U.S. patent application number 12/400509 was filed with the patent office on 2009-10-22 for farnesyl dibenzodiazepinone and processes for its production.
This patent application is currently assigned to THALLION PHARMACEUTICALS INC.. Invention is credited to Chris M. FARNET, Emmanuel ZAZOPOULOS.
Application Number | 20090263886 12/400509 |
Document ID | / |
Family ID | 32777010 |
Filed Date | 2009-10-22 |
United States Patent
Application |
20090263886 |
Kind Code |
A1 |
ZAZOPOULOS; Emmanuel ; et
al. |
October 22, 2009 |
FARNESYL DIBENZODIAZEPINONE AND PROCESSES FOR ITS PRODUCTION
Abstract
This invention relates to a novel farnesylated
dibenzodiazepinone, named ECO-04601, its pharmaceutically
acceptable salts and derivatives, and to methods for obtaining such
compounds. One method of obtaining the ECO-04601 compound is by
cultivation of a novel strain of Micromonospora sp., 046-ECO11;
another method involves expression of biosynthetic pathway genes in
transformed host cells. The present invention further relates to
Micromonospora sp. strain 046-ECO11, to the use of and its
pharmaceutically acceptable salts and derivatives as
pharmaceuticals, in particular to their use as inhibitors of cancer
cell growth, bacterial cell growth, mammalian lipoxygenase, and to
pharmaceutical compositions comprising ECO-04601 or a
pharmaceutically acceptable salt or derivative thereof. Finally,
the invention relates to novel polynucleotide sequences and their
encoded proteins, which are involved in the biosynthesis of
ECO-04601.
Inventors: |
ZAZOPOULOS; Emmanuel;
(Montreal, CA) ; FARNET; Chris M.; (Outremont,
CA) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
THALLION PHARMACEUTICALS
INC.
|
Family ID: |
32777010 |
Appl. No.: |
12/400509 |
Filed: |
March 9, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11511586 |
Aug 28, 2006 |
7521222 |
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12400509 |
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10762107 |
Jan 21, 2004 |
7101872 |
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11511586 |
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60441126 |
Jan 21, 2003 |
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60492997 |
Aug 7, 2003 |
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60518286 |
Nov 10, 2003 |
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Current U.S.
Class: |
435/252.31 ;
435/252.3; 435/252.32; 435/252.33; 435/252.35; 435/320.1;
536/23.1 |
Current CPC
Class: |
C07D 405/06 20130101;
C12N 9/12 20130101; A61P 35/00 20180101; C12P 17/10 20130101; C07D
243/38 20130101; C12N 9/14 20130101; A61P 31/04 20180101; C07D
405/14 20130101; C12N 9/10 20130101; A61K 38/00 20130101; C12N 9/88
20130101; C12R 1/29 20130101; C12N 9/90 20130101; C12N 9/93
20130101; A61P 29/00 20180101; C12N 15/52 20130101; C12N 9/0004
20130101; Y02A 50/30 20180101; Y02A 50/473 20180101 |
Class at
Publication: |
435/252.31 ;
435/320.1; 536/23.1; 435/252.33; 435/252.35; 435/252.3;
435/252.32 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C12N 15/63 20060101 C12N015/63; C07H 21/04 20060101
C07H021/04 |
Claims
1-4. (canceled)
5. Cosmid 046KQ deposited under IDAC accession no. 250203-07.
6. A prokaryotic host cell comprising the cosmid of claim 5.
7. The prokaryotic host cell of claim 6, wherein said host cell is
selected from the group consisting of E. coli, Streptomyces
lividans, Streptomyces griseofuscus, Streptomyces ambofuchsus,
Streptomyces ambofaciens, Actinomycetes, Bacillus, Corynebacteria
and Thermoactinomyces.
8. An isolated DNA molecule having 95% sequence identity over the
entire length of an open reading frame of the cosmid of claim
5.
9-12. (canceled)
Description
RELATED APPLICATIONS
[0001] This Application is a divisional of U.S. Utility application
Ser. No. 11/511,586, filed Aug. 28, 2006, which is a divisional of
U.S. Utility application Ser. No. 10/762,107, filed Jan. 21, 2004,
now issued as U.S. Pat. No. 7,101,872, and which claims priority to
each of U.S. Provisional Application 60/441,126, filed Jan. 21,
2003; U.S. Provisional Application 60/492,997, filed Aug. 7, 2003;
and U.S. Provisional Application 60/518,286, filed Nov. 10, 2003.
The entire teachings of each of the above-noted applications are
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to a novel farnesylated
dibenzodiazepinone, named ECO-04601, its pharmaceutically
acceptable salts and derivatives, and to methods for obtaining the
compound. One method of obtaining the compound is by cultivation of
a novel strain of Micromonospora sp., i.e., 046-ECO11 or [S01]046;
another method involves expression of biosynthetic pathway genes in
transformed host cells. The present invention further relates to
Micromonospora sp. strains 046-ECO11 and [S01]046, to the use of
ECO-04601 and its pharmaceutically acceptable salts and derivatives
as pharmaceuticals, in particular to their use as inhibitors of
cancer cell growth, bacterial cell growth, mammalian lipoxygenase,
and for treating acute and chronic inflammation, and to
pharmaceutical compositions comprising ECO-04601 or a
pharmaceutically acceptable salt or derivative thereof. Finally,
the invention relates to novel polynucleotide sequences and their
encoded proteins, which are involved in the biosynthesis of
ECO-04601.
BACKGROUND OF THE INVENTION
[0003] The euactinomycetes are a subset of a large and complex
group of Gram-positive bacteria known as actinomycetes. Over the
past few decades these organisms, which are abundant in soil, have
generated significant commercial and scientific interest as a
result of the large number of therapeutically useful compounds,
particularly antibiotics, produced as secondary metabolites. The
intensive search for strains able to produce new antibiotics has
led to the identification of hundreds of new species.
[0004] Many of the euactinomycetes, particularly Streptomyces and
the closely related Saccharopolyspora genera, have been extensively
studied. Both of these genera produce a notable diversity of
biologically active metabolites. Because of the commercial
significance of these compounds, much is known about the genetics
and physiology of these organisms.
[0005] Another representative genus of euactinomycetes,
Micromonospora, has also generated commercial interest. For
example, U.S. Pat. No. 5,541,181 (Ohkuma et al.) discloses a
dibenzodiazepinone compound, specifically
5-farnesyl-4,7,9-trihydroxy-dibenzodiazepin-11-one (named
"BU-4664L"), produced by a known euactinomycetes strain,
Micromonospora sp. M990-6 (ATCC 55378). The Ohkurma et al. patent
reports that BU-4664L and its chemically synthesized di- and
tri-alkoxy and acyloxy derivatives possess anti-inflammatory and
anti-tumor cell activities.
[0006] Although many biologically active compounds have been
identified from bacteria, there remains the need to obtain novel
naturally occurring compounds with enhanced properties. Current
methods of obtaining such compounds include screening of natural
isolates and chemical modification of existing compounds, both of
which are costly and time consuming. Current screening methods are
based on general biological properties of the compound, which
require prior knowledge of the structure of the molecules. Methods
for chemically modifying known active compounds exist, but still
suffer from practical limitations as to the type of compounds
obtainable.
[0007] Thus, there exists a considerable need to obtain
pharmaceutically active compounds in a cost-effective manner and
with high yield. The present invention solves these problems by
providing a novel strain of Micromonospora capable of producing a
potent new therapeutic compound, as well as reagents (e.g.,
polynucleotides, vectors comprising the polynucleotides and host
cells comprising the vectors) and methods to generate novel
compounds by de novo biosynthesis rather than by chemical
synthesis.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention relates to a compound of the
formula
##STR00001##
(Formula II) or a pharmaceutically acceptable salt thereof.
[0009] In another aspect, the invention relates to a pharmaceutical
composition comprising a compound of the formula
##STR00002##
or a pharmaceutically acceptable salt thereof, together with a
pharmaceutically acceptable carrier.
[0010] In a further aspect, the invention relates to a class of
compounds represented by Formula I:
##STR00003##
wherein, [0011] W.sup.1, W.sup.2 and W.sup.3 is each independently
selected from
##STR00004##
[0011] or
[0012] the chain from the tricycle may terminate at W.sup.3,
W.sup.2 or W.sup.1 with W.sup.3, W.sup.2 or W.sup.1 respectively
being either --CH.dbd.O or --CH.sub.2OH;
[0013] A is selected from --NH--, --NCH.sub.2R.sup.1,
--NC(O)R.sup.1;
[0014] R.sup.1 is selected from C1-6 alkyl, C2-6 alkene, aryl or
heteroaryl;
[0015] R.sup.2, R.sup.3, and R.sup.4 is each independently selected
from H, R.sup.5, --C(O)R.sup.6
[0016] R.sup.5 is each independently selected from C.sub.1-6 alkyl,
C.sub.2-7 alkalene, aryl or heteroaryl;
[0017] R.sup.6 is each independently selected from H, C.sub.1-6
alkyl, C.sub.2-7 alkalene, aryl or heteroaryl; or a
pharmaceutically acceptable salt thereof.
[0018] In one embodiment, A is NH.
[0019] In another embodiment, A is --NCH.sub.2R.sup.1.
[0020] In another embodiment, A is --NC(O)R.sup.1.
[0021] In another embodiment, R.sup.2 is H.
[0022] In another embodiment, R.sup.3 is H.
[0023] In another embodiment, R.sup.4 is H.
[0024] In another embodiment, R.sup.2, R.sup.3 and R.sup.4 are each
H.
[0025] In another embodiment, R.sup.2, R.sup.3 and R.sup.4 are each
H, and W.sup.1 is --CH.dbd.CH--.
[0026] In another embodiment, R.sup.2, R.sup.3 and R.sup.4 are each
H, and W.sup.2 is --CH.dbd.CH--.
[0027] In another embodiment, R.sup.2, R.sup.3 and R.sup.4 are each
H, and W.sup.3 is --CH.dbd.CH--.
[0028] In another embodiment, A is NH and R.sup.2, R.sup.3 and
R.sup.4 are each H.
[0029] In another embodiment, A is NH, each of W.sup.1, W.sup.2,
and W.sup.3 is --CH.dbd.CH--.
[0030] The invention further encompasses a compound selected from
the group consisting of:
##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009##
##STR00010## ##STR00011## ##STR00012##
[0031] In one embodiment, the invention relates to compositions of
the compounds of Formula I together with a pharmaceutically
acceptable carrier.
[0032] The invention further encompasses a farnesyl
dibenzodiazepinone obtained by a method comprising: a) cultivating
Micromonospora sp. strain [S01]046, wherein the cultivation is
performed under aerobic conditions in a nutrient medium comprising
at least one source of carbon atoms and at least one source of
nitrogen atoms; and b) isolating a farnesyl dibenzodiazepinone from
the bacteria cultivated in step (a). In one embodiment the farnesyl
dibenzodiazapinone is the compound of Formula II.
[0033] In one embodiment, the farnesyl dibenzodiazepinone generates
NMR spectra essentially as shown in FIGS. 3, 4, 5, 6 and 7. In
another embodiment, the farnesyl dibenzodiazepinone generates an
.sup.1H NMR spectrum of FIG. 3.
[0034] The invention further encompasses a process for making a
farnesyl dibenzodiazapinone compound, comprising cultivation of
Micromonospora sp. strain 046-ECO11, in a nutrient medium
comprising at least one source of carbon atoms and at least one
source of nitrogen atoms, and isolation and purification of the
compound.
[0035] The invention further encompasses a process for making a
farnesyl dibenzodiazepinone compound comprising cultivation of
Micromonospora sp. strain [S01]046 in a nutrient medium comprising
at least one source of carbon atoms and at least one source of
nitrogen atoms, and isolation and purification of the compound.
[0036] In one embodiment, the cultivation occurs under aerobic
conditions.
[0037] In another embodiment, the carbon atom and nitrogen atom
sources are chosen from the components shown in Table 16.
[0038] In another embodiment, the cultivation is carried out at a
temperature ranging from 18.degree. C. to 40.degree. C. In a
further embodiment, the temperature range is 18.degree. C. to
29.degree. C.
[0039] In another embodiment, the cultivation is carried out at a
pH ranging from 6 to 9.
[0040] The invention further encompasses the Micromonospora sp.
having IDAC Accession No. 231203-01.
[0041] The invention further encompasses a method of inhibiting the
growth of a cancer cell, the method comprising contacting the
cancer cell with a compound of Formula I, such that growth of the
cancer cell is inhibited.
[0042] In one embodiment, the compound is ECO-04601.
[0043] The invention further encompasses a method of inhibiting the
growth of a cancer cell in a mammal, the method comprising
administering a compound of Formula I to a mammal comprising a
cancer cell, such that growth of the cancer cell is inhibited in
the mammal.
[0044] In one embodiment, the compound is ECO-04601.
[0045] The invention further encompasses a method of treating a
pre-cancerous or cancerous condition in a mammal, comprising the
step of administering to the mammal a therapeutically effective
amount of a compound of Formula I, such that a pre-cancerous or
cancerous condition is treated.
[0046] In one embodiment, the compound is ECO-04601.
[0047] The invention further encompasses a method of treating a
bacterial infection in a mammal, comprising administering a
therapeutically effective amount of a compound of Formula I to a
mammal having a bacterial infection, such that the bacterial
infection is treated.
[0048] In one embodiment, the compound is ECO-04601.
[0049] The invention further encompasses a method of reducing
inflammation in a mammal, comprising administering to a mammal
having inflammation a therapeutically effective amount of a
compound of Formula I, such that the inflammation is reduced.
[0050] In one embodiment, the compound is ECO-04601.
[0051] The invention further encompasses an isolated polynucleotide
comprising one or more of SEQ ID NOs. 1, 64 and 73, wherein the
polynucleotide encodes a polypeptide that participates in a
biosynthetic pathway for a farnesyl dibenzodiazepinone.
[0052] The invention further encompasses an isolated polynucleotide
comprising SEQ ID NOs. 1, 64 and 73, wherein the polynucleotide
encodes a polypeptide that participates in a biosynthetic pathway
for a farnesyl dibenzodiazepinone.
[0053] The invention further encompasses an isolated polynucleotide
that encodes a polypeptide selected from the group consisting of
SEQ ID NOs. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67,
69, 71, 74, 76, 78, 80, 82, 84, 86 and 88.
[0054] In one embodiment, the isolated polynucleotide comprising
SEQ ID No. 1 encodes a polypeptide selected from the group
consisting of SEQ ID Nos. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58,
60 and 62.
[0055] In another embodiment, the isolated polynucleotide
comprising SEQ ID No. 64 encodes a polypeptide selected from the
group consisting of SEQ ID NOS: 65, 67, 69 and 71.
[0056] In another embodiment, the isolated polynucleotide
comprising SEQ ID No. 73, encodes a polypeptide selected from the
group consisting of SEQ ID NOS: 74, 76, 78, 80, 82 84, 86 and
88.
[0057] The invention further encompasses an isolated polypeptide of
SEQ ID NO. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67,
69, 71, 74, 76, 78, 80, 82, 84, 86 or 88.
[0058] In one embodiment, the polypeptide participates in a
biosynthetic pathway for a farnesyl dibenzodiazepinone.
[0059] The invention further encompasses an expression vector
comprising one or more of the polynucleotides described herein.
[0060] The invention further encompasses a recombinant prokaryotic
organism comprising one or more such expression vectors.
[0061] In one embodiment, the organism is an actinomycete.
[0062] In another embodiment, the organism requires the expression
vector to synthesize a farnesyl dibenzodiazepinone. That is, the
organism is deficient in the ability to synthesize a farnesyl
dibenzodiazepinone before transformation with a polynucleotide as
described herein.
[0063] The invention further encompasses a method of making a
farnesyl dibenzodiazepinone de novo in a prokaryote, comprising the
steps of: (a) providing a prokaryote that is incapable of
synthesizing a farnesyl dibenzodiazepinone; (b) transforming the
prokaryote with an expression vector as described herein; and (c)
culturing the prokaryote; wherein the culturing results in the
synthesis of a farnesyl dibenzodiazepinone in the prokaryote.
[0064] In one embodiment, the prokaryote is an actinomycete.
[0065] In another embodiment, the vector expresses a polypeptide of
SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 56, 58, 60, 62, 65, 67,
69, 71, 74, 76, 78, 80, 82, 84, 86 or 88.
BRIEF DESCRIPTION OF THE FIGURES
[0066] FIG. 1 shows the mass of ECO-04601 determined by
electrospray mass spectrometry to be 462.6.
[0067] FIG. 2 shows the absorption spectrum of purified ECO-04601
with a UVmax at 230 nm and a shoulder at 290 nm.
[0068] FIG. 3 shows proton NMR data for the compound dissolved in
MeOH-d.sub.4.
[0069] FIG. 4 shows multidimensional pulse sequences gDQCOSY.
[0070] FIG. 5 shows multidimensional pulse sequences gHSQC.
[0071] FIG. 6 shows multidimensional pulse sequences gHMBC.
[0072] FIG. 7 shows multidimensional pulse sequences NOESY.
[0073] FIG. 8 shows the in vitro anti-inflammatory activity of
ECO-04601. Graph shows percent inhibition of 5-lipoxygenase
activity plotted against the Log .mu.M concentration of ECO-04601
and NDGA. Graph shows the EC.sub.50 of ECO-04601 to be 0.93
.mu.M.
[0074] FIG. 9 shows inhibition of tumor growth resulting from
administration of 10 to 30 mg/kg of ECO-04601 to
glioblastoma-bearing mice beginning one day after tumor cell
inoculation.
[0075] FIG. 10 shows inhibition of tumor growth resulting from
administration of 20-30 mg/kg of ECO-04601 to glioblastoma-bearing
mice beginning ten days after tumor cell inoculation.
[0076] FIG. 11 shows micrographs of tumor sections from mice
bearing glioblastoma tumors and treated with saline or ECO-04601.
The cell density of tumor treated with ECO-04601 appears decreased
and nuclei from ECO-04601-treated tumor cells are larger and
pynotic suggesting a cytotoxic effect.
[0077] FIG. 12 shows the biosynthetic locus of ECO-04601, isolated
from Micromonospora sp. strain 046-ECO11, including the positions
of cosmids 046KM and 046KQ.
[0078] FIG. 13 shows a schematic diagram of the biosynthetic
pathway for the production of the farnesyl-diphosphate group of
ECO-04601 with biosynthetic enzymes indicated by their ORF number
and family designation.
[0079] FIG. 14 shows a schematic diagram of the biosynthetic
pathway for the production of (a) 3-hydroxy-anthranilate-adenylate,
and (b) 2-amino-6-hydroxy-[1,4]benzoquinone components as specified
by ORFs present in the locus encoding ECO-04601. Biosynthetic
enzymes are indicated by their ORF number and family
designation.
[0080] FIG. 15 shows a schematic diagram of the biosynthetic
pathway for the assembly of the ECO-04601 precursors,
farnesyl-diphosphate, 3-hydroxy-anthranilate-adenylate and
2-amino-6-hydroxy-[1,4]benzoquinone. Biosynthetic enzymes are
indicated by their ORF number and family designation.
[0081] FIG. 16 shows a sequence listing table indicating the SEQ ID
NO. and function for each of the open reading frames (ORFs) of the
046D biosynthetic locus and the corresponding gene product.
[0082] FIG. 17 shows results of the fatty acid analysis of
Micromonospora sp. strain 046ECO11 (Accession No. IDAC 070303-01).
Analysis was conducted using gas chromatography on fatty acid
methyl esters (FAME).
[0083] FIG. 18 illustrates the 16 S ribosomal RNA analysis of
Micromonospora sp. strain 046ECO11 (Accession No. IDAC 070303-01).
Alignment of 16 S ribosomal RNA sequences demonstrates the
phylogenetic relatedness of Micromonospora sp. strain 046ECO11
(indicated as MID352 ECOPIA#1 con) to Micromonospora chalcea.
[0084] FIG. 19 shows the complete .sup.1H and .sup.13C NMR
assignments for ECO-04601 when measured in MeOH-d4.
DETAILED DESCRIPTION OF THE INVENTION
[0085] The present invention relates to a novel farnesyl
dibenzodiazepinone, referred to herein as "ECO-04601," which was
isolated from novel strains of actinomycetes, Micromonospora sp.
strain 046-ECO11 and strain [S01]046. These microorganisms were
analysed using gas chromatography as Fatty acid methyl esters
(FAME) (FIG. 17) 6 S ribosomal RNA determination (FIG. 18) and were
found to belong to the genus of Micromonospora. These organisms
were deposited on Mar. 7, 2003, and Dec. 23, 2003, respectively,
with the International Depository Authority of Canada (IDAC),
Bureau of Microbiology, Health Canada, 1015 Arlington Street,
Winnipeg, Manitoba, Canada R3E 3R2, under Accession Nos. IDAC
070303-01 and IDAC 231203-01, respectively.
[0086] The invention further relates to pharmaceutically acceptable
salts and derivatives of ECO-04601, and to methods for obtaining
such compounds. One method of obtaining the compound is by
cultivating Micromonospora sp. strain 046-ECO11, or a mutant or a
variant thereof, under suitable Micromonospora culture conditions,
preferably using the fermentation protocol described
hereinbelow.
[0087] The invention also relates to a method for producing novel
polyketide compounds, namely farnesyl dibenzodiazepinones, by
selectively altering the genetic information of an organism. The
present invention further provides isolated and purified
polynucleotides that encode farnesyl dibenzodiazepinone domains,
i.e., polypeptides from farnesyl dibenzodiazepinone-producing
microorganisms, fragments thereof, vectors containing those
polynucleotides, and host cells transformed with those vectors.
These polynucleotides, fragments thereof, and vectors comprising
the polynucleotides can be used as reagents in the above described
method. Portions of the polynucleotide sequences disclosed herein
are also useful as primers for the amplification of DNA or as
probes to identify related domains from other farnesyl
dibenzodiazepinone producing microorganisms.
[0088] The present invention also relates to pharmaceutical
compositions comprising ECO-04601 and its pharmaceutically
acceptable salts and derivatives. ECO-04601 is useful as a
pharmaceutical, in particular for use as an inhibitor of cancer
cell growth, bacterial cell growth, and mammalian lipoxygenase. The
invention also relates to novel polynucleotide sequences and their
encoded proteins, which are involved in the biosynthesis of
ECO-04601.
[0089] The following detailed description discloses how to make and
use ECO-04601 and compositions containing this compound to inhibit
microbial growth and/or specific disease pathways.
[0090] Accordingly, certain aspects of the present invention relate
to pharmaceutical compositions comprising the farnesylated
dibenzodiazepinone compounds of the present invention together with
a pharmaceutically acceptable carrier, methods of using the
compositions to inhibit bacterial growth, and methods of using the
pharmaceutical compositions to treat diseases, including cancer,
and chronic and acute inflammation.
I. Definitions
[0091] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below.
[0092] As used herein, the term "farnesyl dibenzodiazepinone"
refers to a class of dibenzodiazepinone compounds containing a
farnesyl moiety. The term includes, but is not limited to, the
exemplified compound of the present invention,
10-farnesyl-4,6,8-trihydroxy-dibenzodiazepin-11-one, which is
referred to herein as "ECO-04601." As used herein, the term
"farnesyl dibenzodiazepinone" includes compounds of this class that
can be used as intermediates in chemical syntheses. As used herein,
the term "alkyl" refers to linear or branched hydrocarbon groups.
Examples of alkyl groups include, without limitation, methyl,
ethyl, n-propyl, isopropyl, n-butyl, pentyl, hexyl, heptyl,
cyclopentyl, cyclohexyl, cyclohexymethyl, and the like. Alkyl may
optionally be substituted with substituents selected from acyl,
amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,
cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy,
sulfinyl, sulfonyl, oxo, guanidino and formyl.
[0093] The term "alkenyl" refers to linear, branched or cyclic
hydrocarbon groups containing at least one carbon-carbon double
bond. Examples of alkenyl groups include, without limitation,
vinyl, 1-propen-2-yl, 1-buten-4-yl, 2-buten-4-yl, 1-penten-5-yl and
the like. Alkenyl may optionally be substituted with substituents
selected from acyl, amino, acylamino, acyloxy, carboalkoxy,
carboxy, carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl,
alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl,
alkoxy, aryloxy, sulfinyl, sulfonyl, formyl, oxo and guanidino. The
double bond portion(s) of the unsaturated hydrocarbon chain may be
either in the cis or trans configuration.
[0094] The terms "cycloalkyl" and "cycloalkyl ring" refer to a
saturated or partially unsaturated carbocyclic ring in a single or
fused carbocyclic ring system having from three to fifteen ring
members. Examples of cycloalkyl groups include, without limitation,
cyclopropyl, cyclobutyl, cyclohexyl, and cycloheptyl. Cycloalkyl
may optionally be substituted with substituents selected from acyl,
amino, acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido,
cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy,
sulfinyl, sulfonyl and formyl.
[0095] The terms "heterocyclyl" and "heterocyclic" refer to a
saturated or partially unsaturated ring containing one to four
hetero atoms or hetero groups selected from O, N, NH, NRx, PO2, S,
SO or SO.sub.2 in a single or fused heterocyclic ring system having
from three to fifteen ring members. Examples of a heterocyclyl or
heterocyclic ring include, without limitation, morpholinyl,
piperidinyl, and pyrrolidinyl. Heterocyclyl, heterocyclic or
heterocyclyl ring may optionally be substituted with substituents
selected from acyl, amino, acylamino, acyloxy, oxo, thiocarbonyl,
imino, carboalkoxy, carboxy, carboxyamido, cyano, halo, hydroxyl,
nitro, thio, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl,
aryl, heteroaryl, alkoxy, aryloxy, sulfinyl, sulfonyl and
formyl.
[0096] The term "amino acid" refers to any natural amino acid, all
natural amino acids are well known to a person skilled in the
art.
[0097] The term "halo" refers to a halogen atom, e.g., bromine,
chlorine, fluorine and iodine.
[0098] The terms "aryl" and "aryl ring" refer to aromatic groups in
a single or fused ring system, having from five to fifteen ring
members. Examples of aryl include, without limitation, phenyl,
naphthyl, biphenyl, terphenyl. Aryl may optionally be substituted
with one or more substituent group selected from acyl, amino,
acylamino, acyloxy, azido, alkylthio, carboalkoxy, carboxy,
carboxyamido, cyano, halo, hydroxyl, nitro, thio, alkyl, alkenyl,
alkynyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy,
aryloxy, sulfinyl, sulfonyl and formyl.
[0099] The terms "heteroaryl" and "heteroaryl ring" refer to
aromatic groups in a single or fused ring system, having from five
to fifteen ring members and containing at least one hetero atom
such as O, N, S, SO and SO.sub.2. Examples of heteroaryl groups
include, without limitation, pyridinyl, thiazolyl, thiadiazoyl,
isoquinolinyl, pyrazolyl, oxazolyl, oxadiazoyl, triazolyl, and
pyrrolyl groups. Heteroaryl groups may optionally be substituted
with one or more substituent group selected from acyl, amino,
acylamino, acyloxy, carboalkoxy, carboxy, carboxyamido, cyano,
halo, hydroxyl, nitro, thio, thiocarbonyl, alkyl, alkenyl, alkynyl,
cycloalkyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy,
sulfinyl, sulfonyl, and formyl.
[0100] The terms "aralkyl" and "heteroaralkyl" refer to an aryl
group or a heteroaryl group, respectively bonded directly through
an alkyl group, such as benzyl. Aralkyl and heteroaralkyl may be
optionally substituted as the aryl and heteroaryl groups.
[0101] Similarly, the terms "aralkenyl" and "heteroaralkenyl" refer
to an aryl group or a heteroaryl group, respectively bonded
directly through an alkene group, such as benzyl. Aralkenyl and
heteroaralkenyl may be optionally substituted as the aryl and
heteroaryl groups.
[0102] The compounds of the present invention can possess one or
more asymmetric carbon atoms and can exist as optical isomers
forming mixtures of racemic or non-racemic compounds. The compounds
of the present invention are useful as single isomers or as a
mixture of stereochemical isomeric forms. Diastereoisomers, i.e.,
nonsuperimposable stereochemical isomers, can be separated by
conventional means such as chromatography, distillation,
crystallization or sublimation. The optical isomers can be obtained
by resolution of the racemic mixtures according to conventional
processes.
[0103] The invention encompasses isolated or purified compounds. An
"isolated" or "purified" compound refers to a compound which
represents at least 10%, 20%, 50%, 80% or 90% of the compound of
the present invention present in a mixture, provided that the
mixture comprising the compound of the invention has demonstrable
(i.e. statistically significant) biological activity including
antibacterial, cytostatic, cytotoxic, antiinflammatory or enzyme
inhibitory action when tested in conventional biological assays
known to a person skilled in the art.
[0104] The terms "farnesyl dibenzodiazepinone-producing
microorganism" and "producer of farnesyl dibenzodiazepinone," as
used herein, refer to a microorganism that carries genetic
information necessary to produce a farnesyl dibenzodiazepinone
compound, whether or not the organism naturally produces the
compound. The terms apply equally to organisms in which the genetic
information to produce the farnesyl dibenzodiazepinone compound is
found in the organism as it exists in its natural environment, and
to organisms in which the genetic information is introduced by
recombinant techniques.
[0105] Specific organisms contemplated herein include, without
limitation, organisms of the family Micromonosporaceae, of which
preferred genera include Micromonospora, Actinoplanes and
Dactylosporangium; the family Streptomycetaceae, of which preferred
genera include Streptomyces and Kitasatospora; the family
Pseudonocardiaceae, of which preferred genera are Amycolatopsis and
Saccharopolyspora; and the family Actinosynnemataceae, of which
preferred genera include Saccharothrix and Actinosynnema; however
the terms are intended to encompass all organisms containing
genetic information necessary to produce a farnesyl
dibenzodiazepinone compound. A preferred producer of a farnesyl
dibenzodiazepinone compound includes microbial strain 046-ECO11, a
deposit of which was made on Mar. 7, 2003, with the International
Depository Authority of Canada (IDAC), Bureau of Microbiology,
Health Canada, 1015 Arlington Street, Winnipeg, Manitoba, Canada
R3E, 3R2, under Accession No. IDAC 070303-01.
[0106] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as, where
applicable, intervening regions (introns) between individual coding
segments (exons).
[0107] The terms "gene locus, "gene cluster," and "biosynthetic
locus" refer to a group of genes or variants thereof involved in
the biosynthesis of a farnesyl benzodiazepinone compound. The
biosynthetic locus in strain 046-ECO11 that directs the production
of ECO-04601 is often referred to herein, in both the written
description and Figures, as "046D." Genetic modification of gene
locus, gene cluster or biosynthetic locus refers to any genetic
recombinant techniques known in the art including mutagenesis,
inactivation, or replacement of nucleic acids that can be applied
to generate variants of ECO-04601.
[0108] A DNA or nucleotide "coding sequence" or "sequence encoding"
a particular polypeptide or protein, is a DNA sequence which is
transcribed and translated into a polypeptide or protein when
placed under the control of an appropriate regulatory sequence.
[0109] "Oligonucleotide" refers to a nucleic acid, generally of at
least 10, preferably 15 and more preferably at least 20 nucleotides
in length, preferably no more than 100 nucleotides in length, that
are hybridizable to a genomic DNA molecule, a cDNA molecule, or an
mRNA molecule encoding a gene, mRNA, cDNA or other nucleic acid of
interest.
[0110] A promoter sequence is "operably linked to" a coding
sequence recognized by RNA polymerase which initiates transcription
at the promoter and transcribes the coding sequence into mRNA.
[0111] The term "replicon" as used herein means any genetic
element, such as a plasmid, cosmid, chromosome or virus, that
behaves as an autonomous unit of polynucleotide replication within
a cell. A "expression vector" or "vector" is a replicon in which
another polynucleotide fragment is attached, such as to bring about
the replication and/or expression of the attached fragment.
"Plasmids" are designated herein by a lower case "p" preceded or
followed by capital letters and/or numbers. The starting plasmids
disclosed herein are commercially available, publicly available on
an unrestricted basis, or can be constructed from available
plasmids in accordance with published procedures. In addition,
equivalent plasmids to those described herein are known in the art
and will be apparent to the skilled artisan.
[0112] The terms "express" and "expression" means allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g. the resulting protein, may also be
said to be "expressed" by the cell. An expression product can be
characterized as intracellular, extracellular or secreted.
[0113] "Digestion" of DNA refers to enzymatic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinary
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular enzymes
are specified by the manufacturer. Incubation times of about 1 hour
at 37.degree. C. are ordinarily used, but may vary in accordance
with the supplier's instructions. After digestion the gel
electrophoresis may be performed to isolate the desired
fragment.
[0114] The term "isolated" as used herein means that the material
is removed from its original environment (e.g. the natural
environment where the material is naturally occurring). For
example, a naturally occurring polynucleotide or polypeptide
present in a living animal is not isolated, but the same
polynucleotide or polypeptide, which is separated from some or all
of the coexisting materials in the natural system, is isolated.
Such polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a composition, and
still be isolated in that the vector or composition is not part of
the natural environment.
[0115] The term "restriction fragment" as used herein refers to any
linear DNA generated by the action of one or more restriction
enzymes.
[0116] The term "transformation" means the introduction of a
foreign gene, foreign nucleic acid, DNA or RNA sequence to a host
cell, so that the host cell will express the introduced gene or
sequence to produce a desired substance, typically a protein or
enzyme coded by the introduced gene or sequence. The introduced
gene or sequence may also be called a "cloned" or "foreign" gene or
sequence, may include regulatory or control sequences, such as
start, stop, promoter, signal, secretion, or other sequences used
by a cell's genetic machinery. The gene or sequence may include
nonfunctional sequences or sequences with no known function. A host
cell that receives and expresses introduced DNA or RNA has been
"transformed" and is a "transformant" or a "clone" or
"recombinant". The DNA or RNA introduced to a host cell can come
from any source, including cells of the same genus or species as
the host cell, or cells of a different genus or species.
[0117] The terms "recombinant polynucleotide" and "recombinant
polypeptide" as used herein mean a polynucleotide or polypeptide
which by virtue of its origin or manipulation is not associated
with all or a portion of the polynucleotide or polypeptide with
which it is associated in nature and/or is linked to a
polynucleotide or polypeptide other than that to which it is linked
in nature.
[0118] The term "host cell" as used herein, refer to both
prokaryotic and eukaryotic cells which are used as recipients of
the recombinant polynucleotides and vectors provided herein. In one
embodiment, the host cell is a prokaryote.
[0119] The terms "open reading frame" and "ORF" as used herein
refers to a region of a polynucleotide sequence which encodes a
polypeptide; this region may represent a portion of a coding
sequence or a total coding sequence.
[0120] As used herein and as known in the art, the term "identity"
is the relationship between two or more polynucleotide sequences,
as determined by comparing the sequences. Identity also means the
degree of sequence relatedness between polynucleotide sequences, as
determined by the match between strings of such sequences. Identity
can be readily calculated (see, e.g., Computation Molecular
Biology, Lesk, A. M., eds., Oxford University Press, New York
(1998), and Biocomputing: Informatics and Genome Projects, Smith,
D. W., ed., Academic Press, New York (1993), both of which are
incorporated by reference herein). While there exist a number of
methods to measure identity between two polynucleotide sequences,
the term is well known to skilled artisans (see, e.g., Sequence
Analysis in Molecular Biology, von Heinje, G., Academic Press
(1987); and Sequence Analysis Primer, Gribskov., M. and Devereux,
J., eds., M. Stockton Press, New York (1991)). Methods commonly
employed to determine identity between sequences include, for
example, those disclosed in Carillo, H., and Lipman, D., SIAM J.
Applied Math. (1988) 48:1073. "Substantially identical," as used
herein, means there is a very high degree of homology (preferably
100% sequence identity) between subject polynucleotide sequences.
However, polynucleotides having greater than 90%, or 95% sequence
identity may be used in the present invention, and thus sequence
variations that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence can be tolerated.
[0121] As used herein, the term "treatment" refers to the
application or administration of a therapeutic agent to a patient,
or application or administration of a therapeutic agent to an
isolated tissue or cell line from a patient, who has a disorder,
e.g., a disease or condition, a symptom of disease, or a
predisposition toward a disease, with the purpose to cure, heal,
alleviate, relieve, alter, remedy, ameliorate, improve, or affect
the disease, the symptoms of disease, or the predisposition toward
disease.
[0122] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a farnesyl dibenzodiazepinone
and a pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of a
farnesyl dibenzodiazepinone effective to produce the intended
pharmacological, therapeutic or preventive result. For example, if
a given clinical treatment is considered effective when there is at
least a 25% reduction in a measurable parameter associated with a
disease or disorder, a therapeutically effective amount of a drug
for the treatment of that disease or disorder is the amount
necessary to effect at least a 25% reduction in that parameter.
[0123] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0124] The term "pharmaceutically acceptable salt" refers to both
acid addition salts and base addition salts. The nature of the salt
is not critical, provided that it is pharmaceutically acceptable.
Exemplary acid addition salts include, without limitation,
hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulphuric,
phosphoric, formic, acetic, citric, tartaric, succinic, oxalic,
malic, glutamic, propionic, glycolic, gluconic, maleic, embonic
(pamoic), methanesulfonic, ethanesulfonic, 2-hydroxyethanesulfonic,
pantothenic, benzenesulfonic, toluenesulfonic, sulfanilic, mesylic,
cyclohexylaminosulfonic, stearic, algenic, .beta.-hydroxybutyric,
malonic, galactaric, galacturonic acid and the like. Suitable
pharmaceutically acceptable base addition salts include, without
limitation, metallic salts made from aluminium, calcium, lithium,
magnesium, potassium, sodium and zinc or organic salts made from
N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, ethylenediamine, N-methylglucamine, lysine,
procaine and the like. Additional examples of pharmaceutically
acceptable salts are listed in Journal of Pharmaceutical Sciences
(1977) 66:2. All of these salts may be prepared by conventional
means from a farnesyl dibenzodiazepinone by treating the compound
with the appropriate acid or base.
II. Farnesylated Dibenzodiazepinone Compounds
[0125] In one aspect, the invention relates to a novel farnesyl
dibenzodiazepinone, referred to herein as "ECO-04601" and having
the chemical structure represented by the following formula:
##STR00013##
[0126] ECO-04601 may be described as a new dibenzodiazepinone
having a 10-farnesyl substituent located on the nitrogen atom in
the 10 position of the dibenzodiazepine ring (i.e., the amide
nitrogen in the diazepinone ring), and three phenolic hydroxy
substituents in the 4,6 and 8 positions of the dibenzodiazepinone
ring. ECO-04601 may be characterized by any one or more of its
physicochemical and spectral properties given below, such as its
mass, UV, and NMR spectroscopic data. Mass was determined by
electrospray mass spectrometry to be 462.6 (FIG. 1); UV=230 nm with
a shoulder at 290 nm (FIG. 2). NMR data were collected using
MeOH-d4, including proton (FIG. 3), and multidimensional pulse
sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5), gHMBC (FIG. 6), and
NOESY (FIG. 7).
[0127] In another aspect, the invention relates to a novel class of
farnesyl dibenzodiazepinone compounds represented by Formula I:
##STR00014##
wherein, [0128] W.sup.1, W.sup.2 and W.sup.3 is each independently
selected from
##STR00015##
[0128] or
[0129] the chain from the tricycle may terminate at W.sup.3,
W.sup.2 or W.sup.1 with W.sup.3, W.sup.2 or W.sup.1 respectively
being either --CH.dbd.O or --CH.sub.2OH;
[0130] A is selected from --NH--, --NCH.sub.2R.sup.1,
--NC(O)R.sup.1;
[0131] R.sup.1 is selected from C1-6 alkyl, C2-6 alkene, aryl or
heteroaryl;
[0132] R.sup.2, R.sup.3, and R.sup.4 is each independently selected
from H, R.sup.5, --C(O)R.sup.6
[0133] R.sup.5 is each independently selected from C.sub.1-6 alkyl,
C.sub.2-7 alkalene, aryl or heteroaryl;
[0134] R.sup.6 is each independently selected from H, C.sub.1-6
alkyl, C.sub.2-7 alkalene, aryl or heteroaryl; or a
pharmaceutically acceptable salt thereof.
[0135] In other embodiments, the invention provides compounds of
Formula I, wherein A is selected from the group consisting of NH,
NCH2R1, and NC(O)R1; wherein R2 is H; R3 is H; and R4 is H. In
another embodiment, R2, R3 and R4 are each H; and all other groups
are as previously defined. In a further embodiment, R2, R3 and R4
are each H; and W1 is --CH.dbd.CH-- and all other groups are as
previously defined. In a further embodiment, R2, R3 and R4 are each
H, and W2 is --CH.dbd.CH-- and all other groups are as previously
defined. In a further embodiment, R2, R3 and R4 are each H; and W3
is --CH.dbd.CH--; and all other groups are as previously defined.
In a further embodiment, A is NH; R2, R3 and R4 are each H; and all
other groups are as previously defined. In a further embodiment, A
is NH; each of W1, W2, and W3 is --CH .dbd.CH--; and all other
groups are as previously defined. The invention encompasses all
pharmaceutically acceptable salts of the foregoing compounds.
[0136] The following are exemplary compounds of the invention:
##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020##
##STR00021## ##STR00022## ##STR00023## ##STR00024##
[0137] Certain embodiments expressly exclude one or more of the
compounds of Formula I. In one embodiment, the compound of Formula
II is excluded.
[0138] The compounds of this invention may be formulated into
pharmaceutical compositions comprised of compounds of Formula I in
combination with a pharmaceutical acceptable carrier, as discussed
in Section V below.
III. Method of Making a Farnesyl Dibenzodiazepinone by
Fermentation
[0139] In one embodiment, ECO-04601 is obtained by cultivating a
novel strain of Micromonospora, namely Micromonospora sp. strain
046-ECO11. Strain 046-ECO11 was deposited on Mar. 7, 2003, with the
International Depositary Authority of Canada (IDAC), Bureau of
Microbiology, Health Canada, 1015 Arlington Street, Winnipeg,
Manitoba, Canada R3E 3R2, under Accession No. 070303-01. The
deposit of the strain was made under the terms of the Budapest
Treaty on the International Recognition of the Deposit of
Microorganisms for Purposes of Patent Procedure. The deposited
strains will be irrevocably and without restriction or condition
released to the public upon the issuance of a patent. The deposited
strains are provided merely as convenience to those skilled in the
art and are not an admission that a deposit is required for
enablement, such as that required under 35 U.S.C. .sctn.112.
[0140] It is to be understood that the present invention is not
limited to use of the particular strain 046-ECO11. Rather, the
present invention contemplates the use of other ECO-04601 producing
organisms, such as mutants or variants of 046-ECO11 that can be
derived from this organism by known means such as X-ray
irradiation, ultraviolet irradiation, treatment with nitrogen
mustard, phage exposure, antibiotic selection and the like; or
through the use of recombinant genetic engineering techniques, as
described in Section IV below.
[0141] The farnesyl dibenzodiazepinone compounds of the present
invention may be biosynthesized by various microorganisms.
Microorganisms that may synthesize the compounds of the present
invention include but are not limited to bacteria of the order
Actinomycetales, also referred to as actinomycetes. Non-limiting
examples of members belonging to the genera of Actinomycetes
include Nocardia, Geodermatophilus, Actinoplanes, Micromonospora,
Nocardioides, Saccharothrix, Amycolatopsis, Kutzneria,
Saccharomonospora, Saccharopolyspora, Kitasatospora, Streptomyces,
Microbispora, Streptosporangium, and Actinomadura. The taxonomy of
actinomycetes is complex and reference is made to Goodfellow,
Suprageneric Classification of Actinomycetes (1989); Bergey's
Manual of Systematic Bacteriology, Vol. 4 (Williams and Wilkins,
Baltimore, pp. 2322-2339); and to Embley and Stackebrandt, "The
molecular phylogeny and systematics of the actinomycetes," Annu.
Rev. Microbiol. (1994) 48:257-289, each of which is hereby
incorporated by reference in its entirety, for genera that may
synthesize the compounds of the invention.
[0142] Farnesyl dibenzodiazepinone-producing microorganisms are
cultivated in culture medium containing known nutritional sources
for actinomycetes. Such media having assimilable sources of carbon,
nitrogen, plus optional inorganic salts and other known growth
factors at a pH of about 6 to about 9. Suitable media include,
without limitation, the growth media provided in Table 16.
Microorganisms are cultivated at incubation temperatures of about
18.degree. C. to about 40.degree. C. for about 3 to about 40
days.
[0143] The culture media inoculated with the farnesyl
dibenzodiazepinone-producing microorganisms may be aerated by
incubating the inoculated culture media with agitation, for
example, shaking on a rotary shaker, or a shaking water bath.
Aeration may also be achieved by the injection of air, oxygen or an
appropriate gaseous mixture to the inoculated culture media during
incubation. Following cultivation, the farnesyl dibenzodiazepinone
compounds can be extracted and isolated from the cultivated culture
media by techniques known to a skilled person in the art and/or
disclosed herein, including for example centrifugation,
chromatography, adsorption, filtration. For example, the cultivated
culture media can be mixed with a suitable organic solvent such as
n-butanol, n-butyl acetate or 4-methyl-2-pentanone, the organic
layer can be separated for example, by centrifugation followed by
the removal of the solvent, by evaporation to dryness or by
evaporation to dryness under vacuum. The resulting residue can
optionally be reconstituted with for example water, ethanol, ethyl
acetate, methanol or a mixture thereof, and re-extracted with a
suitable organic solvent such as hexane, carbon tetrachloride,
methylene chloride or a mixture thereof. Following removal of the
solvent, the compounds may be further purified by the use of
standard techniques, such as chromatography.
[0144] The farnesyl dibenzodiapezinones biosynthesized by
microorganisms may optionally be subjected to random and/or
directed chemical modifications to form compounds that are
derivatives or structural analogs. Such derivatives or structural
analogs having similar functional activities are within the scope
of the present invention. Farnesyl dibenzodiapezinone compounds may
optionally be modified using methods known in the art and described
herein.
IV. Method of Making a Farnesyl Dibenzodiazepinone by Recombinant
Technology
[0145] In another embodiment, the present invention relates to
nucleic acid molecules that encode proteins useful in the
production of farnesyl benzodiazepinones. Specifically, the present
invention provides recombinant DNA vectors and nucleic acid
molecules that encode all or part of the biosynthetic locus in
strain 046-ECO11, which directs the production of ECO-04601, and is
referred to herein as "046D." The invention further includes
genetic modification of 046D using conventional genetic recombinant
techniques, such as mutagenesis, inactivation, or replacement of
nucleic acids, to produce chemical variants of ECO-04601.
[0146] The invention thus provides a method for making a farnesyl
benzodiazepinone compound using a transformed host cell comprising
a recombinant DNA vector that encodes one or more of the
polypeptides of the present invention, and culturing the host cell
under conditions such that farnesyl benzodiazepinone is produced.
The host cell is a prokaryote. In one embodiment, the host cell is
an actinomycete. In another embodiment, the host cell is a
Streptomyces host cell.
[0147] The invention provides recombinant nucleic acids that
produce a variety of farnesyl dibenzodiazepinone compounds that
cannot be readily synthesized by chemical methodology alone. The
invention allows direct manipulation of 046D biosynthetic locus via
genetic engineering of the enzymes involved in the biosynthesis of
a farnesyl benzodiazepinone according to the invention. The 046A
biosynthetic locus is described in Example 11.
Recombinant DNA Vectors
[0148] Vectors of the invention typically comprise the DNA of a
transmissible agent, into which foreign DNA is inserted. A common
way to insert one segment of DNA into another segment of DNA
involves the use of specific enzymes called restriction enzymes
that cleave DNA at specific sites (specific groups of nucleotides)
called restriction sites. A "cassette" refers to a DNA coding
sequence or segment of DNA that codes for an expression product
that can be inserted into a vector at defined restriction sites.
The cassette restriction sites are designed to ensure insertion of
the cassette in the proper reading frame. Generally, a nucleic acid
molecule that encodes a protein useful in the production of a
farnesyl benzodiazepinone is inserted at one or more restriction
sites of the vector DNA, and then is carried by the vector into a
prokaryote e.g. actinomycte, by transformation (see below). A
segment or sequence of DNA having inserted or added DNA, such as an
expression vector, can also be called a "DNA construct". A common
type of vector is a "plasmid" which generally is a self-contained
molecule of double-stranded DNA, usually of bacterial origin, that
can readily accept additional (foreign) DNA and which can be
readily introduced into a suitable host cell. A plasmid vector
often contains coding DNA and promoter DNA and has one or more
restriction sites suitable for inserting foreign DNA. Coding DNA is
a DNA sequence that encodes a particular amino acid sequence for a
particular protein or enzyme. In one embodiment of the invention,
the coding DNA encodes for polypeptides of SEQ ID NOs. 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79,
81, 83, 85, 87 or 89 that are required for the biosynthesis of a
farnesyl benzodiazepinone.
[0149] Promoter DNA of a recombinant vector is a DNA sequence that
initiates, regulates, or otherwise mediates or controls the
expression of the coding DNA. Promoter DNA and coding may be from
the same or different organisms. Recombinant cloning vectors will
often include one or more replication systems for cloning or
expression, one or more markers for selection in the host, e.g.
antibiotic resistance, and one or more expression cassettes. Vector
constructs may be produced using conventional molecular biology and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory
Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (herein "Sambrook et al., 1989"); DNA
Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed.
1985); F. M. Ausubel et al. (eds.), Current Protocols in Molecular
Biology, John Wiley & Sons, Inc. (1994).
[0150] Examples of promoters that function in actinomycetes, e.g.
Streptomyces, are taught in U.S. Pat. Nos. 5,830,695 and 5,466,590.
Another example of a transcription promoter useful in Actinomycetes
expression vectors is tipA, a promoter inducible by the antibiotic
thiostrepton [c.f. Murakami, T., et al., (1989), J. Bacteriol.,
171,1459].
Transformation of Actinomycetes
[0151] A suitable transformation method for use with an
actinomycete comprises forming the actinomycete culture into
spheroplasts using lysozyme. A buffer solution containing
recombinant DNA vectors and polyethylene glycol is then added, in
order to introduce the vector into the host cells, by using either
of the methods of Thompson or Keiser [c. f. Thompson, C. J., et
al., (1982), J. Bacteriol., 151, 668-677 or Keiser, T. et al.
(2000), "Practical Streptomyces Genetics", The John Innes
Foundation, Norwich], for example. A thiostrepton-resistance gene
is frequently used as a selective marker in the transformation
plasmid [c.f. Hopwood, D. A., et al., (1987), "Methods in
Enzymology" 153, 116, Academic Press, New York], but the present
invention is not limited thereto. Additional methods for the
transformation of actinomycetes are taught in U.S. Pat. No.
5,393,665.
Assay for Farnesyl Dibenzodiazepinone or Biosynthetic
Intermediates
[0152] Actinomycetes defective in farnesyl dibenzodiazepinone
biosynthesis are transformed with one or more expression vectors
encoding one or more proteins in the farnesyl benzodiazepinone
biosynthetic pathway, thus restoring farnesyl benzodiazepinone
biosynthesis by genetic complementation of the specific defect.
[0153] The presence or absence of farnesyl dibenzodiazepinone or
intermediates in the biosynthetic pathway (see FIGS. 13, 14 and 15)
in a recombinant actinomycete can be determined using methodologies
that are well known to persons of skill in the art. For example,
ethyl acetate extracts of fermentation media used for the culture
of a recombinant actinomycete are processed as described in Example
2 and fractions containing farnesyl dibenzodiazepinone or
intermediates detected by TLC on commercial Kieselgel 60F.sub.254
plates. Farnesyl dibenzodiazepinone and intermediate compounds are
visualized by inspection of dried plates under UV light or by
spraying the plates with a spray containing vanillin (0.75%) and
concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently
heating the plate. The exact identity of the compounds separated by
TLC is then determined using gas chromatography-mass spectroscopy.
Methods of mass spectroscopy are taught in the published U.S.
Patent Application No. US2003/0052268.
Mutagenesis
[0154] The invention allows direct manipulation of 046D
biosynthetic locus via genetic engineering of the enzymes involved
in the biosynthesis of a farnesyl benzodiazepinone according to the
invention.
[0155] A number of methods are known in the art that permit the
random as well as targeted mutation of the DNA sequences of the
invention (see for example, Ausubel et. al. Short Protocols in
Molecular Biology (1995) 3rd Ed. John Wiley & Sons, Inc.). In
addition, there are a number of commercially available kits for
site-directed mutagenesis, including both conventional and
PCR-based methods. Examples include the EXSITE.TM. PCR-Based
Site-directed Mutagenesis Kit available from Stratagene (Catalog
No. 200502) and the QUIKCHANGE.TM. Site-directed mutagenesis Kit
from Stratagene (Catalog No. 200518), and the CHAMELEON.RTM.
double-stranded Site-directed mutagenesis kit, also from Stratagene
(Catalog No. 200509).
[0156] In addition the nucleotides of the invention may be
generated by insertional mutation or truncation (N-terminal,
internal or C-terminal) according to methodology known to a person
skilled in the art.
[0157] Older methods of site-directed mutagenesis known in the art
rely on sub-cloning of the sequence to be mutated into a vector,
such as an M13 bacteriophage vector, that allows the isolation of
single-stranded DNA template. In these methods, one anneals a
mutagenic primer (i.e., a primer capable of annealing to the site
to be mutated but bearing one or more mismatched nucleotides at the
site to be mutated) to the single-stranded template and then
polymerizes the complement of the template starting from the 3' end
of the mutagenic primer. The resulting duplexes are then
transformed into host bacteria and plaques are screened for the
desired mutation.
[0158] More recently, site-directed mutagenesis has employed PCR
methodologies, which have the advantage of not requiring a
single-stranded template. In addition, methods have been developed
that do not require sub-cloning. Several issues must be considered
when PCR-based site-directed mutagenesis is performed. First, in
these methods it is desirable to reduce the number of PCR cycles to
prevent expansion of undesired mutations introduced by the
polymerase. Second, a selection must be employed in order to reduce
the number of non-mutated parental molecules persisting in the
reaction. Third, an extended-length PCR method is preferred in
order to allow the use of a single PCR primer set. And fourth,
because of the non-template-dependent terminal extension activity
of some thermostable polymerases it is often necessary to
incorporate an end-polishing step into the procedure prior to
blunt-end ligation of the PCR-generated mutant product.
[0159] The protocol described below accommodates these
considerations through the following steps. First, the template
concentration used is approximately 1000-fold higher than that used
in conventional PCR reactions, allowing a reduction in the number
of cycles from 25-30 down to 5-10 without dramatically reducing
product yield. Second, the restriction endonuclease Dpn I
(recognition target sequence: 5-Gm6ATC-3, where the A residue is
methylated) is used to select against parental DNA, since most
common strains of E. coli Dam methylate their DNA at the sequence
5-GATC-3. Third, Taq Extender is used in the PCR mix in order to
increase the proportion of long (i.e., full plasmid length) PCR
products. Finally, Pfu DNA polymerase is used to polish the ends of
the PCR product prior to intramolecular ligation using T4 DNA
ligase.
[0160] A non-limiting example for the isolation of mutant
polynucleotides is described in detail as follows:
[0161] Plasmid template DNA (approximately 0.5 pmole) is added to a
PCR cocktail containing: 1.times. mutagenesis buffer (20 mM Tris
HCl, pH 7.5; 8 mM MgCl2; 40 g/ml BSA); 12-20 pmole of each primer
(one of skill in the art may design a mutagenic primer as
necessary, giving consideration to those factors such as base
composition, primer length and intended buffer salt concentrations
that affect the annealing characteristics of oligonucleotide
primers; one primer must contain the desired mutation, and one (the
same or the other) must contain a 5' phosphate to facilitate later
ligation), 250 M each dNTP, 2.5 U Taq DNA polymerase, and 2.5 U of
Taq Extender (Available from Stratagene; See Nielson et al. (1994)
Strategies 7: 27, and U.S. Pat. No. 5,556,772). Primers can be
prepared using the triester method of Matteucci et al., 1981, J.
Am. Chem. Soc. 103:3185-3191, incorporated herein by reference.
Alternatively automated synthesis may be preferred, for example, on
a Biosearch 8700 DNA Synthesizer using cyanoethyl phosphoramidite
chemistry.
[0162] The PCR cycling is performed as follows: 1 cycle of 4 min at
94.degree. C., 2 min at 50.degree. C. and 2 min at 72.degree. C.;
followed by 5-10 cycles of 1 min at 94.degree. C., 2 min at
54.degree. C. and 1 min at 72.degree. C. The parental template DNA
and the linear, PCR-generated DNA incorporating the mutagenic
primer are treated with DpnI (10 U) and Pfu DNA polymerase (2.5 U).
This results in the DpnI digestion of the in vivo methylated
parental template and hybrid DNA and the removal, by Pfu DNA
polymerase, of the non-template-directed Taq DNA
polymerase-extended base(s) on the linear PCR product. The reaction
is incubated at 37.degree. C. for 30 min and then transferred to
72.degree. C. for an additional 30 min. Mutagenesis buffer (115 ul
of 1.times.) containing 0.5 mM ATP is added to the DpnI-digested,
Pfu DNA polymerase-polished PCR products. The solution is mixed and
10 ul are removed to a new microfuge tube and T4 DNA ligase (2-4 U)
is added. The ligation is incubated for greater than 60 min at
37.degree. C. Finally, the treated solution is transformed into
competent E. coli according to standard methods.
[0163] Methods of random mutagenesis, which will result in a panel
of mutants bearing one or more randomly situated mutations, exist
in the art. Such a panel of mutants may then be screened for those
exhibiting reduced uracil detection activity relative to the
wild-type polymerase (e.g., by measuring the incorporation of 10
nmoles of dNTPs into polymeric form in 30 minutes in the presence
of 200 M dUTP and at the optimal temperature for a given DNA
polymerase). An example of a method for random mutagenesis is the
so-called "error-prone PCR method". As the name implies, the method
amplifies a given sequence under conditions in which the DNA
polymerase does not support high fidelity incorporation. The
conditions encouraging error-prone incorporation for different DNA
polymerases vary, however one skilled in the art may determine such
conditions for a given enzyme. A key variable for many DNA
polymerases in the fidelity of amplification is, for example, the
type and concentration of divalent metal ion in the buffer. The use
of manganese ion and/or variation of the magnesium or manganese ion
concentration may therefore be applied to influence the error rate
of the polymerase.
[0164] Genes for desired mutant polypeptides generated by
mutagenesis may be sequenced to identify the sites and number of
mutations. For those mutants comprising more than one mutation, the
effect of a given mutation may be evaluated by introduction of the
identified mutation to the wild-type gene by site-directed
mutagenesis in isolation from the other mutations borne by the
particular mutant. Screening assays of the single mutant thus
produced will then allow the determination of the effect of that
mutation alone.
V. Genes and Proteins for the Production of ECO-04601
[0165] As discussed in more detail below, the isolated, purified or
enriched nucleic acids of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83,
85, 87 and 89 may be used to prepare one of the polypeptides of SEQ
ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67,
69, 71, 74, 76, 78, 80, 82, 84, 86 and 88, respectively, or
fragments comprising at least 50, 75, 100, 200, 300, 500 or more
consecutive amino acids of one of the polypeptides of SEQ ID NO: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71,
74, 76, 78, 80, 82, 84, 86 and 88.
[0166] Accordingly, another aspect of the present invention is an
isolated, purified or enriched nucleic acid which encodes one of
the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88
or fragments comprising at least 50, 75, 100, 150, 200, 300 or more
consecutive amino acids of one of the polypeptides of SEQ ID NOS:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71,
74, 76, 78, 80, 82, 84, 86 and 88. The coding sequences of these
nucleic acids may be identical to one of the coding sequences of
one of the nucleic acids of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87
and 89 or a fragment thereof, or may be different coding sequences
which encode one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80,
82, 84, 86 and 88 or fragments comprising at least 50, 75, 100,
150, 200, 300 consecutive amino acids of one of the polypeptides of
SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65,
67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 as a result of the
redundancy or degeneracy of the genetic code. The genetic code is
well known to those of skill in the art and can be obtained, for
example, from Stryer, Biochemistry, 3.sup.rd edition, W.H. Freeman
& Co., New York.
[0167] The isolated, purified or enriched nucleic acid which
encodes one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82,
84, 86 and 88 may include, but is not limited to: (1) only the
coding sequences of one of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87
and 89; (2) the coding sequences of SEQ ID NOS: 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83,
85, 87 and 89 and additional coding sequences, such as leader
sequences or proprotein; and (3) the coding sequences of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 and non-coding sequences,
such as non-coding sequences 5' and/or 3' of the coding sequence.
Thus, as used herein, the term "polynucleotide encoding a
polypeptide" encompasses a polynucleotide that includes only coding
sequence for the polypeptide as well as a polynucleotide that
includes additional coding and/or non-coding sequence.
[0168] The invention relates to polynucleotides based on SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89 but having polynucleotide
changes that are "silent", for example changes which do not alter
the amino acid sequence encoded by the polynucleotides of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The invention also
relates to polynucleotides which have nucleotide changes which
result in amino acid substitutions, additions, deletions, fusions
and truncations of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80,
82, 84, 86 and 88. Such nucleotide changes may be introduced using
techniques such as site directed mutagenesis, random chemical
mutagenesis, exonuclease III deletion, and other recombinant DNA
techniques.
[0169] The isolated, purified or enriched nucleic acids of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences
complementary thereto, or a fragment comprising at least 100, 150,
200, 300, 400 or more consecutive bases of one of the sequence of
SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66,
68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences
complementary thereto may be used as probes to identify and isolate
DNAs encoding the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82,
84, 86 and 88 respectively. In such procedures, a genomic DNA
library is constructed from a sample microorganism or a sample
containing a microorganism capable of producing a farnesyl
dibenzodiazepinone. The genomic DNA library is then contacted with
a probe comprising a coding sequence or a fragment of the coding
sequence, encoding one of the polypeptides of SEQ ID NOS: 2, 4, 6,
8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40,
42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76,
78, 80, 82, 84, 86 and 88, or a fragment thereof under conditions
which permit the probe to specifically hybridize to sequences
complementary thereto. In a preferred embodiment, the probe is an
oligonucleotide of about 10 to about 30 nucleotides in length
designed based on a nucleic acid of SEQ ID NOS: 3, 5, 7, 9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47,
49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83,
85, 87 and 89. Genomic DNA clones which hybridize to the probe are
then detected and isolated. Procedures for preparing and
identifying DNA clones of interest are disclosed in Ausubel et al.,
Current Protocols in Molecular Biology, John Wiley 503 Sons, Inc.
1997; and Sambrook et al., Molecular Cloning: A Laboratory Manual
2d Ed., Cold Spring Harbor Laboratory Press, 1989. In another
embodiment, the probe is a restriction fragment or a PCR amplified
nucleic acid derived from SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51,
53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77, 79, 81, 83, 85, 87
and 89.
[0170] The isolated, purified or enriched nucleic acids of SEQ ID
NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, the sequences
complementary thereto, or a fragment comprising at least 10, 15,
20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500
consecutive bases of one of the sequences of SEQ ID NOS: 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68, 70, 72, 75, 77,
79, 81, 83, 85, 87 and 89 or the sequences complementary thereto
may be used as probes to identify and isolate related nucleic
acids. In some embodiments, the related nucleic acids may be
genomic DNAs (or cDNAs) from potential farnesyl dibenzodiazepinone
producers. In such procedures, a nucleic acid sample containing
nucleic acids from a potential farnesyl dibenzodiazepinone producer
is contacted with the probe under conditions that permit the probe
to specifically hybridize to related sequences. The nucleic acid
sample may be a genomic DNA (or cDNA) library from the potential
farnesyl dibenzodiazepinone-producer. Hybridization of the probe to
nucleic acids is then detected using any of the methods described
above.
[0171] Hybridization may be carried out under conditions of low
stringency, moderate stringency or high stringency. As an example
of nucleic acid hybridization, a polymer membrane containing
immobilized denatured nucleic acids is first prehybridized for 30
minutes at 45.degree. C. in a solution consisting of 0.9 M NaCl, 50
mM NaH.sub.2PO.sub.4, pH 7.0, 5.0 mM Na.sub.2EDTA, 0.5% SDS,
10.times.Denhardt's, and 0.5 mg/ml polyriboadenylic acid.
Approximately 2.times.10.sup.7 cpm (specific activity
4-9.times.10.sup.8 cpm/ug) of .sup.32P end-labeled oligonucleotide
probe are then added to the solution. After 12-16 hours of
incubation, the membrane is washed for 30 minutes at room
temperature in 1.times.SET (150 mM NaCl, 20 mM Tris hydrochloride,
pH 7.8, 1 mM Na.sub.2EDTA) containing 0.5% SDS, followed by a 30
minute wash in fresh 1.times.SET at Tm-10.degree. C. for the
oligonucleotide probe where Tm is the melting temperature. The
membrane is then exposed to autoradiographic film for detection of
hybridization signals.
