion with heterologous sequences or
substituted with a variant nucleic acid sequence of the invention,
preferably so as to result in altered mitomycin synthesis, such as
an increase in mitomycin synthesis, and/or production of a novel
compound. For example, the invention includes a recombinant host
cell in which the mmcW gene is disrupted, for example, by
replacement with a selectable marker gene, so as to yield a
recombinant host cell having an increase in mitomycin
production.
[0024] Another embodiment of the invention is a recombinant host
cell, the genome of which is augmented by an expression cassette,
e.g., via an extrachromosomal element such as a plasmid or by
stable integration of the cassette into the host chromosome. Thus,
the genome of the recombinant host cell is augmented with at least
one mitomycin biosynthetic gene, polyketide biosynthetic gene or a
sugar biosynthetic gene of the invention so as to yield an altered
level of mitomycin and/or a novel compound(s) relative to the
corresponding non-recombinant host cell.
[0025] Alternatively, the genome of a recombinant host cell is
augmented with a non-mitomycin biosynthetic gene and, optionally,
at least one mitomycin biosynthetic gene, polyketide biosynthetic
gene or a sugar biosynthetic gene of the invention so as to yield
an altered level of mitomycin and/or a novel compound(s) relative
to the corresponding non-recombinant host cell. For example, the
recombinant host cell may be augmented with pikA (see U.S.
application Ser. No. 09/105,537, filed Jun. 26, 1998, the
disclosure of which is incorporated by reference herein) and pikA
expressed in an amount effective to yield a novel compound(s).
[0026] Host cells useful to prepare the recombinant host cells of
the invention include cells which do not express or do not comprise
nucleic acid corresponding to the nucleic acid molecules of the
invention, e.g., mitomycin biosynthetic genes, as well as cells
which naturally produce mitomycin.
[0027] Thus, the invention also provides isolated and purified
polypeptides encoded by a nucleic acid molecule of the invention.
Preferably, the polypeptide of the invention is obtained from
recombinant host cells, e.g., the genome of which is augmented by a
nucleic acid molecule of the invention. In addition, expression
cassettes and host cells comprising antisense sequences of at least
a portion of the mitomycin biosynthetic gene cluster of the
invention are envisioned.
[0028] In another embodiment of the invention, the isolated and
purified nucleic acid molecule which is linked to a mitomycin
biosynthetic gene cluster and which encodes polyketide biosynthetic
enzymes, e.g., a polyketide synthase, is useful in methods to
prepare recombinant polyhydroxyalkanoate monomer synthases and
polymers.
[0029] Thus, the present invention provides a method of preparing a
polyhydroxyalkanoate synthase. The method comprises introducing an
expression cassette into a host cell. The expression cassette
comprises a DNA molecule encoding a polyketide synthase, operably
linked to a promoter functional in the host cell. The DNA molecule
is preferably obtained from a mitomycin-producing organism, e.g., a
Streptomyces spp. such as S. lavendulae. The DNA molecule encoding
the polyketide synthase is then expressed in the cell. Thus,
another embodiment of the invention provides a purified recombinant
polyketide isolated from a host cell which expresses the
synthase.
[0030] Another embodiment of the invention is a method of preparing
a polyhydroxyalkanoate polymer. The method comprises introducing a
first expression cassette and a second expression cassette into a
host cell. The first expression cassette comprises a DNA segment
encoding a fatty acid synthase in which the dehydrase activity has
been inactivated that is operably linked to a promoter functional
in the host cell, e.g., an insect cell. The inactivation preferably
is via a mutation in the catalytic site of the dehydrase. The
second expression cassette comprises a DNA segment encoding a
polyketide synthase that is preferably obtained from a
mitomycin-producing organism operably linked to a promoter
functional in the host cell. The expression cassettes may be on the
same or separate molecules. The DNA segments in the expression
cassettes are expressed in the cell so as to yield a
polyhydroxyalkanoate polymer.
[0031] The present invention also provides an expression cassette
comprising a nucleic acid molecule encoding a polyhydroxyalkanoate
monomer synthase operably linked to a promoter functional in a host
cell. The nucleic acid molecule comprises a plurality of DNA
segments. Thus, the nucleic acid molecule comprises at least a
first and a second DNA segment. The first DNA segment encodes a
first module and the second DNA segment encodes a second module,
wherein the DNA segments together encode a polyhydroxyalkanoate
monomer synthase. No more than one DNA segment is derived from the
eryA gene cluster of Saccharopolyspora erythraea. It is also
preferred that the first DNA segment comprises a module from a
mitomycin-producing organism, e.g., Streptomyces spp. The nucleic
acid molecule may optionally further comprise a third DNA segment
encoding a polyhydroxyalkanoate synthase. Alternatively, a second
nucleic acid molecule encoding a polyhydroxyalkanoate synthase may
be introduced into the host cell.
[0032] Also provided is an isolated and purified DNA molecule. The
DNA molecule comprises a plurality of DNA segments. Thus, the DNA
molecule comprises at least a first and a second DNA segment. The
first DNA segment encodes a first module and the second DNA segment
encodes a second module. Together the DNA segments encode a
recombinant polyhydroxyalkanoate monomer synthase. It is preferred
that no more than one DNA segment is derived from the eryA gene
cluster of Saccharopolyspora erythraea. Also, it is preferred that
no more than one module is derived from the gene cluster from
Streptomyces hygroscopicus that encodes rapamycin or the gene
cluster that encodes spiramycin. A preferred embodiment of the
invention employs a first DNA segment comprising a module from a
mitomycin-producing organism. A further preferred embodiment of the
isolated DNA molecule of the invention includes a DNA segment
encoding a polyhydroxyalkanoate synthase.
[0033] Further provided is a method of preparing a
polyhydroxyalkanoate polymer. The method comprises introducing a
first DNA molecule and a second DNA molecule into a host cell. The
first DNA molecule comprises a DNA segment encoding a recombinant
polyhydroxyalkanoate monomer synthase. The recombinant
polyhydroxyalkanoate monomer synthase comprises a plurality of
modules. Thus, the monomer synthase comprises at least a first
module and a second module. The first DNA molecule is operably
linked to a promoter functional in a host cell. The second DNA
molecule comprises a DNA segment encoding a polyhydroxyalkanoate
synthase operably linked to a promoter functional in the host cell.
It is preferred that at least one module is from a
mitomycin-producing organism. The DNAs encoding the recombinant
polyhydroxyalkanoate monomer synthase and polyhydroxyalkanoate
synthase are expressed in the host cell so as to generate a
polyhydroxyalkanoate polymer.
[0034] Yet another embodiment of the invention is an isolated and
purified DNA molecule. The DNA molecule comprises a plurality of
DNA segments. That is, the DNA molecule comprises at least a first
and a second DNA segment. The first DNA segment encodes a fatty
acid synthase and the second DNA segment encodes a module of a
polyketide synthase. A preferred embodiment of the invention
employs a second DNA segment comprising a module of a polyketide
synthase from a mitomycin-producing organism such as
Streptomyces.
[0035] Also provided is a method of providing a
polyhydroxyalkanoate monomer synthase. The method comprises
introducing an expression cassette into a host cell. The expression
cassette comprises a DNA molecule encoding a polyhydroxyalkanoate
monomer synthase operably linked to a promoter functional in the
host cell. The monomer synthase comprises a plurality of modules.
Thus, the monomer synthase comprises at least a first and second
module which together encode the monomer synthase. A preferred
embodiment of the invention employs a module from a
mitomycin-producing organism. Optionally, the expression cassette
further comprises a second DNA molecule encoding a
polyhydroxyalkanoate synthase.
[0036] The invention also provides an isolated and purified DNA
molecule comprising a first DNA segment encoding a first module and
a second DNA segment encoding a second module, wherein the DNA
segments together encode a recombinant polyhydroxyalkanoate monomer
synthase. Preferably, at least one DNA segment is derived from DNA
which is linked to the mitomycin gene cluster of S. lavendulae.
Also preferably, no more than one DNA segment is derived from the
eryA gene cluster of Saccharopolyspora erythraea. In one embodiment
of the invention, the 3' most DNA segment of the isolated DNA
molecule of the invention encodes a thioesterase II. Also provided
is an expression cassette comprising a nucleic acid molecule
encoding the polyhydroxyalkanoate monomer synthase operably linked
to a promoter functional in a host cell.
[0037] Yet another embodiment of the invention is a method of
providing a polyhydroxyalkanoate monomer. The method comprises
introducing into a host cell a DNA molecule comprising a DNA
segment encoding a recombinant polyhydroxyalkanoate monomer
synthase operably linked to a promoter functional in the host cell.
Preferably, the second DNA molecule is derived from DNA which is
linked to the mitomycin gene cluster. The recombinant
polyhydroxyalkanoate monomer synthase comprises a first module and
a second module, wherein at least one DNA segment is derived from
DNA which is linked to a mitomycin gene cluster, e.g., the
mitomycin gene cluster of S. lavendulae. The DNA encoding the
recombinant polyhydroxyalkanoate monomer synthase is then expressed
in the host cell so as to generate a polyhydroxyalkanoate monomer.
Optionally, a second DNA molecule may be introduced into the host
cell. The second DNA molecule comprises a DNA segment encoding a
polyhydroxyalkanoate synthase operably linked to a promoter
functional in the host cell. The two DNA molecules are expressed in
the host cell so as to generate a polyhydroxyalkanoate polymer.
[0038] Another embodiment of the invention is an isolated and
purified DNA molecule comprising a first DNA segment encoding a
fatty acid synthase and a second DNA segment encoding a module from
the DNA which is linked to the mitomycin gene cluster of S.
lavendulae. Such a DNA molecule can be employed in a method of
providing a polyhydroxyalkanoate monomer. Thus, a DNA molecule
comprising a first DNA segment encoding a fatty acid synthase and a
second DNA segment encoding a polyketide synthase is introduced
into a host cell. The first DNA segment is 5' to the second DNA
segment and the first DNA segment is operably linked to a promoter
functional in the host cell. The first DNA segment is linked to the
second DNA segment so that the linked DNA segments express a fusion
protein. The DNA molecule is expressed in the host cell so as to
generate a polyhydroxyalkanoate monomer.
[0039] Further provided is a method of providing a
polyhydroxyalkanoate monomer synthase. The method comprises
introducing an expression cassette comprising a DNA molecule
encoding a polyhydroxyalkanoate synthase operably linked to a
promoter functional in a host cell. The DNA molecule comprises a
first DNA segment encoding a first module and a second DNA segment
encoding a second module wherein the DNA segments together encode a
polyhydroxyalkanoate monomer synthase. At least one DNA segment is
derived from DNA which is linked to the mitomycin gene cluster of
S. lavendulae. The DNA molecule is expressed in the host cell.
Optionally, the DNA molecule further comprises a DNA segment
encoding a polyhydroxyalkanoate synthase. Alternatively, a second,
separate DNA molecule encoding a polyhydroxyalkanoate synthase is
introduced into the host cell.
[0040] Thus, the invention provides an isolated and purified DNA
molecule comprising a first DNA segment encoding a first module and
a second DNA segment encoding a second module, wherein the DNA
segments together encode a recombinant polyhydroxyalkanoate monomer
synthase, and wherein at least one DNA segment is derived from the
mit/mmc gene cluster of S. lavendulae. Preferably, no more than one
DNA segment is derived from the eryA gene cluster of
Saccharopolyspora erythraea. In one embodiment of the invention,
the 3' most DNA segment of the isolated DNA molecule of the
invention encodes a thioesterase II. Also provided is an expression
cassette comprising a nucleic acid molecule encoding the
polyhydroxyalkanoate monomer synthase operably linked to a promoter
functional in a host cell.
[0041] Yet another embodiment of the invention is a method of
providing a polyhydroxyalkanoate monomer. The method comprises
introducing into a host cell a DNA molecule comprising a DNA
segment encoding a recombinant polyhydroxyalkanoate monomer
synthase operably linked to a promoter functional in the host cell.
The recombinant polyhydroxyalkanoate monomer synthase comprises a
first module and a second module, wherein at least one DNA segment
is derived from the mit/mmc gene cluster of S. lavendulae. The DNA
encoding the recombinant polyhydroxyalkanoate monomer synthase is
then expressed in the host cell so as to generate a
polyhydroxyalkanoate monomer. Optionally, a a second DNA molecule
may be introduced into the host cell. The second DNA molecule
comprises a DNA segment encoding a polyhydroxyalkanoate synthase
operably linked to a promoter functional in the host cell. The two
DNA molecules are expressed in the host cell so as to generate a
polyhydroxyalkanoate polymer.
[0042] Another embodiment of the invention is an isolated and
purified DNA molecule comprising a first DNA segment encoding a
fatty acid synthase and a second DNA segment encoding a module from
the mit/mmc gene cluster of S. lavendulae. Such a DNA molecule can
be employed in a method of providing a polyhydroxyalkanoate
monomer. Thus, a DNA molecule comprising a first DNA segment
encoding a fatty acid synthase and a second DNA segment encoding a
polyketide synthase is introduced into a host cell. The first DNA
segment is 5' to the second DNA segment and the first DNA segment
is operably linked to a promoter functional in the host cell. The
first DNA segment is linked to the second DNA segment so that the
linked DNA segments express a fusion protein. The DNA molecule is
expressed in the host cell so as to generate a polyhydroxyalkanoate
monomer.
[0043] Further provided is a method of providing a
polyhydroxyalkanoate monomer synthase. The method comprises
introducing an expression cassette comprising a DNA molecule
encoding a polyhydroxyalkanoate synthase operably linked to a
promoter functional in a host cell. The DNA molecule comprises a
first DNA segment encoding a first module and a second DNA segment
encoding a second module wherein the DNA segments together encode a
polyhydroxyalkanoate monomer synthase. At least one DNA segment is
derived from the mit/mmc gene cluster of S. lavendulae. The DNA
molecule is expressed in the host cell. Optionally, the DNA
molecule further comprises a DNA segment encoding a
polyhydroxyalkanoate synthase. Alternatively, a second, separate
DNA molecule encoding a polyhydroxyalkanoate synthase is introduced
into the host cell.
[0044] Also provided is a method for directing the biosynthesis of
specific sugar-modified polyketides by genetic manipulation of a
polyketide-producing microorganism. The method comprises
introducing into a polyketide-producing microorganism a DNA
sequence encoding enzymes in sugar biosynthesis, e.g., a DNA
sequence comprising SEQ ID NO:75, a variant or fragment thereof, so
as to yield a microorganism that produces specific sugar-modified
polyketides. Alternatively, an anti-sense DNA sequence of the
invention may be employed. Then the sugar-modified polyketides are
isolated from the microorganism. It is preferred that the DNA
sequence is modified so as to result in the inactivation of at
least one enzymatic activity in sugar biosynthesis or in the
attachment of the sugar to a polyketide
[0045] Thus, the modules encoded by the nucleic acid segments of
the invention may be employed in the methods described hereinabove
to prepare polyhydroxyalkanoates of varied chain length or having
various side chain substitutions.
[0046] The compounds produced by the recombinant host cells of the
invention are preferably biologically active agents such as
antibiotics, anti-inflammatory agents, anti-cancer agents,
antibiotics, immune-enhancers, immunosuppressants, agents to treat
asthma, chronic obstructive pulmonary disease as well as other
diseases involving respiratory inflammation, or
cholesterol-lowering agents; or as crop protection agents (e.g.,
fungicides or insecticides), as well as biopolymers, e.g., in
packaging or biomedical applications, or to engineer PHA monomer
synthases. Methods employing these compounds, e.g., to treat a
mammal, e.g., a human, bird or fish in need of such therapy, are
also envisioned.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1. The biosynthetic pathway for mitomycin
antibiotics.
[0048] FIG. 2. Organization of the mitomycin gene cluster. The
deduced ORFs are drawn to scale, and their corresponding genes are
marked in italics. The filled bars indicate the location of the
mitomycin cluster. Abbreviations of the restriction enzymes: B:
BamHI, S: SphI, P: PstI, E: EcoRI, X: XhoI, K: KpnI.
[0049] FIG. 3. The three SAM dependent methyltransferase conserved
motifs can be found in MitM (SEQ ID NO:1), MitN (SEQ ID NO:2), and
MmcR (SEQ ID NO:3). DmpM (SEQ ID NO:4; Kim et al., 1998), TcmN (SEQ
ID NO:5; Shikano et al., 1998), ORF14 (SEQ ID NO:6; August et al.,
1998), EryG (SEQ ID NO:7; Hardwick and Pelham, 1994) are
O-methyltransferases for puromycin, tetracenomycin C, rifamycin,
and erythromycin biosynthesis, respectively. Consen=consensus
sequence (SEQ ID NO:8).
[0050] FIG. 4. Sequence similarity of MitM, MitN, and MmcR with
other O-methyltransferases: DmpM (Kim et al., 1998), TcmN (Shikano
et al., 1998), ORF14 (August et al., 1998), EryG (Hardwick and
Pelham, 1994), RdmB (Mazodier et al., 1989), DnrK (Lee and Stock,
1996), and DauK (Devereux et al. 1984)); and C-methyltransferases:
SMT (Schaferjohann et al., 1993), ESMT1 (Floss, 1997), SMT1
(Blattner et al., 1997), and SED6 (Guilfoile and Hutchinson,
1992)). The dendrogram was constructed with the program PILEUP
(Denis and Brzezinki, 1992).
[0051] FIG. 5. MC genes and deduced enzyme functions.
[0052] FIG. 6. Bacterial strains and plasmids. Strains DH5.alpha.
and DH5.alpha.F' are available from Gibco BRL (Gaithersburg, Md.),
ATCC 27643 and NRRL 2564 are available from the American Type
Culture Collection, and strain S17-1 is described in Hidaka et al.
(1995). Plasmids pNJ1, pUC119, pKC 1139, pDHS3001, pKN108, and
pFD666 are described in Kuzuyama et al. (1995), Madduri et al.
(1993), Boxer (1997), Kagan and Clarke (1994), Kim et al. (1998),
and Coque et al. (1995), respectively.
[0053] FIG. 7. Biosynthetic pathway leading to mitomycin C.
[0054] FIG. 8. Southern hybridization and restriction-enzyme map of
the mrd and rifK hybridizing regions from S. lavendulae. A)
Southern hybridization with the rifK gene probe (Kim et al., 1998).
Lane 1, A. mediterranei ATCC 27643 genomic DNA digested with BamHI;
Lane 2, S. lavendulae NRRL 2564 genomic DNA digested with BamHI; B)
Physical map showing the mitABC genes. The location of mrd and rifK
hybridizing genes in cosmid pDHS7529 are indicated by solid bars.
Enzymes: E, EcoRI; B, BamHI. The sequenced 3.8 kb BamHI fragment
containing mitA, mitB, mitC is enlarged (wide arrows). Thin arrows
below show sites of resistance gene integration for disruption
experiments.
[0055] FIG. 9. Nucleotide sequence of the 3.8 kb DNA fragment
containing mitABC (SEQ ID NO:9). The deduced gene products are
indicated in the one-letter code under the DNA sequence (SEQ ID
NO:10, MitA; SEQ ID NO:11, MitB; SEQ ID NO:12, MitC). Possible
ribosome binding sites are marked in the boxed regions. The
presumed translational start site and direction of transcription
for each ORF is indicated by an arrow and marked accordingly.
[0056] FIG. 10. Alignment of MitA with three other AHBA synthases.
The deduced amino acid sequence comparison from AHBAS genes derived
from Streptomyces lavendulae (SEQ ID NO:10). Streptomyces collinus
(Z54208; SEQ ID NO:13), Actinosynnema pretiosum (I39657; SEQ ID
NO:14), and Amycolatopsis mediterranei (I39657; SEQ ID NO:15) is
shown with the conserved lysine in the PLP-binding motif
underlined.
[0057] FIG. 11. Southern blot analysis of the mitA mutant strain.
A) Construction of mitA disruption mutant and restriction map of
the wild-type and mitA disruption mutant showing expected band
sizes in the Southern blot. Maps are not drawn to scale. B) S.
lavendulae genomic DNA from wild-type (lanes 1 and 2) and double
crossover mutant (lanes 3 and 4) were digested with BamHI (lane 1
and 3) and SphI (lane 2 and lane 4), respectively. The 4.9 kb
EcoRI-HindIII fragment from pDHS2001 containing tsr-disrupted mitA
was used as the probe.
[0058] FIG. 12. Southern blot analysis of mitB mutant MM101. A)
Construction of mitB disruption mutant and restriction map of the
wild-type and mitB disruption mutant showing the expected sites in
the Southern blot. B) S. lavendulae genomic DNA from wild-type
(lane 1 and 3) and mitB mutant (lane 2 and 4) were digested with
BamHI (lane 1 and 2) and SacI (lane 3 and 4). DNA probe: 3.8 kb
BamHI fragment insert from pDHS7601.
[0059] FIG. 13. Chemical analysis and biological activity of
extracts from S. lavendulae wild-type and mutant strains. A) HPLC
analysis of authentic mitomycin C standard, mitomycin C production
in the wild-type S. lavendulae, mitA (AHBAS) and mitB (gtf)
disruption mutants of S. lavendulae. One mg of crude extract
injected, 1 .mu.g of MC injected as standard. B) Bacillus subtilis
bioassay of mitomycin C production in mitA disruption mutant strain
of S. lavendulae. Filter discs: 1) 100 .mu.g injection of
wild-type--collected 12.5-13.5 minutes; 2) 100 .mu.g injection of
mitA (ahbas) disruption mutant--collected 12.5-13.5 minutes; 3) 100
.mu.g injection of W. T. containing vector--collected 12.5-13.5
minutes; 4) One .mu.g of mitomycin C collected from HPLC from
12.5-13.5 minutes; 5) Tris buffer negative control; 6) methanol
solvent negative control.
[0060] FIG. 14. Strains and plasmids employed in Example 3. BL21
(DE3) and pET17b are available from Novagen (Madison, Wis.).
pDH57006 is described in Sheldon et al. (1997).
[0061] FIG. 15. Genetic map showing the physical linkage of the mct
and mrd genes within the MC biosynthetic gene cluster. The expanded
box shows the line plot of the met ORF.
[0062] FIG. 16. The nucleotide sequence of mct (SEQ ID NO:16). The
deduced amino acid sequence of mct is indicated under the
nucleotide sequence with the one letter designation (SEQ ID NO:17).
A conserved motif characteristic of 14 TMS proteins is boxed while
the invariant beta-turn motif is denoted with a dashed underline.
The putative ribosome binding site is marked with a solid
underline.
[0063] FIG. 17. Dot matrix alignment of the deduced amino acid
sequence of mct with other actinomycete antibiotic efflux proteins.
Comparable parameters were utilized in generating the
alignments.
[0064] FIG. 18. Hydropathy analysis of the deduced amino acid
sequence of MC-translocase. A) Hydropathy plot obtained from
prediction of Kyte and Doolittle (1982). B) Schematic
representation of MC-translocase protein topology. The
transmembrane spanning regions are marked (1-14). The initial and
final amino acid positions of each transmembrane domain are
indicated by small numbers. The relative position of positively (H,
R, K) and negatively (D, Q) charged amino acids are indicated by a
plus and minus, respectively.
[0065] FIG. 19. Creation of the mct disruption mutant. A) The
chromosomal mct gene (black bar) was disrupted by inserting a
neomycin resistance marker (shaded) within the gene. Following
double crossover recombination, specific restriction bands are
predicted to be shifted in the mct mutant genome compared to the
wild-type strain. B) Southern blot analysis of the mct mutant. As
expected, when probed with the 4.0 kb BamHI insert from pDHS7661,
the 4.0 kb BamHI hybridization band in wild-type S. lavendulae was
shifted to 5.4 kb in mct knockouts, while a 1.65 kb SacI
hybridization band was shifted to 3.0 kb in size. Lane 1 and 5:
wild-type genomic DNA digested with BamHI. Lane 2, 3, 4, and 6:
Four double crossover colonies genomic DNA digested with BamHI.
Lane 7:wild-type genomic DNA digested with SstI. Lane 8: double
crossover clone 6 genomic DNA digested with SstI.
[0066] FIG. 20. MC uptake analysis of strains PJS100, PJS102, and
PJS103. BL21(DE3)::pET17b vector control strain, (.circle-solid.);
strain PJS100, (.box-solid.); strain PJS102, (.diamond-solid.);
strain PJS103, (.times.).
[0067] FIG. 21. Complete nucleotide sequence of the mitomycin gene
cluster (SEQ ID NO:96).
[0068] FIG. 22. Complete nucleotide sequence of ORFs 1-9 (SEQ ID
NO:74).
[0069] FIG. 23. Complete nucleotide sequence of ORFs 11-22 (SEQ ID
NO:75).
[0070] FIG. 24. Codons for various amino acids.
[0071] FIG. 25. Exemplary amino acid substitutions.
[0072] FIG. 26. Complete nucleotide sequence of the mitomycin
biosynthetic genes (SEQ ID NO:76).
DETAILED DESCRIPTION OF THE INVENTION
[0073] Definitions
[0074] As used herein, a "Type I polyketide synthase" is a single
polypeptide with a single set of iteratively used active sites.
This is in contrast to a Type II polyketide synthase which employs
active sites on a series of polypeptides.
[0075] As used herein, a "linker region" is an amino acid sequence
present in a multifunctional protein which is less well conserved
in an amino acid sequence than an amino acid sequence with
catalytic activity.
[0076] As used herein, an "extender unit" catalytic or enzymatic
domain is an acyl transferase in a module that catalyzes chain
elongation by adding 2-4 carbon units to an acyl chain and is
located carboxy-terminal to another acyl transferase. For example,
an extender unit with methylmalonylCoA specificity adds acyl groups
to a methylmalonylCoA molecule.
[0077] As used herein, a "polyhydroxyalkanoate" or "PHA" polymer
includes, but is not limited to, linked units of related,
preferably heterologous, hydroxyalkanoates such as
3-hydroxybutyrate, 3-hydroxyvalerate, 3-hydroxycaproate,
3-hydroxyheptanoate, 3-hydroxyhexanoate, 3-hydroxyoctanoate,
3-hydroxyundecanoate, and 3-hydroxydodecanoate, and their 4-hydroxy
and 5-hydroxy counterparts.
[0078] As used herein, a "recombinant" nucleic acid or protein
molecule is a molecule where the nucleic acid molecule which
encodes the protein has been modified in vitro, so that its
sequence is not naturally occurring, or corresponds to naturally
occurring sequences that are not positioned as they would be
positioned in a genome which has not been modified.
[0079] As used herein, a "multifunctional protein" is one where two
or more enzymatic activities are present on a single
polypeptide.
[0080] As used herein, a "module" is one of a series of repeated
units in a multifunctional protein, such as a Type I polyketide
synthase or a fatty acid synthase.
[0081] As used herein, a "premature termination product" is a
product which is produced by a recombinant multifunctional protein
which is different than the product produced by the non-recombinant
multifunctional protein. In general, the product produced by the
recombinant multifunctional protein has fewer acyl groups.
[0082] As used herein, a DNA that is "derived from" a gene cluster
is a DNA that has been isolated and purified in vitro from genomic
DNA, or synthetically prepared on the basis of the sequence of
genomic DNA.
[0083] An "antibiotic" as used herein is a substance produced by a
microorganism which, either naturally or with limited chemical
modification, will inhibit the growth of or kill another
microorganism or eukaryotic cell.
[0084] An "antibiotic biosynthetic gene" is a nucleic acid, e.g.,
DNA, segrnent or sequence that encodes an enzymatic activity which
is necessary for an enzymatic reaction in the process of converting
primary metabolites into antibiotics.
[0085] An "antibiotic biosynthetic pathway" includes the entire set
of antibiotic biosynthetic genes necessary for the process of
converting primary metabolites into antibiotics. These genes can be
isolated by methods well known to the art, e.g., see U.S. Pat. No.
4,935,340.
[0086] Antibiotic-producing organisms include any organism,
including, but not limited to, Actinoplanes, Actinomadura,
Bacillus, Cephalosporium, Micromonospora, Penicilliurn, Nocardia,
and Streptomyces, which either produces an antibiotic or contains
genes which, if expressed, would produce an antibiotic.
[0087] The term "polyketide" as used herein refers to a large and
diverse class of natural products, including but not limited to
antibiotic, antifungal, anticancer, and anti-helminthic
compounds.
[0088] The term "polyketide-associated sugar" refers to a sugar
that is known to attach to polyketides or that can be attached to
polyketides by the processes described herein.
[0089] The term "sugar derivative" refers to a sugar which is
naturally associated with a polyketide but which is altered
relative to the unmodified or native.
[0090] The term "sugar intermediate" refers to an intermediate
compound produced in a sugar biosynthesis pathway.
[0091] A "recombinant" host cell of the invention has a genome that
has been manipulated in vitro so as to alter, e.g., decrease or
disrupt, or, alternatively, increase, the function or activity of
at least one gene, e.g., in the mitomycin biosynthetic gene
cluster, of the invention.
[0092] As used herein, the "mit/mmc" or "mitomycin" gene cluster
includes sequences encoding enzymes for mitosane precursor
formation, mitosane ring assembly, regulation of mitomycin
biosynthesis, functionalization, and resistance to mitomycin, as
well as closely linked sequences encoding polyketide and sugar
biosynthetic enzyes.
[0093] As used herein, the terms "isolated and/or purified" refer
to in vitro isolation of a RNA, DNA or polypeptide molecule from
its natural cellular environment, and from association with other
components of the cell, such as nucleic acid or polypeptide, so
that is can be sequenced, replicated and/or expressed. Moreover,
the nucleic acid may encode more than one polypeptide. For example,
"an isolated DNA molecule encoding an AUBA synthase" is RNA or DNA
containing greater than 7, preferably 15, and more preferably 20 or
more sequential nucleotide bases that preferably encode a
biologically active polypeptide, or a fragment or variant thereof,
that is complementary to the non-coding, or complementary to the
coding strand, of an AHBA synthase RNA, or hybridizes to the RNA or
DNA encoding the AHBA synthase and remains stably bound under low,
moderate, or stringent conditions, as defined by methods well known
to the art, e.g., in Sambrook et al., supra.
[0094] The term "polyketide-producing microorganism" as used herein
includes any microorganism that can produce a polyketide naturally
or after being suitably engineered (i.e., genetically). Examples of
actinomycetes that naturally produce polyketides include but are
not limited to Micromonospora rosaria, Micromonospora megalomicea,
Saccharopolyspora erythraea, Streptomyces antibioticus,
Streptomyces albereticuli, Streptomyces ambofaciens, Streptomyces
avermitilis, Streptomycesfradiae, Streptomyces griseus,
Streptomyces hydroscopicus, Streptomyces tsukulubaensis,
Streptomyces mycarofasciens, Streptomyces platenesis, Streptomyces
violaceoniger, Streptomyces violaceoniger, Streptomyces
thermotolerans, Streptomyces rimosus, Streptomyces peucetius,
Streptomyces coelicolor, Streptomyces glaucescens, Streptomyces
roseofulvus, Streptomyces cinnamonensis, Streptomyces curacoi, and
Amycolatopsis mediterranei (see Hopwood, D. A. and Sherman, D. H.,
Annu. Rev. Genet., 24:37-66 (1990), incorporated herein by
reference). Other examples of polyketide-producing microorganisms
that produce polyketides naturally include various Actinomadura,
Dactylosporangium and Nocardia strains.
[0095] The term "glycosylated polyketide" refers to any polyketide
that contains one or more sugar residues.
[0096] The term "glycosylation-modified polyketide" refers to a
polyketide having a changed glycosylation pattern or configuration
relative to that particular polyketide's unmodified or native
state.
[0097] The term "sugar biosynthesis genes" as used herein refers to
nucleic acid sequences from organisms such as S. lavendulae that
encode sugar biosynthesis enzymes and is intended to include
sequences of DNA from other polyketide-producing microorganisms
which are identical or analogous to those obtained from S.
lavendulae.
[0098] The term "sugar biosynthesis enzymes" as used herein refers
to polypeptides which are involved in the biosynthesis and/or
attachment of polyketide-associated sugars and their derivatives
and intermediates.
[0099] An antibiotic resistance-conferring gene is a nucleic acid
segment that encodes an enzymatic or other activity which alone or
in combination with other gene products, confers resistance to an
antibiotic.
[0100] As used herein, "mitomycin" includes, but is not limited to,
mitomycin A, mitomycin B, mitomycin C, porfiromycin, mitiromycin,
mitomycin D, mitomycin E, mitomycin F, mitomycin G, mitomycin H,
mitomycin I, mitomycin J, mitomycin L, mitomycin M, mitomycin K,
albomitomycin A, isomitomycin A, KW2149, KW2149 metabolites such as
M-16 and M-18, FR66979, FK973, FK317, and FR900482, as well as
structural or functional equivalents thereof ("analogs"), or
derivatives thereof.
[0101] As used herein, the term "derivative" means that a
particular compound produced by a host cell of the invention or
prepared in vitro using polypeptides encoded by the nucleic acid
molecules of the invention, is modified so that it comprises other
moieties, e.g., peptide or polypeptide molecules, such as
antibodies or fragments thereof, nucleic acid molecules, sugars,
lipids, fats, a detectable signal molecule such as a radioisotope,
e.g., gamma emitters, small chemicals, metals, salts, synthetic
polymers, e.g., polylactide and polyglycolide, surfactants and
glycosaminoglycans, which are covalently or non-covalently attached
or linked to the compound.
[0102] It will be appreciated by those skilled in the art that each
atom of the compounds of the invention having a chiral center may
exist in and be isolated in optically active and racemic forms.
Some compounds may exhibit polymorphism. It is to be understood
that the present invention encompasses any racemic, optically
active, polymorphic or stereoisomeric form, or mixtures thereof, of
a compound of the invention, which possess the useful properties
described herein, it being well known in the art how to prepare
optically active forms (for example, by resolution of the racemic
form by recrystallization techniques, by synthesis from optically
active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase) and how
to determine activity using the standard tests described herein, or
using other similar tests which are well known in the art.
[0103] The term "sequence homology" or "sequence identity" means
the proportion of base matches between two nucleic acid sequences
or the proportion amino acid matches between two amino acid
sequences. When sequence homology is expressed as a percentage,
e.g., 50%, the percentage denotes the proportion of matches over
the length of sequence that is compared to some other sequence.
Gaps (in either of the two sequences) are permitted to maximize
matching; gap lengths of 15 bases or less are usually used, 6 bases
or less are preferred with 2 bases or less more preferred. When
using oligonucleotides as probes, the sequence homology between the
target nucleic acid and the oligonucleotide sequence is generally
not less than 17 target base matches out of 20 possible
oligonucleotide base pair matches (85%); preferably not less than 9
matches out of 10 possible base pair matches (90%), and more
preferably not less than 19 matches out of 20 possible base pair
matches (95%).
[0104] Two amino acid sequences are homologous if there is a
partial or complete identity between their sequences. For example,
85% homology means that 85% of the amino acids are identical when
the two sequences are aligned for maximum matching. Gaps (in either
of the two sequences being matched) are allowed in maximizing
matching; gap lengths of 5 or less are preferred with 2 or less
being more preferred. Alternatively and preferably, two protein
sequences (or polypeptide sequences derived from them of at least
30 amino acids in length) are homologous, as this term is used
herein, if they have an alignment score of at more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, 1972, volume 5,
National Biomedical Research Foundation, pp. 101-101, and
Supplement 2 to this volume, pp. 1-10. The two sequences or parts
thereof are more preferably homologous if their amino acids are
greater than or equal to 50% identical when optimally aligned using
the ALIGN program.
[0105] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence", "comparison window", "sequence identity", "percentage of
sequence identity", and "substantial identity". A "reference
sequence" is a defined sequence used as a basis for a sequence
comparison; a reference sequence may be a subset of a larger
sequence, for example, as a segment of a full-length cDNA or gene
sequence given in a sequence listing, or may comprise a complete
cDNA or gene sequence. Generally, a reference sequence is at least
20 nucleotides in length, frequently at least 25 nucleotides in
length, and often at least 50 nucleotides in length. Since two
polynucleotides may each (1) comprise a sequence (i.e., a portion
of the complete polynucleotide sequence) that is similar between
the two polynucleotides, and (2) may further comprise a sequence
that is divergent between the two polynucleotides, sequence
comparisons between two (or more) polynucleotides are typically
performed by comparing sequences of the two polynucleotides over a
"comparison window" to identify and compare local regions of
sequence similarity.
[0106] A "comparison window", as used herein, refers to a
conceptual segment of at least 20 contiguous nucleotides and
wherein the portion of the polynucleotide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
of 20 percent or less as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. Optimal alignment of sequences for aligning a
comparison window may be conducted by the local homology algorithm
of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the
homology alignment algorithm of Needleman and Wunsch (1970) J. Mol.
Biol. 48: 443, by the search for similarity method of Pearson and
Lipman (1988) Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by
computerized implementations of these algorithms (GAP, BESTFIT,
FASTA, and TFASTA in the Wisconsin Genetics Software Package
Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,
Wis.), or by inspection, and the best alignment (i.e., resulting in
the highest percentage of homology over the comparison window)
generated by the various methods is selected.
[0107] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over the window of comparison. The term "percentage of sequence
identity" means that two polynucleotide sequences are identical
(i.e., on a nucleotide-by-nucleotide basis) over the window of
comparison. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs
in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison (i.e., the window size), and
multiplying the result by 100 to yield the percentage of sequence
identity. The terms "substantial identity" as used herein denote a
characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence that has at least 85 percent
sequence identity, preferably at least 90 to 95 percent sequence
identity, more usually at least 99 percent sequence identity as
compared to a reference sequence over a comparison window of at
least 20 nucleotide positions, frequently over a window of at least
20-50 nucleotides, wherein the percentage of sequence identity is
calculated by comparing the reference sequence to the
polynucleotide sequence which may include deletions or additions
which total 20 percent or less of the reference sequence over the
window of comparison.
[0108] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least about 80 percent sequence identity, preferably at least about
90 percent sequence identity, more preferably at least about 95
percent sequence identity, and most preferably at least about 99
percent sequence identity.
[0109] In accordance with the present invention, there is provided
a purified and isolated nucleic acid molecule which encodes the
entire pathway for the biosynthesis of mitomycin, as well as
polyketide biosynthetic and sugar biosynthetic genes that are
linked to the mitomycin biosynthetic genes. Desirably, the nucleic
acid molecule is a DNA isolated from Streptomyces spp. The present
invention further includes isolated and purified DNA sequences
which hybridize under standard or stringent conditions to the the
nucleic acid molecules of the invention. It should be understood to
those skilled in the art that the invention also encompasses the
purified and isolated polypeptides which may be encoded by the
sequences of the nucleic acid molecules of this invention.
[0110] The invention described herein can be used for the
production of mitomycin, analogs or derivatives thereof, or novel
compounds. Commercial chemical syntheses of mitomycin are not
feasible. The gene cluster described herein contains all the genes
required for the production of the mitosane group of antibiotics,
compounds which are clinically prescribed antitumor compounds
employed in the treatment of a wide variety of cancers including
non-small cell lung cancer, metastatic breast cancer, esophageal,
gastric, pancreatic, and anal canal carcinomas. Thus, the isolation
and characterization of this gene cluster allows for the selective
production of mitomycin antibiotics, the overproduction or under
production of particular compounds, e.g., overproduction of certain
mitomycin antibiotics, and the production of novel compounds, e.g.,
mitomycin-derived compounds as well as the production of novel
non-mitomycin related compounds. For example, combinational
biosynthetic-based modification of mitomycin antibiotics may be
accomplished by selective activation or disruption of specific
genes within the cluster or incorporation of the genes into biased
biosynthetic libraries which are assayed for a wide range of
biological activities, to derive greater chemical diversity in the
mitomycins. A further example includes the introduction of a
mitomycin biosynthetic gene(s) into a particular host cell so as to
result in the production of a novel non-mitomycin related compound
due to the activity of the mitomycin biosynthetic gene(s) on other
metabolites, intermediates or components of the host cells. The in
vitro expression of polypeptides from this gene cluster also
provides an enzymatic route to the production of known mitomycin
compounds that are produced in low quantities, or conversion of
currently available mitomycins to other known or novel mitomycins,
e.g., the bioconversion of mitomycin C to porfiromycin.
[0111] The mitomycin resistance genes may also be used to provide
higher mitomycin resistance to cancer patients undergoing treatment
and for clonal selection purposes (e.g., using mrd). For example,
the resistance gene(s) may be inserted into human bone marrow cell
lines to confer higher resistance to non-cancerous cells, thus
allowing higher doses of mitomycins to be administered to cancer
patients. Moreover, because mitomycin acts directly upon DNA
itself, its toxicity is extremely broad, and therefore the
resistance genes could be used for efficient selection in
prokaryotes, fungi, plants, mammalian cell culture, and insect cell
culture. Further, the regulatory resistance and transport genes may
be used to create higher producing strains capable of synthesizing
more mitomycin than can currently be obtained through traditional
fermentation strategies.
[0112] In addition, the invention described herein can be used for
the production of novel compounds which include a diverse range of
biodegradable PHA polymers through genetic redesign of DNA such as
that found in Streptomyces spp. Different PHA synthases can then be
tested for their ability to polymerize the monomers produced by the
recombinant PHA synthase into a biodegradable polymer. PHA
synthases can be tested for their specificity with respect to
different monomer substrates by methods well known to the art.
[0113] The potential uses and applications of PHAs produced by PHA
monomer synthases and PHA synthases include both medical and
industrial applications. Medical applications of PHAs include
surgical pins, sutures, staples, swabs, wound dressings, blood
vessel replacements, bone replacements and plates, stimulation of
bone growth by piezoelectric properties, and biodegradable carrier
for long-term dosage of pharmaceuticals. Industrial applications of
PHAs include disposable items such as baby diapers, packaging
containers, bottles, wrappings, bags, and films, and biodegradable
carriers for long-term dosage of herbicides, fungicides,
insecticides, or fertilizers.
[0114] In animals, the biosynthesis of fatty acids de novo from
malonyl-CoA is catalyzed by FAS. For example, the rat FAS is a
homodimer with a subunit structure consisting of 2505 amino acid
residues having a molecular weight of 272,340 Da. Each subunit
consists of seven catalytic activities in separate physical domains
(Amy et al., Proc. Natl. Acad. Sci. USA, 86, 3114 (1989)). The
physical location of six of the catalytic activities, ketoacyl
synthase (KS), malonyl/acetyltransferase (M/AT), enoyl reductase
(ER), ketoreductase (KR), acyl carrier protein (ACP), and
thioesterase (TE), has been established by (1) the identification
of the various active site residues within the overall amino acid
sequence by isolation of catalytically active fragments from
limited proteolytic digests of the whole FAS, (2) the
identification of regions within the FAS that exhibit sequence
similarity with various monofunctional proteins, (3) expression of
DNA encoding an amino acid sequence with catalytic activity to
produce recombinant proteins, and (4) the identification of DNA
that does not encode catalytic activity, i.e., DNA encoding a
linker region. (Smith et al., Proc. Natl. Acad. Sci. USA, 73, 1184
(1976); Tsukamoto et al., J. Biol. Chem., 263, 16225 (1988); Rangan
et al., J. Biol. Chem., 266, 19180 (1991)).
[0115] The seventh catalytic activity, dehydrase (DH), was
identified as physically residing between AT and ER by an amino
acid comparison of FAS with the amino acid sequences encoded by the
three open reading frames of the eryA polyketide synthase (PKS)
gene cluster of Saccharopolyspora erythraea. The three polypeptides
that comprise this PKS are constructed from "modules" which
resemble animal FAS, both in terms of their amino acid sequence and
in the ordering of the constituent domains (Donadio et al., Gene,
111, 51 (1992); Benh et al., Eur. J. Biochem., 204, 39 (1992)).
[0116] One embodiment of the invention employs a FAS in which the
DH is inactivated (FAS DH-). The FAS DH-employed in this embodiment
of the invention is preferably a eukaryotic FAS DH- and, more
preferably, a mammalian FAS DH-. The most preferred embodiment of
the invention is a FAS where the active site in the DH has been
inactivated by mutation. For example, Joshi et al. (J. Biol. Chem.,
268, 22508 (1993)) changed the His.sup.878 residue in the rat FAS
to an alanine residue by site-directed mutagenesis. In vitro
studies showed that a FAS with this change (ratFAS206) produced
3-hydroxybutyrylCoA as a premature termination product from
acetyl-CoA, malonyl-CoA and NADPH.
[0117] A FAS DH-effectively replaces the .beta.-ketothiolase and
acetoacetyl-CoA reductase activities of the natural pathway by
producing D(-)-3-hydroxybutyrate as a premature termination
product, rather than the usual 16-carbon product, palmitic acid.
This premature termination product can then be incorporated into
PHB by a PHB synthase.
[0118] Another embodiment of the invention employs a recombinant
Streptomyces spp. PKS to produce a variety of .beta.-hydroxyCoA
esters that can serve as monomers for a PHA synthase. One example
of a DNA encoding a Type I PKS is the eryA gene cluster, which
governs the synthesis of erythromycin aglycone deoxyerythronolide B
(DEB). The gene cluster encodes six repeated units, termed modules
or synthase units (SUs). Each module or SU, which comprises a
series of putative FAS-like activities, is responsible for one of
the six elongation cycles required for DEB formation. Thus, the
processive synthesis of asymmetric acyl chains found in complex
polyketides is accomplished through the use of a programmed protein
template, where the nature of the chemical reactions occurring at
each point is determined by the specificities in each SU.
[0119] Two other Type I PKS are encoded by the tyl (tylosin) and
met (methymycin) gene clusters (see U.S. application Ser. No.
09/108,537, the disclosure of which is incorporated by reference
herein). The macrolide multifunctional synthases encoded by tyl and
met provide a greater degree of metabolic diversity than that found
in the eryA gene cluster. The PKSs encoded by the eryA gene cluster
only catalyze chain elongation with methylmalonylCoA, as opposed to
tyl and met PKSs, which catalyze chain elongation with malonylCoA,
methylmalonylCoA and ethylmalonylCoA. Specifically, the tyl PKS
includes two malonylCoA extender units and one ethylmalonylCoA
extender unit, and the met PKS includes one malonylCoA extender
unit.
[0120] In order to manipulate the catalytic specificities within
each module, DNA encoding a catalytic activity must remain
undisturbed. To identify the amino acid sequences between the amino
acid sequences with catalytic activity, the "linker regions," amino
acid sequences of related modules, preferably those encoded by more
than one gene cluster, are compared. Linker regions are amino acid
sequences which are less well conserved than amino acid sequences
with catalytic activity. Witkowski et al., Eur. J. Biochem., 198,
571 (1991).
[0121] In an alternative embodiment of the invention, to provide a
DNA encoding a Type I PKS module with a TE and lacking a functional
DH, a DNA encoding a module F, containing KS, MT, KR, ACP, and TE
catalytic activities, is introduced at the 3' end of a DNA encoding
a first module. Module F introduces the final (R)-3-hydroxyl acyl
group at the final step of PHA monomer synthesis, as a result of
the presence of a TE domain. DNA encoding a module F is not present
in the eryA PKS gene cluster (Donadio et al., supra, 1991).
[0122] A DNA encoding a recombinant monomer synthase is inserted
into an expression vector. The expression vector employed varies
depending on the host cell to be transformed with the expression
vector. That is, vectors are employed with transcription,
translation and/or post-translational signals, such as targeting
signals, necessary for efficient expression of the genes in various
host cells into which the vectors are introduced. Such vectors are
constructed and transformed into host cells by methods well known
in the art. See Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor (1989). Preferred host cells for the
vectors of the invention include insect, bacterial, and plant
cells. Preferred insect cells include Spodoptera frugiperda cells
such as Sf21, and Trichoplusia ni cells. Preferred bacterial cells
include Escherichia coli, Streptomyces and Pseudomonas. Preferred
plant cells include monocot and dicot cells, such as maize, rice,
wheat, tobacco, legumes, carrot, squash, canola, soybean, potato,
and the like.
[0123] Moreover, the appropriate subcellular compartment in which
to locate the enzyme in eukaryotic cells must be considered when
constructing eukaryotic expression vectors. Two factors are
important: the site of production of the acetyl-CoA substrate, and
the available space for storage of the PHA polymer. To direct the
enzyme to a particular subcellular location, targeting sequences
may be added to the sequences encoding the recombinant
molecules.
[0124] The baculovirus system is particularly amenable to the
introduction of DNA encoding a recombinant FAS or a PKS monomer
synthase because an increasing variety of transfer plasmids are
becoming available which can accommodate a large insert, and the
virus can be propagated to high titers. Moreover, insect cells are
adapted readily to suspension culture, facilitating relatively
large-scale recombinant protein production. Further, recombinant
proteins tend to be produced exclusively as soluble proteins in
insect cells, thus, obviating the need for refolding, a task that
might be particularly daunting in the case of a large
multifunctional protein. The Sf21 /baculovirus system has routinely
expressed milligram quantities of catalytically active recombinant
fatty acid synthase. Finally, the baculovirus/insect cell system
provides the ability to construct and analyze different synthase
proteins for the ability to polymerize monomers into unique
biodegradable polymers.
[0125] A further embodiment of the invention is the introduction of
at least one DNA encoding a PHA synthase and a DNA encoding a PHA
monomer synthase into a host cell. Such synthases include, but are
not limited to, A. eutrophus 3-hydroxy, 4-hydroxy, and 5-hydroxy
alkanoate synthases, Rhodococcus ruber C.sub.3-C.sub.5
hydroxyalkanoate synthases, Pseudomonas oleororans C.sub.6-C.sub.14
hydroxyalkanoate synthases, P. putida C.sub.6-C.sub.14
hydroxyalkanoate synthases, P. aeruginosa C.sub.5-C.sub.10
hydroxyalkanoate synthases, P. resinovorans C.sub.4-C.sub.10
hydroxyalkanoate synthases, Rhodospirillum rubrum C.sub.4-C.sub.7
hydroxyalkanoate syntheses, R. gelatinorus C.sub.4-C.sub.7,
Thiocapsa pfennigii C.sub.4-C.sub.8 hydroxyalkanoate synthases, and
Bacillus megaterium C.sub.4-C.sub.5 hydroxyalkanoate synthases.
[0126] The introduction of DNA(s) encoding more than one PHA
synthase may be necessary to produce a particular PHA polymer due
to the specificities exhibited by different PHA synthases. As
multifunctional proteins are altered to produce unusual monomeric
structures, synthase specificity may be problematic for particular
substrates. Although the A. eutrophus PHB synthase utilizes only C4
and C5 compounds as substrates, it appears to be a good prototype
synthase for initial studies since it is known to be capable of
producing copolymers of 3-hydroxybutyrate and 4-hydroxybutyrate
(Kunioka et al., Macromolecules, 22, 694 (1989)) as well as
copolymers of 3-hydroxyvalerate, 3-hydroxybutyrate, and
5-hydroxyvalerate (Doi et al., Macromolecules, 19, 2860 (1986)).
Other synthases, especially those of Pseudomonas aeruginosa (Timm
et al., Eur. J. Biochem., 209, 15 (1992)) and Rhodococcus ruber
(Pieper et al., FEMS Microbiol. Lett., 96, 73 (1992)), can also be
employed in the practice of the invention. Synthase specificity may
be alterable through molecular biological methods.
[0127] In yet another embodiment of the invention, a DNA encoding a
FAS and a PHA synthase can be introduced into a single expression
vector, obviating the need to introduce the genes into a host cell
individually.