[0172] By varying the stringency of the hybridization conditions
used to identify nucleic acids, such as genomic DNAs or cDNAs,
which hybridize to the detectable probe, nucleic acids having
different levels of homology to the probe can be identified and
isolated. Stringency may be varied by conducting the hybridization
at varying temperatures below the melting temperatures of the
probes. The melting temperature of the probe may be calculated
using the following formulas:
[0173] For oligonucleotide probes between 14 and 70 nucleotides in
length the melting temperature (Tm) in degrees Celcius may be
calculated using the formula: T.sub.m=81.5+16.6(log
[Na.sup.+])+0.41 (fraction G+C)-(600/N) where N is the length of
the oligonucleotide.
[0174] If the hybridization is carried out in a solution containing
formamide, the melting temperature may be calculated using the
equation Tm=81.5+16.6(log [Na+])+0.41 (fraction G+C)-(0.63%
formamide)-(600/N) where N is the length of the probe.
[0175] Prehybridization may be carried out in 6.times.SSC,
5.times.Denhardt's reagent, 0.5% SDS, 0.1 mg/ml denatured
fragmented salmon sperm DNA or 6.times.SSC, 5.times.Denhardt's
reagent, 0.5% SDS, 0.1 mg/ml denatured fragmented salmon sperm DNA,
50% formamide. The composition of the SSC and Denhardt's solutions
are listed in Sambrook et al., supra.
[0176] Hybridization is conducted by adding the detectable probe to
the hybridization solutions listed above. Where the probe comprises
double stranded DNA, it is denatured by incubating at elevated
temperatures and quickly cooling before addition to the
hybridization solution. It may also be desirable to similarly
denature single stranded probes to eliminate or diminish formation
of secondary structures or oligomerization. The filter is contacted
with the hybridization solution for a sufficient period of time to
allow the probe to hybridize to cDNAs or genomic DNAs containing
sequences complementary thereto or homologous thereto. For probes
over 200 nucleotides in length, the hybridization may be carried
out at 15-25.degree. C. below the Tm. For shorter probes, such as
oligonucleotide probes, the hybridization may be conducted at
5-10.degree. C. below the Tm. Preferably, the hybridization is
conducted in 6.times.SSC, for shorter probes. Preferably, the
hybridization is conducted in 50% formamide containing solutions,
for longer probes. All the foregoing hybridizations would be
considered to be examples of hybridization performed under
conditions of high stringency.
[0177] Following hybridization, the filter is washed for at least
15 minutes in 2.times.SSC, 0.1% SDS at room temperature or higher,
depending on the desired stringency. The filter is then washed with
0.1.times.SSC, 0.5% SDS at room temperature (again) for 30 minutes
to 1 hour. Nucleic acids which have hybridized to the probe are
identified by conventional autoradiography and non-radioactive
detection methods.
[0178] The above procedure may be modified to identify nucleic
acids having decreasing levels of homology to the probe sequence.
For example, to obtain nucleic acids of decreasing homology to the
detectable probe, less stringent conditions may be used. For
example, the hybridization temperature may be decreased in
increments of 5.degree. C. from 68.degree. C. to 42.degree. C. in a
hybridization buffer having a Na+ concentration of approximately
1M. Following hybridization, the filter may be washed with
2.times.SSC, 0.5% SDS at the temperature of hybridization. These
conditions are considered to be "moderate stringency" conditions
above 50.degree. C. and "low stringency" conditions below
50.degree. C. A specific example of "moderate stringency"
hybridization conditions is when the above hybridization is
conducted at 55.degree. C. A specific example of "low stringency"
hybridization conditions is when the above hybridization is
conducted at 45.degree. C.
[0179] Alternatively, the hybridization may be carried out in
buffers, such as 6.times.SSC, containing formamide at a temperature
of 42.degree. C. In this case, the concentration of formamide in
the hybridization buffer may be reduced in 5% increments from 50%
to 0% to identify clones having decreasing levels of homology to
the probe. Following hybridization, the filter may be washed with
6.times.SSC, 0.5% SDS at 50.degree. C. These conditions are
considered to be "moderate stringency" conditions above 25%
formamide and "low stringency" conditions below 25% formamide. A
specific example of "moderate stringency" hybridization conditions
is when the above hybridization is conducted at 30% formamide. A
specific example of "low stringency" hybridization conditions is
when the above hybridization is conducted at 10% formamide. Nucleic
acids which have hybridized to the probe are identified by
conventional autoradiography and non-radioactive detection
methods.
[0180] The preceding methods may be used to isolate nucleic acids
having at least 97%, at least 95%, at least 90%, at least 85%, at
least 80%, or at least 70% sequence identity to a nucleic acid
sequence selected from the group consisting of the sequences of SEQ
ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66, 68,
70, 72, 75, 77, 79, 81, 83, 85, 87 and 89. The isolated nucleic
acid may have a coding sequence that is a naturally occurring
allelic variant of one of the coding sequences described herein.
Such allelic variant may have a substitution, deletion or addition
of one or more nucleotides when compared to the nucleic acids of
SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 66,
68, 70, 72, 75, 77, 79, 81, 83, 85, 87 and 89, or the sequences
complementary thereto.
[0181] Additionally, the above procedures may be used to isolate
nucleic acids which encode polypeptides having at least 99%, at
least 95%, at least 90%, at least 85%, at least 80%, or at least
70% identity to a polypeptide having the sequence of one of SEQ ID
NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67,
69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising
at least 50, 75, 100, 150, 200, 300 consecutive amino acids
thereof.
[0182] Another aspect of the present invention is an isolated or
purified polypeptide comprising the sequence of one of SEQ ID NOS:
2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36,
38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71,
74, 76, 78, 80, 82, 84, 86 and 88 or fragments comprising at least
50, 75, 100, 150, 200 or 300 consecutive amino acids thereof. As
discussed herein, such polypeptides may be obtained by inserting a
nucleic acid encoding the polypeptide into a vector such that the
coding sequence is operably linked to a sequence capable of driving
the expression of the encoded polypeptide in a suitable host cell.
For example, the expression vector may comprise a promoter, a
ribosome binding site for translation initiation and a
transcription terminator. The vector may also include appropriate
sequences for modulating expression levels, an origin of
replication and a selectable marker.
[0183] Promoters suitable for expressing the polypeptide or
fragment thereof in bacteria include the E. coli lac or trp
promoters, the lad promoter, the lacZ promoter, the T3 promoter,
the T7 promoter, the gpt promoter, the lambda P.sub.R promoter, the
lambda P.sub.L promoter, promoters from operons encoding glycolytic
enzymes such as 3-phosphoglycerate kinase (PGK), and the acid
phosphatase promoter. Fungal promoters include the .alpha. factor
promoter. Eukaryotic promoters include the CMV immediate early
promoter, the HSV thymidine kinase promoter, heat shock promoters,
the early and late SV40 promoter, LTRs from retroviruses, and the
mouse metallothionein-I promoter. Other promoters known to control
expression of genes in prokaryotic or eukaryotic cells or their
viruses may also be used.
[0184] Mammalian expression vectors may also comprise an origin of
replication, any necessary ribosome binding sites, a
polyadenylation site, splice donors and acceptor sites,
transcriptional termination sequences, and 5' flanking
nontranscribed sequences. In some embodiments, DNA sequences
derived from the SV40 splice and polyadenylation sites may be used
to provide the required nontranscribed genetic elements.
[0185] Vectors for expressing the polypeptide or fragment thereof
in eukaryotic cells may also contain enhancers to increase
expression levels. Enhancers are cis-acting elements of DNA,
usually from about 10 to about 300 bp in length that act on a
promoter to increase its transcription. Examples include the SV40
enhancer on the late side of the replication origin bp 100 to 270,
the cytomegalovirus early promoter enhancer, the polyoma enhancer
on the late side of the replication origin, and the adenovirus
enhancers.
[0186] In addition, the expression vectors preferably contain one
or more selectable marker genes to permit selection of host cells
containing the vector. Examples of selectable markers that may be
used include genes encoding dihydrofolate reductase or genes
conferring neomycin resistance for eukaryotic cell culture, genes
conferring tetracycline or ampicillin resistance in E. coli, and
the S. cerevisiae TRP1 gene.
[0187] The appropriate DNA sequence may be inserted into the vector
by a variety of procedures. In general, the DNA sequence is ligated
to the desired position in the vector following digestion of the
insert and the vector with appropriate restriction endonucleases.
Alternatively, appropriate restriction enzyme sites can be
engineered into a DNA sequence by PCR. A variety of cloning
techniques are disclosed in Ausbel et al. Current Protocols in
Molecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et
al., Molecular Cloning: A Laboratory Manual 2d Ed., Cold Spring
Harbour Laboratory Press, 1989. Such procedures and others are
deemed to be within the scope of those skilled in the art.
[0188] The vector may be, for example, in the form of a plasmid, a
viral particle, or a phage. Other vectors include derivatives of
chromosomal, nonchromosomal and synthetic DNA sequences, viruses,
bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors
derived from combinations of plasmids and phage DNA, viral DNA such
as vaccinia, adenovirus, fowl pox virus, and pseudorabies. A
variety of cloning and expression vectors for use with prokaryotic
and eukaryotic hosts are described by Sambrook et al., Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor,
N.Y., (1989).
[0189] Particular bacterial vectors which may be used include the
commercially available plasmids comprising genetic elements of the
well known cloning vector pBR322 (ATCC 37017), pKK223-3 (Pharmacia
Fine Chemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison,
Wis., USA) pQE70, pQE60, pQE-9 (Qiagen), pD10, phiX174,
pBluescript.TM. II KS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene),
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia), pKK232-8
and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,
pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).
However, any other vector may be used as long as it is replicable
and stable in the host cell.
[0190] The host cell may be any of the host cells familiar to those
skilled in the art, including prokaryotic cells or eukaryotic
cells. As representative examples of appropriate hosts, there may
be mentioned: bacteria cells, such as E. coli, Streptomyces
lividans, Streptomyces griseofuscus, Streptomyces ambofaciens,
Bacillus subtilis, Salmonella typhimurium and various species
within the genera Pseudomonas, Streptomyces, Bacillus, and
Staphylococcus, fungal cells, such as yeast, insect cells such as
Drosophila S2 and Spodoptera Sf9, animal cells such as CHO, COS or
Bowes melanoma, and adenoviruses. The selection of an appropriate
host is within the abilities of those skilled in the art.
[0191] The vector may be introduced into the host cells using any
of a variety of techniques, including electroporation
transformation, transfection, transduction, viral infection, gene
guns, or Ti-mediated gene transfer. Where appropriate, the
engineered host cells can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or amplifying the genes of the present invention.
Following transformation of a suitable host strain and growth of
the host strain to an appropriate cell density, the selected
promoter may be induced by appropriate means (e.g., temperature
shift or chemical induction) and the cells may be cultured for an
additional period to allow them to produce the desired polypeptide
or fragment thereof.
[0192] Cells are typically harvested by centrifugation, disrupted
by physical or chemical means, and the resulting crude extract is
retained for further purification. Microbial cells employed for
expression of proteins can be disrupted by any convenient method,
including freeze-thaw cycling, sonication, mechanical disruption,
or use of cell lysing agents. Such methods are well known to those
skilled in the art. The expressed polypeptide or fragment thereof
can be recovered and purified from recombinant cell cultures by
methods including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. Protein refolding steps
can be used, as necessary, in completing configuration of the
polypeptide. If desired, high performance liquid chromatography
(HPLC) can be employed for final purification steps.
[0193] Various mammalian cell culture systems can also be employed
to express recombinant protein. Examples of mammalian expression
systems include the COS-7 lines of monkey kidney fibroblasts
(described by Gluzman, Cell, 23:175 (1981)), and other cell lines
capable of expressing proteins from a compatible vector, such as
the C127, 3T3, CHO, HeLa and BHK cell lines. The constructs in host
cells can be used in a conventional manner to produce the gene
product encoded by the recombinant sequence. Polypeptides of the
invention may or may not also include an initial methionine amino
acid residue.
[0194] Alternatively, the polypeptides of SEQ ID NOS: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,
80, 82, 84, 86 and 88 or fragments comprising at least 50, 75, 100,
150, 200 or 300 consecutive amino acids thereof can be
synthetically produced by conventional peptide synthesizers. In
other embodiments, fragments or portions of the polynucleotides may
be employed for producing the corresponding full-length polypeptide
by peptide synthesis; therefore, the fragments may be employed as
intermediates for producing the full-length polypeptides.
[0195] Cell-free translation systems can also be employed to
produce one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82,
84, 86 and 88 or fragments comprising at least 50, 75, 100, 150,
200 or 300 consecutive amino acids thereof using mRNAs transcribed
from a DNA construct comprising a promoter operably linked to a
nucleic acid encoding the polypeptide or fragment thereof. In some
embodiments, the DNA construct may be linearized prior to
conducting an in vitro transcription reaction. The transcribed mRNA
is then incubated with an appropriate cell-free translation
extract, such as a rabbit reticulocyte extract, to produce the
desired polypeptide or fragment thereof.
[0196] The present invention also relates to variants of the
polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or
fragments comprising at least 50, 75, 100, 150, 200 or 300
consecutive amino acids thereof. The term "variant" includes
derivatives or analogs of these polypeptides. In particular, the
variants may differ in amino acid sequence from the polypeptides of
SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65,
67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 by one or more
substitutions, additions, deletions, fusions and truncations, which
may be present in any combination.
[0197] The variants may be naturally occurring or created in vitro.
In particular, such variants may be created using genetic
engineering techniques such as site directed mutagenesis, random
chemical mutagenesis, exonuclease III deletion procedures, and
standard cloning techniques. Alternatively, such variants,
fragments, analogs, or derivatives may be created using chemical
synthesis or modification procedures.
[0198] Other methods of making variants are also familiar to those
skilled in the art. These include procedures in which nucleic acid
sequences obtained from natural isolates are modified to generate
nucleic acids that encode polypeptides having characteristics which
enhance their value in industrial or laboratory applications. In
such procedures, a large number of variant sequences having one or
more nucleotide differences with respect to the sequence obtained
from the natural isolate are generated and characterized.
Preferably, these nucleotide differences result in amino acid
changes with respect to the polypeptides encoded by the nucleic
acids from the natural isolates.
[0199] The variants of the polypeptides of SEQ ID NOS: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,
80, 82, 84, 86 and 88 may be variants in which one or more of the
amino acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78,
80, 82, 84, 86 and 88 are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code.
[0200] Conservative substitutions are those that substitute a given
amino acid in a polypeptide by another amino acid of like
characteristics. Typically seen as conservative substitutions are
the following replacements: replacements of an aliphatic amino acid
such as Ala, Val, Leu and IIe with another aliphatic amino acid;
replacement of a Ser with a Thr or vice versa; replacement of an
acidic residue such as Asp or Glu with another acidic residue;
replacement of a residue bearing an amide group, such as Asn or
Gln, with another residue bearing an amide group; exchange of a
basic residue such as Lys or Arg with another basic residue; and
replacement of an aromatic residue such as Phe or Tyr with another
aromatic residue.
[0201] Other variants are those in which one or more of the amino
acid residues of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80,
82, 84, 86 and 88 include a substituent group. Still other variants
are those in which the polypeptide is associated with another
compound, such as a compound to increase the half-life of the
polypeptide (for example, polyethylene glycol). Additional variants
are those in which additional amino acids are fused to the
polypeptide, such as leader sequence, a secretory sequence, a
proprotein sequence or a sequence that facilitates purification,
enrichment, or stabilization of the polypeptide.
[0202] In some embodiments, the fragments, derivatives and analogs
retain the same biological function or activity as the polypeptides
of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88. In other
embodiments, the fragment, derivative or analogue includes a fused
heterologous sequence that facilitates purification, enrichment,
detection, stabilization or secretion of the polypeptide that can
be enzymatically cleaved, in whole or in part, away from the
fragment, derivative or analogue.
[0203] Another aspect of the present invention are polypeptides or
fragments thereof which have at least 70%, at least 80%, at least
85%, at least 90%, or more than 95% identity to one of the
polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a
fragment comprising at least 50, 75, 100, 150, 200 or 300
consecutive amino acids thereof. It will be appreciated that amino
acid "substantially identity" includes conservative substitutions
such as those described above.
[0204] The polypeptides or fragments having homology to one of the
polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,
58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 or a
fragment comprising at least 50, 75, 100, 150, 200 or 300
consecutive amino acids thereof may be obtained by isolating the
nucleic acids encoding them using the techniques described
above.
[0205] Alternatively, the homologous polypeptides or fragments may
be obtained through biochemical enrichment or purification
procedures. The sequence of potentially homologous polypeptides or
fragments may be determined by proteolytic digestion, gel
electrophoresis and/or microsequencing. The sequence of the
prospective homologous polypeptide or fragment can be compared to
one of the polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86
and 88 or a fragment comprising at least 5, 10, 15, 20, 25, 30, 35,
40, 50, 75, 100, or 150 consecutive amino acids thereof.
[0206] The polypeptides of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,
52, 54, 56, 58, 60, 62, 65, 67, 69, 71, 74, 76, 78, 80, 82, 84, 86
and 88 or fragments, derivatives or analogs thereof comprising at
least 40, 50, 75, 100, 150, 200 or 300 consecutive amino acids
thereof invention may be used in a variety of applications. For
example, the polypeptides or fragments, derivatives or analogs
thereof may be used to catalyze biochemical reactions as described
elsewhere in the specification.
[0207] (a)
VI. Pharmaceutical Compositions Comprising Farnesyl
Dibenzodiazepinones
[0208] In another embodiment, the invention relates to a
pharmaceutical composition comprising a farnesyl
dibenzodiazepinone, as described in the preceding section, and a
pharmaceutically acceptable carrier, as described below. The
pharmaceutical composition comprising the farnesyl
dibenzodiazepinone is useful for treating a variety of diseases and
disorders, including cancer, inflammation and bacterial
infections.
[0209] The compounds of the present invention, or pharmaceutically
acceptable salts thereof, can be formulated for oral, intravenous,
intramuscular, subcutaneous, topical or parenteral administration
for the therapeutic or prophylactic treatment of diseases,
particularly bacterial infections, acute and chronic inflammation
and cancer. For oral or parental administration, compounds of the
present invention can be mixed with conventional pharmaceutical
carriers and excipients and used in the form of tablets, capsules,
elixirs, suspensions, syrups, wafers and the like. The compositions
comprising a compound of this present invention will contain from
about 0.1% to about 99.9%, about 1% to about 98%, about 5% to about
95%, about 10% to about 80% or about 15% to about 60% by weight of
the active compound.
[0210] The pharmaceutical preparations disclosed herein are
prepared in accordance with standard procedures and are
administered at dosages that are selected to reduce, prevent, or
eliminate bacterial infection, cancer or inflammation. (See, e.g.,
Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa.; and Goodman and Gilman, Pharmaceutical Basis of
Therapeutics, Pergamon Press, New York, N.Y., the contents of which
are incorporated herein by reference, for a general description of
the methods for administering various antimicrobial agents for
human therapy). The compositions of the present invention can be
delivered using controlled (e.g., capsules) or sustained release
delivery systems (e.g., bioerodable matrices). Exemplary delayed
release delivery systems for drug delivery that are suitable for
administration of the compositions of the invention (preferably of
Formula I) are described in U.S. Pat. Nos. 4,452,775 (issued to
Kent), 5,239,660 (issued to Leonard), 3,854,480 (issued to
Zaffaroni).
[0211] The pharmaceutically acceptable compositions of the present
invention comprise one or more compounds of the present invention
in association with one or more non-toxic, pharmaceutically
acceptable carriers and/or diluents and/or adjuvants and/or
excipients, collectively referred to herein as "carrier" materials,
and if desired other active ingredients. The compositions may
contain common carriers and excipients, such as corn starch or
gelatin, lactose, sucrose, microcrystalline cellulose, kaolin,
mannitol, dicalcium phosphate, sodium chloride and alginic acid.
The compositions may contain crosarmellose sodium, microcrystalline
cellulose, sodium starch glycolate and alginic acid.
[0212] Tablet binders that can be included are acacia,
methylcellulose, sodium carboxymethylcellulose,
polyvinylpyrrolidone (Providone), hydroxypropyl methylcellulose,
sucrose, starch and ethylcellulose.
[0213] Lubricants that can be used include magnesium stearate or
other metallic stearates, stearic acid, silicon fluid, talc, waxes,
oils and colloidal silica.
[0214] Flavouring agents such as peppermint, oil of wintergreen,
cherry flavouring or the like can also be used. It may also be
desirable to add a coloring agent to make the dosage form more
aesthetic in appearance or to help identify the product comprising
a compound of the present invention.
[0215] For oral use, solid formulations such as tablets and
capsules are particularly useful. Sustained released or enterically
coated preparations may also be devised. For pediatric and
geriatric applications, suspension, syrups and chewable tablets are
especially suitable. For oral administration, the pharmaceutical
compositions are in the form of, for example, a tablet, capsule,
suspension or liquid. The pharmaceutical composition is preferably
made in the form of a dosage unit containing a
therapeutically-effective amount of the active ingredient. Examples
of such dosage units are tablets and capsules. For therapeutic
purposes, the tablets and capsules which can contain, in addition
to the active ingredient, conventional carriers such as binding
agents, for example, acacia gum, gelatin, polyvinylpyrrolidone,
sorbitol, or tragacanth; fillers, for example, calcium phosphate,
glycine, lactose, maize-starch, sorbitol, or sucrose; lubricants,
for example, magnesium stearate, polyethylene glycol, silica or
talc: disintegrants, for example, potato starch, flavoring or
coloring agents, or acceptable wetting agents. Oral liquid
preparations generally are in the form of aqueous or oily
solutions, suspensions, emulsions, syrups or elixirs and may
contain conventional additives such as suspending agents,
emulsifying agents, non-aqueous agents, preservatives, coloring
agents and flavoring agents. Examples of additives for liquid
preparations include acacia, almond oil, ethyl alcohol,
fractionated coconut oil, gelatin, glucose syrup, glycerin,
hydrogenated edible fats, lecithin, methyl cellulose, methyl or
propyl para-hydroxybenzoate, propylene glycol, sorbitol, or sorbic
acid.
[0216] For intravenous (iv) use, compounds of the present invention
can be dissolved or suspended in any of the commonly used
intravenous fluids and administered by infusion. Intravenous fluids
include, without limitation, physiological saline or Ringer's
solution.
[0217] Formulations for parental administration can be in the form
of aqueous or non-aqueous isotonic sterile injection solutions or
suspensions. These solutions or suspensions can be prepared from
sterile powders or granules having one or more of the carriers
mentioned for use in the formulations for oral administration. The
compounds can be dissolved in polyethylene glycol, propylene
glycol, ethanol, corn oil, benzyl alcohol, sodium chloride, and/or
various buffers.
[0218] For intramuscular preparations, a sterile formulation of
compounds of the present invention or suitable soluble salts
forming the compound, can be dissolved and administered in a
pharmaceutical diluent such as Water-for-Injection (WFI),
physiological saline or 5% glucose. A suitable insoluble form of
the compound may be prepared and administered as a suspension in an
aqueous base or a pharmaceutically acceptable oil base, e.g. an
ester of a long chain fatty acid such as ethyl oleate.
[0219] For topical use the compounds of present invention can also
be prepared in suitable forms to be applied to the skin, or mucus
membranes of the nose and throat, and can take the form of creams,
ointments, liquid sprays or inhalants, lozenges, or throat paints.
Such topical formulations further can include chemical compounds
such as dimethylsulfoxide (DMSO) to facilitate surface penetration
of the active ingredient.
[0220] For application to the eyes or ears, the compounds of the
present invention can be presented in liquid or semi-liquid form
formulated in hydrophobic or hydrophilic bases as ointments,
creams, lotions, paints or powders.
[0221] For rectal administration the compounds of the present
invention can be administered in the form of suppositories admixed
with conventional carriers such as cocoa butter, wax or other
glyceride.
[0222] Alternatively, the compound of the present invention can be
in powder form for reconstitution in the appropriate
pharmaceutically acceptable carrier at the time of delivery. In
another embodiment, the unit dosage form of the compound can be a
solution of the compound or a salt thereof in a suitable diluent in
sterile, hermetically sealed ampoules.
[0223] The amount of the compound of the present invention in a
unit dosage comprises a therapeutically-effective amount of at
least one active compound of the present invention which may vary
depending on the recipient subject, route and frequency of
administration. A recipient subject refers to a plant, a cell
culture or an animal such as an ovine or a mammal including a
human.
[0224] According to this aspect of the present invention, the novel
compositions disclosed herein are placed in a pharmaceutically
acceptable carrier and are delivered to a recipient subject
(including a human subject) in accordance with known methods of
drug delivery. In general, the methods of the invention for
delivering the compositions of the invention in vivo utilize
art-recognized protocols for delivering the agent with the only
substantial procedural modification being the substitution of the
compounds of the present invention for the drugs in the
art-recognized protocols.
[0225] Likewise, the methods for using the claimed composition for
treating cells in culture, for example, to eliminate or reduce the
level of bacterial contamination of a cell culture, utilize
art-recognized protocols for treating cell cultures with
antibacterial agent(s) with the only substantial procedural
modification being the substitution of the compounds of the present
invention for the agents used in the art-recognized protocols.
[0226] The compounds of the present invention provide a method for
treating bacterial infections, pre-cancerous or cancerous
conditions, and acute or chronic inflammatory disease. As used
herein, the term "unit dosage" refers to a quantity of a
therapeutically effective amount of a compound of the present
invention that elicits a desired therapeutic response. As used
herein, the phrase "therapeutically effective amount" means an
amount of a compound of the present invention that prevents the
onset, alleviates the symptoms, or stops the progression of a
bacterial infection, inflammatory condition, or pre-cancerous or
cancerous condition. The term "treating" is defined as
administering, to a subject, a therapeutically effective amount of
at least one compound of the present invention, both to prevent the
occurrence of a bacterial infection, inflammation or pre-cancer or
cancer condition, or to control or eliminate a bacterial infection,
inflammation or pre-cancer or cancer condition. The term "desired
therapeutic response" refers to treating a recipient subject with a
compound of the present invention such that a bacterial or
inflammatory condition or pre-cancer or cancer condition is
reversed, arrested or prevented in a recipient subject.
[0227] The compounds of the present invention can be administered
as a single daily dose or in multiple doses per day. The treatment
regime may require administration over extended periods of time,
e.g., for several days or for from two to four weeks. The amount
per administered dose or the total amount administered will depend
on such factors as the nature and severity of the disease
condition, the age and general health of the recipient subject, the
tolerance of the recipient subject to the compound and the type of
the bacterial infection, inflammatory disorder, or type of
cancer.
[0228] A compound according to this invention may also be
administered in the diet or feed of a patient or animal. The diet
for animals can be normal foodstuffs to which the compound can be
added or it can be added to a premix.
[0229] The compounds of the present invention may be taken in
combination, together or separately with any known clinically
approved antibiotic, inflammation or anti-cancer agent to treat a
recipient subject in need of such treatment.
VII. Method of Inhibiting Tumor Growth
[0230] In another embodiment, the present invention relates to a
method of inhibiting tumor growth. Compounds as described herein
can possess antitumor activity. The compounds are effective against
mammalian tumor cells such as leukemia cells, melanoma cells,
breast carcinoma cells, lung carcinoma cells, pancreatic carcinoma
cells, ovarian carcinoma cells, renal carcinoma cells, colon
carcinoma cells prostate carcinoma cells and glioma cells. The
antitumor method of the invention results in inhibition of tumor
cells. The term "inhibition", when used in conjunction with the
antitumor method refers to suppression, killing, stasis, or
destruction of tumor cells. The antitumor method preferably results
in prevention, reduction or elimination of invasive activity and
related metastasis of tumor cells. The term "effective amount" when
used in conjunction with the antitumor cell method refers to the
amount of the compound sufficient to result in the inhibition of
mammalian tumor cells.
[0231] The inhibition of mammalian tumor growth according to this
method can be monitored in several ways. First, tumor cells grown
in vitro can be treated with the compound and monitored for growth
or death relative to the same cells cultured in the absence of the
compound. A cessation of growth or a slowing of the growth rate
(i.e., the doubling rate), e.g., by 10% or more, is indicative of
tumor cell inhibition. Alternatively, tumor cell inhibition can be
monitored by administering the compound to an animal model of the
tumor of interest. Examples of experimental animal tumor models are
known in the art and described in the examples herein. A cessation
of tumor growth (i.e., no further increase in size) or a reduction
in tumor size (i.e., tumor volume) or cell number (e.g., at least a
10% decrease in either) in animals treated with a compound as
described herein relative to tumors in control animals not treated
with the compound is indicative of tumor growth inhibition.
[0232] To monitor the efficacy of tumor treatment in a human, tumor
size or tumor cell titer is measured before and after initiation of
the treatment, and treatment is considered effective if either the
tumor size or titer ceases further growth, or if the tumor is
reduced in size or titer, e.g., by at least 10% or more (e.g., 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100%, that is, the
absence of the tumor). Methods of determining the size or cell
titer of a tumor in vivo vary with the type of tumor, and include,
for example, various imaging techniques well known to those in the
medical imaging or oncology fields (MRI, CAT, PET, etc.), as well
as histological techniques and flow cytometry.
[0233] For the antitumor method of the invention, a typical
effective dose of the compounds given orally or parenterally would
be from about 5 to about 100 mg/kg of body weight of the subject
with a daily dose ranging from about 15 to about 300 mg/kg of body
weight of the subject.
VIII. Method of Inhibiting Lipoxygenase
[0234] In another embodiment, the present invention also provides
for a method of treating diseased states, in particular
inflammation, caused by the 5-lipoxygenase system and/or by the
synthesis of the Leukotrienes C.sub.4, D.sub.4, E.sub.4 and F.sub.4
as well as Leukotriene B.sub.4 in mammals, especially in human
subjects. This method comprises administering to a subject an
effective amount of ECO-04601. Compound ECO-04601 may be used alone
or in combination with other anti-inflammatory compounds to treat
or prevent disease states related to inflammation including
pulmonary conditions, inflammation, cardiovascular conditions,
central nervous system conditions or skin conditions. More specific
diseases include gastritis; erosive esophagitis; inflammatory bowel
disease; ethanol-induced hemorrhagic erosions; hepatic ischemia;
ischemic neuronal injury; noxious agent induced damage or necrosis
of hepatic, pancreatic, renal, neuronal or myocardial tissue; liver
parenchymal damage caused by hepatoxic agents such as CCl.sub.4 and
D-galactosamine; ischemic renal failure; disease-induced hepatic
damage; trauma- or stress-induced cell damage; asthma; multiple
sclerosis; ischemic reperfusion; edema; rheumatoid arthritis; viral
encephalitis; bacterial pneumonia; neurodegeneration; Alzheimer's
disease and glycerol-induced renal failure.
[0235] For the method of the invention related to the
5-lipoxygenase system and/or the biosynthesis of Leukotrienes, a
typical effective unit dose of ECO-04601 given orally or
parenterally would be from about 5 to about 100 mg/kg of body
weight of the subject with a daily dose ranging from about 15 to
about 300 mg/kg of body weight of the subject.
[0236] The inhibition of lipoxygenase enzymes is monitored using
methods well known in the art and as described in the examples
herein. A decrease in enzyme activity by at least 10%, relative to
the activity in the absence of a compound as described herein is
indicative of effective inhibition of lipoxygenase activity.
[0237] Farnesyl dibenzodiazepinone compounds useful according to
the invention can be used to reduce or prevent inflammation. Among
the hallmarks of local acute inflammation are heat, redness,
swelling, pain and loss of function. These changes are induced
largely by changes in vascular flow and caliber, changes in
vascular permeability and leukocyte exudation (Robbins et al.,
"Pathologic Basis of Disease", 6.sup.th Ed., W.B. Saunders Co.,
Philadelphia, Pa.). Anti-inflammatory therapy performed using
compounds useful according to the invention can be monitored for
success by tracking any of these changes. For example, a decrease
in swelling (e.g., at least 10% decrease following treatment) or
reported pain (e.g., a sustained decrease of 1 point or more on a
1-10 scale reported by the patient, with 10 being the worst pain
experienced in association with this disorder prior to treatment,
and 0 being no pain) can be used to indicate successful
treatment.
[0238] Other measurable hallmarks of inflammation include leukocyte
infiltration and inflammatory cytokine levels. These hallmarks can
be monitored by biopsy of the affected tissue. A decrease of 10% or
more in leukocyte infiltration in fixed, stained tissue relative to
infiltration in similar tissue prior to treatment can be used to
indicate successful treatment, as can a decrease of 10% or more in
the level of any given inflammatory cytokine, relative to the level
before treatment. Those skilled in the art can readily assay for
inflammatory cytokine levels in tissue, blood, or other fluid
samples. Alternatively, the level of systemic indicators of
inflammation such as C reactive protein levels and erythrocyte
sedimentation rate can be monitored. Each of these has established
normal ranges in medicine, and treatment is considered successful
if one or more of such indicators goes from outside the normal
range to inside the normal range after the initiation of
treatment.
IX. Method of Inhibiting Bacterial Growth
[0239] In another embodiment, the present invention relates to a
method for treating bacterial infection in a mammalian subject in
need thereof, comprising the step of administering to the mammal a
therapeutically effective amount of compound ECO-04601, a compound
as described herein, or a pharmaceutically acceptable derivative or
prodrug thereof.
[0240] According to another embodiment, the invention provides a
method of decreasing bacterial quantity in a biological sample.
This method comprises the step of contacting the biological sample
with a compound ECO-04601, a compound as described herein, or a
pharmaceutically acceptable derivative or prodrug thereof. This
method is effective if the number of bacteria decreases by at least
10%, and preferably more, e.g., 25%, 50%, 75% or even 100% after
contacting the biological sample with compound ECO-04601, a
compound as described herein, or a pharmaceutically acceptable
derivative or prodrug thereof.
[0241] These pharmaceutical compositions effective to treat or
prevent a bacterial infection which comprise ECO-04601, a compound
as described herein, or a pharmaceutically acceptable derivative or
prodrug thereof in an amount sufficient to measurably decrease
bacterial quantity, and a pharmaceutically acceptable carrier, are
another embodiment of the present invention. The term "measurably
decrease bacterial quantity", as used herein means a measurable
change in the number of bacteria between a sample containing the
inhibitor and a sample not containing the inhibitor.
[0242] Agents which increase the susceptibility of bacterial
organisms to antibiotics are known. For example, U.S. Pat. No.
5,523,288, U.S. Pat. No. 5,783,561 and U.S. Pat. No. 6,140,306
describe methods of using bactericidal/permeability-increasing
protein (BPI) for increasing antibiotic susceptibility of
gram-positive and gram-negative bacteria. Agents that increase the
permeability of the outer membrane of bacterial organisms have been
described by Vaara, M. in Microbiological Reviews (1992) pp.
395-411, and the sensitization of gram-negative bacteria has been
described by Tsubery, H., et al, in J. Med. Chem. (2000) pp.
3085-3092.
[0243] For the method of the invention related to treatment of
subjects with a bacterial infection, a typical effective unit dose
of ECO-04601, a compound described herein or a pharmaceutically
acceptable derivative or prodrug thereof given orally or
parenterally would be from about 5 to about 100 mg/kg of body
weight of the subject with a daily dose ranging from about 15 to
about 300 mg/kg of body weight of the subject.
[0244] Another preferred embodiment of this invention relates to a
method, as described above, of treating a bacterial infection in a
mammal in need thereof, but further comprising the step of
administering to the mammal an agent which increases the
susceptibility of bacterial organisms to antibiotics.
[0245] According to another preferred embodiment, the invention
provides a method, as described above, of decreasing bacterial
quantity in a biological sample, but further comprising the step of
contacting the biological sample with an agent which increases the
susceptibility of bacterial organisms to antibiotics.
[0246] Methods of decreasing bacterial quantity are effective if
the number of bacteria decreases at least 10%, and preferably more,
e.g., 25%, 50%, 75% or even 100% after contacting the biological
sample with compound ECO-04601, a compound as described herein, or
a pharmaceutically acceptable derivative or prodrug thereof.
[0247] The pharmaceutical compositions and methods of this
invention will be useful generally for controlling bacterial
infections in vivo. Examples of bacterial organisms that may be
controlled by the compositions and methods of this invention
include, but are not limited to the following organisms:
Streptococcus pneumoniae, Streptococcus pyrogenes, Enterococcus
fecalis, Enterococcus faecium, Klebsiella pneumoniae, Enterobacter
spp., Proteus spp., Pseudomonas aeruginosa, E. coli, Serratia
marcesens, Staphylococcus aureus, Coagulase negative
Staphylococcus, Haemophilus infuenzae, Bacillus anthracis,
Mycoplasma pneumoniae, and Staphylococcus epidermidis. The
compositions and methods will therefore be useful for controlling,
treating or reducing the advancement, severity or effects of
nosocomial or non-nosocomial infections. Examples of nosocomial
uses include, but are not limited to, urinary tract infections,
pneumonia, surgical wound infections, bacteremia and therapy for
febrile neutropenic patients. Examples of non-nosocomial uses
include but are not limited to urinary tract infections, pneumonia,
prostatitis, skin and soft tissue infections and intra-abdominal
infections.
[0248] In addition to the compounds of this invention,
pharmaceutically acceptable derivatives or prodrugs of the
compounds of this invention may also be employed in compositions to
treat or prevent the above-identified disorders.
[0249] A "pharmaceutically acceptable derivative or prodrug" means
any pharmaceutically acceptable salt, ester, salt of an ester or
other derivative of a compound of this invention which, upon
administration to a recipient, is capable of providing, either
directly or indirectly, a compound of this invention or an
inhibitorily active metabolite or residue thereof. Particularly
favored derivatives or prodrugs are those that increase the
bioavailability of the compounds of this invention when such
compounds are administered to a mammal (e.g., by allowing an orally
administered compound to be more readily absorbed into the blood)
or which enhance delivery of the parent compound to a biological
compartment (e.g., the brain or lymphatic system) relative to the
parent species.
[0250] Pharmaceutically acceptable prodrugs of the compounds of
this invention include, without limitation, esters, amino acid
esters, phosphate esters, metal salts and sulfonate esters.
[0251] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, IC.sub.50 and so forth used in the
specification and claims are to be understood as being modified in
all instances by the term "about". Accordingly, unless indicated to
the contrary, the numerical parameters set forth in the present
specification and attached claims are approximations. At the very
least, and not as an attempt to limit the application of the
doctrine of equivalents to the scope of the claims, each numerical
parameter should at least be construed in light of the number of
significant figures and by applying ordinary rounding techniques.
Notwithstanding that the numerical ranges and parameters setting
forth the broad scope of the invention are approximations, the
numerical values set in the examples, Tables and Figures are
reported as precisely as possible. Any numerical values may
inherently contain certain errors resulting from variations in
experiments, testing measurements, statistical analyses and
such.
[0252] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
Example 1
Preparation of Production Culture
[0253] Unless otherwise noted, all reagents were purchased from
Sigma Chemical Co. (St. Louis, Mo.), (Aldrich). Micromonospora spp.
(deposit accession number IDAC 070303-01) was maintained on agar
plates of ISP2 agar (Difco Laboratories, Detroit, Mich.). An
inoculum for the production phase was prepared by transferring the
surface growth of the Micromonospora spp. from the agar plates to
125-mL flasks containing 25 mL of sterile medium comprised of 24 g
potato dextrin, 3 g beef extract, 5 g Bacto-casitone, 5 g glucose,
5 g yeast extract, and 4 g CaCO.sub.3 made up to one liter with
distilled water (pH 7.0). The culture was incubated at about
28.degree. C. for approximately 60 hours on a rotary shaker set at
250 rpm. Following incubation, 10 mL of culture was transferred to
a 2 L baffled flask containing 500 mL of sterile production medium
containing 20 g/L potato dextrin, 20 g/L glycerol, 10 g/L Fish
meal, 5 g/L Bacto-peptone, 2 g/L CaCO.sub.3, and 2 g/L
(NH.sub.4).sub.2SO.sub.4, pH 7.0. Fermentation broth was prepared
by incubating the production culture at 28.degree. C. in a rotary
shaker set at 250 rpm for one week.
Example 2
Isolation
[0254] 500 mL ethyl acetate was added to 500 mL of fermentation
broth prepared as described in Example 1 above. The mixture was
agitated for 30 minutes on an orbital shaker at 200 rpm to create
an emulsion. The phases were separated by centrifugation and
decantation. Between 4 and 5 g of anhydrous MgSO.sub.4 was added to
the organic phase, which was then filtered and the solvents removed
in vacuo.
[0255] An ethyl acetate extract from 2 L fermentation was mixed
with HP-20 resin (100 mL; Mitsubishi Casei Corp., Tokyo, Japan) in
water (300 mL). Ethyl acetate was removed in vacuo, the resin was
filtered on a Buchner funnel and the filtrate was discarded. The
adsorbed HP-20 resin was then washed successively with 2.times.125
mL of 50% acetonitrile in water, 2.times.125 mL of 75% acetonitrile
in water and 2.times.125 mL of acetonitrile.
[0256] Fractions containing the compound of Formula II were
evaporated to dryness and 100 mg was digested in the 5 mL of the
upper phase of a mixture prepared from chloroform, cyclohexane,
methanol, and water in the ratios, by volume, of 5:2:10:5. The
sample was subjected to centrifugal partition chromatography using
a High Speed Countercurrent (HSCC) system (Kromaton Technologies,
Angers, France) fitted with a 200 mL cartridge and prepacked with
the upper phase of this two-phase system. The HSCC was run with the
lower phase mobile and the compound of Formula II was eluted at
approximately one-half column volume. Fractions were collected and
the compound of Formula II was detected by TLC of aliquots of the
fractions on commercial Kieselgel 60F.sub.254 plates. Compound
could be visualized by inspection of dried plates under UV light or
by spraying the plates with a spray containing vanillin (0.75%) and
concentrated sulfuric acid (1.5%, v/v) in ethanol and subsequently
heating the plate. Fractions contained substantially pure compound
of Formula II, although highly colored. A buff-colored sample could
be obtained by chromatography on HPLC as follows.
[0257] 6 mg of sample was dissolved in acetonitrile and injected
onto a preparative HPLC column (XTerra ODS (10 .mu.m), 19.times.150
mm, Waters Co., Milford, Mass.), with a 9 mL/min flow rate and UV
peak detection at 300 nm. The column was eluted with
acetonitrile/buffer (20 mM of NH.sub.4HCO.sub.3) according to the
following gradient shown in Table 1
TABLE-US-00001 TABLE 1 Time (min) Water (%) Acetonitrile (%) 0 70
30 10 5 95 15 5 95 20 70 30
[0258] Fractions containing the compound of Formula II eluted at
approximately 11:0 min and were combined, concentrated and
lyophilized to give a yield of 3.8 mg compound.
[0259] Alternative Protocol 1
[0260] The compound of Formula II was also isolated using the
following alternative protocol. At the end of the incubation
period, the fermentation broth from the baffled flasks of Example 1
was centrifuged and the supernatant decanted from the pellet
containing the bacterial mycelia. 100 mL of 100% MeOH was added to
the mycelial pellet and the sample was stirred for 10 minutes and
centrifuged for 15 minutes. The methanolic supernatant was decanted
and saved. 100 mL of acetone was then added to the mycelial pellet
and stirred for 10 minutes then centrifuged for 15 minutes. The
acetonic supernatant was decanted and combined with the methanolic
supernatant. Finally, 100 mL of 20% MeOH/H.sub.2O was added to the
mycelial pellet, stirred for 10 minutes and centrifuged for 15
minutes. The supernatant was combined with the acetonic and
methanolic supernatants.
[0261] The combined supernatant was added to 400 ml of HP-20 resin
in 1000 mL of water and the organics were removed in vacuo. The
resulting slurry was filtered on a Buchner funnel and the filtrate
was discarded. Adsorbed HP-20 resin was washed successively with
2.times.500 mL of 50% MeOH/H.sub.2O, 2.times.500 mL of 75%
MeOH/H.sub.2O and 2.times.500 mL of MeOH.
[0262] The individual washes were collected separately and analyzed
by TLC as described above. Those fractions containing the compound
of Formula II were evaporated to near dryness and lyophilized. The
lyophilizate was dissolved in methanol and injected onto a
preparative HPLC column (Xterra ODS (10 .mu.m), 19.times.150 mm,
Waters Co., Milford, Mass.) with a flow rate of 9 mL/min and peak
detection at 300 nm.
[0263] The column was eluted with acetonitrile/buffer (5 mM of
NH.sub.4HCO.sub.3) according to gradient shown in Table 2.
TABLE-US-00002 TABLE 2 Time (min) Buffer (%) Acetonitrile (%) 0 95
5 15 45 55 20 5 95 30 5 95 35 95 5
[0264] Fractions containing the compound of Formula II were
combined, concentrated and lyophilized to yield about 33.7 mg of
compound.
[0265] Alternative Protocol 2
[0266] 10 liters of the whole broth from Example 1 are extracted
twice with equal volumes of ethyl acetate and the two extracts are
combined and concentrated to dryness. The dried extract is weighed,
and for every gram of dry extract, 100 mL of MeOH--H.sub.2O (2:1
v/v) and 100 mL of hexane is added. The mixture is swirled gently
but well to achieve dissolution. The two layers are separated and
the aqueous layer is washed with 100 mL of hexane. The two hexane
layers are combined and the combined hexane solution is washed with
100 mL methanol:water (2:1, v/v). The two methanol:water layers are
combined and treated with 200 mL of EtOAc and 400 mL of water. The
layers are separated and the aqueous layer is extracted twice more
with 200 mL portions of EtOAc. The EtOAc layers are combined and
concentrated. The residue obtained will be suitable for final
purification, either by HSCC or by HPLC as described above. This
extraction process achieves a ten-fold purification when compared
with the extraction protocol used above.
Example 3
Elucidation of the Structure of Compound of Formula II
[0267] The structure of the compound of Formula II was derived from
spectroscopic data, including mass, UV, and NMR spectroscopy. Mass
was determined by electrospray mass spectrometry to be 462.6 (FIG.
1), UVmax 230 nm with a shoulder at 290 nm (FIG. 2). NMR data were
collected dissolved in MeOH-d.sub.4 including proton (FIG. 3), and
multidimensional pulse sequences gDQCOSY (FIG. 4), gHSQC (FIG. 5),
gHMBC (FIG. 6), and NOESY (FIG. 7).
[0268] A number of cross peaks in the 2D spectra of ECO-04601 are
key in the structural determination. For example, the farnesyl
chain is placed on the amide nitrogen by a strong cross peak
between the proton signal of the terminal methylene of that chain
at 4.52 ppm and the amide carbonyl carbon at 170 ppm in the gHMBC
experiment. This conclusion is confirmed by a cross peak in the
NOESY spectrum between the same methylene signals at 4.52 ppm and
the aromatic proton signal at 6.25 ppm from one of the two protons
of the tetra substituted benzenoid ring.
[0269] Based on the mass, UV and NMR spectroscopy data, the
structure of the compound was determined to be the structure of
Formula II.
Example 4
Antibacterial Activity (Minimal Inhibitory Concentration
Determination)
[0270] Minimal Inhibitory Concentration (MIC) is defined as the
lowest concentration of drug that inhibits more than 99% of the
bacterial population. The MIC determination of ECO-04601 against
bacteria strains (Bacillus subtilis--ATCC 23857; Micrococcus luteus
--ATCC 9341) was performed using broth microdilution assay (Methods
for Dilution Antimicrobial Susceptibility Tests for Bacteria That
Grow Aerobically; Approved Standard-Fifth Edition. NCCLS document
M7-A5 (ISBN 1-56238-394-9). NCCLS, 940 West Valley Road, Suite
1400, Wayne, Pa. 19087-1898 USA.).
Test compound preparation: The test article ECO-04601 is prepared
as 100.times. stock solutions in DMSO, with concentrations ranging
from 3.2 mg/ml to 0.0625 mg/ml (a two-fold dilution series over 10
points). The first dilution (3.2 mg/ml) was prepared by
resuspending 0.5 mg of each test article in 156.25 .mu.l of DMSO.
The stock is then serially diluted by two-fold decrement to obtain
the desired concentration range. Inoculum preparation: From an
overnight culture in Mueller Hinton (MH) broth, cell density for
each indicator strain (Bacillus subtilis; Micrococcus luteus) was
adjusted to 0.5 Mc Farland units in 0.85% saline, then further
diluted 1/100 in appropriate assay medium (.about.1.times.10.sup.6
cells/ml). MIC determination: The 100.times.ECO-04601 solutions was
diluted 50 times in MH broth and dispensed in a 96 well plate, one
test concentration per column of wells, 10 columns in total. The
11.sup.th column of wells contained MH broth with 1% DMSO, the
12.sup.th column of wells contained 100 .mu.l of broth alone. 50
.mu.l of the final cell dilution of each indicator strain was added
to each corresponding well of the microplate containing 50 .mu.l of
diluted drug or media alone. Assay plates were incubated at
35.degree. C. for 24 hrs. The results of the MIC for the compound
of ECO-04601, shown in Table 3, demonstrate a range of
antibacterial effects:
TABLE-US-00003 TABLE 3 Indicator strain MIC (.mu.g/mL) Bacillus
subtilis ATCC 23857 12.5 Micrococcus luteus ATCC 9341 6.25
Example 5
Anticancer Activity In Vitro Against Human and Animal Tumor Cell
Lines from Various Tissues
[0271] Culture conditions: The cell lines listed in Table 4 were
used to characterize the cytotoxicity of ECO-04601 against human
and animal tumor cell lines. These cell lines were shown to be free
of mycoplasma infection and were maintained on the appropriate
media (Table 4) supplemented with 10% heat-inactivated fetal bovine
serum and 1% penicillin-streptomycin, under 5% CO.sub.2 at
37.degree. C. Cells were passaged twice to three times per week.
Viability was examined by staining with 0.25% trypan blue and only
flasks where cell viability was >95% were used for this study.
Cell lines amplification and plating: Tumor cells were seeded
(1-3.times.10.sup.3 cells per 100 .mu.L) in 96-wells flat bottom
microtiter plates and incubated at 37.degree. C. and 5% CO.sub.2
for 16 hrs before treatment in drug-free medium supplemented with
10% serum. Evaluation of inhibitory activity on cell proliferation:
Cells were incubated for 96 hrs with 6 log.sub.10-fold
concentrations of the test substance starting at 10 .mu.g/ml (20
.mu.M). The test substance stock solution (5 mg/mL) was initially
diluted at 1/70 fold in medium supplemented with serum. Other
concentrations were then obtained from 1/10 fold successive
dilutions in the same supplemented medium. Cell survival was
evaluated 96 h later by replacing the culture media with 150 .mu.L
fresh medium containing 10 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4.
Next, 50 .mu.L of 2.5 mg/mL of
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
in phosphate buffer solution, pH 7.4, was added. After 3-4 h of
incubation at 37.degree. C., the medium and soluble MTT was
removed, and 200 .mu.L of dimethylsulfoxide was added to dissolve
the precipitate of reduced MTT followed by addition of 25 .mu.L
glycine buffer (0.1 M glycine plus 0.1 M NaCl, pH 10.5). The
absorbance was determined at 570 nm with a microplate reader.
Results were expressed as the concentration of drug which inhibits
50% of the cell growth (IC.sub.50). The IC.sub.50 values shown in
Table 4 demonstrated a pharmacologically relevant cytotoxic
activity of ECO-04601 against a variety of tumor types such as
leukemias, melanomas, pancreatic and breast carcinomas.
TABLE-US-00004 TABLE 4 Cell Culture IC.sub.50 lines Type Origin
Source medium (x10.sup.-5 M) K562 Leukemia Human ATCC RPMI 1640 8.6
myelogeneous P388 Leukemia Mouse ATCC RPMI 1640 10.9 I83 Leukemia
Human ATCC RPMI 1640 2.7 B16 (F10) Melanoma Mouse ATCC RPMI 1640
11.4 SK-MEL 28 Melanoma Human ATCC RPMI 1640 14.0 SK-MEL Melanoma
Human ATCC RPMI 1640 14.3 28VEGF (expressing VEGF) SK-MEL-1
Melanoma Human ATCC EMEM 1% 14.1 non- essential amino acid 1%
Sodium puryvate Panc 96 Pancreatic Human ATCC RPMI 1% 12.5
carcinoma Sodium puryvate Panc Pancreatic Human ATCC RPMI 1% 14.2
10.05 carcinoma Sodium puryvate Insulin MCF-7 Breast Human ATCC
RPMI 1640 9.7 adeno- carcinoma
Example 6
Anticancer Activity In Vitro Against Various Human Tumor Cell Lines
from the U.S. National Cancer Institute Panel
[0272] A study measuring the in vitro antitumor activity of
ECO-04601 was performed by the National Cancer Institute (National
Institutes of Health, Bethesda, Md., USA) against panel of human
cancer cell lines in order to determine the ECO-04601
concentrations needed to obtain a 50% inhibition of cell
proliferation (GI.sub.50). The operation of this unique screen
utilizes 50 different human tumor cell lines, representing
leukemia, melanoma and cancers of the lung, colon, brain, ovary,
breast, prostate, and kidney.
Culture conditions and plating: The human tumor cell lines of the
cancer-screening panel were grown in RPMI 1640 medium containing 5%
fetal bovine serum and 2 mM L-glutamine. For a typical screening
experiment, cells were inoculated into 96 well microtiter plates in
100 .mu.L at plating densities ranging from 5,000 to 40,000
cells/well depending on the doubling time of individual cell lines
(Table 5). After cell inoculation, the microtiter plates were
incubated at 37.degree. C., 5% CO.sub.2, 95% air and 100% relative
humidity for 24 h prior to addition of experimental drugs. After 24
h, two plates of each cell line were fixed in situ with TCA, to
represent a measurement of the cell population for each cell line
at the time of drug addition (Tz).
[0273] Evaluation of inhibitory activity on cell proliferation:
ECO-04601 was provided as a lyophilized powder with an estimated
purity of 90+%. The compound was stored at -20.degree. C. until day
of use. ECO-04601 was solubilized in dimethyl sulfoxide at 400-fold
the desired final maximum test concentration. At the time of drug
addition, an aliquot of frozen concentrate was thawed and diluted
to twice the desired final maximum test concentration with complete
medium containing 50 .mu.g/mL gentamicin. Additional four, 10-fold
or 1/2 log serial dilutions were made to provide a total of five
drug concentrations plus control. Aliquots of 100 .mu.l of these
different drug dilutions were added to the appropriate microtiter
wells already containing 100 .mu.l of medium, resulting in the
required final drug concentrations (8.0.times.10.sup.-5 M to
8.0.times.10.sup.-9 M).
[0274] Following drug addition, the plates were incubated for an
additional 48 h at 37.degree. C., 5% CO.sub.2, 95% air, and 100%
relative humidity. For adherent cells, the assay was terminated by
the addition of cold TCA. Cells were fixed in situ by the gentle
addition of 50 .mu.l of cold 50% (w/v) TCA (final concentration,
10% TCA) and incubated for 60 minutes at 4.degree. C. Supernatants
were discarded, and the plates were washed five times with tap
water and air-dried. Sulforhodamine B (SRB) solution (100 .mu.l) at
0.4% (w/v) in 1% acetic acid was added to each well, and plates
were incubated for 10 minutes at room temperature. After staining,
unbound dye was removed by washing five times with 1% acetic acid
and the plates were air-dried. Bound stain was subsequently
solubilized with 10 mM trizma base, and the absorbance was read on
an automated plate reader at a wavelength of 515 nm. For suspension
cells, the methodology was the same except that the assay was
terminated by fixing settled cells at the bottom of the wells by
gently adding 50 .mu.l of 80% TCA (final concentration, 16%
TCA).
[0275] The growth inhibitory activity of ECO-04601 was measured by
NCl utilizing the GI.sub.50 value, rather than the classical
IC.sub.50 value. The GI.sub.50 value emphasizes the correction for
the cell count at time zero and, using the seven absorbance
measurements [time zero, (Tz), control growth, (C), and test growth
in the presence of drug at the five concentration levels (Ti)],
GI.sub.50 is calculated as [(Ti-Tz)/(C-Tz)].times.100=-50, which is
the drug concentration resulting in a 50% reduction in the net
protein increase (as measured by SRB staining) in control cells
during the drug incubation.
Result: ECO-04601 shows a significant antitumor activity against
several types of tumor as revealed by the NCl screening. Results of
the screen are shown in Table 5, and more detailed results of
activity against gliomas are shown in Example 7 (Table 6).
TABLE-US-00005 TABLE 5 Inoculation Density (number of Cell Line
Name Type Origin cells/well) GI.sub.50 (.times.10.sup.-6 M)
CCRF-CEM Leukemia Human 40,000 1.08 K-562 Leukemia Human 5,000 1.43
RPMI-8226 Leukemia Human 20,000 3.15 A549/ATCC Non-Small Cell Lung
Human 7,500 9.10 EKVX Non-Small Cell Lung Human 20,000 0.23 HOP-62
Non-Small Cell Lung Human 10,000 8.29 NCl-H226 Non-Small Cell Lung
Human 20,000 2.00 NCl-H23 Non-Small Cell Lung Human 20,000 2.02
NCl-H460 Non-Small Cell Lung Human 7,500 13.60 NCl-H522 Non-Small
Cell Lung Human 20,000 3.44 COLO 205 Colon Human 15,000 12.70
HCT-116 Colon Human 5,000 2.92 HCT-15 Colon Human 10,000 9.73 HT29
Colon Human 5,000 20.70 SW-620 Colon Human 10,000 2.72 SF-268 CNS
Human 15,000 4.94 SF-295 CNS Human 10,000 12.70 SF-539 CNS Human
15,000 0.0075 SNB-19 CNS Human 15,000 2.90 SNB-75 CNS Human 20,000
7.71 U251 CNS Human 7,500 2.19 LOX IMVI Melanoma Human 7,500 4.53
M14 Melanoma Human 15,000 4.57 SK-MEL-2 Melanoma Human 20,000 25.0
SK-MEL-28 Melanoma Human 10,000 11.6 SK-MEL-5 Melanoma Human 10,000
7.80 UACC-257 Melanoma Human 20,000 2.31 UACC-62 Melanoma Human
10,000 1.55 IGR-OV1 Ovarian Human 10,000 3.11 OVCAR-3 Ovarian Human
10,000 13.50 OVCAR-4 Ovarian Human 15,000 9.67 OVCAR-5 Ovarian
Human 20,000 2.81 OVCAR-8 Ovarian Human 10,000 2.65 SK-OV-3 Ovarian
Human 20,000 4.00 786-0 Renal Human 10,000 6.99 A498 Renal Human
25,000 22.30 ACHN Renal Human 10,000 3.10 CAKI-1 Renal Human 10,000
15.20 RXF 393 Renal Human 15,000 7.71 SN12C Renal Human 15,000 3.85
UO-31 Renal Human 15,000 19.70 DU-145 Prostate Human 10,000 3.56
MCF7 Breast Human 10,000 10.10 NCI/ADR-RES Breast Human 15,000
18.30 MDA-MB-231/ATCC Breast Human 20,000 2.72 HS 578T Breast Human
20,000 2.76 MDA-MB-435 Breast Human 15,000 15.30 BT-549 Breast
Human 20,000 0.11 T-47D Breast Human 20,000 0.77
[0276] The results indicate that ECO-04601 was effective against
most of the human tumor cell lines that have been assayed in the
NCl screening panel suggesting a broad anticancer activity against
several types of human cancer.
Example 7
In Vitro Antiproliferative Study Against a Panel of Glioma Cell
Lines
[0277] The anticancer activity of ECO-04601 was evaluated using a
panel of glioma cancer cell lines shown in Table 6, and the 50%
inhibition of cell proliferation (IC.sub.50) was determined.
Culture conditions: The cell lines listed in Table 6 were shown to
be free of mycoplasma infection and were maintained on DMEM medium
supplemented with 10% heat-inactivated fetal bovine serum and 1%
penicillin-streptomycin, under 5% CO.sub.2 at 37.degree. C. Cells
were passaged once a week. Prior to use the cells were detached
from the culture flask by treating with trypsin for five to ten
minutes. The cells were counted with a Neubauer glass slide and
viability assessed by 0.25% trypan blue exclusion. Only flasks with
>95% cell viability, were used in the study. Cell lines
amplification and plating: Cells, 5.times.10.sup.3 cells per well
in 100 .mu.L drug-free medium supplemented with 10% serum, were
plated in 96-well flat bottom microtiter plates and incubated at
37.degree. C. for 48 hrs before treatment. Evaluation of inhibitory
activity on cell proliferation: Cells (in triplicate wells) were
incubated 96 hrs with medium containing different concentrations of
ECO-04601, starting at 5.0 .mu.g/ml (10 .mu.M). The compound was
used in a solution of 1% DMSO in D-MEM or RPMI media (or other
equivalent media). The concentrations of ECO-04601 were as follows:
10 .mu.M (5.0 .mu.g/ml), 1 .mu.M (0.50 .mu.g/ml), 0.5 .mu.M (0.25
.mu.g/ml), 0.1 .mu.M (0.050 .mu.g/ml), 0.5 .mu.M (0.025 .mu.g/ml),
0.01 .mu.M (0.0050 .mu.g/ml), 0.001 .mu.M (0.00050 .mu.g/ml).
Negative controls were cells treated with vehicle alone (1% DMSO in
culture medium). Positive controls were cells treated with 4 to 6
increasing concentrations of cisplatin (CDDP) (data not shown). The
optical density was measured before incubation (time 0) and
following 96 hrs of incubation with test compound in order to
measure the growth rate of each cell line.
[0278] At the end of the cell treatment, cell culture media was
replaced with 150 .mu.l of fresh medium containing 10 mM of
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4.
Then 50 .mu.l of 2.5 mg/ml of
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide in PBS
pH 7.4, were added to each well and the culture plates incubated
for 4 hrs at 37.degree. C. The resulting supernatant was removed
and formazan crystals were dissolved with 200 .mu.l of DMSO
followed by 25 .mu.l of glycine buffer (0.1 M glycine plus 0.1 M
NaCl, pH 10.5). The optical density was read in each well using a
single wavelength spectrophotometer plate reader at 570 nm. Results
were expressed as the concentration of drug, which inhibits 50% of
the cell growth (IC.sub.50). Each of the cell lines was tested in
at least 3 independent experiments.
[0279] Results shown in Table 6 confirmed the activity of ECO-04601
against different brain cancer cell lines including gliosarcoma,
which is the most malignant form of type IV glioblastoma multiform.
Gliosarcomas are a mixture of glial and endothelial cells and are
resistant to any chemotherapy.
TABLE-US-00006 TABLE 6 Cell IC.sub.50 lines Type Origin Source (x
10.sup.-6 M) 9L Gliosarcoma Rat ATCC 6.82 .+-. 2.90 GHD Astrocytoma
Human ATCC 6.29 .+-. 2.98 U 373 Astrocytoma Human ATCC 3.83 .+-.
1.37 GL26 Glioblastoma Human ATCC 8.93 .+-. 1.10 C6 Glioblastoma
Rat ATCC 4.28 .+-. 2.82 DN Oligodendro- Human ATCC 3.26 .+-. 0.93
glioma GHA Oligodendro- Human ATCC 1.78 .+-. 0.84 glioma
Example 8
Effect on the Enzymatic Activity of Human Lipoxygenase (5-LO)
[0280] 5-Lipoxygenase catalyzes the oxidative metabolism of
arachidonic acid to 5-hydroxyeicosatetraenoic acid (5-HETE), the
initial reaction leading to formation of leukotrienes. Eicosanoids
derived from arachidonic acid by the action of lipoxygenases or
cycloxygenases have been found to be involved in acute and chronic
inflammatory diseases (i.e. asthma, multiple sclerosis, rheumatoid
arthritis, ischemia, edema) as well in neurodegeneration (Alzheimer
disease), aging and various steps of carcinogenesis, including
tumor promotion, progression and metastasis.
[0281] The aim of this study was to determine whether ECO-04601, is
able to block the formation of leukotrienes by inhibiting the
enzymatic activity of human 5-LO. Methods employed are based on
Carter et al (1991) J. Pharmacol. Exp. Ther. 256(3):929-937, and
Safayhi (2000), Planta Medica 66:110-113 which are incorporated
herein in their entirety by reference.
Experimental Design Human peripheral blood mononuclear cells (PMNs)
were isolated through a Ficoll-Paque density gradient. PMNs were
stimulated by addition A23187 (30 .mu.M final concentration).
Stimulated PMNs were adjusted to a density of 5.times.10.sup.6
cells/mL in HBBS medium and incubated with the vehicle control
(DMSO), ECO-04601 (at final concentrations of 0.1, 0.5, 1, 2.5, 5
and 10 .mu.M) and NDGA as positive control (at final concentrations
of 3, 1, 0.3, 0.1 and 0.03 .mu.M) for 15 minutes at 37.degree. C.