[0128] A further embodiment of the invention is the generation of a
DNA encoding a recombinant multifunctional protein, which comprises
a FAS, of either eukaryotic or prokaryotic origin, and a PKS module
F. Module F will carry out the final chain extension to include two
additional carbons and the reduction of the .beta.-keto group,
which results in a (R)-3-hydroxy acyl CoA moiety.
[0129] To produce this recombinant protein, DNA encoding the FAS TE
is replaced with a DNA encoding a linker region which is normally
found in the ACP-KS interdomain region of bimodular ORFs. DNA
encoding a module F is then inserted 3' to the DNA encoding the
linker region. Different linker regions, such as those described
below which vary in length and amino acid composition, can be
tested to determine which linker most efficiently mediates or
allows the required transfer of the nascent saturated fatty acid
intermediate to module F for the final chain elongation and keto
reduction steps. The resulting DNA encoding the protein can then be
tested for expression of long-chain .beta.-hydroxy fatty acids in
insect cells, such as Sf21 cells, or Streptomyces, or Pseudomonas.
The expected 3-hydroxy C-18 fatty acid can serve as a potential
substrate for PHA synthases which are able to accept long-chain
alkyl groups. A preferred embodiment of the invention is a FAS that
has a chain length specificity between 4-22 carbons.
[0130] Examples of linker regions that can be employed in this
embodiment of the invention include, but are not limited to, the
ACP-KS linker regions encoded by the tyl ORFI (ACP.sub.1-KS.sub.2;
ACP.sub.2-KS.sub.3), and ORF3 (ACP.sub.5-KS.sub.6), and eryA ORFI
(ACP.sub.1-KS.sub.1; ACP.sub.2-KS.sub.2), ORF2 (ACP.sub.3-KS.sub.4)
and ORF3 (ACP.sub.5-KS.sub.6).
[0131] This approach can also be used to produce shorter chain
fatty acid groups by limiting the ability of the FAS unit to
generate long-chain fatty acids. Mutagenesis of DNA encoding
various FAS catalytic activities, starting with the KS, may result
in the synthesis of short-chain (R)-3-hydroxy fatty acids.
[0132] The PHA polymers are then recovered from the biomass.
Large-scale solvent extraction can be used, but is expensive. An
alternative method involving heat shock with subsequent enzymatic
and detergent digestive processes is also available (Byron, Trends
Biotechnical, 5, 246 (1987); Holmes, In: Developments in
Crystalline Polymers, D. C. Bassett (ed.), pp. 1-65 (1988)). PHB
and other PHAs are readily extracted from microorganisms by
chlorinated hydrocarbons. Refluxing with chloroform has been
extensively used; the resulting solution is filtered to remove
debris and concentrated, and the polymer is precipitated with
methanol or ethanol, leaving low-molecular-weight lipids in
solution. Longer side-chain PHAs show a less restricted solubility
than PHB and are, for example, soluble in acetone. Other strategies
adopted include the use of ethylene carbonate and propylene
carbonate as disclosed by Lafferty et al. (Chem. Rundschau, 30, 14
(1977)) to extract PHB from biomass. Scandola et al. (Int. J. Biol.
Microbiol., 10, 373 (1988)) reported that 1 M HCl-chloroform
extraction of Rhizobium meliloti yielded PHB of
M.sub.w=6.times.10.sup.4 compared with 1.4.times.10.sup.6 when
acetone was used.
[0133] Methods are well known in the art for the determination of
the PHB or PHA content of microorganisms, the composition of PHAs,
and the distribution of the monomer units in the polymer. Gas
chromatography and high-pressure liquid chromatography are widely
used for quantitative PHB analysis. See Anderson et al., Microbiol.
Rev., 54, 450 (1990) for a review of such methods. NMR techniques
can also be used to determine polymer composition, and the
distribution of monomer units.
[0134] Variants of the Nucleic Acid Molecules of the Invention
[0135] The present invention contemplates nucleic acid sequences
which hybridize under low, medium or high stringency hybridization
conditions to the exemplified nucleic acid sequences set forth
herein. Hybridization conditions are well known in the art. Thus,
nucleic acid sequences encoding variant polypeptides, i.e., those
having at least one amino acid substitution, insertion, addition or
deletion, or nucleic acid sequences having conservative (e.g.,
silent) nucleotide substitutions (see FIGS. 24-25), are within the
scope of the invention. Preferably, variant polypeptides encoded by
the nucleic acid sequences of the invention are biologically
active. The present invention also contemplates naturally occurring
allelic variations and mutations of the nucleic acid sequences
described herein.
[0136] As is well known in the art, because of the degeneracy of
the genetic code, there are numerous other DNA and RNA molecules
that can code for the same polypeptides as those encoded by the
exemplified biosynthetic genes and fragments thereof. The present
invention, therefore, contemplates those other DNA and RNA
molecules which, on expression, encode the polypeptides of, for
example, portions of SEQ ID NO:96. Having identified the amino acid
residue sequence encoded by a mitomycin, sugar or polyketide
biosynthetic gene, and with knowledge of all triplet codons for
each particular amino acid residue, it is possible to describe all
such encoding RNA and DNA sequences. DNA and RNA molecules other
than those specifically disclosed herein and, which molecules are
characterized simply by a change in a codon for a particular amino
acid, are within the scope of this invention.
[0137] The 20 common amino acids and their representative
abbreviations, symbols and codons are well known in the art (see,
for example, Molecular Biology of the Cell, Second Edition, B.
Alberts et al., Garland Publishing Inc., New York and London,
1989). As is also well known in the art, codons constitute triplet
sequences of nucleotides in mRNA molecules and as such, are
characterized by the base uracil (U) in place of base thymidine (T)
which is present in DNA molecules. A simple change in a codon for
the same amino acid residue within a polynucleotide will not change
the structure of the encoded polypeptide. By way of example, it can
be seen from SEQ ID NO:16 that a TCA codon for serine exists at
nucleotide positions 146-148. However, serine can be encoded by a
TCT codon, and a TCC codon. Substitution of the latter codons for
serine with the TCA codon for serine or vice versa, does not
substantially alter the DNA sequence of SEQ ID NO:16 and results in
production of the same polypeptide. In a similar manner,
substitutions of the recited codons with other equivalent codons
can be made in a like manner without departing from the scope of
the present invention.
[0138] A nucleic acid molecule, segment or sequence of the present
invention can also be an RNA molecule, segment or sequence. An RNA
molecule contemplated by the present invention corresponds to, is
complementary to or hybridizes under low, medium or high stringency
conditions to, any of the DNA sequences set forth herein. Exemplary
and preferred RNA molecules are mRNA molecules that comprise at
least one mitomycin, sugar or polyketide biosynthetic gene of this
invention.
[0139] Mutations can be made to the native nucleic acid sequences
of the invention and such mutants used in place of the native
sequence, so long as the mutants are able to function with other
sequences to collectively catalyze the synthesis of an identifiable
sugar, polyketide or mitomycin. Such mutations can be made to the
native sequences using conventional techniques such as by preparing
synthetic oligonucleotides including the mutations and inserting
the mutated sequence into the gene using restriction endonuclease
digestion. (See, e.g., Kunkel, T. A. Proc. Natl. Acad. Sci. USA
(1985) 82:448; Geisselsoder et al. BioTechniques (1987) 5:786.)
Alternatively, the mutations can be effected using a mismatched
primer (generally 10-30 nucleotides in length) which hybridizes to
the native nucleotide sequence (generally cDNA corresponding to the
RNA sequence), at a temperature below the melting temperature of
the mismatched duplex. The primer can be made specific by keeping
primer length and base composition within relatively narrow limits
and by keeping the mutant base centrally located. Zoller and Smith,
Methods Enzymol., (1983) 100:468. Primer extension is effected
using DNA polymerase, the product cloned and clones containing the
mutated DNA, derived by segregation of the primer extended strand,
selected. Selection can be accomplished using the mutant primer as
a hybridization probe. The technique is also applicable for
generating multiple point mutations. See, e.g., Dalbie-McFarland et
al., Proc. Natl. Acad. Sci. USA (1982) 79:6409. PCR mutagenesis
will also find use for effecting the desired mutations.
[0140] Random mutagenesis of the nucleotide sequence can be
accomplished by several different techniques known in the art, such
as by altering sequences within restriction endonuclease sites,
inserting an oligonucleotide linker randomly into a plasmid, by
irradiation with X-rays or ultraviolet light, by incorporating
incorrect nucleotides during in vitro DNA synthesis, by error-prone
PCR mutagenesis, by preparing synthetic mutants or by damaging
plasmid DNA in vitro with chemicals. Chemical mutagens include, for
example, sodium bisulfite, nitrous acid, hydroxylamine, agents
which damage or remove bases thereby preventing normal base-pairing
such as hydrazine or formnic acid, analogues of nucleotide
precursors such as nitrosoguanidine, 5-bromouracil, 2-aminopurine,
or acridine intercalating agents such as proflavine, acriflavine,
quinacrine, and the like. Generally, plasmid DNA or DNA fragments
are treated with chemicals, transformed into E. coli and propagated
as a pool or library of mutant plasmids.
[0141] Large populations of random enzyme variants can be
constructed in vivo using "recombination-enhanced mutagenesis."
This method employs two or more pools of, for example, 10.sup.6
mutants each of the wild-type encoding nucleotide sequence that are
generated using any convenient mutagenesis technique and then
inserted into cloning vectors.
[0142] Chimeric Expression Cassettes, Vectors and Host Cells of the
Invention
[0143] As used herein, "chimeric" means that a vector comprises DNA
from at least two different species, or comprises DNA from the same
species, which is linked or associated in a manner which does not
occur in the "native" or wild type of the species. The recombinant
DNA sequence or segment, used for transformation herein, may be
circular or linear, double-stranded or single-stranded. Generally,
the DNA sequence or segment is in the form of chimeric DNA, such as
plasmid DNA, that can also contain coding regions flanked by
control sequences which promote the expression of the DNA present
in the resultant transformed (recombinant) host cell. Aside from
DNA sequences that serve as transcription units for the nucleic
acid molecules of the invention or portions thereof, a portion of
the DNA may be untranscribed, serving a regulatory or a structural
function. For example, the preselected DNA may itself comprise a
promoter that is active in a particular host cell.
[0144] Other elements functional in the host cells, such as
introns, enhancers, polyadenylation sequences and the like, may
also be a part of the DNA. Such elements may or may not be
necessary for the function of the DNA, but may provide improved
expression of the DNA by affecting transcription, stability of the
mRNA, or the like. Such elements may be included in the DNA as
desired to obtain the optimal performance of the transforming DNA
in the cell.
[0145] "Control sequences" is defined to mean DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotic cells, for example, include a promoter,
and optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers. Other regulatory sequences may also be
desirable which allow for regulation of expression of the genes
relative to the growth of the host cell. Regulatory sequences are
known to those of skill in the art, and examples include those
which cause the expression of a gene to be turned on or off in
response to a chemical or physical stimulus, including the presence
of a regulatory compound. Other types of regulatory elements may
also be present in the vector, for example, enhancer sequences.
[0146] "Operably linked" is defined to mean that the nucleic acids
are placed in a functional relationship with another nucleic acid
sequence. For example, DNA for a presequence or secretory leader is
operably linked to DNA for a polypeptide if it is expressed as a
preprotein that participates in the secretion of the polypeptide; a
promoter or enhancer is operably linked to a coding sequence if it
affects the transcription of the sequence; or a ribosome binding
site is operably linked to a coding sequence if it is positioned so
as to facilitate translation. Generally, "operably linked" means
that the DNA sequences being linked are contiguous and, in the case
of a secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accord with conventional practice.
[0147] The DNA to be introduced into the cells further will
generally contain either a selectable marker gene or a reporter
gene or both to facilitate identification and selection of
transformed cells from the population of cells sought to be
transformed. Alternatively, the selectable marker may be carried on
a separate piece of DNA and used in a co-transformation procedure.
Both selectable markers and reporter genes may be flanked with
appropriate regulatory sequences to enable expression in the host
cells. Useful selectable markers are well known in the art and
include, for example, antibiotic and herbicide-resistance genes,
such as neo, hpt, dhfr, bar, aroA, dapA and the like. See also, the
genes listed on Table 1 of Lundquist et al. (U.S. Pat. No.
5,848,956).
[0148] Reporter genes are used for identifying potentially
transformed cells and for evaluating the functionality of
regulatory sequences. Reporter genes which encode for easily
assayable proteins are well known in the art. In general, a
reporter gene is a gene which is not present in or expressed by the
recipient organism or tissue and which encodes a protein whose
expression is manifested by some easily detectable property, e.g.,
enzymatic activity. Expression of the reporter gene is assayed at a
suitable time after the DNA has been introduced into the recipient
cells.
[0149] Prokaryotic expression systems are preferred, and in
particular, systems compatible with Streptomyces spp. are of
particular interest. Control elements for use in such systems
include promoters, optionally containing operator sequences, and
ribosome binding sites. Particularly useful promoters include
control sequences derived from the gene clusters of the invention.
However, other bacterial promoters, such as those derived from
sugar metabolizing enzymes, such as galactose, lactose (lac) and
maltose, will also find use in the expression cassettes encoding
desosamine. Preferred promoters are Streptomyces promoters,
including but not limited to the ermE*,pikA and tipA promoters.
Additional examples include promoter sequences derived from
biosynthetic enzymes such as tryptophan (trp), the .beta.-lactamase
(bla) promoter system, bacteriophage lambda PL, and T5. In
addition, synthetic promoters, such as the tac promoter (U.S. Pat.
No. 4,551,433), which do not occur in nature, also function in
bacterial host cells.
[0150] The various nucleic acid molecules of interest can be cloned
into one or more recombinant vectors as individual cassettes, with
separate control elements, or under the control of, e.g., a single
promoter. The nucleic acid molecules can include flanking
restriction sites to allow for the easy deletion and insertion of
other sequences. The design of such unique restriction sites is
known to those of skill in the art and can be accomplished using
the techniques, such as site-directed mutagenesis and PCR.
[0151] For sequences generated by random mutagenesis, the choice of
vector depends on the pool of mutant sequences, i.e., donor or
recipient, with which they are to be employed. Furthermore, the
choice of vector determines the host cell to be employed in
subsequent steps of the claimed method. Any transducible cloning
vector can be used as a cloning vector for the donor pool of
mutants. It is preferred, however, that phagemids, cosmids, or
similar cloning vectors be used for cloning the donor pool of
mutant encoding nucleotide sequences into the host cell. Phagemids
and cosmids, for example, are advantageous vectors due to the
ability to insert and stably propagate therein larger fragments of
DNA than in M13 phage and .lambda. phage, respectively. Phagemids
which will find use in this method generally include hybrids
between plasmids and filamentous phage cloning vehicles. Cosmids
which will find use in this method generally include .lambda.
phage-based vectors into which cos sites have been inserted.
Recipient pool cloning vectors can be any suitable plasmid. The
cloning vectors into which pools of mutants are inserted may be
identical or may be constructed to harbor and express different
genetic markers (see, e.g., Sambrook et al., supra). The utility of
employing such vectors having different marker genes may be
exploited to facilitate a determination of successful
transduction.
[0152] Thus, for example, the cloning vector employed may be an E.
coli/Streptomyces shuttle vector (see, for example, U.S. Pat. Nos.
4,416,994, 4,343,906, 4,477,571, 4,362,816, and 4,340,674), a
cosmid, a plasmid, an artificial bacterial chromosome (see, e.g.,
Zhang and Wing, Plant Mol. Biol., 35, 115 (1997); Schalkwyk et al.,
Curr. Op. Biotech., 6, 37 91995); and Monaco and Lavin, Trends in
Biotech., 12, 280 (1994), or a phagemid, and the host cell may be a
bacterial cell such as E. coli, Penicillium patulum, and
Streptomyces spp. such as S. lividans, S. venezuelae, or S.
lavendulae, or a eukaryotic cell such as fungi, yeast or a plant
cell, e.g., monocot and dicot cells, preferably cells that are
regenerable.
[0153] The general methods for constructing recombinant DNA which
can transform target cells are well known to those skilled in the
art, and the same compositions and methods of construction may be
utilized to produce the DNA useful herein. For example, J. Sambrook
et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press (2d ed., 1989), provides suitable methods of
construction.
[0154] The recombinant DNA can be readily introduced into the host
cells by any procedure useful for the introduction into a
particular cell, e.g., calcium phosphate precipitation, protoplast
fusion, conjugation, lipofection, electroporation, and the
like.
[0155] As used herein, the term "cell line" or "host cell" is
intended to refer to well-characterized homogenous, biologically
pure populations of cells. These cells may be eukaryotic cells that
are neoplastic or which have been "immortalized" in vitro by
methods known in the art, as well as primary cells, or prokaryotic
cells. In particular, the cell line or host cell may be of
mammalian, plant, insect, yeast, fungal or bacterial origin.
[0156] "Transfected" or "transformed" is used herein to include any
host cell or cell line, the genome of which has been altered or
augmented by the presence of at least one DNA sequence, which DNA
is also referred to in the art of genetic engineering as
"heterologous DNA," "recombinant DNA," "exogenous DNA,"
"genetically engineered," "non-native," or "foreign DNA," wherein
said DNA was isolated and introduced into the genome of the host
cell or cell line by the process of genetic engineering. The
transfected DNA may be maintained as an extrachromosomal element or
as an element which is stably integrated into the host
chromosome.
[0157] Moreover, recombinant polypeptides having a particular
activity may be prepared via "gene-shuffling". See, for example,
Crameri et al., Nature, 391, 288 (1998); Patten et al., Curr. Op.
Biotech., 8, 724 (1997), U.S. Pat. Nos. 5,837,458, 5,834,252,
5,830,727, 5,811,238, 5,605,793).
[0158] For phagemids, upon infection of the host cell which
contains a phagemid, single-stranded phagemid DNA is produced,
packaged and extruded from the cell in the form of a transducing
phage in a manner similar to other phage vectors. Thus, clonal
amplification of mutant encoding nucleotide sequences carried by
phagemids is accomplished by propagating the phagemids in a
suitable host cell.
[0159] Following clonal amplification, the cloned donor pool of
mutants is infected with a helper phage to obtain a mixture of
phage particles containing either the helper phage genome or
phagemids mutant alleles of the wild-type encoding nucleotide
sequence.
[0160] Infection, or transfection, of host cells with helper phage
is generally accomplished by methods well known in the art (see.,
e.g., Sambrook et al., supra; and Russell et al. (1986) Gene
45:333-338).
[0161] The helper phage may be any phage which can be used in
combination with the cloning phage to produce an infective
transducing phage. For example, if the cloning vector is a cosmid,
the helper phage will necessarily be a .lambda. phage. Preferably,
the cloning vector is a phagemid and the helper phage is a
filamentous phage, and preferably phage M13.
[0162] If desired after infecting the phagemid with helper phage
and obtaining a mixture of phage particles, the transducing phage
can be separated from helper phage based on size difference (Barnes
et al. (1983) Methods Enzymol. 101:98-122), or other similarly
effective technique.
[0163] The entire spectrum of cloned donor mutations can now be
transduced into clonally amplified recipient cells into which has
been transduced or transformed a pool of mutant encoding nucleotide
sequences. Recipient cells which may be employed in the method
disclosed and claimed herein may be, for example, E. coli, or other
bacterial expression systems which are not recombination deficient.
A recombination deficient cell is a cell in which recombinatorial
events is greatly reduced, such as recv mutants of E. coli (see,
Clark et al. (1965) Proc. Natl. Acad. Sci. USA 53:451-459).
[0164] These transductants can now be selected for the desired
expressed protein property or characteristic and, if necessary or
desirable, amplified. Optionally, if the phagemids into which each
pool of mutants is cloned are constructed to express different
genetic markers, as described above, transductants may be selected
by way of their expression of both donor and recipient plasmid
markers.
[0165] The recombinants generated by the above-described methods
can then be subjected to selection or screening by any appropriate
method, for example, enzymatic or other biological activity.
[0166] The above cycle of amplification, infection, transduction,
and recombination may be repeated any number of times using
additional donor pools cloned on phagemids. As above, the phagemids
into which each pool of mutants is cloned may be constructed to
express a different marker gene. Each cycle could increase the
number of distinct mutants by up to a factor of 10.sup.6. Thus, if
the probability of occurrence of an inter-allelic recombination
event in any individual cell is f (a parameter that is actually a
function of the distance between the recombining mutations), the
transduced culture from two pools of 10.sup.6 allelic mutants will
express up to 10.sup.12 distinct mutants in a population of
10.sup.12/f cells.
[0167] Preparation, Isolation and Modification of the Polypeptides
of the Invention
[0168] The present isolated, purified polypeptides, variants or
fragments thereof, can be synthesized in vitro, e.g., by the solid
phase peptide synthetic method or by recombinant DNA approaches
(see above). The solid phase peptide synthetic method is an
established and widely used method, which is described in the
following references: Stewart et al., Solid Phase Peptide
Synthesis, W. H. Freeman Co., San Francisco (1969); Merrifield, J.
Am. Chem. Soc., 85 2149 (1963); Meienhofer in "Hormonal Proteins
and Peptides," ed.; C. H. Li, Vol. 2 (Academic Press, 1973), pp.
48-267; Bavaay and Merrifield, "The Peptides," eds. E. Gross and F.
Meienhofer, Vol. 2 (Academic Press, 1980) pp. 3-285; and
Clark-Lewis et al., Meth. Enzymol., 287, 233 (1997). These
polypeptides can be further purified by fractionation on
immunoaffinity or ion-exchange columns; ethanol precipitation;
reverse phase HPLC; chromatography on silica or on an
anion-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; or ligand affinity chromatography.
[0169] In particular, fusion polypeptides are prepared which
comprise an amino acid sequence useful in purification, e.g., a His
tag is useful to purify fusion polypeptides on nickel columns. Once
isolated and characterized, derivatives, e.g., chemically derived
derivatives, of a given polypeptide can be readily prepared. For
example, amides of the polypeptides of the present invention may
also be prepared by techniques well known in the art for converting
a carboxylic acid group or precursor, to an amide. A preferred
method for amide formation at the C-terminal carboxyl group is to
cleave the polypeptide from a solid support with an appropriate
amine, or to cleave in the presence of an alcohol, yielding an
ester, followed by aminolysis with the desired amine.
[0170] Salts of carboxyl groups of a polypeptide or polypeptide
variant of the invention may be prepared in the usual manner by
contacting the polypeptide with one or more equivalents of a
desired base such as, for example, a metallic hydroxide base, e.g.,
sodium hydroxide; a metal carbonate or bicarbonate base such as,
for example, sodium carbonate or sodium bicarbonate; or an amine
base such as, for example, triethylamine, triethanolamine, and the
like.
[0171] N-acyl derivatives of an amino group of the polypeptide or
polypeptide variants may be prepared by utilizing an N-acyl
protected amino acid for the final condensation, or by acylating a
protected or unprotected polypeptide. O-acyl derivatives may be
prepared, for example, by acylation of a free hydroxy peptide or
peptide resin. Either acylation may be carried out using standard
acylating reagents such as acyl halides, anhydrides, acyl
imidazoles, and the like. Both N- and O-acylation may be carried
out together, if desired.
[0172] One or more of the residues of the polypeptide can be
altered, so long as the polypeptide variant is biologically active.
For example, it is preferred that the variant has at least about 1%
of the biological activity of the corresponding non-variant
polypeptide, e.g. Conservative amino acid substitutions are
preferred--that is, for example, aspartic-glutamic as acidic amino
acids; lysine/arginine/histidine as basic amino acids;
leucine/isoleucine, methionine/valine, alanine/valine as
hydrophobic amino acids; serine/glycine/alanine/threonine as
hydrophilic amino acids. Conservative amino acid substitution also
includes groupings based on side chains. For example, a group of
amino acids having aliphatic side chains is glycine, alanine,
valine, leucine, and isoleucine; a group of amino acids having
aliphatic-hydroxyl side chains is serine and threonine; a group of
amino acids having amide-containing side chains is asparagine and
glutamine; a group of amino acids having aromatic side chains is
phenylalanine, tyrosine, and tryptophan; a group of amino acids
having basic side chains is lysine, arginine, and histidine; and a
group of amino acids having sulfur-containing side chains is
cysteine and methionine. For example, it is reasonable to expect
that replacement of a leucine with an isoleucine or valine, an
aspartate with a glutamate, a threonine with a serine, or a similar
replacement of an amino acid with a structurally related amino acid
will not have a major effect on the properties of the resulting
variant polypeptide. Whether an amino acid change results in a
functional polypeptide can readily be determined by assaying the
specific activity of the polypeptide variant.
[0173] Conservative substitutions are shown in FIG. 25 under the
heading of exemplary substitutions. More preferred substitutions
are under the heading of preferred substitutions. After the
substitutions are introduced, the variants are screened for
biological activity.
[0174] Amino acid substitutions falling within the scope of the
invention, are, in general, accomplished by selecting substitutions
that do not differ significantly in their effect on maintaining (a)
the structure of the peptide backbone in the area of the
substitution, (b) the charge or hydrophobicity of the molecule at
the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common
side-chain properties:
[0175] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0176] (2) neutral hydrophilic: cys, ser, thr;
[0177] (3) acidic: asp, glu;
[0178] (4) basic: asn, gln, his, lys, arg;
[0179] (5) residues that influence chain orientation: gly, pro;
and
[0180] (6) aromatic; trp, tyr, phe.
[0181] The invention also envisions polypeptide variants with
non-conservative substitutions. Non-conservative substitutions
entail exchanging a member of one of the classes described above
for another.
[0182] Acid addition salts of the polypeptide or variant
polypeptide or of amino residues of the polypeptide or variant
polypeptide may be prepared by contacting the polypeptide or amine
with one or more equivalents of the desired inorganic or organic
acid, such as, for example, hydrochloric acid. Esters of carboxyl
groups of the polypeptides may also be prepared by any of the usual
methods known in the art.