Following incubation, samples were neutralized with NaOH and
centrifuged. Leukotriene B4 content was measured in the supernatant
using an Enzyme Immunosorbant Assay (EIA) assay. Results: Results
shown in FIG. 8 demonstrated that ECO-04601 inhibited the activity
of human 5-LO with an apparent IC.sub.50=0.93 .mu.M (versus 0.1
.mu.M for the positive control NDGA) and therefore displays
anti-inflammatory properties.
Example 9
In Vivo Efficacy in a Glioma Model
[0282] The aim of this study was to test whether ECO-04601
administered by i.p. route prevents or delays tumor growth in C6
glioblastoma cell-bearing mice, and to determine an effective
dosage regimen.
Animals: A total of 60 six-week-old female mice (Mus musculus nude
mice), ranging between 18 to 25 g in weight, were observed for 7
days before treatment. Animal experiments were performed according
to ethical guidelines of animal experimentation (Charte du comite
d'ethique du CNRS, juillet 2003) and the English guidelines for the
welfare of animals in experimental neoplasia (WORKMAN, P.,
TWENTYMAN, P., BALKWILL, F., et al. (1998). United Kingdom
Coordinating Committee on Cancer Research (UKCCCR) Guidelines for
the welfare of animals in experimental neoplasia(Second Edition,
July 1997; British Journal of Cancer 77:1-10). Any dead or
apparently sick mice were promptly removed and replaced with
healthy mice. Sick mice were euthanized upon removal from the cage.
Animals were maintained in rooms under controlled conditions of
temperature (23.+-.2.degree. C.), humidity (45.+-.5%),
photoperiodicity (12 hrs light/12 hrs dark) and air exchange.
Animals were housed in polycarbonate cages (5/single cage) that
were equipped to provide food and water. Animal bedding consisted
of sterile wood shavings that were replaced every other day. Food
was provided ad libitum, being placed in the metal lid on the top
of the cage. Autoclaved tap water was provided ad libitum. Water
bottles were equipped with rubber stoppers and sipper tubes. Water
bottles were cleaned, sterilized and replaced once a week. Two
different numbers engraved on two earrings identified the animals.
Each cage was labelled with a specific code. Tumor Cell Line: The
C6 cell line was cloned from a rat glial tumor induced by
N-nitrosomethyurea (NMU) by Premont et al. (Premont J, Benda P,
Jard S., [3H] norepinephrine binding by rat glial cells in culture.
Lack of correlation between binding and adenylate cyclase
activation. Biochim Biophys Acta. 1975 Feb. 13; 381(2):368-76.)
after series of alternate culture and animal passages.
[0283] Cells were grown as adherent monolayers at 37.degree. C. in
a humidified atmosphere (5% CO.sub.2, 95% air). The culture medium
was DMEM supplemented with 2 mM L-glutamine and 10% fetal bovine
serum. For experimental use, tumor cells were detached from the
culture flask by a 10 min treatment with trypsin-versen. The cells
were counted in a hemocytometer and their viability assessed by
0.25% trypan blue exclusion.
Preparation of the Test Article: for the Test Article, the
Following Procedure was Followed for reconstitution (performed
immediately preceding injection). The vehicle consisted of a
mixture of benzyl alcohol (1.5%), ethanol (8.5%), propylene glycol
(27%), PEG 400 (27%), dimethylacetamide (6%) and water (30%). The
vehicle solution was first vortexed in order to obtain a
homogeneous liquid. 0.6 mL of the vortexed vehicle solution was
added to each vial containing the test article (ECO-04601). Vials
were mixed thoroughly by vortexing for 1 minute and inverted and
shaken vigorously. Vials were mixed again prior to injection into
each animal. Animal Inoculation with tumor cells: Experiment
started at day 0 (D.sub.0). On D.sub.0, mice received a superficial
intramuscular injection of C6 tumor cells (5.times.10.sup.5 cells)
in 0.1 mL of DMEM complete medium into the upper right posterior
leg.
Treatment Regimen and Results
[0284] In a first series of experiments, treatment started 24 hrs
following inoculation of C6 cells. On the day of the treatment,
each mouse was slowly injected with 100 .mu.L of test or control
articles by i.p. route. For all groups, treatment was performed
until the tumor volume of the saline-treated mice (group 1) reached
approximately 3 cm.sup.3 (around day 16). Mice of group 1 were
treated daily with a saline isosmotic solution for 16 days. Mice of
group 2 were treated daily with the vehicle solution for 16 days.
Mice of group 3 were treated daily with 10 mg/kg of ECO-04601 for
16 days. Mice of group 3 were treated every two days with 30 mg/kg
of ECO-04601 and received 8 treatments. Mice of group 5 were
treated every three days with 30 mg/kg of ECO-04601 and received 6
treatments. Measurement of tumor volume started as soon as tumors
became palpable (>100 mm.sup.3; around day 11 post-inoculation)
and was evaluated every second day until the end of the treatment
using callipers. As shown in Table 7 and FIG. 9, the mean value of
the tumor volume of all ECO-04601 treated groups (6 mice/group) was
significantly reduced as demonstrated by the one-way analysis of
variance (Anova) test followed by the non-parametric Dunnett's
multiple comparison test comparing treated groups to the saline
group. An asterisk in the P value column of Table 7 indicates a
statistically significant value, while "ns" signifies not
significant.
TABLE-US-00007 TABLE 7 Tumor volume Treatment (mm.sup.3) % P
Treatment regimen (mean .+-. SEM) Inhibition value Saline Q1
.times. 16 3,004.1 .+-. 249.64 -- -- Vehicle Q1 .times. 16 2,162.0
.+-. 350.0 28.0% >0.05 ns solution ECO-04601 Q1 .times. 16
1,220.4 .+-. 283.46 59.4% <0.01* (10 mg/kg) ECO-04601 Q2 .times.
8 1,236.9 .+-. 233.99 58.8% <0.01* (30 mg/kg) ECO-04601 Q3
.times. 6 1,184.1 .+-. 221.45 60.6% <0.01* (30 mg/kg)
[0285] In a second series of experiments, treatment started at day
10 following inoculation of C6 cells when tumors became palpable
(around 100 to 200 mm.sup.3). Treatment was repeated daily for 5
consecutive days. On the day of the treatment, each mouse was
slowly injected with 100 .mu.L of ECO-04601 by i.p. route. Mice of
group 1 were treated daily with saline isosmotic solution. Mice of
group 2 were treated daily with the vehicle solution. Mice of group
3 were treated daily with 20 mg/kg of ECO-04601. Mice of group 4
were treated daily with 30 mg/kg of ECO-04601. Mice were treated
until the tumor volume of the saline-treated control mice (group 1)
reached around 4 cm.sup.3. Tumor volume was measured every second
day until the end of the treatment using callipers. As shown in
Table 8 and FIG. 10, the mean value of the tumor volume of all
treated groups (6 mice/group) was significantly reduced as
demonstrated by the one-way analysis of variance (Anova) test
followed by the non-parametric Dunnett's multiple comparison test
comparing treated groups to the saline group. An asterisk in the P
value column of Table 8 indicates a statistically significant
value, while "ns" signifies not statistically significant.
[0286] Histological analysis of tumor sections showed pronounced
morphological changes between ECO-04601-treated tumors and control
groups. In tumors treated with ECO-04601 (20-30 mg/kg), cell
density was decreased and the nuclei of remaining tumor cells
appeared larger and pycnotic while no such changes were observed
for vehicle-treated mice (FIG. 11).
TABLE-US-00008 TABLE 8 Tumor volume Treatment (mm.sup.3) % P
Treatment regimen (mean .+-. SEM) Inhibition value Saline Q1
.times. 5 4,363.1 .+-. 614.31 -- -- Vehicle solution Q1 .times. 5
3,205.0 .+-. 632.37 26.5% >0.05 ns ECO-04601 Q1 .times. 5
1,721.5 .+-. 374.79 60.5% <0.01* (20 mg/kg) ECO-04601 Q1 .times.
5 1,131.6 .+-. 525.21 74.1% <0.01* (30 mg/kg)
Example 10
Generation of Variants of Eco-04601 According to the Invention
[0287] Variants of the ECO-04601 molecule, for example those
identified herein as Formulae III-LIX, can be generated by standard
organic chemistry approaches. General principles of organic
chemistry required for making and manipulating the compounds
described herein, including functional moieties, reactivity and
common protocols are described, for example, in "Advanced Organic
Chemistry," 3.sup.rd Edition by Jerry March (1985) which is
incorporated herein by reference in its entirety. In addition, it
will be appreciated by one of ordinary skill in the art that the
synthetic methods described herein may use a variety of protecting
groups, whether or not they are explicitly described. A "protecting
group" as used herein means a moiety used to block one or more
functional moieties such as reactive groups including oxygen,
sulfur or nitrogen, so that a reaction can be carried out
selectively at another reactive site in a polyfunctional compound.
General principles for the use of protective groups, their
applicability to specific functional groups and their uses are
described for example in T. H. Greene and P. G. M. Wuts, Protective
Groups in Organic Synthesis, 3.sup.rd Edition, John Wiley &
Sons, New York (1999).
Scheme 1: Epoxide variants
[0288] The epoxide compounds of the present invention (e.g.,
compounds according to exemplary Formulae VII-XIV) are made from
the compound of Formula II (ECO-04601) by treatment with any of a
number of epoxidizing reagents such as perbenzoic acid,
monoperphthalic acid or more preferably by m-chloroperbenzoic acid
in an inert solvent such as tetrahydrofuran (THF) dichloromethane
or 1,2-dichloroethane. It will be appreciated by one of ordinary
skill in the art that slightly greater than one molecule equivalent
of epoxidizing agent will result in the maximal yield of
mono-epoxides, and that the reagent, solvent, concentration and
temperature of the reaction will dictate the ratio of specific
mono-epoxides formed. It will also be appreciated that the
mono-epoxides will be enantiomeric mixtures, and that the
di-epoxides and the tri-epoxide can be prepared as diastereomers
and that the conditions of the reaction will determine the ratios
of the products. One skilled in the art will appreciate that under
most conditions of reactions the product will be a mixture of all
possible epoxides and that these may be separated by standard
methods of chromatography. Exemplary approaches to the generation
of mono-, di-, and tri-epoxides are provided below. [0289] A)
Mono-Epoxides of the Formulae VII, VIII, and IX by Epoxidation of
the Compound of Formula II:
##STR00025##
[0290] To a solution of the compound of Formula II dissolved in
tetrahydrofuran (THF) is added 1.1 equivalents of
meta-chloroperbenzoic acid. The reaction is cooled in an ice bath
and stirred at 0.degree. C. for 1-2 hours. The reaction mixture is
then evaporated to dryness, re-dissolved in methanol and subjected
to liquid chromatography on a column of Sephadex LH-20 to isolate a
mixture of predominantly the compounds of Formulae VII, VIII and
IX, contaminated with some unchanged starting material and some di-
and tri-epoxides. The compounds of Formulae VII, VIII and XIX are
separated and purified by HPLC using the system described in
Example 2 for the purification of the compound of Formulae II. In a
typical experiment yields of 15% to 25% are obtained for each of
the compounds of Formulae VII, VIII and IX. [0291] B) Synthesis of
Compounds of Formulae X, XI, and XII by Di-Epoxidation of Compound
of Formula II:
##STR00026##
[0292] To a solution of the compound of Formula II dissolved in
tetrahydrofuran (THF) is added 2.3 equivalents of
meta-chloroperbenzoic acid. The reaction is cooled in an ice bath
and stirred at 0.degree. C. for 1-2 hours. The reaction mixture is
then evaporated to dryness, re-dissolved in methanol and subjected
to liquid chromatography on a column of Sephadex LH-20 to isolate a
mixture of predominantly the compounds of Formulae X, XI and XII,
contaminated with traces of unchanged starting material and some
mono- and tri-epoxides. The Compounds of Formulae X, XI and XII are
separated and purified by HPLC using the system described in
Example 2 for the purification of the compound of Formulae II. In a
typical experiment, yields of 15% to 20% are obtained for each of
the compounds of Formulae X, XI and XII. [0293] C) Synthesis of
Compound of Formula XIII by Tri-Epoxidation of Compound of Formula
II:
##STR00027##
[0294] To a solution of the compound of Formula II, dissolved in
tetrahydrofuran (THF), is added 3.5 equivalents of
meta-chloroperbenzoic acid. The reaction is cooled in an ice bath
and stirred at 0.degree. C. for 1-2 hours. The reaction mixture is
then evaporated to dryness, re-dissolved in methanol and subjected
to liquid chromatography on a column of Sephadex LH-20 to isolate
the compound of Formula XIII as a mixture of diasteriomers in a
yield of 80+%.
##STR00028##
[0295] To a solution of Compound of Formula II dissolved in
tetrahydrofuran (THF) is added 1.2 equivalents of acetic anhydride
and a few drops of triethylamine. The reaction mixture allowed to
stand at room temperature for 1-2 hours and then evaporated to
dryness under reduced pressure to obtain the Compound of Formula
III in an essentially pure form in an almost quantitative yield
##STR00029##
[0296] To a solution of Compound of Formula II dissolved in
terachloroethylene is added 1.2 equivalents of the appropriate
alkyl bromide (benzyl bromide for the compound of formula IV or
ethyl bromide for the Compound of Formula V). The reaction mixture
the reaction mixture is heated under reflux for 1-2 hours and then
evaporated to dryness under reduced pressure to obtain the Compound
of Formula IV or the Compound of Formula V respectively, in an
essentially pure form in an almost quantitative yield.
##STR00030##
[0297] A solution of the Compound of Formula II (462 mg) in ethanol
(200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an
hydrogenation apparatus in an atmosphere of hydrogen. The uptake of
hydrogen by the reaction is measured carefully and at the point
where one millimole of hydrogen has been consumed, shaking is
stopped, the vessel is rapidly evacuated and the atmosphere is
replaced with nitrogen. The catalyst is removed by filtration and
the filtrate is concentrated to obtain a crude mixture of the
Compounds of Formulae XL, XLI and XLII contaminated by unreacted
starting material and minor amounts of over reduced products. The
desired products may be separated and purified by HPLC or HSCC
chromatography using the systems as described in Example 2 above,
to obtain approximately 100 mg of each of the Compounds of Formulae
XL, XLI and XLII.
##STR00031##
[0298] A solution of the Compound of Formula II (462 mg) in ethanol
(200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an
hydrogenation apparatus in an atmosphere of hydrogen. The uptake of
hydrogen by the reaction is measured carefully and at the point
where two millimoles of hydrogen has been consumed, shaking is
stopped, the vessel is rapidly evacuated and the atmosphere is
replaced with nitrogen. The catalyst is removed by filtration and
the filtrate is concentrated to obtain a crude mixture of the
Compounds of Formulae XLIII, XLIV and XLV contaminated by trace
amounts unreacted starting material and minor amounts of under and
over reduced products. The desired products may be separated and
purified by HPLC or HSCC chromatography using the systems as
described in Example 2 above, to obtain approximately 100 mg of
each of the Compounds of Formulae XLIII, XLIV and XLV.
##STR00032##
[0299] A solution of the Compound of Formula II (462 mg) in ethanol
(200 ml) with palladium on charcoal (25 mg of 5%) is shaken in an
hydrogenation apparatus in an atmosphere of hydrogen. The uptake of
hydrogen by the reaction is measured carefully and at the point
where three millimoles of hydrogen has been consumed, shaking is
stopped, the vessel is rapidly evacuated and the atmosphere is
replaced with nitrogen. The catalyst is removed by filtration and
the filtrate is concentrated to obtain an essentially pure sample
of the Compound of Formula XLVI
##STR00033##
[0300] A solution of the Compound of Formula II (100 mg) in acetic
anhydride (5 ml) is treated with pyridine (250 ul). The reaction
mixture is allowed to stand overnight at room temperature and is
then diluted with toluene (100 ml). The toluene solution is washed
well with aqueous 5% sodium bicarbonate solutions, then with water
and is finally concentrated under reduced pressure to give an
essentially pure sample of the Compound of Formula VI in almost
quantitative yield.
##STR00034##
[0301] A solution of the Compound of Formula VII (100 mg) in
tetrahydrofuran (50 ml) is treated with 1N aqueous hydrochloric
acid (5 ml). The reaction mixture is stirred overnight at room
temperature and is then diluted with toluene (100 ml) and water
(200 ml). The toluene layer is separated and the aqueous layer is
extracted with a further 100 ml of toluene. The combined toluene
layers are washed once more with water (50 ml) and the separated
and dried under vacuum to give the vicinal glycol Compound of
Formula LI.
##STR00035##
[0302] A solution of the Compound of Formula II (462 mg) in dry
ethyl acetate (200 ml) in an ozonolysis apparatus is cooled to
below -20.degree. C. A stream of ozone-containing oxygen is passed
into the solution from an ozone generator, which has been
precalibrated such that the rate of ozone generation is known. To
obtain predominantly the compound of Formula XLVII the passage of
ozone is halted after 0.9 millimole have been generated. To obtain
predominantly the compound of Formula XLIX the ozone passage is
halted after 2 millimoles have been generated and to obtain the
compound of Formula LI as the predominant product 3.3 millimoles of
ozone are generated.
[0303] At the completion of the ozonolysis, the reaction mixture is
transferred to an hydrogenation apparatus, 5% palladium on calcium
carbonate catalyst (0.2 g) is added to the reaction mixture which
is maintained at less than -20.degree. C. and is hydrogenated. When
hydrogen uptake is complete the hydrogen atmosphere is replaced
with nitrogen and the reaction mixture is allowed to come to room
temperature, filtered to remove catalyst and the filtrate is
concentrated. The crude product may be purified by chromatography
using either HPLC or HSCC with the systems as described in Example
2 to give, dependent on the amount of ozone used, Compounds of
Formulae XLVII, XLIX and LI.
##STR00036##
[0304] A solution of the Compound of Formula XLVIII (50 mg) in
isopropanol (5 ml) is cooled in an ice-salt bath and sodium
borohydride (10 mg) is added and the mixture is stirred for 20
minutes. It is then diluted with water (20 ml) and extracted twice
with toluene (10 ml portions) at ambient temperature. The combined
toluene extracts are filtered and the filtrate is concentrated to
give the Compound of Formula XLVII.
##STR00037##
[0305] To a solution of Compound of Formula XLII dissolved in
tetrahydrofuran (THF) is added 1.1 equivalents of
meta-chloroperbenzoic acid. The reaction is cooled in an ice bath
and stirred at 0.degree. C. for 1-2 hours. The reaction mixture is
then evaporated to dryness, re-dissolved in methanol and subjected
to liquid chromatography on a column of Sephadex LH-20 to isolate a
mixture of predominantly the Compounds of Formulae XIV, and XV,
contaminated with some unchanged starting material and some
diepoxide. The Compounds of Formulae XIV and XV are separated and
purified by HPLC or HSCC using one of the systems described in
Example 2 for the purification of the Compound of Formulae II. In a
typical experiment yields of 35% to 40% are obtained for each of
the Compounds of Formulae XIV and XV.
##STR00038##
[0306] To a solution of Compound of Formula XL dissolved in
tetrahydrofuran (THF) is added 2.2 equivalents of
meta-chloroperbenzoic acid. The reaction is cooled in an ice bath
and stirred at 0.degree. C. for 1-2 hours. The reaction mixture is
then evaporated to dryness, re-dissolved in methanol and subjected
to liquid chromatography on a column of Sephadex LH-20 to isolate
essentially pure Compound of Formulae XIX in good yield.
##STR00039##
[0307] To a solution of Compound of Formula II dissolved in toluene
(9 parts) tetrahydrofuran (1 part), cooled in an ice-bath is added
1.1 equivalents of acetic anhydride and two drops of boron
trifluoride etherate. The reaction is maintained cool in an ice
bath and stirred at 0.degree. C. for 1-2 hours. The reaction
mixture is then poured into aqueous 5% sodium bicarbonate solution
shaken and the toluene layer is removed. The aqueous layer is
re-extracted with toluene and the combined toluene layers are
concentrated to a mixture of predominantly the Compounds of
Formulae XXVI, XXVII and XXVIII, contaminated with some unchanged
starting material and some diacetates. The Compounds of Formulae
XXVI, XXVII and XXVIII are separated and purified by HPLC or HSCC
using one of the systems described in Example 2 for the
purification of the Compound of Formulae II. In a typical
experiment yields of 25% to 30% are obtained for each of the
Compounds of Formulae XXVI, XXVII and XXVIII.
##STR00040##
[0308] A solution of the Compound of Formula II (1 g) in
tetrahydrofuran 50 (ml) is titrated with exactly one equivalent of
sodium methoxide, allowed to stand for 30 minutes at room
temperature and then treated with 1.2 equivalents of
dimethylsulphate. Heat the mixture under reflux for one hour, cool
to room temperature and pour into a mixture of toluene (200 ml) and
water (200 ml). The layers are separated and the aqueous layer is
extracted once more with an equal portion of toluene. The combined
toluene layers are washed once with 1N aqueous acetic acid and then
concentrated to s crude product, which is predominantly a mixture
of the Compounds of Formulae XXXIII, XXXIV and XXXV with some
unchanged starting material and traces of over-methylated
derivatives. The desired products may be separated and purified by
HPLC or HSCC chromatography using the systems as described in
Example 2 above, to obtain approximately 200 mg of each of the
Compounds of Formulae XXXIII, XXXIV and XXXV.
Example 11
Genes and Proteins for the Production of Compounds of Formula
[0309] Micromonospora sp. strain 046-ECO11 is a representative
microorganism useful in the production of the compound of the
invention. Strain 046-ECO11 has been deposited with the
International Depositary Authority of Canada (IDAC), Bureau of
Microbiology, Health Canada, 1015 Arlington Street, Winnipeg,
Manitoba, Canada R3E 3R2 on Mar. 7, 2003 and was assigned IDAC
accession no. 070303-01. The biosynthetic locus for the production
of the compound of Formula II was identified in the genome of
Micromonospora sp. strain 046-ECO11 using the genome scanning
method described in U.S. Ser. No. 10/232,370, CA 2,352,451 and
Zazopoulos et. al., Nature Biotechnol., 21, 187-190 (2003).
[0310] The biosynthetic locus spans approximately 52,400 base pairs
of DNA and encodes 43 proteins. More than 10 kilobases of DNA
sequence were analyzed on each side of the locus and these regions
were deemed to contain primary genes or genes unrelated to the
synthesis of the compound of Formula II. As illustrated in FIG. 12,
the locus is contained within three sequences of contiguous base
pairs, namely Contig 1 having the 36,602 contiguous base pairs of
SEQ ID NO: 1 and comprising ORFs 1 to 31 (SEQ ID NOS: 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61 and 63), Contig 2 having the
5,960 contiguous base pairs of SEQ ID NO: 64 and comprising ORFs 32
to 35 (SEQ ID NOS: 66, 68, 70 and 72), and Contig 3 having the
9,762 base pairs of SEQ ID NO: 73 and comprising ORFs 36 to 43 (SEQ
ID NOS: 75, 77, 79, 81, 83, 85, 87 and 89). The order, relative
position and orientation of the 43 open reading frames representing
the proteins of the biosynthetic locus are illustrated
schematically in FIG. 12. The top line in FIG. 12 provides a scale
in base pairs. The gray bars depict the three DNA contigs (SEQ ID
NOS: 1, 64 and 73) that cover the locus. The empty arrows represent
the 43 open reading frames of this biosynthetic locus. The black
arrows represent the two deposited cosmid clones covering the
locus.
[0311] The biosynthetic locus will be further understood with
reference to the sequence listing which provides contiguous
nucleotide sequences and deduced amino acid sequences of the locus
from Micromonospora sp. strain 046-ECO11. The contiguous nucleotide
sequences are arranged such that, as found within the biosynthetic
locus, Contig 1 (SEQ ID NO: 1) is adjacent to the 5' end of Contig
2 (SEQ ID NO: 64), which in turn is adjacent to Contig 3 (SEQ ID
NO: 73). The ORFs illustrated in FIG. 12 and provided in the
sequence listing represent open reading frames deduced from the
nucleotide sequences of Contigs 1, 2 and 3 (SEQ ID NOS: 1, 64 and
73). Referring to the Sequence Listing, ORF 1 (SEQ ID NO: 3) is the
polynucleotide drawn from residues 2139 to 424 of SEQ ID NO: 1, and
SEQ ID NO: 2 represents that polypeptide deduced from SEQ ID NO: 3.
ORF 2 (SEQ ID NO: 5) is the polynucleotide drawn from residues 2890
to 4959 of SEQ ID NO: 1, and SEQ ID NO: 4 represents the
polypeptide deduced from SEQ ID NO: 5. ORF 3 (SEQ ID NO: 7) is the
polynucleotide drawn from residues 7701 to 5014 of SEQ ID NO: 1,
and SEQ ID NO: 6 represents the polypeptide deduced from SEQ ID NO:
7. ORF 4 (SEQ ID NO: 9) is the polynucleotide drawn from residues
8104 to 9192 of SEQ ID NO: 1, and SEQ ID NO: 8 represents the
polypeptide deduced from SEQ ID NO: 9. ORF 5 (SEQ ID NO: 11) is the
polynucleotide drawn from residues 9192 to 10256 of SEQ ID NO: 1,
and SEQ ID NO: 10 represents the polypeptide deduced from SEQ ID
NO: 11. ORF 6 (SEQ ID NO: 13) is the polynucleotide drawn from
residues 10246 to 11286 of SEQ ID NO: 1, and SEQ ID NO: 12
represents the polypeptide deduced from SEQ ID NO: 13. ORF 7 (SEQ
ID NO: 15) is the polynucleotide drawn from residues 11283 to 12392
of SEQ ID NO: 1, and SEQ ID NO: 14 represents the polypeptide
deduced from SEQ ID NO: 15. ORF 8 (SEQ ID NO: 17) is the
polynucleotide drawn from residues 12389 to 13471 of SEQ ID NO: 1,
and SEQ ID NO: 16 represents the polypeptide deduced from SEQ ID
NO: 17. ORF 9 (SEQ ID NO: 19) is the polynucleotide drawn from
residues 13468 to 14523 of SEQ ID NO: 1, and SEQ ID NO: 18
represents the polypeptide deduced from SEQ ID NO: 19. ORF 10 (SEQ
ID NO: 21) is the polynucleotide drawn from residues 14526 to 15701
of SEQ ID NO: 1, and SEQ ID NO: 20 represents the polypeptide
deduced from SEQ ID NO: 21. ORF 11 (SEQ ID NO: 23) is the
polynucleotide drawn from residues 15770 to 16642 of SEQ ID NO: 1,
and SEQ ID NO: 22 represents the polypeptide deduced from SEQ ID
NO: 23. ORF 12 (SEQ ID NO: 25) is the polynucleotide drawn from
residues 16756 to 17868 of SEQ ID NO: 1, and SEQ ID NO: 24
represents the polypeptide deduced from SEQ ID NO: 25. ORF 13 (SEQ
ID NO: 27) is the polynucleotide drawn from residues 17865 to 18527
of SEQ ID NO: 1, and SEQ ID NO: 26 represents the polypeptide
deduced from SEQ ID NO: 27. ORF 14 (SEQ ID NO: 29) is the
polynucleotide drawn from residues 18724 to 19119 of SEQ ID NO: 1,
and SEQ ID NO: 28 represents the polypeptide deduced from SEQ ID
NO: 29. ORF 15 (SEQ ID NO: 31) is the polynucleotide drawn from
residues 19175 to 19639 of SEQ ID NO: 1, and SEQ ID NO: 30
represents the polypeptide deduced from SEQ ID NO: 31. ORF 16 (SEQ
ID NO: 33) is the polynucleotide drawn from residues 19636 to 21621
of SEQ ID NO: 1, and SEQ ID NO: 32 represents the polypeptide
deduced from SEQ ID NO: 33. ORF 17 (SEQ ID NO: 35) is the
polynucleotide drawn from residues 21632 to 22021 of SEQ ID NO: 1,
and SEQ ID NO: 34 represents the polypeptide deduced from SEQ ID
NO: 35. ORF 18 (SEQ ID NO: 37) is the polynucleotide drawn from
residues 22658 to 22122 of SEQ ID NO: 1, and SEQ ID NO: 36
represents the polypeptide deduced from SEQ ID NO: 37. ORF 19 (SEQ
ID NO: 39) is the polynucleotide drawn from residues 24665 to 22680
of SEQ ID NO: 1, and SEQ ID NO: 38 represents the polypeptide
deduced from SEQ ID NO: 39. ORF 20 (SEQ ID NO: 41) is the
polynucleotide drawn from residues 24880 to 26163 of SEQ ID NO: 1,
and SEQ ID NO: 40 represents the polypeptide deduced from SEQ ID
NO: 41. ORF 21 (SEQ ID NO: 43) is the polynucleotide drawn from
residues 26179 to 27003 of SEQ ID NO: 1, and SEQ ID NO: 42
represents the polypeptide deduced from SEQ ID NO: 43. ORF 22 (SEQ
ID NO: 45) is the polynucleotide drawn from residues 27035 to 28138
of SEQ ID NO: 1, and SEQ ID NO: 44 represents the polypeptide
deduced from SEQ ID NO: 45. ORF 23 (SEQ ID NO: 47) is the
polynucleotide drawn from residues 28164 to 28925 of SEQ ID NO: 1,
and SEQ ID NO: 46 represents the polypeptide deduced from SEQ ID
NO: 47. ORF 24 (SEQ ID NO: 49) is the polynucleotide drawn from
residues 28922 to 30238 of SEQ ID NO: 1, and SEQ ID NO: 48
represents the polypeptide deduced from SEQ ID NO: 49. ORF 25 (SEQ
ID NO: 51) is the polynucleotide drawn from residues 30249 to 31439
of SEQ ID NO: 1, and SEQ ID NO: 50 represents the polypeptide
deduced from SEQ ID NO: 51. ORF 26 (SEQ ID NO: 53) is the
polynucleotide drawn from residues 31439 to 32224 of SEQ ID NO: 1,
and SEQ ID NO: 52 represents the polypeptide deduced from SEQ ID
NO: 53. ORF 27 (SEQ ID NO: 55) is the polynucleotide drawn from
residues 32257 to 32931 of SEQ ID NO: 1, and SEQ ID NO: 54
represents the polypeptide deduced from SEQ ID NO: 55. ORF 28 (SEQ
ID NO: 57) is the polynucleotide drawn from residues 32943 to 33644
of SEQ ID NO: 1, and SEQ ID NO: 56 represents the polypeptide
deduced from SEQ ID NO: 57. ORF 29 (SEQ ID NO: 59) is the
polynucleotide drawn from residues 34377 to 33637 of SEQ ID NO: 1,
and SEQ ID NO: 58 represents the polypeptide deduced from SEQ ID
NO: 59. ORF 30 (SEQ ID NO: 61) is the polynucleotide drawn from
residues 34572 to 34907 of SEQ ID NO: 1, and SEQ ID NO: 60
represents the polypeptide deduced from SEQ ID NO: 61. ORF 31 (SEQ
ID NO: 63) is the polynucleotide drawn from residues 34904 to 36583
of SEQ ID NO: 1, and SEQ ID NO: 62 represents the polypeptide
deduced from SEQ ID NO: 63. ORF 32 (SEQ ID NO: 66) is the
polynucleotide drawn from residues 23 to 1621 of SEQ ID NO: 64, and
SEQ ID NO: 65 represents the polypeptide deduced from SEQ ID NO:
66. ORF 33 (SEQ ID NO: 68) is the polynucleotide drawn from
residues 1702 to 2973 of SEQ ID NO: 64, and SEQ ID NO: 67
represents the polypeptide deduced from SEQ ID NO: 68. ORF 34 (SEQ
ID NO: 70) is the polynucleotide drawn from residues 3248 to 4270
of SEQ ID NO: 64, and SEQ ID NO: 69 represents the polypeptide
deduced from SEQ ID NO: 70. ORF 35 (SEQ ID NO: 72) is the
polynucleotide drawn from residues 4452 to 5933 of SEQ ID NO: 64,
and SEQ ID NO: 71 represents the polypeptide deduced from SEQ ID
NO: 72. ORF 36 (SEQ ID NO: 75) is the polynucleotide drawn from
residues 30 to 398 of SEQ ID NO: 73, and SEQ ID NO: 74 represents
the polypeptide deduced from SEQ ID NO: 75. ORF 37 (SEQ ID NO: 77)
is the polynucleotide drawn from residues 395 to 1372 of SEQ ID NO:
73, and SEQ ID NO: 76 represents the polypeptide deduced from SEQ
ID NO: 77. ORF 38 (SEQ ID NO: 79) is the polynucleotide drawn from
residues 3388 to 1397 of SEQ ID NO: 73, and SEQ ID NO: 78
represents the polypeptide deduced from SEQ ID NO: 79. ORF 39 (SEQ
ID NO: 81) is the polynucleotide drawn from residues 3565 to 5286
of SEQ ID NO: 73, and SEQ ID NO: 80 represents the polypeptide
deduced from SEQ ID NO: 81. ORF 40 (SEQ ID NO: 83) is the
polynucleotide drawn from residues 5283 to 7073 of SEQ ID NO: 73,
and SEQ ID NO: 82 represents the polypeptide deduced from SEQ ID
NO: 83. ORF 41 (SEQ ID NO: 85) is the polynucleotide drawn from
residues 7108 to 8631 of SEQ ID NO: 73, and SEQ ID NO: 84
represents the polypeptide deduced from SEQ ID NO: 85. ORF 42 (SEQ
ID NO: 87) is the polynucleotide drawn from residues 9371 to 8673
of SEQ ID NO: 73, and SEQ ID NO: 86 represents the polypeptide
deduced from SEQ ID NO: 87. ORF 43 (SEQ ID NO: 89) is the
polynucleotide drawn from residues 9762 to 9364 of SEQ ID NO: 73,
and SEQ ID NO: 88 represents the polypeptide deduced from SEQ ID
NO: 89.
[0312] Some open reading frames provided in the Sequence Listing,
namely ORF 2 (SEQ ID NO: 5), ORF 5 (SEQ ID NO: 11), ORF 12 (SEQ ID
NO: 25), ORF 13 (SEQ ID NO: 27), ORF 15 (SEQ ID NO: 31), ORF 17
(SEQ ID NO: 35), ORF 19 (SEQ ID NO: 39), ORF 20 (SEQ ID NO: 41),
ORF 22 (SEQ ID NO: 45), ORF 24 (SEQ ID NO: 49), ORF 26 (SEQ ID NO:
53) and ORF 27 (SEQ ID NO: 55) initiate with non-standard
initiation codons (eg. GTG-Valine, or CTG-Leucine) rather than
standard initiation codon ATG methionine. All ORFs are listed with
the appropriate M, V or L amino acids at the amino-terminal
position to indicate the specificity of the first codon of the ORF.
It is expected, however, that in all cases the biosynthesized
protein will contain a methionine residue, and more specifically a
formylmethionine residue, at the amino terminal position, in
keeping with the widely accepted principle that protein synthesis
in bacteria initiate with methionine (formylmethionine) even when
the encoding gene specifies a non-standard initiation codon (e.g.
Stryer BioChemistry 3.sup.rd edition, 1998, W.H. Freeman and Co.,
New York, pp. 752-754).
[0313] ORF 32 (SEQ ID NO: 65) is incomplete and contains a
truncation of 10 to 20 amino acids from its carboxy terminus. This
is due to incomplete sequence information between Contigs 2 and 3
(SEQ ID NOS: 64 and 73, respectively).
[0314] Deposits of E. coli DH10B vectors, each harbouring a cosmid
clone (designated in FIG. 12 as 046KM and 046KQ respectively) of a
partial biosynthetic locus for the compound of Formula II from
Micromonospora sp. strain 046-ECO11 and together spanning the full
biosynthetic locus for production of the compound of Formula II
have been deposited with the International Depositary Authority of
Canada, Bureau of Microbiology, Health Canada, 1015 Arlington
Street, Winnipeg, Manitoba, Canada R3E 3R2 on Feb. 25, 2003. The
cosmid clone designated 046KM was assigned deposit accession
numbers IDAC 250203-06, and the cosmid clone designated 046KQ was
assigned deposit accession numbers IDAC 250203-07. Cosmid 046KM
covers residue 1 to residue 32,250 of Contig 1 (SEQ ID NO: 1).
Cosmid 046KQ covers residue 21,700 of Contig 1 (SEQ ID NO: 1) to
residue 9,762 of Contig 3 (SEQ ID NO: 73). The sequence of the
polynucleotides comprised in the deposited strains, as well as the
amino acid sequence of any polypeptide encoded thereby are
controlling in the event of any conflict with any description of
sequences herein.
[0315] The deposit of the deposited strains has been made under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for Purposes of Patent Procedure.
The deposited strains will be irrevocably and without restriction
or condition released to the public upon the issuance of a patent.
The deposited strains are provided merely as convenience to those
skilled in the art and are not an admission that a deposit is
required for enablement, such as that required under 35 U.S.C.
.sctn.112. A license may be required to make, use or sell the
deposited strains, and compounds derived therefrom, and no such
license is hereby granted.
[0316] In order to identify the function of the proteins coded by
the genes forming the biosynthetic locus for the production of the
compound of Formula II the gene products of ORFs 1 to 43, namely
SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 65,
67, 69, 71, 74, 76, 78, 80, 82, 84, 86 and 88 were compared, using
the BLASTP version 2.2.10 algorithm with the default parameters, to
sequences in the National Center for Biotechnology Information
(NCBI) nonredundant protein database and the DECIPHER.RTM. database
of microbial genes, pathways and natural products (Ecopia
BioSciences Inc. St.-Laurent, QC, Canada).
[0317] The accession numbers of the top GenBank.TM. hits of this
BLAST analysis are presented in Table 14 along with the
corresponding E values. The E value relates the expected number of
chance alignments with an alignment score at least equal to the
observed alignment score. An E value of 0.00 indicates a perfect
homolog. The E values are calculated as described in Altschul et
al. J. Mol. Biol., 215, 403-410 (1990). The E value assists in the
determination of whether two sequences display sufficient
similarity to justify an inference of homology.
TABLE-US-00009 TABLE 14 ORF Family # aa GenBank homology
Probability % Identity % Similarity Proposed function of GenBank
match 1 ABCC 571 NP_736627.1, 590aa 1.00E-107 222/496 (44.76%)
278/496 (56.05%) ABC transporter Corynebacterium efficiens
NP_600638.1, 510aa 5.00E-80 184/500 (36.8%) 260/500 (52%) ABC
transporter Corynebacterium efficiens NP_600638.1, 510aa 3.00E-12
58/195 (29.74%) 84/195 (43.08%) ABC transporter Corynebacterium
efficiens 2 RECH 689 CAC93719.1, 923aa 3.00E-17 57/158 (36.08%)
87/158 (55.06%) regulator[Lechevalieria aerocolonigenes]
BAC55205.1, 943aa 3.00E-12 51/170 (30%) 81/170 (47.65%)
transcriptional activator [Streptomyces sp. NP_631154.1, 932aa
3.00E-07 29/63 (46.03%) 40/63 (63.49%) regulator. [Streptomyces
coelicolor A3(2) 3 REGD 895 CAC93719.1, 923aa 3.00E-20 92/330
(27.88%) 142/330 (43.03%) regulator [Lechevalieria aerocolonigenes]
BAC55205.1, 943aa 1.00E-15 80/277 (28.88%) 101/277 (36.46%)
activator [Streptomyces sp. TP-A0274] NP_733725.1, 908aa 3.00E-12
95/339 (28.02%) 140/339 (41.3%) regulator [Streptomyces coelicolor
A3(2)] 4 IDSA 362 NP_601376.2, 371aa 2.00E-80 158/321 (49.22%)
208/321 (64.8%) GGPP synthase [Corynebacterium glutamicum
NP_738677.1, 366aa 3.00E-79 158/330 (47.88%) 204/330 (61.82%)
polyprenyl synthase, Corynebacterium efficiens NP_216689.1, 352aa
2.00E-78 153/331 (46.22%) 203/331 (61.33%) idsA2 [Mycobacterium
tuberculosis H37Rv] 5 MVKA 354 BAB07790.1, 345aa 2.00E-71 150/326
(46.01%) 193/326 (59.2%) mevalonate kinase [Streptomyces sp. CL190]
BAB07817.1, 334aa 5.00E-66 145/324 (44.75%) 185/324 (57.1%)
mevalonate kinase [Kitasatospora griseola] NP_720650.1, 332aa
3.00E-36 95/327 (29.05%) 157/327 (48.01%) mevalonate kinase
[Streptococcus mutans 6 DMDA 346 BAB07791.1, 350aa 2.00E-88 177/305
(58.03%) 199/305 (65.25%) diphosphomevalonate decarboxylase
[Streptomyces sp. BAB07818.1, 300aa 2.00E-69 145/275 (52.73%)
168/275 (61.09%) mevalonate diPH decaroboxylase [Kitasatospora
griseola] NP_785307.1, 325aa 3.00E-44 105/307 (34.2%) 141/307
(45.93%) diphosphomevalonate decarboxylase [Lactobacillus plantarum
7 MVKP 369 BAB07792.1, 374aa 4.00E-93 183/365 (50.14%) 220/365
(60.27%) phosphomevalonate kinase [Streptomyces sp. CL190]
BAB07819.1, 360aa 6.00E-77 171/358 (47.77%) 202/358 (56.42%)
phosphomevalonate kinase [Kitasatospora griseola] AAG02442.1, 368aa
2.00E-31 102/354 (28.81%) 149/354 (42.09%) 3 phosphomevalonate
kinase [Enterococcus faecalis] 8 IPPI 360 Q9KWF6, 364aa 1.00E-128
238/361 (65.93%) 269/361 (74.52%) Isopentenyl-diphosphate
delta-isomerase Q9KWG2, 363aa 1.00E-128 230/349 (65.9%) 270/349
(77.36%) Isopentenyl-diphosphate delta-isomerase NP_814639.1, 347aa
5.00E-73 154/348 (44.25%) 212/348 (60.92%) isopentenyl diphosphate
isomerase [Enterococcus faecalis 9 HMGA 351 BAA70975.1, 353aa
1.00E-165 284/348 (81.61%) 317/348 (91.09%)
3-hydroxy-3-methylglutaryl coenzyme A reductase [Streptomyces sp.]
BAA74565.1, 353aa 1.00E-160 282/347 (81.27%) 310/347 (89.34%)
3-hydroxy-3-methylglutaryl coenzyme A reductase [Kitasatospora
griseola] BAA74566.1, 353aa 1.00E-155 277/347 (79.83%) 299/347
(86.17%) 3-hydroxy-3-methylglutaryl coenzyme A reductase
[Streptomyces sp.] 10 KASH 391 BAB07795.1, 389aa 1.00E-148 260/386
(67.36%) 300/386 (77.72%) 3-hydroxy-3-methylglutaryl CoA synthase
[Streptomyces sp. CL190] BAB07822.1, 346aa 1.00E-136 239/343
(69.68%) 268/343 (78.13%) HMG-CoA synthase [Kitasatospora griseola]
CAD24420.1, 388aa 6.00E-79 166/385 (43.12%) 210/385 (54.55%)
HMG-CoA synthase [Paracoccus zeaxanthinifaciens] 11 IPTN 290
NP_631248.1, 295aa 5.00E-22 79/282 (28.01%) 124/282 (43.97%)
hypothetical protein [Streptomyces coelicolor A3(2)] AAN65239.1,
324aa 5.00E-06 70/278 (25.18%) 112/278 (40.29%) cloQ [Streptomyces
roseochromogenes subsp. oscitans] 12 SPKG 370 AAM78435.1, 344aa
5.00E-48 112/208 (53.85%) 131/208 (62.98%) two-component sensor
[Streptomyces coelicolor A3(2)] NP_630507.1, 382aa 5.00E-48 112/208
(53.85%) 131/208 (62.98%) sensor kinase [Streptomyces coelicolor
A3(2)] ZP_00058991.1, 407aa 9.00E-34 88/198 (44.44%) 114/198
(57.58%) Signal transduction histidine kinase [Thermobifida fusca]
13 RREB 220 NP_630508.1, 224aa 3.00E-79 148/220 (67.27%) 179/220
(81.36%) regulatory protein [Streptomyces coelicolor A3(2)]
ZP_00058992.1, 221aa 4.00E-67 129/218 (59.17%) 163/218 (74.77%)
Response regulator [Thermobifida fusca] NP_625364.1, 221aa 6.00E-66
134/222 (60.36%) 164/222 (73.87%) response regulator [Streptomyces
coelicolor A3(2)] 14 UNES 131 No hit -- -- -- -- 15 UNEZ 154
NP_649459.2, 628aa 7.60E-02 21/55 (38.18%) 33/55 (60%) CG1090-PB
[Drosophila melanogaster] NP_730819.1, 473aa 7.60E-02 21/55
(38.18%) 33/55 (60%) CG1090-PA [Drosophila melanogaster]
AAM11079.1, 428aa 7.60E-02 21/55 (38.18%) 33/55 (60%) GH23040p
[Drosophila melanogaster] 16 OXDS 661 NP_242948.1, 500aa 1.00E-52
129/433 (29.79%) 197/433 (45.5%) unknown conserved protein
[Bacillus halodurans] ZP_00091617.1, 480aa 3.00E-32 123/426
(28.87%) 175/426 (41.08%) Putative multicopper oxidases
[Azotobacter vinelandii] NP_252457.1, 463aa 1.00E-31 115/408
(28.19%) 170/408 (41.67%) metallo-oxidoreductase [Pseudomonas
aeruginosa PA01] 17 UNFD 129 NP_437360.1, 127aa 7.00E-33 73/121
(60.33%) 87/121 (71.9%) bleomycin resistance protein family
[Sinorhizobium meliloti] AAO91879.1, 123aa 1.00E-31 68/117 (58.12%)
86/117 (73.5%) unknown [uncultured bacterium] NP_103287.1, 131aa
1.00E-23 59/122 (48.36%) 76/122 (62.3%) unknown protein
[Mesorhizobium loti] 18 UNFA 178 19 CSMB 661 ZP_00137697.1, 769aa
1.00E-166 319/622 (51.29%) 408/622 (65.59%)
Anthranilate/para-aminobenzoate synthase [Pseudomonas aeruginosa
NP_250594.1, 627aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%)
phenazine biosynthesis protein PhzE [Pseudomonas aeruginosa PA01]
ZP_00137701.1, 687aa 1.00E-166 319/622 (51.29%) 408/622 (65.59%)
Anthranilate/para-aminobenzoate synthas [Pseudomonas aeruginosa 20
AAKD 427 P41403, 421aa 1.00E-64 161/420 (38.33%) 214/420 (50.95%)
Aspartokinase (Aspartate kinase) ZP_00057166.1, 445aa 2.00E-64
154/415 (37.11%) 218/415 (52.53%) Aspartokinases [Thermobifida
fusca] AAD49567.1, 421aa 6.00E-64 152/412 (36.89%) 216/412 (52.43%)
aspartokinase subunit A [Amycolatopsis mediterranei] 21 ALDB 274
NP_275722.1, 266aa 2.00E-53 104/231 (45.02%) 147/231 (63.64%)
conserved protein [Methanothermobacter thermautotrophicus]
NP_614692.1, 270aa 2.00E-52 104/240 (43.33%) 146/240 (60.83%)
Fructose-1,6-bisphosphate aldolase [Methanopyrus kandleri AV19]
NP_615406.1, 267aa 2.00E-50 99/231 (42.86%) 141/231 (61.04%)
fructose-bisphosphate aldolase [Methanosarcina acetivorans str.
C2A] 22 UNFC 367 NP_275723.1, 378aa 4.00E-46 116/308 (37.66%)
171/308 (55.52%) conserved protein [Methanothermobacter
thermautotrophicus] NP_614691.1, 402aa 2.00E-45 115/295 (38.98%)
163/295 (55.25%) alternative 3-dehydroquinate synthase
[Methanopyrus kandleri NP_248244.1, 361aa 2.00E-43 103/255 (40.39%)
150/255 (58.82%) conserved hypothetical protein [Methanococcus
jannaschii 23 HYDK 253 NP_577771.1, 247aa 4.00E-14 55/178 (30.9%)
87/178 (48.88%) metal-dependent hydrolase [Pyrococcus furiosus DSM
3638] NP_142108.1, 247aa 1.00E-12 50/151 (33.11%) 78/151 (51.66%)
hypothetical protein PH0093 [Pyrococcus horikoshii] NP_125791.1,
248aa 1.00E-11 42/151 (27.81%) 76/151 (50.33%) hypothetical protein
[Pyrococcus abyssi] 24 ADSA 438 NP_070499.1, 433aa 2.00E-41 122/347
(35.16%) 171/347 (49.28%) coenzyme F390 synthetase [Archaeoglobus
fulgidus NP_618724.1, 434aa 5.00E-41 119/345 (34.49%) 171/345
(49.57%) coenzyme F390 synthetase [Methanosarcina acetivorans
NP_632700.1, 437aa 7.00E-41 121/345 (35.07%) 171/345 (49.57%)
Coenzyme F390 synthetase [Methanosarcina mazei Goe1] 25 HOXV 396
ZP_00027430.1, 442aa 8.00E-76 152/358 (42.46%) 211/358 (58.94%)
2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum]
NP_627457.1, 420aa 1.00E-71 161/420 (38.33%) 216/420 (51.43%)
salicylate hydroxylase [Streptomyces coelicolor A3(2)]
ZP_00033877.1, 403aa 2.00E-68 146/395 (36.96%) 200/395 (50.63%)
2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum] 26
SDRA 261 NP_391080.1, 261aa 6.00E-58 119/261 (45.59%) 149/261
(57.09%) 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase [Bacillus
subtilis] ZP_00059512.1, 260aa 1.00E-55 116/259 (44.79%) 144/259
(55.6%) Dehydrogenase [Thermobifida fusca] AAG31126.1, 257aa
9.00E-55 117/257 (45.53%) 144/257 (56.03%) MxcC [Stigmatella
aurantiaca] 27 DHBS 224 Q51790, 207aa 7.00E-60 110/198 (55.56%)
142/198 (71.72%) isochorismatase Q51518, 207aa 1.00E-58 110/198
(55.56%) 140/198 (70.71%) isochorismatase NP_391077.1, 312aa
2.00E-58 106/203 (52.22%) 139/203 (68.47%) isochorismatase
[Bacillus subtilis] 28 SDRA 233 NP_103491.1, 242aa 9.00E-21 74/230
(32.17%) 112/230 (48.7%) acyl-carrier protein reductase
[Mesorhizobium loti] AAL14912.1, 245aa 1.00E-15 65/229 (28.38%)
100/229 (43.67%) short-chain dehydrogenase [Rhizobium leguminosarum
bv. trifolii] NP_902480.1, 235aa 7.00E-15 67/229 (29.26%) 100/229
(43.67%) oxidoreductase [Chromobacterium violaceum 29 UNIQ 246
S18541, 281aa 4.50E-02 43/146 (29.45%) 63/146 (43.15%) hypothetical
protein 3 - Streptomyces coelicolor NP_629228.1, 281aa 5.90E-02
43/146 (29.45%) 63/146 (43.15%) hypothetical protein [Streptomyces
coelicolor A3(2)] 30 UNFE 111 ZP_00058149.1, 130aa 1.00E-10 35/97
(36.08%) 47/97 (48.45%) membrane protein [Thermobifida fusca]
NP_737701.1, 120aa 1.00E-09 37/111 (33.33%) 51/111 (45.95%)
hypothetical protein [Corynebacterium efficiens NP_827629.1, 118aa
7.00E-09 35/105 (33.33%) 51/105 (48.57%) hypothetical protein
[Streptomyces avermitilis MA-4680] 31 EFFT 559 ZP_00058148.1, 537aa
2.00E-67 165/517 (31.91%) 253/517 (48.94%) Predicted symporter
[Thermobifida fusca] NP_626090.1, 544aa 4.00E-66 162/521 (31.09%)
257/521 (49.33%) transport protein [Streptomyces coelicolor A3(2)]
NP_827630.1, 549aa 7.00E-63 160/523 (30.59%) 256/523 (48.95%)
sodium-dependent symporter [Streptomyces avermitilis 32 HOYH 532
AAM96655.1, 544aa 2.00E-92 206/526 (39.16%) 279/526 (53.04%)
2,4-dihydroxybenzoate monooxygenase [Sphingobium chlorophenolicum]
ZP_00029353.1, 543aa 1.00E-73 188/539 (34.88%) 263/539 (48.79%)
2-polyprenyl-6-methoxyphenol hydroxylase [Burkholderia fungorum]
NP_769326.1, 569aa 5e-62 173/519 (33.33%) 251/519 (48.36%) blr2686
[Bradyrhizobium japonicum] dbj 33 DAHP 423 T03226, 391aa 1.00E-111
207/383 (54.05%) 259/383 (67.62%) hypothetical protein -
Streptomyces hygroscopicus ZP_00137693.1, 405aa 3.00E-87 172/385
(44.68%) 233/385 (60.52%) DAHP synthase [Pseudomonas aeruginosa
UCBPP-PA14] NP_250592.1, 405aa 1.00E-86 169/380 (44.47%) 232/380
(61.05%) phenazine biosynthesis protein PhzC [Pseudomonas
aeruginosa 34 REGG 340 BAC53615.1, 346aa 1.00E-67 142/307 (46.25%)
192/307 (62.54%) regulator protein [Streptomyces kasugaensis]
S44506, 424aa 3.00E-66 141/305 (46.23%) 182/305 (59.67%) regulator
protein - Streptomyces glaucescens AAK81822.1, 348aa 1.00E-65
141/323 (43.65%) 192/323 (59.44%) transcriptional regulator
[Streptomyces lavendulae] 35 UNFJ 493 ZP_00073237.1, 678aa 7.00E-35
124/454 (27.31%) 197/454
(43.39%) RTX toxins [Trichodesmium erythraeum IMS101] NP_484716.1,
433aa 3.00E-05 109/470 (23.19%) 172/470 (36.6%) similar to vanadium
chloroperoxidase [Nostoc sp. ZP_00067005.1, 667aa 7.40E-02 37/139
(26.62%) 52/139 (37.41%) hypothetical protein [Microbulbifer
degradans 2-40] 36 RECI 112 NP_627088.1, 125aa 3.00E-17 48/100
(48%) 59/100 (59%) hypothetical protein [Streptomyces coelicolor
A3(2)] NP_846017.1, 109aa 7.00E-15 40/101 (39.6%) 60/101 (59.41%)
hypothetical protein [Bacillus anthracis str. Ames] NP_241272.1,
174aa 9.00E-15 39/106 (36.79%) 62/106 (58.49%) unknown conserved
protein [Bacillus halodurans] 37 UNIQ 325 NP_422203.1, 187aa
1.00E-03 24/61 (39.34%) 36/61 (59.02%) hypothetical protein
[Caulobacter crescentus CB15] 38 OXAH 663 ZP_00058724.1, 659aa
0.00E+00 370/647 (57.19%) 435/647 (67.23%) Acyl-CoA dehydrogenases
[Thermobifida fusca] AAB97825.1, 433aa 5.00E-93 203/446 (45.52%)
251/446 (56.28%) acyl-CoA oxidase [Myxococcus xanthus] AAF14635.1,
694aa 5.00E-85 211/565 (37.35%) 292/565 (51.68%) 1 acyl-CoA oxidase
[Petroselinum crispum] 39 ABCA 537 T14162, 574aa 9.00E-62 189/509
(37%) 240/509 (47%) hABC transport protein - Mycobacterium
smegmatis NP_624808.1 4.00E-60 184/540 (35%) 251/540 (46%) ABC
transporter [Streptomyces coelicolor A3(2)] NP_822745.1 8.00E-32
124/392 (31%) 168/392 (42%) ABC transportert [Streptomyces
avermitilis MA-4680] 40 ABCA 596 T14180, 1122aa 1.00E-107 236/594
(39.73%) 300/594 (50.51%) exiT protein - Mycobacterium smegmatis
AAC82548.1, 589aa 1.00E-107 234/583 (40.14%) 295/583 (50.6%)
unknown [Mycobacterium smegmatis] NP_624810.1, 601aa 3.00E-97
222/593 (37.44%) 283/593 (47.72%) ABC-transporter [Streptomyces
coelicolor A3(2)] 41 UNIQ 507 NP_831570.1, 676aa 8.00E-07 62/262
(23.66%) 116/262 (44.27%) methyltransferases [Bacillus cereus
NP_655735.1, 676aa 2.00E-06 61/262 (23.28%) 116/262 (44.27%)
ubiE/COQ5 methyltransferase family [Bacillus anthracis NP_844290.1,
681aa 2.00E-06 61/262 (23.28%) 116/262 (44.27%) hypothetical
protein [Bacillus anthracis str. Ames] 42 232 NP_830809.1, 208aa
8.00E-08 46/210 (21.9%) 74/210 (35.24%) Transporter, LysE family
[Bacillus cereus] NP_844737.1, 210aa 2.00E-07 46/210 (21.9%) 74/210
(35.24%) homoserine/threonine efflux protein [Bacillus anthracis
NP_655752.1, 208aa 1.00E-06 47/210 (22.38%) 75/210 (35.71%) LysE,
LysE type translocator [Bacillus anthracis 43 132 NP_827272.1,
127aa 4.00E-09 38/107 (35.51%) 52/107 (48.6%) hypothetical protein
[Streptomyces avermitilis MA-4680] NP_246491.1, 112aa 5.90E-02
21/94 (22.34%) 44/94 (46.81%) unknown [Pasteurella multocida]
[0318] The ORFs encoding proteins involved in the biosynthesis of
compounds of Formula II are assigned a putative function and
grouped together in families based on sequence similarity to known
proteins. To correlate structure and function, the protein families
are given a four-letter designation used throughout the description
and figures as indicated in Table 15. The meaning of the four
letter designations is as follows: AAKD designates an amino acid
kinase; ABCA and ABCC designate ABC transporters; ADSA designates
an amide synthetase; ALDB designates an aldolase function; CSMB
designates a chorismate transaminase; DAHP designates a
3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate
synthase activity; DHBS designates a
2,3-dihydro-2,3-dihydroxybenzoate synthase activity; DMDA
designates a diphosphomevalonate decarboxylase; EFFT designates an
efflux protein; HMGA designates a 3-hydroxy-3-methylglutaryl-CoA
reductase; HOXV designates a monooxygenase activity; HOYH
designates a hydroxylase/decarboxylase activity; HYDK designates a
hydrolase activity; IDSA designates an isopentenyl diphosphate
synthase; IPPI designates an isopentenyl diphosphate isomerase;
IPTN designates an isoprenyltransferase; KASH designates
3-hydroxy-3-methylglutaryl-CoA synthase; MVKA designates a
mevalonate kinase; MVPK designates a phosphomevalonate kinase; OXAH
designates an acylCoA oxidase; OXDS designates an oxidoreductase;
RECH, RECI, REGD, REGG and RREB designate regulators; SDRA
designates a dehydrogenase/ketoreductase, SPKG designates a sensory
protein kinase; UNES, UNEZ, UNFA, UNFC, UNFD, UNFE, UNFJ and UNIQ
designate proteins of unknown function.
TABLE-US-00010 TABLE 15 FAMILY FUNCTION AAKD amino acid kinase;
strong homology to primary aspartate kinases, converting L-
aspartate to 4-phospho-L-aspartate ABCA ABC transporter ABCC ABC
transporter ADSA adenylating amide synthetase ALDB aldolase;
similarity to fructose-1,6-biphosphate aldolase that generates D-
glyceraldehyde-3Ph, precursor of D-erythrose-4Ph involved in the
shikimate pathway CSMB chorismate transaminase, similarity to
anthranilate synthase DAHP DAHP synthase, class II; involved in
formation of aminoDAHP from PEP and erythrose-4-phosphate DHBS
2,3-dihydro-2,3-dihydroxybenzoate synthase (isochorismatase) DMDA
diphosphomevalonate decarboxylase (mevalonate pyrophosphate
decarboxylase) EFFT efflux protein HMGA HMG-CoA reductase; converts
3-hydroxy-3-methylglutaryl-CoA to mevalonate plus CoA in isoprenoid
biosynthesis HOXV FAD monooxygenase; shows homology to a variety of
monooxygenases including salicylate hydroxylases, zeaxanthin
epoxidases HOYH hydroxylase/decarboxylase; FAD-dependent
monooxygenase HYDK hydrolase IDSA isoprenyl diphosphate synthase,
catalyzes the addition of 2 molecules of isopentenyl pyrophosphate
to dimethylallyl pyrophosphate to generate GGPP IPPI isopentenyl
diphosphate isomerase, catalyzes the isomerization of IPP to
produce dimethylallyl diphosphate IPTN isoprenyltransferase;
catalyzes covalent N-terminal attachment of isoprenyl units to
amide groups of nitrogen-containing heterocycle rings KASH HMG-CoA
synthase; condenses acetyl-CoA with acetoacetyl-CoA to form 3-
hydroxy-3-methylglutaryl-CoA MVKA mevalonate kinase; converts
mevalonate to 5-phosphomevalonate in the mevalonate pathway of
isoprenoid biosynthesis MVKP phosphomevalonate kinase; converts
5-phosphomevalonate to 5- diphosphomevalonate in the mevalonate
pathway of isoprenoid biosynyhesis OXAH acyl CoA oxidase OXDS
oxidoreductase RECH regulator RECI regulator; similarity to PadR
transcriptional regulators involved in repression of phenolic acid
metabolism REGD transcriptional regulator; relatively large
regulators with an N-terminal ATP-binding domain containing Walker
A and B motifs and a C-terminal LuxR type DNA-binding domain REGG
regulator RREB response regulator; similar to response regulators
that are known to bind DNA and act as transcriptional activators
SDRA dehydrogenase/ketoreductase, NAD-dependent SPKG sensory
protein kinase, two component system UNES unknown function UNEZ
unknown function UNFA unknown function UNFC unknown function UNFD
unknown function UNFE putative membrane protein UNFJ unknown
function UNIQ unknown function
[0319] Biosynthesis of the compound of Formula II involves the
action of various enzymes that synthesize the three building blocks
of the compound, namely the farnesyl-diphosphate component (FIG.
13), the 3-hydroxy-anthranilate-adenylate component (FIG. 14a) and
the 2-amino-6-hydroxy-benzoquinone component (FIG. 14b) that are
subsequently condensed to form the final compound (FIG. 15).
[0320] The farnesyl-diphosphate biosynthesis involves the concerted
action of seven enzymes (FIG. 13). ORF 10 (KASH) (SEQ ID NO: 20)
encodes a hydroxymethylglutaryl-CoA synthase that catalyzes an
aldol addition of acetyl-CoA onto acetoacyl-CoA to yield
3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). This product is
subsequently reduced through the action of ORF 9 (HMGA) (SEQ ID NO:
18) to form mevalonic acid (MVA). ORF 5 (MVKA) (SEQ ID NO: 10)
phosphorylates mevalonate to 5'-phosphomevalonate using ATP as the
phosphate donor. The next step in the farnesyl-diphosphate
biosynthesis is the phosphorylation reaction of the
5'-phosphomevalonate to 5'-pyrophosphomevalonate (DPMVA) that is
catalyzed by ORF 7 (MVKP) (SEQ ID NO: 14). Subsequent
decarboxylation of 5'-pyrophosphomevalonate catalyzed by ORF 6
(DMDA) (SEQ ID NO: 12) yields isopentenyl diphosphate (IPP) which
is then converted to dimethylallyldiphosphate (DMADP) through the
action of ORF 8 (IPPI) (SEQ ID NO: 16) that has isomerase enzymatic
activity. The final step in the biosynthesis of
farnesyl-diphosphate is the condensation of one molecule of
dimethylallyldiphosphate with two molecules of isopentenyl
diphosphate catalyzed by the isoprenyl diphosphate synthase ORF 4
(IDSA) (SEQ ID NO: 8). The described pathway involved in synthesis
of farnesyl-diphosphate is entirely consistent with related
mevalonate pathways described in other actinomycete species (Takagi
et al., J. Bacteriol. 182, 4153-4157, (2000)).
[0321] Biosynthesis of the 3-hydroxy-anthranilate component
involves the use of precursors derived from the shikimate pathway
(FIG. 14a). Chorismic acid is transaminated through the action of
ORF 19 (CSMB) (SEQ ID NO: 38) to form aminodeoxyisochorismic acid.
This enzyme resembles anthranilate synthases and is likely to
catalyze specifically the transfer of the amino group using
glutamine as the amino donor. The next step involves
isochorismatase activity and is mediated by ORF 27 (DHBS) (SEQ ID
NO: 54). This reaction consists in the removal of the pyruvate side
chain from aminodeoxyisochorismic acid to form
6-amino-5-hydroxy-cyclohexa-1,3-dienecarboxylic acid. This compound
is subsequently oxidized through the action of ORF 26 (SDRA) (SEQ
ID NO: 52) yielding 3-hydroxy-anthranilic acid. ORF 24 (ADSA) (SEQ
ID NO: 48) catalyzes the activation of 3-hydroxy-anthranilic acid
through adenylation generating the 3-hydroxy-anthranilate-adenylate
component (FIG. 14a).
[0322] Biosynthesis of the 2-amino-6-hydroxy-benzoquinone component
of the compound of Formula II, requires components derived from the
aminoshikimate pathway. FIG. 14b depicts the series of enzymatic
reactions involved in the biosynthesis of this constituent. ORF 21
(ALDB) (SEQ ID NO: 42) resembles aldolases involved in the
generation of precursors of D-erythrose-4-phosphate which is part
of the aminoshikimate pathway used for the generation of
2-amino-6-hydroxy-[1,4]-benzoquinone. ORF 33 (DAHP) (SEQ ID NO: 67)
catalyzes the initial step in the aminoshikimate pathway that
corresponds to the formation of
3,4-dideoxy-4-amino-D-arabino-heptulosonic acid 7-phosphate (amino
DAHP) from phosphoenolpyruvate (PEP) and erythrose 4-phosphate
(E-4Ph). Subsequent reactions leading to 3-amino-5-hydroxy-benzoic
acid are catalyzed by enzymes provided by primary metabolism
biosynthetic pathways present in Micromonospora sp. strain
046-ECO11. ORF 25 (HOXV) (SEQ ID NO: 50) hydroxylates
3-amino-5-hydroxy-benzoic acid at position 2, generating
3-amino-2,5-dihydroxy-benzoic acid. This intermediate is further
modified by ORF 32 (HOYH) (SEQ ID NO: 65) that catalyzes a
decarboxylative oxidation reaction yielding
6-amino-benzene-1,2,4-triol. A final oxidation reaction is
performed by ORF 16 (OXDS) (SEQ ID NO: 32) yielding
2-amino-6-hydroxy-[1,4]-benzoquinone (FIG. 14b).
[0323] Assembly of the three components resulting in the compound
of Formula II is catalyzed by ORFs 24 and 11 (FIG. 15). ORF 24
(ADSA) (SEQ ID NO: 48) catalyzes the condensation of the adenylated
3-hydroxy-anthranilate with the
2-amino-6-hydroxy-[1,4]-benzoquinone component. A spontaneous
condensation between the free amino group of the
3-hydroxy-anthranilate and one of the carbonyl groups present on
the 2-amino-6-hydroxy-[1,4]-benzoquinone component occurs yielding
a dibenzodiazepinone intermediate. This compound is further
modified through transfer of the farnesyl group of the
farnesyl-diphosphate intermediate onto the nitrogen of the amide of
the dibenzodiazepinone catalyzed by ORF 11 (IPTN) (SEQ ID NO: 22)
and resulting in the formation of the compound of Formula II (FIG.
15).
[0324] Additional ORFs, namely ORF 2 (RECH) (SEQ ID NO: 4), ORF 3
(REGD) (SEQ ID NO: 6), ORF 12 (SPKG) (SEQ ID NO: 24), ORF 13 (RREB)
(SEQ ID NO: 26), ORF 34 (REGG) (SEQ ID NO: 69) and ORF 36 (RECI)
(SEQ ID NO: 74) are involved in the regulation of the biosynthetic
locus encoding the compound of Formula II. Other ORFs, namely ORF 1
(ABCC) (SEQ ID NO: 2), ORF 31 (EFFT) (SEQ ID NO: 62), ORFs 39 and
40 (ABCA) (SEQ ID NOS: 80 and 82, respectively) and ORF 42 (SEQ ID
NO: 86) are involved in transport. Other ORFs involved in the
biosynthesis of the compound of Formula II include ORF 20 (AAKD)
(SEQ ID NO: 40), ORF 23 (HYDK) (SEQ ID NO: 46), ORF 38 (OXAH) (SEQ
ID NO: 78) as well as ORFs 14, 15, 17, 18, 22, 29, 30, 35, 37, 41
and 43 (SEQ ID NOS: 28, 30, 34, 34, 44, 58, 60, 71, 76, 84 and 88,
respectively) of unknown function.
TABLE-US-00011 TABLE 16 PREFERRED MEDIA COMPOSITION FOR PRODUCTION
OF ECO-04601 COMPONENT QB MA KH RM JA FA pH*.sup.5 7.2 7.5 7 6.85
7.3 7.0 Glucose 12 10 10 10 Sucrose 100 Lactose Cane molasses 15
Corn starch 30 Soluble starch 10 25 Potato dextrin 20 40 Corn steep
solid Corn steep 5 15 Dried yeast 2 Yeast extract 5 Malt extract 35
Pharmamedia .TM. 10 15 Glycerol NZ-Amine 5 10 Soybean 15 Soybean
flour Meat extract Bacto-peptone MgSO.sub.4.cndot.7H.sub.2O 1
MgCl.sub.2.cndot.6H.sub.2O CaCO.sub.3 4 1 2 2 NaCl 5
(NH.sub.4).sub.2 SO.sub.4 2 K.sub.2SO.sub.4 0.25
MnCl.sub.2.cndot.4H.sub.2O MgCl.sub.2.cndot.6H.sub.2O 10
FeCl.sub.2.cndot.4H.sub.2O ZnCl.sub.2 Na.sub.2HPO.sub.4 3 Thiamine
Casamino acid 0.1 Proflo oil 4 MOPS 21 Trace element 2
solution*.sup.3 ml/L Unless otherwise indicated all the ingredients
are in gm/L. *.sup.3Trace elements solution contains: ZnCl.sub.2 40
mg; FeCl.sub.3 6H.sub.2O (200 mg); CuCl.sub.2 2H.sub.2O (10 mg);
MnCl.sub.2.cndot.4H.sub.2O; Na.sub.2B.sub.4O.sub.7.cndot.10H.sub.2O
(10 mg); (NH.sub.4).sub.6 MO.sub.7O.sub.24.cndot.4H.sub.2O (10 mg)
per litre. *.sup.5The pH is to adjusted as marked prior to the
addition of CaCO.sub.3.
[0325] All patents, patent applications, and published references
cited herein are hereby incorporated by reference in their
entirety. While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
89136602DNAMicromonospora sp. strain 046-ECO11 1ccggtgcacc
gggttctcca ggatcgccgt cgcgcccacc ggccccgaca ggtagacgac 60gttcagggac
ttgccgcgcc cttcgtagtt ggcccgcacc acctgcgcgt cgccgatccg
120gcccctggtc tccagcgtgc ggttctccca cacctgccat ccgacgaagg
tcaggaacag 180cgcggtgaac agggacgtga cgagcagcca gaggccagct
gtcagcacgg tcgccccctc 240gccccgtagc aggccgagga cgacctcctc
gtagcgcgag gggcggccga cggggccggt 300gcccgctccg tcgacagcca
tcccgccgct ccttcgccga ctgccccgga catccacggt 360agccagcgag
tccagtccgg tgaggaaggg gtggcgagaa gtcgatatga ctgagaggca
420tatttatgac tcccagtcat atcgctcgga agtgaccgaa cgacctgacg
ccgccggggc 480tgtgagcggc agcgtgggcc aggccgcgag gtcctggagc
atctgccggt cgtgggtggc 540gacgaccacc gccgcccggg tcgtcagcag
ggcggcggtg aggtcgtcga ccagcggcgc 600cgacaggtgg ttcgtcggtt
cgtcgaggat cagcaggtcg ggacgttcgg ccaggcgcag 660cgccaggttc
agccgccgtt gctgtccgtg cgacatccgg ccgacggggg tacgccgggc
720ctcggcgtcg agcaggttcg tcgcgctcag cggcagggcc gtgccggagc
cgacgcgccc 780gctggagcgg agccggccca cgtgctgctc gtacaggtcg
tgcgcgagca gcgccggcgg 840ccagtcgggc acctcctgac cgaggtacgc
gacgcgcgcg ccggacaggt gccggacctc 900cccggtcgac ggcgtgaggt
cgccggccag cacggagagc agcgtcgact tgcccgcgcc 960gttgggtccg
gtcaccagca ggcggtcccc gccgtcgagc gtgagcgtga cccgggtacg
1020caggcgcccg gccaccgtga cgtcgtggca tcgcaggatg ggcagtccgg
cacgggtgtc 1080cagcggcggc cagcgcagcg gctgcggtgg ctccggcacg
gtgacgcggt gcgcgtcgag 1140cgcctcctgc cggcggcgca gcgcctggac
cagtccgggc gcgcgggact ggcgctggtg 1200cttgccgtgc cccttctccg
gccgccagcc ggtgctgagc cggtcccgcg cctcccgtac 1260cccgtcggcc
agccgctggt gctcggcctg ctgcgcctcg tggtcgcgta cccagtgcgc
1320gaagtcgcgg cggcgcccgt cctgccaggc gacgtagtcc ccggcgtagc
ggcgcgggcg 1380cccgtccgcg ctggggtcga ggtccaggaa ctccgtggcg
acgtcccgca gcagggcgcg 1440gtcgtgggtc accagcacga cgccgcccgg
gtggtcgcgt agccgggcgg tgaggaaggc 1500caggctgtcg gcgtcgaggt
ggttcgtcgg ctcgtccagc atcagcaggt cgaccctcgc 1560tcccagcagg
cacgccagcc gtacccggta gcgctggccg acggacaacg tggccagctg
1620ccggtcccgg tccgggcacg cgtcgaggcc ggccagcgcc acgtcgacgc
gccgctgcgc 1680gtcccaggcg tccagccggg tcgccgcgtc gagcgcggcc
gcgtacgcgt cgtccgcgcc 1740cgcccggccc tcggtgagcg cgatcgtcgc
ctcgtcgagc gcccgcagcg cgcgttcgga 1800ctcccggatc gcctcccgga
cgagcgtgcc cactgtctcg ccgtggcgcg actccaggtt 1860ctgccgggcg
acgccgatcg tgcccagccg ttccaccacg ccctggtcgg gcgcgatgag
1920gccggccagc acgtgcagca gggtggtctt gccgcggccg ttctcgccga
cgactgcgag 1980gcgggaagcg gcggagacgg tcacgctgac gtcggacagc
acgacccggc cgccgcgtac 2040gacgcggacg ccgtcggccc gcacgtgcgc
ccggtgcccg gcgggcagcg aaccgcccga 2100ggtggatggg gaggaaggaa
tgttgtcgag gttgtgcaca gtccgctctt cggctcgtcg 2160tggagccggg
cagcgcgagg acaccgcccg gcgggaacgc cgggacggcg gagcagagct
2220ggtacgtcag aagaagccgg tcaccctgcc gccgtcagcg gagggaccag
ggcttcatga 2280cagcggcgta gaacctcatg cggtcaacac tacccggggc
cgggccggag atcgccgcag 2340ttatcggcgg cggcgggcgt cggcctcggc
gtcgagcagg tcgttcaccg ccagcgccga 2400gttgatcaga gcgaggtggc
tgaacgcctg cggatccacg gttgcggtac tccatttgca 2460gtacacctgt
cggtatccgg tcagcgccgt atcctgcgct ttctctgtcg gcagagcggc
2520gcggtcgccc gccgcccgcc gacgtggctg cggggccggt cgggctcgga
ccgctcggtg 2580cggcgtcgcg gcccggccgt agcatgtttc acctgttcag
agcggcttcc gggcgctcgg 2640gccgtcggcc gcggtggtta ccggcgaggg
ctatttcggt catgcgagag ggttctgcca 2700atcgtggcat tgtttagtta
agtccgatat cagcgggatg ctgcctgata tatgacggct 2760gcgcccgggc
ctgccggata gctatgatga gcgacgacgg tgatcgatgg caaatgttgt
2820tgctgtgggg tagcgtcacc gccgagtcca ggcttttctt gagctgtgtg
cgcatattcc 2880ggggggatta tgacaacggg acggccgggg gagaaccggg
cgacagacgc ggcacgaaat 2940ccggggtggg ccgccggggg gccggcgtcc
cagccatggg gcggggggaa cgacgagcag 3000gtcctgcgcg agatcctcgg
ggtcgacgtg caccgcgagc tgattgactt cgcgggtggt 3060gccggcggaa
atccgcacct ggtcgccgaa ctcgcgcgcg ggctcgccga agagggattg
3120attcgggaga caaacggtcg ggcggaattg gtgtcccggc gaattccccg
gcgcgtgctg 3180agttttgtca tgcgtcgatt gaatgatgtc agcgccggct
gccagcagtt cttgaaggtt 3240gccgcggcat tgggcagatc cttcatgctg
gaggacgttt cgagaatgct gggccgatcg 3300tcggcggccc tgctcccgcc
ggtggacgag gcgatcgcat cgggcttcgt cgtcgccgcc 3360gagcatcaac
tcgcctttca gagcgacttc ctgctgcgcg gcatcatcga gtccattccc
3420gggcccgccc gcgacgcctt acgacgtgag gcgatgagcc tttccgggcg
acggcgcccg 3480gcggccgacc agaatcgccg gttggacgcg gcgcctaccg
cgccggtgag cgcgaccggg 3540gaggacgcca ccggatcctg ttcccgggcg
caccgcctga taatgaacgg gaacgcgaag 3600gccggcattc gcgtcgccga
ggcggttctc gccggcccgg ccgcgtcgct cgctgcccgg 3660cgtgacgcgg
aggcgtgtct ggtgctggcc gatctgctgc tcggcgggga gggcggcggc
3720ccgatgaccg aggcgatcct gcgcgaacgc gacgccgagt ccggtgacgc
cgcactggcg 3780atggcgctga ccgcccggtc caccgggctg tggtcggcgg
gaaagctggc ggagggcctg 3840aagctgggac gggcggcggt gcgggcgggc
gcggaggccg aaccggtgtg gcgtctgcac 3900gcccagctcg cgctcgccgg
gaaactcgcg aacctccgcg agttcgacga ggccgaggcg 3960ttgatcaacg
aggcggaagc gggcctgcgc ggactgcccg cgccgatctg gacggccgcg
4020acggcggtga tgcggtcccg gttgctgctc caggcggggc ggatcgggga
ggcgcgtcgg 4080gaggcggcgc tggccaccac cgccgtggag ggggacgcgg
tgccgatgct gcggcctctc 4140gcctacgcgg tgctcagcac cgcctccttc
tacatggggg acctgcccgc cgcgatcgag 4200tacctcaggc gggggcagcg
ggacgcggac cgccacgtgg tcctcgactc ggtgcagtac 4260tcgtgggcgg
aagtgctgat cacggtcaag caggaaggcc cgcgggccgc cgcccagctg
4320ctcgcgggca agcaccaccg cctgcccacg cagcgccgcc tctacgtcga
ggtgccgagc 4380gccgccgcct tcctggtcct gctcgcccgc gacgtggacg
accgtgacct cgaacgccgc 4440gtcctcgaca cggtcaacgg gctcgccgcg
gacaacccca ggatccaggt cgtcagcctc 4500accgccatgc acgcccacgc
gctggcgaac agcgctccgg ccgccctggc gctcatcatc 4560gtgcagtcac
gggacccgat ctcggtggcg ctggccaccg aggaactcgc caagctctac
4620gccgcgcagg cccaggcggg gggacggccg gcgacgccgg cccgcgccga
ggaggccgcc 4680accccgccgg cgagctgctg gtcgaccctg tccgacatgg
agcagcggat cgcctacctg 4740gtgagcgtgg gtctgacgaa ccggcagatc
gccaagcagg tccacctgtc cgcgcacacc 4800gtcaactacc acctgcggaa
gatctaccgg aaactgggtt tcaacacccg ggccgagctg 4860gcgcacgccg
cggccacgta ctccggccgg gcggcgatct actccatgag cggcgaccag
4920gactggggcg ccggatccat gaccggcaag gccagctgaa ccgcattccc
ggcgtccgcc 4980ggctgaaccg cgccccggcg tacgccggcc ggttcagccg
gcggacgccg gctggcgtgt 5040ggtggccagc gccggccgga ccgcctcgtg
cgcgatgaag cagcgggtca gttccacccg 5100gctgttgatg tcgagcttgg
agaagacgcg ccgcaggtga ctgtcgacgg tgtgcgggga 5160caggaacagc
gaactcgccg cctcgcggtt ggtcatcccg tccacgatgg cccgcacgac
5220ccgcagctcc gcgctggtca ggctctccca ccccgaccgg ggccggtcgg
ggaccagcgg 5280gcggacgttg tgagccggca ggccacgcag ctcggcctcc
acgcgctcca ggtcgcgtcg 5340cgcgccgcac tcccggtagc cgtccgtcgc
ggcctcgagc agacgggtgg cctcggcccg 5400gtcccgggtg ctgcgggccg
cgtcctccac cgcgccggcc gccgcgagcg tacggccggc 5460gagccggtgc
agatccgcgg cccgcagcag cgccgccgga tcgtcgcgca ggagacccgc
5520ggcgtgttcc gccgccgccg ccagcgactg gacgaacggg ttgccgcggg
cgacgcgccg 5580ggcgacctcc acggcgcgct cggcctccgc gtcgagcccc
gcccggcggg cctggcgtac 5640gagcgtcgcc gcagcggccg gcgcctcggt
gaacagcagc ggatcgggtg cgacctgtcc 5700ggcgacgttg atcagcgtct
gcaccatcat cgccggacgg ccgctggcag cgtggaaccg 5760ggccagcgcc
cagtccatcc gcgccgagtc gtcggcggag gccagccgct cggccgcccg
5820caactggtcg ctggccgtgg cgaggtcacc gtggtgcacg ccgaggtggg
ccaggaccag 5880gcgcgccggc acgcagtcgc ccggccggga gtggtcggcg
gctcgcagcg ccgcctccgc 5940ctcggcgcgt gcctcgtcca gccgtccggc
cgctgccagc agctcggccc ggtggccgcg 6000ccagagcgac tccgagccgg
tgtgactggg ctcctgcgcc agcggtcgta cggtgtccag 6060caccgcctgc
gcctcgtcga gctgatcggc cgcgcccagc gcccggacca gccaggtcca
6120cagcggccgc cggccgggcg cgcagcccgg ggactggtgc cggggctcca
gctcggcgga 6180ggaggcaccg cccaggtgct tcgtggtgtc cgcgagcgcc
cggtccagct tggcgcggtc 6240cagctcgcac acgtcgtggc gggcctgcgt
ccggcgcagg aagccggccg ccaggcggtg 6300gctgccggcg gcccgcatcc
cgtgtcccag ttcgagcacg agctgcgcct cgacgtccgc 6360cgcgaggtcg
cggcggagca tcacctccgc gaggcggccg gcctcggcgg cccgccccgc
6420cccggccagc aggcgcagcg cacgggccag tgctcgtggc gcctcggcgg
atccgttctc 6480caggtgggac acggcggctg ccgccacgtc gtcgcacccg
caccggcccg agcgcggccc 6540cgccgtcgcg gcgggcgtgg ccgcgtccgg
cgcggagcgc gtgacgcgta cgccggcggg 6600ggagtggggc gtcccgggcc
gcggatcggg ccgcccgcgc cggaccgggt cgcccgccgc 6660cggtgccggc
gcggatccgg gctcggcacg ctccggttcc gggtacgcgg cgtggcgaag
6720cgcctctccg agcaccgggt gggcgaaggt cagctccgcg ccgtcgcgtc
gtatcagccc 6780gacccgcacc gcctcgtcga tcgcggcgga cacgtcggcg
gccgagccgt ccagcaggcc 6840cgtcacccgg tcgacgggaa acgtgtggcc
gagccggccg ccggccgcga gcaggcgccg 6900cagcgggggc ggcagctcct
ccagcagccc gcgaacggcg gcgaggacac cgtcgggcag 6960ctcgtcggac
accaccgacg ccgccccgtc cacgatgatc atctggccgg ccttgatgaa
7020cgcgctgaag acgatctcca tcaccttcgg gttgccgccg cagcgggccg
cccagcgcag 7080gacggaggcg tccggccggg cgccgaggat gccggcgcac
aggtcggcca ccgcctcctc 7140gcccggctcg cgcagccgta cccgtaccgc
gacgtgctcg gccagccagt cgacggcgtg 7200ctgagcgatc gacccggcgg
cgaccggccg gcgggccagc agccagagca ccggcgagga 7260cgccaggcgc
ggcacgagcc cgcgcagggc cagggcgctg acgtcgtcga tgcgctgggc
7320gtcgtccagg gcgaccacga gcgggcgccg gcgcgccgcg acctcgacca
gatcgccgac 7380ccggtcgatc agccagaacg ggttggcgcc cggcagggcg
agctgctcga ccgccgcttc 7440gccgggcatc gcgtggcgca ggaagttgac
gagcaggtgt acgggcaccg gctgatccgt 7500gacgcttgcc cgcccggcca
ccactgtcag cccgcgggcc gccgcctcca ggccggtgac 7560cttcagcagg
tgggtcttgc cgatgccgaa cggcccgtcg acgacgacgc agcccccgga
7620tccccgcatg gtggcgtcga gcagttcccc caatgaggac aattcctgcc
cgcgccccgc 7680catgcgattc atgatgacca tcccgttttc ctctgctgaa
tcgtccgacg tgcgccgcga 7740gccgatgtcc caccgcgttc gaccgtccgt
tctggacagt tgaacgccgg atcggggcgg 7800gctactcagt tatacgggat
ctgcggccgt tcgtcggcga cgtcgctggc agcgcgcact 7860actcgcgtga
gtagtgggca gggtgtcagg ccgcgattac tgtcaggcca tgccgggctc
7920ggcgtgccgg cgcggacgaa atggcgacgc cgatggggag atcggcgtcg
tttccgcgcc 7980ggcgcaaaac gtccggaacg gaatcgacta atcgccgctc
gacgcgactg gtccagcgaa 8040tccaggggag tccgagatgc gtgagtgtaa
tggtgaccgc cgtcttgatc gggagacgcg 8100ggcatgaccg tcggatatct
cgggacggtc accgactcgg cgcccgtcga cgccgcgctg 8160cgcgacttct
tcgccgagcg ccgcgccgag gcacgcgagc tcggcgacga cttcgcggcc
8220ctggtcgccg agctggagag ctacgtcctg cggggcggca agcgcatccg
gcccgccttc 8280gcctggctgg gctggatcgg cgccggcggc gacccggagg
acccggtggc gaccgcggtg 8340ctgaacgcct gcgccgggtt cgagctgctg
cacgcgtccg gcctcatcca cgacgacatc 8400atcgacgcgt cgcagacccg
ccgcggccat cccgccgcgc acgtcgcgta cgccgaacgg 8460catcgggcgc
ggcgcttctc cggtgacccg ggaacgttcg gcaccggcac cgccatcctg
8520atcggagacc tcgtcctgat ctgggccgac gtcctggtcc gcgcctccgg
cctgccggcc 8580gacgcgcacg tgcgggtctc gccggtgtgg tcggcggtgc
gctccgaggt catgtacggc 8640cagctgctcg atctgatcag ccaggtgagc
cggagcgagg acgtcgacgc ggcgctgcgc 8700atcaaccagt acaagaccgc
gtcgtacacg gtggagcggc cactgcagtt cggcgcggcg 8760atcgccggcg
cggacgacga cctcttcgcg gcctaccgcg ccttcggcgc cgacgtgggt
8820attgccttcc agctgcgcga cgacctgctc ggcgtgttcg gcgacccggt
ggtgacgggc 8880aagccgtccg gcgacgacct gcgggagggc aagcggacgg
tcctgctcgc cacggcgctc 8940aagcgcgccg acgaacggga cccggacgcg
gcggcctacc tgcgggcgaa ggtcggcacg 9000gacctcgcgg acgaggagat
cgcccgcatc cgcgccatct tccgcgacgt cggcgcggtc 9060gaggagatcg
agcggcagat ctcgcagcgc accgaccggg cgctggccgc gctggaggcg
9120agcagcgcca ccgcccccgc gaagcatcag ctcgccgaca tggcgatcaa
ggccacccag 9180cgggcccagt gatgtccacg gaaccggtga ccgtcgtcgc
ccgcggcgtt ctcgacggcc 9240ggggtgacgg gccgggccgc ctcggcaccg
gccgcgccca cggcaaggcc atcctgctgg 9300gcgaacacgc cgtcgtgtac
ggcgctccgg cgctcgccgt cccggtgccg caactgaccg 9360ccgtggccaa
ggcgcggcgg gccggcggcg acggcggcga cgaggtctcc ttcgccatcg
9420ccgggctgga gagcccggag gtgacgtcgc ttccgaccga cggcctgcaa
catctggtga 9480cggagttccg gcagcgggcc gccgtcaccg agccgatgcg
cgtcgacgtg ctcgtggact 9540gcgccatccc gcagggccgg gggctcgggt
cgagcgccgc ctgcgcccgc gccgcggtgc 9600tggccctcgc ggacgcgttc
gaccgccgcc tcgacgccgc cacggtgttc gatctggtgc 9660agacctcgga
gaacgtggcg cacggccggg ccagcggcat cgacgccctg gccaccggtg
9720cgaccgcgcc gctgatcttc cgcaacggcg tgggccggga actgccggtc
gccatggcgg 9780gcgccgcgcg tgccgcgcga gggtcgggcc cggccggctt
cgacgcggtg ctcgtcatcg 9840ccgacagcgg cgtcagcggc agcacccggg
acgcggtgga gctgctgcgg ggtgccttcg 9900agcgctcccc gcgcacgcgc
gacgagttcg tcagccgggt gaccagcctg accgaggcgg 9960cggcgcacga
cctgctccag ggccgggtcg ccgacttcgg cgcgcggctg accgagaacc
10020accggctgtt gcgcgaggtc ggcatcagca ccgaacggat cgaccggatg
gtcgacgccg 10080cgctcgcggc gggcagcccg ggcgccaaga tcagcggcgg
tggcctgggc ggctgcatga 10140tcgcactggc ccgggaccgc caggaatccg
cggcggtggt gcggagcgtc cagcaggccg 10200gcgccgtccg cacctggacc
gtcccgatgg ggaggttcac cggccatgac gactgaccac 10260cgggcggagc
cgtccgagcc ggcgctcgac cggcccgcga ccgccgtggc ccatccgaac
10320atcgcgctga tcaagtactg gggcaagcgc gacgagcagc tgatgatccc
gtacgccgac 10380agcctgtcga tgacgctcga cgtcttcccg accaccacca
ccgtccggat cgacagcggc 10440gcggcggccg acgaggtcgt cctcgacggc
tcgcccgccg acggcgaacg gcgacagcgc 10500gtcgtcacct tcctggacct
ggtacgcaag ctggccgggc gcacggaacg ggcctgcgtc 10560gacacccgca
actccgtgcc caccggcgcc ggcctggcgt cctcggcgag cggattcgcc
10620gccctcgccc tcgccggcgc cgccgcgtac ggcctcgacc tggacaccac
cgcgctgtcc 10680cgcctggccc ggcggggatc cgtgtcggcc tcccggtcgg
tcttcggcgg cttcgcgatg 10740tgccacgcag gccccggcgc cgggaccgcc
gcggacctcg gctcctacgc cgagccggtg 10800cccgtcgcgc ccctcgacgt
cgcgctggtg atcgcgatcg tcgacgccgg gccgaaggcg 10860gtgtcgagcc
gcgaggggat gcggcgaacc gtccggacct ccccgctcta tcagtcgtgg
10920gtcgcctccg gccgcgccga cctggccgag atgcgggccg cgctgctcca
gggagacctg 10980gacgcggtcg gcgagatcgc cgaacgcaac gccctcggca
tgcacgccac catgctggcc 11040gcccggccgg cggtgcgcta cctggcgccg
gtcactgtcg ccgtgctcga cagcgtgctg 11100cgcctgcgcg ccgacggcgt
ctccgcctac gccacgatgg acgcgggacc gaacgtcaag 11160gtgctctgcc
gccgcgcgga cgccgaccgg gtcgccgaca ccctgcgcga cgccgcgccg
11220agctgcgccg tggtcgtcgc cggaccgggg ccggcggccc ggccggaccc
gggcagccgg 11280ccgtgaccgg cccgggcgcc gtgcgccgcc acgcgccggg
caagctgttc gtcgccggtg 11340agtacgcggt gctggagccg ggccacccgg
cgctgctggt ggcggtcgac aggggagtgg 11400acgtcaccgt ctccggcgcc
gacgcccacc tcgttgtcga ctccgacctc tgcccggagc 11460aggcgtgcct
gcggtggcag gacggccggc tcgtcggcgc gggcgacggg cagccggcgc
11520ccgacgccct cggcgccgtg gtctcggcga tcgaggtggt cggcgaactc
ctgaccggac 11580gagggctgcg cccgctgccc atgcgggtgg cgatcaccag
ccggctgcac cgcgacggca 11640cgaagttcgg cctcgggtcg agcggggcgg
tgacagtcgc cacggtgacc gcagtggccg 11700cgtaccacgg ggtggagctg
tcgctcgaat cgcggttccg gctggcgatg ctggcgacgg 11760tgcgtgacgg
cgccgacgcc tccggcggtg atctggccgc gagcgtctgg ggcggctgga
11820tcgcctacca ggcgcccgac cgcgcggccg tgcgcgagat ggcgcggcgg
cgcggcgtcg 11880aggagacgat gcgcgcgccc tggccgggcc tgcgggtccg
gcggctgcca ccaccgcgtg 11940gcctcgcgct ggaggtgggc tggaccggcg
agccggcgag cagcagctcg ttgaccgggc 12000ggctggccgc ctcccggtgg
cggggcagcc cggcgcggtg gagcttcacc agccgtagcc 12060aggagtgtgt
gcgtaccgcc atcgacgcgc tggagcgggg cgacgaccag gaactgctgc
12120accaggtccg gcgggcccgg cacgtgcttg ccgagctgga cgacgaggtc
cggctcggga 12180tcttcacccc ccggctgacg gcgctgtgcg acgccgccga
gaccgtcggc ggcgcggcca 12240aaccgtccgg cgccggtggc ggggactgcg
gcatcgcgtt gctggacgcc accgccgcga 12300cgcggaccgc gcggctgcgc
gagcagtggg ccgccgccgg ggtgctcccc atgccgatcc 12360aggtccatca
gacgaacggg agcgcgcgat gatcgccaac cgcaaggacg accacgtccg
12420gctcgccgcc gagcagcagg gccggctcgg cggtcaccac gagttcgacg
acgtgtcctt 12480cgtgcaccac gccctggccg gcatcgaccg gtccgacgtc
tcgctggcca cgtcgttcgg 12540cggcatcgac tggccggtgc cgctgtgcat
caacgcgatg accggcggca gcaccaagac 12600cggcctgatc aaccgggacc
tggcgatcgc ggcccgggag accggcgtac cgatcgccac 12660cgggtcgatg
agcgcctact tcgccgacga gtcggtggcc gagagtttca gcgtgatgcg
12720ccgggagaac cccgacgggt tcatcatggc caacgtcaac gccaccgcct
ccgtcgaacg 12780ggcccggcgg gctgtcgacc tgatgcgggc cgacgcgctg
cagatccacc tgaacaccat 12840ccaggagacg gtgatgccgg agggggaccg
gtcgttcgcc gcctgggggc cgcggatcga 12900acagatcgtc gccggcgtcg
gtgtgccggt gatcgtcaag gaggtcggct tcgggctcag 12960ccgcgaaacg
ctgctgcggc tgcgggacat gggcgtccgg gtggccgacg tcgccggccg
13020cggcggcacg aacttcgcgc gcatcgagaa cgaccggcgg gacgccgccg
actactcctt 13080cctcgacggg tggggacagt cgacacccgc ctgcctgctg
gacgcccagg gcgtggacct 13140gcccgtgctg gcctccggcg gcatccgcaa
cccgctcgac gtggtccgcg ggctggcgct 13200cggcgccggc gcggccgggg
tgtccggact gttcctgcgc acgctcctgg acggcggcgt 13260gccggcgctg
ctgtcgctgc tgtccacctg gctcgaccag atcgaagccc tgatgaccgc
13320cctgggcgcg cggaccccgg ccgacctgac ccgctgcgac ctgctgatcc
agggtcggct 13380gagcgcgttc tgcgcggccc ggggcatcga cacccaccgc
ctcgccaccc gttccggcgc 13440cacccacgag atgatcggag gcattcgatg
aacgacgcga tcgccggtgt gcccatgaaa 13500tgggtaggtc ccgtgcggat
ctcgggaaac gtggcgcaga tcgagacgga ggttccgctc 13560gccacgtacg
agtcgccgct ctggccgtcc gtcggccggg gcgcgaagat ctcccggatg
13620gtcgaggcgg gcatcgtcgc cacgctcgtc gacgagcgca tgacccgctc
ggtgttcgtg 13680cgcgccaagg acgcgcagac cgcctacctg gcctcgcttg
aggtcgacgc gcggttcgac 13740gaactgcgtg acatcgtgcg cacctgcggc
aggttcgtcg agctgatcgg gttccaccac 13800gagatcaccg cgaacctgct
gttcctgcgg ttcagtttca ccaccggcga cgcgtccggg 13860cacaacatgg
cgacgctggc cgccgacgcg ctgctgaagc acatcctgga caccattccg
13920ggcatctcgt acggctcgat ctcgggcaac tactgcaccg acaagaaggc
caccgcgata 13980aacggcattc tcggccgggg caagaacgtg gtcaccgagc
tggtcgtgcc gcgggagatc 14040gtccacgaca gcctgcacac gacggcggcg
gcgatcgccc agctgaacgt gcacaagaac 14100atgatcggca cgttgctcgc
cggcggtatc cgctcggcca acgcccacta cgcgaacatg 14160ctgctcgggt
tctacctggc cacgggtcag gacgccgcga acatcgtcga gggctcccag
14220ggcgtgacgg tcgccgagga ccgcgacggc gacctctact tctcctgcac
gctgcccaac 14280ctgatcgtgg gcaccgtcgg caacggcaag gggctcggct
tcgtcgagga gaacctggag 14340cggctcggct gccgcgcctc gcgtgatccg
ggcgagaacg cccggcggct cgcggtcatc 14400gcggccgcga cggtgctctg
cggcgagctg tccctgctcg ccgcgcagac caacccgggc 14460gagctgatgc
gggcgcacgt ccggctcgaa cgcccgaccg agaccacgaa gatcggagcc
14520tgacgatggc cgagagaccc gccgtcggca tccacgacct gtccgccgcg
acggcgcatc 14580acgtgctgac acacgagacc ctggccgcga gcaacggcgc
cgacgtggcc aagtaccacc 14640gtggcatcgg gctgcgggcg atgagcgtgc
ccgccccgga cgaggacatc gtgacgatgg 14700ctgctgccgc cgccgcgccg
gtggtcgccc gccacggcac cgaccggatc cggaccgtcg 14760tgttcgccac
ggagtcgtcg gtcgaccagg cgaaggcggc cgggatacac gtccactccc
14820tgctcggcct cccctcggcc acccgggtgg tcgagctgaa gcaggcctgc
tacggcggta 14880cggcgggact gcagttcgcc atcggcctgg tgcaccgtga
cccgtcgcag caggtcctgg 14940tgatcgccag cgacgtgtcg aagtacgcgc
tgggtgagcc cggcgaggcg acccagggcg 15000ccgcggcggt cgccatgctc
gtcggcgcgg acccggcgct ggtacgcgtc gaggacccgt 15060cgggcatgtt
caccgccgac gtcatggact tctggcggcc gaactaccgc accaccgccc
15120tggtcgacgg gcacgagtcc atctccgcct acctgcaggc gctggagggc
tcgtggaagg 15180actacaccga gcgcggcggt cgcaccctgg acgagttcgg
cgcgttctgc taccaccagc 15240cgttcccgag gatggccgac aaggcgcacc
ggcacctgct caactactgc gggcgcgacg 15300tcgacgacgc gctggtggcc
ggggccatcg ggcacaccac cgcgtacaac gccgagatcg 15360gcaacagcta
cacggcgtcg atgtatctcg ggctcgcggc actgctcgac accgccgacg
15420acctgaccgg ccggaccgtc ggcttcctca gctacgggtc cggcagcgtc
gccgagttct 15480tcgccggcac tgtcgtgccc gggtaccgcg cgcacacgcg
acccgaccag caccgcgcgg 15540cgatcgaccg gcggcaggag atcgactacg
cgacgtaccg ggagttgcac gagcacgcct 15600tcccggtcga cggcggcgac
tatccggcgc cggaggtgac caccgggccg taccggctgg 15660ccgggctctc
cggtcacaag cgcgtctacg agccgcgata ggaccggcca cgccggccgc
15720cctgaccgaa cgaaccatgc ttggaggatc gatgtccgga actcccgagg
tggccgagct 15780ctactcgacc atcgaggaat cggcccggca actggacgtg
ccgtgttcgc gcgaccgggt 15840ctggcccatc ctgtccgcgt acggcgacgc
gttcgcccat cccgaggcgg tggtcgcctt 15900ccgggtggcg accgcgctgc
gtcacgcggg cgagctggac tgccggttcc ggacgcatcc 15960ggacgaccgg
gacccgtacg cctcggcgct cgcccggggc ctcaccccgc gcacggacca
16020ccccgtcggc gcgctgctct ccgaggtcca ccggcgctgc ccggtggaga
gccacggcat 16080cgacttcggg gtggtcggcg gcttcaagaa gatctacgcg
gccttcgccc cggacgagct 16140gcaggtggcc acgtcgctcg ccggcattcc
ggcgatgccc cgcagcctcg ccgcgaacgc 16200cgacttcttc acccggcacg
gcctcgacga ccgggtcggc gtgctgggat tcgactaccc 16260ggcccggacc
gtgaacgtct acttcaacga cgtgccgcgt gagtgcttcg agccggagac
16320catccggtcg acgctgcgcc ggaccgggat ggccgagccg agcgagcaga
tgctccggct 16380cggcaccggg gcgttcgggc tctacgtcac gctgggctgg
gactccccgg agatcgagcg 16440gatctgctac gccgcggcga ccacggacct
gaccacgctt ccggtacccg tggaaccgga 16500gatcgagaag ttcgtgaaaa
gcgttccgta cggcggcggg gaccggaagt tcgtctacgg 16560cgtggcgctg
acccccaagg gggagtacta caaactcgag tcgcactaca aatggaagcc
16620gggcgcggtg aacttcattt gaacagcggc cggttccgcc gcccgggcgg
cggaaccggg 16680atcaatgcct gttcgctcgg gttcaacact ggcgcgctcc
gctaaagtgc gaacatgacg 16740actggactgt ccagtgtgtg ggcccgggtg
aagaactggg tcgtcgcgtt ggctgtggcg 16800gcggtgctga tgatcagcgc
gctggccggt gaccatcctg cccccgaggg cctcggtctg 16860ctcggcttcg
cgctggtggc ggcgagcggc ctggcgctgg ccgccagtcg tcgggccccg
16920atcgccgtgc tggtcgccac cgggctgtgc gtggtgggct acaacgcgat
cggcttcggg 16980gtgcccgcca tcgcgtacct gttcgcggtc tacgcggcgg
tccgggccgg gcaccggctc 17040gtcacgctcg gggcgagcgc cgccctgctc
gtcgtcctgc cgctggcgat catggtctcg 17100cccgcggacg gcgccctcaa
ggaggcgctc gcgcagtcgc ggggcgtgct ggaactggcc 17160tggctgatcg
ccgcggcggc ggccggtgag gcgctgcggc aggccgaacg gcgagcggac
17220gaggcggaac ggacccgcga ggagaccgcc cggctgcgcg ccacccagga
gcggctgcac 17280atcgcacggg agctgcacga ctcgctcacc caccagatct
cgatcatcaa ggtgcaggcg 17340gaggtggcgg tccacctggc ccgcaagcgg
ggcgagcagg tgccggagtc gctgctggcg 17400atccaggagg ccggccgggc
ggcgactcgc gagctgcgcg cgaccctgga gacgctgcgt 17460gacctgacca
agtccccgtc gcacgggctc gaccacctcc cggagctgct ggccggggcc
17520gagaagatcg gcctggccac cacgctgacc atcgagggcg accagcggga
cgtgccggag 17580gcggtgggcc gcaccgcgta ccggatcgtg caggagtcgc
tcaccaacac cgcccggcac 17640gcctccgccg cggccgccgc ggtccggatc
gactaccgcc cggacgcgct gagcatccgg 17700atcgacgacg acgggacggc
ccggccgggc gccgccccgg tgcccggcgt cgggctgctg 17760gggatgcacg
agcgcgtcct cgcgctgggc ggccggctgc gggcggaacc ccgcaccggc
17820ggaggcttca ccgtccaggc cgaactcccg gtggtgcgcg tcccatgatc
aggatcatgc 17880tgctcgacga ccagccgctg ctgcgcagcg ggttccgcgc
gctcctcgac gccgaggacg 17940acatcgaggt ggtggccgag ggcgggaacg
gccgggaggg cctggcgctg gcccggcagc 18000acctgcccga tctcgccctg
atcgacatcc agatgccggt catggacggc gtcgagacga 18060cccggcagat
cgtcgcggat ccggcgctgg ccggggtacg cgtcgtcatc ctcaccaact
18120acggcctcga cgagtacgtc ttccacgcgc tgcgcgccgg cgccaccggc
ttcctggtca 18180aggacatcga gccggacgac ctgctgcacg ccgtgcgggt
cgccgcgcgc ggtgacgcgc 18240tgctcgcgcc gtcgatcacc cggatgctga
tcaacaggta cgtgtcggag ccgctctgcg 18300cggacgtcac gcccggcatg
gaggagctga ccaaccggga acgcgaggcg gtcgccctgg 18360ccgcccgggg
cctgtccaac gacgagatcg ccgatcgcat ggtgatcagc ccgctgaccg
18420cgaagaccca cgtcaaccgc gccatgacca agctgcaggc ccgcgaccgc
gcccagctgg 18480tggtgttcgc ctacgagtcc ggcctggtgt cacccggcaa
tcgctgaccg ggcagcccgc 18540ccggtctgtc gcctcggcag tgctgcggct
gcggtatgcg gctgctcccg gcgcagacgc 18600cggagcccgt ggataccgtc
accgcagtag atcgatcgat tgtctccttc ggcatgacga 18660cccgtagcgg
ggtcgttacc tacgctggcg cagatgcctg ttcccgcagc cgaaggggct
18720tccatgttca tccgtcgttt gctcaccgcc gccgcagccg gcgtcctcgg
tgggctcgca 18780ctcgtcgcac cggcggccgc gcaggtgacg gccgccgacg
gtgacggtgg ttccggccgc 18840gccggatccg tgctggcgct cgcgctcgcg
ttgctcggcc tcgtcctggg cgggtgggcg 18900ttgcgctccg cggggcgcgg
cggcggtcgt ggcaacgcga tcgccgcgct ggtgctcgcg 18960gtggccggcc
tgatcgccgg cgtggtcgcc ctggccggct ccgacggtgg tgtcggcagc
19020ggcaacggcc gtggtggcgc catcgtggcc gtcgtgctgg cgctgatcgg
gatcgccgtc 19080ggcggcctgg cattcacccg ctcccggcgc gccgcctgac
cggcgctgcc gaccgaacac 19140cccggtgacc caaccgaacc cgaaggggag
tcccatgcgc aaagtgttcg ccggactggc 19200agcgttcctg ctgctcgtgc
tcgtggtgca gttcttcctg gccgccagcg gcgcgttcag 19260caacgaggcc
aacgaggagg cgttccgccc tcaccggatc ctgggcctgg ggagcatcct
19320cgtcgccgtg gtgctgacgg tggccgccgc ggtgatgcgg atgcccggcc
ggatcatcgg 19380cctgtccggc ctggtcgccg ggctgggcat cctgcaggcc
ctgatcgcgg tcatcgccaa 19440ggcgttcggc gactcggccg gtgactcggc
cgtcggccgg tacgtgttcg gcctgcacgc 19500ggtcaacgga ctggtgatgg
tggccgtcgc ccgcgtcatc ctgcgcagcg tccgggcggc 19560gccggacacg
accaccacgc ccggcgtgga cacgacggtc accggtccgg cggccgactc
19620ggcgcgaacg gcgtcatgag cacgctccaa tggatcctcg tggaccacgt
cgtggcgctg 19680ctcggtgtcg cgacgtggtt cgcaacgggt gtcacggcag
ctctcggccg ccaccggatc 19740gcgttggcgc tcctcggcgc cgcggtgctg
gtgacagtcg cccgcctggg caccgtggcg 19800ctgctggccg accgcggctg
gtggttcgtc caggagaagg ttctgctggg gctgccgatg 19860ctcggcgccg
cggggctcgt cgcggtgctc ctggccggcc cgcgcctgct cgcggcccgg
19920cagtcaccgg cggcggacct gccggccggc gcgctggtcg cggtgctgac
cgccggcttc 19980gccgcgctgg ccggcctggt ggtgacgttc accgccgggt
acccgctgac gtggagcacc 20040gcgctgatcg ccgtcgccct cgtctgcgcc
gccgcgctgc tcaccgcgcg ggtggtcgga 20100cgacccgccg ccccggccgc
ggaggccggc tccccggagc acacgccggc ggcggccggg 20160cccacggcgc
tgtcccgccg ccggttcctc ggcgtggccg ggggagtggt cgcggcgggc
20220gccggcgcca ccggcgtcgg cctgctcttc cgcgacccgg aggcgatggt
caccggaggc 20280ggccccggac acgccggtgg cgcccgcccc aaggtctccg
tggcggacct gcgcggcccc 20340ggcgctccgg cggcgggcgg cacggcgcga
cgccacgtgc tcaccgcccg gacgggcacc 20400gtcacgattc cgtccggacg
tccgatcgac gcctggagct acgagggccg cctgcccggg 20460ccggccatca
ccgcgaccga gggcgacctg atcgaggtga cgctccgcaa cgccgacatc
20520gaggacggcg tcaccgtgca ctggcacggg tacgacgtgc cgtgcggcga
ggacggcgcg 20580ccgggcgcca cgcagcacgc ggtgcagccc ggcggcgagt
tcgtctaccg gttccaggcg 20640gaccaggtgg ggacgtactg gtaccacacc
caccaggcgt cgcaccccgc cgtgcgcaaa 20700gggctgtacg ggacgctcgt
cgtgacgccg cgcgaggacc ggccggaagc ggagcgcggg 20760ctggacctga
cgctgccggt gcacacgttc gacgacgtca cgatcctcgg cgaccaggag
20820ggacgcgccg tccacgacgt ccgccccggc cagccggtgc gactgcgtct
gatcaacacc 20880gactccaacc cgcactggtt cgccgtcgtc ggctcgccct
tccgcgtggt ggccgtcgac 20940ggccgcgacc tcaaccagcc gggcgaggta
cgcgaggtcg ggctccgcct gcccgccgga 21000ggccggtacg acctgaccct
ggccatgccg gacgccaagg tcacgctgct gctcgacaac 21060gactccgacc
agggcgtcct gctgcgcccg ccgggcgtcg gcggtggtga ccgcccgctg
21120ccggacaccg ccgactggcc cgagttcgac ctgctgggct acggcgagcc
ggcgcccgtg 21180ccgttcgacg ccgacgacgc cgaccgccac ttcaccatcg
tcctcgaccg ggccctggcc 21240atggtcgacg gcaagcccgc gtacgcccag
accgtcgacg gtcgcgcaca tccctccgtc 21300cccgaccagc tcgtccggga
gggggacgtc gtgcgcttca cggtggtcaa ccggagcctc 21360gaaacccacc
cgtggcacct gcacggccat ccggtgctga tcctgtcccg cgacggccgg
21420ccgtactccg gcagcccgct gtggatggac accttcgacg tgcggccggg
agaggtgtgg 21480gaggtggcgt tccgggcgga caatccgggt gtctggatga
accactgcca caacctgccg 21540caccaggagc agggcatgat gctgcggctc
gtctacgacg gtgtcaccac gcccttcgcc 21600agcacgagcc acgcacactg
aggggactcg catgaccgca gacctgcacg gcctggccag 21660cgtccgctac
atcgtcgacg acgtgtcggc ggcgatcgag ttctacacca cccacctggg
21720tttcacggtg tcgaccgcgt tcccgccggc cttcgccgac gtggtgcgcg
ggccgctgcg 21780gctcctgctg tccgggccga ccagctcggg cgcccgggtc
accccggcgg acgcggccgg 21840gtgcgggcgc aaccgcatcc acctgatcgt
cgacgatctc gacgccgaac gggagcggct 21900ggagcgcgcc ggggtgacgt
tgcgcagcga cgtcgtggcc gggccgggcg gccgtcagtt 21960cctgatcgcc
gacccggcgg gcaacctggt cgaggtgttc gagccggcag cccgcggctg
22020aaccgccgac ggacgccctc ccacctcgcg acgcccgaag cccgacacct
ggccgcgtcg 22080cggccacgat caccgtggcc gcgacgcggt gacggggtgc
cttaccgggg cggggtgggc 22140gcggcgagcc gcgcggccag gatggagatg
atcacggcgc cggcgatcac gtgggtgccg 22200gcgaggacga gctgcgtcga
caccggggtg tcccgggcga aggcgggcgc ggcgagggac 22260agcacggtga
acgcgacggt gccggccacg aaggcacgca cgggccgccg ggcccgccgc
22320gccacgacca ccgccaggac gattccgccg atcgaccaga gcacgacgct
gcgggcgatg 22380gcccccaccg ggatcgcctg cgcctgctcc tcccagacgc
cggccgcctc catcggtacg 22440ccgaagcccc gggcggcgag cgtgaacgcc
tccgcggcca cggccccggc gagggtggcc 22500agcacgccga ccagccacac
cggagcggtg gccggcgacc aggtgggccg tgccgcgacg 22560ggagttcggg
gagtggcctc atccacggcg tcgcctccgg tcgggtgcct cgatgtgttc
22620tcgggagaat gcggggacgc cacgacggca gtcaacatgg acagttgaac
gccctggcgt 22680cacgggcggt tcccgcgccg gcccgccgcc tcggccgcgg
cggcggccgt gccgtcggcg 22740agcagggaga ccagcaggtc gcccaggatc
cgtgggccgt gctgggtgag gacggactcc 22800aggtggaact ggacggaacg
gaatcccggg ccgcgcagcg cgtgcacgtc cccgctgtcc 22860gggctgcggc
tgatctcgat cgggccccgc cggccaccgg ccaccacgtc gtgcgcggag
22920cgggcggtgt aggtgttgta gaaccccacg agttccggcc ggccgaacag
gtcgatccgc 22980ttctgcacac cctggttggg caccgcgcgc cgggcgaggg
ggaaccccag ttcggcggcg 23040agcacctggt ggcccaggca gatggacagg
aacggcaccg ttccggcgag caggtcgcgg 23100gtgagcccgc gcagggtccg
catacgcggg tcggtcaggt cgcccgggtc gccggggccg 23160ggaccgacga
cgacgaggtc gtgtccgtcc ggccgcagcc ggctgtcgaa ccgggcgatg
23220ctcgaccgca gcccgagggc ccgcaactgg tggtcgagca tggccatgaa
cgtgtcctcg 23280ttgtcgacga cgagcacgcg gcgtccggtc agcgccgggt
tcggggtgcg ccgctccgcg 23340ccgtcgagcc agaacctcga cagtgtggtg
ttgcgctcgc gcaacgcccg ccgtacccgg 23400gggtcggtgg ccagggacga
acgagcccgc gcggccgtgg tccgcccgcc gtccgggccg 23460tccgggtcga
cgccgaggcc gagcgccgcg cgcatggcgc ccgccttggc ccgcgtctcg
23520gccacctccg actccggctt ggagtcccgc acgagggtgg cgccgacgcc
caggcgcagc 23580gtgcccgcgt cgtcgatctc ggcggtgcgg atcatgatgg
ccgagtcgag cgtacggctg 23640ccggccgagt cacggcccat caacgcgagc
acgccgccgt agtagccgcg gccggtcgtc 23700tcgtggcggg tgatgacccg
gaacgcgttc tcgatcgggc tgccggtgac cgtcggcgcg 23760agcagggtct
cccgcagcac gtcgcgcacg tccaggtcgc tgcggccggt caggatgtac
23820tcggagtgcg tcacccgcgc catttccttg aggaacgggc cgtgcacctg
gccgccggag 23880gcgcacatcc gcgccatcat tttcagttcc tcgtcgacga
ccatgtagag ttcgttagcc 23940tctttcgggt cgttcaggaa ttccagcaga
ccggaaacgg ccgggccgtt cggggggtgc 24000cggtaggtcc cgctgatggg
attcatcgag acggttccgt cgatcatgct gacgtgtcgt 24060tccggtgacg
cgccgatgaa cgtgccggcg ccggagtgga acagaaacgt ccagtaggaa
24120cccagttcgc cggtcagcaa ccggcggaag agcgccagtt ccgtggcgat
cgagtagtcg 24180gccagccgcg cggtgaaggt gcgccggatg acgaagttgg
atccggcgcc cagcccgatc 24240tcgtcaccca ccacccgctt gacgatcgcg
gcgtagtcct cgtcgctgag gtcgaagtcg 24300gcgtcggtca ccggcacacc
gcgttcgggc aggcccgcca gcgcctgtcc gcggtcgagc 24360ccgaactgct
cgtggacgcg catcgcgagc agcggcgcgc cgtcgtcgtg gcagtcgaac
24420ccccgttcgg tgacctgccg gtacggcacc gccacgagca ggtcgtgccg
cgcgccggtc 24480gccggctcgg tgggcagggg cagctcgccg agagtgtcca
cgtcgcacac ctcgccggtc 24540agaacctcca cgtacgcgca cccggccgcg
ccgggccggt gcagcagggc gaaggcgcgc 24600ccgtcgccgc cgagaccgga
cagcagatcg gggaatccgg tcacgttcga ttccgtcccg 24660tccatgtcgc
tccctttgcc tgagagatcg cctgtcgata ctgcgtccgg caaaaggcgt
24720cgcacatgac gtgaagtcgc cgacggcatc acgtgtttcc ggtaacgcgc
cgacgttatg 24780gcgtgaacga ctgaatcggc gggctactac tcgggcgagt
agtgcccacg cagatcgacc 24840gcgattactg tcgaccgcaa tgccgatacg
acgagggcgg tgaagacgac tgtggacgtg 24900ctggtccaga aatacggggg
cacctcgctg cagaccctcg accgcgttcg gcacgccgcg 24960ctgcggatcg
ccgaggcgcg gcggcacggc tccgccgtga cagtggtcgt gtcggcgcgc
25020ggcagccgga ccgacgacct gctgcggctg gcggccgacg tcggcgccgc
gggtccgtcc 25080cgggaactcg accagttgct cgcagtcggc gagtccgagt
cggcggcgct gatggcgctg 25140gcgttgaccg ggctgggagt gccggccgtc
tcgctgaccg ggcaccaggc ggagatccac 25200accaccgacc ggcacggcga
cgcgctgatc tcgcggatcg gggcggcgcg ggtggaagcg 25260gcgctgggcc
gtggcgaggt cgccgtggtc accggattcc agggcatcga ccgggccggt
25320gacgtcgcca cgctggggcg cggcggctcc gacacgacag cggtggcgct
cgcggcccgg 25380ctccgcgcgt cggcgtgcga gatctacacc gacgtggacg
gcgtcttcag cgccgacccc 25440cgcatccttc cggcggcgcg ttgcctgccg
tgggtggagc ccggcgtcat ggcggagatg 25500gcgttcgccg gcgcgcgggt
cctgcacacc cgatgcatcg agctggccgc catggaaggg 25560gtcgaagtgc
gcgtgcgcaa cgcgtcgtcg caggcgcccg gaacgatagt cgtggaccgg
25620cccgacgacc ggccgctgga gacccggcgg gccgtggtgg cggtcaccca
cgacaccgat 25680gtcgtccgcg tgctggtgca ctgccgcgac ggccgccggg
acatggcacc cgacgtgttc 25740gaggtgctgg ccgcccatgg ggcggtggcg
gacctggtgg cccggtccgg gccctacgag 25800agcgagttcc ggatggggtt
caccatccgc cgcagccagg ccgaagcggt gcggaccgcg 25860ctgcacgacc
tcaccgcgtc cttcgacggc ggggtccact tcgacgagaa cgtcggcaag
25920gtgtccgtgg tcggcatggg cctgctcagc cgccccgagc acacggcccg
gctgatggcg 25980gcgctggccg cggcggggat ctcgacgagc tggatctcca
cctcccagat gcggctgtcg 26040gtgatcgtgt cgcgggaccg caccgtcgac
gccgtcgaag ccctgcaccg cgcgttccgc 26100ctggaccggt ccgagccggc
ggacgccacg tccctgacct cccgccgttc cgccaccgcc 26160tgagagaggt
aggaaaccgt ggccgtactc aacgcttcgt tcgctcgtgg cctgcgtctg
26220cgccgactgt tccgacgcgg cgacggacgc ctgctcgtcg tcccgctcga
ccactccgtc 26280accgacgggc cgctgcgccg cggcgacctg aactcgctgc
tcggtgagct cgccggcacc 26340ggcgtggacg ccgtggtgct gcacaagggc
agcctgcggc acgtcgacca cggctggttc 26400ggcgacatgt cgctgatcgt
gcatctgagc gtgagcaccc ggcacgcccc ggacccggac 26460gcgaagtacc
tggtcgcgca cgtggaggag gcgctgcggc tgggcgccga cgcggtcagc
26520gtgcacgtca acctcggctc accgcaggag gcgcggcaga tcgccgacct
ggcggcggtg 26580gcgggggagt gcgaccgctg gaacgtcccg ctgctggcca
tggtgtacgc ccgcgggccg 26640cagatcaccg actcccgggc accggagctg
gtggcgcacg ccgcgacgct cgccgcggac 26700ctcggcgccg acatcgtcaa
gaccgactac gtgggcacgc ccgagcagat ggccgaggtg 26760gtgcgcggct
gcccgatccc gctgatcgtg gccggcggcc cgcgctcggc cgacactccg
26820acggtgctcg cctacgtctc ggacgcgctg cgcggcggcg tggccgggat
ggccatgggc 26880cgcaacgtgt tccaggccga gcagcccggc ctgatggccg
ccgccgtggc acggctggtg 26940cacgagccac ggcacgtgcc ggaccggtac
gacgtcgacg accggctcgc ccttacgtcc 27000tgagactccc tgaccgtcca
ccgaggagaa acccgtgaag ctgtgctggc tggacatccg 27060taacgtcaac
ggcgccaagg aggcaatcgt cgaggaggcg gtccaccagc gggtggacgc
27120cgtcgtggcg gccgatccgg ccgacctgga gacgcttccc ccgacggtga
agaaggtgct 27180gttcccgcag ggcgggccgc tgccggagaa gctggaaccg
gccgacctgg tgatcgtcga 27240gccggcccgg cacggcgagc ccgccgagct
ggcggcccgg tacccggagg tggagttcgg 27300ccggttcgtc gagatcgtcg
acgcggacag cctggaggac gcctgccggt ccgcgcgcca 27360cgaccggtgg
agcctgctgt acttccgcga ccccaccaag atcccgctgg agatcgtgct
27420ggcggccgcg gcgggcgcgg agggcagcat catcacccag gtcgccgacg
tcgaggaggc 27480ggagatcgtc ttcggcgtcc tggagcacgg ctcggacgga
gtgatgctgg cgccccgcgc 27540cgtgggggag gccaccgagc tgcggaccgc
cgcggtgagc acggcggcgg acctgtcgct 27600cgtggagctg gaggtcaccg
gcatccggcg ggtgggcatg ggcgagcgcg cctgcgtcga 27660cacgtgcacg
aacttccgtc tggacgaggg catcctggtc ggctcgcact ccaccggcat
27720gatcctgtgc tgcagcgaga cgcatccgct gccgtacatg ccgacccggc
cgttccgggt 27780caacgccggc gcgctgcact cgtacacgct ctccgccggc
gggcggacca actacctcag 27840cgagctggtc tccggcggcc gggtgctcgc
cgtggactcg caggggaagt cccgcgtcgt 27900cacagtggga cgggtcaaga
tcgagacgcg tccgctgctg gcgatcgacg cggtctcccc 27960ctccgggaca
cgcgtcaacc tcatcgtcca ggacgactgg cacgtgcgcg tgctcgggcc
28020gggcggcacc gtgctcaacg tgaccgagct gaccgccggc acgaaggtgc
tcggttacct 28080gccggtggag aagcggcacg tcggctaccc gatcgacgag
ttctgcatcg agaagtgaca 28140ggcggcggga aggggagcgg gcgatgaccg
cgcagccggt gctggacttc cacgtacgcc 28200tggcgccccg gcccggggcg
cgggagcggc tgctcgccgc gctgcgcgag tgcgggctgg 28260cgcgggcggt
ggtgtgcgcg ggcggcacca tcgacctgga ccggctgtcc cgccagctcg
28320tcaccggcgg ccacgtcgag accgacgccg acaacgacgc ggtggcggcg
gcctgcgccg 28380gcaccgacgg ccggctggtg ccgttcttct tcgccaaccc
gcaccggccg gccgaggcgt 28440accgggcccg cgccgccgag ttccgcggcc
tggagatctc acccgccgtc cacggcgtcg 28500ccctgaccga cccgcgggtc
gccgacctcg tggccgtggc ggcggagttc gaccatccgg 28560tgtacgtggt
ctgcctggac cgacccggcg cgggcgtggc cgacctggtc ggcctgagcc
28620gccggttccc gcaggtgagc ttcgtgctcg ggcacagcgg cgtcggcaac
atcgacctct 28680acgccctgac cctgatccag gacgagccga acatctcgct
ggagacctcc ggcggctaca 28740cctgcgtggc cgaggcggcg ctacgccgcc
tcggcgacga ccgggtggtg ttcggctccg 28800agtacccgct gcagcacccg
gccgtggaac tggccaagtt ccaggcgttg cgactgccgc 28860cggagcggtg
gcggcggatc gcctgggaca acgcgcatcg actgctagga gaggagaagc
28920ggtgagcgag ccaagttcga gcctgccccg gctcggccag tggcacggcc
tcgaggacct 28980gcggcgcctc caggagaagc aactggcgga gacgttcacc
tgggcggccc ggtcgccgtt 29040ctaccgggcg cggctggcct ccggcgcgcc
gccggtgacg cccgccgacc tggccgacct 29100gccgctgacc accaagcagg
acctgcggga caactacccc ttcggcatgc tcgccgtgcc 29160ccgcgaacgg
ctggcgacct accacgagtc gagcgggacc gccgggaagc ccaccccctc
29220ctactacacc gcggaggact ggaccgacct ggcggagcgc ttcgcccgca
agtggatcgg 29280catgtccgcc gacgacgtct tcctggtccg cacgccgtac
gcgctgctgc tgaccgggca 29340tctcgcccac gccgcagccc ggctgcgtgg
ggccacggtg gtacctggcg acaaccggtc 29400gctggcgatg ccgtacgccc
gggtggtccg ggtgatgcac gacctggacg tcacgctcac 29460ctggtcggtg
ccgacggagt gcctgatctg ggccgccgcg gcgatcgcgg ccgggcaccg
29520gcccgacatc gacttcccgg cgctgcgcgc gctgttcgtc ggcggcgagc
cgatgaccga 29580cgcccgccgg cggcggatca gccgcctgtg gggggtgccg
gtcatcgagg agtacggctc 29640gacggagacc ggcagcctgg ccggggagtg
ccccgaggga cgcctgcacc tgtgggccga 29700ccgggcgctg ttcgaggtgt
acgacccgga caccggcgcc gtccgcgcgg acggcgacgg 29760ccagctcgtg
gtcacgccgc tgttccggga ggcgatgccg ctgctgcggt acaacctgga
29820ggacaacgtg tcggtctcct acgacgactg cggatgcggc tggaagctgc
ccaccgtgcg 29880ggtgctcggc cggtcggcgt tcggctaccg ggtcggcggc
accaccatca cccagcacca 29940gctggaggaa ctggtcttct ccctgccgga
ggcgcaccgg gtgatgttct ggcgggccaa 30000ggcggagccg gcgctgttgc
gggtcgagat cgaggtggcc gccgcgcacc gggtcgccgc 30060cgaggcggag
ctgaccgccg
cgatccgggc cgccttcggc gtggacagcg aggtcaccgg 30120cctggcgccg
ggaaccctga tcccgctcga cgcgctgacc agcatgccgg acgtggtgaa
30180gccacgcagc ctgttcggtc cggacgagga ctggagcaaa gcgctcctct
actactgagg 30240gaaccgacat gccgcagatg agggtcgccg tggccggcgc
cggcatcgcc gggctcgcct 30300tcgccgccgc cctgcgccgg accgggatcg
actgccacgt gtacgaacag gccgaccagc 30360tcatggaggt gggcgcgggc
gtgcaggtcg cgccgaacgc cacccggctg ctgcaccggc 30420tgggcctgcg
tgaccgcctg cgtacggtgg ctgtcgcgcc gcaggcgatc gagatgcgcc
30480gctgggacga cggcacgctg ctgcaacgca cccagctggg cagcgtgtgc
ggacgccgct 30540tcggcgcgcc gtactacgtg gtgcaccgcg cggacctgca
cagcagcctg ctgtcgctgg 30600tgccgccgga ccgggtgcac ctgggcgccc
gcctcaccgc cgtgacgcag accgccgacg 30660aggcgtacct gcacctgtcc
aacggcacca cggtcgcggc ggatctcgtc gtgggcgccg 30720acggcatcca
ctcggtcgcg cgggagcaga tcgtggcgga ccggccgcgc ttctccggac
30780agtccatcta ccgcgggctg gtgccggccg agcgggtgcc gttcctgctc
accgaacccc 30840gggtgcagtt gtggttcggg ccggaccagc actgcgtctg
ctacccggtg tccgccggcc 30900ggcaggtgag cttcggcgcg acggtgcccg
ccaccgactg gcggcaggag tcgtggtcgg 30960gccggggcga cgtgacgcaa
ctcgcggccg cgtacgcggg ctggcacccg gacgtcaccc 31020ggctgatcgc
cgcggccgac cgggtcggca ggtgggcgct gcacgaccgg gacagcatcg
31080accggctcag cgcgggacgg gtgaccctga tcggcgacgc cgcgcacccg
atgctgccgt 31140tccaggcgca gggcgcgaac caggccgtcg aggacgcggt
ggtgctcgcg gtctgcctgg 31200ccggcgtgga accggcgggc ctgggcgccg
cgctgcgccg ctacgaacgg atccgcctgc 31260cccggaccac ccggatccag
cggcagtccc gggccaacgc cgagatgttc cacctggccg 31320acggcgccga
ccagcgccgc cgggacgtcg ccgcacaatc ctcgtccggc ctggaccgcc
31380acgaatggct cttcgggtac gacgccgaga aagccaccac gaccagcggg
agcgcctgat 31440ggaactgacc ggaatcgagt cgaaggtcgc cctggtcacg
ggcgcggggc agggcatcgg 31500cgccgccgtg gccggtgtcc tggcgagggc
gggcgcgcag gtggcggcgg tggaccgcaa 31560cgccgaggcg ctgaccaccg
tcgtgacgaa gctcgccgcc gagggcgact cggcgcgcgc 31620ctactgcgtc
gacgtgtgcg acagcgaggc ggtggacgcg ctggtgcgcc gggtcgagga
31680cgagatgggg ccggtcgcca tcctggtcaa cgccgccggc gtgctgcaca
ccggacgggt 31740cgtcgagctg tcggaccggc agtggcgccg gaccttctcg
gtgaacgccg acggcgtgtt 31800ccacgtgtcc cgggcggtgg cgcggcggat
ggtgggccgc cgtcgtggcg cgatcgtcac 31860cgtggcgtcg aacgccgccg
gggtgccgcg taccgagatg gccgcgtacg ccgcctccaa 31920ggccgcgtcc
gcgcagttca cccgctgcct ggggcttgag ctgtccggct acggcatccg
31980gtgcaacgtg gtctcgcccg gctccaccga cacccccatg ctgcgggcca
tgctcggcga 32040gggcgccgac ccgagcgcgg tgatcgaggg cacgccgggc
gcgtaccgcg tcggcatccc 32100gctgcgcaag ctggcccagc cgcgcgacgt
ggccgaggcg gtcgcctatc tggtgtccga 32160ccaggcgggc cacgtgacca
tgcacgacct gtacgtcgac ggcggcgcgg ccctgcacgt 32220gtgacgccct
cgcacggaaa ccggaggcga gaaccgatgg ccatgacccc gatcgcgccg
32280taccgcatgc ccggcgacgg cgacctgccc ggcaccgcgc tgccctggcg
tccgcacccg 32340gaccgggccg ccgtgctggt gcacgacctg caacgctact
tcctgcgccc gttcgaggcc 32400ggggagtccc cgatggccga actgctcccc
aacgtcgcga agctgctcgc cacggcgcgg 32460gcggccggcg tgccggtgct
gtacaccgcg cagcccggcg gcatgagccg gcaggaccgc 32520gggttgctgc
acgacctgtg gggccccggc atgagcagcg ccgaggacga ccggggcatc
32580gtcgacgacg tcgccccgca gccgggcgac acggtgctga ccaagtggcg
ctacagcgcg 32640ttcttccgca gcgacctgga ggagcgactg cgcggtgcgg
gacgggacca gctcgtggtc 32700tgcggcgtgt acgcgcacat ggggtgcctg
atcaccgcct gcgacgcgtt cagccgcgac 32760atcgaggcgt tcctggtggc
ggacgcgctg gccgacctat cgcgcgagga ccacctgatg 32820gcgctgcgct
acgccgcgga ccgctgcgcg gtgccgttgt ggacggcgga tgtgctggac
32880gggctggcgg acgccgccgg gcgtccggat cagagcagca cccaacgatg
aggagaacat 32940cgatgtcgga tcggacccgg gtcgtggtcg tcggcggaac
ctcggggatc gggcggcact 33000tcgcccgatt ctgcgccgaa cgcggagacg
acgtggtgat caccggccgt tcggcggccc 33060ggaccaagac cgtggcggac
gagatcggcg ggcggacccg tgggctcgct ctcgacctgg 33120ccgagccgga
gacgatcgcg gacgcgctcg ccgacgtgcc gcacgtcgac cggctcgtgg
33180tcgcggcgct ggaccgcgac tacaacaccg tccgcgcgta ccggccgggc
gacgcggcgc 33240ggctgctgac cgtcaagctg gtcggctaca cggcggtcct
gcacgccctc gccccgcgga 33300tgaccgacga gagcgcagtc gtgctgctcg
gcggcctggc cagccaccgg ccgtatcccg 33360gctccacctc cgtcacgacc
gccaacggcg ggatcagcgc gctggtgcgg accctggctg 33420tggaactctc
gccggtccgg gtcaacgccc tgcacccgag catcgtctcc gacacgccgt
33480tctggagcga caagcccgcc gcgcgggagg ccgccgcgac ccgcgcgctc
agccgacggc 33540cggtcaccat gcaggactgc gccgaggcga tcgacttcct
gctgacgaac cgctcgataa 33600acggggtcaa cctgaacatc gacggcgggg
acgtgctcat ctgacgccgg aggcgatccg 33660ccacggcccc caccacccgg
tcgcgccctg cccgtgctcc cgctgctcgc gggggtaccg 33720ggccaggtcg
cgggcggaga agagcgccat gccggcgtgg aatccggtca ccggcaccgg
33780gacccgcgcc cagtaggcga gccggccgtc gacgtggaac tccacctccg
acgtcggcgc 33840ccggtaggtg atggcgtatc cgtgcgcccg gcccggctcc
gtcggcacgt ccaggaccac 33900ccggtggatg tagtgctcgt gcggctgggt
cacgccgggc agcaccaggc gctcgaccgt 33960cgcgtacacg gtgtcgttcg
tggcggcggc gttgaacacg acgccggtct ccaggtcgaa 34020caggttcacc
gtgccgaacg cgtccagcag gtcgtgcggg atctgccggt acgtccgcac
34080gcccatctcc acctcgacgg tcagcgagcc ctccgccggc acggcgaagc
gccgcaccga 34140ccggtacatc tgcttggcgt tgttctgccg gggatcggtg
tcgtggaagc gggtgaacgg 34200gtcgacggtc agctccagcc gcccgtcgcc
ggtgcggacc tgggcgttgc ggtcctggta 34260cctgtgggtc tgcccgtccg
cgccggcgat cgacatgatc gcccagcggg cggggtccag 34320ctcgcggctg
gtgaagtcgt cgtacgtcca cgcgctggtt ctcagtgccg acgtcatgca
34380gtcaccatcg gacgccggcc gggcgcgggc atcacccgtt cacgcggttc
ggccggaccc 34440ggcacgccaa tgcgccggcc acgccccgga aatcccgtga
ttaagccatg ccggagcgtg 34500aacggtcgcc gagactgacg ccgcacccat
ctccgcatcg tctgcgacgt tctcaccagg 34560gggagagagc aatggacacg
gcagctccgg caacggacgg cggtcgctac ctcgccgtcc 34620atcacagcgc
agagttcagg gaactacggc gacgatcgag cacgttcacg ctctgggcca
34680gcgtcgcctt cttcggctgg tggttcctcg gcagcctgct cgccacctac
gcgccggact 34740tcttccggga gaaggtggcc ggcccggtca acgtgggtct
gctcttcgtc ttcctgtcgt 34800tcgccttcgt ggtgacgctc gccgccttct
acctgcgtta cgcccgcacg catctcgatc 34860cgctcagcga gaagatccgt