[0183] Antibodies of the Invention
[0184] The antibodies of the invention are prepared by using
standard techniques. To prepare polyclonal antibodies or
"antisera," an animal is inoculated with an antigen that is an
isolated and purified polypeptide of the invention, and
immunoglobulins are recovered from a fluid, such as blood serum,
that contains the immunoglobulins, after the animal has had an
immune response. For inoculation, the antigen is preferably bound
to a carrier peptide and emulsified using a biologically suitable
emulsifying agent, such as Freund's incomplete adjuvant. A variety
of mammalian or avian host organisms may be used to prepare
polyclonal antibodies
[0185] Following immunization, Ig is purified from the immunized
bird or mammal, e.g., goat, rabbit, mouse, rat, or donkey and the
like. For certain applications, it is preferable to obtain a
composition in which the antibodies are essentially free of
antibodies that do not react with the immunogen. This composition
is composed virtually entirely of the high titer, monospecific,
purified polyclonal antibodies to the antigen. Antibodies can be
purified by affinity chromatography. Purification of antibodies by
affinity chromatography is generally known to those skilled in the
art (see, for example, U.S. Pat. No. 4,533,630). Briefly, the
purified antibody is contacted with the purified polypeptide, or a
peptide thereof, bound to a solid support for a sufficient time and
under appropriate conditions for the antibody to bind to the
polypeptide or peptide. Such time and conditions are readily
determinable by those skilled in the art. The unbound, unreacted
antibody is then removed, such as by washing. The bound antibody is
then recovered from the column by eluting the antibodies, so as to
yield purified, monospecific polyclonal antibodies.
[0186] Monoclonal antibodies can be also prepared, using known
hybridoma cell culture techniques. In general, this method involves
preparing an antibody-producing fused cell line, e.g., of primary
spleen cells fused with a compatible continuous line of myeloma
cells, and growing the fused cells either in mass culture or in an
animal species, such as a murine species, from which the myeloma
cell line used was derived or is compatible. Such antibodies offer
many advantages in comparison to those produced by inoculation of
animals, as they are highly specific and sensitive and relatively
"pure" immunochemically. Inmunologically active fragments of the
present antibodies are also within the scope of the present
invention, e.g., the F(ab) fragment, scFv antibodies, as are
partially humanized monoclonal antibodies.
[0187] Thus, it will be understood by those skilled in the art that
the hybridomas herein referred to may be subject to genetic
mutation or other changes while still retaining the ability to
produce monoclonal antibody of the same desired specificity. The
present invention encompasses mutants, other derivatives and
descendants of the hybridomas.
[0188] It will be further understood by those skilled in the art
that a monoclonal antibody may be subjected to the techniques of
recombinant DNA technology to produce other derivative antibodies,
humanized or chimeric molecules or antibody fragments which retain
the specificity of the original monoclonal antibody. Such
techniques may involve combining DNA encoding the immunoglobulin
variable region, or the complementarity determining regions (CDRs),
of the monoclonal antibody with DNA coding the constant regions, or
constant regions plus framework regions, of a different
immunoglobulin, for example, to convert a mouse-derived monoclonal
antibody into one having largely human immunoglobulin
characteristics (see EP 184187A, 2188638A, herein incorporated by
reference).
[0189] The antibodies of the invention are useful for detecting or
determining the presence or amount of a polypeptide of the
invention in a sample. The antibodies are contacted with the sample
for a period of time and under conditions sufficient for antibodies
to bind to the polypeptide so as to form a binary complex between
at least a portion of said antibodies and said polypeptide. Such
times, conditions and reaction media can be readily determined by
persons skilled in the art.
[0190] For example, the cells are lysed to yield an extract which
comprises cellular proteins. Alternatively, intact cells are
permeabilized in a manner which permits macromolecules, i.e.,
antibodies, to enter the cell. The antibodies of the invention are
then incubated with the protein extract, e.g., in a Western blot,
or permeabilized cells, e.g., prior to flow cytometry, so as to
form a complex. The presence or amount of the complex is then
determined or detected.
[0191] The antibodies of the invention may also be coupled to an
insoluble or soluble substrate. Soluble substrates include proteins
such as bovine serum albumin. Preferably, the antibodies are bound
to an insoluble substrate, i.e., a solid support. The antibodies
are bound to the support in an amount and manner that allows the
antibodies to bind the polypeptide (ligand). The amount of the
antibodies used relative to a given substrate depends upon the
particular antibody being used, the particular substrate, and the
binding efficiency of the antibody to the ligand. The antibodies
may be bound to the substrate in any suitable manner. Covalent,
noncovalent, or ionic binding may be used. Covalent bonding can be
accomplished by attaching the antibodies to reactive groups on the
substrate directly or through a linking moiety.
[0192] The solid support may be any insoluble material to which the
antibodies can be bound and which may be conveniently used in an
assay of the invention. Such solid supports include permeable and
semipermeable membranes, glass beads, plastic beads, latex beads,
plastic microtiter wells or tubes, agarose or dextran particles,
sepharose, and diatomaceous earth. Alternatively, the antibodies
may be bound to any porous or liquid permeable material, such as a
fibrous (paper, felt etc.) strip or sheet, or a screen or net. A
binder may be used as long as it does not interfere with the
ability of the antibodies to bind the ligands.
[0193] The invention will be further described by the following
examples.
EXAMPLE 1
Molecular Characterization and Analysis of the mit/mmc Biosynthetic
Gene Cluster
[0194] Materials and Methods
[0195] Bacterial Strains and Cloning Vectors
[0196] S. lavendulae NRRL 2564 was used as the source strain for
cosmid library construction and the creation of gene disruption
mutants. E. coli DH5.alpha. was used as the host strain for
constructing the library and subsequent DNA manipulation. E. coli
strain S17-1 (Mazodier et al., 1989) served as the conjugative host
for introducing foreign DNA into S. lavendulae. The cosmid library
was constructed with the E. coli/Streptomyces shuttle vector pNJ1
(Tuan et al., 1990), and pUC119 was routinely used as a vector for
subcloning and sequencing. The conjugative E. coli/Streptomyces
shuttle vector pKC 1139 (Bierman et al., 1992) was used for gene
disruption in S. lavendulae.
[0197] DNA Manipulation
[0198] Standard in vitro techniques were used for DNA manipulation
(Sambrook et al., 1989). S. lavendulae genomic DNA was harvested by
standard procedures (Hopwood et al., 1985).
[0199] A library of size-fractionated genomic DNA in pNJ1 (Tuan et
al., 1990) was screened with the rifamycin AHBA synthase (rifK)
gene probe from Amycolatopsis mediterranei (Kim et al., 1998).
Through subsequent cosmid walking, a contiguous 120 kb region of S.
lavendulae chromosomal DNA containing the putative mitomycin
biosynthetic genes was mapped. M13 forward and reverse primers were
used for sequencing (Gibco BRL, Gaithersburg, Md.). To accomplish
this, individual fragments of less than 5 kb were subcloned into
pUC 119 and serial deletion subdlones were generated using the
exonuclease III Erase-a Base System (Promega, Madison, Wis.).
[0200] DNA Sequencing and Analysis
[0201] Automatic DNA sequencing was done with the ABI PRISM.TM. Dye
Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems,
Warrington, U.K.), and analyzed on an Applied Biosystems mode 377
DNA Sequencer at the University of Minnesota Advanced Genetic
Analysis Center. Both DNA strands were sequenced redundantly a
minimum of three times. Sequence compilation was performed with
MacVector (Oxford Molecular Group, Mountain View, Calif.) and
GeneWorks (Oxford Molecular Group) software, and sequence homology
analysis was accomplished with Blast (Altschul et al., 1990) and
GCG programs (Devereux et al., 1984).
[0202] Disruption Mutants Construction
[0203] A 1.4 kb ApaL1-HindIII fragment from pFD666 (Denis and
Brzezinski, 1998) containing the aphII gene for kanamycin
resistance was routinely used as the selection marker for the
creation of gene disruption constructs. The target genes were
subcloned into pUC 119, cut at a unique internal restriction site,
blunt-ended, and ligated with the end-blunted selection marker. The
inserts were then transferred from pUC119 to pKC1139, and
conjugated into wild-type S. lavendulae. Transconjugants were
selected on AS1 plates (Baltz, 1980), overlaid with apramycin,
kanamycin, and nalidixic acid followed by propagation on R5T plates
(g/L: sucrose 121.1, K.sub.2SO.sub.40.3, MgCl.sub.2. 6H.sub.2O
11.92, glucose 11.8, yeast extract 5.89, casamino acids 0.12, trace
elements 2.35 ml (Hopwood et al., 1985), agar 25.9, after
autoclaving the following solutions were added: 0.5%
KH.sub.2PO.sub.4 11.8 ml, 5 M CaCl.sub.2 4.71 ml, 1 N NaOH 8.25 ml)
at 37.degree. C. for several generations. Disruption mutants were
selected based on the phenotype changing from apramycin and
kanamycin resistant to apramycin sensitive and kanamycin resistant.
Replacement of the chromosomal copy of the target gene with the
disrupted plasmid-born copy was confirmed by Southern blot
hybridization.
[0204] Mitomycin C Analysis
[0205] MC production was evaluated using 3-day cultures in
Nishikohri media (Nishikohri and Fukui, 1978). The culture broth
was extracted twice with equal volumes of ethyl acetate. After
removing the chemical solvent by vacuum, the crude broth extract
was dissolved in 50% methanol and 50% 50 mM pH 7.2 Tris buffer and
monitored by HPLC (C.sub.18 reverse phase column) at 363 nm. A
continuous methanol gradient from 20% to 60% in methanol/50 mM pH
7.2 Tris buffer system over 24 minutes was employed to resolve MC
from other crude extract components. A 90% CHCl.sub.3/10% MeOH
solvent system was used to resolve and detect MC on TLC plates.
[0206] Results
[0207] Identification of the Mitomycin Biosynthetic Gene
Cluster
[0208] The mitomycin cluster was identified by linkage of a cosmid
clone containing mrd and a gene (mitA) that hybridized with the
rifK gene encoding the rifamycin AHBA synthase (Kim et al., 1998)
from Amycolatopsis mediterranei. mitA was subsequently shown to be
essential for mitomycin biosynthesis since genetic disruption of
the chromosomal copy blocked MC production, and could be
complemented with exogenous AHBA (Example 2). Linkage of mitA with
one of the mitomycin resistance genes (mrd) implied that the
corresponding biosynthetic genes were adjacent to mitA. Cosmid
walking was used to obtain overlapping DNA fragments spanning more
than 120 kb of the S. lavendulae chromosome adjacent to mitA.
Subsequent nucleotide sequence analysis included 55 kb of
contiguous DNA, revealing 47 genes involved in mitomycin assembly,
regulation and resistance (FIGS. 2 and 5).
1TABLE 1 MC production in wild-type S. lavendulae and gene
disruption mutants MC No. gene disrupted production 0.0 Wild-type
control ++ 0.1 additional copy ++ of orf1 in wild-type 1 orf8 ++ 2
orf4 ++ 3 orf1 ++ 4 mitR + 5 mitM - 6 mitI - 7 mitH - 8 mitE - 9
mitB - 10 mitA - 11 mmcA - 12 mmcB - 13 mmcM ++ 14 mmcP - 15 mmcR -
16 mmcT - 17 mmcW ++++ 18 mmcX ++++ 19 orf11 ++ 20 orf12 ++ 21
orf16 ++ 22 orf19 ++
[0209] mitT Defines the Left-hand Boundary of the Mitomycin
Cluster
[0210] Nucleotide sequence analysis extended 30 kb downstream of
mitA and revealed a set of genes corresponding to a type I
polyketide synthase (PKS, orf9, SEQ ID NO:18; orf8, SEQ ID NO:19)
and thioesterase (TEII, orf7, SEQ ID NO:20). MC is not derived from
the polyketide pathway, and thus an orf8 disruption mutant showed
normal MC production as expected (Table 1). Approximately 20 kb
downstream of mitA, two genes (mitT, SEQ ID NO:29 and mitS, SEQ ID
NO:30) encoding a putative aminoquinate dehydrogenase and glucose
kinase, respectively, were located. Both are believed to be
involved in AHBA biosynthesis since their equivalents are also
present in the rifamycin biosynthetic gene cluster (rif cluster)
(August et al., 1998). However, whether the six genes between orf7
and mitT are involved in MC biosynthesis remained unclear, since
the two putative hydroxylases (orf3, SEQ ID NO:24 and orf4, SEQ ID
NO:22) and the candidate activator gene (orf1, SEQ ID NO:26) could
play a role in MC production. Both orf3 and orf4 are predicted to
encode cytochrome P450 monooxygenases with Orf4 most similar to
OleP and RapN (50% identity, 63% similarity) for oleandomycin and
rapamycin biosynthesis, respectively (Rodriguez et al., 1995;
Schwecke et al., 1995). Orf3 shows a high degree of similarity to
cytochrome P450 105C1(49% identity, 64% similarity) in Streptomyces
sp. and cytochrome P450-SU2 in Streptomyces griseolus (Horii et
al., 1990; Omer et al., 1990).
[0211] Database analysis revealed that Orf1 belonged to the
ActII-ORF4, RedD, DnrI and CcaR family of Streptomyces antibiotic
pathway specific activators regulating the production of
actinorhodin, undecylprodigiosin, daunorubicin, and cephamycin,
respectively (Fernandez-Moreno et al., 1991; Perez-Laraine et al.,
1997; Takano et al., 1992; Tang et al., 1996; Wietzorrek and Bibb;
1997). A common feature of this group of activators is that
disruption of the corresponding gene abolishes the production of
the corresponding antibiotic while overexpression results in a
several-fold increase in metabolite production. However, when orf1
was disrupted, the mutant strain showed normal MC production (Table
1). Moreover, the wild-type MC producer containing an additional
copy of orf1 in pKC1139 also had a normal MC production profile
(Table 1). Interestingly, orf4, one of the cytochrome P450
monooxygenase encoding genes adjacent to orf1 also showed normal MC
production when disrupted (Table 1). Thus, mitT appears to map to
the left-hand end of the mitomycin cluster, while orf1 to orf9
presumably specify biosynthesis of a polyketide product.
[0212] mmcY Defines the Right-hand Boundary of the Mitomycin
Cluster
[0213] Nucleotide sequence analysis of the mitomycin biosynthetic
gene cluster extended 30 kb upstream of mitA and several orfs
corresponding to genes involved in sugar metabolism were
identified. They included an acid trehalase (orf12, SEQ ID NO:28),
one ABC type transporter (orf16, SEQ ID NO:79), and four adjacent
.alpha.-amylases (orf19, SEQ ID NO:82; orf20, SEQ ID NO:83; orf21,
SEQ ID NO:84; orf22, SEQ ID NO:85) for starch degradation spanning
more than 18 kb (FIG. 2). Disruption of four genes (orf19, SEQ ID
NO:27; orf12, SEQ ID NO:28; orf16, SEQ ID NO:79; orf19, SEQ ID
NO:82) within this region resulted in mutants with wild-type level
MC production profiles, indicating that they fall outside of the
mitomycin cluster (Table 1). At the beginning of this group of
sugar metabolism genes, a gene (mmcY, SEQ ID NO:75) encoding a
presumed chitinase is proposed to be the upstream terminus of the
mitomycin cluster. This is evident because mitomycin requires
D-glucosamine as a biosynthetic precursor, and MmcY shows 75%
identity (85% similarity) with the chitinase C gene (chiC) product
from S. griseus that generates N-acetylglucosamine from chitin
(Ohno et al., 1996). In addition, mutants with disrupted orf11 and
orf12 genes had no effect on MC production, while disruption of
mmcW (SEQ ID NO:71) and mmcX (SEQ ID NO:72) both affected MC
production significantly (Table 1).
[0214] Mitomycin Resistance Genes
[0215] Antibiotic biosynthetic gene clusters typically include one
or more genes for cellular self-protection (Seno and Baltz, 1989).
Previous work has identified two mitomycin C resistance genes (mcr
and mrd) with mrd linked to mitA (August et al., 1994; Sheldon et
al., 1997; Example 2). Subsequent analysis showed that MRD is a
resistance protein that binds mitomycin C with 1:1 stoichiometry
(Sheldon et al., 1997). However, this resistance mechanism would be
extremely inefficient unless the bound drug is transported out of
the cell. Indeed, 5 kb upstream of mrd, the mct gene (SEQ ID NO:16,
putative mitomycin translocase) encoding a presumed antibiotic
transporter was found and shown to be a third resistance component
(Example 3). mct encodes 484 amino-acid protein with 14 predicted
transmembrane domains. Disruption of mct resulted in a mutant S.
lavendulae strain substantially more sensitive to MC, while
coexpression of mct with mrd in E. coli dramatically increased MC
resistance levels compared to individual expression of the genes
(Example 3). In contrast, the high-level MC resistance gene (mcrA)
that encodes an MC oxidase (MCRA) capable of re-oxidizing activated
MC (Johnson et al., 1997) is not linked with this cluster (August
et al., 1990; Example 2). Interestingly, database searches
identified two McrA homologues (MitR, MmcM) within the MC cluster,
both of which encode putative flavoproteins conserved in the
FMN/FAD binding motif. MitR displayed weak similarity with McrA
(26% identity, 33% similarity), while MmcM showed end-to-end (54%
identity, 69% similarity) alignment with the protein. mitR (SEQ ID
NO:31) and mmcM (SEQ ID NO:61) were genetically disrupted giving
substantially decreased MC production in the mitR mutant strain, in
contrast to the mmcM mutant which displayed wild type MC production
levels (Table 1).
[0216] Regulatory Genes
[0217] Two genes, mitQ (SEQ ID NO:32) and mmcW (SEQ ID NO:71), were
identified in the mitomycin cluster and are presumed to be
pathway-specific regulators. MitQ belongs to the OmpR-PhoB
subfamily of DNA binding regulators in the two-component regulatory
system, with the greatest similarity to members of the phosphate
assimilation pathway (PhoR-PhoB) (Makino et al., 1986), ferric
enterobactin response pair (PfeR-PfeS) (Dean et al., 1996), and one
histidine protein kinase--response regulator system (HpkA-DrrA)
from Thermotoga maritima (Lee and Stock, 1996). In contrast to the
MitQ group of regulators that typically serve as transcriptional
activators (Mizuno and Tanaka, 1997), MmcW showed high sequence
similarity with the MarR groups of repressors. The most significant
similarity corresponds to EmrR, the negative regulator of the E.
coli multidrug resistance pump EmrAB (Lomovskaya et al., 1995), and
Pacs, a repressor for pectinase, cellulase, and blue pigment
production in Erwinia chrysanthemi (Praillet et al., 1996).
Significantly, the mmcW disruption mutant displayed a several-fold
increase in MC production (Table 1).
[0218] AHBA Biosynthetic Genes
[0219] Precursor incorporation studies previously demonstrated that
AHBA is an intermediate for both the ansamycin and mitomycin
natural products (Becker et al., 1983; Example 2). Combining the
biochemical, enzymatic and molecular genetic results on the
biosynthesis of the ansamycin antibiotic rifamycin, Floss has
proposed that AHBA is derived from the ammoniated shikimate pathway
via phosphenolpyruvate (PEP) and erythose 4-phosphate (E4P) by the
early incorporation of nitrogen (Kim et al., 1996). In the
shikimate pathway, PEP and E4P is first converted to
3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP) then
stepwise transformed to 3-dehydroquinate (DHQ), 3-dehydroshikimate
(DHS) and shikimate, catalyzed by DAHP synthase, DHQ synthase, DHQ
dehydratase, and shikimate dehydrogenase, respectively (Dewick,
1998). Quinate can also enter the pathway by the action of quinate
dehydrogenase to generate DHQ.
[0220] Evidence to support this new variant of the shikimate
pathway includes the following experimental observations. First,
all proposed ammoniated shikimate pathway compounds including PEP,
E4P, 3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate
(aminoDAHP), 5-deoxy-5-amino-3-dehydroquinic acid (aminoDHQ), and
5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS) can be readily
converted into AHBA by cell-free extracts from the ansamycin
producers, while none of the early shikimate pathway intermediates,
DAHP, DHQ, DHS, quinic acid, shikimic acid can be incorporated into
AHBA under the same conditions (Homemann, 1981; Kim et al., 1996).
Second, the rifamycin biosynthetic gene cluster (rif cluster) has
been sequenced, and all of the genes encoding early shikimate
pathway enzymes are found within the cluster (August et al., 1998).
Finally, the ability of the rifamycin AHBA synthase (RifK) to
catalyze dehydration of aminoDHS to AHBA has been previously
demonstrated (Kim et al., 1998). As described in Example 2, the
AHBA synthase gene (mitA) in S. lavendulae is required for AHBA
biosynthesis.
[0221] A group of AHBA biosynthetic genes similar to those
described for rif have been identified in the mitomycin cluster. In
addition to AHBA synthase, six gene products in the cluster showed
high sequence similarity (over 43% identity) with their rifamycin
AHBA biosynthetic gene homologs. These gene products include
aminoDHQ synthase (MitP, RifG equivalent), aminoquinate
dehydrogenase (MitT, Rift equivalent), oxidoreductase (MitG, RifL
equivalent), phosphatase (MitJ, RifM equivalent), kinase (MitS,
RifN equivalent), and aminoDHQ dehydratase (MmcF, RifJ equivalent).
In addition to the significant sequence similarity to rifamycin
counterparts, all three putative mitomycin shikimate pathway
enzymes displayed significant alignment with microbial primary
shikimate metabolic enzymes including MitT with the quinate
dehydrogenase (AroE) from Methanococcus jannaschii (28% identity,
46% similarity) (Bult et al., 1996), MitP with the DHQ synthase
(AroB) from Mycobacterium tuberculosis (46% identity, 61%
similarity) (Cole et al., 1998), and MmcF with the DHQ dehydratase
from S. coelicolor (50% identity, 62% similarity) (White et al.,
1990). Despite extensive sequencing of 15 kb on either side of the
mapped right- and left-hand ends of the mitomycin cluster, an
aminoDAHP synthase gene corresponding to RifH (the proposed first
enzyme in the de novo biosynthesis from PEP and E4P in the rif
cluster), was not found (FIG. 2). Interestingly, a rifH homologue
has been cloned from S. lavendulae genomic DNA through Southern
hybridization and shown to be unlinked to the mitomycin
cluster.
[0222] The existence of non-shikimate pathway-related
phosphatase/kinase pair in the mitomycin cluster (MitJ/MitS) and
the rif cluster (RifM/RifN) further support the finding that these
two genes are required for AHBA biosynthesis (Floss, 1997). In
addition to the strong homology to RifM, MitJ also showed 56%
identity (69% similarity) with ORF8 from the ansamycin antibiotic
ansamitocin producer Actinosynnema pretiosum auranticum. Other
polypeptides with considerable sequence similarity belong to the
CBBY family of phosphoglycolate phosphatases in glycolate oxidation
(Schafejohann et al., 1993). MitS, most similar to RifN (53%
identity, 63% similarity), also showed significant similarity with
the glucose kinase (involved in glucose repression) from S.
coelicolor and Bacillus megaterium (Angell et al., 1992; Spath et
al., 1997). mitG, the third non-shikimate pathway-related AHBA
biosynthetic gene in this cluster is also worthy of note since it
shows exclusive similarity (46% identity, 61% similarity) with
oxidoreductase RifL and its equivalent in Actinosynnema pretiosum
auranticum.
[0223] Mitosane Formation Genes
[0224] Precursor incorporation studies established that the
mitosane core is assembled form the condensation of AHBA and
D-glucosamine. Although no specific gene products can be assigned
for forming the three bonds bridging AHBA and D-glucosamine, two
genes downstream of mitA (SEQ ID NO:97), mitb (SEQ ID NO:99), and
mitE (SEQ ID NO:44) likely encode enzymes that mediate one of these
reactions. MitB shows local sequence similarity with a group of
glycosyltransferases involved in glycopeptide antibiotic and
polysaccharide biosynthesis, the typical function of which is to
attach an activated sugar residue to a core compound (Yamazaki et
al., 1996; Example 2). Meanwhile, MitE showed weak similarity (22%
identity and 45% similarity) to the two cloned
4-hydroxybenzoate-CoA ligases from Rhodopseudomonas palustris in
the anaerobic degradation of aromatic compounds (Gibson et al.,
1994). It also showed similarity to a group of long chain fatty
acid CoA ligases, as well as to the O-succinylbenzoic acid CoA
synthetase in Vitamin K2 biosynthesis (Kwon et al., 1996). mitB and
mitE disruption mutants both had a MC deficient phenotype (Table
1).
[0225] The condensation of AHBA with D-glucosamine may be initiated
in two different ways. This includes either initial formation of a
C.sub.8a-C.sub.9 bond by an acylation or alkylation reaction, or
formation of a Schiff base between the AHBA nitrogen and
D-glucosamine C1 aldehyde, followed by the ring closure at
C.sub.8a-C.sub.9.mitR (SEQ ID NO:31), one of the two McrA
homologues may be involved in one of the ring closure reactions.
Interesting, MitR showed high sequence homology with the plant
berberine bridge enzyme (BBE) (30% identity, 37% similarity) in
benzophenanthridine alkaloid formation, where it catalyzes an
unusual C--C bond formation of the berberine bridgehead carbon of
(S)-scoulerine from the N-methyl carbon of (S)-reticuline (Dittrich
and Kutchan, 1991). Using a mechanism similar to BBE, it is
possible that MitB is involved in C.sub.8a-C.sub.9 bond formation.
The decreased MC production in the mitR disruption mutant may be
due to the existence of isoenzymes (e.g., MmcM) that could catalyze
the reaction in the absence of a functional MitR.
[0226] Side Group Modification Genes
[0227] Complete assembly of MC requires functionalization of
several sites on the core mitosane ring system. First, complete
reduction of the carbonyl group at C-6 must occur. Second,
hydroxylation at C-5 and C9a must proceed followed by methylation
at C-9a. Third, amination at C-7 must occur presumably through
initial hydroxylation followed by transamination. Fourth, oxidation
of the hydroxyl groups at C-5 and C-8 to form the benzoquinone are
required. Fifth, intramolecular amination of C-1 by N-1a to form
the aziridine ring must be completed and finally, carbamoylation at
C-10 completes assembly of the molecule. Several enzymes found in
this cluster likely catalyze these modifications and are discussed
below.