gccgacctgg aaggagcgtc ccgatgagcg tcatcctcgc 34920cgacccgcca
cccccggtcg acaacacgtg ggcgacgccc gcgatcgccg tgccggtcac
34980catcgtcctc gcgctcgcgg tgctctacct ggtccggtcg gcgcgcgcca
gcaccaccac 35040cgcggacggc ttcctgctgg ccgaccggcg gatcgggccg
gtgcagaacg cgctggcggt 35100ggcctccgcg ccgctgatgt actcgacgat
gtacatcatc accggccaca tcgcgctcag 35160cggctacgac gccatcctgc
tgatgaccgc cttcaccatg ggcaccatgc tcgcgctgtt 35220cctcttcgcc
gggccggtgc gcaacgtggg cggctacacg ctcggtgacc tgctcgcggt
35280ccgtacccgg gagcggccgg cgcggatcgc gtcggcggtg ctcacgctgc
tgacgtacgt 35340catgctgacg gtgatcatga tggccgccat cgcgttcatc
ttcaaccgct ggttcggcgt 35400cgacgccctc gtcggcctgg tcctcccggt
gttcgtcgtc ggtctgatca cggtggggta 35460cgtgtacctc ggcgggatgc
tcggggtcac ccgcatcctg gtgttcaagc tggtgctgtc 35520ggtggtcgtc
gtgggcgtgc tgaccgcctg ggtgctggcc cgcttcgacc tgaacctctt
35580cagcctgctg gagcgggccg aggcgaacgc ggcgccggtg cccagcggca
gcgacctgct 35640gggcccgggc cggctgttcg gcgagggcgc gaccacgctc
gtgcacctgt cgaagctgtt 35700cgccatcgcc gtcggagtgg cggccattcc
gttcctgttc atgcgcaact tcgcggtgac 35760cagcgggcgg gacgcgcgcc
ggtcgaccgg gtgggcgtcg atgatcatcg tcgggttcta 35820cctgtgcctg
tccgtcgtcg ggctcggtgc cgtcgcgatc ctcggccggg acaacatcgg
35880cgtcatcaag gcccaccgcg acatcagctt ccccaagctc gccgacgagc
tcggcggtcc 35940ggtgatggtc ggctccctgg ccggcgtcgc ggtcctgacg
atcgtcggcg tcttcgcgcc 36000gctgctgcac agcgccgtga cgacggtgac
caaggacctg aacgtgatcc gcggccggcg 36060gctggatccg gccgccgagc
tgcgggacat caagcgcaac accctgatca tcggcgtcgg 36120ctccgtgctg
ctggcggtcg tgatgctgcc ggtacggacc cacatcttca tcccgacctc
36180gatcgacatt gccggcgcgg tggtcctgcc gatcgtcgtc tacgcgttgt
tctggcggcg 36240tttcaacacc cgcggactgc agtggacggt ctacggcggc
ctcgcgctca ccgcgttcct 36300ggtgctgttc tccaacggtg tctcgggcga
gccggacgcc atcttcccgg accgcaactt 36360caagttcgtg gacgtcgagc
ccgcgctgat cacggtgccg gtcggcttcc tgctcggcta 36420cctcggctcg
atcaccagcc gggagcgcga cgacgccgcg ttcgccgaga tgcaggtccg
36480gtccctcacc ggagctgtcg tcacgggacc gccgcggccg gccgccgtgg
acgacgagga 36540ccgcgacggc cgccaggacc gggcgcccag cccggtgagc
tgaacatccg caacggtgtg 36600gg 366022571PRTMicromonospora sp. strain
046-ECO11 2Val His Asn Leu Asp Asn Ile Pro Ser Ser Pro Ser Thr Ser
Gly Gly1 5 10 15Ser Leu Pro Ala Gly His Arg Ala His Val Arg Ala Asp
Gly Val Arg 20 25 30Val Val Arg Gly Gly Arg Val Val Leu Ser Asp Val
Ser Val Thr Val 35 40 45Ser Ala Ala Ser Arg Leu Ala Val Val Gly Glu
Asn Gly Arg Gly Lys 50 55 60Thr Thr Leu Leu His Val Leu Ala Gly Leu
Ile Ala Pro Asp Gln Gly65 70 75 80Val Val Glu Arg Leu Gly Thr Ile
Gly Val Ala Arg Gln Asn Leu Glu 85 90 95Ser Arg His Gly Glu Thr Val
Gly Thr Leu Val Arg Glu Ala Ile Arg 100 105 110Glu Ser Glu Arg Ala
Leu Arg Ala Leu Asp Glu Ala Thr Ile Ala Leu 115 120 125Thr Glu Gly
Arg Ala Gly Ala Asp Asp Ala Tyr Ala Ala Ala Leu Asp 130 135 140Ala
Ala Thr Arg Leu Asp Ala Trp Asp Ala Gln Arg Arg Val Asp Val145 150
155 160Ala Leu Ala Gly Leu Asp Ala Cys Pro Asp Arg Asp Arg Gln Leu
Ala 165 170 175Thr Leu Ser Val Gly Gln Arg Tyr Arg Val Arg Leu Ala
Cys Leu Leu 180 185 190Gly Ala Arg Val Asp Leu Leu Met Leu Asp Glu
Pro Thr Asn His Leu 195 200 205Asp Ala Asp Ser Leu Ala Phe Leu Thr
Ala Arg Leu Arg Asp His Pro 210 215 220Gly Gly Val Val Leu Val Thr
His Asp Arg Ala Leu Leu Arg Asp Val225 230 235 240Ala Thr Glu Phe
Leu Asp Leu Asp Pro Ser Ala Asp Gly Arg Pro Arg 245 250 255Arg Tyr
Ala Gly Asp Tyr Val Ala Trp Gln Asp Gly Arg Arg Arg Asp 260 265
270Phe Ala His Trp Val Arg Asp His Glu Ala Gln Gln Ala Glu His Gln
275 280 285Arg Leu Ala Asp Gly Val Arg Glu Ala Arg Asp Arg Leu Ser
Thr Gly 290 295 300Trp Arg Pro Glu Lys Gly His Gly Lys His Gln Arg
Gln Ser Arg Ala305 310 315 320Pro Gly Leu Val Gln Ala Leu Arg Arg
Arg Gln Glu Ala Leu Asp Ala 325 330 335His Arg Val Thr Val Pro Glu
Pro Pro Gln Pro Leu Arg Trp Pro Pro 340 345 350Leu Asp Thr Arg Ala
Gly Leu Pro Ile Leu Arg Cys His Asp Val Thr 355 360 365Val Ala Gly
Arg Leu Arg Thr Arg Val Thr Leu Thr Leu Asp Gly Gly 370 375 380Asp
Arg Leu Leu Val Thr Gly Pro Asn Gly Ala Gly Lys Ser Thr Leu385 390
395 400Leu Ser Val Leu Ala Gly Asp Leu Thr Pro Ser Thr Gly Glu Val
Arg 405 410 415His Leu Ser Gly Ala Arg Val Ala Tyr Leu Gly Gln Glu
Val Pro Asp 420 425 430Trp Pro Pro Ala Leu Leu Ala His Asp Leu Tyr
Glu Gln His Val Gly 435 440 445Arg Leu Arg Ser Ser Gly Arg Val Gly
Ser Gly Thr Ala Leu Pro Leu 450 455 460Ser Ala Thr Asn Leu Leu Asp
Ala Glu Ala Arg Arg Thr Pro Val Gly465 470 475 480Arg Met Ser His
Gly Gln Gln Arg Arg Leu Asn Leu Ala Leu Arg Leu 485 490 495Ala Glu
Arg Pro Asp Leu Leu Ile Leu Asp Glu Pro Thr Asn His Leu 500 505
510Ser Ala Pro Leu Val Asp Asp Leu Thr Ala Ala Leu Leu Thr Thr Arg
515 520 525Ala Ala Val Val Val Ala Thr His Asp Arg Gln Met Leu Gln
Asp Leu 530 535 540Ala Ala Trp Pro Thr Leu Pro Leu Thr Ala Pro Ala
Ala Ser Gly Arg545 550 555 560Ser Val Thr Ser Glu Arg Tyr Asp Trp
Glu Ser 565 57031716DNAMicromonospora sp. strain 046-ECO11 3
gtgcacaacc tcgacaacat tccttcctcc ccatccacct cgggcggttc gctgcccgcc
60gggcaccggg cgcacgtgcg ggccgacggc gtccgcgtcg tacgcggcgg ccgggtcgtg
120ctgtccgacg tcagcgtgac cgtctccgcc gcttcccgcc tcgcagtcgt
cggcgagaac 180ggccgcggca agaccaccct gctgcacgtg ctggccggcc
tcatcgcgcc cgaccagggc 240gtggtggaac ggctgggcac gatcggcgtc
gcccggcaga acctggagtc gcgccacggc 300gagacagtgg gcacgctcgt
ccgggaggcg atccgggagt ccgaacgcgc gctgcgggcg 360ctcgacgagg
cgacgatcgc gctcaccgag ggccgggcgg gcgcggacga cgcgtacgcg
420gccgcgctcg acgcggcgac ccggctggac gcctgggacg cgcagcggcg
cgtcgacgtg 480gcgctggccg gcctcgacgc gtgcccggac cgggaccggc
agctggccac gttgtccgtc 540ggccagcgct accgggtacg gctggcgtgc
ctgctgggag cgagggtcga cctgctgatg 600ctggacgagc cgacgaacca
cctcgacgcc gacagcctgg ccttcctcac cgcccggcta 660cgcgaccacc
cgggcggcgt cgtgctggtg acccacgacc gcgccctgct gcgggacgtc
720gccacggagt tcctggacct cgaccccagc gcggacgggc gcccgcgccg
ctacgccggg 780gactacgtcg cctggcagga cgggcgccgc cgcgacttcg
cgcactgggt acgcgaccac 840gaggcgcagc aggccgagca ccagcggctg
gccgacgggg tacgggaggc gcgggaccgg 900ctcagcaccg gctggcggcc
ggagaagggg cacggcaagc accagcgcca gtcccgcgcg 960cccggactgg
tccaggcgct gcgccgccgg caggaggcgc tcgacgcgca ccgcgtcacc
1020gtgccggagc caccgcagcc gctgcgctgg ccgccgctgg acacccgtgc
cggactgccc 1080atcctgcgat gccacgacgt cacggtggcc gggcgcctgc
gtacccgggt cacgctcacg 1140ctcgacggcg gggaccgcct gctggtgacc
ggacccaacg gcgcgggcaa gtcgacgctg 1200ctctccgtgc tggccggcga
cctcacgccg tcgaccgggg aggtccggca cctgtccggc 1260gcgcgcgtcg
cgtacctcgg tcaggaggtg cccgactggc cgccggcgct gctcgcgcac
1320gacctgtacg agcagcacgt gggccggctc cgctccagcg ggcgcgtcgg
ctccggcacg 1380gccctgccgc tgagcgcgac gaacctgctc gacgccgagg
cccggcgtac ccccgtcggc 1440cggatgtcgc acggacagca acggcggctg
aacctggcgc tgcgcctggc cgaacgtccc 1500gacctgctga tcctcgacga
accgacgaac cacctgtcgg cgccgctggt cgacgacctc 1560accgccgccc
tgctgacgac ccgggcggcg gtggtcgtcg ccacccacga ccggcagatg
1620ctccaggacc tcgcggcctg gcccacgctg ccgctcacag ccccggcggc
gtcaggtcgt 1680tcggtcactt ccgagcgata tgactgggag tcataa
17164689PRTMicromonospora sp. strain 046-ECO11 4Met Thr Thr Gly Arg
Pro Gly Glu Asn Arg Ala Thr Asp Ala Ala Arg1 5 10 15Asn Pro Gly Trp
Ala Ala Gly Gly Pro Ala Ser Gln Pro Trp Gly Gly 20 25 30Gly Asn Asp
Glu Gln Val Leu Arg Glu Ile Leu Gly Val Asp Val His 35 40 45Arg Glu
Leu Ile Asp Phe Ala Gly Gly Ala Gly Gly Asn Pro His Leu 50 55 60Val
Ala Glu Leu Ala Arg Gly Leu Ala Glu Glu Gly Leu Ile Arg Glu65 70 75
80Thr Asn Gly Arg Ala Glu Leu Val Ser Arg Arg Ile Pro Arg Arg Val
85 90 95Leu Ser Phe Val Met Arg Arg Leu Asn Asp Val Ser Ala Gly Cys
Gln 100 105 110Gln Phe Leu Lys Val Ala Ala Ala Leu Gly Arg Ser Phe
Met Leu Glu 115 120 125Asp Val Ser Arg Met Leu Gly Arg Ser Ser Ala
Ala Leu Leu Pro Pro 130 135 140Val Asp Glu Ala Ile Ala Ser Gly Phe
Val Val Ala Ala Glu His Gln145 150 155 160Leu Ala Phe Gln Ser Asp
Phe Leu Leu Arg Gly Ile Ile Glu Ser Ile 165 170 175Pro Gly Pro Ala
Arg Asp Ala Leu Arg Arg Glu Ala Met Ser Leu Ser 180 185 190Gly Arg
Arg Arg Pro Ala Ala Asp Gln Asn Arg Arg Leu Asp Ala Ala 195 200
205Pro Thr Ala Pro Val Ser Ala Thr Gly Glu Asp Ala Thr Gly Ser Cys
210 215 220Ser Arg Ala His Arg Leu Ile Met Asn Gly Asn Ala Lys Ala
Gly Ile225 230 235 240Arg Val Ala Glu Ala Val Leu Ala Gly Pro Ala
Ala Ser Leu Ala Ala 245 250 255Arg Arg Asp Ala Glu Ala Cys Leu Val
Leu Ala Asp Leu Leu Leu Gly 260 265 270Gly Glu Gly Gly Gly Pro Met
Thr Glu Ala Ile Leu Arg Glu Arg Asp 275 280 285Ala Glu Ser Gly Asp
Ala Ala Leu Ala Met Ala Leu Thr Ala Arg Ser 290 295 300Thr Gly Leu
Trp Ser Ala Gly Lys Leu Ala Glu Gly Leu Lys Leu Gly305 310 315
320Arg Ala Ala Val Arg Ala Gly Ala Glu Ala Glu Pro Val Trp Arg Leu
325 330 335His Ala Gln Leu Ala Leu Ala Gly Lys Leu Ala Asn Leu Arg
Glu Phe 340 345 350Asp Glu Ala Glu Ala Leu Ile Asn Glu Ala Glu Ala
Gly Leu Arg Gly 355 360 365Leu Pro Ala Pro Ile Trp Thr Ala Ala Thr
Ala Val Met Arg Ser Arg 370 375 380Leu Leu Leu Gln Ala Gly Arg Ile
Gly Glu Ala Arg Arg Glu Ala Ala385 390 395 400Leu Ala Thr Thr Ala
Val Glu Gly Asp Ala Val Pro Met Leu Arg Pro 405 410 415Leu Ala Tyr
Ala Val Leu Ser Thr Ala Ser Phe Tyr Met Gly Asp Leu 420 425 430Pro
Ala Ala Ile Glu Tyr Leu Arg Arg Gly Gln Arg Asp Ala Asp Arg 435 440
445His Val Val Leu Asp Ser Val Gln Tyr Ser Trp Ala Glu Val Leu Ile
450 455 460Thr Val Lys Gln Glu Gly Pro Arg Ala Ala Ala Gln Leu Leu
Ala Gly465 470 475 480Lys His His Arg Leu Pro Thr Gln Arg Arg Leu
Tyr Val Glu Val Pro 485 490 495Ser Ala Ala Ala Phe Leu Val Leu Leu
Ala Arg Asp Val Asp Asp Arg 500 505 510Asp Leu Glu Arg Arg Val
Leu Asp Thr Val Asn Gly Leu Ala Ala Asp 515 520 525Asn Pro Arg Ile
Gln Val Val Ser Leu Thr Ala Met His Ala His Ala 530 535 540Leu Ala
Asn Ser Ala Pro Ala Ala Leu Ala Leu Ile Ile Val Gln Ser545 550 555
560Arg Asp Pro Ile Ser Val Ala Leu Ala Thr Glu Glu Leu Ala Lys Leu
565 570 575Tyr Ala Ala Gln Ala Gln Ala Gly Gly Arg Pro Ala Thr Pro
Ala Arg 580 585 590Ala Glu Glu Ala Ala Thr Pro Pro Ala Ser Cys Trp
Ser Thr Leu Ser 595 600 605Asp Met Glu Gln Arg Ile Ala Tyr Leu Val
Ser Val Gly Leu Thr Asn 610 615 620Arg Gln Ile Ala Lys Gln Val His
Leu Ser Ala His Thr Val Asn Tyr625 630 635 640His Leu Arg Lys Ile
Tyr Arg Lys Leu Gly Phe Asn Thr Arg Ala Glu 645 650 655Leu Ala His
Ala Ala Ala Thr Tyr Ser Gly Arg Ala Ala Ile Tyr Ser 660 665 670Met
Ser Gly Asp Gln Asp Trp Gly Ala Gly Ser Met Thr Gly Lys Ala 675 680
685Ser 52070DNAMicromonospora sp. strain 046-ECO11 5atgacaacgg
gacggccggg ggagaaccgg gcgacagacg cggcacgaaa tccggggtgg 60gccgccgggg
ggccggcgtc ccagccatgg ggcgggggga acgacgagca ggtcctgcgc
120gagatcctcg gggtcgacgt gcaccgcgag ctgattgact tcgcgggtgg
tgccggcgga 180aatccgcacc tggtcgccga actcgcgcgc gggctcgccg
aagagggatt gattcgggag 240acaaacggtc gggcggaatt ggtgtcccgg
cgaattcccc ggcgcgtgct gagttttgtc 300atgcgtcgat tgaatgatgt
cagcgccggc tgccagcagt tcttgaaggt tgccgcggca 360ttgggcagat
ccttcatgct ggaggacgtt tcgagaatgc tgggccgatc gtcggcggcc
420ctgctcccgc cggtggacga ggcgatcgca tcgggcttcg tcgtcgccgc
cgagcatcaa 480ctcgcctttc agagcgactt cctgctgcgc ggcatcatcg
agtccattcc cgggcccgcc 540cgcgacgcct tacgacgtga ggcgatgagc
ctttccgggc gacggcgccc ggcggccgac 600cagaatcgcc ggttggacgc
ggcgcctacc gcgccggtga gcgcgaccgg ggaggacgcc 660accggatcct
gttcccgggc gcaccgcctg ataatgaacg ggaacgcgaa ggccggcatt
720cgcgtcgccg aggcggttct cgccggcccg gccgcgtcgc tcgctgcccg
gcgtgacgcg 780gaggcgtgtc tggtgctggc cgatctgctg ctcggcgggg
agggcggcgg cccgatgacc 840gaggcgatcc tgcgcgaacg cgacgccgag
tccggtgacg ccgcactggc gatggcgctg 900accgcccggt ccaccgggct
gtggtcggcg ggaaagctgg cggagggcct gaagctggga 960cgggcggcgg
tgcgggcggg cgcggaggcc gaaccggtgt ggcgtctgca cgcccagctc
1020gcgctcgccg ggaaactcgc gaacctccgc gagttcgacg aggccgaggc
gttgatcaac 1080gaggcggaag cgggcctgcg cggactgccc gcgccgatct
ggacggccgc gacggcggtg 1140atgcggtccc ggttgctgct ccaggcgggg
cggatcgggg aggcgcgtcg ggaggcggcg 1200ctggccacca ccgccgtgga
gggggacgcg gtgccgatgc tgcggcctct cgcctacgcg 1260gtgctcagca
ccgcctcctt ctacatgggg gacctgcccg ccgcgatcga gtacctcagg
1320cgggggcagc gggacgcgga ccgccacgtg gtcctcgact cggtgcagta
ctcgtgggcg 1380gaagtgctga tcacggtcaa gcaggaaggc ccgcgggccg
ccgcccagct gctcgcgggc 1440aagcaccacc gcctgcccac gcagcgccgc
ctctacgtcg aggtgccgag cgccgccgcc 1500ttcctggtcc tgctcgcccg
cgacgtggac gaccgtgacc tcgaacgccg cgtcctcgac 1560acggtcaacg
ggctcgccgc ggacaacccc aggatccagg tcgtcagcct caccgccatg
1620cacgcccacg cgctggcgaa cagcgctccg gccgccctgg cgctcatcat
cgtgcagtca 1680cgggacccga tctcggtggc gctggccacc gaggaactcg
ccaagctcta cgccgcgcag 1740gcccaggcgg ggggacggcc ggcgacgccg
gcccgcgccg aggaggccgc caccccgccg 1800gcgagctgct ggtcgaccct
gtccgacatg gagcagcgga tcgcctacct ggtgagcgtg 1860ggtctgacga
accggcagat cgccaagcag gtccacctgt ccgcgcacac cgtcaactac
1920cacctgcgga agatctaccg gaaactgggt ttcaacaccc gggccgagct
ggcgcacgcc 1980gcggccacgt actccggccg ggcggcgatc tactccatga
gcggcgacca ggactggggc 2040gccggatcca tgaccggcaa ggccagctga
20706895PRTMicromonospora sp. strain 046-ECO11 6Met Val Ile Met Asn
Arg Met Ala Gly Arg Gly Gln Glu Leu Ser Ser1 5 10 15Leu Gly Glu Leu
Leu Asp Ala Thr Met Arg Gly Ser Gly Gly Cys Val 20 25 30Val Val Asp
Gly Pro Phe Gly Ile Gly Lys Thr His Leu Leu Lys Val 35 40 45Thr Gly
Leu Glu Ala Ala Ala Arg Gly Leu Thr Val Val Ala Gly Arg 50 55 60Ala
Ser Val Thr Asp Gln Pro Val Pro Val His Leu Leu Val Asn Phe65 70 75
80Leu Arg His Ala Met Pro Gly Glu Ala Ala Val Glu Gln Leu Ala Leu
85 90 95Pro Gly Ala Asn Pro Phe Trp Leu Ile Asp Arg Val Gly Asp Leu
Val 100 105 110Glu Val Ala Ala Arg Arg Arg Pro Leu Val Val Ala Leu
Asp Asp Ala 115 120 125Gln Arg Ile Asp Asp Val Ser Ala Leu Ala Leu
Arg Gly Leu Val Pro 130 135 140Arg Leu Ala Ser Ser Pro Val Leu Trp
Leu Leu Ala Arg Arg Pro Val145 150 155 160Ala Ala Gly Ser Ile Ala
Gln His Ala Val Asp Trp Leu Ala Glu His 165 170 175Val Ala Val Arg
Val Arg Leu Arg Glu Pro Gly Glu Glu Ala Val Ala 180 185 190Asp Leu
Cys Ala Gly Ile Leu Gly Ala Arg Pro Asp Ala Ser Val Leu 195 200
205Arg Trp Ala Ala Arg Cys Gly Gly Asn Pro Lys Val Met Glu Ile Val
210 215 220Phe Ser Ala Phe Ile Lys Ala Gly Gln Met Ile Ile Val Asp
Gly Ala225 230 235 240Ala Ser Val Val Ser Asp Glu Leu Pro Asp Gly
Val Leu Ala Ala Val 245 250 255Arg Gly Leu Leu Glu Glu Leu Pro Pro
Pro Leu Arg Arg Leu Leu Ala 260 265 270Ala Gly Gly Arg Leu Gly His
Thr Phe Pro Val Asp Arg Val Thr Gly 275 280 285Leu Leu Asp Gly Ser
Ala Ala Asp Val Ser Ala Ala Ile Asp Glu Ala 290 295 300Val Arg Val
Gly Leu Ile Arg Arg Asp Gly Ala Glu Leu Thr Phe Ala305 310 315
320His Pro Val Leu Gly Glu Ala Leu Arg His Ala Ala Tyr Pro Glu Pro
325 330 335Glu Arg Ala Glu Pro Gly Ser Ala Pro Ala Pro Ala Ala Gly
Asp Pro 340 345 350Val Arg Arg Gly Arg Pro Asp Pro Arg Pro Gly Thr
Pro His Ser Pro 355 360 365Ala Gly Val Arg Val Thr Arg Ser Ala Pro
Asp Ala Ala Thr Pro Ala 370 375 380Ala Thr Ala Gly Pro Arg Ser Gly
Arg Cys Gly Cys Asp Asp Val Ala385 390 395 400Ala Ala Ala Val Ser
His Leu Glu Asn Gly Ser Ala Glu Ala Pro Arg 405 410 415Ala Leu Ala
Arg Ala Leu Arg Leu Leu Ala Gly Ala Gly Arg Ala Ala 420 425 430Glu
Ala Gly Arg Leu Ala Glu Val Met Leu Arg Arg Asp Leu Ala Ala 435 440
445Asp Val Glu Ala Gln Leu Val Leu Glu Leu Gly His Gly Met Arg Ala
450 455 460Ala Gly Ser His Arg Leu Ala Ala Gly Phe Leu Arg Arg Thr
Gln Ala465 470 475 480Arg His Asp Val Cys Glu Leu Asp Arg Ala Lys
Leu Asp Arg Ala Leu 485 490 495Ala Asp Thr Thr Lys His Leu Gly Gly
Ala Ser Ser Ala Glu Leu Glu 500 505 510Pro Arg His Gln Ser Pro Gly
Cys Ala Pro Gly Arg Arg Pro Leu Trp 515 520 525Thr Trp Leu Val Arg
Ala Leu Gly Ala Ala Asp Gln Leu Asp Glu Ala 530 535 540Gln Ala Val
Leu Asp Thr Val Arg Pro Leu Ala Gln Glu Pro Ser His545 550 555
560Thr Gly Ser Glu Ser Leu Trp Arg Gly His Arg Ala Glu Leu Leu Ala
565 570 575Ala Ala Gly Arg Leu Asp Glu Ala Arg Ala Glu Ala Glu Ala
Ala Leu 580 585 590Arg Ala Ala Asp His Ser Arg Pro Gly Asp Cys Val
Pro Ala Arg Leu 595 600 605Val Leu Ala His Leu Gly Val His His Gly
Asp Leu Ala Thr Ala Ser 610 615 620Asp Gln Leu Arg Ala Ala Glu Arg
Leu Ala Ser Ala Asp Asp Ser Ala625 630 635 640Arg Met Asp Trp Ala
Leu Ala Arg Phe His Ala Ala Ser Gly Arg Pro 645 650 655Ala Met Met
Val Gln Thr Leu Ile Asn Val Ala Gly Gln Val Ala Pro 660 665 670Asp
Pro Leu Leu Phe Thr Glu Ala Pro Ala Ala Ala Ala Thr Leu Val 675 680
685Arg Gln Ala Arg Arg Ala Gly Leu Asp Ala Glu Ala Glu Arg Ala Val
690 695 700Glu Val Ala Arg Arg Val Ala Arg Gly Asn Pro Phe Val Gln
Ser Leu705 710 715 720Ala Ala Ala Ala Glu His Ala Ala Gly Leu Leu
Arg Asp Asp Pro Ala 725 730 735Ala Leu Leu Arg Ala Ala Asp Leu His
Arg Leu Ala Gly Arg Thr Leu 740 745 750Ala Ala Ala Gly Ala Val Glu
Asp Ala Ala Arg Ser Thr Arg Asp Arg 755 760 765Ala Glu Ala Thr Arg
Leu Leu Glu Ala Ala Thr Asp Gly Tyr Arg Glu 770 775 780Cys Gly Ala
Arg Arg Asp Leu Glu Arg Val Glu Ala Glu Leu Arg Gly785 790 795
800Leu Pro Ala His Asn Val Arg Pro Leu Val Pro Asp Arg Pro Arg Ser
805 810 815Gly Trp Glu Ser Leu Thr Ser Ala Glu Leu Arg Val Val Arg
Ala Ile 820 825 830Val Asp Gly Met Thr Asn Arg Glu Ala Ala Ser Ser
Leu Phe Leu Ser 835 840 845Pro His Thr Val Asp Ser His Leu Arg Arg
Val Phe Ser Lys Leu Asp 850 855 860Ile Asn Ser Arg Val Glu Leu Thr
Arg Cys Phe Ile Ala His Glu Ala865 870 875 880Val Arg Pro Ala Leu
Ala Thr Thr Arg Gln Pro Ala Ser Ala Gly 885 890
89572688DNAMicromonospora sp. strain 046-ECO11 7atggtcatca
tgaatcgcat ggcggggcgc gggcaggaat tgtcctcatt gggggaactg 60ctcgacgcca
ccatgcgggg atccgggggc tgcgtcgtcg tcgacgggcc gttcggcatc
120ggcaagaccc acctgctgaa ggtcaccggc ctggaggcgg cggcccgcgg
gctgacagtg 180gtggccgggc gggcaagcgt cacggatcag ccggtgcccg
tacacctgct cgtcaacttc 240ctgcgccacg cgatgcccgg cgaagcggcg
gtcgagcagc tcgccctgcc gggcgccaac 300ccgttctggc tgatcgaccg
ggtcggcgat ctggtcgagg tcgcggcgcg ccggcgcccg 360ctcgtggtcg
ccctggacga cgcccagcgc atcgacgacg tcagcgccct ggccctgcgc
420gggctcgtgc cgcgcctggc gtcctcgccg gtgctctggc tgctggcccg
ccggccggtc 480gccgccgggt cgatcgctca gcacgccgtc gactggctgg
ccgagcacgt cgcggtacgg 540gtacggctgc gcgagccggg cgaggaggcg
gtggccgacc tgtgcgccgg catcctcggc 600gcccggccgg acgcctccgt
cctgcgctgg gcggcccgct gcggcggcaa cccgaaggtg 660atggagatcg
tcttcagcgc gttcatcaag gccggccaga tgatcatcgt ggacggggcg
720gcgtcggtgg tgtccgacga gctgcccgac ggtgtcctcg ccgccgttcg
cgggctgctg 780gaggagctgc cgcccccgct gcggcgcctg ctcgcggccg
gcggccggct cggccacacg 840tttcccgtcg accgggtgac gggcctgctg
gacggctcgg ccgccgacgt gtccgccgcg 900atcgacgagg cggtgcgggt
cgggctgata cgacgcgacg gcgcggagct gaccttcgcc 960cacccggtgc
tcggagaggc gcttcgccac gccgcgtacc cggaaccgga gcgtgccgag
1020cccggatccg cgccggcacc ggcggcgggc gacccggtcc ggcgcgggcg
gcccgatccg 1080cggcccggga cgccccactc ccccgccggc gtacgcgtca
cgcgctccgc gccggacgcg 1140gccacgcccg ccgcgacggc ggggccgcgc
tcgggccggt gcgggtgcga cgacgtggcg 1200gcagccgccg tgtcccacct
ggagaacgga tccgccgagg cgccacgagc actggcccgt 1260gcgctgcgcc
tgctggccgg ggcggggcgg gccgccgagg ccggccgcct cgcggaggtg
1320atgctccgcc gcgacctcgc ggcggacgtc gaggcgcagc tcgtgctcga
actgggacac 1380gggatgcggg ccgccggcag ccaccgcctg gcggccggct
tcctgcgccg gacgcaggcc 1440cgccacgacg tgtgcgagct ggaccgcgcc
aagctggacc gggcgctcgc ggacaccacg 1500aagcacctgg gcggtgcctc
ctccgccgag ctggagcccc ggcaccagtc cccgggctgc 1560gcgcccggcc
ggcggccgct gtggacctgg ctggtccggg cgctgggcgc ggccgatcag
1620ctcgacgagg cgcaggcggt gctggacacc gtacgaccgc tggcgcagga
gcccagtcac 1680accggctcgg agtcgctctg gcgcggccac cgggccgagc
tgctggcagc ggccggacgg 1740ctggacgagg cacgcgccga ggcggaggcg
gcgctgcgag ccgccgacca ctcccggccg 1800ggcgactgcg tgccggcgcg
cctggtcctg gcccacctcg gcgtgcacca cggtgacctc 1860gccacggcca
gcgaccagtt gcgggcggcc gagcggctgg cctccgccga cgactcggcg
1920cggatggact gggcgctggc ccggttccac gctgccagcg gccgtccggc
gatgatggtg 1980cagacgctga tcaacgtcgc cggacaggtc gcacccgatc
cgctgctgtt caccgaggcg 2040ccggccgctg cggcgacgct cgtacgccag
gcccgccggg cggggctcga cgcggaggcc 2100gagcgcgccg tggaggtcgc
ccggcgcgtc gcccgcggca acccgttcgt ccagtcgctg 2160gcggcggcgg
cggaacacgc cgcgggtctc ctgcgcgacg atccggcggc gctgctgcgg
2220gccgcggatc tgcaccggct cgccggccgt acgctcgcgg cggccggcgc
ggtggaggac 2280gcggcccgca gcacccggga ccgggccgag gccacccgtc
tgctcgaggc cgcgacggac 2340ggctaccggg agtgcggcgc gcgacgcgac
ctggagcgcg tggaggccga gctgcgtggc 2400ctgccggctc acaacgtccg
cccgctggtc cccgaccggc cccggtcggg gtgggagagc 2460ctgaccagcg
cggagctgcg ggtcgtgcgg gccatcgtgg acgggatgac caaccgcgag
2520gcggcgagtt cgctgttcct gtccccgcac accgtcgaca gtcacctgcg
gcgcgtcttc 2580tccaagctcg acatcaacag ccgggtggaa ctgacccgct
gcttcatcgc gcacgaggcg 2640gtccggccgg cgctggccac cacacgccag
ccggcgtccg ccggctga 26888362PRTMicromonospora sp. strain 046-ECO11
8Met Thr Val Gly Tyr Leu Gly Thr Val Thr Asp Ser Ala Pro Val Asp1 5
10 15Ala Ala Leu Arg Asp Phe Phe Ala Glu Arg Arg Ala Glu Ala Arg
Glu 20 25 30Leu Gly Asp Asp Phe Ala Ala Leu Val Ala Glu Leu Glu Ser
Tyr Val 35 40 45Leu Arg Gly Gly Lys Arg Ile Arg Pro Ala Phe Ala Trp
Leu Gly Trp 50 55 60Ile Gly Ala Gly Gly Asp Pro Glu Asp Pro Val Ala
Thr Ala Val Leu65 70 75 80Asn Ala Cys Ala Gly Phe Glu Leu Leu His
Ala Ser Gly Leu Ile His 85 90 95Asp Asp Ile Ile Asp Ala Ser Gln Thr
Arg Arg Gly His Pro Ala Ala 100 105 110His Val Ala Tyr Ala Glu Arg
His Arg Ala Arg Arg Phe Ser Gly Asp 115 120 125Pro Gly Thr Phe Gly
Thr Gly Thr Ala Ile Leu Ile Gly Asp Leu Val 130 135 140Leu Ile Trp
Ala Asp Val Leu Val Arg Ala Ser Gly Leu Pro Ala Asp145 150 155
160Ala His Val Arg Val Ser Pro Val Trp Ser Ala Val Arg Ser Glu Val
165 170 175Met Tyr Gly Gln Leu Leu Asp Leu Ile Ser Gln Val Ser Arg
Ser Glu 180 185 190Asp Val Asp Ala Ala Leu Arg Ile Asn Gln Tyr Lys
Thr Ala Ser Tyr 195 200 205Thr Val Glu Arg Pro Leu Gln Phe Gly Ala
Ala Ile Ala Gly Ala Asp 210 215 220Asp Asp Leu Phe Ala Ala Tyr Arg
Ala Phe Gly Ala Asp Val Gly Ile225 230 235 240Ala Phe Gln Leu Arg
Asp Asp Leu Leu Gly Val Phe Gly Asp Pro Val 245 250 255Val Thr Gly
Lys Pro Ser Gly Asp Asp Leu Arg Glu Gly Lys Arg Thr 260 265 270Val
Leu Leu Ala Thr Ala Leu Lys Arg Ala Asp Glu Arg Asp Pro Asp 275 280
285Ala Ala Ala Tyr Leu Arg Ala Lys Val Gly Thr Asp Leu Ala Asp Glu
290 295 300Glu Ile Ala Arg Ile Arg Ala Ile Phe Arg Asp Val Gly Ala
Val Glu305 310 315 320Glu Ile Glu Arg Gln Ile Ser Gln Arg Thr Asp
Arg Ala Leu Ala Ala 325 330 335Leu Glu Ala Ser Ser Ala Thr Ala Pro
Ala Lys His Gln Leu Ala Asp 340 345 350Met Ala Ile Lys Ala Thr Gln
Arg Ala Gln 355 36091089DNAMicromonospora sp. strain 046-ECO11
9atgaccgtcg gatatctcgg gacggtcacc gactcggcgc ccgtcgacgc cgcgctgcgc
60gacttcttcg ccgagcgccg cgccgaggca cgcgagctcg gcgacgactt cgcggccctg
120gtcgccgagc tggagagcta cgtcctgcgg ggcggcaagc gcatccggcc
cgccttcgcc 180tggctgggct ggatcggcgc cggcggcgac ccggaggacc
cggtggcgac cgcggtgctg 240aacgcctgcg ccgggttcga gctgctgcac
gcgtccggcc tcatccacga cgacatcatc 300gacgcgtcgc agacccgccg
cggccatccc gccgcgcacg tcgcgtacgc cgaacggcat 360cgggcgcggc
gcttctccgg tgacccggga acgttcggca ccggcaccgc catcctgatc
420ggagacctcg tcctgatctg ggccgacgtc ctggtccgcg cctccggcct
gccggccgac 480gcgcacgtgc gggtctcgcc ggtgtggtcg gcggtgcgct
ccgaggtcat gtacggccag 540ctgctcgatc tgatcagcca ggtgagccgg
agcgaggacg tcgacgcggc gctgcgcatc 600aaccagtaca agaccgcgtc
gtacacggtg gagcggccac tgcagttcgg cgcggcgatc 660gccggcgcgg
acgacgacct cttcgcggcc taccgcgcct tcggcgccga cgtgggtatt
720gccttccagc tgcgcgacga cctgctcggc gtgttcggcg acccggtggt
gacgggcaag 780ccgtccggcg acgacctgcg ggagggcaag cggacggtcc
tgctcgccac ggcgctcaag 840cgcgccgacg aacgggaccc ggacgcggcg
gcctacctgc gggcgaaggt cggcacggac 900ctcgcggacg aggagatcgc
ccgcatccgc gccatcttcc gcgacgtcgg cgcggtcgag 960gagatcgagc
ggcagatctc gcagcgcacc gaccgggcgc tggccgcgct ggaggcgagc
1020agcgccaccg cccccgcgaa gcatcagctc gccgacatgg cgatcaaggc
cacccagcgg 1080gcccagtga 108910354PRTMicromonospora sp. strain
046-ECO11 10Met Ser Thr Glu Pro Val Thr Val Val Ala Arg Gly Val Leu
Asp Gly1 5 10 15Arg Gly Asp Gly Pro Gly Arg Leu Gly Thr Gly Arg Ala
His Gly Lys 20 25
30Ala Ile Leu Leu Gly Glu His Ala Val Val Tyr Gly Ala Pro Ala Leu
35 40 45Ala Val Pro Val Pro Gln Leu Thr Ala Val Ala Lys Ala Arg Arg
Ala 50 55 60Gly Gly Asp Gly Gly Asp Glu Val Ser Phe Ala Ile Ala Gly
Leu Glu65 70 75 80Ser Pro Glu Val Thr Ser Leu Pro Thr Asp Gly Leu
Gln His Leu Val 85 90 95Thr Glu Phe Arg Gln Arg Ala Ala Val Thr Glu
Pro Met Arg Val Asp 100 105 110Val Leu Val Asp Cys Ala Ile Pro Gln
Gly Arg Gly Leu Gly Ser Ser 115 120 125Ala Ala Cys Ala Arg Ala Ala
Val Leu Ala Leu Ala Asp Ala Phe Asp 130 135 140Arg Arg Leu Asp Ala
Ala Thr Val Phe Asp Leu Val Gln Thr Ser Glu145 150 155 160Asn Val
Ala His Gly Arg Ala Ser Gly Ile Asp Ala Leu Ala Thr Gly 165 170
175Ala Thr Ala Pro Leu Ile Phe Arg Asn Gly Val Gly Arg Glu Leu Pro
180 185 190Val Ala Met Ala Gly Ala Ala Arg Ala Ala Arg Gly Ser Gly
Pro Ala 195 200 205Gly Phe Asp Ala Val Leu Val Ile Ala Asp Ser Gly
Val Ser Gly Ser 210 215 220Thr Arg Asp Ala Val Glu Leu Leu Arg Gly
Ala Phe Glu Arg Ser Pro225 230 235 240Arg Thr Arg Asp Glu Phe Val
Ser Arg Val Thr Ser Leu Thr Glu Ala 245 250 255Ala Ala His Asp Leu
Leu Gln Gly Arg Val Ala Asp Phe Gly Ala Arg 260 265 270Leu Thr Glu
Asn His Arg Leu Leu Arg Glu Val Gly Ile Ser Thr Glu 275 280 285Arg
Ile Asp Arg Met Val Asp Ala Ala Leu Ala Ala Gly Ser Pro Gly 290 295
300Ala Lys Ile Ser Gly Gly Gly Leu Gly Gly Cys Met Ile Ala Leu
Ala305 310 315 320Arg Asp Arg Gln Glu Ser Ala Ala Val Val Arg Ser
Val Gln Gln Ala 325 330 335Gly Ala Val Arg Thr Trp Thr Val Pro Met
Gly Arg Phe Thr Gly His 340 345 350Asp Asp 111065DNAMicromonospora
sp. strain 046-ECO11 11atgtccacgg aaccggtgac cgtcgtcgcc cgcggcgttc
tcgacggccg gggtgacggg 60ccgggccgcc tcggcaccgg ccgcgcccac ggcaaggcca
tcctgctggg cgaacacgcc 120gtcgtgtacg gcgctccggc gctcgccgtc
ccggtgccgc aactgaccgc cgtggccaag 180gcgcggcggg ccggcggcga
cggcggcgac gaggtctcct tcgccatcgc cgggctggag 240agcccggagg
tgacgtcgct tccgaccgac ggcctgcaac atctggtgac ggagttccgg
300cagcgggccg ccgtcaccga gccgatgcgc gtcgacgtgc tcgtggactg
cgccatcccg 360cagggccggg ggctcgggtc gagcgccgcc tgcgcccgcg
ccgcggtgct ggccctcgcg 420gacgcgttcg accgccgcct cgacgccgcc
acggtgttcg atctggtgca gacctcggag 480aacgtggcgc acggccgggc
cagcggcatc gacgccctgg ccaccggtgc gaccgcgccg 540ctgatcttcc
gcaacggcgt gggccgggaa ctgccggtcg ccatggcggg cgccgcgcgt
600gccgcgcgag ggtcgggccc ggccggcttc gacgcggtgc tcgtcatcgc
cgacagcggc 660gtcagcggca gcacccggga cgcggtggag ctgctgcggg
gtgccttcga gcgctccccg 720cgcacgcgcg acgagttcgt cagccgggtg
accagcctga ccgaggcggc ggcgcacgac 780ctgctccagg gccgggtcgc
cgacttcggc gcgcggctga ccgagaacca ccggctgttg 840cgcgaggtcg
gcatcagcac cgaacggatc gaccggatgg tcgacgccgc gctcgcggcg
900ggcagcccgg gcgccaagat cagcggcggt ggcctgggcg gctgcatgat
cgcactggcc 960cgggaccgcc aggaatccgc ggcggtggtg cggagcgtcc
agcaggccgg cgccgtccgc 1020acctggaccg tcccgatggg gaggttcacc
ggccatgacg actga 106512346PRTMicromonospora sp. strain 046-ECO11
12Met Thr Thr Asp His Arg Ala Glu Pro Ser Glu Pro Ala Leu Asp Arg1
5 10 15Pro Ala Thr Ala Val Ala His Pro Asn Ile Ala Leu Ile Lys Tyr
Trp 20 25 30Gly Lys Arg Asp Glu Gln Leu Met Ile Pro Tyr Ala Asp Ser
Leu Ser 35 40 45Met Thr Leu Asp Val Phe Pro Thr Thr Thr Thr Val Arg
Ile Asp Ser 50 55 60Gly Ala Ala Ala Asp Glu Val Val Leu Asp Gly Ser
Pro Ala Asp Gly65 70 75 80Glu Arg Arg Gln Arg Val Val Thr Phe Leu
Asp Leu Val Arg Lys Leu 85 90 95Ala Gly Arg Thr Glu Arg Ala Cys Val
Asp Thr Arg Asn Ser Val Pro 100 105 110Thr Gly Ala Gly Leu Ala Ser
Ser Ala Ser Gly Phe Ala Ala Leu Ala 115 120 125Leu Ala Gly Ala Ala
Ala Tyr Gly Leu Asp Leu Asp Thr Thr Ala Leu 130 135 140Ser Arg Leu
Ala Arg Arg Gly Ser Val Ser Ala Ser Arg Ser Val Phe145 150 155
160Gly Gly Phe Ala Met Cys His Ala Gly Pro Gly Ala Gly Thr Ala Ala
165 170 175Asp Leu Gly Ser Tyr Ala Glu Pro Val Pro Val Ala Pro Leu
Asp Val 180 185 190Ala Leu Val Ile Ala Ile Val Asp Ala Gly Pro Lys
Ala Val Ser Ser 195 200 205Arg Glu Gly Met Arg Arg Thr Val Arg Thr
Ser Pro Leu Tyr Gln Ser 210 215 220Trp Val Ala Ser Gly Arg Ala Asp
Leu Ala Glu Met Arg Ala Ala Leu225 230 235 240Leu Gln Gly Asp Leu
Asp Ala Val Gly Glu Ile Ala Glu Arg Asn Ala 245 250 255Leu Gly Met
His Ala Thr Met Leu Ala Ala Arg Pro Ala Val Arg Tyr 260 265 270Leu
Ala Pro Val Thr Val Ala Val Leu Asp Ser Val Leu Arg Leu Arg 275 280
285Ala Asp Gly Val Ser Ala Tyr Ala Thr Met Asp Ala Gly Pro Asn Val
290 295 300Lys Val Leu Cys Arg Arg Ala Asp Ala Asp Arg Val Ala Asp
Thr Leu305 310 315 320Arg Asp Ala Ala Pro Ser Cys Ala Val Val Val
Ala Gly Pro Gly Pro 325 330 335Ala Ala Arg Pro Asp Pro Gly Ser Arg
Pro 340 345131041DNAMicromonospora sp. strain 046-ECO11
13atgacgactg accaccgggc ggagccgtcc gagccggcgc tcgaccggcc cgcgaccgcc
60gtggcccatc cgaacatcgc gctgatcaag tactggggca agcgcgacga gcagctgatg
120atcccgtacg ccgacagcct gtcgatgacg ctcgacgtct tcccgaccac
caccaccgtc 180cggatcgaca gcggcgcggc ggccgacgag gtcgtcctcg
acggctcgcc cgccgacggc 240gaacggcgac agcgcgtcgt caccttcctg
gacctggtac gcaagctggc cgggcgcacg 300gaacgggcct gcgtcgacac
ccgcaactcc gtgcccaccg gcgccggcct ggcgtcctcg 360gcgagcggat
tcgccgccct cgccctcgcc ggcgccgccg cgtacggcct cgacctggac
420accaccgcgc tgtcccgcct ggcccggcgg ggatccgtgt cggcctcccg
gtcggtcttc 480ggcggcttcg cgatgtgcca cgcaggcccc ggcgccggga
ccgccgcgga cctcggctcc 540tacgccgagc cggtgcccgt cgcgcccctc
gacgtcgcgc tggtgatcgc gatcgtcgac 600gccgggccga aggcggtgtc
gagccgcgag gggatgcggc gaaccgtccg gacctccccg 660ctctatcagt
cgtgggtcgc ctccggccgc gccgacctgg ccgagatgcg ggccgcgctg
720ctccagggag acctggacgc ggtcggcgag atcgccgaac gcaacgccct
cggcatgcac 780gccaccatgc tggccgcccg gccggcggtg cgctacctgg
cgccggtcac tgtcgccgtg 840ctcgacagcg tgctgcgcct gcgcgccgac
ggcgtctccg cctacgccac gatggacgcg 900ggaccgaacg tcaaggtgct
ctgccgccgc gcggacgccg accgggtcgc cgacaccctg 960cgcgacgccg
cgccgagctg cgccgtggtc gtcgccggac cggggccggc ggcccggccg
1020gacccgggca gccggccgtg a 104114369PRTMicromonospora sp. strain
046-ECO11 14Val Thr Gly Pro Gly Ala Val Arg Arg His Ala Pro Gly Lys
Leu Phe1 5 10 15Val Ala Gly Glu Tyr Ala Val Leu Glu Pro Gly His Pro
Ala Leu Leu 20 25 30Val Ala Val Asp Arg Gly Val Asp Val Thr Val Ser
Gly Ala Asp Ala 35 40 45His Leu Val Val Asp Ser Asp Leu Cys Pro Glu
Gln Ala Cys Leu Arg 50 55 60Trp Gln Asp Gly Arg Leu Val Gly Ala Gly
Asp Gly Gln Pro Ala Pro65 70 75 80Asp Ala Leu Gly Ala Val Val Ser
Ala Ile Glu Val Val Gly Glu Leu 85 90 95Leu Thr Gly Arg Gly Leu Arg
Pro Leu Pro Met Arg Val Ala Ile Thr 100 105 110Ser Arg Leu His Arg
Asp Gly Thr Lys Phe Gly Leu Gly Ser Ser Gly 115 120 125Ala Val Thr
Val Ala Thr Val Thr Ala Val Ala Ala Tyr His Gly Val 130 135 140Glu
Leu Ser Leu Glu Ser Arg Phe Arg Leu Ala Met Leu Ala Thr Val145 150
155 160Arg Asp Gly Ala Asp Ala Ser Gly Gly Asp Leu Ala Ala Ser Val
Trp 165 170 175Gly Gly Trp Ile Ala Tyr Gln Ala Pro Asp Arg Ala Ala
Val Arg Glu 180 185 190Met Ala Arg Arg Arg Gly Val Glu Glu Thr Met
Arg Ala Pro Trp Pro 195 200 205Gly Leu Arg Val Arg Arg Leu Pro Pro
Pro Arg Gly Leu Ala Leu Glu 210 215 220Val Gly Trp Thr Gly Glu Pro
Ala Ser Ser Ser Ser Leu Thr Gly Arg225 230 235 240Leu Ala Ala Ser
Arg Trp Arg Gly Ser Pro Ala Arg Trp Ser Phe Thr 245 250 255Ser Arg
Ser Gln Glu Cys Val Arg Thr Ala Ile Asp Ala Leu Glu Arg 260 265
270Gly Asp Asp Gln Glu Leu Leu His Gln Val Arg Arg Ala Arg His Val
275 280 285Leu Ala Glu Leu Asp Asp Glu Val Arg Leu Gly Ile Phe Thr
Pro Arg 290 295 300Leu Thr Ala Leu Cys Asp Ala Ala Glu Thr Val Gly
Gly Ala Ala Lys305 310 315 320Pro Ser Gly Ala Gly Gly Gly Asp Cys
Gly Ile Ala Leu Leu Asp Ala 325 330 335Thr Ala Ala Thr Arg Thr Ala
Arg Leu Arg Glu Gln Trp Ala Ala Ala 340 345 350Gly Val Leu Pro Met
Pro Ile Gln Val His Gln Thr Asn Gly Ser Ala 355 360 365Arg
151110DNAMicromonospora sp. strain 046-ECO11 15gtgaccggcc
cgggcgccgt gcgccgccac gcgccgggca agctgttcgt cgccggtgag 60tacgcggtgc
tggagccggg ccacccggcg ctgctggtgg cggtcgacag gggagtggac
120gtcaccgtct ccggcgccga cgcccacctc gttgtcgact ccgacctctg
cccggagcag 180gcgtgcctgc ggtggcagga cggccggctc gtcggcgcgg
gcgacgggca gccggcgccc 240gacgccctcg gcgccgtggt ctcggcgatc
gaggtggtcg gcgaactcct gaccggacga 300gggctgcgcc cgctgcccat
gcgggtggcg atcaccagcc ggctgcaccg cgacggcacg 360aagttcggcc
tcgggtcgag cggggcggtg acagtcgcca cggtgaccgc agtggccgcg
420taccacgggg tggagctgtc gctcgaatcg cggttccggc tggcgatgct
ggcgacggtg 480cgtgacggcg ccgacgcctc cggcggtgat ctggccgcga
gcgtctgggg cggctggatc 540gcctaccagg cgcccgaccg cgcggccgtg
cgcgagatgg cgcggcggcg cggcgtcgag 600gagacgatgc gcgcgccctg
gccgggcctg cgggtccggc ggctgccacc accgcgtggc 660ctcgcgctgg
aggtgggctg gaccggcgag ccggcgagca gcagctcgtt gaccgggcgg
720ctggccgcct cccggtggcg gggcagcccg gcgcggtgga gcttcaccag
ccgtagccag 780gagtgtgtgc gtaccgccat cgacgcgctg gagcggggcg
acgaccagga actgctgcac 840caggtccggc gggcccggca cgtgcttgcc
gagctggacg acgaggtccg gctcgggatc 900ttcacccccc ggctgacggc
gctgtgcgac gccgccgaga ccgtcggcgg cgcggccaaa 960ccgtccggcg
ccggtggcgg ggactgcggc atcgcgttgc tggacgccac cgccgcgacg
1020cggaccgcgc ggctgcgcga gcagtgggcc gccgccgggg tgctccccat
gccgatccag 1080gtccatcaga cgaacgggag cgcgcgatga
111016360PRTMicromonospora sp. strain 046-ECO11 16Met Ile Ala Asn
Arg Lys Asp Asp His Val Arg Leu Ala Ala Glu Gln1 5 10 15Gln Gly Arg
Leu Gly Gly His His Glu Phe Asp Asp Val Ser Phe Val 20 25 30His His
Ala Leu Ala Gly Ile Asp Arg Ser Asp Val Ser Leu Ala Thr 35 40 45Ser
Phe Gly Gly Ile Asp Trp Pro Val Pro Leu Cys Ile Asn Ala Met 50 55
60Thr Gly Gly Ser Thr Lys Thr Gly Leu Ile Asn Arg Asp Leu Ala Ile65
70 75 80Ala Ala Arg Glu Thr Gly Val Pro Ile Ala Thr Gly Ser Met Ser
Ala 85 90 95Tyr Phe Ala Asp Glu Ser Val Ala Glu Ser Phe Ser Val Met
Arg Arg 100 105 110Glu Asn Pro Asp Gly Phe Ile Met Ala Asn Val Asn
Ala Thr Ala Ser 115 120 125Val Glu Arg Ala Arg Arg Ala Val Asp Leu
Met Arg Ala Asp Ala Leu 130 135 140Gln Ile His Leu Asn Thr Ile Gln
Glu Thr Val Met Pro Glu Gly Asp145 150 155 160Arg Ser Phe Ala Ala
Trp Gly Pro Arg Ile Glu Gln Ile Val Ala Gly 165 170 175Val Gly Val
Pro Val Ile Val Lys Glu Val Gly Phe Gly Leu Ser Arg 180 185 190Glu
Thr Leu Leu Arg Leu Arg Asp Met Gly Val Arg Val Ala Asp Val 195 200
205Ala Gly Arg Gly Gly Thr Asn Phe Ala Arg Ile Glu Asn Asp Arg Arg
210 215 220Asp Ala Ala Asp Tyr Ser Phe Leu Asp Gly Trp Gly Gln Ser
Thr Pro225 230 235 240Ala Cys Leu Leu Asp Ala Gln Gly Val Asp Leu
Pro Val Leu Ala Ser 245 250 255Gly Gly Ile Arg Asn Pro Leu Asp Val
Val Arg Gly Leu Ala Leu Gly 260 265 270Ala Gly Ala Ala Gly Val Ser
Gly Leu Phe Leu Arg Thr Leu Leu Asp 275 280 285Gly Gly Val Pro Ala
Leu Leu Ser Leu Leu Ser Thr Trp Leu Asp Gln 290 295 300Ile Glu Ala
Leu Met Thr Ala Leu Gly Ala Arg Thr Pro Ala Asp Leu305 310 315
320Thr Arg Cys Asp Leu Leu Ile Gln Gly Arg Leu Ser Ala Phe Cys Ala
325 330 335Ala Arg Gly Ile Asp Thr His Arg Leu Ala Thr Arg Ser Gly
Ala Thr 340 345 350His Glu Met Ile Gly Gly Ile Arg 355
360171083DNAMicromonospora sp. strain 046-ECO11 17atgatcgcca
accgcaagga cgaccacgtc cggctcgccg ccgagcagca gggccggctc 60ggcggtcacc
acgagttcga cgacgtgtcc ttcgtgcacc acgccctggc cggcatcgac
120cggtccgacg tctcgctggc cacgtcgttc ggcggcatcg actggccggt
gccgctgtgc 180atcaacgcga tgaccggcgg cagcaccaag accggcctga
tcaaccggga cctggcgatc 240gcggcccggg agaccggcgt accgatcgcc
accgggtcga tgagcgccta cttcgccgac 300gagtcggtgg ccgagagttt
cagcgtgatg cgccgggaga accccgacgg gttcatcatg 360gccaacgtca
acgccaccgc ctccgtcgaa cgggcccggc gggctgtcga cctgatgcgg
420gccgacgcgc tgcagatcca cctgaacacc atccaggaga cggtgatgcc
ggagggggac 480cggtcgttcg ccgcctgggg gccgcggatc gaacagatcg
tcgccggcgt cggtgtgccg 540gtgatcgtca aggaggtcgg cttcgggctc
agccgcgaaa cgctgctgcg gctgcgggac 600atgggcgtcc gggtggccga
cgtcgccggc cgcggcggca cgaacttcgc gcgcatcgag 660aacgaccggc
gggacgccgc cgactactcc ttcctcgacg ggtggggaca gtcgacaccc
720gcctgcctgc tggacgccca gggcgtggac ctgcccgtgc tggcctccgg
cggcatccgc 780aacccgctcg acgtggtccg cgggctggcg ctcggcgccg
gcgcggccgg ggtgtccgga 840ctgttcctgc gcacgctcct ggacggcggc
gtgccggcgc tgctgtcgct gctgtccacc 900tggctcgacc agatcgaagc
cctgatgacc gccctgggcg cgcggacccc ggccgacctg 960acccgctgcg
acctgctgat ccagggtcgg ctgagcgcgt tctgcgcggc ccggggcatc
1020gacacccacc gcctcgccac ccgttccggc gccacccacg agatgatcgg
aggcattcga 1080tga 108318351PRTMicromonospora sp. strain 046-ECO11
18Met Asn Asp Ala Ile Ala Gly Val Pro Met Lys Trp Val Gly Pro Val1
5 10 15Arg Ile Ser Gly Asn Val Ala Gln Ile Glu Thr Glu Val Pro Leu
Ala 20 25 30Thr Tyr Glu Ser Pro Leu Trp Pro Ser Val Gly Arg Gly Ala
Lys Ile 35 40 45Ser Arg Met Val Glu Ala Gly Ile Val Ala Thr Leu Val
Asp Glu Arg 50 55 60Met Thr Arg Ser Val Phe Val Arg Ala Lys Asp Ala
Gln Thr Ala Tyr65 70 75 80Leu Ala Ser Leu Glu Val Asp Ala Arg Phe
Asp Glu Leu Arg Asp Ile 85 90 95Val Arg Thr Cys Gly Arg Phe Val Glu
Leu Ile Gly Phe His His Glu 100 105 110Ile Thr Ala Asn Leu Leu Phe
Leu Arg Phe Ser Phe Thr Thr Gly Asp 115 120 125Ala Ser Gly His Asn
Met Ala Thr Leu Ala Ala Asp Ala Leu Leu Lys 130 135 140His Ile Leu
Asp Thr Ile Pro Gly Ile Ser Tyr Gly Ser Ile Ser Gly145 150 155
160Asn Tyr Cys Thr Asp Lys Lys Ala Thr Ala Ile Asn Gly Ile Leu Gly
165 170 175Arg Gly Lys Asn Val Val Thr Glu Leu Val Val Pro Arg Glu
Ile Val 180 185 190His Asp Ser Leu His Thr Thr Ala Ala Ala Ile Ala
Gln Leu Asn Val 195 200 205His Lys Asn Met Ile Gly Thr Leu Leu Ala
Gly Gly Ile Arg Ser Ala 210 215 220Asn Ala His Tyr Ala Asn Met Leu
Leu Gly Phe Tyr Leu Ala Thr Gly225 230 235 240Gln Asp Ala Ala Asn
Ile Val Glu Gly Ser Gln Gly Val Thr Val Ala 245 250 255Glu Asp Arg
Asp Gly Asp Leu Tyr Phe Ser Cys Thr Leu Pro Asn Leu 260 265 270Ile
Val Gly Thr Val Gly Asn Gly Lys Gly Leu Gly Phe Val Glu Glu 275 280
285Asn Leu Glu Arg Leu Gly Cys Arg Ala Ser Arg Asp Pro Gly Glu Asn
290
295 300Ala Arg Arg Leu Ala Val Ile Ala Ala Ala Thr Val Leu Cys Gly
Glu305 310 315 320Leu Ser Leu Leu Ala Ala Gln Thr Asn Pro Gly Glu
Leu Met Arg Ala 325 330 335His Val Arg Leu Glu Arg Pro Thr Glu Thr
Thr Lys Ile Gly Ala 340 345 350191056DNAMicromonospora sp. strain
046-ECO11 19atgaacgacg cgatcgccgg tgtgcccatg aaatgggtag gtcccgtgcg
gatctcggga 60aacgtggcgc agatcgagac ggaggttccg ctcgccacgt acgagtcgcc
gctctggccg 120tccgtcggcc ggggcgcgaa gatctcccgg atggtcgagg
cgggcatcgt cgccacgctc 180gtcgacgagc gcatgacccg ctcggtgttc
gtgcgcgcca aggacgcgca gaccgcctac 240ctggcctcgc ttgaggtcga
cgcgcggttc gacgaactgc gtgacatcgt gcgcacctgc 300ggcaggttcg
tcgagctgat cgggttccac cacgagatca ccgcgaacct gctgttcctg
360cggttcagtt tcaccaccgg cgacgcgtcc gggcacaaca tggcgacgct
ggccgccgac 420gcgctgctga agcacatcct ggacaccatt ccgggcatct
cgtacggctc gatctcgggc 480aactactgca ccgacaagaa ggccaccgcg
ataaacggca ttctcggccg gggcaagaac 540gtggtcaccg agctggtcgt
gccgcgggag atcgtccacg acagcctgca cacgacggcg 600gcggcgatcg
cccagctgaa cgtgcacaag aacatgatcg gcacgttgct cgccggcggt
660atccgctcgg ccaacgccca ctacgcgaac atgctgctcg ggttctacct
ggccacgggt 720caggacgccg cgaacatcgt cgagggctcc cagggcgtga
cggtcgccga ggaccgcgac 780ggcgacctct acttctcctg cacgctgccc
aacctgatcg tgggcaccgt cggcaacggc 840aaggggctcg gcttcgtcga
ggagaacctg gagcggctcg gctgccgcgc ctcgcgtgat 900ccgggcgaga
acgcccggcg gctcgcggtc atcgcggccg cgacggtgct ctgcggcgag
960ctgtccctgc tcgccgcgca gaccaacccg ggcgagctga tgcgggcgca
cgtccggctc 1020gaacgcccga ccgagaccac gaagatcgga gcctga
105620391PRTMicromonospora sp. strain 046-ECO11 20Met Ala Glu Arg
Pro Ala Val Gly Ile His Asp Leu Ser Ala Ala Thr1 5 10 15Ala His His
Val Leu Thr His Glu Thr Leu Ala Ala Ser Asn Gly Ala 20 25 30Asp Val
Ala Lys Tyr His Arg Gly Ile Gly Leu Arg Ala Met Ser Val 35 40 45Pro
Ala Pro Asp Glu Asp Ile Val Thr Met Ala Ala Ala Ala Ala Ala 50 55
60Pro Val Val Ala Arg His Gly Thr Asp Arg Ile Arg Thr Val Val Phe65
70 75 80Ala Thr Glu Ser Ser Val Asp Gln Ala Lys Ala Ala Gly Ile His
Val 85 90 95His Ser Leu Leu Gly Leu Pro Ser Ala Thr Arg Val Val Glu
Leu Lys 100 105 110Gln Ala Cys Tyr Gly Gly Thr Ala Gly Leu Gln Phe
Ala Ile Gly Leu 115 120 125Val His Arg Asp Pro Ser Gln Gln Val Leu
Val Ile Ala Ser Asp Val 130 135 140Ser Lys Tyr Ala Leu Gly Glu Pro
Gly Glu Ala Thr Gln Gly Ala Ala145 150 155 160Ala Val Ala Met Leu
Val Gly Ala Asp Pro Ala Leu Val Arg Val Glu 165 170 175Asp Pro Ser
Gly Met Phe Thr Ala Asp Val Met Asp Phe Trp Arg Pro 180 185 190Asn
Tyr Arg Thr Thr Ala Leu Val Asp Gly His Glu Ser Ile Ser Ala 195 200
205Tyr Leu Gln Ala Leu Glu Gly Ser Trp Lys Asp Tyr Thr Glu Arg Gly
210 215 220Gly Arg Thr Leu Asp Glu Phe Gly Ala Phe Cys Tyr His Gln
Pro Phe225 230 235 240Pro Arg Met Ala Asp Lys Ala His Arg His Leu
Leu Asn Tyr Cys Gly 245 250 255Arg Asp Val Asp Asp Ala Leu Val Ala
Gly Ala Ile Gly His Thr Thr 260 265 270Ala Tyr Asn Ala Glu Ile Gly
Asn Ser Tyr Thr Ala Ser Met Tyr Leu 275 280 285Gly Leu Ala Ala Leu
Leu Asp Thr Ala Asp Asp Leu Thr Gly Arg Thr 290 295 300Val Gly Phe
Leu Ser Tyr Gly Ser Gly Ser Val Ala Glu Phe Phe Ala305 310 315
320Gly Thr Val Val Pro Gly Tyr Arg Ala His Thr Arg Pro Asp Gln His
325 330 335Arg Ala Ala Ile Asp Arg Arg Gln Glu Ile Asp Tyr Ala Thr
Tyr Arg 340 345 350Glu Leu His Glu His Ala Phe Pro Val Asp Gly Gly
Asp Tyr Pro Ala 355 360 365Pro Glu Val Thr Thr Gly Pro Tyr Arg Leu
Ala Gly Leu Ser Gly His 370 375 380Lys Arg Val Tyr Glu Pro Arg385
390211176DNAMicromonospora sp. strain 046-ECO11 21atggccgaga
gacccgccgt cggcatccac gacctgtccg ccgcgacggc gcatcacgtg 60ctgacacacg
agaccctggc cgcgagcaac ggcgccgacg tggccaagta ccaccgtggc
120atcgggctgc gggcgatgag cgtgcccgcc ccggacgagg acatcgtgac
gatggctgct 180gccgccgccg cgccggtggt cgcccgccac ggcaccgacc
ggatccggac cgtcgtgttc 240gccacggagt cgtcggtcga ccaggcgaag
gcggccggga tacacgtcca ctccctgctc 300ggcctcccct cggccacccg
ggtggtcgag ctgaagcagg cctgctacgg cggtacggcg 360ggactgcagt
tcgccatcgg cctggtgcac cgtgacccgt cgcagcaggt cctggtgatc
420gccagcgacg tgtcgaagta cgcgctgggt gagcccggcg aggcgaccca
gggcgccgcg 480gcggtcgcca tgctcgtcgg cgcggacccg gcgctggtac
gcgtcgagga cccgtcgggc 540atgttcaccg ccgacgtcat ggacttctgg
cggccgaact accgcaccac cgccctggtc 600gacgggcacg agtccatctc
cgcctacctg caggcgctgg agggctcgtg gaaggactac 660accgagcgcg
gcggtcgcac cctggacgag ttcggcgcgt tctgctacca ccagccgttc
720ccgaggatgg ccgacaaggc gcaccggcac ctgctcaact actgcgggcg
cgacgtcgac 780gacgcgctgg tggccggggc catcgggcac accaccgcgt
acaacgccga gatcggcaac 840agctacacgg cgtcgatgta tctcgggctc
gcggcactgc tcgacaccgc cgacgacctg 900accggccgga ccgtcggctt
cctcagctac gggtccggca gcgtcgccga gttcttcgcc 960ggcactgtcg
tgcccgggta ccgcgcgcac acgcgacccg accagcaccg cgcggcgatc
1020gaccggcggc aggagatcga ctacgcgacg taccgggagt tgcacgagca
cgccttcccg 1080gtcgacggcg gcgactatcc ggcgccggag gtgaccaccg
ggccgtaccg gctggccggg 1140ctctccggtc acaagcgcgt ctacgagccg cgatag
117622290PRTMicromonospora sp. strain 046-ECO11 22Val Ala Glu Leu
Tyr Ser Thr Ile Glu Glu Ser Ala Arg Gln Leu Asp1 5 10 15Val Pro Cys
Ser Arg Asp Arg Val Trp Pro Ile Leu Ser Ala Tyr Gly 20 25 30Asp Ala
Phe Ala His Pro Glu Ala Val Val Ala Phe Arg Val Ala Thr 35 40 45Ala
Leu Arg His Ala Gly Glu Leu Asp Cys Arg Phe Arg Thr His Pro 50 55
60Asp Asp Arg Asp Pro Tyr Ala Ser Ala Leu Ala Arg Gly Leu Thr Pro65
70 75 80Arg Thr Asp His Pro Val Gly Ala Leu Leu Ser Glu Val His Arg
Arg 85 90 95Cys Pro Val Glu Ser His Gly Ile Asp Phe Gly Val Val Gly
Gly Phe 100 105 110Lys Lys Ile Tyr Ala Ala Phe Ala Pro Asp Glu Leu
Gln Val Ala Thr 115 120 125Ser Leu Ala Gly Ile Pro Ala Met Pro Arg
Ser Leu Ala Ala Asn Ala 130 135 140Asp Phe Phe Thr Arg His Gly Leu
Asp Asp Arg Val Gly Val Leu Gly145 150 155 160Phe Asp Tyr Pro Ala
Arg Thr Val Asn Val Tyr Phe Asn Asp Val Pro 165 170 175Arg Glu Cys
Phe Glu Pro Glu Thr Ile Arg Ser Thr Leu Arg Arg Thr 180 185 190Gly
Met Ala Glu Pro Ser Glu Gln Met Leu Arg Leu Gly Thr Gly Ala 195 200
205Phe Gly Leu Tyr Val Thr Leu Gly Trp Asp Ser Pro Glu Ile Glu Arg
210 215 220Ile Cys Tyr Ala Ala Ala Thr Thr Asp Leu Thr Thr Leu Pro
Val Pro225 230 235 240Val Glu Pro Glu Ile Glu Lys Phe Val Lys Ser
Val Pro Tyr Gly Gly 245 250 255Gly Asp Arg Lys Phe Val Tyr Gly Val
Ala Leu Thr Pro Lys Gly Glu 260 265 270Tyr Tyr Lys Leu Glu Ser His
Tyr Lys Trp Lys Pro Gly Ala Val Asn 275 280 285Phe Ile
29023873DNAMicromonospora sp. strain 046-ECO11 23gtggccgagc
tctactcgac catcgaggaa tcggcccggc aactggacgt gccgtgttcg 60cgcgaccggg
tctggcccat cctgtccgcg tacggcgacg cgttcgccca tcccgaggcg
120gtggtcgcct tccgggtggc gaccgcgctg cgtcacgcgg gcgagctgga
ctgccggttc 180cggacgcatc cggacgaccg ggacccgtac gcctcggcgc
tcgcccgggg cctcaccccg 240cgcacggacc accccgtcgg cgcgctgctc
tccgaggtcc accggcgctg cccggtggag 300agccacggca tcgacttcgg
ggtggtcggc ggcttcaaga agatctacgc ggccttcgcc 360ccggacgagc
tgcaggtggc cacgtcgctc gccggcattc cggcgatgcc ccgcagcctc
420gccgcgaacg ccgacttctt cacccggcac ggcctcgacg accgggtcgg
cgtgctggga 480ttcgactacc cggcccggac cgtgaacgtc tacttcaacg
acgtgccgcg tgagtgcttc 540gagccggaga ccatccggtc gacgctgcgc
cggaccggga tggccgagcc gagcgagcag 600atgctccggc tcggcaccgg
ggcgttcggg ctctacgtca cgctgggctg ggactccccg 660gagatcgagc
ggatctgcta cgccgcggcg accacggacc tgaccacgct tccggtaccc
720gtggaaccgg agatcgagaa gttcgtgaaa agcgttccgt acggcggcgg
ggaccggaag 780ttcgtctacg gcgtggcgct gacccccaag ggggagtact
acaaactcga gtcgcactac 840aaatggaagc cgggcgcggt gaacttcatt tga
87324370PRTMicromonospora sp. strain 046-ECO11 24Val Trp Ala Arg
Val Lys Asn Trp Val Val Ala Leu Ala Val Ala Ala1 5 10 15Val Leu Met
Ile Ser Ala Leu Ala Gly Asp His Pro Ala Pro Glu Gly 20 25 30Leu Gly
Leu Leu Gly Phe Ala Leu Val Ala Ala Ser Gly Leu Ala Leu 35 40 45Ala
Ala Ser Arg Arg Ala Pro Ile Ala Val Leu Val Ala Thr Gly Leu 50 55
60Cys Val Val Gly Tyr Asn Ala Ile Gly Phe Gly Val Pro Ala Ile Ala65
70 75 80Tyr Leu Phe Ala Val Tyr Ala Ala Val Arg Ala Gly His Arg Leu
Val 85 90 95Thr Leu Gly Ala Ser Ala Ala Leu Leu Val Val Leu Pro Leu
Ala Ile 100 105 110Met Val Ser Pro Ala Asp Gly Ala Leu Lys Glu Ala
Leu Ala Gln Ser 115 120 125Arg Gly Val Leu Glu Leu Ala Trp Leu Ile
Ala Ala Ala Ala Ala Gly 130 135 140Glu Ala Leu Arg Gln Ala Glu Arg
Arg Ala Asp Glu Ala Glu Arg Thr145 150 155 160Arg Glu Glu Thr Ala
Arg Leu Arg Ala Thr Gln Glu Arg Leu His Ile 165 170 175Ala Arg Glu
Leu His Asp Ser Leu Thr His Gln Ile Ser Ile Ile Lys 180 185 190Val
Gln Ala Glu Val Ala Val His Leu Ala Arg Lys Arg Gly Glu Gln 195 200
205Val Pro Glu Ser Leu Leu Ala Ile Gln Glu Ala Gly Arg Ala Ala Thr
210 215 220Arg Glu Leu Arg Ala Thr Leu Glu Thr Leu Arg Asp Leu Thr
Lys Ser225 230 235 240Pro Ser His Gly Leu Asp His Leu Pro Glu Leu
Leu Ala Gly Ala Glu 245 250 255Lys Ile Gly Leu Ala Thr Thr Leu Thr
Ile Glu Gly Asp Gln Arg Asp 260 265 270Val Pro Glu Ala Val Gly Arg
Thr Ala Tyr Arg Ile Val Gln Glu Ser 275 280 285Leu Thr Asn Thr Ala
Arg His Ala Ser Ala Ala Ala Ala Ala Val Arg 290 295 300Ile Asp Tyr
Arg Pro Asp Ala Leu Ser Ile Arg Ile Asp Asp Asp Gly305 310 315
320Thr Ala Arg Pro Gly Ala Ala Pro Val Pro Gly Val Gly Leu Leu Gly
325 330 335Met His Glu Arg Val Leu Ala Leu Gly Gly Arg Leu Arg Ala
Glu Pro 340 345 350Arg Thr Gly Gly Gly Phe Thr Val Gln Ala Glu Leu
Pro Val Val Arg 355 360 365Val Pro 370251113DNAMicromonospora sp.