[0228] Methylation
[0229] In contrast to MC which has an O-methyl group at C-9a,
mitomycin A and mitomycin B also contain a C-7 O-methyl group,
while mitomycin B, mitomycin D and porfiromycin have an N-methyl on
the aziridine ring (FIG. 1). Radio-labeled precursor incorporation
studies showed that all of the O- and N-methyl (but not the
C-methyl) groups in the mitomycin molecules are derived from
L-methionine (Bezanson and Vining, 1971). Typically, the methyl
donor for most C1 reactions is S-adenosyl-L-methionine (SAM), which
can be formed through activation of L-methionine by ATP. Three SAM
dependent methyltransferase genes were identified in this cluster
(encoding MitM, MitN, and MmcR), all of which have three conserved
S-adenosylmethionine or S-adenosylhomocysteine binding motifs
(Kagan and Clarke, 1994) (FIG. 3). Interestingly, database searches
of MitM and MitN (likely responsible for the MC C-9a side chain
methylation) revealed a group of plant .delta.-(24)-sterol
C-methyltransferases that have a closer phylogenetic relationship
with the rifamycin O-methyltransferase (ORF14) and erythromycin
O-methyltransferase (EryG) (5, 86) (FIG. 4). In contrast, protein
database searches revealed that MmcR is most related to other
Streptomyces antibiotic biosynthetic O-methyltransferases with
greatest similarity to O-demethylpuromycin O-methyltransferase (44%
identity, 60% similarity) from S. anulatus and carminomycin
4-O-methyltransferase from S. peucetius (Lacalle et al., 1991;
Madduri et al., 1963). MmcR may be involved in the O-methylation of
the phenol ring of MC before oxidation to the quinone. Both mmcR
(SEQ ID NO:67) and mitM (SEQ ID NO:36) were shown to be essential
for MC biosynthesis since disruption of each one completely
abolished MC production (Table 1).
[0230] A SAM-independent methyltransferase, MmcD, was also
identified in the mitomycin cluster. MmcD revealed strong sequence
homology with the magnesium-protoporphyrin IX monomethyl ester
oxidative cyclase (34% identity, 53% similarity) from
Methanobacterium thermoautotrophicum (Accession Number 2622915), as
well as the phosphonoacetaldehyde methyltransferase from
Streptomyces wedmorensis (Hidaka et al., 1995), the
P-methyltransferase from Streptomyces hygroscopicus (Hidaka et al.,
1995) and the fortimicin KL methyltransferase from Micromonospora
olivasterospora (Kuzuyama et al., 1995). Instead of SAM, this group
of methyltransferases uses methylcobalamine or a structurally
related protoporphyrin as the direct methyl donor. While the
greatest number of matches were made to protoporphyrin
methyltransferases, it is expected that this enzyme has another
function in the mitomycin C biosynthetic pathway as all the O- and
N-methyl groups of MC have been shown to be derived from
SAM-dependent methyltransferases.
[0231] C-6 Carbonyl Reduction
[0232] The C-6 methyl group was previously shown to be derived from
the reduction of the carboxylic acid of AHBA, since
[carboxy-.sup.13C] AHBA can be efficiently, and specifically
incorporated into the C-6 methyl group of porfiromycin (Anderson et
al., 1980). In the mitomycin cluster, four F420-dependent
tetrahydromethanopterin (H.sub.4MPT) reductase genes (encoding
MitH, MitK, Mmcl, MmcJ) and one H.sub.4MPT:CoM methyltransferase
gene (encoding MmcE) are candidates for the C-6 carbonyl reduction.
In the methanogenesis pathway of Methanobacterium
thermoautotrophicum, two cofactor F420-dependent H.sub.4MPT
reductases, and one cofactor CoM dependent methyltransferase are
required in the seven step reduction from CO.sub.2 to CH.sub.4.
Steps 4 to 6 from CH-H.sub.4MPT to CH.sub.2-H.sub.4MPT, and
CH.sub.3-H.sub.4MPT to CH.sub.3-CoM are catalyzed by N.sup.5,
N.sup.10-methylene-H.sub.4MPT dehydrogenase, N.sup.5,
N.sup.10-methylene-H.sub.4MPT reductase, and
N.sup.5-methyl-H.sub.4MPT:CoM methyltransferase, respectively
(Deppenmeier et al., 1996; Thauer et al., 1993). All four enzymes
(MitH, MitK, Mmcl, MmcJ) in this cluster showed local sequence
similarity with the cloned F420 dependent H.sub.4MPT reductase (42%
identity, 62% similarity in several 50 amino-acid fragments)
(Nolling et al., 1995; Vaupel and Thauer 1995). One of these genes,
mitH (SEQ ID NO:41) was disrupted, and the mutant strain displayed
a MC deficient phenotype (Table 1). MmcE is notable since the
deduced protein sequence contains two domains showing significant
alignment (33% identity, 56% similarity) to the N-terminus of
H.sub.4MPT:CoM methyltransferase from Methanobacterium
thermoautotrophicum (Stupperich et al., 1993), while the remaining
C-terminus is related to fatty acid biosynthetic acyl carrier
proteins (ACP) (Morbidoni et al., 1996; Platt et al., 1990). The
potential function of this ACP-like domain in MC biosynthesis
remains unknown, as does the role of a distinct gene (mmcB, SEQ ID
NO:50) encoding a putative ACP identified just upstream of mmcE
(SEQ ID NO:53). Significantly, the disruption of mmcB resulted in
total abrogation of MC production (Table 1).
[0233] Hydroxylation
[0234] The two putative hydroxylases (encoded by mmcN, SEQ ID
NO:62; and mmcT, SEQ ID NO:69) identified in the mitomycin cluster
are candidates for catalyzing hydroxylation at the C-5, C-7, and
C-9a positions on the mitosane system. MmcN belongs to the
cytochrome P450 family of monooxygenases, with greatest homology
(37% identity, 56% similarity) to the two herbicide-inducible
cytochrome P450s (P450-SU1 and P450-SU2) from S. griseolus, as well
as to RapJ and RapN in the rapamycin biosynthetic gene cluster from
S. hygroscopicus (Omer et al., 1990; Schwecke et al., 1995). MmcT
showed highest similarity to the tetracenomycin C hydroxylase
(TcmG) in Streptomyces glaucescens (38% identity, 55% similarity),
with lower but significant sequence similarity to a group of phenol
or hydroxybenzoate hydroxylases (Decker et al., 1993). Genetic
disruption of mmcT completely blocked MC biosynthesis (Table
1).
[0235] Carbamoylation
[0236] The carbamoyl group of MC is derived intact from
L-citrulline or L-arginine with carbamoyl phosphate as the
incorporated precursor (Homemann, 1981). In eubacteria, carbamoyl
phosphate can be generated from L-glutamine, HCO.sub.3, and ATP by
the enzyme carbamoyl phosphate synthetase, which is indispensable
for pyrimidine biosynthesis. One candidate carbamoyl transferase
gene (mmcS, SEQ ID NO:68) was identified directly upstream of mmcT.
MmcS belongs to the NodU/CmcH family of O-carbamoylation enzymes,
with the greatest similarity (35% identity, 44% similarity) to No1O
from Rhizobium sp. (Jabbouri et al., 1998). Other members with
significant alignment in this family include No1O from
Bradyrhizobium japonicum (Luka et al., 1993) and NodU from
Rhizobium sp. for 6-O-carbamoylation of Nod-factors (Jabbouri et
al., 1995) and CmcH from Nocardia lactamdurans and S. clavuligerus
for 3'-hydroxymethylcephem O-carbamoylation in cephamycin
biosynthesis (Coque et al., 1995).
[0237] Discussion
[0238] Bridging Primary and Secondary Metabolism
[0239] The shikimate pathway is an essential metabolic route in
microorganisms and plants for aromatic amino acid biosynthesis.
Genes encoding the early shikimate pathway enzymes from various
organisms have been well studied and are often dispersed along the
chromosome as revealed by genome sequencing projects (Blattner et
al., 1997; Bult et al., 1996; Cole et al., 1998). The finding that
the ansamycin and mitomycin natural products are derived in part
from an ammoniated shikimate pathway whose genes are clustered on
the bacterial chromosome is a significant difference to the primary
metabolic network, and may suggest an important evolutionary bridge
leading to secondary metabolism. The lack of incorporation of early
shikimate pathway intermediates into mitomycin and ansamycin
metabolites indicated the existence and ultimate substrate
specificity of the alternate ammoniated shikimate pathway enzymes.
However, the conversion of aminoDA-HP and aminoshikimic acid by the
corresponding primary shikimate pathway enzymes to aminoDHQ and
aminoDHS, respectively (Kim et al., 1996), suggested that the
substrates specificity in primary metabolic shikimate pathway is
mainly determined by the initial reaction step. This notion is
further supported by the disruption results for rifG and rifI
mutants showing only slightly affected rifamycin production (Floss,
1997).
[0240] In addition to the absence of an aminoDAHP synthase gene,
the organization of the AHBA biosynthetic genes in the MC cluster
is quite different compared to the rif cluster. In rif (with the
exception rifJ), all AHBA biosynthetic genes are found within a
defined sub-cluster that are organized into a single apparent
operon. In contrast, almost all of the mit/mmc encoded AHBA genes
are scattered within the 55 kb MC cluster. Thus, as opposed to the
multifunctional polyketide gene clusters whose linearity of
architecture reflects a precise pattern of biosynthesis, the MC
cluster is biochemically less transparent based on a similar
primary analysis. In addition, the MC cluster provides a good model
for analyzing genetic evolution both vertically, from the primary
metabolic shikimate pathway to the secondary shikimate pathway
related route, and horizontally by comparing different groups of
secondary metabolic biosynthetic clusters.
[0241] The MC Biosynthetic Network
[0242] In a typical liquid culture of S. lavendulae, MC production
initiates 24 hours after inoculating the seed culture, reaches
maximum production in two days, and maintains drug synthesis during
stationary phase for another two days. Compared to high level MC
resistance of the wild-type S. lavendulae (>150 .mu.g/ml), MC
production is relatively low (<5 .mu.g/ml MC). The significant
gap between the self-resistance and production levels makes it
possible to improve drug production through genetic engineering. As
described herein, disruption of the candidate repressor gene (mmcW)
and downstream mmcX (encoding a putative membrane protein) in the
mitomycin cluster resulted in a several-fold increase in MC
production. The existence of a repressor gene(s) is not uncommon in
Streptomyces antibiotic biosynthetic gene clusters. Previous
examples include, mmyR from the methylenomycin cluster (Chater and
Briton, 1985), actII-orfI in the actinorhodin cluster (Caballero et
al., 1991), jadR (Anderson et al., 1980) in jadomycin biosynthesis
(Yang et al., 1995), and dnrO in the daunorubicin cluster (Otten,
1995). Disruption of jadR and mmyR also resulted in increased
levels of the corresponding antibiotic (Chater and Bruton, 1985;
Yang et al., 1995).
[0243] In order to avoid auto-toxicity, drug-producing
microorganisms must evolve self-protection systems. Currently,
three types of self-protection mechanisms have been identified in
S. lavendulae for mitomycin resistance including, MC binding (MRD),
efflux (MCT), and reversing MC reductive activation (MCRA). In
principle, resistance genes must be expressed before drug
formation. In this respect, it is interesting to note the linkage
of the mitomycin resistance genes with the regulatory genes.
Expression of the high-level resistance gene mcrA has been
demonstrated to be regulated by the downstream gene mcrB which is
presumably cotranscribed with mcrA (August et al., 1994). Though
the function of the McrA homolog MitR in the mitomycin cluster
remains unknown, mitR is also followed by a cotranscribed
regulatory gene (mitQ). Meanwhile, the putative mitomycin
translocase gene, mct is followed by the repressor gene, mmcW.
Genetic linkage of membrane transporter/resistance and repressor
genes have been described in a number of cases, including tetA/tetR
in tetracycline resistance (Guilfoile and Hutchinson, 1992),
tcmA/tcmR in tetracenomycin C resistance (Guilfoile and Hutchinson,
1992), actII-orf2/actII-orf1 in actinorhodin resistance (Caballero
et al., 1991), and the qacA/qacR pair for multidrug resistance in
S. aureus (Grkovic et al., 1998).
[0244] Conclusion
[0245] Although MC was first isolated more than 40 years ago and
has been used in anti-cancer chemotherapy since the 1960s, the
mechanistic details and order of its biosynthesis has remained
unclear. The results described herein are clearly consistent with
precursor incorporation studies gathered in the 1970s, showing that
MC is biosynthetically derived from D-glucosamine, L-methionine,
carbamoyl phosphate, and AHBA, and also support the use of the
variant de novo shikimate pathway leading to AHBA (Homemann, 1981;
Kim et al., 1996). Many, if not all, of the genes responsible for
the formation of the mitosane and aziridine rings are evidently
located within the boundary of the 55 kb mitomycin cluster. These
genes are of special interest since they may be useful as probes
for identification of related natural product biosynthetic genes
from other microorganisms and plants.
[0246] The cloned genes presented here are useful to study
mitomycin biosynthesis and natural product assembly. The advantage
of having this information has already been demonstrated through
genetic disruption of the candidate repressor gene (mmcW) that
provided a several-fold increase in MC production. In addition,
expression and genetic disruption of selected genes should be
useful for engineering the biosynthesis of clinically valuable
mitomycin analogues, as well as more complex hybrid natural product
systems. Finally, the MC resistance and regulatory genes identified
in this cluster provide important insight into the mitomycin
biosynthetic and regulatory network in the S. lavendulae.
EXAMPLE 2
Genetic Localization and Molecular Characterization of Two Genes
Required for MC Biosynthesis
[0247] Materials and Methods
[0248] Strains and culture conditions. E. coli DH5.alpha. was grown
in either Luria broth (LB) or tryptic soy broth (TSB) (Difco) as
liquid medium or agar plates. E. coli DH5.alpha.F', the host for
harvesting single-stranded DNA, was grown at 37.degree. C. on TBG
(1.2% tryptone, 2.4% yeast extract, 0.4% glycerol, 17 mM
KH.sub.2PO.sub.4, 55 mM K.sub.2HPO.sub.4, and 20 mM glucose). E.
coli S17-1 (Mazodier et al., 1989) used for conjugation was grown
in TSB with 10 ug/ml of streptomycin. S. lavendulae was grown in
TSB or on R5T plates. For MC production, S. lavendulae was grown in
Nishikohri media (g/L: glucose 15, soluble starch 5, NaCl 5,
CaCO.sub.3 3, yeast extract 5) for 72 hours from a 1% v/v inoculum
of frozen mycelia. Pulse feeding of AHBA to the disruption mutant,
MV100, and the site-directed mutant, MV102, occurred with feedings
of 2.5 mg of a 20 mg/mL solution of the sodium salt of AHBA at pH
7.1 in three pulses at 24, 43, and 57 hours of a culture that was
harvested at 76 hours.
[0249] DNA preparation and amplification. Isolation and
purification of DNA was performed using standard methods (Sambrook
et al., 1989). S. lavendulae NRRL 2564 genomic DNA was isolated by
using the modified Chater protocol (Hopwood et al., 1988). Plasmid
DNA was isolated from E. coli by using the alkaline-sodium dodecyl
sulfate method.
[0250] pDHS2002 was constructed as follows: The 1.1 kb thiostrepton
resistance gene (tsr) fragment was removed from pDHS5000 with a
SmaI-BamHI digestion, blunt-ended with the large fragment of DNA
polymerase (Gibco BRL), and ligated to MscI restriction enzyme
digested pDHS7601 to yield pDHS20001. MscI digestion of pDHS7601
resulted in the removal of 155 nucleotides at the C-terminus of the
mitA gene, and ligation of the blunt-ended BamHI site of the tsr
adjacent to the MscI site of pDHS7601 resulted in regeneration of
the BamHI site in pDHS2001. The 4.9 kb EcoRI-HindIII fragment from
pDHS2001 containing the tsr disrupted mitA gene was removed and
ligated into EcoRI-HindIII digested pKC1139 to yield pDHS2002.
[0251] Primer-mediated site-directed mutagenesis (SDM) was employed
to construct pDHS2015 containing a K191A mutation in mitA. Primer
1: 5'-GGCAAGGCATGCGAGGGTCGC-3' (SEQ ID NO:46) and primer 2:
5'-TTCCAGAACGGCGCCCTGATGACCGCCGGC-3' (SEQ ID NO:47) were used to
amplify the 691 bp fragment of the 5' end of mitA. The 3' end of
mitA was amplified with primer 3:
5'-GCCGGCGGTCATCAGGGCGCCGTTCTGGAA-3' (SEQ ID NO:48) and primer 4:
5'-TCAGAATTCGGATCCGAGGGCCGGAGT-3' (SEQ ID NO:86) to generate a 1151
bp band (see amplification reaction conditions in Example 3). A
second round of PCR was performed using the overlapping 691 and
1151 bp units as the initial templates with primer 1 and primer 4
to yield a 1.8 kb fragment. The final product containing
mutagenized mitA was digested with EcoRI-Sph1, ligated to the 2.1
kb HindlII-SphI fragment from pDHS7601 and the EcoRI-HindIII
digested pKC1139 to yield pDSH2015. The site-directed mutation of
MitA K191A in pDHS2015 was confirmed by sequencing with forward
primer:
2 5'-ACCTACTGCCTCGATGCC-3' (SEQ ID NO:87) and reverse primer:
5'-CTGATCCTTCAAGCG-3'. (SEQ ID NO:88)
[0252] The mitB disruption vector pDHS7702 was constructed as
follows. pDHS7601 was digested with BstBI, blunt-ended, and ligated
with the 1.4 kb neomycin-resistant gene fragment from pFD666 (Denis
and Brzezinski et al., 1992) (ApaL1-HindIII digestion,
blunt-ended). The 5.2 kb EcoRI-HindIII fragment from the resulting
construct pDHS7701 was subdloned into pKC1139 to create
pDHS7702.
[0253] DNA library construction and screening. S. lavendulae NRRL
2564 genomic DNA was partially digested with Sau3AI, and a fraction
containing 30-50 kb fragments was recovered by sucrose gradient
centrifugation and ligated into the calf intestinal alkaline
phosphatase (CIP) treated BglII site of the E. coli-Streptomyces
shuttle vector pNJ1 (Tuan et al., 1990), then packaged with the
Packagene Lambda DNA Packaging System (Promega). The cosmid library
was constructed by transfecting E. coli DH5.alpha., and colonies
that appeared on the LB plates containing 100 ug/ml of ampicillin
were transferred to a BioTrace NT nitrocellulose blotting membrane
(Gelman Sciences, Ann Arbor, Mich.). Colony hybridization was
performed as specified by the manufacturer. A PCR-amplified 0.7 kb
DNA fragment from plasmid pKN108 (FIG. 6) was used to screen the
library. The primers used for PCR were: 5'-GCGTCCGTGCTGCGCGCGCA-3'
(SEQ ID NO:89), and 5'-TGCGCGCGCAGCACGGACGC-3' (SEQ ID NO:90). The
cosmids from the positive colonies were confirmed by Southern blot
hybridization, and a 1.7 kb AflIII-BamHI fragment from pDHS3001
containing the mitomycin resistance determinant (mrd) (Sheldon et
al., 1997) was used as a probe to establish genetic linkage.
[0254] DNA sequencing and analysis. Deletion subdlones from
pDHS7601 were made with exonuclease III Erase-a-Base System
(Promega). Sequencing was accomplished with the ABI PRISM.TM. Dye
Terminator Cycle Sequencing Ready Reaction Kit (Applied
Biosystems), and analyzed on an Applied Biosystems 377 DNA
Sequencer at the University of Minnesota Advanced Genetic Analysis
Center. For generating single-stranded DNA, deletion subclones in
pUC119 were transformed into E. coli DH5.alpha.F', and M13K07
Helper Phage was used (GIBCO BRL). Nucleotide sequence data were
analyzed using Wisconsin Genetics Computer Group software (version
9.0) (Devereux et al., 1984), and GeneWorks software version 2.51
(Oxford Molecular Group). The GenBank accession number for mitABC
is AF 115779.
[0255] Conjugation from E. coli S17-1 to S. lavendulae. The
procedure of Bierman et al. (Bierinan et al., 1992) was used with
the following modification. A single colony of E. coli S17-1
/pDHS2002 was used to inoculate 2 ml of TSB containing 100 .mu.g/ml
of apramycin and 10 .mu.g/ml of streptomycin. Following overnight
incubation at 37.degree. C. a 1:100 inoculation was made into TSB
broth with 100 .mu.g/ml of apramycin and 10 .mu.g/ml of
streptomycin. This culture was grown for 3 hours at 37.degree. C.,
and the cells were washed twice with TSB and resuspended in 2 ml of
TSB to provide the donor E. coli culture. The recipient S.
lavendulae culture was generated by inoculating 9 ml of TSB with 1
ml of frozen wild-type culture. Following overnight (16 hour)
incubation at 29.degree. C., the culture was homogenized by
sonication and 2 ml of this culture was used to inoculate 18 ml of
TSB. Following overnight growth at 29.degree. C. and sonication
treatment to homogenize the culture, a 1 ml inoculum was placed in
9 ml of TSB. This culture was grown for 3 hours, the mycelia were
washed with TSB and resuspended in 2 ml of TSB to provide the stock
recipient culture.
[0256] The donor and recipient cultures were mixed together in 9:1,
1:1, and 1:1/10 donor:recipient ratios, and 100 .mu.l of the cell
mixture was spread on AS1 plates (Baltz, 1980). The plates were
incubated overnight at 29.degree. C. and overlaid with 1 ml of
water containing a suspension of 500 .mu.g/ml each of thiostrepton,
apramycin and nalidixic acid. For the pKC1139 control, only
apramycin and nalidixic acid were overlaid, while for pDHS7702, 500
.mu.g/ml of kanamycin was used instead of thiostrepton. S.
lavendulae exconjugates appeared in approximately 11-13 days at a
frequency ranging from 10.sup.-7-10.sup.-5. pKC 1139 has a
temperature-sensitive Streptomyces replication origin, which is
unable to replicate at temperatures above 34.degree. C. (Muth et
al., 1989), while the S. lavendulae host grows well at 42.degree.
C. Thus, after propagating the conjugants at 39.degree. C. for
several generations, double crossover mutants were readily
generated. Presence of plasmid was determined by transformation of
E. coli DH5.alpha. with plasmid extracts from S. lavendulae
transconjugants.
[0257] Double-crossover selection procedure. A single colony of S.
lavendulae/pDHS2002 grown on R5T plates (50 .mu.g/ml of
thiostrepton and apramycin) was used to inoculate TSB broth
containing 20 .mu.g/ml of thiostrepton. After 72 hours of
incubation at 39.degree. C., 10.sup.-4, 10.sup.-5 and 10.sup.-6
diluted aliquots were used to inoculate R5T plates containing 50
.mu.g/ml of thiostrepton. Following 48 hours of growth at
39.degree. C., 84 colonies were picked randomly and each colony was
patched out on separate 50 .mu.g/ml of thiostrepton and 50 .mu.g/ml
of apramycin containing R5T plates. One of the 84 colonies
displayed the double crossover phenotype of thiostrepton resistance
and apramycin sensitivity. Integration of the tsr disrupted mitA
gene and loss of plasmid pDHS2002 was confirmed by Southern
hybridization analysis.
[0258] MitA K191A site-directed mutants (MV102) were selected by
propagating MV100/pDHS2015 on R5T plates for two generations at
37.degree. C. Colonies were replicated to plates containing 50
.mu.g/ml of thiostrepton and plates without antibiotics. Of the 108
colonies replicated in the first round, one had the correct
(thiostrepton sensitive) phenotype. To confirm the K191A mutation,
the mitA gene was amplified from the chromosome with primers 1 and
4. Mutation of the conserved lysine codon (AAG) to an alanine codon
(GCC) was verified with the same sequencing primers employed to
confirm the correct construction of pDHS2015. The alanine codon was
observed in both the forward and reverse sequence data.
[0259] Mutants for mitB (MM101) were selected as follows: S.
lavendulae/pDHS7702 was propagated on R5T plates for five
generations at 39.degree. C. before single colonies were replicated
on R5T plates as described above. Of the 300 colonies tested, 12
clones displayed the correct phenotype (kanamycin resistance and
apramycin sensitivity). The genotype of selected mitB mutants was
confirmed by Southern blot hybridization of S. lavendulae genomic
DNA.
[0260] Analysis of MC production. All cultures intended for MC
extraction were grown in Nishikohri media (Nishikohri and Fukui,
1975) for a period of 72 hours. In all cases a wild-type S.
lavendulae culture was grown concurrently with the mutant cultures
to provide a MC production reference point. A 72 hours, 50 ml
culture (250 ml flask) of the MitA K191A MV102 mutant strain was
supplemented with 125 .mu.l of a 20 mg/ml solution of the sodium
salt of AHBA (pH 7.05) at 24, 43 and 55 hours. In each case, the
culture broth was separated from mycelia by centrifugation and then
extracted three times with equal volumes of ethyl acetate. The
ethyl acetate extracts were pooled and solvent was removed by
vacuum to provide the crude broth extract. The preliminary screen
for MC production involved thin layer chromatography (TLC) on
silica gel plates (Whatman K6) eluted with 9:1 chloroform:methanol.
Production of MC was monitored by HPLC (C.sub.18 reverse phase
column) using a gradient of 80% 50 mM Tris buffer (pH 7.1)/20%
methanol to 40% 50 mM Tris buffer (pH 7.2)/60% methanol with the UV
detector set to 363 nm.