strain 046-ECO11 25gtgtgggccc gggtgaagaa ctgggtcgtc gcgttggctg
tggcggcggt gctgatgatc 60agcgcgctgg ccggtgacca tcctgccccc gagggcctcg
gtctgctcgg cttcgcgctg 120gtggcggcga gcggcctggc gctggccgcc
agtcgtcggg ccccgatcgc cgtgctggtc 180gccaccgggc tgtgcgtggt
gggctacaac gcgatcggct tcggggtgcc cgccatcgcg 240tacctgttcg
cggtctacgc ggcggtccgg gccgggcacc ggctcgtcac gctcggggcg
300agcgccgccc tgctcgtcgt cctgccgctg gcgatcatgg tctcgcccgc
ggacggcgcc 360ctcaaggagg cgctcgcgca gtcgcggggc gtgctggaac
tggcctggct gatcgccgcg 420gcggcggccg gtgaggcgct gcggcaggcc
gaacggcgag cggacgaggc ggaacggacc 480cgcgaggaga ccgcccggct
gcgcgccacc caggagcggc tgcacatcgc acgggagctg 540cacgactcgc
tcacccacca gatctcgatc atcaaggtgc aggcggaggt ggcggtccac
600ctggcccgca agcggggcga gcaggtgccg gagtcgctgc tggcgatcca
ggaggccggc 660cgggcggcga ctcgcgagct gcgcgcgacc ctggagacgc
tgcgtgacct gaccaagtcc 720ccgtcgcacg ggctcgacca cctcccggag
ctgctggccg gggccgagaa gatcggcctg 780gccaccacgc tgaccatcga
gggcgaccag cgggacgtgc cggaggcggt gggccgcacc 840gcgtaccgga
tcgtgcagga gtcgctcacc aacaccgccc ggcacgcctc cgccgcggcc
900gccgcggtcc ggatcgacta ccgcccggac gcgctgagca tccggatcga
cgacgacggg 960acggcccggc cgggcgccgc cccggtgccc ggcgtcgggc
tgctggggat gcacgagcgc 1020gtcctcgcgc tgggcggccg gctgcgggcg
gaaccccgca ccggcggagg cttcaccgtc 1080caggccgaac tcccggtggt
gcgcgtccca tga 111326220PRTMicromonospora sp. strain 046-ECO11
26Met Ile Arg Ile Met Leu Leu Asp Asp Gln Pro Leu Leu Arg Ser Gly1
5 10 15Phe Arg Ala Leu Leu Asp Ala Glu Asp Asp Ile Glu Val Val Ala
Glu 20 25 30Gly Gly Asn Gly Arg Glu Gly Leu Ala Leu Ala Arg Gln His
Leu Pro 35 40 45Asp Leu Ala Leu Ile Asp Ile Gln Met Pro Val Met Asp
Gly Val Glu 50 55 60Thr Thr Arg Gln Ile Val Ala Asp Pro Ala Leu Ala
Gly Val Arg Val65 70 75 80Val Ile Leu Thr Asn Tyr Gly Leu Asp Glu
Tyr Val Phe His Ala Leu 85 90 95Arg Ala Gly Ala Thr Gly Phe Leu Val
Lys Asp Ile Glu Pro Asp Asp 100 105 110Leu Leu His Ala Val Arg Val
Ala Ala Arg Gly Asp Ala Leu Leu Ala 115 120 125Pro Ser Ile Thr Arg
Met Leu Ile Asn Arg Tyr Val Ser Glu Pro Leu 130 135 140Cys Ala Asp
Val Thr Pro Gly Met Glu Glu Leu Thr Asn Arg Glu Arg145 150 155
160Glu Ala Val Ala Leu Ala Ala Arg Gly Leu Ser Asn Asp Glu Ile Ala
165 170 175Asp Arg Met Val Ile Ser Pro Leu Thr Ala Lys Thr His Val
Asn Arg 180 185 190Ala Met Thr Lys Leu Gln Ala Arg Asp Arg Ala Gln
Leu Val Val Phe 195 200 205Ala Tyr Glu Ser Gly Leu Val Ser Pro Gly
Asn Arg 210 215 22027663DNAMicromonospora sp. strain 046-ECO11
27atgatcagga tcatgctgct cgacgaccag ccgctgctgc gcagcgggtt ccgcgcgctc
60ctcgacgccg aggacgacat cgaggtggtg gccgagggcg ggaacggccg ggagggcctg
120gcgctggccc ggcagcacct gcccgatctc gccctgatcg acatccagat
gccggtcatg 180gacggcgtcg agacgacccg gcagatcgtc gcggatccgg
cgctggccgg ggtacgcgtc 240gtcatcctca ccaactacgg cctcgacgag
tacgtcttcc acgcgctgcg cgccggcgcc 300accggcttcc tggtcaagga
catcgagccg gacgacctgc tgcacgccgt gcgggtcgcc 360gcgcgcggtg
acgcgctgct cgcgccgtcg atcacccgga tgctgatcaa caggtacgtg
420tcggagccgc tctgcgcgga cgtcacgccc ggcatggagg agctgaccaa
ccgggaacgc 480gaggcggtcg ccctggccgc ccggggcctg tccaacgacg
agatcgccga tcgcatggtg 540atcagcccgc tgaccgcgaa gacccacgtc
aaccgcgcca tgaccaagct gcaggcccgc 600gaccgcgccc agctggtggt
gttcgcctac gagtccggcc tggtgtcacc cggcaatcgc 660tga
66328131PRTMicromonospora sp. strain 046-ECO11 28Met Phe Ile Arg
Arg Leu Leu Thr Ala Ala Ala Ala Gly Val Leu Gly1 5 10 15Gly Leu Ala
Leu Val Ala Pro Ala Ala Ala Gln Val Thr Ala Ala Asp 20 25 30Gly Asp
Gly Gly Ser Gly Arg Ala Gly Ser Val Leu Ala Leu Ala Leu 35 40 45Ala
Leu Leu Gly Leu Val Leu Gly Gly Trp Ala Leu Arg Ser Ala Gly 50 55
60Arg Gly Gly Gly Arg Gly Asn Ala Ile Ala Ala Leu Val Leu Ala Val65
70 75 80Ala Gly Leu Ile Ala Gly Val Val Ala Leu Ala Gly Ser Asp Gly
Gly 85 90 95Val Gly Ser Gly Asn Gly Arg Gly Gly Ala Ile Val Ala Val
Val Leu 100 105 110Ala Leu Ile Gly Ile Ala Val Gly Gly Leu Ala Phe
Thr Arg Ser Arg 115 120 125Arg Ala Ala 13029396DNAMicromonospora
sp. strain 046-ECO11 29atgttcatcc gtcgtttgct caccgccgcc gcagccggcg
tcctcggtgg gctcgcactc 60gtcgcaccgg cggccgcgca ggtgacggcc gccgacggtg
acggtggttc cggccgcgcc 120ggatccgtgc tggcgctcgc gctcgcgttg
ctcggcctcg tcctgggcgg gtgggcgttg 180cgctccgcgg ggcgcggcgg
cggtcgtggc aacgcgatcg ccgcgctggt gctcgcggtg 240gccggcctga
tcgccggcgt ggtcgccctg gccggctccg acggtggtgt cggcagcggc
300aacggccgtg gtggcgccat cgtggccgtc gtgctggcgc tgatcgggat
cgccgtcggc 360ggcctggcat tcacccgctc ccggcgcgcc gcctga
39630154PRTMicromonospora sp. strain 046-ECO11 30Met Arg Lys Val
Phe Ala Gly Leu Ala Ala Phe Leu Leu Leu Val Leu1 5 10 15Val Val Gln
Phe Phe Leu Ala Ala Ser Gly Ala Phe Ser Asn Glu Ala 20 25 30Asn Glu
Glu Ala Phe Arg Pro His Arg Ile Leu Gly Leu Gly Ser Ile 35 40
45Leu Val Ala Val Val Leu Thr Val Ala Ala Ala Val Met Arg Met Pro
50 55 60Gly Arg Ile Ile Gly Leu Ser Gly Leu Val Ala Gly Leu Gly Ile
Leu65 70 75 80Gln Ala Leu Ile Ala Val Ile Ala Lys Ala Phe Gly Asp
Ser Ala Gly 85 90 95Asp Ser Ala Val Gly Arg Tyr Val Phe Gly Leu His
Ala Val Asn Gly 100 105 110Leu Val Met Val Ala Val Ala Arg Val Ile
Leu Arg Ser Val Arg Ala 115 120 125Ala Pro Asp Thr Thr Thr Thr Pro
Gly Val Asp Thr Thr Val Thr Gly 130 135 140Pro Ala Ala Asp Ser Ala
Arg Thr Ala Ser145 15031465DNAMicromonospora sp. strain 046-ECO11
31atgcgcaaag tgttcgccgg actggcagcg ttcctgctgc tcgtgctcgt ggtgcagttc
60ttcctggccg ccagcggcgc gttcagcaac gaggccaacg aggaggcgtt ccgccctcac
120cggatcctgg gcctggggag catcctcgtc gccgtggtgc tgacggtggc
cgccgcggtg 180atgcggatgc ccggccggat catcggcctg tccggcctgg
tcgccgggct gggcatcctg 240caggccctga tcgcggtcat cgccaaggcg
ttcggcgact cggccggtga ctcggccgtc 300ggccggtacg tgttcggcct
gcacgcggtc aacggactgg tgatggtggc cgtcgcccgc 360gtcatcctgc
gcagcgtccg ggcggcgccg gacacgacca ccacgcccgg cgtggacacg
420acggtcaccg gtccggcggc cgactcggcg cgaacggcgt catga
46532661PRTMicromonospora sp. strain 046-ECO11 32Met Ser Thr Leu
Gln Trp Ile Leu Val Asp His Val Val Ala Leu Leu1 5 10 15Gly Val Ala
Thr Trp Phe Ala Thr Gly Val Thr Ala Ala Leu Gly Arg 20 25 30His Arg
Ile Ala Leu Ala Leu Leu Gly Ala Ala Val Leu Val Thr Val 35 40 45Ala
Arg Leu Gly Thr Val Ala Leu Leu Ala Asp Arg Gly Trp Trp Phe 50 55
60Val Gln Glu Lys Val Leu Leu Gly Leu Pro Met Leu Gly Ala Ala Gly65
70 75 80Leu Val Ala Val Leu Leu Ala Gly Pro Arg Leu Leu Ala Ala Arg
Gln 85 90 95Ser Pro Ala Ala Asp Leu Pro Ala Gly Ala Leu Val Ala Val
Leu Thr 100 105 110Ala Gly Phe Ala Ala Leu Ala Gly Leu Val Val Thr
Phe Thr Ala Gly 115 120 125Tyr Pro Leu Thr Trp Ser Thr Ala Leu Ile
Ala Val Ala Leu Val Cys 130 135 140Ala Ala Ala Leu Leu Thr Ala Arg
Val Val Gly Arg Pro Ala Ala Pro145 150 155 160Ala Ala Glu Ala Gly
Ser Pro Glu His Thr Pro Ala Ala Ala Gly Pro 165 170 175Thr Ala Leu
Ser Arg Arg Arg Phe Leu Gly Val Ala Gly Gly Val Val 180 185 190Ala
Ala Gly Ala Gly Ala Thr Gly Val Gly Leu Leu Phe Arg Asp Pro 195 200
205Glu Ala Met Val Thr Gly Gly Gly Pro Gly His Ala Gly Gly Ala Arg
210 215 220Pro Lys Val Ser Val Ala Asp Leu Arg Gly Pro Gly Ala Pro
Ala Ala225 230 235 240Gly Gly Thr Ala Arg Arg His Val Leu Thr Ala
Arg Thr Gly Thr Val 245 250 255Thr Ile Pro Ser Gly Arg Pro Ile Asp
Ala Trp Ser Tyr Glu Gly Arg 260 265 270Leu Pro Gly Pro Ala Ile Thr
Ala Thr Glu Gly Asp Leu Ile Glu Val 275 280 285Thr Leu Arg Asn Ala
Asp Ile Glu Asp Gly Val Thr Val His Trp His 290 295 300Gly Tyr Asp
Val Pro Cys Gly Glu Asp Gly Ala Pro Gly Ala Thr Gln305 310 315
320His Ala Val Gln Pro Gly Gly Glu Phe Val Tyr Arg Phe Gln Ala Asp
325 330 335Gln Val Gly Thr Tyr Trp Tyr His Thr His Gln Ala Ser His
Pro Ala 340 345 350Val Arg Lys Gly Leu Tyr Gly Thr Leu Val Val Thr
Pro Arg Glu Asp 355 360 365Arg Pro Glu Ala Glu Arg Gly Leu Asp Leu
Thr Leu Pro Val His Thr 370 375 380Phe Asp Asp Val Thr Ile Leu Gly
Asp Gln Glu Gly Arg Ala Val His385 390 395 400Asp Val Arg Pro Gly
Gln Pro Val Arg Leu Arg Leu Ile Asn Thr Asp 405 410 415Ser Asn Pro
His Trp Phe Ala Val Val Gly Ser Pro Phe Arg Val Val 420 425 430Ala
Val Asp Gly Arg Asp Leu Asn Gln Pro Gly Glu Val Arg Glu Val 435 440
445Gly Leu Arg Leu Pro Ala Gly Gly Arg Tyr Asp Leu Thr Leu Ala Met
450 455 460Pro Asp Ala Lys Val Thr Leu Leu Leu Asp Asn Asp Ser Asp
Gln Gly465 470 475 480Val Leu Leu Arg Pro Pro Gly Val Gly Gly Gly
Asp Arg Pro Leu Pro 485 490 495Asp Thr Ala Asp Trp Pro Glu Phe Asp
Leu Leu Gly Tyr Gly Glu Pro 500 505 510Ala Pro Val Pro Phe Asp Ala
Asp Asp Ala Asp Arg His Phe Thr Ile 515 520 525Val Leu Asp Arg Ala
Leu Ala Met Val Asp Gly Lys Pro Ala Tyr Ala 530 535 540Gln Thr Val
Asp Gly Arg Ala His Pro Ser Val Pro Asp Gln Leu Val545 550 555
560Arg Glu Gly Asp Val Val Arg Phe Thr Val Val Asn Arg Ser Leu Glu
565 570 575Thr His Pro Trp His Leu His Gly His Pro Val Leu Ile Leu
Ser Arg 580 585 590Asp Gly Arg Pro Tyr Ser Gly Ser Pro Leu Trp Met
Asp Thr Phe Asp 595 600 605Val Arg Pro Gly Glu Val Trp Glu Val Ala
Phe Arg Ala Asp Asn Pro 610 615 620Gly Val Trp Met Asn His Cys His
Asn Leu Pro His Gln Glu Gln Gly625 630 635 640Met Met Leu Arg Leu
Val Tyr Asp Gly Val Thr Thr Pro Phe Ala Ser 645 650 655Thr Ser His
Ala His 660331986DNAMicromonospora sp. strain 046-ECO11
33atgagcacgc tccaatggat cctcgtggac cacgtcgtgg cgctgctcgg tgtcgcgacg
60tggttcgcaa cgggtgtcac ggcagctctc ggccgccacc ggatcgcgtt ggcgctcctc
120ggcgccgcgg tgctggtgac agtcgcccgc ctgggcaccg tggcgctgct
ggccgaccgc 180ggctggtggt tcgtccagga gaaggttctg ctggggctgc
cgatgctcgg cgccgcgggg 240ctcgtcgcgg tgctcctggc cggcccgcgc
ctgctcgcgg cccggcagtc accggcggcg 300gacctgccgg ccggcgcgct
ggtcgcggtg ctgaccgccg gcttcgccgc gctggccggc 360ctggtggtga
cgttcaccgc cgggtacccg ctgacgtgga gcaccgcgct gatcgccgtc
420gccctcgtct gcgccgccgc gctgctcacc gcgcgggtgg tcggacgacc
cgccgccccg 480gccgcggagg ccggctcccc ggagcacacg ccggcggcgg
ccgggcccac ggcgctgtcc 540cgccgccggt tcctcggcgt ggccggggga
gtggtcgcgg cgggcgccgg cgccaccggc 600gtcggcctgc tcttccgcga
cccggaggcg atggtcaccg gaggcggccc cggacacgcc 660ggtggcgccc
gccccaaggt ctccgtggcg gacctgcgcg gccccggcgc tccggcggcg
720ggcggcacgg cgcgacgcca cgtgctcacc gcccggacgg gcaccgtcac
gattccgtcc 780ggacgtccga tcgacgcctg gagctacgag ggccgcctgc
ccgggccggc catcaccgcg 840accgagggcg acctgatcga ggtgacgctc
cgcaacgccg acatcgagga cggcgtcacc 900gtgcactggc acgggtacga
cgtgccgtgc ggcgaggacg gcgcgccggg cgccacgcag 960cacgcggtgc
agcccggcgg cgagttcgtc taccggttcc aggcggacca ggtggggacg
1020tactggtacc acacccacca ggcgtcgcac cccgccgtgc gcaaagggct
gtacgggacg 1080ctcgtcgtga cgccgcgcga ggaccggccg gaagcggagc
gcgggctgga cctgacgctg 1140ccggtgcaca cgttcgacga cgtcacgatc
ctcggcgacc aggagggacg cgccgtccac 1200gacgtccgcc ccggccagcc
ggtgcgactg cgtctgatca acaccgactc caacccgcac 1260tggttcgccg
tcgtcggctc gcccttccgc gtggtggccg tcgacggccg cgacctcaac
1320cagccgggcg aggtacgcga ggtcgggctc cgcctgcccg ccggaggccg
gtacgacctg 1380accctggcca tgccggacgc caaggtcacg ctgctgctcg
acaacgactc cgaccagggc 1440gtcctgctgc gcccgccggg cgtcggcggt
ggtgaccgcc cgctgccgga caccgccgac 1500tggcccgagt tcgacctgct
gggctacggc gagccggcgc ccgtgccgtt cgacgccgac 1560gacgccgacc
gccacttcac catcgtcctc gaccgggccc tggccatggt cgacggcaag
1620cccgcgtacg cccagaccgt cgacggtcgc gcacatccct ccgtccccga
ccagctcgtc 1680cgggaggggg acgtcgtgcg cttcacggtg gtcaaccgga
gcctcgaaac ccacccgtgg 1740cacctgcacg gccatccggt gctgatcctg
tcccgcgacg gccggccgta ctccggcagc 1800ccgctgtgga tggacacctt
cgacgtgcgg ccgggagagg tgtgggaggt ggcgttccgg 1860gcggacaatc
cgggtgtctg gatgaaccac tgccacaacc tgccgcacca ggagcagggc
1920atgatgctgc ggctcgtcta cgacggtgtc accacgccct tcgccagcac
gagccacgca 1980cactga 198634129PRTMicromonospora sp. strain
046-ECO11 34Met Thr Ala Asp Leu His Gly Leu Ala Ser Val Arg Tyr Ile
Val Asp1 5 10 15Asp Val Ser Ala Ala Ile Glu Phe Tyr Thr Thr His Leu
Gly Phe Thr 20 25 30 Val Ser Thr Ala Phe Pro Pro Ala Phe Ala Asp
Val Val Arg Gly Pro 35 40 45Leu Arg Leu Leu Leu Ser Gly Pro Thr Ser
Ser Gly Ala Arg Val Thr 50 55 60Pro Ala Asp Ala Ala Gly Cys Gly Arg
Asn Arg Ile His Leu Ile Val65 70 75 80Asp Asp Leu Asp Ala Glu Arg
Glu Arg Leu Glu Arg Ala Gly Val Thr 85 90 95Leu Arg Ser Asp Val Val
Ala Gly Pro Gly Gly Arg Gln Phe Leu Ile 100 105 110Ala Asp Pro Ala
Gly Asn Leu Val Glu Val Phe Glu Pro Ala Ala Arg 115 120
125Gly35390DNAMicromonospora sp. strain 046-ECO11 35atgaccgcag
acctgcacgg cctggccagc gtccgctaca tcgtcgacga cgtgtcggcg 60gcgatcgagt
tctacaccac ccacctgggt ttcacggtgt cgaccgcgtt cccgccggcc
120ttcgccgacg tggtgcgcgg gccgctgcgg ctcctgctgt ccgggccgac
cagctcgggc 180gcccgggtca ccccggcgga cgcggccggg tgcgggcgca
accgcatcca cctgatcgtc 240gacgatctcg acgccgaacg ggagcggctg
gagcgcgccg gggtgacgtt gcgcagcgac 300gtcgtggccg ggccgggcgg
ccgtcagttc ctgatcgccg acccggcggg caacctggtc 360gaggtgttcg
agccggcagc ccgcggctga 39036178PRTMicromonospora sp. strain
046-ECO11 36Met Leu Thr Ala Val Val Ala Ser Pro His Ser Pro Glu Asn
Thr Ser1 5 10 15Arg His Pro Thr Gly Gly Asp Ala Val Asp Glu Ala Thr
Pro Arg Thr 20 25 30Pro Val Ala Ala Arg Pro Thr Trp Ser Pro Ala Thr
Ala Pro Val Trp 35 40 45Leu Val Gly Val Leu Ala Thr Leu Ala Gly Ala
Val Ala Ala Glu Ala 50 55 60Phe Thr Leu Ala Ala Arg Gly Phe Gly Val
Pro Met Glu Ala Ala Gly65 70 75 80Val Trp Glu Glu Gln Ala Gln Ala
Ile Pro Val Gly Ala Ile Ala Arg 85 90 95Ser Val Val Leu Trp Ser Ile
Gly Gly Ile Val Leu Ala Val Val Val 100 105 110Ala Arg Arg Ala Arg
Arg Pro Val Arg Ala Phe Val Ala Gly Thr Val 115 120 125Ala Phe Thr
Val Leu Ser Leu Ala Ala Pro Ala Phe Ala Arg Asp Thr 130 135 140Pro
Val Ser Thr Gln Leu Val Leu Ala Gly Thr His Val Ile Ala Gly145 150
155 160Ala Val Ile Ile Ser Ile Leu Ala Ala Arg Leu Ala Ala Pro Thr
Pro 165 170 175Pro Arg37537DNAMicromonospora sp. strain 046-ECO11
37atgttgactg ccgtcgtggc gtccccgcat tctcccgaga acacatcgag gcacccgacc
60ggaggcgacg ccgtggatga ggccactccc cgaactcccg tcgcggcacg gcccacctgg
120tcgccggcca ccgctccggt gtggctggtc ggcgtgctgg ccaccctcgc
cggggccgtg 180gccgcggagg cgttcacgct cgccgcccgg ggcttcggcg
taccgatgga ggcggccggc 240gtctgggagg agcaggcgca ggcgatcccg
gtgggggcca tcgcccgcag cgtcgtgctc 300tggtcgatcg gcggaatcgt
cctggcggtg gtcgtggcgc ggcgggcccg gcggcccgtg 360cgtgccttcg
tggccggcac cgtcgcgttc accgtgctgt ccctcgccgc gcccgccttc
420gcccgggaca ccccggtgtc gacgcagctc gtcctcgccg gcacccacgt
gatcgccggc 480gccgtgatca tctccatcct ggccgcgcgg ctcgccgcgc
ccaccccgcc ccggtaa 53738661PRTMicromonospora sp. strain 046-ECO11
38Met Asp Gly Thr Glu Ser Asn Val Thr Gly Phe Pro Asp Leu Leu Ser1
5 10 15Gly Leu Gly Gly Asp Gly Arg Ala Phe Ala Leu Leu His Arg Pro
Gly 20 25 30Ala Ala Gly Cys Ala Tyr Val Glu Val Leu Thr Gly Glu Val
Cys Asp 35 40 45Val Asp Thr Leu Gly Glu Leu Pro Leu Pro Thr Glu Pro
Ala Thr Gly 50 55 60Ala Arg His Asp Leu Leu Val Ala Val Pro Tyr Arg
Gln Val Thr Glu65 70 75 80Arg Gly Phe Asp Cys His Asp Asp Gly Ala
Pro Leu Leu Ala Met Arg 85 90 95Val His Glu Gln Phe Gly Leu Asp Arg
Gly Gln Ala Leu Ala Gly Leu 100 105 110Pro Glu Arg Gly Val Pro Val
Thr Asp Ala Asp Phe Asp Leu Ser Asp 115 120 125Glu Asp Tyr Ala Ala
Ile Val Lys Arg Val Val Gly Asp Glu Ile Gly 130 135 140Leu Gly Ala
Gly Ser Asn Phe Val Ile Arg Arg Thr Phe Thr Ala Arg145 150 155
160Leu Ala Asp Tyr Ser Ile Ala Thr Glu Leu Ala Leu Phe Arg Arg Leu
165 170 175Leu Thr Gly Glu Leu Gly Ser Tyr Trp Thr Phe Leu Phe His
Ser Gly 180 185 190Ala Gly Thr Phe Ile Gly Ala Ser Pro Glu Arg His
Val Ser Met Ile 195 200 205Asp Gly Thr Val Ser Met Asn Pro Ile Ser
Gly Thr Tyr Arg His Pro 210 215 220Pro Asn Gly Pro Ala Val Ser Gly
Leu Leu Glu Phe Leu Asn Asp Pro225 230 235 240Lys Glu Ala Asn Glu
Leu Tyr Met Val Val Asp Glu Glu Leu Lys Met 245 250 255Met Ala Arg
Met Cys Ala Ser Gly Gly Gln Val His Gly Pro Phe Leu 260 265 270Lys
Glu Met Ala Arg Val Thr His Ser Glu Tyr Ile Leu Thr Gly Arg 275 280
285Ser Asp Leu Asp Val Arg Asp Val Leu Arg Glu Thr Leu Leu Ala Pro
290 295 300Thr Val Thr Gly Ser Pro Ile Glu Asn Ala Phe Arg Val Ile
Thr Arg305 310 315 320His Glu Thr Thr Gly Arg Gly Tyr Tyr Gly Gly
Val Leu Ala Leu Met 325 330 335Gly Arg Asp Ser Ala Gly Ser Arg Thr
Leu Asp Ser Ala Ile Met Ile 340 345 350Arg Thr Ala Glu Ile Asp Asp
Ala Gly Thr Leu Arg Leu Gly Val Gly 355 360 365Ala Thr Leu Val Arg
Asp Ser Lys Pro Glu Ser Glu Val Ala Glu Thr 370 375 380Arg Ala Lys
Ala Gly Ala Met Arg Ala Ala Leu Gly Leu Gly Val Asp385 390 395
400Pro Asp Gly Pro Asp Gly Gly Arg Thr Thr Ala Ala Arg Ala Arg Ser
405 410 415Ser Leu Ala Thr Asp Pro Arg Val Arg Arg Ala Leu Arg Glu
Arg Asn 420 425 430Thr Thr Leu Ser Arg Phe Trp Leu Asp Gly Ala Glu
Arg Arg Thr Pro 435 440 445Asn Pro Ala Leu Thr Gly Arg Arg Val Leu
Val Val Asp Asn Glu Asp 450 455 460Thr Phe Met Ala Met Leu Asp His
Gln Leu Arg Ala Leu Gly Leu Arg465 470 475 480Ser Ser Ile Ala Arg
Phe Asp Ser Arg Leu Arg Pro Asp Gly His Asp 485 490 495Leu Val Val
Val Gly Pro Gly Pro Gly Asp Pro Gly Asp Leu Thr Asp 500 505 510Pro
Arg Met Arg Thr Leu Arg Gly Leu Thr Arg Asp Leu Leu Ala Gly 515 520
525Thr Val Pro Phe Leu Ser Ile Cys Leu Gly His Gln Val Leu Ala Ala
530 535 540Glu Leu Gly Phe Pro Leu Ala Arg Arg Ala Val Pro Asn Gln
Gly Val545 550 555 560Gln Lys Arg Ile Asp Leu Phe Gly Arg Pro Glu
Leu Val Gly Phe Tyr 565 570 575Asn Thr Tyr Thr Ala Arg Ser Ala His
Asp Val Val Ala Gly Gly Arg 580 585 590Arg Gly Pro Ile Glu Ile Ser
Arg Ser Pro Asp Ser Gly Asp Val His 595 600 605Ala Leu Arg Gly Pro
Gly Phe Arg Ser Val Gln Phe His Leu Glu Ser 610 615 620Val Leu Thr
Gln His Gly Pro Arg Ile Leu Gly Asp Leu Leu Val Ser625 630 635
640Leu Leu Ala Asp Gly Thr Ala Ala Ala Ala Ala Glu Ala Ala Gly Arg
645 650 655Arg Gly Asn Arg Pro 660391986DNAMicromonospora sp.
strain 046-ECO11 39atggacggga cggaatcgaa cgtgaccgga ttccccgatc
tgctgtccgg tctcggcggc 60gacgggcgcg ccttcgccct gctgcaccgg cccggcgcgg
ccgggtgcgc gtacgtggag 120gttctgaccg gcgaggtgtg cgacgtggac
actctcggcg agctgcccct gcccaccgag 180ccggcgaccg gcgcgcggca
cgacctgctc gtggcggtgc cgtaccggca ggtcaccgaa 240cgggggttcg
actgccacga cgacggcgcg ccgctgctcg cgatgcgcgt ccacgagcag
300ttcgggctcg accgcggaca ggcgctggcg ggcctgcccg aacgcggtgt
gccggtgacc 360gacgccgact tcgacctcag cgacgaggac tacgccgcga
tcgtcaagcg ggtggtgggt 420gacgagatcg ggctgggcgc cggatccaac
ttcgtcatcc ggcgcacctt caccgcgcgg 480ctggccgact actcgatcgc
cacggaactg gcgctcttcc gccggttgct gaccggcgaa 540ctgggttcct
actggacgtt tctgttccac tccggcgccg gcacgttcat cggcgcgtca
600ccggaacgac acgtcagcat gatcgacgga accgtctcga tgaatcccat
cagcgggacc 660taccggcacc ccccgaacgg
cccggccgtt tccggtctgc tggaattcct gaacgacccg 720aaagaggcta
acgaactcta catggtcgtc gacgaggaac tgaaaatgat ggcgcggatg
780tgcgcctccg gcggccaggt gcacggcccg ttcctcaagg aaatggcgcg
ggtgacgcac 840tccgagtaca tcctgaccgg ccgcagcgac ctggacgtgc
gcgacgtgct gcgggagacc 900ctgctcgcgc cgacggtcac cggcagcccg
atcgagaacg cgttccgggt catcacccgc 960cacgagacga ccggccgcgg
ctactacggc ggcgtgctcg cgttgatggg ccgtgactcg 1020gccggcagcc
gtacgctcga ctcggccatc atgatccgca ccgccgagat cgacgacgcg
1080ggcacgctgc gcctgggcgt cggcgccacc ctcgtgcggg actccaagcc
ggagtcggag 1140gtggccgaga cgcgggccaa ggcgggcgcc atgcgcgcgg
cgctcggcct cggcgtcgac 1200ccggacggcc cggacggcgg gcggaccacg
gccgcgcggg ctcgttcgtc cctggccacc 1260gacccccggg tacggcgggc
gttgcgcgag cgcaacacca cactgtcgag gttctggctc 1320gacggcgcgg
agcggcgcac cccgaacccg gcgctgaccg gacgccgcgt gctcgtcgtc
1380gacaacgagg acacgttcat ggccatgctc gaccaccagt tgcgggccct
cgggctgcgg 1440tcgagcatcg cccggttcga cagccggctg cggccggacg
gacacgacct cgtcgtcgtc 1500ggtcccggcc ccggcgaccc gggcgacctg
accgacccgc gtatgcggac cctgcgcggg 1560ctcacccgcg acctgctcgc
cggaacggtg ccgttcctgt ccatctgcct gggccaccag 1620gtgctcgccg
ccgaactggg gttccccctc gcccggcgcg cggtgcccaa ccagggtgtg
1680cagaagcgga tcgacctgtt cggccggccg gaactcgtgg ggttctacaa
cacctacacc 1740gcccgctccg cgcacgacgt ggtggccggt ggccggcggg
gcccgatcga gatcagccgc 1800agcccggaca gcggggacgt gcacgcgctg
cgcggcccgg gattccgttc cgtccagttc 1860cacctggagt ccgtcctcac
ccagcacggc ccacggatcc tgggcgacct gctggtctcc 1920ctgctcgccg
acggcacggc cgccgccgcg gccgaggcgg cgggccggcg cgggaaccgc 1980ccgtga
198640427PRTMicromonospora sp. strain 046-ECO11 40Val Lys Thr Thr
Val Asp Val Leu Val Gln Lys Tyr Gly Gly Thr Ser1 5 10 15Leu Gln Thr
Leu Asp Arg Val Arg His Ala Ala Leu Arg Ile Ala Glu 20 25 30Ala Arg
Arg His Gly Ser Ala Val Thr Val Val Val Ser Ala Arg Gly 35 40 45Ser
Arg Thr Asp Asp Leu Leu Arg Leu Ala Ala Asp Val Gly Ala Ala 50 55
60Gly Pro Ser Arg Glu Leu Asp Gln Leu Leu Ala Val Gly Glu Ser Glu65
70 75 80Ser Ala Ala Leu Met Ala Leu Ala Leu Thr Gly Leu Gly Val Pro
Ala 85 90 95Val Ser Leu Thr Gly His Gln Ala Glu Ile His Thr Thr Asp
Arg His 100 105 110Gly Asp Ala Leu Ile Ser Arg Ile Gly Ala Ala Arg
Val Glu Ala Ala 115 120 125Leu Gly Arg Gly Glu Val Ala Val Val Thr
Gly Phe Gln Gly Ile Asp 130 135 140Arg Ala Gly Asp Val Ala Thr Leu
Gly Arg Gly Gly Ser Asp Thr Thr145 150 155 160Ala Val Ala Leu Ala
Ala Arg Leu Arg Ala Ser Ala Cys Glu Ile Tyr 165 170 175Thr Asp Val
Asp Gly Val Phe Ser Ala Asp Pro Arg Ile Leu Pro Ala 180 185 190Ala
Arg Cys Leu Pro Trp Val Glu Pro Gly Val Met Ala Glu Met Ala 195 200
205Phe Ala Gly Ala Arg Val Leu His Thr Arg Cys Ile Glu Leu Ala Ala
210 215 220Met Glu Gly Val Glu Val Arg Val Arg Asn Ala Ser Ser Gln
Ala Pro225 230 235 240Gly Thr Ile Val Val Asp Arg Pro Asp Asp Arg
Pro Leu Glu Thr Arg 245 250 255Arg Ala Val Val Ala Val Thr His Asp
Thr Asp Val Val Arg Val Leu 260 265 270Val His Cys Arg Asp Gly Arg
Arg Asp Met Ala Pro Asp Val Phe Glu 275 280 285Val Leu Ala Ala His
Gly Ala Val Ala Asp Leu Val Ala Arg Ser Gly 290 295 300Pro Tyr Glu
Ser Glu Phe Arg Met Gly Phe Thr Ile Arg Arg Ser Gln305 310 315
320Ala Glu Ala Val Arg Thr Ala Leu His Asp Leu Thr Ala Ser Phe Asp
325 330 335Gly Gly Val His Phe Asp Glu Asn Val Gly Lys Val Ser Val
Val Gly 340 345 350Met Gly Leu Leu Ser Arg Pro Glu His Thr Ala Arg
Leu Met Ala Ala 355 360 365Leu Ala Ala Ala Gly Ile Ser Thr Ser Trp
Ile Ser Thr Ser Gln Met 370 375 380Arg Leu Ser Val Ile Val Ser Arg
Asp Arg Thr Val Asp Ala Val Glu385 390 395 400Ala Leu His Arg Ala
Phe Arg Leu Asp Arg Ser Glu Pro Ala Asp Ala 405 410 415Thr Ser Leu
Thr Ser Arg Arg Ser Ala Thr Ala 420 425411284DNAMicromonospora sp.
strain 046-ECO11 41gtgaagacga ctgtggacgt gctggtccag aaatacgggg
gcacctcgct gcagaccctc 60gaccgcgttc ggcacgccgc gctgcggatc gccgaggcgc
ggcggcacgg ctccgccgtg 120acagtggtcg tgtcggcgcg cggcagccgg
accgacgacc tgctgcggct ggcggccgac 180gtcggcgccg cgggtccgtc
ccgggaactc gaccagttgc tcgcagtcgg cgagtccgag 240tcggcggcgc
tgatggcgct ggcgttgacc gggctgggag tgccggccgt ctcgctgacc
300gggcaccagg cggagatcca caccaccgac cggcacggcg acgcgctgat
ctcgcggatc 360ggggcggcgc gggtggaagc ggcgctgggc cgtggcgagg
tcgccgtggt caccggattc 420cagggcatcg accgggccgg tgacgtcgcc
acgctggggc gcggcggctc cgacacgaca 480gcggtggcgc tcgcggcccg
gctccgcgcg tcggcgtgcg agatctacac cgacgtggac 540ggcgtcttca
gcgccgaccc ccgcatcctt ccggcggcgc gttgcctgcc gtgggtggag
600cccggcgtca tggcggagat ggcgttcgcc ggcgcgcggg tcctgcacac
ccgatgcatc 660gagctggccg ccatggaagg ggtcgaagtg cgcgtgcgca
acgcgtcgtc gcaggcgccc 720ggaacgatag tcgtggaccg gcccgacgac
cggccgctgg agacccggcg ggccgtggtg 780gcggtcaccc acgacaccga
tgtcgtccgc gtgctggtgc actgccgcga cggccgccgg 840gacatggcac
ccgacgtgtt cgaggtgctg gccgcccatg gggcggtggc ggacctggtg
900gcccggtccg ggccctacga gagcgagttc cggatggggt tcaccatccg
ccgcagccag 960gccgaagcgg tgcggaccgc gctgcacgac ctcaccgcgt
ccttcgacgg cggggtccac 1020ttcgacgaga acgtcggcaa ggtgtccgtg
gtcggcatgg gcctgctcag ccgccccgag 1080cacacggccc ggctgatggc
ggcgctggcc gcggcgggga tctcgacgag ctggatctcc 1140acctcccaga
tgcggctgtc ggtgatcgtg tcgcgggacc gcaccgtcga cgccgtcgaa
1200gccctgcacc gcgcgttccg cctggaccgg tccgagccgg cggacgccac
gtccctgacc 1260tcccgccgtt ccgccaccgc ctga
128442274PRTMicromonospora sp. strain 046-ECO11 42Val Ala Val Leu
Asn Ala Ser Phe Ala Arg Gly Leu Arg Leu Arg Arg1 5 10 15Leu Phe Arg
Arg Gly Asp Gly Arg Leu Leu Val Val Pro Leu Asp His 20 25 30Ser Val
Thr Asp Gly Pro Leu Arg Arg Gly Asp Leu Asn Ser Leu Leu 35 40 45Gly
Glu Leu Ala Gly Thr Gly Val Asp Ala Val Val Leu His Lys Gly 50 55
60Ser Leu Arg His Val Asp His Gly Trp Phe Gly Asp Met Ser Leu Ile65
70 75 80Val His Leu Ser Val Ser Thr Arg His Ala Pro Asp Pro Asp Ala
Lys 85 90 95Tyr Leu Val Ala His Val Glu Glu Ala Leu Arg Leu Gly Ala
Asp Ala 100 105 110Val Ser Val His Val Asn Leu Gly Ser Pro Gln Glu
Ala Arg Gln Ile 115 120 125Ala Asp Leu Ala Ala Val Ala Gly Glu Cys
Asp Arg Trp Asn Val Pro 130 135 140Leu Leu Ala Met Val Tyr Ala Arg
Gly Pro Gln Ile Thr Asp Ser Arg145 150 155 160Ala Pro Glu Leu Val
Ala His Ala Ala Thr Leu Ala Ala Asp Leu Gly 165 170 175Ala Asp Ile
Val Lys Thr Asp Tyr Val Gly Thr Pro Glu Gln Met Ala 180 185 190Glu
Val Val Arg Gly Cys Pro Ile Pro Leu Ile Val Ala Gly Gly Pro 195 200
205Arg Ser Ala Asp Thr Pro Thr Val Leu Ala Tyr Val Ser Asp Ala Leu
210 215 220Arg Gly Gly Val Ala Gly Met Ala Met Gly Arg Asn Val Phe
Gln Ala225 230 235 240Glu Gln Pro Gly Leu Met Ala Ala Ala Val Ala
Arg Leu Val His Glu 245 250 255Pro Arg His Val Pro Asp Arg Tyr Asp
Val Asp Asp Arg Leu Ala Leu 260 265 270Thr Ser
43825DNAMicromonospora sp. strain 046-ECO11 43gtggccgtac tcaacgcttc
gttcgctcgt ggcctgcgtc tgcgccgact gttccgacgc 60ggcgacggac gcctgctcgt
cgtcccgctc gaccactccg tcaccgacgg gccgctgcgc 120cgcggcgacc
tgaactcgct gctcggtgag ctcgccggca ccggcgtgga cgccgtggtg
180ctgcacaagg gcagcctgcg gcacgtcgac cacggctggt tcggcgacat
gtcgctgatc 240gtgcatctga gcgtgagcac ccggcacgcc ccggacccgg
acgcgaagta cctggtcgcg 300cacgtggagg aggcgctgcg gctgggcgcc
gacgcggtca gcgtgcacgt caacctcggc 360tcaccgcagg aggcgcggca
gatcgccgac ctggcggcgg tggcggggga gtgcgaccgc 420tggaacgtcc
cgctgctggc catggtgtac gcccgcgggc cgcagatcac cgactcccgg
480gcaccggagc tggtggcgca cgccgcgacg ctcgccgcgg acctcggcgc
cgacatcgtc 540aagaccgact acgtgggcac gcccgagcag atggccgagg
tggtgcgcgg ctgcccgatc 600ccgctgatcg tggccggcgg cccgcgctcg
gccgacactc cgacggtgct cgcctacgtc 660tcggacgcgc tgcgcggcgg
cgtggccggg atggccatgg gccgcaacgt gttccaggcc 720gagcagcccg
gcctgatggc cgccgccgtg gcacggctgg tgcacgagcc acggcacgtg
780ccggaccggt acgacgtcga cgaccggctc gcccttacgt cctga
82544367PRTMicromonospora sp. strain 046-ECO11 44Val Lys Leu Cys
Trp Leu Asp Ile Arg Asn Val Asn Gly Ala Lys Glu1 5 10 15Ala Ile Val
Glu Glu Ala Val His Gln Arg Val Asp Ala Val Val Ala 20 25 30Ala Asp
Pro Ala Asp Leu Glu Thr Leu Pro Pro Thr Val Lys Lys Val 35 40 45Leu
Phe Pro Gln Gly Gly Pro Leu Pro Glu Lys Leu Glu Pro Ala Asp 50 55
60Leu Val Ile Val Glu Pro Ala Arg His Gly Glu Pro Ala Glu Leu Ala65
70 75 80Ala Arg Tyr Pro Glu Val Glu Phe Gly Arg Phe Val Glu Ile Val
Asp 85 90 95Ala Asp Ser Leu Glu Asp Ala Cys Arg Ser Ala Arg His Asp
Arg Trp 100 105 110Ser Leu Leu Tyr Phe Arg Asp Pro Thr Lys Ile Pro
Leu Glu Ile Val 115 120 125Leu Ala Ala Ala Ala Gly Ala Glu Gly Ser
Ile Ile Thr Gln Val Ala 130 135 140Asp Val Glu Glu Ala Glu Ile Val
Phe Gly Val Leu Glu His Gly Ser145 150 155 160Asp Gly Val Met Leu
Ala Pro Arg Ala Val Gly Glu Ala Thr Glu Leu 165 170 175Arg Thr Ala
Ala Val Ser Thr Ala Ala Asp Leu Ser Leu Val Glu Leu 180 185 190Glu
Val Thr Gly Ile Arg Arg Val Gly Met Gly Glu Arg Ala Cys Val 195 200
205Asp Thr Cys Thr Asn Phe Arg Leu Asp Glu Gly Ile Leu Val Gly Ser
210 215 220His Ser Thr Gly Met Ile Leu Cys Cys Ser Glu Thr His Pro
Leu Pro225 230 235 240Tyr Met Pro Thr Arg Pro Phe Arg Val Asn Ala
Gly Ala Leu His Ser 245 250 255Tyr Thr Leu Ser Ala Gly Gly Arg Thr
Asn Tyr Leu Ser Glu Leu Val 260 265 270Ser Gly Gly Arg Val Leu Ala
Val Asp Ser Gln Gly Lys Ser Arg Val 275 280 285Val Thr Val Gly Arg
Val Lys Ile Glu Thr Arg Pro Leu Leu Ala Ile 290 295 300Asp Ala Val
Ser Pro Ser Gly Thr Arg Val Asn Leu Ile Val Gln Asp305 310 315
320Asp Trp His Val Arg Val Leu Gly Pro Gly Gly Thr Val Leu Asn Val
325 330 335Thr Glu Leu Thr Ala Gly Thr Lys Val Leu Gly Tyr Leu Pro
Val Glu 340 345 350Lys Arg His Val Gly Tyr Pro Ile Asp Glu Phe Cys
Ile Glu Lys 355 360 365451104DNAMicromonospora sp. strain 046-ECO11
45gtgaagctgt gctggctgga catccgtaac gtcaacggcg ccaaggaggc aatcgtcgag
60gaggcggtcc accagcgggt ggacgccgtc gtggcggccg atccggccga cctggagacg
120cttcccccga cggtgaagaa ggtgctgttc ccgcagggcg ggccgctgcc
ggagaagctg 180gaaccggccg acctggtgat cgtcgagccg gcccggcacg
gcgagcccgc cgagctggcg 240gcccggtacc cggaggtgga gttcggccgg
ttcgtcgaga tcgtcgacgc ggacagcctg 300gaggacgcct gccggtccgc
gcgccacgac cggtggagcc tgctgtactt ccgcgacccc 360accaagatcc
cgctggagat cgtgctggcg gccgcggcgg gcgcggaggg cagcatcatc
420acccaggtcg ccgacgtcga ggaggcggag atcgtcttcg gcgtcctgga
gcacggctcg 480gacggagtga tgctggcgcc ccgcgccgtg ggggaggcca
ccgagctgcg gaccgccgcg 540gtgagcacgg cggcggacct gtcgctcgtg
gagctggagg tcaccggcat ccggcgggtg 600ggcatgggcg agcgcgcctg
cgtcgacacg tgcacgaact tccgtctgga cgagggcatc 660ctggtcggct
cgcactccac cggcatgatc ctgtgctgca gcgagacgca tccgctgccg
720tacatgccga cccggccgtt ccgggtcaac gccggcgcgc tgcactcgta
cacgctctcc 780gccggcgggc ggaccaacta cctcagcgag ctggtctccg
gcggccgggt gctcgccgtg 840gactcgcagg ggaagtcccg cgtcgtcaca
gtgggacggg tcaagatcga gacgcgtccg 900ctgctggcga tcgacgcggt
ctccccctcc gggacacgcg tcaacctcat cgtccaggac 960gactggcacg
tgcgcgtgct cgggccgggc ggcaccgtgc tcaacgtgac cgagctgacc
1020gccggcacga aggtgctcgg ttacctgccg gtggagaagc ggcacgtcgg
ctacccgatc 1080gacgagttct gcatcgagaa gtga
110446253PRTMicromonospora sp. strain 046-ECO11 46Met Thr Ala Gln
Pro Val Leu Asp Phe His Val Arg Leu Ala Pro Arg1 5 10 15Pro Gly Ala
Arg Glu Arg Leu Leu Ala Ala Leu Arg Glu Cys Gly Leu 20 25 30Ala Arg
Ala Val Val Cys Ala Gly Gly Thr Ile Asp Leu Asp Arg Leu 35 40 45Ser
Arg Gln Leu Val Thr Gly Gly His Val Glu Thr Asp Ala Asp Asn 50 55
60Asp Ala Val Ala Ala Ala Cys Ala Gly Thr Asp Gly Arg Leu Val Pro65
70 75 80Phe Phe Phe Ala Asn Pro His Arg Pro Ala Glu Ala Tyr Arg Ala
Arg 85 90 95Ala Ala Glu Phe Arg Gly Leu Glu Ile Ser Pro Ala Val His
Gly Val 100 105 110Ala Leu Thr Asp Pro Arg Val Ala Asp Leu Val Ala
Val Ala Ala Glu 115 120 125Phe Asp His Pro Val Tyr Val Val Cys Leu
Asp Arg Pro Gly Ala Gly 130 135 140Val Ala Asp Leu Val Gly Leu Ser
Arg Arg Phe Pro Gln Val Ser Phe145 150 155 160Val Leu Gly His Ser
Gly Val Gly Asn Ile Asp Leu Tyr Ala Leu Thr 165 170 175Leu Ile Gln
Asp Glu Pro Asn Ile Ser Leu Glu Thr Ser Gly Gly Tyr 180 185 190Thr
Cys Val Ala Glu Ala Ala Leu Arg Arg Leu Gly Asp Asp Arg Val 195 200
205Val Phe Gly Ser Glu Tyr Pro Leu Gln His Pro Ala Val Glu Leu Ala
210 215 220Lys Phe Gln Ala Leu Arg Leu Pro Pro Glu Arg Trp Arg Arg
Ile Ala225 230 235 240Trp Asp Asn Ala His Arg Leu Leu Gly Glu Glu
Lys Arg 245 25047762DNAMicromonospora sp. strain 046-ECO11
47atgaccgcgc agccggtgct ggacttccac gtacgcctgg cgccccggcc cggggcgcgg
60gagcggctgc tcgccgcgct gcgcgagtgc gggctggcgc gggcggtggt gtgcgcgggc
120ggcaccatcg acctggaccg gctgtcccgc cagctcgtca ccggcggcca
cgtcgagacc 180gacgccgaca acgacgcggt ggcggcggcc tgcgccggca
ccgacggccg gctggtgccg 240ttcttcttcg ccaacccgca ccggccggcc
gaggcgtacc gggcccgcgc cgccgagttc 300cgcggcctgg agatctcacc
cgccgtccac ggcgtcgccc tgaccgaccc gcgggtcgcc 360gacctcgtgg
ccgtggcggc ggagttcgac catccggtgt acgtggtctg cctggaccga
420cccggcgcgg gcgtggccga cctggtcggc ctgagccgcc ggttcccgca
ggtgagcttc 480gtgctcgggc acagcggcgt cggcaacatc gacctctacg
ccctgaccct gatccaggac 540gagccgaaca tctcgctgga gacctccggc
ggctacacct gcgtggccga ggcggcgcta 600cgccgcctcg gcgacgaccg
ggtggtgttc ggctccgagt acccgctgca gcacccggcc 660gtggaactgg
ccaagttcca ggcgttgcga ctgccgccgg agcggtggcg gcggatcgcc
720tgggacaacg cgcatcgact gctaggagag gagaagcggt ga
76248438PRTMicromonospora sp. strain 046-ECO11 48Val Ser Glu Pro
Ser Ser Ser Leu Pro Arg Leu Gly Gln Trp His Gly1 5 10 15Leu Glu Asp
Leu Arg Arg Leu Gln Glu Lys Gln Leu Ala Glu Thr Phe 20 25 30Thr Trp
Ala Ala Arg Ser Pro Phe Tyr Arg Ala Arg Leu Ala Ser Gly 35 40 45Ala
Pro Pro Val Thr Pro Ala Asp Leu Ala Asp Leu Pro Leu Thr Thr 50 55
60Lys Gln Asp Leu Arg Asp Asn Tyr Pro Phe Gly Met Leu Ala Val Pro65
70 75 80Arg Glu Arg Leu Ala Thr Tyr His Glu Ser Ser Gly Thr Ala Gly
Lys 85 90 95Pro Thr Pro Ser Tyr Tyr Thr Ala Glu Asp Trp Thr Asp Leu
Ala Glu 100 105 110Arg Phe Ala Arg Lys Trp Ile Gly Met Ser Ala Asp
Asp Val Phe Leu 115 120 125Val Arg Thr Pro Tyr Ala Leu Leu Leu Thr
Gly His Leu Ala His Ala 130 135 140Ala Ala Arg Leu Arg Gly Ala Thr
Val Val Pro Gly Asp Asn Arg Ser145 150 155 160Leu Ala Met Pro Tyr
Ala Arg Val Val Arg Val Met His Asp Leu Asp 165 170 175Val Thr Leu
Thr Trp Ser Val Pro Thr Glu Cys Leu Ile Trp Ala Ala 180 185 190Ala
Ala Ile Ala Ala Gly His Arg Pro Asp Ile Asp Phe Pro Ala Leu 195
200 205Arg Ala Leu Phe Val Gly Gly Glu Pro Met Thr Asp Ala Arg Arg
Arg 210 215 220Arg Ile Ser Arg Leu Trp Gly Val Pro Val Ile Glu Glu
Tyr Gly Ser225 230 235 240Thr Glu Thr Gly Ser Leu Ala Gly Glu Cys
Pro Glu Gly Arg Leu His 245 250 255Leu Trp Ala Asp Arg Ala Leu Phe
Glu Val Tyr Asp Pro Asp Thr Gly 260 265 270Ala Val Arg Ala Asp Gly
Asp Gly Gln Leu Val Val Thr Pro Leu Phe 275 280 285Arg Glu Ala Met
Pro Leu Leu Arg Tyr Asn Leu Glu Asp Asn Val Ser 290 295 300Val Ser
Tyr Asp Asp Cys Gly Cys Gly Trp Lys Leu Pro Thr Val Arg305 310 315
320Val Leu Gly Arg Ser Ala Phe Gly Tyr Arg Val Gly Gly Thr Thr Ile
325 330 335Thr Gln His Gln Leu Glu Glu Leu Val Phe Ser Leu Pro Glu
Ala His 340 345 350Arg Val Met Phe Trp Arg Ala Lys Ala Glu Pro Ala
Leu Leu Arg Val 355 360 365Glu Ile Glu Val Ala Ala Ala His Arg Val
Ala Ala Glu Ala Glu Leu 370 375 380Thr Ala Ala Ile Arg Ala Ala Phe
Gly Val Asp Ser Glu Val Thr Gly385 390 395 400Leu Ala Pro Gly Thr
Leu Ile Pro Leu Asp Ala Leu Thr Ser Met Pro 405 410 415Asp Val Val
Lys Pro Arg Ser Leu Phe Gly Pro Asp Glu Asp Trp Ser 420 425 430Lys
Ala Leu Leu Tyr Tyr 435491317DNAMicromonospora sp. strain 046-ECO11
49gtgagcgagc caagttcgag cctgccccgg ctcggccagt ggcacggcct cgaggacctg
60cggcgcctcc aggagaagca actggcggag acgttcacct gggcggcccg gtcgccgttc
120taccgggcgc ggctggcctc cggcgcgccg ccggtgacgc ccgccgacct
ggccgacctg 180ccgctgacca ccaagcagga cctgcgggac aactacccct
tcggcatgct cgccgtgccc 240cgcgaacggc tggcgaccta ccacgagtcg
agcgggaccg ccgggaagcc caccccctcc 300tactacaccg cggaggactg
gaccgacctg gcggagcgct tcgcccgcaa gtggatcggc 360atgtccgccg
acgacgtctt cctggtccgc acgccgtacg cgctgctgct gaccgggcat
420ctcgcccacg ccgcagcccg gctgcgtggg gccacggtgg tacctggcga
caaccggtcg 480ctggcgatgc cgtacgcccg ggtggtccgg gtgatgcacg
acctggacgt cacgctcacc 540tggtcggtgc cgacggagtg cctgatctgg
gccgccgcgg cgatcgcggc cgggcaccgg 600cccgacatcg acttcccggc
gctgcgcgcg ctgttcgtcg gcggcgagcc gatgaccgac 660gcccgccggc
ggcggatcag ccgcctgtgg ggggtgccgg tcatcgagga gtacggctcg
720acggagaccg gcagcctggc cggggagtgc cccgagggac gcctgcacct
gtgggccgac 780cgggcgctgt tcgaggtgta cgacccggac accggcgccg
tccgcgcgga cggcgacggc 840cagctcgtgg tcacgccgct gttccgggag
gcgatgccgc tgctgcggta caacctggag 900gacaacgtgt cggtctccta
cgacgactgc ggatgcggct ggaagctgcc caccgtgcgg 960gtgctcggcc
ggtcggcgtt cggctaccgg gtcggcggca ccaccatcac ccagcaccag
1020ctggaggaac tggtcttctc cctgccggag gcgcaccggg tgatgttctg
gcgggccaag 1080gcggagccgg cgctgttgcg ggtcgagatc gaggtggccg
ccgcgcaccg ggtcgccgcc 1140gaggcggagc tgaccgccgc gatccgggcc
gccttcggcg tggacagcga ggtcaccggc 1200ctggcgccgg gaaccctgat
cccgctcgac gcgctgacca gcatgccgga cgtggtgaag 1260ccacgcagcc
tgttcggtcc ggacgaggac tggagcaaag cgctcctcta ctactga
131750396PRTMicromonospora sp. strain 046-ECO11 50Met Pro Gln Met
Arg Val Ala Val Ala Gly Ala Gly Ile Ala Gly Leu1 5 10 15Ala Phe Ala
Ala Ala Leu Arg Arg Thr Gly Ile Asp Cys His Val Tyr 20 25 30Glu Gln
Ala Asp Gln Leu Met Glu Val Gly Ala Gly Val Gln Val Ala 35 40 45Pro
Asn Ala Thr Arg Leu Leu His Arg Leu Gly Leu Arg Asp Arg Leu 50 55
60Arg Thr Val Ala Val Ala Pro Gln Ala Ile Glu Met Arg Arg Trp Asp65
70 75 80Asp Gly Thr Leu Leu Gln Arg Thr Gln Leu Gly Ser Val Cys Gly
Arg 85 90 95Arg Phe Gly Ala Pro Tyr Tyr Val Val His Arg Ala Asp Leu
His Ser 100 105 110Ser Leu Leu Ser Leu Val Pro Pro Asp Arg Val His
Leu Gly Ala Arg 115 120 125Leu Thr Ala Val Thr Gln Thr Ala Asp Glu
Ala Tyr Leu His Leu Ser 130 135 140Asn Gly Thr Thr Val Ala Ala Asp
Leu Val Val Gly Ala Asp Gly Ile145 150 155 160His Ser Val Ala Arg
Glu Gln Ile Val Ala Asp Arg Pro Arg Phe Ser 165 170 175Gly Gln Ser
Ile Tyr Arg Gly Leu Val Pro Ala Glu Arg Val Pro Phe 180 185 190Leu
Leu Thr Glu Pro Arg Val Gln Leu Trp Phe Gly Pro Asp Gln His 195 200
205Cys Val Cys Tyr Pro Val Ser Ala Gly Arg Gln Val Ser Phe Gly Ala
210 215 220Thr Val Pro Ala Thr Asp Trp Arg Gln Glu Ser Trp Ser Gly
Arg Gly225 230 235 240Asp Val Thr Gln Leu Ala Ala Ala Tyr Ala Gly
Trp His Pro Asp Val 245 250 255Thr Arg Leu Ile Ala Ala Ala Asp Arg
Val Gly Arg Trp Ala Leu His 260 265 270Asp Arg Asp Ser Ile Asp Arg
Leu Ser Ala Gly Arg Val Thr Leu Ile 275 280 285Gly Asp Ala Ala His
Pro Met Leu Pro Phe Gln Ala Gln Gly Ala Asn 290 295 300Gln Ala Val
Glu Asp Ala Val Val Leu Ala Val Cys Leu Ala Gly Val305 310 315
320Glu Pro Ala Gly Leu Gly Ala Ala Leu Arg Arg Tyr Glu Arg Ile Arg
325 330 335Leu Pro Arg Thr Thr Arg Ile Gln Arg Gln Ser Arg Ala Asn
Ala Glu 340 345 350Met Phe His Leu Ala Asp Gly Ala Asp Gln Arg Arg
Arg Asp Val Ala 355 360 365Ala Gln Ser Ser Ser Gly Leu Asp Arg His
Glu Trp Leu Phe Gly Tyr 370 375 380Asp Ala Glu Lys Ala Thr Thr Thr
Ser Gly Ser Ala385 390 395511191DNAMicromonospora sp. strain
046-ECO11 51atgccgcaga tgagggtcgc cgtggccggc gccggcatcg ccgggctcgc
cttcgccgcc 60gccctgcgcc ggaccgggat cgactgccac gtgtacgaac aggccgacca
gctcatggag 120gtgggcgcgg gcgtgcaggt cgcgccgaac gccacccggc
tgctgcaccg gctgggcctg 180cgtgaccgcc tgcgtacggt ggctgtcgcg
ccgcaggcga tcgagatgcg ccgctgggac 240gacggcacgc tgctgcaacg
cacccagctg ggcagcgtgt gcggacgccg cttcggcgcg 300ccgtactacg
tggtgcaccg cgcggacctg cacagcagcc tgctgtcgct ggtgccgccg
360gaccgggtgc acctgggcgc ccgcctcacc gccgtgacgc agaccgccga
cgaggcgtac 420ctgcacctgt ccaacggcac cacggtcgcg gcggatctcg
tcgtgggcgc cgacggcatc 480cactcggtcg cgcgggagca gatcgtggcg
gaccggccgc gcttctccgg acagtccatc 540taccgcgggc tggtgccggc
cgagcgggtg ccgttcctgc tcaccgaacc ccgggtgcag 600ttgtggttcg
ggccggacca gcactgcgtc tgctacccgg tgtccgccgg ccggcaggtg
660agcttcggcg cgacggtgcc cgccaccgac tggcggcagg agtcgtggtc
gggccggggc 720gacgtgacgc aactcgcggc cgcgtacgcg ggctggcacc
cggacgtcac ccggctgatc 780gccgcggccg accgggtcgg caggtgggcg
ctgcacgacc gggacagcat cgaccggctc 840agcgcgggac gggtgaccct
gatcggcgac gccgcgcacc cgatgctgcc gttccaggcg 900cagggcgcga
accaggccgt cgaggacgcg gtggtgctcg cggtctgcct ggccggcgtg
960gaaccggcgg gcctgggcgc cgcgctgcgc cgctacgaac ggatccgcct
gccccggacc 1020acccggatcc agcggcagtc ccgggccaac gccgagatgt
tccacctggc cgacggcgcc 1080gaccagcgcc gccgggacgt cgccgcacaa
tcctcgtccg gcctggaccg ccacgaatgg 1140ctcttcgggt acgacgccga
gaaagccacc acgaccagcg ggagcgcctg a 119152261PRTMicromonospora sp.
strain 046-ECO11 52Met Glu Leu Thr Gly Ile Glu Ser Lys Val Ala Leu
Val Thr Gly Ala1 5 10 15Gly Gln Gly Ile Gly Ala Ala Val Ala Gly Val
Leu Ala Arg Ala Gly 20 25 30Ala Gln Val Ala Ala Val Asp Arg Asn Ala
Glu Ala Leu Thr Thr Val 35 40 45Val Thr Lys Leu Ala Ala Glu Gly Asp
Ser Ala Arg Ala Tyr Cys Val 50 55 60Asp Val Cys Asp Ser Glu Ala Val
Asp Ala Leu Val Arg Arg Val Glu65 70 75 80Asp Glu Met Gly Pro Val
Ala Ile Leu Val Asn Ala Ala Gly Val Leu 85 90 95His Thr Gly Arg Val
Val Glu Leu Ser Asp Arg Gln Trp Arg Arg Thr 100 105 110Phe Ser Val
Asn Ala Asp Gly Val Phe His Val Ser Arg Ala Val Ala 115 120 125Arg
Arg Met Val Gly Arg Arg Arg Gly Ala Ile Val Thr Val Ala Ser 130 135
140Asn Ala Ala Gly Val Pro Arg Thr Glu Met Ala Ala Tyr Ala Ala
Ser145 150 155 160Lys Ala Ala Ser Ala Gln Phe Thr Arg Cys Leu Gly
Leu Glu Leu Ser 165 170 175Gly Tyr Gly Ile Arg Cys Asn Val Val Ser
Pro Gly Ser Thr Asp Thr 180 185 190Pro Met Leu Arg Ala Met Leu Gly
Glu Gly Ala Asp Pro Ser Ala Val 195 200 205Ile Glu Gly Thr Pro Gly
Ala Tyr Arg Val Gly Ile Pro Leu Arg Lys 210 215 220Leu Ala Gln Pro
Arg Asp Val Ala Glu Ala Val Ala Tyr Leu Val Ser225 230 235 240Asp
Gln Ala Gly His Val Thr Met His Asp Leu Tyr Val Asp Gly Gly 245 250
255Ala Ala Leu His Val 26053786DNAMicromonospora sp. strain
046-ECO11 53atggaactga ccggaatcga gtcgaaggtc gccctggtca cgggcgcggg
gcagggcatc 60ggcgccgccg tggccggtgt cctggcgagg gcgggcgcgc aggtggcggc
ggtggaccgc 120aacgccgagg cgctgaccac cgtcgtgacg aagctcgccg
ccgagggcga ctcggcgcgc 180gcctactgcg tcgacgtgtg cgacagcgag
gcggtggacg cgctggtgcg ccgggtcgag 240gacgagatgg ggccggtcgc
catcctggtc aacgccgccg gcgtgctgca caccggacgg 300gtcgtcgagc
tgtcggaccg gcagtggcgc cggaccttct cggtgaacgc cgacggcgtg
360ttccacgtgt cccgggcggt ggcgcggcgg atggtgggcc gccgtcgtgg
cgcgatcgtc 420accgtggcgt cgaacgccgc cggggtgccg cgtaccgaga
tggccgcgta cgccgcctcc 480aaggccgcgt ccgcgcagtt cacccgctgc
ctggggcttg agctgtccgg ctacggcatc 540cggtgcaacg tggtctcgcc
cggctccacc gacaccccca tgctgcgggc catgctcggc 600gagggcgccg
acccgagcgc ggtgatcgag ggcacgccgg gcgcgtaccg cgtcggcatc
660ccgctgcgca agctggccca gccgcgcgac gtggccgagg cggtcgccta
tctggtgtcc 720gaccaggcgg gccacgtgac catgcacgac ctgtacgtcg
acggcggcgc ggccctgcac 780gtgtga 78654224PRTMicromonospora sp.
strain 046-ECO11 54Met Ala Met Thr Pro Ile Ala Pro Tyr Arg Met Pro
Gly Asp Gly Asp1 5 10 15Leu Pro Gly Thr Ala Leu Pro Trp Arg Pro His
Pro Asp Arg Ala Ala 20 25 30Val Leu Val His Asp Leu Gln Arg Tyr Phe
Leu Arg Pro Phe Glu Ala 35 40 45Gly Glu Ser Pro Met Ala Glu Leu Leu
Pro Asn Val Ala Lys Leu Leu 50 55 60Ala Thr Ala Arg Ala Ala Gly Val
Pro Val Leu Tyr Thr Ala Gln Pro65 70 75 80Gly Gly Met Ser Arg Gln
Asp Arg Gly Leu Leu His Asp Leu Trp Gly 85 90 95Pro Gly Met Ser Ser
Ala Glu Asp Asp Arg Gly Ile Val Asp Asp Val 100 105 110Ala Pro Gln
Pro Gly Asp Thr Val Leu Thr Lys Trp Arg Tyr Ser Ala 115 120 125Phe
Phe Arg Ser Asp Leu Glu Glu Arg Leu Arg Gly Ala Gly Arg Asp 130 135
140Gln Leu Val Val Cys Gly Val Tyr Ala His Met Gly Cys Leu Ile
Thr145 150 155 160Ala Cys Asp Ala Phe Ser Arg Asp Ile Glu Ala Phe
Leu Val Ala Asp 165 170 175Ala Leu Ala Asp Leu Ser Arg Glu Asp His
Leu Met Ala Leu Arg Tyr 180 185 190Ala Ala Asp Arg Cys Ala Val Pro
Leu Trp Thr Ala Asp Val Leu Asp 195 200 205Gly Leu Ala Asp Ala Ala
Gly Arg Pro Asp Gln Ser Ser Thr Gln Arg 210 215
22055675DNAMicromonospora sp. strain 046-ECO11 55atggccatga
ccccgatcgc gccgtaccgc atgcccggcg acggcgacct gcccggcacc 60gcgctgccct
ggcgtccgca cccggaccgg gccgccgtgc tggtgcacga cctgcaacgc
120tacttcctgc gcccgttcga ggccggggag tccccgatgg ccgaactgct
ccccaacgtc 180gcgaagctgc tcgccacggc gcgggcggcc ggcgtgccgg
tgctgtacac cgcgcagccc 240ggcggcatga gccggcagga ccgcgggttg
ctgcacgacc tgtggggccc cggcatgagc 300agcgccgagg acgaccgggg
catcgtcgac gacgtcgccc cgcagccggg cgacacggtg 360ctgaccaagt
ggcgctacag cgcgttcttc cgcagcgacc tggaggagcg actgcgcggt
420gcgggacggg accagctcgt ggtctgcggc gtgtacgcgc acatggggtg
cctgatcacc 480gcctgcgacg cgttcagccg cgacatcgag gcgttcctgg
tggcggacgc gctggccgac 540ctatcgcgcg aggaccacct gatggcgctg
cgctacgccg cggaccgctg cgcggtgccg 600ttgtggacgg cggatgtgct
ggacgggctg gcggacgccg ccgggcgtcc ggatcagagc 660agcacccaac gatga
67556233PRTMicromonospora sp. strain 046-ECO11 56Met Ser Asp Arg
Thr Arg Val Val Val Val Gly Gly Thr Ser Gly Ile1 5 10 15Gly Arg His
Phe Ala Arg Phe Cys Ala Glu Arg Gly Asp Asp Val Val 20 25 30Ile Thr
Gly Arg Ser Ala Ala Arg Thr Lys Thr Val Ala Asp Glu Ile 35 40 45Gly
Gly Arg Thr Arg Gly Leu Ala Leu Asp Leu Ala Glu Pro Glu Thr 50 55
60Ile Ala Asp Ala Leu Ala Asp Val Pro His Val Asp Arg Leu Val Val65
70 75 80Ala Ala Leu Asp Arg Asp Tyr Asn Thr Val Arg Ala Tyr Arg Pro
Gly 85 90 95Asp Ala Ala Arg Leu Leu Thr Val Lys Leu Val Gly Tyr Thr
Ala Val 100 105 110Leu His Ala Leu Ala Pro Arg Met Thr Asp Glu Ser
Ala Val Val Leu 115 120 125Leu Gly Gly Leu Ala Ser His Arg Pro Tyr
Pro Gly Ser Thr Ser Val 130 135 140Thr Thr Ala Asn Gly Gly Ile Ser
Ala Leu Val Arg Thr Leu Ala Val145 150 155 160Glu Leu Ser Pro Val
Arg Val Asn Ala Leu His Pro Ser Ile Val Ser 165 170 175Asp Thr Pro
Phe Trp Ser Asp Lys Pro Ala Ala Arg Glu Ala Ala Ala 180 185 190Thr
Arg Ala Leu Ser Arg Arg Pro Val Thr Met Gln Asp Cys Ala Glu 195 200
205Ala Ile Asp Phe Leu Leu Thr Asn Arg Ser Ile Asn Gly Val Asn Leu
210 215 220Asn Ile Asp Gly Gly Asp Val Leu Ile225
23057702DNAMicromonospora sp. strain 046-ECO11 57atgtcggatc
ggacccgggt cgtggtcgtc ggcggaacct cggggatcgg gcggcacttc 60gcccgattct
gcgccgaacg cggagacgac gtggtgatca ccggccgttc ggcggcccgg
120accaagaccg tggcggacga gatcggcggg cggacccgtg ggctcgctct
cgacctggcc 180gagccggaga cgatcgcgga cgcgctcgcc gacgtgccgc
acgtcgaccg gctcgtggtc 240gcggcgctgg accgcgacta caacaccgtc
cgcgcgtacc ggccgggcga cgcggcgcgg 300ctgctgaccg tcaagctggt
cggctacacg gcggtcctgc acgccctcgc cccgcggatg 360accgacgaga
gcgcagtcgt gctgctcggc ggcctggcca gccaccggcc gtatcccggc
420tccacctccg tcacgaccgc caacggcggg atcagcgcgc tggtgcggac
cctggctgtg 480gaactctcgc cggtccgggt caacgccctg cacccgagca
tcgtctccga cacgccgttc 540tggagcgaca agcccgccgc gcgggaggcc
gccgcgaccc gcgcgctcag ccgacggccg 600gtcaccatgc aggactgcgc
cgaggcgatc gacttcctgc tgacgaaccg ctcgataaac 660ggggtcaacc
tgaacatcga cggcggggac gtgctcatct ga 70258246PRTMicromonospora sp.
strain 046-ECO11 58Met Thr Ser Ala Leu Arg Thr Ser Ala Trp Thr Tyr
Asp Asp Phe Thr1 5 10 15Ser Arg Glu Leu Asp Pro Ala Arg Trp Ala Ile
Met Ser Ile Ala Gly 20 25 30Ala Asp Gly Gln Thr His Arg Tyr Gln Asp
Arg Asn Ala Gln Val Arg 35 40 45Thr Gly Asp Gly Arg Leu Glu Leu Thr
Val Asp Pro Phe Thr Arg Phe 50 55 60His Asp Thr Asp Pro Arg Gln Asn
Asn Ala Lys Gln Met Tyr Arg Ser65 70 75 80Val Arg Arg Phe Ala Val
Pro Ala Glu Gly Ser Leu Thr Val Glu Val 85 90 95Glu Met Gly Val Arg
Thr Tyr Arg Gln Ile Pro His Asp Leu Leu Asp 100 105 110Ala Phe Gly
Thr Val Asn Leu Phe Asp Leu Glu Thr Gly Val Val Phe 115 120 125Asn
Ala Ala Ala Thr Asn Asp Thr Val Tyr Ala Thr Val Glu Arg Leu 130 135
140Val Leu Pro Gly Val Thr Gln Pro His Glu His Tyr Ile His Arg
Val145 150 155 160Val Leu Asp Val Pro Thr Glu Pro Gly Arg Ala His
Gly Tyr Ala Ile 165 170 175Thr Tyr Arg Ala Pro Thr Ser Glu Val Glu
Phe His Val Asp Gly Arg 180 185 190Leu Ala Tyr Trp Ala Arg Val Pro
Val Pro Val Thr Gly Phe His Ala 195 200 205Gly Met Ala Leu Phe Ser
Ala Arg Asp Leu Ala Arg Tyr Pro Arg Glu 210 215 220Gln Arg Glu His
Gly Gln Gly Ala Thr Gly Trp Trp Gly Pro Trp Arg225 230 235 240Ile
Ala Ser Gly Val Arg 24559741DNAMicromonospora sp. strain 046-ECO11
59atgacgtcgg cactgagaac cagcgcgtgg acgtacgacg acttcaccag ccgcgagctg
60gaccccgccc gctgggcgat catgtcgatc gccggcgcgg acgggcagac ccacaggtac
120caggaccgca acgcccaggt ccgcaccggc gacgggcggc tggagctgac
cgtcgacccg 180ttcacccgct tccacgacac cgatccccgg cagaacaacg
ccaagcagat gtaccggtcg 240gtgcggcgct tcgccgtgcc ggcggagggc
tcgctgaccg tcgaggtgga gatgggcgtg 300cggacgtacc ggcagatccc
gcacgacctg ctggacgcgt tcggcacggt gaacctgttc 360gacctggaga
ccggcgtcgt gttcaacgcc gccgccacga acgacaccgt gtacgcgacg
420gtcgagcgcc tggtgctgcc cggcgtgacc cagccgcacg agcactacat
ccaccgggtg 480gtcctggacg tgccgacgga gccgggccgg gcgcacggat
acgccatcac ctaccgggcg 540ccgacgtcgg aggtggagtt ccacgtcgac
ggccggctcg cctactgggc gcgggtcccg 600gtgccggtga ccggattcca
cgccggcatg gcgctcttct ccgcccgcga cctggcccgg 660tacccccgcg
agcagcggga gcacgggcag ggcgcgaccg ggtggtgggg gccgtggcgg
720atcgcctccg gcgtcagatg a 74160111PRTMicromonospora sp. strain
046-ECO11 60Met Asp Thr Ala Ala Pro Ala Thr Asp Gly Gly Arg Tyr Leu
Ala Val1 5 10 15His His Ser Ala Glu Phe Arg Glu Leu Arg Arg Arg Ser
Ser Thr Phe 20 25 30Thr Leu Trp Ala Ser Val Ala Phe Phe Gly Trp Trp
Phe Leu Gly Ser 35 40 45Leu Leu Ala Thr Tyr Ala Pro Asp Phe Phe Arg
Glu Lys Val Ala Gly 50 55 60Pro Val Asn Val Gly Leu Leu Phe Val Phe
Leu Ser Phe Ala Phe Val65 70 75 80Val Thr Leu Ala Ala Phe Tyr Leu
Arg Tyr Ala Arg Thr His Leu Asp 85 90 95Pro Leu Ser Glu Lys Ile Arg
Ala Asp Leu Glu Gly Ala Ser Arg 100 105 11061336DNAMicromonospora
sp. strain 046-ECO11 61atggacacgg cagctccggc aacggacggc ggtcgctacc
tcgccgtcca tcacagcgca 60gagttcaggg aactacggcg acgatcgagc acgttcacgc
tctgggccag cgtcgccttc 120ttcggctggt ggttcctcgg cagcctgctc
gccacctacg cgccggactt cttccgggag 180aaggtggccg gcccggtcaa
cgtgggtctg ctcttcgtct tcctgtcgtt cgccttcgtg 240gtgacgctcg
ccgccttcta cctgcgttac gcccgcacgc atctcgatcc gctcagcgag
300aagatccgtg ccgacctgga aggagcgtcc cgatga
33662559PRTMicromonospora sp. strain 046-ECO11 62Met Ser Val Ile
Leu Ala Asp Pro Pro Pro Pro Val Asp Asn Thr Trp1 5 10 15Ala Thr Pro
Ala Ile Ala Val Pro Val Thr Ile Val Leu Ala Leu Ala 20 25 30Val Leu
Tyr Leu Val Arg Ser Ala Arg Ala Ser Thr Thr Thr Ala Asp 35 40 45Gly
Phe Leu Leu Ala Asp Arg Arg Ile Gly Pro Val Gln Asn Ala Leu 50 55
60Ala Val Ala Ser Ala Pro Leu Met Tyr Ser Thr Met Tyr Ile Ile Thr65
70 75 80Gly His Ile Ala Leu Ser Gly Tyr Asp Ala Ile Leu Leu Met Thr
Ala 85 90 95Phe Thr Met Gly Thr Met Leu Ala Leu Phe Leu Phe Ala Gly
Pro Val 100 105 110Arg Asn Val Gly Gly Tyr Thr Leu Gly Asp Leu Leu
Ala Val Arg Thr 115 120 125Arg Glu Arg Pro Ala Arg Ile Ala Ser Ala
Val Leu Thr Leu Leu Thr 130 135 140Tyr Val Met Leu Thr Val Ile Met
Met Ala Ala Ile Ala Phe Ile Phe145 150 155 160Asn Arg Trp Phe Gly
Val Asp Ala Leu Val Gly Leu Val Leu Pro Val 165 170 175Phe Val Val
Gly Leu Ile Thr Val Gly Tyr Val Tyr Leu Gly Gly Met 180 185 190Leu
Gly Val Thr Arg Ile Leu Val Phe Lys Leu Val Leu Ser Val Val 195 200
205Val Val Gly Val Leu Thr Ala Trp Val Leu Ala Arg Phe Asp Leu Asn
210 215 220Leu Phe Ser Leu Leu Glu Arg Ala Glu Ala Asn Ala Ala Pro
Val Pro225 230 235 240Ser Gly Ser Asp Leu Leu Gly Pro Gly Arg Leu
Phe Gly Glu Gly Ala 245 250 255Thr Thr Leu Val His Leu Ser Lys Leu
Phe Ala Ile Ala Val Gly Val 260 265 270Ala Ala Ile Pro Phe Leu Phe
Met Arg Asn Phe Ala Val Thr Ser Gly 275 280 285Arg Asp Ala Arg Arg
Ser Thr Gly Trp Ala Ser Met Ile Ile Val Gly 290 295 300Phe Tyr Leu
Cys Leu Ser Val Val Gly Leu Gly Ala Val Ala Ile Leu305 310 315
320Gly Arg Asp Asn Ile Gly Val Ile Lys Ala His Arg Asp Ile Ser Phe
325 330 335Pro Lys Leu Ala Asp Glu Leu Gly Gly Pro Val Met Val Gly
Ser Leu 340 345 350Ala Gly Val Ala Val Leu Thr Ile Val Gly Val Phe
Ala Pro Leu Leu 355 360 365His Ser Ala Val Thr Thr Val Thr Lys Asp
Leu Asn Val Ile Arg Gly 370 375 380Arg Arg Leu Asp Pro Ala Ala Glu
Leu Arg Asp Ile Lys Arg Asn Thr385 390 395 400Leu Ile Ile Gly Val
Gly Ser Val Leu Leu Ala Val Val Met Leu Pro 405 410 415Val Arg Thr
His Ile Phe Ile Pro Thr Ser Ile Asp Ile Ala Gly Ala 420 425 430Val
Val Leu Pro Ile Val Val Tyr Ala Leu Phe Trp Arg Arg Phe Asn 435 440
445Thr Arg Gly Leu Gln Trp Thr Val Tyr Gly Gly Leu Ala Leu Thr Ala
450 455 460Phe Leu Val Leu Phe Ser Asn Gly Val Ser Gly Glu Pro Asp
Ala Ile465 470 475 480Phe Pro Asp Arg Asn Phe Lys Phe Val Asp Val
Glu Pro Ala Leu Ile 485 490 495Thr Val Pro Val Gly Phe Leu Leu Gly
Tyr Leu Gly Ser Ile Thr Ser 500 505 510Arg Glu Arg Asp Asp Ala Ala
Phe Ala Glu Met Gln Val Arg Ser Leu 515 520 525Thr Gly Ala Val Val
Thr Gly Pro Pro Arg Pro Ala Ala Val Asp Asp 530 535 540Glu Asp Arg
Asp Gly Arg Gln Asp Arg Ala Pro Ser Pro Val Ser545 550
555631680DNAMicromonospora sp. strain 046-ECO11 63atgagcgtca
tcctcgccga cccgccaccc ccggtcgaca acacgtgggc gacgcccgcg 60atcgccgtgc
cggtcaccat cgtcctcgcg ctcgcggtgc tctacctggt ccggtcggcg
120cgcgccagca ccaccaccgc ggacggcttc ctgctggccg accggcggat
cgggccggtg 180cagaacgcgc tggcggtggc ctccgcgccg ctgatgtact
cgacgatgta catcatcacc 240ggccacatcg cgctcagcgg ctacgacgcc
atcctgctga tgaccgcctt caccatgggc 300accatgctcg cgctgttcct
cttcgccggg ccggtgcgca acgtgggcgg ctacacgctc 360ggtgacctgc
tcgcggtccg tacccgggag cggccggcgc ggatcgcgtc ggcggtgctc
420acgctgctga cgtacgtcat gctgacggtg atcatgatgg ccgccatcgc
gttcatcttc 480aaccgctggt tcggcgtcga cgccctcgtc ggcctggtcc
tcccggtgtt cgtcgtcggt 540ctgatcacgg tggggtacgt gtacctcggc
gggatgctcg gggtcacccg catcctggtg 600ttcaagctgg tgctgtcggt
ggtcgtcgtg ggcgtgctga ccgcctgggt gctggcccgc 660ttcgacctga
acctcttcag cctgctggag cgggccgagg cgaacgcggc gccggtgccc
720agcggcagcg acctgctggg cccgggccgg ctgttcggcg agggcgcgac
cacgctcgtg 780cacctgtcga agctgttcgc catcgccgtc ggagtggcgg
ccattccgtt cctgttcatg 840cgcaacttcg cggtgaccag cgggcgggac
gcgcgccggt cgaccgggtg ggcgtcgatg 900atcatcgtcg ggttctacct
gtgcctgtcc gtcgtcgggc tcggtgccgt cgcgatcctc 960ggccgggaca
acatcggcgt catcaaggcc caccgcgaca tcagcttccc caagctcgcc
1020gacgagctcg gcggtccggt gatggtcggc tccctggccg gcgtcgcggt
cctgacgatc 1080gtcggcgtct tcgcgccgct gctgcacagc gccgtgacga
cggtgaccaa ggacctgaac 1140gtgatccgcg gccggcggct ggatccggcc
gccgagctgc gggacatcaa gcgcaacacc 1200ctgatcatcg gcgtcggctc
cgtgctgctg gcggtcgtga tgctgccggt acggacccac 1260atcttcatcc
cgacctcgat cgacattgcc ggcgcggtgg tcctgccgat cgtcgtctac
1320gcgttgttct ggcggcgttt caacacccgc ggactgcagt ggacggtcta
cggcggcctc 1380gcgctcaccg cgttcctggt gctgttctcc aacggtgtct
cgggcgagcc ggacgccatc 1440ttcccggacc gcaacttcaa gttcgtggac
gtcgagcccg cgctgatcac ggtgccggtc 1500ggcttcctgc tcggctacct
cggctcgatc accagccggg agcgcgacga cgccgcgttc 1560gccgagatgc
aggtccggtc cctcaccgga gctgtcgtca cgggaccgcc gcggccggcc
1620gccgtggacg acgaggaccg cgacggccgc caggaccggg cgcccagccc
ggtgagctga 1680645960DNAMicromonospora sp. strain 046-ECO11
64ccacacccct cgggaggcaa ctgtggatcc ggtaccggtt ctggtcgtgg gcgcgggccc
60ggtcggcatg gtcaccgcgc tggcgctcgc ccgtcacggc gtcgcctgcg tcctcgtcga
120ccagggcttc gagacgtcgg tccatcccaa gctggactac gtcaacgccc
gcagcatgga 180gttcctccgc cagttcggcc tcgccgacga cgtccgtgcc
gccggcgtcg cgcccgagca 240ccgggccgac gtcatctggt cgaccggcct
ggccggtgag ccgatcacca ggtgggggct 300gccctcggtg acgcaggagt
ggcgccgcat cgccgagcac aacgacggca cccagccggc 360cgagcccggc
cagcggatct cccagatcga cctggaaccg gtcctgcggg cccgctgccg
420gcgggagccc cttgtcgacc tgcgcctcgg cgtacggttc gactcgctga
cccaggacga 480cgcgggggtc accagcgtcc tcgccgacga caccggcggc
gaggtccggg tgcggtcgga 540gtacgtggtc gggtgcgacg gcgcgtcgag
ccaggtccgc cgggccgtgg gcatcggtga 600ggaggggttc gacgtgcccg
gcctgccggg cgccttcatg gtgcacttca ccagccggga 660cctggacagc
ctgcaccggc acggccggtt ctggcactac ttcgcgttcc ggtacgtgat
720catcgcccag gacgaggtcg acacctggac cgcgcacgtc aacggcgtcg
acccgaacga 780gttcgacgag ccgccggccg acccggaggc gttcctgctc
gacacgatcc gcaccgagct 840gcggatcgac aaggtgctgc tcacctcgcg
ctggcgtccc ggcttcatgc tcgccgacag 900gtaccgcgcc ggccgggtgc
tgctcgccgg tgactcggcc caccggatgt tccccaccgg 960cgcgtacggc
atgaacaccg gcatcggcga cgccgtcgac gtggcctgga agctggccgc
1020tgtcgtccgg ggcttcggcg gccccgggct gctcgacagc tacgacgccg
aacgccgccc 1080ggtggggcgg cgcaacatgc gcacctcgca ccggcacctg
ggcgtgcacc tgcgggcggg 1140cgagctcctg cgcggcggcg ccccgctgcc
gtccgtcgcg gccttcctcg acgccgagcg 1200gggcgagaac gagtaccggg
ggatcgagct cggctaccgc tactccggct cgccggtgct 1260ctggccggag
ggcccggggg agccctcgga cgacccgcgg gcgtacgccc cgacgacctg
1320gcccggcgcc cgtccgccca gcctcctgct gagcgacggg cagcagatct
tcgaccggtt 1380cgacccggcc tcgttcaccc tcgtggactt caccggtgac
ggcgccgccg gtccgctgct 1440ggcggcggcg gccgcgcggg ggctcccggt
cacccacacc gtggtgaccg acccccgggc 1500tcgtgagctg tgggaacgcg
acctcgtcct gctgcggccg gaccaccacg tcgcctggcg 1560gggaaacacc
gtgccgccgg accccgacgc cgtggtccag cgcgtgcggg gtggcggata
1620ggcgcgacgt gccgtcaccg gcggcccggg tcacgcgcac acgcgaccgg
ccggtccggc 1680tgactctcga ctggaggaca gatgcagcaa tccggttcaa
cggcggaacg cagcccactc 1740gggccgtggg agggcatgcc ggcggtccag
caaccggact ggcaggacca cccggcgtac 1800gcggagacct gtcaggcgtt
ggcgtcggcc ccgccgctgg tcccacccgg ggaggtacgg 1860gggttccggc
agctgttgtc ggagctggcg tcgaccgacg ggctcctgct gcagttgggc
1920gactgcgccg agagcctcta cgagtgcacc ccccggcaca cctcggacaa
gatcgaggtc 1980atcgaccggc tgggggaccg gctcagcgag ctcaccgggc
gcaacgtgct gcgggtgggc 2040cggatggccg ggcagttcgc caagccccgg
tcgcaggcga cggagtggca cgacgcgctg 2100agcatcccct ccttccgcgg
ccacatgatc aattccgagc tggccgcgcc cggtacgcgc 2160aaggccgacc
ctcgccgcat gtggtgggcg tacgaggcga gcgaccgggt gcagcgggtc
2220ctgcgcgccc accgggaggg caaccggcgt gccgcgcgga ccgaggggcc
gtggtcgagc 2280cacgaggccc tggtcgtcga ctacgagtcc cgcctgatcc
gccgggaccc ggacacgggc 2340gagcactacc tggcgtcgac ccacctgccg
tgggtggggg agcggacccg ccggtccgcc 2400gaggcgcacg tggccatgct
gtccacggtg gtgaacccgg tcggctgcaa gatcgggccg 2460gacgccgacc
cggacgacgt cctgcgggtg tgcgaggcgc tcgacccgcg gcgcgatccg
2520ggccgtctcg tcctgatccc gcggatgggc cgggaccgga tccgggagtc
cctgccgccg 2580atcgtccgcg cggtggtgaa cgcggggcac cccgtgctct
ggctgagcga tcccatgcac 2640ggcaacaccg tcaaggcctc ggtcggcctg
aagacgcgcc acctctccga cgtggtcacc 2700gaggcgctgt ggttccgcga
catcctcgac cagcagcggc agcacgccgc cgggctgcac 2760atcgaggtcg
ccgccaccga cgtgaccgag tgcgtcggcg gttcggtggc cggcgaggag
2820gacctggcgc ggcactacac ctcgctgtgc gacccgcggc tcaacccggg
tcaggccacc 2880gagctgatcg aagcgtgggc caaggacacc gcgacggtcg
gcccgggacc gcggcgctcc 2940ggcccttcgg cgcggccgga ggtcgccgcc
tgacgtcgcc ggtctttgcg ccggccgttt 3000ccgaactgcg ggaaaattga
cagaaggaga cctgccggag caaattcggc caggctagcc 3060gcgccgtagt
tcgtcgtcca ctacttgcgt gggtagtgtc aactacccgt gccgggaccg
3120tcggtggtgt tgctcagcag gaatcccatc gcaatgatgt gtgagaaggc
gtaatccttc 3180gatcggtgac gcgcgtacct catcctatcc gcactgaatc
ctgtctcagc tgaagcgagt 3240gtttccaatg tggggcagct caaacacgct
ggaagtgaag ggcaacgacg agagattccc 3300cctgcccgat gcagctacgg
aggatcggtc tgtgcttggc gagacggttc cggtttccgc 3360gctgctgccc
ggtgactccc cgcggctggc gggcgagaac gtcgagcaca tccggctgct
3420ggccgcgatg cacgacctcc cgccgatcct ggtgcaacgc ggcacgatgc
gggtgatcga 3480cggcatgcac cggctgcggg ccgccaagct gcgcggcgac
gagaccgtgc gggtgacgtt 3540cttcgacggg gacgacgccg cggcgttcct
gctctcggtc gacgccaaca tcaaacacgg 3600gctgccgttg tcccgcgccg
accgggaggc cgccgccacc cgcatcctgc ggttgtatcc 3660gcagtggtcg
gaccgcgccg tcgccgcggc ggccgggctg tcaccgacca cggcgagcgg
3720catccggcgc cgcctgctgc aaccggcggc gcgggagggc agccgggtgg
gacgggacgg 3780gcgggtgcgc ccgctggacg gctcggcggg ccgacggcgg
gccagcgcgg tcatcgcgct 3840ccggccggac gcgcccctgc gtgccatcgc
gcaggaggcc ggggtgtcgg tgggcacggc 3900gcgggacgtg cgcgcccggt
tgcaggcggg ccgggacccc gtcctgacct cgcagcgacc 3960ggcggccgag
cccgagccgg ccgccgacga cgggccggag gcgcgcagac gccggctcgg
4020ccagccctcc gtgccgcctg tcgactggcc ggcggtacgg ggcaacctga
tccgggaccc 4080cgcggtgaag tacgccgagc tgggccgggc cttcgtccgc
tgggccgacg ggcacgtggt 4140ggatccggcg gcctggcgcg agttcgtcga
cgccgtgccg ccgtactggc gcaaatcggt 4200ggccgagctg gcccgttcgt
gcgccagcgc ctggctggcg ttcgcccagg aactggagga 4260ccgggcgtga
aaatggcggc cggcatattt acggtggttg ccgacagcgc gtcgcattcc
4320actgtcgcgg ccactacccg atcgagtagt ggaccggctt gaataacgcg
cgttaatgtt 4380ccttcgatcc gctgccctca tttttcggtg agcacatttt
tgcggcggtc caatggagag 4440gagaattccc ggtgaacatt ctgaggcggc
cgcggaaacg gcatctcggg ggtgtcgcgg 4500ccgtcgccgc ggcgatcgcc
ctggtggcgt cgctgacaaa cggtgtggcg gctgccccgc 4560aggcgccgac
cttcgacctc gacaacggga acgccctgac cgacgtcatc tacccggccc
4620tcaacaccga gccgcgggtc gagtacagcg gccggcccgg gtcctgggcc
gcggaccgcg 4680ccatgctcat cgaactgccg tggttcgacg ccctggcggc
gtaccacccc accgcggtcg 4740gcatcttctc caccatcggc cgccgtcccg
ccgaggagca cacgacgcgc aacaagaaca 4800tcgccgtcat ctactcggcc
tacacctcgc tcagcaagct ctacccccag cacgaggcga 4860cctggcagcg
gatgatggcc accgcgggcc tggacccggc cgtcaccgcg gaggaccgga
4920ccaccgccag cggcatcggc atcctcgcct cgaagaacgc gatggcggcg
cgccggaacg 4980acggcacgaa ccgcgacggc gacgcgggcg gccgtcgcta
caaccgtgag ccgtacgccg 5040accacaccgg ctaccggccg gtcaacagcc
cgtacgagct gcgcttcccg tcgcgctggc 5100agccgaacac catctccaag
cgcgaggtcg tcctgacgca ggagttcgcg acgccccagt 5160tcggccgggt
caagccgatc accttcgagc ggcccgagca gttccggctc accccgccgc
5220cgaaccacca cctgttgaac ccgaagggct accggaagca ggccgacgag
gtgctgcgcg 5280cctcggcggg cctggacgac cgcaagaaga tgagcgcgga
gatcttcagc gacaacatca 5340cgccgtacgg cgccatcgcg cacacgctcc
tgcggggccg gtacaacacc gaggactccg 5400tccggttcat cgtgatgact
gacgtcgccg ggttcgacgt ggcgatcgcg tcctggtact 5460acatgcgcaa
gtacgactcg gtgcagccgt tcagcgcgat ccgccacctg tacccgaaca
5520agaagctgac cgcgtggggc ggcccgggcc ggggcaccgt caacgacatc
accggcaccc 5580agtggcgcag ctacctcagc tcggtcgcca tcgcggctcc
ggattacccg tcggtcaacg 5640cggcggtctg cgtcgcctac gcccaggtcg
cgcgccggtt caccggcacg gacaagctga 5700ccgtcgtgat cccggtccgc
aagggctcct cgatcgtgga accgggcgtg accccggccg 5760ccgacatgat
gctcacctgg aacagctact cggagtgggc cgccgagtgc gggcagagcc
5820gggtctgggc cggcgagaac ttccccgcct cggtcgcggc cgccgaccag
tacgcgccgc 5880agatcggcga ccgtgccttc gacttcgtcc agagcaagct
gaacgggcgc tgacgcccgc 5940gtaccggtcc gtgctgccgg
596065532PRTMicromonospora sp. strain 046-ECO11 65Val Asp Pro Val
Pro Val Leu Val Val Gly Ala Gly Pro Val Gly Met1 5 10 15Val Thr Ala
Leu Ala Leu Ala Arg His Gly Val Ala Cys Val Leu Val 20 25 30Asp Gln
Gly Phe Glu Thr Ser Val His Pro Lys Leu Asp Tyr Val Asn 35 40 45Ala
Arg Ser Met Glu Phe Leu Arg Gln Phe Gly Leu Ala Asp Asp Val 50 55
60Arg Ala Ala Gly Val Ala Pro Glu His Arg Ala Asp Val Ile Trp Ser65
70 75 80Thr Gly Leu Ala Gly Glu Pro Ile Thr Arg Trp Gly Leu Pro Ser
Val 85 90 95Thr Gln Glu Trp Arg Arg Ile Ala Glu His Asn Asp Gly Thr
Gln Pro 100 105 110Ala Glu Pro Gly Gln Arg Ile Ser Gln Ile Asp Leu
Glu Pro Val Leu 115 120 125Arg Ala Arg Cys Arg Arg Glu Pro Leu Val
Asp Leu Arg Leu Gly Val 130 135 140Arg Phe Asp Ser Leu Thr Gln Asp
Asp Ala Gly Val Thr Ser Val Leu145 150 155 160Ala Asp Asp Thr Gly
Gly Glu Val Arg Val Arg Ser Glu Tyr Val Val 165 170 175Gly Cys Asp
Gly Ala Ser Ser Gln Val Arg Arg Ala Val Gly Ile Gly 180 185 190Glu
Glu Gly Phe Asp Val Pro Gly Leu Pro Gly Ala Phe Met Val His 195 200
205Phe Thr Ser Arg Asp Leu Asp Ser Leu His Arg His Gly Arg Phe Trp
210 215 220His Tyr Phe Ala Phe Arg Tyr Val Ile Ile Ala Gln Asp Glu
Val Asp225 230 235 240Thr Trp Thr Ala His Val Asn Gly Val Asp Pro
Asn Glu Phe Asp Glu 245 250 255Pro Pro Ala Asp Pro Glu Ala Phe Leu
Leu Asp Thr Ile Arg Thr Glu 260 265 270Leu Arg Ile Asp Lys Val Leu
Leu Thr Ser Arg Trp Arg Pro Gly Phe 275 280 285Met Leu Ala Asp Arg
Tyr Arg Ala Gly Arg Val Leu Leu Ala Gly Asp 290 295 300Ser Ala His
Arg Met Phe Pro Thr Gly Ala Tyr Gly Met Asn Thr Gly305 310 315
320Ile Gly Asp Ala Val Asp Val Ala Trp Lys Leu Ala Ala Val Val Arg
325 330 335Gly Phe Gly Gly Pro Gly Leu Leu Asp Ser Tyr Asp Ala Glu
Arg Arg
340 345 350Pro Val Gly Arg Arg Asn Met Arg Thr Ser His Arg His Leu
Gly Val 355 360 365His Leu Arg Ala Gly Glu Leu Leu Arg Gly Gly Ala
Pro Leu Pro Ser 370 375 380Val Ala Ala Phe Leu Asp Ala Glu Arg Gly
Glu Asn Glu Tyr Arg Gly385 390 395 400Ile Glu Leu Gly Tyr Arg Tyr
Ser Gly Ser Pro Val Leu Trp Pro Glu 405 410 415Gly Pro Gly Glu Pro
Ser Asp Asp Pro Arg Ala Tyr Ala Pro Thr Thr 420 425 430Trp Pro Gly
Ala Arg Pro Pro Ser Leu Leu Leu Ser Asp Gly Gln Gln 435 440 445Ile
Phe Asp Arg Phe Asp Pro Ala Ser Phe Thr Leu Val Asp Phe Thr 450 455
460Gly Asp Gly Ala Ala Gly Pro Leu Leu Ala Ala Ala Ala Ala Arg
Gly465 470 475 480Leu Pro Val Thr His Thr Val Val Thr Asp Pro Arg
Ala Arg Glu Leu 485 490 495Trp Glu Arg Asp Leu Val Leu Leu Arg Pro
Asp His His Val Ala Trp 500 505 510Arg Gly Asn Thr Val Pro Pro Asp
Pro Asp Ala Val Val Gln Arg Val 515 520 525Arg Gly Gly Gly
530661599DNAMicromonospora sp. strain 046-ECO11 66gtggatccgg
taccggttct ggtcgtgggc gcgggcccgg tcggcatggt caccgcgctg 60gcgctcgccc
gtcacggcgt cgcctgcgtc ctcgtcgacc agggcttcga gacgtcggtc
120catcccaagc tggactacgt caacgcccgc agcatggagt tcctccgcca
gttcggcctc 180gccgacgacg tccgtgccgc cggcgtcgcg cccgagcacc
gggccgacgt catctggtcg 240accggcctgg ccggtgagcc gatcaccagg
tgggggctgc cctcggtgac gcaggagtgg 300cgccgcatcg ccgagcacaa
cgacggcacc cagccggccg agcccggcca gcggatctcc 360cagatcgacc
tggaaccggt cctgcgggcc cgctgccggc gggagcccct tgtcgacctg
420cgcctcggcg tacggttcga ctcgctgacc caggacgacg cgggggtcac
cagcgtcctc 480gccgacgaca ccggcggcga ggtccgggtg cggtcggagt
acgtggtcgg gtgcgacggc 540gcgtcgagcc aggtccgccg ggccgtgggc
atcggtgagg aggggttcga cgtgcccggc 600ctgccgggcg ccttcatggt
gcacttcacc agccgggacc tggacagcct gcaccggcac 660ggccggttct
ggcactactt cgcgttccgg tacgtgatca tcgcccagga cgaggtcgac
720acctggaccg cgcacgtcaa cggcgtcgac ccgaacgagt tcgacgagcc
gccggccgac 780ccggaggcgt tcctgctcga cacgatccgc accgagctgc
ggatcgacaa ggtgctgctc 840acctcgcgct ggcgtcccgg cttcatgctc
gccgacaggt accgcgccgg ccgggtgctg 900ctcgccggtg actcggccca
ccggatgttc cccaccggcg cgtacggcat gaacaccggc 960atcggcgacg
ccgtcgacgt ggcctggaag ctggccgctg tcgtccgggg cttcggcggc
1020cccgggctgc tcgacagcta cgacgccgaa cgccgcccgg tggggcggcg
caacatgcgc 1080acctcgcacc ggcacctggg cgtgcacctg cgggcgggcg
agctcctgcg cggcggcgcc 1140ccgctgccgt ccgtcgcggc cttcctcgac
gccgagcggg gcgagaacga gtaccggggg 1200atcgagctcg gctaccgcta
ctccggctcg ccggtgctct ggccggaggg cccgggggag 1260ccctcggacg
acccgcgggc gtacgccccg acgacctggc ccggcgcccg tccgcccagc
1320ctcctgctga gcgacgggca gcagatcttc gaccggttcg acccggcctc
gttcaccctc 1380gtggacttca ccggtgacgg cgccgccggt ccgctgctgg
cggcggcggc cgcgcggggg 1440ctcccggtca cccacaccgt ggtgaccgac
ccccgggctc gtgagctgtg ggaacgcgac 1500ctcgtcctgc tgcggccgga
ccaccacgtc gcctggcggg gaaacaccgt gccgccggac 1560cccgacgccg
tggtccagcg cgtgcggggt ggcggatag 159967423PRTMicromonospora sp.