[0261] Bioassay detection of MC was performed by loading a 1 cm
disk with fractions eluting at the mitomycin retention time from
HPLC injections of wild-type, MV100, pKC1139 vector control crude
extracts and MC standards. The disks were placed on antibiotic
media number 2 agar plates (Difco) with Bacillus subtilis spores
added directly to the media. The plates were incubated overnight at
29.degree. C. and examined for zones of inhibition. To confirm the
production of MC by MV102 in the presence of exogenous AHBA the
fraction eluting at the MC retention time was collected, dried
down, desalted and submitted for desorption ionization mass
spectrometric analysis on a Bio-Ion 20R DS-MS instrument (Applied
Biosystems). The MC (M.W.=334)-sodium (M.W.=23) adduct peak,
[M+Na].sup.+=357, was diagnostic for the presence of MC in the AHBA
supplemented culture.
[0262] Results
[0263] The mrd and ahbas genes are linked in the S. lavendulae
genome. Southern blot analysis with the A. mediterranei AHBA
synthase (rifK) gene probe (Kim et al., 1998) showed a single 3.8
kb band that hybridized with BamHI digested S. lavendulae genomic
DNA (FIG. 8). Subsequently, a S. lavendulae genomic DNA library was
constructed using the E. coli-Streptomyces shuttle cosmid pNJ1. Of
the 5,000 colonies screened, 21 positive clones were identified
with six of these hybridizing with the mrd gene probe (none
hybridized with the mcr gene probe described in August et al.,
1994). Restriction-enzyme mapping and reciprocal hybridization of
the cosmid clones established that the mrd and S. mediterranei AHBA
synthase homologous genes were about 20 kb apart in the S.
lavendulae genome. The 3.8 kb BamHI fragment bearing a putative S.
lavendulae AHBA synthase gene was subdloned and its nucleotide
sequence determined.
[0264] Three ORFs are identified within the 3.8 kb BamHI fragment.
Three ORFs (mitA, mitB, mitC) were identified within the sequenced
3.8 kb BamHI fragment (FIGS. 8 and 9). mitA comprises 1164
nucleotides and starts from ATG (position 579 of the sequenced
fragment) that is preceded by a potential ribosome binding site
(RBS), GAAAGG (SEQ ID NO:91). The deduced product of the mitA gene
encodes a hydrophilic protein of 388 amino acids with a predicted
M.sub.r of 41,949 Da and a calculated pI of 5.62. A BLAST (Altschul
et al., 1990) search showed that the predicted MitA protein has
high sequence similarity (about 71% identity, 80% similarity) with
AHBA synthases (AHBASs), both from the rifamycin producer A.
mediterranei (Kim et al., 1998) and other ansamycin-producing
actinomycetes, including Actinosynnema pretiosum (ansamitocin) and
Streptomyces collinus (naphthomycin A and ansatrienin) (FIG. 10). A
conserved pyridoxal phosphate (PLP) coenzyme binding motif
(GX.sub.3DX.sub.7AX.sub.8EDX.sub.14GX.sub.13KX.sub.4-5geGGX.sub.19G)
(SEQ ID NO:92) including the conserved lysine residue can also be
found in these four proteins (Piepersberg, 1994).
[0265] The mitB gene is predicted to start at a GTG (position 1879)
that is preceded by a presumed RBS (GGAACG) (SEQ ID NO:93). This
gene encodes a 272 amino acid protein with a deduced M.sub.r or
28,648 Da and a deduced pI of 6.06. Database sequence homology
searches revealed that the product of mitB shows local sequence
similarity with a group of O-glycosyltransferases involved in
polysaccharide biosynthesis. One segment of 70 amino acid residues
at the N-terminus of MitB has 43% similarity (36% identity) with
the two glycosyltransferases SpsL and SpsQ from Sphingomonas S88,
and ExoO form Rhizobium meliloti involved in polysaccharide (S88)
and succinoglycan biosynthesis, respectively (Becker et al., 1963).
Another 60 amino acid residues located at the C-terminus displayed
30% identity with UDP-GalNAc:polypeptide
N-acetylgalactosaminyltransferase from Mus musculus and Homo
sapiens (Bennett et al., 1996).
[0266] The third ORF, mitC, starts from the ATG at position 2694,
which is coupled to the stop codon TGA of mitB and encodes a
putative protein of 260 amino acids with a molecular mass of 27,817
Da and a pI of 10.45. Database searches with the deduced protein
product showed significant similarity over the first 90 amino acids
(38% identity, 40% similarity) with the ImbE gene product (unknown
function) from Mycobacterium leprae (U15183).
[0267] Insertional disruption of the mitA and mitB genes in
Streptomyces lavendulae. To test the dependence of functional mitA
and mitB genes for MC biosynthesis, gene disruption constructs were
generated for subsequent isolation of the corresponding S.
lavendulae isogenic mutant strains.
[0268] The mitA disruption construct was made by replacing a 155 bp
fragment between the two MscI sites (located at the C-terminus of
the mitA gene in pDHS7601) with the 1.1 kb SmaI-BamHI fragment
containing a thiostrepton resistance gene from pDHS5000 (FIG. 11A).
This replacement regenerated a BamHI site at the junction and the
resulting construct was then subcloned into the E.
coli-Streptomyces conjugative shuttle plasmid pKC1139, followed by
conjugation into S. lavendulae. A double crossover mutant strain
(MV100) was selected based on the expected phenotype (thiostrepton
resistant, apramycin sensitive), and further confirmed by Southern
blot hybridization. Genomic DNA from wild-type S. lavendulae and
MV100 was digested with BamHI and SphI, and hybridized with the 4.9
kb EcoRI-HindIII tsr-disrupted mitA fragment from pDHS2001. As
expected, the 4.0 kb SphI hybridized band in the wild-type strain
was shifted to 4.9 kb in MV100, whereas the 3.8 kb BamHI
hybridization and in the wild-type was converted to two bands (2.2
kb and 2.5 kb) in the mutant (FIG. 11B).
[0269] The mitB gene was disrupted by inserting a neomycin
resistance gene (aphII) into the BstBI site (located at the 5'-end
of mitB) (FIG. 12A). Transconjugants were selected on
kanamycin/apramycin plates, and a double crossover mutant strain
(MM101) was identified with a kanamycin-resistant,
apramycin-sensitive phenotype and subsequently confirmed by
Southern blot hybridization. As expected, the 3.8 kb BamHI
hybridization band in wild-type S. lavendulae was shifted to 5.2 kb
in MM101, whereas a 5.2 kb SacI hybridization band was shifted to
6.6 kb (FIG. 12B).
[0270] mitA and mitB disrupted strains (MV100 MM101) are blocked in
MC biosynthesis. The growth characteristics and morphology of MV100
and MM1001 in liquid media and on agar plates was identical to
wild-type S. lavendulae. HPLC was used to quantify production of MC
in MV100 and MM101 (FIG. 13A), and culture extracts were used in a
biological assay to test for presence of the drug (FIG. 13B).
Injection of one mg of wild-type S. lavendulae culture extract gave
a peak in the HPLC that eluted with the same retention time as the
MC standard. Upon injection of one mg of culture extract from the
mitA or mitB disrupted strains (MV100, MM101) no MC peak was
observed. To corroborate the lack of production of MC, the HPLC
eluant obtained from the MV100 culture extracts was collected over
the retention time range determined for MC. This eluant completely
lacked biological activity against Bacillus subtilis (the MC target
strain) while the fraction collected from the same retention time
region of wild-type S. lavendulae and the vector control strain
culture extracts showed substantial levels of biological activity
(FIG. 13B).
[0271] It is important to note that the presence of the vector
pKC1139 in S. lavendulae reduced the percentage of MC in the total
crude extract while simultaneously increasing the total amount of
material extractable by ethyl acetate. The combination of these two
effects reduces the absolute amount of MC by approximately 25% in
the vector control culture crude extract compared to the wild-type
crude extract.
[0272] Exogenous AHBA can restore MC production in the MC-deficient
MitA K191A mutant. Although complementation of MV100 (mitA
insertional disruptant) was attempted by providing exogenous
3-amino-5-hydroxybenzoic acid in the culture medium, MC production
was not restored as measured by HPLC or biological assay. A polar
effect on genes downstream of tsr-disrupted mitA in MV100 appeared
likely since supplying mitA in trans on a medium copy number
plasmid (MV103) also failed to restore MC production. Therefore,
site-directed mutagenesis was employed to generate a MitA K191A
mutant resulting in strain MV102. Kim et al. (1998) had
demonstrated that the AHBA synthase from A. mediterranei is PLP
dependent and catalyzes the aromatization of
5-deoxy-5-amino-3-dehydroshikimic acid (aminoDHS). Thus, the
nitrogen of the conserved lysine 191 is supposed to form a Schiff
base with the PLP cofactor. Replacement of lysine 191 with alanine
prevents binding of the cofactor and eliminates enzymatic activity.
Replacement of the AGG encoding lysine 191 in wild-type S.
lavendulae with a GCC codon in MV102 was confirmed by nucleotide
sequence analysis. As expected, MV102 did not produce MC, however,
when the culture medium was supplemented with exogenous AHBA, MC
production was restored as determined by MS ([M+Na].sup.+=357),
HPLC and TLC analysis (Table 2).
3TABLE 2 Complementation results with (+) or without (-) AHBA. S.
lavendulae MC production strains -AHBA +AHBA Wild-type + + MV100 -
- MV103 - - MV102 - +
[0273] Discussion
[0274] An effective strategy for the identification of natural
product biosynthetic gene clusters in actinomycetes has included
cloning of antibiotic resistance genes followed by investigation of
adjacent DNA for the presence of structural and regulatory genes
(Butler et al., 1989, Donadio et al., 1991; Motamedi and
Hutchinson, 1987; Vara et al., 1985). Although linkage of
antibiotic resistance and biosynthetic genes appears to be a
general feature in prokaryotes, a growing number of examples
involve the existence of multiple resistance loci that may be
linked or unlinked to the biosynthetic gene cluster (Vara et al.,
1985; Seno and Baltz, 1989; Smith et al., 1995). The identification
and characterization of two genetically unlinked resistance loci
(August et al, 1994; Sheldon et al., 1997) for MC created a dilemma
for mounting an effective search for the MC biosynthetic gene
cluster. However, the use of the AHBA synthase gene from A.
mediterranei provided an effective probe to identify cosmid clones
bearing a linked MC resistance gene. Thus, the isolation of several
cosmid clones form an S. lavendulae genomic DNA library that
hybridized to both the A. mediterranei AHBA synthase gene and the
S. lavendulae mrd gene indicated that the MC biosynthetic gene
cluster resided on DNA adjacent to mrd. DNA sequence analysis of
the 3.8 kb BamHI fragment revealed three ORFs whose deduced protein
sequences corresponded to an AHBA synthase, a glycosyltransferase,
and a ImbE-like product.
[0275] As determined by precursor feeding experiments, the mitosane
core is formed through the condensation of AHBA and D-glucosamine
(Hornemann, 1981). AHBA is thought to be derived from the
ammoniated shikimate pathway from PEP and E4P, in which the last
step from aminoDHS to AHBA is catalyzed by AHBA synthase (FIG. 7)
(Kim et al., 1996; Kim et al., 1998). Meanwhile, the reaction of
attaching an activated sugar residue to a core compound is usually
catalyzed by a group of enzymes called glycosyltransferases as
specified by macrolide, glycopeptide antibiotic and polysaccharide
biosynthesis (Kahler et al., 1996; Otten et al., 1995b; Solenberg
et al., 1997; Yamazaki et al., 1996). In principle, the
condensation of AHBA with D-glucosamine can be initiated in two
different ways (FIG. 7). One would involve the formation of the
C.sub.8a-C.sub.9 bond by an electrophilic aromatic alkylation or
acylation. A second possibility would be formation of a Schiff base
between the nitrogen of AHBA and the D-glucosamine C1 aldehyde,
followed by ring closure at C.sub.8a-C.sub.9. In either case, a C-
or N- instead of O-glycosyltransferase is expected. Although
previously described glycosyltransferases display a high degree of
sequence divergence (Yamazaki et al., 1996), the mechanistic
similarity with O-glycosyl transfer may suggest that mitB encodes a
N-glycosyltransferase that initiates the formation of the mitosane
system by linking glucosamine to AHBA. The mitA and mitB genes and
their corresponding products are likely candidates to mediate
formation of AHBA and the mitosane ring system, respectively.
However, the possible function of the lmbE-like protein remains
unclear, since its current role within lincomycin biosynthetic
pathway of S. lincolnensis is not known (Peschke, 1995).
[0276] The involvement of AHBA synthase (mitA) and the putative
glycosyltransferase (mitB) in MC biosynthesis was established by
gene disruption to create mutants blocked in MC biosynthesis. This
required development of a method to introduce DNA into S.
lavendulae NRRL 2564 since the strain remains refractory to
traditional Streptomyces protoplast and electroporation-mediated
transformation procedures. Other such refractory strains include,
but are not limited to, ATCC 27422. The modified Bierman protocol
(Bierman et al., 1992) was used to affect efficient conjugative
transfer into S. lavendulae using the E. coli-Streptomyces shuttle
plasmid pKC1139. This result is significant because it permits the
development of an effective system for analyzing in detail the
genes involved in mitomycin biosynthesis.
[0277] The function of mitA was probed by providing strains MV100
and MV102 with exogenous 3-amino-5-hydroxybenzoic acid in the
culture medium. Despite repeated attempts to complement MV100, MC
production was not restored as measured by HPLC or biological
assay. It is believed that insertion of the tsr gene into mitA
resulted in disruption of biosynthetic genes immediately
downstream, since supplying mitA in trans on a medium copy number
plasmid also failed to restore MC production to MV100. This
putative polar effect was eliminated by generating the MitA K191A
mutant strain MV102. Providing exogenous 3-amino-5-hydroxybenzoic
acid to this mutant strain of S. lavendulae restored production of
MC as shown by TLC, HPLC and mass spectrometry. When MV102 was
grown in the absence of AHBA, there was no detectable production of
MC. The ability of 3-amino-5-hydroxybenzoic acid to complement the
mutant MitA protein further supports the function of MitA as an
AHBA synthase as indicated by the database protein sequence
alignment and previous studies on rifK (Kim et al., 1998).
EXAMPE 3
Mitomycin Resistance in Streptomyces lavendulae Includes a
Drug-Binding Protein-Dependent Export System
[0278] As a prodrug, MC is unreactive until chemical or enzymatic
reduction renders the molecule a highly effective alkylating agent
(Iyer and Szybalski, 1964). The molecular basis of MC bioactivity
derives mainly from its propensity to covalently interact with DNA
at 5'-CpG sequences, causing lethal intra- and inter-strand
crosslinks as well as monofunctional alkylation (Tomasz, 1995).
[0279] S. lavendulae encounters a daunting challenge in avoiding
potentially lethal MC-mediated crosslinks since it has a
chromosomal G+C content of over 70%, which translates into at least
one million potential drug target sites per cell. Indeed, two
genetic loci that mediate mitomycin resistance have been reported
in this organism. One locus (mcr) encodes a protein (MCRA) that
catalyzes oxidation of the reduced, bioactivated species of MC via
a redox relay mechanism (August et al., 1994; Johnson et al.,
1997). The second locus (mnrd) encodes MRD that functions to
sequester the prodrug by a specific mitomycin-binding protein
(Sheldon et al., 1997). A paradox of current knowledge regarding
mitomycin resistance has been the lack of a clear mechanism for
drug transport. Indeed, the observed stoichiometry suggests that it
would be ineffective for S. lavendulae to utilize MRD as a solo
mechanism for cellular self-protection. Pathogenic bacteria
(Nikaido, 1994), and antibiotic-producing microorganisms
(Cundliffe, 1992; Mendez and Salas, 1998), employ export of toxic
compounds as a means of resistance.
[0280] Materials and Methods
[0281] Bacterial strains, culture conditions, and media. E. coli
DH5.alpha. used as a host for generation of double-stranded plasmid
DNA, was grown at 37.degree. C. on LB medium. E. coli BL21 (DE3),
used as host for protein expression, was grown at 37.degree. C. in
NZCYM medium (Sambrook et al., 1989). S. lavendulae NRRL 2564 was
grown on YEME medium (Hopwood et al., 1985) at 30.degree. C. for
preparation of genomic DNA.
[0282] DNA preparation and amplification. S. lavendulae genomic DNA
was isolated by the lysozyyne-2X Kirby mix method (Hopwood et al.,
1988). General DNA manipulation was performed as described
previously (August et al., 1994). Oligonucleotides for PCR and
sequencing were obtained from Gibco BRL. PCR amplifications were
carried out using a Hybaid thermal cycler (Hybaid Ltd., Teddington,
U.K.).
[0283] Cloning and sequencing of mct. A S. lavendulae NRRL 2564
genomic DNA library was constructed in the cosmid vector pNJ1 (Tuan
et al., 1990) as previously described (August et al., 1994). The
insert DNA of a cosmid clone containing sequences flanking mrd was
digested with BamHI and subcloned into the BamHI site of pUC119.
Using exonuclease III (Erase-A-Base kit, Promega, Madison, Wis.), a
set of nested deletion clones was generated and both strands of the
insert DNA were sequenced by the dideoxy chain termination method
using the ABI Prism kit (PE Applied Biosystems) in coordination
with an ABI 373 automated sequencer. 10% DMSO was added to the
reactions to reduce compressions. Sequence data was analyzed using
the GeneWorks (Oxford Molecular) software package. Deduced amino
acid sequence data were compared to the available databases using
the BLAST program of the Genetics Computer Group version 9.0
software (Oxford Molecular Group). The met gene has been deposited
in the GenBank database under Accession No. AF120930.
[0284] Construction of the met mutant strain of S. lavendulae. The
mct disruption vector pDHS7704 was constructed as follows. pDHS7661
was digested with EcoRI, blunt-ended, and ligated with the 1.4 kb
neomycin resistance gene fragment from pFD666 (ApaLI-HindIII
digestion, blunt-ended) (Ames, 1986). The 5.4 kb EcoRI-HindIII
fragment from the resulting construct (pDHS7703) was subcloned into
pKC1139 to create pDHS7704, and conjugated into S. lavendulae
according to Bierman et al. (1992). A met double crossover mutant
was selected after propagating transconjugants on R5T plates for
five generations at 39.degree. C. Kanamycin-resistant and
apramycin-sensitive colonies were further tested by Southern blot
to confirm the desired double crossover genotype.
[0285] Construction of mct expression plasmid. For the construction
of the E. coli expression plasmid NdeI and HindIII sites were
introduced at the translational start codon and downstream of the
translational stop codon of mct, respectively. The primers used for
PCR were 5'-GGGAATTCCATATGATGCAGTCCATGTCAC-3' (SEQ ID NO:94) and
5'-GGGAATTCAAGCTTTCATTCCGCCGGGGTC-3' (SEQ ID NO:95). The PCR was
carried out using 2.5 U of Taq polymerase, 0.4 .mu.g of each
primer, 1 .mu.g of pDHS7661 DNA as template, 10 mM each of
dATP-dGTP-dCTP-dTTP, 1.5 mM MgCl.sub.2, and 10 .mu.l of
10.times.Promega PCR buffer in a total volume of 100 .mu.l.
Amplification was achieved with 30 cycles of denaturation at
94.degree. C. for 30 seconds, annealing at 37.degree. C. for 1
minute, and extension at 70.degree. C. for 2 minutes. The 1.45 kb
PCR product was recovered by 0.8% agarose gel electrophoresis,
digested with NdeI-HindIII and ligated into the T7 expression
plasmid pET17b (Novagen), which had been similarly cut with
EcoRI-HindIII, to give pDHS7023. pDHS7023 was introduced by
transformation into E. coli BL21(DE3) to provide strain PJS102.
[0286] Construction of mct-mrd co-expression plasmid. From plasmid
pDHS7006 (Sheldon et al., 1997), a 2.1 kb SspI fragment was
isolated. The fragment contained the mrd gene under the control of
the T7 promoter, including transcriptional terminator sequences
(rrnB T1) upstream and downstream of mrd. The fragment was ligated
into the MC-translocase construct pDHS7023, which had been cut with
MscI, to give pDHS7024. pDHS7024 was introduced by transformation
into E. coli BL21 (DE3) to result in strain PJS103.
[0287] MC resistance phenotype of E. coli. To analyze resistance
conferred by the expression of the MC-translocase in E. coli, 10
.mu.l of strain PJS102 was spread on LB agar medium containing 100
.infin.g/ml of ampicillin, IPTG to a final concentration of 1.0 mM,
and various concentrations of MC. The cultures were grown overnight
at 37.degree. C. and colony-forming units (CFUs) were deternined.
Similarly, the MC resistance phenotype of strain PJS103 (mcr-mrd
co-expression strain) was quantified.
[0288] [.sup.3H]-MC uptake assay of strains PJS102 and PJS103.
[.sup.3H]-MC was obtained from Kyowa Hakko Kogyo, Ltd. Uptake
studies were performed for whole cells of PJS100, PJS102, PJS103
and E. coli BL21(DE3)::pT7SC and pET17b. PJS100, PJS102, and PJS103
as well as vector-only cultures were cultured (37.degree. C.) in 5
ml of NCZYM medium with IPTG added to a final concentration of 1 mM
(at approximately 3 hours growth). At 9 hours (late exponential
phase), cells were harvested by centrifugation and resuspended in 1
ml NCZYM broth (5.times.concentration). The concentrated suspension
of late-exponential growth phase cells was exposed to [.sup.3H]-MC
(59 Ci/mmol) at a final concentration of 0.022 .mu.g/ml (0.0655
nmol). Aliquots (100 .mu.l) were removed at frequent intervals,
placed on 1.2 .mu.M GF/C filters (Whatman International, Maidstone,
U.K.) and washed once with 6 ml of 0.85% NaCl poured over the
filters under vacuum pressure. Additional aliquots were
simultaneously removed for determination of protein content
(protein assay kit, Bio-Rad Laboratories, Richmond, Calif.).
Radioactivity on the filters was quantified using a Beckman LS7000
scintillation counter. Results were expressed as nanograms of
mitomycin per milligram of cell protein.
[0289] Results
[0290] A gene encoding a transmembrane protein is physically linked
to mrd. DNA sequence analysis of a cosmid clone containing the mrd
locus, a previously characterized MC resistance determinant
(Sheldon et al., 1997), identified an open reading frame (ORF)
encoding a polypeptide predicted to be highly hydrophobic that
shows similarity to a variety of antibiotic export proteins in
drug-producing actinomycetes. Significantly, the gene (mct, SEQ ID
NO:72) encoding the putative mitomycin exporter (MC-translocase;
MCT) protein is located within 5 kb of mrd (SEQ ID NO:64) and is
physically linked to the mitomycin biosynthetic gene cluster (FIG.
15).
[0291] Sequence analysis of the mct locus. Nucleotide sequence
analysis of cosmid clone pDHS7547 revealed an ORF predicted to
start with the ATG codon at position 132 and end with the TGA codon
at nucleotide 1587 (FIG. 16), resulting in a 484 amino acid
polypeptide with a predicted molecular weight of 50,023 daltons.
Comparison of the deduced amino acid sequence of the mct gene with
proteins in the available databases revealed significant similarity
to several integral membrane proteins that confer drug resistance.
These include the CmcT protein from the cephamycin producer,
Nocardia lactamdurans (Coque et al., 1993), the Pur8 protein from
the puromycin producer, Streptomyces alboniger (Tercero et al,
1993), the Mmr protein from the methylenomycin producer,
Streptomyces coelicolor (Neal and Chater, 1987), and the LmrA
protein from the lincomycin producer, Streptomyces lincolnensis
(Zhang et al., 1992). The similarities of the mct gene product and
related proteins extend over the entire sequences, with the highest
levels of similarity found within the amino-terninal regions (FIG.
17).
[0292] Within the N-terminal regions of several antibiotic efflux
proteins, including Mmr and LmrA, several highly conserved
structural motifs have been noted. The .beta.-turn motif
(VxGxLxDxxGRKxxxL), found within the highly conserved cytoplasmic
loop sequence separating transmembrane domains two and three of
most eukaryotic and prokaryotic transport proteins, is clearly
evident in MCT at positions 79-95 (FIG. 16). A motif (LDxTVxNVALP)
found at the end of transmembrane domain one, specific to the 14
transmembrane segment family (Paulson and Skurray (1993)) is
present in MCT at positions 41-51 (FIG. 16). In addition, several
other invariant motifs are apparent in the MCT sequence.
[0293] Transmembrane proteins that mediate resistance to
antibiotics and antiseptics by active efflux are highly related,
usually containing 12 or 14 transmembrane regions. Notably, the
actinomycete drug transport proteins that share homology with MCT
appear to contain 14 transmembrane spanning regions and constitute
a family of drug resistance translocases. Utilizing the membrane
structure and topology program MEMSAT (University College, London),
and hydropathy analyses based on the algorithm of Kyte and
Doolittle (1982), a prediction of 14 transmembrane spanning domains
was made for the deduced amino acid sequence of MCT (FIG. 18).
[0294] Inactivation of met results in greater sensitivity to MC. To
establish a physiological role for MCT in S. lavendulae, the
corresponding gene (mct) was inactivated by insertion of the aphII
gene from transposon Tn5 to give pDHS7704. After conjugal transfer
of pDHS7704 from E. coli to S. lavendulae and growth of the
transconjugants under selective conditions, targeted replacement of
native met was achieved by double crossover homologous
recombination. Gene disruption was confirmed by Southern blot
hybridization of total DNA from the S. lavendulae wild-type and
mutant with a DNA probe that included the mct locus. Analytical
digests of the genomic DNA resulted in detection of the predicted
band shifts in the mutant and wild-type strains (FIG. 19). The S.
lavendulae met disruption mutant strain (MM105) exhibited an
approximately 25-fold reduction in resistance to MC when exposed to
100 .mu.g of MC per ml of medium (Table 3). In media lacking MC,
the growth kinetics of the strain MM105 was comparable to the
wild-type S. lavendulae strain.
4TABLE 3 Resistance of S. lavendulae strains to varying
concentrations of MC Plate count CFU/ml Strain Concentration S.
lavendulae mct mutant of MC (.mu.g/ml) S. lavendulae wild-type (MM
105) 10 >10.sup.7 >10.sup.7 20 >10.sup.7 >10.sup.7 40
5.3 .times. 10.sup.3 2.6 .times. 10.sup.3 80 2.6 .times. 10.sup.3
2.4 .times. 10.sup.2 100 2.0 .times. 10.sup.3 8.0 .times.
10.sup.1
[0295] Expression of mct in E. coli. To investigate further the
function of mct, heterologous expression of the gene in E. coli was
pursued. mct was amplified by PCR and cloned into the protein
expression vector pET17b to give pDHS7023. pDHS7023 was then
introduced into E. coli BL21(DE3) to give strain PJS102. After
disruption of the cells by sonication, MCT was found to be
associated mainly with the membrane fraction of the cell lysate, as
expected for an integral membrane protein. To determine if strain
PJS102 was resistant to MC, cultures were grown up and plated on
agar medium containing various concentrations of MC. Significantly,
IPTG-induced cultures of PJS102 exhibited resistance to MC at drug
concentrations 5-fold greater than those for E. coli BL21(DE3)
containing vector alone (Table 4).
5TABLE 4 MC resistance of mct, mrd expressing E. coli strains Plate
count CFU/ml Strain Concentration BL21(DE3):: of MC(.mu.g/ml)
pET17b PJS100 PJS102 PJS103 0.0 >10.sup.7 >10.sup.7
>10.sup.7 >10.sup.7 0.01 >10.sup.7 >10.sup.7
>10.sup.7 >10.sup.7 0.1 7.3 .times. 10.sup.3 >10.sup.7
>10.sup.7 >10.sup.7 0.5 3.2 .times. 10.sup.2 >10.sup.7 2.1
.times. 10.sup.5 >10.sup.7 1.0 0.0 3.3 .times. 10.sup.6 5.9
.times. 10.sup.4 >10.sup.7 2.5 -- NA.sup.a 2.0 .times. 10.sup.2
>10.sup.7 5.0 -- 2.7 .times. 10.sup.6 0.0 >10.sup.7 10 -- 6.1
.times. 10.sup.5 -- >10.sup.7 20 -- 2.5 .times. 10.sup.5 --
>10.sup.7 30 -- 5.0 .times. 10.sup.2 -- >10.sup.7 60 -- 0.0
-- >10.sup.7 80 -- -- -- 1.4 .times. 10.sup.5 100 -- -- -- 9.6
.times. 10.sup.3 150 -- -- -- 3.0 .times. 10.sup.1 Mitomycin B --
>10.sup.7b NA.sup.c >10.sup.7d .sup.aDid not test strain
against this concentration of MC .sup.bMitomycin B tested at a
concentration of 1.0 .mu.g/ml .sup.cDid not test strain against
mitomycin B .sup.dMitomycin B tested at a concentration of 15
.mu.g/ml
[0296] Co-expression of mct and mrd in E. coli. To address the
notion that MRD and MCT proteins participate as components of a
binding protein-dependent drug export system, the mct and mrd genes
were co-expressed in E. coli. From plasmid pDHS7006 (mrd expression
construct) (Sheldon et al., 1997), a DNA fragment containing the
mrd gene under the control of the T7 promoter was ligated into
pDHS7023 to give pDHS7024. pDHS7024 was then introduced into E.
coli BL21(DE3) to give strain PJS103. To determine if strain PJS103
was resistant to MC, cultures were grown up and plated on agar
medium containing various concentrations of MC. Significantly,
EPTG-induced cultures of PJS103 exhibited resistance to MC at drug
concentrations 300-fold greater than those for E. coli BL21(DE3)
containing vector alone (150 .mu.g/ml vs. 0.5 .mu.g/ml of MC; Table
4). In addition to PJS103 maintaining levels of resistance over
that of the vector control strain, co-expression of mct and mrd
confers MC resistance at drug concentrations 5 and 60-fold greater
compared to PJS100 (containing the mrd gene alone) (Sheldon et al.,
1997) or strain PJS102 (containing the mct gene alone),
respectively. Strain PJS103 also displayed high-level resistance to
mitomycin B (Table 4), a mitomycin also produced by S.
lavendulae.
[0297] MC uptake by E. coli cells expressing mct, mrd or mct/mrd.
Since the deduced amino acid sequence of the mct gene was similar
to antibiotic export proteins, reduced accumulation of MC in
MCT-expressing cells would be expected. An assay, modeled after
experiments used to study tetracycline efflux-mediated resistance
in E. coli (Levy and McMurry, 1978), was designed to study the
uptake of [.sup.3H]-MC by the susceptible vector control and
resistant mct, mrd and mct/mrd expressing E. coli strains.
[0298] MC accumulation by the susceptible vector control strain
(BL21(DE3)::pET17b) was found to reach a maximum level at 5 minutes
and thereafter maintained at constant concentrations. In contrast,
the quantity of MC accumulation in the resistant, mct-expressing
strain (PJS102) was only 25% of the susceptible control at 5
minutes, and thereafter remained at reduced concentrations (FIG.
21). Reduced accumulation of drug in PJS102 suggests that mct
encodes a protein that facilitates MC export from the cell. To
determine if the co-expression of mct and mrd in E. coli also
resulted in reduced accumulation of MC, strain PJS103 was analyzed
using the [.sup.3H]-MC uptake assay. Analyses of drug uptake by
cultures of strain PJS100 (Sheldon et al., 1997) were also
performed to determine drug accumulation levels in this MC
resistant E. coli strain.
[0299] The results show a clear difference in MC accumulation
between the MC sensitive and resistant strains. Compared to E. coli
cells bearing vector alone, MC accumulation in PJS103 was only 35%
at 5 minutes and thereafter remained at reduced concentrations. The
accumulation of drug in strain PJS103 was found to parallel that of
strain PJS102, albeit at slightly higher levels (about 23% greater)
of drug over the course of the experiment. Interestingly, strain
PJS100, although resistant to significant concentrations of MC,
accumulated drug to levels 42% higher than the drug-sensitive
vector control at 30 minutes (FIG. 20).
[0300] Discussion
[0301] Most antibiotics inhibit bacterial growth by binding to
proteins or other macromolecular components that involve essential
metabolic processes of the cell (Cundliffe, 1992). For instance,
DNA alkylation by MC results in disruption of chromosomal
replication leading to cell death (Iyer and Szybalski, 1964). In
many antibiotic-producing streptomycetes, macromolecular target
site(s) are likewise susceptible to endogenous cytotoxic compounds
(that is certainly the case in S. lavendulae). Thus, pumping the
antibiotic out of the cell at a rate equal to its production and/or
re-uptake would prevent drug access to intracellular target sites.
Based on the levels of drug found in most antibiotic fermentation
broths (concentrations of intracellular drug being low), it is
apparent that drug-producing organisms often depend on efficient
antibiotic transport mechanisms. Indeed, a growing number of
membrane systems implicated in transport (and therefore resistance)
of a variety of antibiotics have been discovered in drug-producing
streptomycetes (Mendez and Salas, 1998; Paulsen et al., 1996).
[0302] In general, genes coding for drug export proteins are
physically linked to the corresponding biosynthetic genes within
the genome of the antibiotic-producing microorganism. Presumably,
the tight linkage of antibiotic export and biosynthetic genes
ensures coordinate gene regulation. Interestingly, the presence of
back-to-back and overlapping divergent promoters of antibiotic
export and regulatory genes has been observed within the
tetracenomycin (Guilfoile and Hutchinson, 1992) and actinorhodin
(Caballero et al, 1991) biosynthetic gene clusters. Conforming to
this example, S. lavendulae possesses a gene coding for an integral
membrane drug export protein within the mitomycin biosynthetic gene
cluster. Analysis of the deduced amino acid sequence of MCT
revealed several similarities with actinomycete proteins predicted
to function as drug exporters. By virtue of homology to
tetracycline resistance proteins, which have been shown to use
proton motive force to energize transport (Littlejohn et al.,
1992), the actinomycete drug resistance translocases cited in this
study are predicted to power excretion by a proton-dependent
electrochemical gradient. It has been suggested that highly
conserved sequences within the amino-terminal regions of these
proteins play a role in proton translocation (Rouch et al., 1990),
while the less well-conserved C-terminal regions may be involved in
drug binding (Paulsen et al., 1996; and references therein) or
recognition of a protein-drug complex.
[0303] Disruption of met in S. lavendulae resulted in a 25-fold
increase in sensitivity to exogenously added MC, providing evidence
that MCT maintains a role in providing drug resistance in S.
lavendulae. Although the effect is significant, alternative
mechanisms of cellular self-protection clearly continue to operate.
This evidently includes MCRA, the novel redox-relay protein that
re-oxidizes activated MC in S. lavendulae. It is also likely that
unidentified xenobiotic transporters provide an alternative mode of
drug transport in the absence of MCT, albeit with lower
efficiency.
[0304] In order to probe the ability of MCT to transport drug in
the presence and absence of the MC binding protein, accumulation of
[.sup.3H]-MC in E. coli was analyzed. Expression of met in E. coli
resulted in MC-resistant cultures that accumulated lower levels of
drug than strains bearing vector control (FIG. 20). Interestingly,
strain PJS102 (expressing mct only) accumulates less drug
intracellularly than strain PJS103 (expressing mrd and met) (FIG.
20). Increased drug accumulation in strain PJS100 may lend support
to the model of equimolar binding between MRD and MC (Sheldon et
al., 1997). Significantly higher levels of drug accumulation in
strain PJS100 may be the result of intracellular sequestration of
MC by MRD. Accordingly, the presence of MRD could also account for
the slightly greater levels of MC accumulation in strain PJS103
(co-expressing mct-mrd) as compared to strain PJS102 (expressing
met alone). Comparable to binding protein-dependent import systems
(Miller et al., 1983), the binding of MC by MRD may be
rate-limiting in the drug excretion process.
[0305] Taken together, these results suggest that cellular
protection afforded by MCT is a function of drug transport from the
cytoplasm. Interestingly, co-expression of mrd and met in E. coli
led to cultures that are dramatically more resistant to exogenously
added drug. While normally required for the transport systems with
which they are associated, in many instances binding proteins are
not integral to the process of solute translocation (Higgins et
al., 1990). Similarly, the presence of MRD is not required for MC
translocation but dramatically enhances drug tolerance. Hence, the
binding protein (MRD) may be considered an accessory component, a
rather specific adaptation required for optimal drug resistance.
The drug-resistance phenotype of E. coli strains expressing met
alone and in combination with mrd along with the MC uptake analysis
of these strains provides evidence that MRD and MCT are components
of a novel drug transport system. Such a resistance mechanism,
sequestering the intact drug for efficient excretion to the
environment, represents a unique cellular strategy for
self-preservation by the MC-producing organism.
[0306] References
[0307] Alderson, G., D. A. Ritchie, C. Caballero, R. H. Cool, N. M.
Ivanova, A. S. Huddleston, C. S. Flaxman, V. Kristufek, and A.
Lounes. Physiology and genetics of antibiotic production and
resistance. Res. Microbiol., 144, 665-672 (1993).
[0308] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and D.
J. Lipman. Basic local alignment search tool, J. Mol. Biol., 215,
403-410 (1990).
[0309] Ames, G. Bacterial periplasmic transport systems: structure,
mechanism, and evolution. Ann. Rev. Biochem., 55, 397-425
(1986).
[0310] Anderson, M. G., Kibby, J. J., Rickards, R. W. and J. M.
Rothschild. Biosynthesis of the mitomycin antibiotics from
3-amino-5-hydroxybenzoic acid, J. Chem. Soc. Chem. Commun.,
1277-1278 (1980).
[0311] Angell, S., Schwarz, A., and M. J. Bibb. The glucose kinase
gene of Streptomyces coelicolor A3(2): its nucleotide sequence,
transcriptional analysis and role in glucose repression, Mol.
Microbiol., 6, 2833-2844 (1992).
[0312] August, P. R., Flickinger, M. C. and D. H. Sherman. Cloning
and analysis of a locus (mcr) involved in mitomycin C resistance in
Streptomyces lavendulae, J. Bacteriol., 176, 4448-4454 (1994).
[0313] August, P. R., Tang, L., Yoon, Y. J., Ning, S., Muller, R.,
Yu, T. W., Taylor, M., Hoffhann, D., Kim, C. G., Zhang, X.,
Hutchinson, C. R. and H. G. Floss. Biosynthesis of the ansamycin
antibiotic rifamycin: deductions from the molecular analysis of the
rif biosynthetic gene cluster of Amycolatopsis mediterranei S699,
Chem. Biol., 5, 69-70 (1998).
[0314] Baltz, R. H. Genetic recombination in Streptomyces fradiae
by protoplast fusion and cell regeneration, Dev. Ind. Microbiol.,
21, 43-54 (1980).
[0315] Baltz, R. H., and T. J. Hosted. Molecular genetic methods
for improving secondary-metabolite production in actinomycetes,
Trends Biotech., 14:245-250 (1996).
[0316] Beck, A., A. Kleickmann, M. Keller, W. Arnold, and A.
Puhler. Identification and analysis of the Rhizobium meliloti
exoAMONP genes involved in exopolysaccharide biosynthesis and
mapping of promoters located on the exoHKLAMONP fragment, Mol. Gen.
Genet., 241, 367-379 (1993).
[0317] Becker, A. M., Herlt, A. J., Hilton, G. L., Kibby, J. J. and
R. W. Rickards. 3-Amino-5-hydroxybenzoic acid in antibiotic
biosynthesis, VI. Directed biosynthesis studies with ansamycin
antibiotics, J. Antibiot., 36, 1323-1328 (1983).
[0318] Bennett, E. P., H. Hassan, and H. Clausen. cDNA cloning and
expression of a novel human UDP-N-acetyl-alpha-D-galactosamine.
Polypeptide N-acetylgalatosaminyltransferase, GalNAc-t3. J. Biol.
Chem., 271, 17006-17012 (1996).
[0319] Berdy, J. Are actinomycetes exhausted as a source of
secondary metabolites?, p. 13-14. In V. Debabov, Dudnik, Y. And
Danlienko, V. (eds.), Ninth International Symposium on the Biology
of Actinomycetes. All-Russia Scientific Research Institute for
Genetics and Selection of Industrial Microorganisms, Moscow
(1995).
[0320] Bezanson, G. S. and L. C. Vining. Studies on the
biosynthesis of mitomycin C by Streptomyces verticillatus, Can. J.
Biochem., 49, 911-918 (1971).
[0321] Biermnan, M., Logan, R., O'Brien, K., Seno, E. T., Rao, R.
N. and B. E. Schoner. Plasmid cloning vectors for the conjugal
transfer of DNA from Escherichia coli to Streptomyces spp., Gene,
116, 43-49 (1992).
[0322] Blattner, F. R., Plunkett, G. R., Bloch, C. A., Perna, N.
T., Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D.,
Rode, C. K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick,
H. A., Goeden, M. A., Rose, D. J., Mau, B. and Shao, Y., the
complete genome sequence of Escherichia coli K-12, Science, 277,
1453-74 (1997).
[0323] Bouvier-Nave, P., Husselstein, T., Desprez, T. and
Benveniste, P., Identification of cDNAs encoding sterol
methyl-transferases involved in the second methylation step of
plant sterol biosynthesis, Euro. J. Biochem., 246, 518-29
(1997).
[0324] Boyer, M. J., Bioreductive agents: a clinical update, Oncol.
Res., 9, 391-395 (1997).
[0325] Brown, W. C., and J. L. Campbell. A new cloning vector and
expression strategy for genes encoding proteins toxic to
Escherichia coli, Gene, 127:99-103 (1993).
[0326] Bult, C. J., White, O., Olsen, G. J., Zhou, L., Flesichmann,
R. D., Sutton, G. G., Blake, J. A., FitzGerald, L. M., Clayton, R.
A., Gocayne, J. D., Kerlavage, A. R., Dougherty, B. A., Tomb, J.
F., Adams, M. D., Reich, C. I., Overbeek, R., Kirkness, E. F.,
Weinstock, K. G., Merrick, J. M., Glodek, A., Scott, J. L.,
Geoghagen, N. and J. C. Venter. Complete genome sequence of the
methanogenic archaeon, Methanococcus jannaschii, Science, 273,
1058-1073 (1996).
[0327] Butler, M. J., E. J. Friend, I. S. Hunter, F. S. Kaczmarek,
D. A. Sugden, and M. Warren. Molecular cloning of resistance genes
and architecture of a linked gene cluster involved in biosynthesis
of oxytetracycline by Streptomyces rimosus. Mol. Gen. Genet., 215,
231-238 (1989).
[0328] Caballero, J. L., Malpartida, F. and D. A. Hopwood.
Transcriptional organization and regulation of an antibiotic export
complex in the producing Streptomyces culture, Mol. Gen. Genet.,
228, 372-380 (1991).
[0329] Chater, K. F. Genetic regulation of secondary metabolic
pathways in Streptomyces. Ciba Foundation Symposium, 171, 144-156
(1992).
[0330] Chater, K. F. and C. J. Bruton. Resistance, regulatory and
production genes for the antibiotic methylenomycin are clustered,
Embo Journal, 4, 1893-7 (1985).
[0331] Chiao, J. S., T. H. Xia, B. G. Mei, Z. K. Jin, and W. L. Gu.
Rifamycin SV and related ansamycins, p. 477-498. In L. C. Vining
and Stuttard, C. (Eds.), Genetics and biochemistry and antibiotic
production. Butterworth-Heinemann, Newton, Mass. (1995).
[0332] Cole, S. T., Brosch, R., Parkhill, J., Garnier, T.,
Churcher, C., Harris, D., Gordon, S. V., Eiglmeier, K., Gas, S.,
Barry, C. R., Tekaia, F., Badcock, K., Basham, D., Brown, D.,
Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell,
T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K.,
Barrell, B. G. and et al., Deciphering the biology of Mycobacterium
tuberculosis from the complete genome sequence, Nature, 393,
537-544 (1998).
[0333] Coque, J., P. Liras, and J. Martin. Genes for a
.beta.-lactamase, a penicillin-binding protein and a transmembrane
protein are clustered with the cephamycin biosynthetic genes in
Nocardia lactamdurans. EMBO. J., 12, 631-639 (1993).
[0334] Coque, J. J., Perez-Laraine, F. J., Enguita, F. J., Fuente,
J. L., Martin, J. F. and P. Liras. Characterization of the cmcH
genes of Nocardia lactamdurans and Streptomyces clavuligerus
encoding a functional 3'-hydroxymethylcephem O-carbamoyltransferase
for cephamycin biosynthesis, Gene, 162, 21-27 (1995).
[0335] Cummings, J., Spanswick, V. J., Tomasz, M. and J .F. Smyth.
Enzymology of mitomycin C metabolic activation in tumor
tissue--implications for enzyme-directed bioreductive drug
development, Biochemical Pharmacology, 56, 405-414 (1998).
[0336] Cundliffe, E. Self-protection mechanisms in antibiotic
producers. Ciba Found. Symp., 171, 199-208 (1992).
[0337] Cundliffe, E., L. A. Merson-Davies, and G. H. Keleman.
Aspects of tylosin production and resistance in Streptomyces
fradiae, p. 235-243, Industrial microorganisms: basic and applied
molecular genetics. American Society for Microbiology, Washington,
D.C. (1993).
[0338] Dean, C. R., Neshat, S. and K. Poole. PfeR, an
enterobactin-responsive activator of ferric enterobactin receptor
gene expression in Pseudomonas aeruginosa, J. Bacteriol., 178,
5361-5369 (1996).
[0339] Decker, H., Motamedi, H. and C. R. Hutchinson, Nucleotide
sequences and heterologous expression of tcmG and tcmP,
biosynthetic genes for tetracenomycin C in Streptomyces
glaucescens, J. Bacteriol., 175, 3876-3886 (1993).
[0340] Denis, F. and R. Brzezinski, A versatile shuttle cosmid
vector for use in Escherichia coli and Actinomycetes, Gene, 111,
115-118 (1992).
[0341] Deppenmeier, U., Muller, V. and G. Gottschalk. Pathways of
energy conservation in methanogenic archaea, Arch. Microbiol., 165,
149-163 (1996).
[0342] Devereux, J., Haeberli, P. and O. Smithies. A comprehensive
set of sequence analysis programs for the VAX, Nucleic Acids Res.,
12, 387-395 (1984).
[0343] Dewick, P. M., The biosynthesis of shikimate metabolites,
Nat. Prod. Rep., 15, 17-58 (1995).
[0344] Dickens, M. L., Ye, J. and W. R. Strohl. Analysis of
clustered genes encoding both early and late steps in daunomycin
biosynthesis by Streptomyces sp. strain C5. J. Bacteriol., 177,
536-543 (1995).
[0345] Dittrich, H. and T. M. Kutchan. Molecular cloning,
expression, and induction of berberine bridge enzyme, an enzyme
essential to the formation of benzophenanthridine alkaloids in the
response of plants to pathogenic attach, Proc. Natl. Acad. Sci.
USA, 88, 9969-9973 (1991).
[0346] Donadio, S., M. J. Staver, J. B. McAlpine, S. J. Swanson,
and L. Katz. Modular organization of genes required for complex
polyketide biosynthesis. Science, 252, 675-679 (1991).
[0347] Evans, J. W., Yudoh, K., Delahoussaye, Y. M. and J. M.
Brown. Tirpazamine is metabolized to its DNA-damaging radical by
intranuclear enzymes, Cancer Research, 58, 2098-2101 (1998).
[0348] Femandez-Moreno, M. A., Caballero, J. L., Hopwood, D. A. and
F. Malpartida. The act cluster contains regulatory and antibiotic
export genes, direct targets for translational control by the bldA
tRNA gene of Streptomyces, Cell, 66, 769-80 (1991).
[0349] Floss, H. G. Natural products derived from unusual variants
of the shikimate pathway, Nat. Prod. Rep., 14, 433-52 (1997).
[0350] Ghisalba, O., and N. Nuesch. A genetic approach to the
biosynthesis of the rifamycin-chromophore in Nocardia
mediterraniae. IV. Identification of 3-amino-5-hydroxybenzoic acid
as a direct precursor of the seven-carbon amino starter-unit. J.
Antibiot., 34, 64-71 (1981).
[0351] Gibson, J., Dispensa, M., Fogg, G. C., Evans, D. T. and C.
S. Harwood. 4-Hydroxybenzoate-coenzyme A ligase from
Rhodopseudomonas palustris: purification, gene sequence, and role
in anaerobic degradation, J. Bacteriol., 176, 634-641 (1994).
[0352] Grebenok, R. J., Galbraith, D. W. and D. D. Penna.
Characterization of Zea mays endosperm C-24 sterol
methyltransferase: one of two types of sterol methyltransferase in
higher plants, Plant Mol. Biol., 34, 891-6 (1997).
[0353] Grkovic, S., Brown, M. H., Roberts, N. J., Paulsen, I. T.
and R. A. Skurray. QacR is a repressor protein that regulates
expression of the Staphylococcus aureus multidrug efflux pump QacA,
J. Biol. Chem., 273, 18665-73 (1998).
[0354] Guilfoile, P. G. and C. R. Hutchinson. Sequence and
transcriptional analysis of the Streptomyces glaucescens tcmAR
tetracenomycin C resistance and repressor gene loci, Journal of
Bacteriology, 174, 3651-3658 (1992).
[0355] Guilfoile, P. G. and C. R. Hutchinson. The Streptomyces
glaucescens TcmR protein represses transcription of the divergently
oriented tcmR and tcmA genes by binding to an intergenic operator
region, Journal of Bacteriology, 174, 3659-66 (1992).
[0356] Hardwick, K. G. and H. R. Pelham. SED6 is identical to ERG6,
and encodes a putative methyltransferase required for ergosterol
synthesis, Yeast, 10, 265-269 (1994).
[0357] Hata, T., Sano, Y., Sugawara, R., Matsumae, A., Kanamori,
K., Shima, T. and T. Hoshi. Mitomycin, a new antibiotic from
Streptomyces, J. Antibiot. Ser. A, 9, 141-146 (1956).
[0358] Hatano, K., S. Akiyama, M. Asai, and R. W. Richards.
Biosynthetic origin of amino benzenoid nucleus (C.sub.7N-unit) of
ansamitocin, a group of novel maytansinoid antibiotics. J.
Antibiot., 35, 1415-1417 (1982).
[0359] Haydock, S. F., Dowson, J. A., Dhillon, N., Roberts, G. A.,
Cortes, J. and P. F. Leadlay. Cloning and sequence analysis of
genes involved in erythromycin biosynthesis in Saccharopolyspora
erythraea: sequence similarities between EryG and a family of
S-adenosylmethionine-dependent methyltransferases, Mol. Gen.
Genet., 230, 120-128 (1991).
[0360] Henderson, I. C., Recent Advances in the Usage of Mitomycin,
Proceedings of a symposium, Hawaii, March 21-24, Oncology, 1, 1-83
(1993).
[0361] Henderson, C. I., Recent advances in the usage of mitomycin,
Oncology, 50:(Suppl. 1), 1-84 (1993).
[0362] Hidaka, T., Goda, M., Kuzuyama, T., Takei, N., Hidaka, M.
and H. Seto. Cloning and nucleotide sequence of fosfomycin
biosynthetic genes of Streptomyces wedmorensis, Mol. Gen. Genet.,
249, 274-280 (1995).
[0363] Hidaka, T., Hidaka, M., Kuzuyama, T. and H. Seto. Sequence
of a P-methyltransferase-encoding gene isolated from a
bialaphos-producing Streptomyces hygroscopicus, Gene, 158, 149-150
(1995).
[0364] Higgins, C., S. Hyde, M. Mimmack, U. Gileadi, D. Gill, and
M. Gallagher. Binding protein-dependent transport systems. J.
Bioenerg. Biomem., 22, 571-592 (1990).
[0365] Hirai, O., Miyamae, Y., Hattori, Y., Takashima, M.,
Miyamoto, A., Zaizen, K. and Y. Mine. Microbial mutagenicity an in
vitro chromosome aberration induction by fk973, a new antitumor
agent, Mutation Res., 324, 43-50 (1994).
[0366] Hopwood, D. A. Genetic contributions to understanding
polyketide synthases. Chem. Rev., 97, 2465-2497 (1997).
[0367] 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. and H. S. Schrempf. Genetic manipulation of Streptomvces: a
laboratory manual, John Innes Institute, Norwich, United Kingdom,
1985.
[0368] Horii, M., Ishizaki, T., Paik, S. Y., Manome, T. and Y.
Murooka. An operon containing the genes for cholesterol oxidase and
a cytochrome P-450-like protein from a Streptomyces sp., J.
Bacteriol., 172, 3644-3653 (1990).
[0369] Hornemann, U., Biosynthesis of the mitomycins, 1981.
[0370] Hornemann, U. and J. H. Eggert. Utilization of the intact
carbamoyl group of L-(NH.sub.2CO-.sup.13C,.sup.15N) citrulline in
mitomycin biosynthesis by Streptomyces verticillatus, Journal of
Antibiotics, 28, 841-843 (1975).
[0371] Hornemann, Y., Kehrer, J. P., Nunez, C. S. and R. L.
Ranieri. D-glucosamine and L-citrulline, precursors in mitomycin
biosynthesis by Streptomyces verticillatus, Journal of the American
Chemical Society, 96, 320-322 (1974).
[0372] Iyer, N., and W. Szybalski. Mitomycin or porfiromycin:
chemical mechanism of activation and cross-linking of DNA. Science,
145, 55-56 (1964).
[0373] Jabbouri, S., Fellay, R., Talmont, F., Kamalaprija, P.,
Burger, U., Relic, B., Prome, J. C. and W. J. Broughton.
Involvement of nodS in N-methylation and nodU in 6-O-carbamoylation
of Rhizobium sp. NGR234 nod factors, J. Biol. Chem., 270,
22968-22073 (1995).
[0374] Jabbouri, S., Relic, B., Hanin, M., Kamalaprija, P., Burger,
U., Prome, D., Prome, J. C. and W. J. Broughton. nolO and noeI
(HsnIII) of Rhizobium sp. NGR234 are involved in 3-O-carbamoylation
and 2-O-methylation of Nod factors, J. Biol. Chem., 273,
12047-12055 (1998).
[0375] Johnson, D. A., August, P. R., Shackleton, C., Liu, H. W.
and D. H. Sherman. Microbial resistance to mitomycins involves a
redox relay mechanism, J. Am. Chem. Soc., 119, 2576-2577
(1997).
[0376] Kagan, R. M. and S. Clarke. Widespread occurrence of three
sequence motifs in diverse S-adenosylmethionine-dependent
methyltransferases suggests a common structure for these enzymes,
Arch. Biochem. Biophy., 310, 417-27 (1994).
[0377] Kahler, C. M., R. W. Carlson, M. M. Rahman, L. E. Martin, D.
S. Stephens. Two glycosyltransferase genes, IgtF and rfaK,
constitute the lipooligosaccharide ice (inner core extension)
biosynthesis operon of Neisseria meningitidis. J. Bacteriol., 178,
6677-6684 (1996).
[0378] Kasai, M. and H. Arai. Novel mitomycin derivatives, Exp.
Opin. Ther. Patents, 5, 757-770 (1995).
[0379] Kibby, J. J., I. A. McDonald, and R. W. Rickards.
3-amino-5-hydroxybenzoic acid as a key intermediate in ansamycin
and maytansinoid biosynthesis. J. Chem. Soc. Chem. Comm., 1980,
768-769 (1980).
[0380] Kibby, J. J. and R. W. Rickards. The identification of
3-amino-5-hydroxybenzoic acid as a new natural aromatic amino acid,
J. Antibiot., 34, 605-607 (1981).
[0381] Kim, C. G., Kirschning, A., Bergon, P., Zhou, P., Su, E.,
Sauerbrei, B., Ning, S., Ahn, Y., Breuer, M., Leistner, E. and H.
G. Floss. Biosynthesis of 3-amino-5-hydroxybenzoic acid, the
precursor of mC.sub.7N units in ansamycin antibiotics, J. Am. Chem.
Soc., 188, 7486-7491 (1996).
[0382] Kim, C. G., A. Kirschning, P. Bergon, Y. Ahn, J. J. Wang, M.
Shibuya, and H. G. Floss. Formation of 3-amino-5-hydroxybenzoic
acid, the precursor of mC.sub.7N units in ansamycin antibiotics, by
a new variant of the shikimate pathway. J. Am. Chem. Soc., 114,
4941-4943 (1992).
[0383] Kim, C. G., Yu, T. W., Fryhle, C. B., Handa, S. and H. G.
Floss. 3-Amino-5-hydroxybenzoic acid synthase, the terminal enzyme
in the formation of the precursor of mC.sub.7N units in rifamycin
and related antibiotics, J. Biol. Chem., 273, 6030-6040 (1998).
[0384] Kuzuyama, T., Seki, T., Dairi, T., Hidaka, T. and H. Seto.
Nucleotide sequence of fortimicin KL1 methyltransferase gene
isolated from Micromonospora olivasterospora and comparison of its
deduced amino acid sequence with those of methyltransferases
involved in the biosynthesis of bialaphos and fosfomycin, J.
Antibiot., 48, 1191-3 (1995).
[0385] Kwon, O., Bhattacharyya, D. K. and R. Meganathan.
Menaquinone (vitamin K2) biosynthesis: overexpression,
purification, and properties of o-succinylbenzoyl-coenzyme A
synthetase from Escherichia coli, J. Bacteriol., 178, 6778-6781
(1996).
[0386] Kyte, J., and R. F. Doolittle. A simple method for
displaying the hydropathic character of a protein. J. Mol. Biol.,
157, 105-132 (1982).
[0387] Lacalle, R. A., Ruiz, D. and A. Jimenez. Molecular analysis
of the dmpM gene encoding an O-dimethyl puromycin
O-methyltransferase from Streptomyces alboniger, Gene, 109, 55-61
(1991).
[0388] Lee, J. P., S. W. Tsao, C. J. Chang, X. G. He, and H. G.
Floss. Biosynthesis of naphthomycin A in Streptomyces collinus.
Can. J. Chem., 72, 182-187 (1994).
[0389] Lee, P. J. and A. M. Stock. Characterization of the genes
and proteins of a two-component system from the hyperthermophilic
bacterium Thermotoga maritima, J. Bacteriol., 178, 5579-5585
(1996).
[0390] Levy, S., and L. McMurry. Plasmid-mediated tetracycline
resistance involves alternative transport systems for tetracycline.
Nature, 276, 90-92 (1978).
[0391] Littlejohn, T., I. Paulsen, M. Gillespie, J. Tennant, M.
Midgley, T. Jones, A. Purewal, and R. Skurray. Substrate
specificity and energetics of antiseptic and disinfectant
resistance in Staphylococcus aureus. FEMS Microbiol. Lett., 95,
259-266 (1992).
[0392] Lomovskaya, O., Lewis, K. and A. Matin. EmrR is a negative
regulator of the Escherichia coli multidrug resistance pump EmrAB,
J. Bacteriol., 177, 2328-2334 (1995).
[0393] Luka, S., Sanjuan, J., Carlson, R. W. and G. Stacey. nolMNO
genes of Bradyrhizobium japonicum are co-transcribed with
nodYABCSUIJ, and nolO is involved in the synthesis of the
lipo-oligosaccharide nodulation signals, J. Biol. Chem., 268,
27053-27059 (1993).
[0394] Madduri, K., Torti, F., Colombo, A. L. and C. R. Hutchinson.
Cloning and sequencing of a gene encoding carminomycin
4-O-methyltransferase from Streptomyces peucetius and its
expression in Escherichia coli, J. Bacteriol., 175, 3900-3904
(1993).
[0395] Makino, K., Shinagawa, H., Amemura, M. and A. Nakata.
Nucleotide sequence of the phoB gene, the positive regulatory gene
for the phosphate regulon of Escherichia coli K-12, J. Mol. Biol.,
190, 37-44 (1986).
[0396] Martin, J. F. Clusters of genes for the biosynthesis of
antibiotics: regulatory genes and overproduction of
pharmaceuticals. J. Ind. Microbiol., 9, 73-90 (1992).
[0397] Mazodier, P., Petter, R. and C. Thomson. Intergeneric
conjugation between Escherichia coli and Streptomyces species, J.
Bacteriol., 171, 3583-3585 (1989).
[0398] Mendez, C., and J. A. Salas. ABC transporters in
antibiotic-producing actinomycetes. FEMS Microb. Lett., 158, 1-8
(1998).
[0399] Miller, J., J. Olson, J. Plfugrath, and F. Quiocho. Rates of
ligand binding to periplasmic proteins involved in bacterial
transport and chemotaxis. J. Biol. Chem., 238, 13665-13672
(1983).
[0400] Mizuno, T. and I. Tanaka. Structure of the DNA-binding
domain of the OmpR family of response regulators, Mol. Microbiol.,
24, 665-667 (1997).
[0401] Morbidoni, H. R., de Mondoza, D. and J. Cronan Jr. Bacillus
subtilis acyl carrier protein is encoded in a cluster of lipid
biosynthesis genes, J. Bacteriol., 178, 4794-800 (1996).
[0402] Motamedi, H., and C. R. Hutchinson. Cloning and heterologous
expression of a gene cluster for the biosynthesis of tetracenomycin
C, the anthracycline antitumor antibiotic of Streptomyces
glaucescens. Proc. Natl. Acad. Sci. USA, 84, 4445-4449 (1987).
[0403] Muth, G., B. Nussbaumer, W. Wohileben, and A. Publer. A
vector system with temperature-sensitive replication for gene
disruption and mutational cloning in streptomycetes. Mol. Gen.
Genet., 219, 341-348 (1989).
[0404] Naoe, Y., Inami, M., Matsumoto, S., Nishigaki, F.,
Tsujimoto, S., Kawamura, I., Miyayasu, K., Manda, T. and K.
Shimomura. Fk317--a novel substituted dihydrobenzoxazine with
potent antitumor activity which does not induce vascular leak
syndrome, Cancer Chemo. Pharmacol., 42, 31-36 (1998).
[0405] Neal, R. J., and K. F. Chater. Nucleotide sequence analysis
reveals similarities between proteins determining methylenomycin A
resistance in Streptomyces and tetracycline resistance in
eubacteria. Gene, 58 229-241 (1987).
[0406] Niemi, J. and Mantsala, P., Nucleotide sequences and
expression of genes from Streptomyces purpurascens that cause the
production of new anthracyclines in Streptomyces galilaeus, J.
Bacteriol., 177, 2942-2945 (1995).
[0407] Nikaido, H. Prevention of drug access to bacterial targets:
Permeability barriers and active efflux. Science, 264, 382-388
(1994).
[0408] Nishikohri, K. and S, Fukui. Biosynthesis of mitomycin in
Streptomyces caespitosus. Relationship of intracellular vitamin
B.sub.12 level to mitomycin synthesis and enzymatic methylation of
a demethylated derivative of mitomycin, Eur. J. Appl. Microbiol.,
2, 129-145 (1975).
[0409] Nolling, J., Pihl, T. D. and J. N. Reeve. Cloning,
sequencing, and growth phase-dependent transcription of the
coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin
reductase-encoding genes from Methanobacterium thermoautotrophicum
delta H and Methanopyrus kandleri, J. Bacteriol., 177, 7238-7244
(1995).
[0410] Ohno, T., Armand, S., Hata, T., Nikaidou, N., Henrissat, B.,
Mitsutomi, M. and T. Watanabe. A modular family 19 chitinase found
in the prokaryotic organism Streptomyces griseus HUT 6037, J.
Bacteriol., 178, 5065-5070 (1996).
[0411] Omer, C. A., Lenstra, R., Little, P. J., Dean, C.,
Tepperman, J. M., Leto, K. J., Romesser, J. A. and D. P. O'Keefe.
Genes for two herbicide-inducible cytochromes P-450 from
Streptomyces griseolus, J. Bacteriol., 172, 3335-3345 (1990).
[0412] Otten, S. L., X. Liu, J. Ferguson, and C. R. Hutchinson.
Cloning and characterization of the Streptomyces peucetius dnrQS
genes encoding a daunosamine biosynthesis enzyme and a glycosyl
transferase involved in daunorubicin biosynthesis. J. Bacteriol.,
177, 6688-6692 (1995b).
[0413] Otten, S. L., Ferguson, J. and C. R. Hutchinson. Regulation
of daunorubicin production in Streptomyces peucetius by the dnrR2
locus, J. Bacteriol., 177, 1216-1224 (1995a).
[0414] Pan, S. S. and T. Iracki. Metabolites and DNA adduct
formation from flavoenzyme-activated porfiromycin, Molecular
Pharmacology, 34, 223-228 (1988).
[0415] Paulsen, I., and R. Skurray. Topology, structure and
evolution of two families of proteins involved in antibiotic and
antiseptic resistance in eukaryotes and prokaryotes--an analysis.
Gene, 124:1-11 (1993).
[0416] Paulsen, I., M. Brown, and R. Skurray. Proton-dependent
multidrug efflux pumps. Microbiol. Rev., 60, 575-608 (1996).
[0417] Paz, M. M. and P. B. Hopkins. DNA-DNA interstrand
cross-linking by FR66979-intermediates in the activation cascade,
J. Am. Chem. Soc., 119, 5999-6005 (1997).
[0418] Perez-Laraine, F. J., Liras, P., Rodriguez-Garcia, A. and J.
F. Martin. A regulatory gene (ccaR) required for cephamycin and
clavulanic acid production in Streptomyces clavuligerus:
amplification results in overproduction of both beta-lactam
compounds, J. Bacteriol., 179, 2053-2059 (1997).
[0419] Peschke, U., H. Schmidt, H. Z. Zhang, and W. Piepersberg.
Molecular characterization of the lincomycin-production gene
cluster of Streptomyces lincolnensis. 78-11. Mol. Microbiol., 16,
1137-1156 (1995).
[0420] Piepersberg, W. Pathway engineering in secondary
metabolite-producing actinomycetes, Crit. Rev. Biotechnol.,
14:251-285 (1994).
[0421] Platt, M. W., Miller, K. J., Lane, W. S. and E. P. Kennedy.
Isolation and characterization of the constitutive acyl carrier
protein from Rhozobium meliloti, J. Bacteriol., 172, 5440-4
(1990).
[0422] Potgieter, M. Biosynthetic studies on geldanamycin and
pactamycin. Ph.D. thesis. Univ. Illinois (1983).
[0423] Praillet, T., Nasser, W., Robert-Baudouy, J. and S.
Reverchon. Purification and functional characterization of Pacs, a
regulator of virulence-factor synthesis in Erwinia chrysanthemi,
Molecular Microbiology, 20, 391-402 (1996).
[0424] Rodriguez, A. M., Olano, C., Mendez, C., Hutchinson, C. R.
and J. A. Salas. A cytochrome P450-like gene possibly involved in
oleandomycin biosynthesis by Streptomyces antibioticus, FEMS
Microbiol. Lett., 127, 117-20 (1995).
[0425] Rouch, D., D. Cram, D. DiBerardino, T. Littlejohn and R.
Skurray. Efflux-mediated antiseptic resistance gene qacA from
Staphylococcus aureus: common ancestry with tetracycline and
sugar-transport proteins. Mol. Microbiol., 4, 2051-2062 (1990).
[0426] Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular
cloning: a laboratory manual, 2nd ed, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y. (1989).
[0427] Sartorelli, A. C., W. F. Hodnick, M. F. Belcourt, M. Tomasz,
B. Haffty, J. J. Fischer, and S. Rockwell. Mitomycin C: a prototype
bioreductive agent, Oncol. Res., 6:501-508 (1994).
[0428] Schaferjohann, J., Yoo, J. G., Kusian, B. and B. Bowien. The
cbb operons of the facultative chemoautotroph Alcaligenes eutrophus
encode phosphoglycolate phosphatase, J. Bacteriol., 175, 7329-40
(1993).
[0429] Schwecke, T., Aparicio, J. F., Molnar, I., Konig, A., Khaw,
L. E., Haydock, S. F., Oliynyk, M., Caffrey, P., Cortes, J.,
Lester, J. B. and et al., The biosynthetic gene cluster for the
polyketide immunosuppressant rapamycin, Proc. Natl. Acad. Sci. USA,
92, 7839-43 (1995).
[0430] Seno, E. T. and R. H. Baltz. Structural organization and
regulation of antibiotic biosynthesis and resistance genes in
actinomycetes, CRC Press, Boca Raton, Fla. (1989).
[0431] Sheldon, P. J., Johnson, D. A., August, P. J., Liu, H. W.
and D. H. Sherman. Characterization of a mitomycin-binding drug
resistance mechanism from the producing organism, Streptomyces
lavendulae, J. Bacteriol., 179, 1796-1804 (1997).
[0432] Shi, J., Gonzales, R. A. and Bhattacharyya, M. K.,
Identification and characterization of an S-adenosyl-L-methionine:
delta 24-sterol-C-methyltransferase cDNA from soybean, J. Biol.
Chem., 271, 9384-9389 (1996).
[0433] Shikano, M., Onimura, K., Fukai, Y., Hori, M., Fukazawa, H.,
Mizuno, S., Yazawa, K. and Y. Uehara. 1a-docosahexaenoyl mitomycin
C: a novel inhibitor of protein tyrosine kinase, Biochem. Biophys.
Res. Commun., 248, 858-863 (1998).
[0434] Simon, R., U. Priefer, and A. Puhler, A broad host range
mobilization system for in vivo genetic engineering: Transposon
mutagenesis in Gram negative bacteria, Bio/Technology, 1:784-791
(1983).
[0435] Smith, T. M., Y. F. Jiang, P. Shipley, and H. G. Floss. The
thiostrepton-resistance encoding gene in Streptomyces laurentii is
located within a cluster of ribosomal protein operons. Gene, 164,
137-142 (1995).
[0436] Smitskampwilms, E., Hendriks, H. R. and Peters, G. J.,
Development, pharmacology, role of DT-diaphorase and prospects of
the indoloquinone EO9, Gen. Pharmacol., 27, 421-429 (1996).
[0437] Solenberg, P. J., P. Matsushima, D. R. Stack, S. C. Wilkie,
R. C. Thompson, and R. H. Baltz. Production of hybrid glycopeptide
antibiotics in vitro and in Streptomyces toyocaensis. Chem. Biol.,
4, 195-202 (1997).
[0438] Spanswick, V. J., Cummings, J. and J. F. Smyth. Current
issues in the enzymology of mitomycin C metabolic activation, Gen.
Pharmacol., 31, 539-544 (1998).
[0439] Spath, C., Kraus, A. and W. Hillen. Contribution of glucose
kinase to glucose repression of xylose utilization in Bacillus
megaterium, J. Bacteriol., 179, 7603-7605 (1997).
[0440] Stackebrandt, E., and C. R. Woese. Towards a phylogeny of
the actinomycetes and related organisms. Curr. Microbiol., 5,
197-202 (1981).
[0441] Staley, A. L., and K. L. Rinehart. Biosynthesis of the
streptovaricins: 3-amino-5-hydroxybenzoic acid as a precursor to
the meta-C.sub.7N unit. J. Antibiot., 44, 218-224 (1991).
[0442] Stupperich, E., Juza, A., Hoppert, M. and F. Mayer. Cloning,
sequencing and immunological characterization of the
corrinoid-containing subunit of the
N5-methyltetrahydromethanopterin: coenzyme-M methyltransferase from
Methanobacterium thermoautotrophicum, Euro. J Biochem., 217,
115-121 (1993).
[0443] Summers, R. G., Wendt-Pienkowski, E., Motamedi, H. and C. R.
Hutchinson. Nucleotide sequence of the tcmII-tcmIV region of the
tetracenomycin C biosynthetic gene cluster of Streptomyces
glaucescens and evidence that the tcmN gene encodes a
multifunctional cyclase-dehydratase-O-methyl transferase, J.
Bacteriol., 174, 1810-1820 (1992).
[0444] Takano, E., Gramajo, H. C., Strauch, E., Andres, N., White,
J. and M. J. Bibb. Transcriptional regulation of the redD
transcriptional activator gene accounts for growth-phase-dependent
production of the antibiotic undecylprodigiosin in Streptomyces
coelicolor A3(2), Molecular Microbiology, 6, 2797-2804 (1992).
[0445] Tang, L., Grimm, A., Zhang, Y. X. and C. R. Hutchinson.
Purification and characterization of the DNA-binding protein DnrI,
a transcriptional factor of daunorubicin biosynthesis in
Streptomyces peucetius, Molecular Microbiology, 22, 801-13
(1996).
[0446] Tercero, J., R. Lacalle, and A. Jimenez. The pur8 gene from
the pur cluster of Streptomyces alboniger encodes a highly
hydrophobic polypeptide which confers resistance to puromycin. Eur.
J. Biochem., 218, 963-971 (1993).
[0447] Thauer, R. K., Hedderich, R. and R. Fischer. Reactions and
enzymes involved in methanogenesis from CO.sub.2 and H.sub.2,
Chapman and Hall, New York, N.Y., 1993.
[0448] Tomasz, M. Mitomycin C: small fast and deadly (but very
selective), Chemistry and Biology, 2, 575-579 (1995).
[0449] Tomasz, M. and Y. Palom. The mitomycin bioreductive
antitumor agents: cross-linking and alkylation of DNA as the
molecular basis of their activity, Pharmacol. Therap., 76, 73-87
(1997).
[0450] Tuan, J. S., Weber, J. M., Staver, M. J., Leung, J. O.,
Donadio, S. and L. Katz. Cloning of the genes involved in
erythromycin biosynthesis from Saccaropolyspora erythraea using a
novel Actinomycete-Escherichia coli cosmid, Gene, 90, 21-29
(1990).
[0451] Turgay, K., and M. A. Marahiel. A general approach for
identifying and cloning peptide synthetase genes. Peptide Res., 7,
238-241 (1994).
[0452] Vara, J., F. Malpartida, D. A. Hopwood, and A. Jimenez.
Cloning and expression of a puromycin N-acetyl transferase gene
from Streptomyces alboniger in Streptomyces lividans and
Escherichia coli Gene. 33, 197-206 (1985).
[0453] Vaupel, M. and R. K. Thauer. Coenzyme F420-dependent
N5,N10-methylenetetrahydromethanopterin reductase (Mer) from
Methanobacterium thermoautotrophicum strain Marburg. Cloning,
sequencing, transcriptional analysis, and functional expression in
Escherichia coli of the mer gene, Euro. J. Biochem., 231, 773-8
(1995).
[0454] Verweij, J. Mitomycins, Cancer Chemotherapy and Biological
Response Modifiers, 17, 46-58 (1997).
[0455] Wakaki, K., Harumo, H., Tomioka, K., Shimizu, G., Kato, E.,
Kamada, H., Kudo, S. and Y. Fujimoto. Isolation of new fractions of
antitumor mitomycins, Antibiot. Chemother., 8, 228-240 (1958).
[0456] Webb, J. S., D. B. Cosalich, T. H. Mowat, J. B. Patrick, R.
W. Broschard, W. E. Meyor, R. P. Williams, C. F. Wolf, W. Fulmore,
C. Pidacks, and J. E. Lancaster. The structure of Mitomycins A, B,
and C and Porfiromycin-Part 1. J. Am. Chem. Soc., 84, 3185-3188
(1962).
[0457] White, P. J., Young, J., Hunter, I. S., Nimmo, H. G. and J.
R. Coggins. The purification and characterization of
3-dehydroquinase from Streptomyces coelicolor, Biochem. J., 265,
735-8 (1990).
[0458] Wietzorrek, A. and M. Bibb. A novel family of proteins that
regulates antibiotic production in streptomycetes appears to
contain an OmpR-like DNA-binding fold, Molecular Microbiology, 25,
1181-4 (1997).
[0459] Williams, R. M., Raj ski, S. R. and S. B. Rollins. FR900482,
a close cousin of mitomycin C that exploits mitosene-based DNA
cross-linking, Chemistry and Biology, 4, 127-137 (1997).
[0460] Wu, T. S., J. Duncan, S. W. Tsao, C. J. Chang, P. J. Keller,
and H. G. Floss. Biosynthesis of the ansamycin antibiotic
assatrienin (mycotrienin) by Streptomyces collinus. J. Nat. Prod.,
50, 108-118 (1987).
[0461] Yamazaki, M., Thome, L., Mikolajczak, M., Armentrout, R. W.
and T. J. Pollock. Linkage of genes essential for synthesis of a
polysaccharide capsule in Sphingomonas strain S88, J. Bacteriol.,
178, 2676-87 (1996).
[0462] Yang, K., Han, L. and L. C. Vining. Regulation of jadomycin
B production in Streptomyces venezuelae ISP5230: involvement of a
repressor gene, jadR2, Journal of Bacteriology, 177, 6111-7
(1995).
[0463] Yanisch-Perron, C., J. Vieira, and J. Messing. Improved M13
phage cloning vectors and host strains: nucleotide sequences of the
M13mp18 and pUC19 vectors, Gene, 33:103-119 (1985).
[0464] Zhang, H. Z., H. Schmidt, and W. Piepersberg. Molecular
cloning and characterization of two lincomycin-resistance genes,
ImrA and ImrB, from Streptomyces lincolnensis 78-11. Mol.
Microbiol., 6, 2147-2157 (1992).
[0465] While the present invention has been described in connection
with the preferred embodiment thereof, it will be understood many
modifications will be readily apparent to those skilled in the art,
and this application is intended to cover any adaptations or
variations thereof. It is manifestly intended this invention be
limited only by the claims and equivalents thereof.
Sequence CWU 0
0
* * * * *