strain 046-ECO11 67Met Gln Gln Ser Gly Ser Thr Ala Glu Arg Ser Pro
Leu Gly Pro Trp1 5 10 15Glu Gly Met Pro Ala Val Gln Gln Pro Asp Trp
Gln Asp His Pro Ala 20 25 30Tyr Ala Glu Thr Cys Gln Ala Leu Ala Ser
Ala Pro Pro Leu Val Pro 35 40 45Pro Gly Glu Val Arg Gly Phe Arg Gln
Leu Leu Ser Glu Leu Ala Ser 50 55 60Thr Asp Gly Leu Leu Leu Gln Leu
Gly Asp Cys Ala Glu Ser Leu Tyr65 70 75 80Glu Cys Thr Pro Arg His
Thr Ser Asp Lys Ile Glu Val Ile Asp Arg 85 90 95Leu Gly Asp Arg Leu
Ser Glu Leu Thr Gly Arg Asn Val Leu Arg Val 100 105 110Gly Arg Met
Ala Gly Gln Phe Ala Lys Pro Arg Ser Gln Ala Thr Glu 115 120 125Trp
His Asp Ala Leu Ser Ile Pro Ser Phe Arg Gly His Met Ile Asn 130 135
140Ser Glu Leu Ala Ala Pro Gly Thr Arg Lys Ala Asp Pro Arg Arg
Met145 150 155 160Trp Trp Ala Tyr Glu Ala Ser Asp Arg Val Gln Arg
Val Leu Arg Ala 165 170 175His Arg Glu Gly Asn Arg Arg Ala Ala Arg
Thr Glu Gly Pro Trp Ser 180 185 190Ser His Glu Ala Leu Val Val Asp
Tyr Glu Ser Arg Leu Ile Arg Arg 195 200 205Asp Pro Asp Thr Gly Glu
His Tyr Leu Ala Ser Thr His Leu Pro Trp 210 215 220Val Gly Glu Arg
Thr Arg Arg Ser Ala Glu Ala His Val Ala Met Leu225 230 235 240Ser
Thr Val Val Asn Pro Val Gly Cys Lys Ile Gly Pro Asp Ala Asp 245 250
255Pro Asp Asp Val Leu Arg Val Cys Glu Ala Leu Asp Pro Arg Arg Asp
260 265 270Pro Gly Arg Leu Val Leu Ile Pro Arg Met Gly Arg Asp Arg
Ile Arg 275 280 285Glu Ser Leu Pro Pro Ile Val Arg Ala Val Val Asn
Ala Gly His Pro 290 295 300Val Leu Trp Leu Ser Asp Pro Met His Gly
Asn Thr Val Lys Ala Ser305 310 315 320Val Gly Leu Lys Thr Arg His
Leu Ser Asp Val Val Thr Glu Ala Leu 325 330 335Trp Phe Arg Asp Ile
Leu Asp Gln Gln Arg Gln His Ala Ala Gly Leu 340 345 350His Ile Glu
Val Ala Ala Thr Asp Val Thr Glu Cys Val Gly Gly Ser 355 360 365Val
Ala Gly Glu Glu Asp Leu Ala Arg His Tyr Thr Ser Leu Cys Asp 370 375
380Pro Arg Leu Asn Pro Gly Gln Ala Thr Glu Leu Ile Glu Ala Trp
Ala385 390 395 400Lys Asp Thr Ala Thr Val Gly Pro Gly Pro Arg Arg
Ser Gly Pro Ser 405 410 415Ala Arg Pro Glu Val Ala Ala
420681272DNAMicromonospora sp. strain 046-ECO11 68atgcagcaat
ccggttcaac ggcggaacgc agcccactcg ggccgtggga gggcatgccg 60gcggtccagc
aaccggactg gcaggaccac ccggcgtacg cggagacctg tcaggcgttg
120gcgtcggccc cgccgctggt cccacccggg gaggtacggg ggttccggca
gctgttgtcg 180gagctggcgt cgaccgacgg gctcctgctg cagttgggcg
actgcgccga gagcctctac 240gagtgcaccc cccggcacac ctcggacaag
atcgaggtca tcgaccggct gggggaccgg 300ctcagcgagc tcaccgggcg
caacgtgctg cgggtgggcc ggatggccgg gcagttcgcc 360aagccccggt
cgcaggcgac ggagtggcac gacgcgctga gcatcccctc cttccgcggc
420cacatgatca attccgagct ggccgcgccc ggtacgcgca aggccgaccc
tcgccgcatg 480tggtgggcgt acgaggcgag cgaccgggtg cagcgggtcc
tgcgcgccca ccgggagggc 540aaccggcgtg ccgcgcggac cgaggggccg
tggtcgagcc acgaggccct ggtcgtcgac 600tacgagtccc gcctgatccg
ccgggacccg gacacgggcg agcactacct ggcgtcgacc 660cacctgccgt
gggtggggga gcggacccgc cggtccgccg aggcgcacgt ggccatgctg
720tccacggtgg tgaacccggt cggctgcaag atcgggccgg acgccgaccc
ggacgacgtc 780ctgcgggtgt gcgaggcgct cgacccgcgg cgcgatccgg
gccgtctcgt cctgatcccg 840cggatgggcc gggaccggat ccgggagtcc
ctgccgccga tcgtccgcgc ggtggtgaac 900gcggggcacc ccgtgctctg
gctgagcgat cccatgcacg gcaacaccgt caaggcctcg 960gtcggcctga
agacgcgcca cctctccgac gtggtcaccg aggcgctgtg gttccgcgac
1020atcctcgacc agcagcggca gcacgccgcc gggctgcaca tcgaggtcgc
cgccaccgac 1080gtgaccgagt gcgtcggcgg ttcggtggcc ggcgaggagg
acctggcgcg gcactacacc 1140tcgctgtgcg acccgcggct caacccgggt
caggccaccg agctgatcga agcgtgggcc 1200aaggacaccg cgacggtcgg
cccgggaccg cggcgctccg gcccttcggc gcggccggag 1260gtcgccgcct ga
127269340PRTMicromonospora sp. strain 046-ECO11 69Met Trp Gly Ser
Ser Asn Thr Leu Glu Val Lys Gly Asn Asp Glu Arg1 5 10 15Phe Pro Leu
Pro Asp Ala Ala Thr Glu Asp Arg Ser Val Leu Gly Glu 20 25 30Thr Val
Pro Val Ser Ala Leu Leu Pro Gly Asp Ser Pro Arg Leu Ala 35 40 45Gly
Glu Asn Val Glu His Ile Arg Leu Leu Ala Ala Met His Asp Leu 50 55
60Pro Pro Ile Leu Val Gln Arg Gly Thr Met Arg Val Ile Asp Gly Met65
70 75 80His Arg Leu Arg Ala Ala Lys Leu Arg Gly Asp Glu Thr Val Arg
Val 85 90 95Thr Phe Phe Asp Gly Asp Asp Ala Ala Ala Phe Leu Leu Ser
Val Asp 100 105 110Ala Asn Ile Lys His Gly Leu Pro Leu Ser Arg Ala
Asp Arg Glu Ala 115 120 125Ala Ala Thr Arg Ile Leu Arg Leu Tyr Pro
Gln Trp Ser Asp Arg Ala 130 135 140Val Ala Ala Ala Ala Gly Leu Ser
Pro Thr Thr Ala Ser Gly Ile Arg145 150 155 160Arg Arg Leu Leu Gln
Pro Ala Ala Arg Glu Gly Ser Arg Val Gly Arg 165 170 175Asp Gly Arg
Val Arg Pro Leu Asp Gly Ser Ala Gly Arg Arg Arg Ala 180 185 190Ser
Ala Val Ile Ala Leu Arg Pro Asp Ala Pro Leu Arg Ala Ile Ala 195 200
205Gln Glu Ala Gly Val Ser Val Gly Thr Ala Arg Asp Val Arg Ala Arg
210 215 220Leu Gln Ala Gly Arg Asp Pro Val Leu Thr Ser Gln Arg Pro
Ala Ala225 230 235 240Glu Pro Glu Pro Ala Ala Asp Asp Gly Pro Glu
Ala Arg Arg Arg Arg 245 250 255Leu Gly Gln Pro Ser Val Pro Pro Val
Asp Trp Pro Ala Val Arg Gly 260 265 270Asn Leu Ile Arg Asp Pro Ala
Val Lys Tyr Ala Glu Leu Gly Arg Ala 275 280 285Phe Val Arg Trp Ala
Asp Gly His Val Val Asp Pro Ala Ala Trp Arg 290 295 300Glu Phe Val
Asp Ala Val Pro Pro Tyr Trp Arg Lys Ser Val Ala Glu305 310 315
320Leu Ala Arg Ser Cys Ala Ser Ala Trp Leu Ala Phe Ala Gln Glu Leu
325 330 335Glu Asp Arg Ala 340701023DNAMicromonospora sp. strain
046-ECO11 70atgtggggca gctcaaacac gctggaagtg aagggcaacg acgagagatt
ccccctgccc 60gatgcagcta cggaggatcg gtctgtgctt ggcgagacgg ttccggtttc
cgcgctgctg 120cccggtgact ccccgcggct ggcgggcgag aacgtcgagc
acatccggct gctggccgcg 180atgcacgacc tcccgccgat cctggtgcaa
cgcggcacga tgcgggtgat cgacggcatg 240caccggctgc gggccgccaa
gctgcgcggc gacgagaccg tgcgggtgac gttcttcgac 300ggggacgacg
ccgcggcgtt cctgctctcg gtcgacgcca acatcaaaca cgggctgccg
360ttgtcccgcg ccgaccggga ggccgccgcc acccgcatcc tgcggttgta
tccgcagtgg 420tcggaccgcg ccgtcgccgc ggcggccggg ctgtcaccga
ccacggcgag cggcatccgg 480cgccgcctgc tgcaaccggc ggcgcgggag
ggcagccggg tgggacggga cgggcgggtg 540cgcccgctgg acggctcggc
gggccgacgg cgggccagcg cggtcatcgc gctccggccg 600gacgcgcccc
tgcgtgccat cgcgcaggag gccggggtgt cggtgggcac ggcgcgggac
660gtgcgcgccc ggttgcaggc gggccgggac cccgtcctga cctcgcagcg
accggcggcc 720gagcccgagc cggccgccga cgacgggccg gaggcgcgca
gacgccggct cggccagccc 780tccgtgccgc ctgtcgactg gccggcggta
cggggcaacc tgatccggga ccccgcggtg 840aagtacgccg agctgggccg
ggccttcgtc cgctgggccg acgggcacgt ggtggatccg 900gcggcctggc
gcgagttcgt cgacgccgtg ccgccgtact ggcgcaaatc ggtggccgag
960ctggcccgtt cgtgcgccag cgcctggctg gcgttcgccc aggaactgga
ggaccgggcg 1020tga 102371493PRTMicromonospora sp. strain 046-ECO11
71Val Asn Ile Leu Arg Arg Pro Arg Lys Arg His Leu Gly Gly Val Ala1
5 10 15Ala Val Ala Ala Ala Ile Ala Leu Val Ala Ser Leu Thr Asn Gly
Val 20 25 30Ala Ala Ala Pro Gln Ala Pro Thr Phe Asp Leu Asp Asn Gly
Asn Ala 35 40 45Leu Thr Asp Val Ile Tyr Pro Ala Leu Asn Thr Glu Pro
Arg Val Glu 50 55 60Tyr Ser Gly Arg Pro Gly Ser Trp Ala Ala Asp Arg
Ala Met Leu Ile65 70 75 80Glu Leu Pro Trp Phe Asp Ala Leu Ala Ala
Tyr His Pro Thr Ala Val 85 90 95Gly Ile Phe Ser Thr Ile Gly Arg Arg
Pro Ala Glu Glu His Thr Thr 100 105 110Arg Asn Lys Asn Ile Ala Val
Ile Tyr Ser Ala Tyr Thr Ser Leu Ser 115 120 125Lys Leu Tyr Pro Gln
His Glu Ala Thr Trp Gln Arg Met Met Ala Thr 130 135 140Ala Gly Leu
Asp Pro Ala Val Thr Ala Glu Asp Arg Thr Thr Ala Ser145 150 155
160Gly Ile Gly Ile Leu Ala Ser Lys Asn Ala Met Ala Ala Arg Arg Asn
165 170 175Asp Gly Thr Asn Arg Asp Gly Asp Ala Gly Gly Arg Arg Tyr
Asn Arg 180 185 190Glu Pro Tyr Ala Asp His Thr Gly Tyr Arg Pro Val
Asn Ser Pro Tyr 195 200 205Glu Leu Arg Phe Pro Ser Arg Trp Gln Pro
Asn Thr Ile Ser Lys Arg 210 215 220Glu Val Val Leu Thr Gln Glu Phe
Ala Thr Pro Gln Phe Gly Arg Val225 230 235 240Lys Pro Ile Thr Phe
Glu Arg Pro Glu Gln Phe Arg Leu Thr Pro Pro 245 250 255Pro Asn His
His Leu Leu Asn Pro Lys Gly Tyr Arg Lys Gln Ala Asp 260 265 270Glu
Val Leu Arg Ala Ser Ala Gly Leu Asp Asp Arg Lys Lys Met Ser 275 280
285Ala Glu Ile Phe Ser Asp Asn Ile Thr Pro Tyr Gly Ala Ile Ala His
290 295 300Thr Leu Leu Arg Gly Arg Tyr Asn Thr Glu Asp Ser Val Arg
Phe Ile305 310 315 320Val Met Thr Asp Val Ala Gly Phe Asp Val Ala
Ile Ala Ser Trp Tyr 325 330 335Tyr Met Arg Lys Tyr Asp Ser Val Gln
Pro Phe Ser Ala Ile Arg His 340 345 350Leu Tyr Pro Asn Lys Lys Leu
Thr Ala Trp Gly Gly Pro Gly Arg Gly 355 360 365Thr Val Asn Asp Ile
Thr Gly Thr Gln Trp Arg Ser Tyr Leu Ser Ser 370 375 380Val Ala Ile
Ala Ala Pro Asp Tyr Pro Ser Val Asn Ala Ala Val Cys385 390 395
400Val Ala Tyr Ala Gln Val Ala Arg Arg Phe Thr Gly Thr Asp Lys Leu
405 410 415Thr Val Val Ile Pro Val Arg Lys Gly Ser Ser Ile Val Glu
Pro Gly 420 425 430Val Thr Pro Ala Ala Asp Met Met Leu Thr Trp Asn
Ser Tyr Ser Glu 435 440 445Trp Ala Ala Glu Cys Gly Gln Ser Arg Val
Trp Ala Gly Glu Asn Phe 450 455 460Pro Ala Ser Val Ala Ala Ala Asp
Gln Tyr Ala Pro Gln Ile Gly Asp465 470 475 480Arg Ala Phe Asp Phe
Val Gln Ser Lys Leu Asn Gly Arg 485 490721482DNAMicromonospora sp.
strain 046-ECO11 72gtgaacattc tgaggcggcc gcggaaacgg catctcgggg
gtgtcgcggc cgtcgccgcg 60gcgatcgccc tggtggcgtc gctgacaaac ggtgtggcgg
ctgccccgca ggcgccgacc 120ttcgacctcg acaacgggaa cgccctgacc
gacgtcatct acccggccct caacaccgag 180ccgcgggtcg agtacagcgg
ccggcccggg tcctgggccg cggaccgcgc catgctcatc 240gaactgccgt
ggttcgacgc cctggcggcg taccacccca ccgcggtcgg catcttctcc
300accatcggcc gccgtcccgc cgaggagcac acgacgcgca acaagaacat
cgccgtcatc 360tactcggcct acacctcgct cagcaagctc tacccccagc
acgaggcgac ctggcagcgg 420atgatggcca ccgcgggcct ggacccggcc
gtcaccgcgg aggaccggac caccgccagc 480ggcatcggca tcctcgcctc
gaagaacgcg atggcggcgc gccggaacga cggcacgaac 540cgcgacggcg
acgcgggcgg ccgtcgctac aaccgtgagc cgtacgccga ccacaccggc
600taccggccgg tcaacagccc gtacgagctg cgcttcccgt cgcgctggca
gccgaacacc 660atctccaagc gcgaggtcgt cctgacgcag gagttcgcga
cgccccagtt cggccgggtc 720aagccgatca ccttcgagcg gcccgagcag
ttccggctca ccccgccgcc gaaccaccac 780ctgttgaacc cgaagggcta
ccggaagcag gccgacgagg tgctgcgcgc ctcggcgggc 840ctggacgacc
gcaagaagat gagcgcggag atcttcagcg acaacatcac gccgtacggc
900gccatcgcgc acacgctcct gcggggccgg tacaacaccg aggactccgt
ccggttcatc 960gtgatgactg acgtcgccgg gttcgacgtg gcgatcgcgt
cctggtacta catgcgcaag 1020tacgactcgg tgcagccgtt cagcgcgatc
cgccacctgt acccgaacaa gaagctgacc 1080gcgtggggcg gcccgggccg
gggcaccgtc aacgacatca ccggcaccca gtggcgcagc 1140tacctcagct
cggtcgccat cgcggctccg gattacccgt cggtcaacgc ggcggtctgc
1200gtcgcctacg cccaggtcgc gcgccggttc accggcacgg acaagctgac
cgtcgtgatc 1260ccggtccgca agggctcctc gatcgtggaa ccgggcgtga
ccccggccgc cgacatgatg 1320ctcacctgga acagctactc ggagtgggcc
gccgagtgcg ggcagagccg ggtctgggcc 1380ggcgagaact tccccgcctc
ggtcgcggcc gccgaccagt acgcgccgca gatcggcgac 1440cgtgccttcg
acttcgtcca gagcaagctg aacgggcgct ga 1482739762DNAMicromonospora sp.
strain 046-ECO11 73cagccacggc gttccgaccc cccgcaagat ggcttgtata
gcaaggtatc ttgcgatgca 60tggacggggc acgtgagcgg atcactacga acatccgcaa
gggcgtgctg gagtactgcg 120tgctcgccct gctctcgcgg cgcgacatgt
acggcctgga actggccgac tggctcgccg 180tccgcggtct gaccgcgagc
gagggcagcc tgtatccgct gctcgcccgc atgcggcagg 240ccggctccgt
gcagacccgg tgggtggccc ccgagcaggg gcacgcccgg cggtactacg
300cgatcaccga ccaggggcgg gcgcacctgc gggtgttcgc ggcggtgtgg
caggagatcc 360agccgcacgt ggacgacctg atgggggagg aagcatgagc
gacgacggcc tcccggaggc 420ggcgtggacc
tatctgcgcg cgctcgacgc ggagttgtcc gacgtcccgt ccggcacggc
480ggaggagatc gtcgcggatg tccgcgcgca catcgccgac gccctcgaca
gcggacggag 540cgcccacgag atcctcgccg gcctcggcgc cgcgcgggac
gtggcccggc aggcgcgcga 600ggagctgggg ctgccggccc aggaccgccc
ggcccgggcc ggccggaccc tgtccctggc 660cgcggtggcg gtcggcgtgc
tgatcgccgt gtgcgtgagc ttcctgctgc cgtccgcagt 720gccggtggag
ccgatccagg ccggccccgg cgagcagggc gtcctccgcc ggctcggccc
780cggaatcgcg ctgctcacgc tgctgccggc gctcgtcgcg gccgcgccgc
tcgtggcgcc 840cgcccgggca cgtgccgggg tacggttcgc cggcgcggcg
gtcctgacga tgttcgcctg 900cgcggccggc gagacgggcc tgtactactt
cccgctcgcg ctgatggcct gggcggcggc 960gatcgtgccg tgggccctgc
ggcgcggagc cggtggacgg tggtggcgct atctgaccgg 1020tggattcgtg
gcgatgcccg gcgtgctggt ggcggtcgcg tcggccggtg gctcggtcgg
1080cgtcggctgg gtcggcgcgg cgctgtggat cgccgggccg ctcgcggccg
gcgcgctgtg 1140cgcctacggg atccgggccg gctacgccgt gaccgcgctg
gccggcgcgc tggccatagc 1200gctctcgatg gccgagcgcg gcttcctgtt
cgccgccttc tggctgttcg gcgggctgta 1260cctggcgctc ggcgccgctg
cgtacaccgc ctcgcgggcc gtcgacggcg acgccgccgc 1320gacgcccggc
ccgccggccc ggccggaacc cgcgccggcc cccggaggct gacccggggg
1380ccgtggcgcc ggccggctag gcggggacgg cctgcgggtc gccggcggcg
tcgtgcgcgg 1440ccatcgtctc ctgccggacg ggctcctcgc gcaggatcgc
cgcgtgcagc cacgcgtccg 1500ggatggcgaa gccgtccacg agcgtgcgca
tgtccgggcg cagctccttg agcagcccgt 1560tcaccacgct ggtgatggtc
ttcgagcggg ccggggtgag ccggccgtgc tcgagcagcc 1620agcccttgtt
cgcctcgatc acggtgagcg cgtacaggtc gcagacccgg gacagcagtt
1680ccttgaccgc cgggtcggcg atggcgtcga tcccggcgac gaacgcctcc
agcgtcaccc 1740ggtcgatgtg cgccgcggcg acggcgagga cgtggtcctg
gacgtcgttg aagatgtcga 1800aggggcggtc cttcttggtg gacgcgccac
cgcgcaggcg gcggaccgcg ctgtcgagca 1860ggtgctcctc gcggtcctcg
aagagcttga gctgccagcc ccggtcggtg acggcgacct 1920cgtcgtcgcg
cccgggcacg gcgctgacca gacgtgcgat cagcgcccgc gcggcggtgc
1980gttccagcac catctcgcgt acctgctcgg ccacgaagga ggcgcgtccc
cagccgtcga 2040gcgagccgaa ctcgtcccgg tagccggtca gcagcccctt
ggcgaccagt tgcagcagca 2100ccgtgttgtc gccctcgaag gtggtgaaga
catcggtgtc ggccttgagg ctgggcaggc 2160ggttctcgga caggtagccg
gcgccgccac acgcctcccg gcagatctgg atggtgcggg 2220tggcgtgcca
ggtctgcgcc gccttcagac cggcggcccg ggactccagc tcccgctgcc
2280ggtgctcgtc gaccggcccg tcgccgccct ggatgtcgtc gagcgccgcg
accagctccg 2340cctgggcgaa ggtcagcgcg tacgtggtgg ccagcgcggg
cagcagcttg cgctggtgcg 2400ccaggtagtc gttgagcagc acctcgcggt
cgccgtcggc gtcggcgaac tgccggcgga 2460tgtcgccgta gcgcaccgcg
atggccagcg ccgacttggt ggccgccgac gcggcgccgc 2520ccacgctcac
ccggccccgg accagggtgc ccagcatggt gaagaagcgc cgggagtcgt
2580tctcgatcgg gctggagtac gtgccgtcct cggcgacctg cgcgtactgg
tccagcagca 2640tctcccgcgg cacccgcacg tggtcgaagc tgagccgccc
gttgtccacg ccgagcaggc 2700cggccttggg cccggcgtcg ccgatggtca
cgccgggcat cggcttgccg tgctcgtcgc 2760ggatcggcac cagccaggcg
tgcaccccgt ggcggcgccc gccggtgacg agctgggcga 2820acaccacagc
catccgcccg tcccgggccg cgttgccgat gtagtccttg cgcgcggcct
2880cgtgcggggt gtgcaggtcg aaggtctgcg tctgcgggtc gtagacgcag
gtggtgcgca 2940gttgctgcac gtccgagccg tggccggtct cggtcatcgc
gaagcagccg aagagccggc 3000ccgcgacgat gtcccgcagg taggcgtcgt
ggtgccgctt cgtgccgagg gcggcgaccg 3060cgccgccgaa caggccccac
tgcacgccgg ccttcaccat cagtgacagg tccacctggg 3120ccagcatctc
ggtggcgacg atcgaggcgc ccacgtcgcc gcggccgccg tactcggcgg
3180ggaaaccgga ggcgatgccc agctcgacgg ggagttcgga cagcagccgg
gtgatgcgct 3240cgcgggcctg gtcaccggtc tcgccgtaca ccgggaggaa
gcgttcgtcg aggtgttcgc 3300ggtgcgcccg gcggacctcg gcccaccggc
cgtcgagcgc ttcccgcagg cgtgtgacgt 3360cgatgcggcc ggatgcgtga
tcgagcattg tcactcctcg gggcagcgga catttgcgta 3420tactctcggc
ctgatcaaca ttaccggcgg tgatcgcacc ccgctggcgg agcgcgtggt
3480gagcccggcc acccccggcg gttcggccac ccgtgaagct gaggttaggc
tgtcctcact 3540tcacagcact ggaggcatcc cctcgtgtcc ccgcttcccc
ccggcagcgc cgtcaccgcc 3600cggcacgtgc tccgccaggc gctgcgccgc
cagcgccgcc cggtgctgat cggcgtgacc 3660ctgctcgggc tgcaccaggt
caccgaggcg ctcgtgccgg tggcgatcgg cgtcatcatc 3720gaccgggccg
tggtgaccgg cgacccgtgg gcgctcgcgt actccgtcgc cggcctcgcc
3780gccctgttca ccgtgctggc gttcgcctac cgcaacggcg cccgccaggc
gttcgcggcg 3840gtggaacggg aggcgcacct gctgcgggtc gagctggccg
agcgcgcgct cgacccgcgc 3900gggcaccgct ccggcctgcg cgacggcgag
ctgctctcgg tcgccgcctc cgacgccgaa 3960ctctccgcgt acgtggtccg
ggtggccggc ttcggcgtcg ccgcggtgag cgcgctgacc 4020gtcgcggcgg
tcgcgctgct ggtcatcgac gtcccgctcg gactcggcgt gctcatcggc
4080gtaccggtgc tggtcctggc gctgcaacgg atggcgccgc tgctgtcccg
gcgcagcgcc 4140tcccagcagg aggccctcgc ggagaccacg gcgctcgccg
tggacctcgt ctccggcctg 4200cgcgtgctgc gcggcatcgg cgcccagcac
cacgccgccg gccggtacgc cgaggccagc 4260cgacgcgccc tcgccgtgac
gctgcgcgcc gccaacacca agggcctgca cctcgggctc 4320accaccgccg
cgaacggcct cttcctcgcc gccgtcgccg gggtcgccgg ctggctcgcg
4380ctgcgcggcc ggctcaccat cggcgagctg gtcaccgtgg tcgggctcgc
gcagttcgtc 4440gccgagccgg tgcagacgct gggctactgc gtgcagctgt
tcgcgatggc ccgcgcctcc 4500gccgcccggg tcgggcgcgt gctcggcgcc
gagccgctga cccggccggg cagcgcgccc 4560cggccggacc gcacggacgg
gccgcggctc gtcctcgacc acgtcggcca cgccgcgctg 4620gacggggtgt
gcctgcgcgt cgacccggga gagatcgtcg gcgtcctggc gtacgacccg
4680gccgacgcgg acgcgctggt ggcgctgctg tccgggcggg tgcccgcgga
ccggcgccgg 4740ggcacggtac gcgtcgacgg ggtacccgcc gacgacctgg
acgtcgacgc gctgcgcggc 4800gccgtcctgg tcgagccgca cgacgtgacg
ctgttcgagg gaaccgtggc cgccaacctc 4860gccgccggga gcaggaccga
ggaggggcgc ctgcgcgccg cggtccgggc ggccgcggcg 4920gacgacgtgg
tggacgcgca ccccggcggc ctcggccacc ggctcgtcga gcggggcgcc
4980aacctctccg gcgggcagcg ccagcggctc gggctggcgc gggcgctgca
cgccgacccg 5040ccggtgctgg tgctgcacga ccccaccacc gccgtggacg
cggccaccga ggcccaactc 5100gccgacggac tggccggcgc gcgccgcgaa
gcgccccggg gcacgctgct ggtcaccagc 5160agccccgccc tgctgcggat
caccgaccgg gtggtggtga tcgccgacgg ccgggtgacc 5220gccgagggga
cgcacgagca cctgctggcc accgacgccc gctaccgcga ggagacactg
5280cggtgaccgc tgacccgcgt accgccgaac ccacccgggt gttgctgccc
accgcgaccg 5340cccggcggac ctggacgacg ctcggcgcgg agttccgccg
gcggcccggc ctcagcgccg 5400ccgcgaccgc cgtgctcgtc gccgccgcca
ccggcgggct ggtcgcgccc tgggtgctcg 5460gccgcctcgt cgacgacgtc
atcgccgacg ccccggtctc ccggatcgcc ggccgggtgg 5520cggtgatcgc
cggcgcggca gtgctcaccg gactgctcac cgccgccggg gccgcgctcg
5580cgtcccgcct gggggagacg gtgctggccc ggctgcgcga gcgggtcctc
gaccgggcgc 5640tgcacctgcc ctcggcgacg ctggaacggg ccggcaccgg
cgacctgctg gcccgggtcg 5700gcgacgacgt ggcggtggtg acgaacgtga
tcgcggtcag cggcccggcg ttcgtcggcg 5760cgctgctgtc cgtggtgctg
accgtgttcg ggctggtcgc gctcgactgg cggctcggcc 5820tcgccgggct
ggtcgccgcg cccgcctacg cgctggcgct gcgctggtac ctgcgccggt
5880cggcgccgta ctacgcccgc gagcgcgtcg ccaccggcga gcggacgcag
gcgatggccg 5940gcgcgctgcg tggcgcggcc accgtgcgcg cgtaccggac
cgaggacgcg cacgtcgcgg 6000cgatcgccga gcgctccggc gtggcgcgcg
acctgtcgct ggagatcttc aacctgcaca 6060cccggttcgg gctgcggatc
aacaggtcgg agttcctcgg cctggccgcg gtgctcgtcg 6120ccgggttctt
cctggtccgc gccgacctgg tcacagtggg cgcggcgacc accgccgcgc
6180tctacttcca ccggctgttc aacccgatcg gcctgctgct gatggagtcc
gactcggtgc 6240tgcaggccgg cgcgagcctc gcccggctgg tcggcgtggc
cacgctgccc gacaccgccc 6300cgtccgggcc cgcgccgtcg gcggccgggc
ggcgcggccc ggcggcgctg gacgtcacgg 6360tccgccggca ccgctacgac
gacgacggcc ctctggtcct ggccgacgtc gacctgcgcc 6420tggccccggg
cgagcgggtc gcgctcgtgg gcgccagcgg cgcgggcaag agcacgctcg
6480ccggcatcgc cgccgggatc atcgcgccca ccgacgggtc ggtacgcctg
ggcggcgtgc 6540cgctgaccga gcggggcgag cacgccgtgc ggcgcgacgt
cgcgctggtc agccaggagg 6600tgcacgtctt cgctggaccg ctcgccgagg
atctgcgcct ggctgccccg gacgccaccg 6660acgccgaact gctcgacgcg
ctggaccggg tcggcgccac cacctggctg cgcgcgctgc 6720cggacgggct
ggccacagcg gtcggcgagg gcggccaccg gctcaccgcc gcgcaggccc
6780agcaggtcgc cctggcccgg ctggtgctgg ccgcgcccgc cgtcgccgtg
ctggacgagg 6840ccaccgccga ggccggcagc gccggagcgc gtgacctgga
ccgggcggcg ctggccgcca 6900ccgagggacg gaccacgctg atcgtggcgc
accggctcag ccaggcggtc gccgccgacc 6960ggatcgtcct gctcgaccac
gggcggatcg tggagcaggg cacgcactcg gaactgctcg 7020ccgccgacgg
ccggtacggg catctgtggc gctcctggag cgtcccggta tgatcgcgca
7080ccgcccatcg gcccaggtga ggggaacatg accgacgcgc cggcccgctt
cgtgctcttc 7140ccggggcggc accacctgct gacccggttc caggccgact
acctgcggcg gctggccggg 7200gacgacgcca cagtggtctg ggcggtgacg
tcggccaacc acgagaacac caggcgcaac 7260ccggtgccct accaccggcg
ggaggccgcg atcgaacgat tcagcgtgct gagcgggctg 7320cgctcggtgg
tggtgccgat cttcgacacc gcgtacaccg acgcgttcgc cgaggtgacg
7380ctgaagtcca tcgcggtggc caccgggctc gaactcaccc ccgccgacac
cgtgctggcc 7440tgctccacgc cggaggtcgc gaagctgtac gagcagctcg
gcttttcgat cgcgccggtc 7500gaggcggacc cggacctgcc cgagccgccc
gaacggccgt gggacgtgct gctgcgcctg 7560gccgccgggg acgagacctg
gcgcgcgctc acccacccgg ccaccatcga cgtgttcgag 7620cgctaccgcc
tggtcgagtc gatccggtcg gtggtgaacg acccgctcgt cggcgacgag
7680ggcggtctca cagtgacccg cgactaccgg acctacgtcg aggcgttcgc
cacggccgcg 7740cagcgcaagt gggactcggt acgccggtac gtgcagcccg
gccgcatcgt ggacatcggc 7800tgcggcgcgg gcgccgtcct ggaactcgcc
gaccgggagg ccgcgctgcg tgagagcgac 7860ctgatcggcg tggaggtcgc
ccgccacctc taccaggagt gcctgcacaa gaaggcgcag 7920ggcgtgttcc
gcaacgccaa cgtctacttc ttccaccgca acgtcctcgg cggcgcggtg
7980ttcaaggacc gctcggtcga caccacgctc acgttcgcgc tgacccacga
gatctggtcg 8040tacgggcggc ggcgggagtc gctgctgcag ttcgcccgcc
gcatccacga ccacacggtg 8100cccggcggcg tctggatcaa cagcgacgtg
tgcggtccgg acgacccccg gcggcaggtg 8160ctcctgcgac tgtccaccga
cgacggcgac aacccggccg cgccccgccc cgacctcgcc 8220gagctgacct
cggcggaggt ccggcgttac gtcggcgggc tgtcgacgcg ggcgcggctg
8280gaccagttcg ccgtcgactt cgcgttcgac ttcgactacg agccgctccc
cgacggcgcg 8340gtacgcctga cgctgggcgc cgcgatggac tacctgaccc
gcaaggacta cacggacaac 8400tggctgtcgg agacgcagga gcagttctgc
ggcctgagct tcgccgactg gacggacctg 8460ctcaccgagg cggggttcga
gatcggcccg gcgtcggcgc cggtgcgcaa cgagtgggtg 8520atcgacaacc
ggatcgcgcc agtcgcgtcc ctcaccgacc tcgacggccg gccgctggac
8580tggccgacca cccacgtcct caccgtcgcc caccgccccc gcaaccagtg
agaccgacgg 8640cgcccgccgc gttcggcggg cgccgtcgtc gctcaccggc
tcagcgcgat ccggatcgcc 8700aggacgatca ggatgagccc ggtcagccgt
tcgatcacca gcagcacgga cggccgggtc 8760agccagggct gcaacctgtc
gatgagcatg atgtagcagg cccaccagag caccgcgagg 8820ccgatgaacg
tggcggcgag caccgccgta cgggccgccg ccccctcgcc gggcttgacg
8880aactgcggca cgaacgagac gtagaagacg accaccttga cgttcagcag
ctggctggtg 8940acgcccatga cgaacgagcg gcgggccacg tgcggctcgt
cggcggccgg ggtgtccggc 9000accggcgcgg ggccggtgtc cgtgtccggc
ccggcgccgc ccgcgccgac agtgaccggc 9060tgcgccgccg ggaccgtccg
gcgcggccgg gtcgcccaga ggatcgtgcc gcccaggtag 9120agcaggtaca
gcgcgccggc gacgcgcagc accgtgtaga gcgtcggcga ggagaccagc
9180agggcggaca ggccggcggt cgcgaacgac gcgtgcacca gcgcggcgac
gaacagcccg 9240gccagcacca cgaacccggc ccgccggccg tacctgacgg
tctgccgggt gacgagcgcg 9300aagtcgacgc ccggcacgat gatgatgagc
aggctggcgg cgacgaaact gatgatctgg 9360atgtcagaca cgacgccggc
tctcctgtcc tccggcgagc gccggcactg cctcctcgat 9420gacggagacg
ccgctgtcct ggcgtggtcc gtgccggcgc cactgttccc gcagccggat
9480ccggccgtcc ggcagccgtt cgggccggga ctcgcactcg ccgatgacta
tggtgccgtc 9540ggtgagcacc tccaggtagg cgaagcgcac gacgccctgc
gcgtcgcagg tgccggccag 9600ccggccgtgc cggaccgggc cgccggtgat
ctccgcccag accaggtcgc cacgctggtg 9660gtagtgcccc cgcagcggct
cggcgccgtc accggcgtcg tggtccaccg agacgaagac 9720gcggccgtcg
tagtcgaatg tcgtcatcgc gctcacgccc ac 976274112PRTMicromonospora sp.
strain 046-ECO11 74Met Asp Gly Ala Arg Glu Arg Ile Thr Thr Asn Ile
Arg Lys Gly Val1 5 10 15Leu Glu Tyr Cys Val Leu Ala Leu Leu Ser Arg
Arg Asp Met Tyr Gly 20 25 30Leu Glu Leu Ala Asp Trp Leu Ala Val Arg
Gly Leu Thr Ala Ser Glu 35 40 45Gly Ser Leu Tyr Pro Leu Leu Ala Arg
Met Arg Gln Ala Gly Ser Val 50 55 60Gln Thr Arg Trp Val Ala Pro Glu
Gln Gly His Ala Arg Arg Tyr Tyr65 70 75 80Ala Ile Thr Asp Gln Gly
Arg Ala His Leu Arg Val Phe Ala Ala Val 85 90 95Trp Gln Glu Ile Gln
Pro His Val Asp Asp Leu Met Gly Glu Glu Ala 100 105
11075339DNAMicromonospora sp. strain 046-ECO11 75atggacgggg
cacgtgagcg gatcactacg aacatccgca agggcgtgct ggagtactgc 60gtgctcgccc
tgctctcgcg gcgcgacatg tacggcctgg aactggccga ctggctcgcc
120gtccgcggtc tgaccgcgag cgagggcagc ctgtatccgc tgctcgcccg
catgcggcag 180gccggctccg tgcagacccg gtgggtggcc cccgagcagg
ggcacgcccg gcggtactac 240gcgatcaccg accaggggcg ggcgcacctg
cgggtgttcg cggcggtgtg gcaggagatc 300cagccgcacg tggacgacct
gatgggggag gaagcatga 33976325PRTMicromonospora sp. strain 046-ECO11
76Met Ser Asp Asp Gly Leu Pro Glu Ala Ala Trp Thr Tyr Leu Arg Ala1
5 10 15Leu Asp Ala Glu Leu Ser Asp Val Pro Ser Gly Thr Ala Glu Glu
Ile 20 25 30Val Ala Asp Val Arg Ala His Ile Ala Asp Ala Leu Asp Ser
Gly Arg 35 40 45Ser Ala His Glu Ile Leu Ala Gly Leu Gly Ala Ala Arg
Asp Val Ala 50 55 60Arg Gln Ala Arg Glu Glu Leu Gly Leu Pro Ala Gln
Asp Arg Pro Ala65 70 75 80Arg Ala Gly Arg Thr Leu Ser Leu Ala Ala
Val Ala Val Gly Val Leu 85 90 95Ile Ala Val Cys Val Ser Phe Leu Leu
Pro Ser Ala Val Pro Val Glu 100 105 110Pro Ile Gln Ala Gly Pro Gly
Glu Gln Gly Val Leu Arg Arg Leu Gly 115 120 125Pro Gly Ile Ala Leu
Leu Thr Leu Leu Pro Ala Leu Val Ala Ala Ala 130 135 140Pro Leu Val
Ala Pro Ala Arg Ala Arg Ala Gly Val Arg Phe Ala Gly145 150 155
160Ala Ala Val Leu Thr Met Phe Ala Cys Ala Ala Gly Glu Thr Gly Leu
165 170 175Tyr Tyr Phe Pro Leu Ala Leu Met Ala Trp Ala Ala Ala Ile
Val Pro 180 185 190Trp Ala Leu Arg Arg Gly Ala Gly Gly Arg Trp Trp
Arg Tyr Leu Thr 195 200 205Gly Gly Phe Val Ala Met Pro Gly Val Leu
Val Ala Val Ala Ser Ala 210 215 220Gly Gly Ser Val Gly Val Gly Trp
Val Gly Ala Ala Leu Trp Ile Ala225 230 235 240Gly Pro Leu Ala Ala
Gly Ala Leu Cys Ala Tyr Gly Ile Arg Ala Gly 245 250 255Tyr Ala Val
Thr Ala Leu Ala Gly Ala Leu Ala Ile Ala Leu Ser Met 260 265 270Ala
Glu Arg Gly Phe Leu Phe Ala Ala Phe Trp Leu Phe Gly Gly Leu 275 280
285Tyr Leu Ala Leu Gly Ala Ala Ala Tyr Thr Ala Ser Arg Ala Val Asp
290 295 300Gly Asp Ala Ala Ala Thr Pro Gly Pro Pro Ala Arg Pro Glu
Pro Ala305 310 315 320Pro Ala Pro Gly Gly 32577978DNAMicromonospora
sp. strain 046-ECO11 77atgagcgacg acggcctccc ggaggcggcg tggacctatc
tgcgcgcgct cgacgcggag 60ttgtccgacg tcccgtccgg cacggcggag gagatcgtcg
cggatgtccg cgcgcacatc 120gccgacgccc tcgacagcgg acggagcgcc
cacgagatcc tcgccggcct cggcgccgcg 180cgggacgtgg cccggcaggc
gcgcgaggag ctggggctgc cggcccagga ccgcccggcc 240cgggccggcc
ggaccctgtc cctggccgcg gtggcggtcg gcgtgctgat cgccgtgtgc
300gtgagcttcc tgctgccgtc cgcagtgccg gtggagccga tccaggccgg
ccccggcgag 360cagggcgtcc tccgccggct cggccccgga atcgcgctgc
tcacgctgct gccggcgctc 420gtcgcggccg cgccgctcgt ggcgcccgcc
cgggcacgtg ccggggtacg gttcgccggc 480gcggcggtcc tgacgatgtt
cgcctgcgcg gccggcgaga cgggcctgta ctacttcccg 540ctcgcgctga
tggcctgggc ggcggcgatc gtgccgtggg ccctgcggcg cggagccggt
600ggacggtggt ggcgctatct gaccggtgga ttcgtggcga tgcccggcgt
gctggtggcg 660gtcgcgtcgg ccggtggctc ggtcggcgtc ggctgggtcg
gcgcggcgct gtggatcgcc 720gggccgctcg cggccggcgc gctgtgcgcc
tacgggatcc gggccggcta cgccgtgacc 780gcgctggccg gcgcgctggc
catagcgctc tcgatggccg agcgcggctt cctgttcgcc 840gccttctggc
tgttcggcgg gctgtacctg gcgctcggcg ccgctgcgta caccgcctcg
900cgggccgtcg acggcgacgc cgccgcgacg cccggcccgc cggcccggcc
ggaacccgcg 960ccggcccccg gaggctga 97878663PRTMicromonospora sp.
strain 046-ECO11 78Met Leu Asp His Ala Ser Gly Arg Ile Asp Val Thr
Arg Leu Arg Glu1 5 10 15Ala Leu Asp Gly Arg Trp Ala Glu Val Arg Arg
Ala His Arg Glu His 20 25 30Leu Asp Glu Arg Phe Leu Pro Val Tyr Gly
Glu Thr Gly Asp Gln Ala 35 40 45Arg Glu Arg Ile Thr Arg Leu Leu Ser
Glu Leu Pro Val Glu Leu Gly 50 55 60Ile Ala Ser Gly Phe Pro Ala Glu
Tyr Gly Gly Arg Gly Asp Val Gly65 70 75 80Ala Ser Ile Val Ala Thr
Glu Met Leu Ala Gln Val Asp Leu Ser Leu 85 90 95Met Val Lys Ala Gly
Val Gln Trp Gly Leu Phe Gly Gly Ala Val Ala 100 105 110Ala Leu Gly
Thr Lys Arg His His Asp Ala Tyr Leu Arg Asp Ile Val 115 120 125Ala
Gly Arg Leu Phe Gly Cys Phe Ala Met Thr Glu Thr Gly His Gly 130 135
140Ser Asp Val Gln Gln Leu Arg Thr Thr Cys Val Tyr Asp Pro Gln
Thr145 150 155 160Gln Thr Phe Asp Leu His Thr Pro His Glu Ala Ala
Arg Lys Asp Tyr 165 170 175Ile Gly Asn Ala Ala Arg Asp Gly Arg Met
Ala Val Val Phe Ala Gln 180 185 190Leu Val Thr Gly Gly Arg Arg His
Gly Val His Ala Trp Leu Val Pro 195 200 205Ile Arg Asp Glu His Gly
Lys Pro Met Pro Gly Val Thr Ile Gly Asp 210 215 220Ala Gly Pro Lys
Ala Gly Leu Leu Gly Val Asp Asn Gly Arg Leu Ser225 230 235
240Phe Asp His Val Arg Val Pro Arg Glu Met Leu Leu Asp Gln Tyr Ala
245 250 255Gln Val Ala Glu Asp Gly Thr Tyr Ser Ser Pro Ile Glu Asn
Asp Ser 260 265 270Arg Arg Phe Phe Thr Met Leu Gly Thr Leu Val Arg
Gly Arg Val Ser 275 280 285Val Gly Gly Ala Ala Ser Ala Ala Thr Lys
Ser Ala Leu Ala Ile Ala 290 295 300Val Arg Tyr Gly Asp Ile Arg Arg
Gln Phe Ala Asp Ala Asp Gly Asp305 310 315 320Arg Glu Val Leu Leu
Asn Asp Tyr Leu Ala His Gln Arg Lys Leu Leu 325 330 335Pro Ala Leu
Ala Thr Thr Tyr Ala Leu Thr Phe Ala Gln Ala Glu Leu 340 345 350Val
Ala Ala Leu Asp Asp Ile Gln Gly Gly Asp Gly Pro Val Asp Glu 355 360
365His Arg Gln Arg Glu Leu Glu Ser Arg Ala Ala Gly Leu Lys Ala Ala
370 375 380Gln Thr Trp His Ala Thr Arg Thr Ile Gln Ile Cys Arg Glu
Ala Cys385 390 395 400Gly Gly Ala Gly Tyr Leu Ser Glu Asn Arg Leu
Pro Ser Leu Lys Ala 405 410 415Asp Thr Asp Val Phe Thr Thr Phe Glu
Gly Asp Asn Thr Val Leu Leu 420 425 430Gln Leu Val Ala Lys Gly Leu
Leu Thr Gly Tyr Arg Asp Glu Phe Gly 435 440 445Ser Leu Asp Gly Trp
Gly Arg Ala Ser Phe Val Ala Glu Gln Val Arg 450 455 460Glu Met Val
Leu Glu Arg Thr Ala Ala Arg Ala Leu Ile Ala Arg Leu465 470 475
480Val Ser Ala Val Pro Gly Arg Asp Asp Glu Val Ala Val Thr Asp Arg
485 490 495Gly Trp Gln Leu Lys Leu Phe Glu Asp Arg Glu Glu His Leu
Leu Asp 500 505 510Ser Ala Val Arg Arg Leu Arg Gly Gly Ala Ser Thr
Lys Lys Asp Arg 515 520 525Pro Phe Asp Ile Phe Asn Asp Val Gln Asp
His Val Leu Ala Val Ala 530 535 540Ala Ala His Ile Asp Arg Val Thr
Leu Glu Ala Phe Val Ala Gly Ile545 550 555 560Asp Ala Ile Ala Asp
Pro Ala Val Lys Glu Leu Leu Ser Arg Val Cys 565 570 575Asp Leu Tyr
Ala Leu Thr Val Ile Glu Ala Asn Lys Gly Trp Leu Leu 580 585 590Glu
His Gly Arg Leu Thr Pro Ala Arg Ser Lys Thr Ile Thr Ser Val 595 600
605Val Asn Gly Leu Leu Lys Glu Leu Arg Pro Asp Met Arg Thr Leu Val
610 615 620Asp Gly Phe Ala Ile Pro Asp Ala Trp Leu His Ala Ala Ile
Leu Arg625 630 635 640Glu Glu Pro Val Arg Gln Glu Thr Met Ala Ala
His Asp Ala Ala Gly 645 650 655Asp Pro Gln Ala Val Pro Ala
660791992DNAMicromonospora sp. strain 046-ECO11 79atgctcgatc
acgcatccgg ccgcatcgac gtcacacgcc tgcgggaagc gctcgacggc 60cggtgggccg
aggtccgccg ggcgcaccgc gaacacctcg acgaacgctt cctcccggtg
120tacggcgaga ccggtgacca ggcccgcgag cgcatcaccc ggctgctgtc
cgaactcccc 180gtcgagctgg gcatcgcctc cggtttcccc gccgagtacg
gcggccgcgg cgacgtgggc 240gcctcgatcg tcgccaccga gatgctggcc
caggtggacc tgtcactgat ggtgaaggcc 300ggcgtgcagt ggggcctgtt
cggcggcgcg gtcgccgccc tcggcacgaa gcggcaccac 360gacgcctacc
tgcgggacat cgtcgcgggc cggctcttcg gctgcttcgc gatgaccgag
420accggccacg gctcggacgt gcagcaactg cgcaccacct gcgtctacga
cccgcagacg 480cagaccttcg acctgcacac cccgcacgag gccgcgcgca
aggactacat cggcaacgcg 540gcccgggacg ggcggatggc tgtggtgttc
gcccagctcg tcaccggcgg gcgccgccac 600ggggtgcacg cctggctggt
gccgatccgc gacgagcacg gcaagccgat gcccggcgtg 660accatcggcg
acgccgggcc caaggccggc ctgctcggcg tggacaacgg gcggctcagc
720ttcgaccacg tgcgggtgcc gcgggagatg ctgctggacc agtacgcgca
ggtcgccgag 780gacggcacgt actccagccc gatcgagaac gactcccggc
gcttcttcac catgctgggc 840accctggtcc ggggccgggt gagcgtgggc
ggcgccgcgt cggcggccac caagtcggcg 900ctggccatcg cggtgcgcta
cggcgacatc cgccggcagt tcgccgacgc cgacggcgac 960cgcgaggtgc
tgctcaacga ctacctggcg caccagcgca agctgctgcc cgcgctggcc
1020accacgtacg cgctgacctt cgcccaggcg gagctggtcg cggcgctcga
cgacatccag 1080ggcggcgacg ggccggtcga cgagcaccgg cagcgggagc
tggagtcccg ggccgccggt 1140ctgaaggcgg cgcagacctg gcacgccacc
cgcaccatcc agatctgccg ggaggcgtgt 1200ggcggcgccg gctacctgtc
cgagaaccgc ctgcccagcc tcaaggccga caccgatgtc 1260ttcaccacct
tcgagggcga caacacggtg ctgctgcaac tggtcgccaa ggggctgctg
1320accggctacc gggacgagtt cggctcgctc gacggctggg gacgcgcctc
cttcgtggcc 1380gagcaggtac gcgagatggt gctggaacgc accgccgcgc
gggcgctgat cgcacgtctg 1440gtcagcgccg tgcccgggcg cgacgacgag
gtcgccgtca ccgaccgggg ctggcagctc 1500aagctcttcg aggaccgcga
ggagcacctg ctcgacagcg cggtccgccg cctgcgcggt 1560ggcgcgtcca
ccaagaagga ccgccccttc gacatcttca acgacgtcca ggaccacgtc
1620ctcgccgtcg ccgcggcgca catcgaccgg gtgacgctgg aggcgttcgt
cgccgggatc 1680gacgccatcg ccgacccggc ggtcaaggaa ctgctgtccc
gggtctgcga cctgtacgcg 1740ctcaccgtga tcgaggcgaa caagggctgg
ctgctcgagc acggccggct caccccggcc 1800cgctcgaaga ccatcaccag
cgtggtgaac gggctgctca aggagctgcg cccggacatg 1860cgcacgctcg
tggacggctt cgccatcccg gacgcgtggc tgcacgcggc gatcctgcgc
1920gaggagcccg tccggcagga gacgatggcc gcgcacgacg ccgccggcga
cccgcaggcc 1980gtccccgcct ag 199280573PRTMicromonospora sp. strain
046-ECO11 80Val Ser Pro Leu Pro Pro Gly Ser Ala Val Thr Ala Arg His
Val Leu1 5 10 15Arg Gln Ala Leu Arg Arg Gln Arg Arg Pro Val Leu Ile
Gly Val Thr 20 25 30Leu Leu Gly Leu His Gln Val Thr Glu Ala Leu Val
Pro Val Ala Ile 35 40 45Gly Val Ile Ile Asp Arg Ala Val Val Thr Gly
Asp Pro Trp Ala Leu 50 55 60Ala Tyr Ser Val Ala Gly Leu Ala Ala Leu
Phe Thr Val Leu Ala Phe65 70 75 80Ala Tyr Arg Asn Gly Ala Arg Gln
Ala Phe Ala Ala Val Glu Arg Glu 85 90 95Ala His Leu Leu Arg Val Glu
Leu Ala Glu Arg Ala Leu Asp Pro Arg 100 105 110Gly His Arg Ser Gly
Leu Arg Asp Gly Glu Leu Leu Ser Val Ala Ala 115 120 125Ser Asp Ala
Glu Leu Ser Ala Tyr Val Val Arg Val Ala Gly Phe Gly 130 135 140Val
Ala Ala Val Ser Ala Leu Thr Val Ala Ala Val Ala Leu Leu Val145 150
155 160Ile Asp Val Pro Leu Gly Leu Gly Val Leu Ile Gly Val Pro Val
Leu 165 170 175Val Leu Ala Leu Gln Arg Met Ala Pro Leu Leu Ser Arg
Arg Ser Ala 180 185 190Ser Gln Gln Glu Ala Leu Ala Glu Thr Thr Ala
Leu Ala Val Asp Leu 195 200 205Val Ser Gly Leu Arg Val Leu Arg Gly
Ile Gly Ala Gln His His Ala 210 215 220Ala Gly Arg Tyr Ala Glu Ala
Ser Arg Arg Ala Leu Ala Val Thr Leu225 230 235 240Arg Ala Ala Asn
Thr Lys Gly Leu His Leu Gly Leu Thr Thr Ala Ala 245 250 255Asn Gly
Leu Phe Leu Ala Ala Val Ala Gly Val Ala Gly Trp Leu Ala 260 265
270Leu Arg Gly Arg Leu Thr Ile Gly Glu Leu Val Thr Val Val Gly Leu
275 280 285Ala Gln Phe Val Ala Glu Pro Val Gln Thr Leu Gly Tyr Cys
Val Gln 290 295 300Leu Phe Ala Met Ala Arg Ala Ser Ala Ala Arg Val
Gly Arg Val Leu305 310 315 320Gly Ala Glu Pro Leu Thr Arg Pro Gly
Ser Ala Pro Arg Pro Asp Arg 325 330 335Thr Asp Gly Pro Arg Leu Val
Leu Asp His Val Gly His Ala Ala Leu 340 345 350Asp Gly Val Cys Leu
Arg Val Asp Pro Gly Glu Ile Val Gly Val Leu 355 360 365Ala Tyr Asp
Pro Ala Asp Ala Asp Ala Leu Val Ala Leu Leu Ser Gly 370 375 380Arg
Val Pro Ala Asp Arg Arg Arg Gly Thr Val Arg Val Asp Gly Val385 390
395 400Pro Ala Asp Asp Leu Asp Val Asp Ala Leu Arg Gly Ala Val Leu
Val 405 410 415Glu Pro His Asp Val Thr Leu Phe Glu Gly Thr Val Ala
Ala Asn Leu 420 425 430Ala Ala Gly Ser Arg Thr Glu Glu Gly Arg Leu
Arg Ala Ala Val Arg 435 440 445Ala Ala Ala Ala Asp Asp Val Val Asp
Ala His Pro Gly Gly Leu Gly 450 455 460His Arg Leu Val Glu Arg Gly
Ala Asn Leu Ser Gly Gly Gln Arg Gln465 470 475 480Arg Leu Gly Leu
Ala Arg Ala Leu His Ala Asp Pro Pro Val Leu Val 485 490 495Leu His
Asp Pro Thr Thr Ala Val Asp Ala Ala Thr Glu Ala Gln Leu 500 505
510Ala Asp Gly Leu Ala Gly Ala Arg Arg Glu Ala Pro Arg Gly Thr Leu
515 520 525Leu Val Thr Ser Ser Pro Ala Leu Leu Arg Ile Thr Asp Arg
Val Val 530 535 540Val Ile Ala Asp Gly Arg Val Thr Ala Glu Gly Thr
His Glu His Leu545 550 555 560Leu Ala Thr Asp Ala Arg Tyr Arg Glu
Glu Thr Leu Arg 565 570811722DNAMicromonospora sp. strain 046-ECO11
81gtgtccccgc ttccccccgg cagcgccgtc accgcccggc acgtgctccg ccaggcgctg
60cgccgccagc gccgcccggt gctgatcggc gtgaccctgc tcgggctgca ccaggtcacc
120gaggcgctcg tgccggtggc gatcggcgtc atcatcgacc gggccgtggt
gaccggcgac 180ccgtgggcgc tcgcgtactc cgtcgccggc ctcgccgccc
tgttcaccgt gctggcgttc 240gcctaccgca acggcgcccg ccaggcgttc
gcggcggtgg aacgggaggc gcacctgctg 300cgggtcgagc tggccgagcg
cgcgctcgac ccgcgcgggc accgctccgg cctgcgcgac 360ggcgagctgc
tctcggtcgc cgcctccgac gccgaactct ccgcgtacgt ggtccgggtg
420gccggcttcg gcgtcgccgc ggtgagcgcg ctgaccgtcg cggcggtcgc
gctgctggtc 480atcgacgtcc cgctcggact cggcgtgctc atcggcgtac
cggtgctggt cctggcgctg 540caacggatgg cgccgctgct gtcccggcgc
agcgcctccc agcaggaggc cctcgcggag 600accacggcgc tcgccgtgga
cctcgtctcc ggcctgcgcg tgctgcgcgg catcggcgcc 660cagcaccacg
ccgccggccg gtacgccgag gccagccgac gcgccctcgc cgtgacgctg
720cgcgccgcca acaccaaggg cctgcacctc gggctcacca ccgccgcgaa
cggcctcttc 780ctcgccgccg tcgccggggt cgccggctgg ctcgcgctgc
gcggccggct caccatcggc 840gagctggtca ccgtggtcgg gctcgcgcag
ttcgtcgccg agccggtgca gacgctgggc 900tactgcgtgc agctgttcgc
gatggcccgc gcctccgccg cccgggtcgg gcgcgtgctc 960ggcgccgagc
cgctgacccg gccgggcagc gcgccccggc cggaccgcac ggacgggccg
1020cggctcgtcc tcgaccacgt cggccacgcc gcgctggacg gggtgtgcct
gcgcgtcgac 1080ccgggagaga tcgtcggcgt cctggcgtac gacccggccg
acgcggacgc gctggtggcg 1140ctgctgtccg ggcgggtgcc cgcggaccgg
cgccggggca cggtacgcgt cgacggggta 1200cccgccgacg acctggacgt
cgacgcgctg cgcggcgccg tcctggtcga gccgcacgac 1260gtgacgctgt
tcgagggaac cgtggccgcc aacctcgccg ccgggagcag gaccgaggag
1320gggcgcctgc gcgccgcggt ccgggcggcc gcggcggacg acgtggtgga
cgcgcacccc 1380ggcggcctcg gccaccggct cgtcgagcgg ggcgccaacc
tctccggcgg gcagcgccag 1440cggctcgggc tggcgcgggc gctgcacgcc
gacccgccgg tgctggtgct gcacgacccc 1500accaccgccg tggacgcggc
caccgaggcc caactcgccg acggactggc cggcgcgcgc 1560cgcgaagcgc
cccggggcac gctgctggtc accagcagcc ccgccctgct gcggatcacc
1620gaccgggtgg tggtgatcgc cgacggccgg gtgaccgccg aggggacgca
cgagcacctg 1680ctggccaccg acgcccgcta ccgcgaggag acactgcggt ga
172282596PRTMicromonospora sp. strain 046-ECO11 82Val Thr Ala Asp
Pro Arg Thr Ala Glu Pro Thr Arg Val Leu Leu Pro1 5 10 15Thr Ala Thr
Ala Arg Arg Thr Trp Thr Thr Leu Gly Ala Glu Phe Arg 20 25 30Arg Arg
Pro Gly Leu Ser Ala Ala Ala Thr Ala Val Leu Val Ala Ala 35 40 45Ala
Thr Gly Gly Leu Val Ala Pro Trp Val Leu Gly Arg Leu Val Asp 50 55
60Asp Val Ile Ala Asp Ala Pro Val Ser Arg Ile Ala Gly Arg Val Ala65
70 75 80Val Ile Ala Gly Ala Ala Val Leu Thr Gly Leu Leu Thr Ala Ala
Gly 85 90 95Ala Ala Leu Ala Ser Arg Leu Gly Glu Thr Val Leu Ala Arg
Leu Arg 100 105 110Glu Arg Val Leu Asp Arg Ala Leu His Leu Pro Ser
Ala Thr Leu Glu 115 120 125Arg Ala Gly Thr Gly Asp Leu Leu Ala Arg
Val Gly Asp Asp Val Ala 130 135 140Val Val Thr Asn Val Ile Ala Val
Ser Gly Pro Ala Phe Val Gly Ala145 150 155 160Leu Leu Ser Val Val
Leu Thr Val Phe Gly Leu Val Ala Leu Asp Trp 165 170 175Arg Leu Gly
Leu Ala Gly Leu Val Ala Ala Pro Ala Tyr Ala Leu Ala 180 185 190Leu
Arg Trp Tyr Leu Arg Arg Ser Ala Pro Tyr Tyr Ala Arg Glu Arg 195 200
205Val Ala Thr Gly Glu Arg Thr Gln Ala Met Ala Gly Ala Leu Arg Gly
210 215 220Ala Ala Thr Val Arg Ala Tyr Arg Thr Glu Asp Ala His Val
Ala Ala225 230 235 240Ile Ala Glu Arg Ser Gly Val Ala Arg Asp Leu
Ser Leu Glu Ile Phe 245 250 255Asn Leu His Thr Arg Phe Gly Leu Arg
Ile Asn Arg Ser Glu Phe Leu 260 265 270Gly Leu Ala Ala Val Leu Val
Ala Gly Phe Phe Leu Val Arg Ala Asp 275 280 285Leu Val Thr Val Gly
Ala Ala Thr Thr Ala Ala Leu Tyr Phe His Arg 290 295 300Leu Phe Asn
Pro Ile Gly Leu Leu Leu Met Glu Ser Asp Ser Val Leu305 310 315
320Gln Ala Gly Ala Ser Leu Ala Arg Leu Val Gly Val Ala Thr Leu Pro
325 330 335Asp Thr Ala Pro Ser Gly Pro Ala Pro Ser Ala Ala Gly Arg
Arg Gly 340 345 350Pro Ala Ala Leu Asp Val Thr Val Arg Arg His Arg
Tyr Asp Asp Asp 355 360 365Gly Pro Leu Val Leu Ala Asp Val Asp Leu
Arg Leu Ala Pro Gly Glu 370 375 380Arg Val Ala Leu Val Gly Ala Ser
Gly Ala Gly Lys Ser Thr Leu Ala385 390 395 400Gly Ile Ala Ala Gly
Ile Ile Ala Pro Thr Asp Gly Ser Val Arg Leu 405 410 415Gly Gly Val
Pro Leu Thr Glu Arg Gly Glu His Ala Val Arg Arg Asp 420 425 430Val
Ala Leu Val Ser Gln Glu Val His Val Phe Ala Gly Pro Leu Ala 435 440
445Glu Asp Leu Arg Leu Ala Ala Pro Asp Ala Thr Asp Ala Glu Leu Leu
450 455 460Asp Ala Leu Asp Arg Val Gly Ala Thr Thr Trp Leu Arg Ala
Leu Pro465 470 475 480Asp Gly Leu Ala Thr Ala Val Gly Glu Gly Gly
His Arg Leu Thr Ala 485 490 495Ala Gln Ala Gln Gln Val Ala Leu Ala
Arg Leu Val Leu Ala Ala Pro 500 505 510Ala Val Ala Val Leu Asp Glu
Ala Thr Ala Glu Ala Gly Ser Ala Gly 515 520 525Ala Arg Asp Leu Asp
Arg Ala Ala Leu Ala Ala Thr Glu Gly Arg Thr 530 535 540Thr Leu Ile
Val Ala His Arg Leu Ser Gln Ala Val Ala Ala Asp Arg545 550 555
560Ile Val Leu Leu Asp His Gly Arg Ile Val Glu Gln Gly Thr His Ser
565 570 575Glu Leu Leu Ala Ala Asp Gly Arg Tyr Gly His Leu Trp Arg
Ser Trp 580 585 590Ser Val Pro Val 595831791DNAMicromonospora sp.
strain 046-ECO11 83gtgaccgctg acccgcgtac cgccgaaccc acccgggtgt
tgctgcccac cgcgaccgcc 60cggcggacct ggacgacgct cggcgcggag ttccgccggc
ggcccggcct cagcgccgcc 120gcgaccgccg tgctcgtcgc cgccgccacc
ggcgggctgg tcgcgccctg ggtgctcggc 180cgcctcgtcg acgacgtcat
cgccgacgcc ccggtctccc ggatcgccgg ccgggtggcg 240gtgatcgccg
gcgcggcagt gctcaccgga ctgctcaccg ccgccggggc cgcgctcgcg
300tcccgcctgg gggagacggt gctggcccgg ctgcgcgagc gggtcctcga
ccgggcgctg 360cacctgccct cggcgacgct ggaacgggcc ggcaccggcg
acctgctggc ccgggtcggc 420gacgacgtgg cggtggtgac gaacgtgatc
gcggtcagcg gcccggcgtt cgtcggcgcg 480ctgctgtccg tggtgctgac
cgtgttcggg ctggtcgcgc tcgactggcg gctcggcctc 540gccgggctgg
tcgccgcgcc cgcctacgcg ctggcgctgc gctggtacct gcgccggtcg
600gcgccgtact acgcccgcga gcgcgtcgcc accggcgagc ggacgcaggc
gatggccggc 660gcgctgcgtg gcgcggccac cgtgcgcgcg taccggaccg
aggacgcgca cgtcgcggcg 720atcgccgagc gctccggcgt ggcgcgcgac
ctgtcgctgg agatcttcaa cctgcacacc 780cggttcgggc tgcggatcaa
caggtcggag ttcctcggcc tggccgcggt gctcgtcgcc 840gggttcttcc
tggtccgcgc cgacctggtc acagtgggcg cggcgaccac cgccgcgctc
900tacttccacc ggctgttcaa cccgatcggc ctgctgctga tggagtccga
ctcggtgctg 960caggccggcg cgagcctcgc ccggctggtc ggcgtggcca
cgctgcccga caccgccccg 1020tccgggcccg cgccgtcggc ggccgggcgg
cgcggcccgg cggcgctgga cgtcacggtc 1080cgccggcacc gctacgacga
cgacggccct ctggtcctgg ccgacgtcga cctgcgcctg 1140gccccgggcg
agcgggtcgc gctcgtgggc gccagcggcg cgggcaagag cacgctcgcc
1200ggcatcgccg ccgggatcat cgcgcccacc gacgggtcgg tacgcctggg
cggcgtgccg 1260ctgaccgagc ggggcgagca cgccgtgcgg cgcgacgtcg
cgctggtcag ccaggaggtg 1320cacgtcttcg ctggaccgct cgccgaggat
ctgcgcctgg ctgccccgga cgccaccgac 1380gccgaactgc tcgacgcgct
ggaccgggtc ggcgccacca cctggctgcg cgcgctgccg 1440gacgggctgg
ccacagcggt cggcgagggc ggccaccggc tcaccgccgc gcaggcccag
1500caggtcgccc tggcccggct ggtgctggcc gcgcccgccg tcgccgtgct
ggacgaggcc 1560accgccgagg ccggcagcgc cggagcgcgt gacctggacc
gggcggcgct ggccgccacc 1620gagggacgga ccacgctgat cgtggcgcac
cggctcagcc aggcggtcgc cgccgaccgg 1680atcgtcctgc tcgaccacgg
gcggatcgtg gagcagggca cgcactcgga actgctcgcc 1740gccgacggcc
ggtacgggca tctgtggcgc tcctggagcg tcccggtatg a
179184507PRTMicromonospora sp. strain 046-ECO11 84Met Thr Asp Ala
Pro Ala Arg Phe Val Leu Phe Pro Gly Arg His His1 5 10 15Leu Leu Thr
Arg Phe Gln Ala Asp Tyr Leu Arg Arg Leu Ala Gly Asp 20 25 30Asp Ala
Thr Val Val Trp Ala Val Thr Ser Ala Asn His Glu Asn Thr 35 40 45Arg
Arg Asn Pro Val Pro Tyr His Arg Arg Glu Ala Ala Ile Glu Arg 50 55
60Phe Ser Val Leu Ser Gly Leu Arg Ser Val Val Val Pro Ile Phe Asp65
70 75 80Thr Ala Tyr Thr Asp Ala Phe Ala Glu Val Thr Leu Lys Ser Ile
Ala 85 90 95Val Ala Thr Gly Leu Glu Leu Thr Pro Ala Asp Thr Val Leu
Ala Cys 100 105 110Ser Thr Pro Glu Val Ala Lys Leu Tyr Glu Gln Leu
Gly Phe Ser Ile 115 120 125Ala Pro Val Glu Ala Asp Pro Asp Leu Pro
Glu Pro Pro Glu Arg Pro 130 135 140Trp Asp Val Leu Leu Arg Leu Ala
Ala Gly Asp Glu Thr Trp Arg Ala145 150 155 160Leu Thr His Pro Ala
Thr Ile Asp Val Phe Glu Arg Tyr Arg Leu Val 165 170 175Glu Ser Ile
Arg Ser Val Val Asn Asp Pro Leu Val Gly Asp Glu Gly 180 185 190Gly
Leu Thr Val Thr Arg Asp Tyr Arg Thr Tyr Val Glu Ala Phe Ala 195 200
205Thr Ala Ala Gln Arg Lys Trp Asp Ser Val Arg Arg Tyr Val Gln Pro
210 215 220Gly Arg Ile Val Asp Ile Gly Cys Gly Ala Gly Ala Val Leu
Glu Leu225 230 235 240Ala Asp Arg Glu Ala Ala Leu Arg Glu Ser Asp
Leu Ile Gly Val Glu 245 250 255Val Ala Arg His Leu Tyr Gln Glu Cys
Leu His Lys Lys Ala Gln Gly 260 265 270Val Phe Arg Asn Ala Asn Val
Tyr Phe Phe His Arg Asn Val Leu Gly 275 280 285Gly Ala Val Phe Lys
Asp Arg Ser Val Asp Thr Thr Leu Thr Phe Ala 290 295 300Leu Thr His
Glu Ile Trp Ser Tyr Gly Arg Arg Arg Glu Ser Leu Leu305 310 315
320Gln Phe Ala Arg Arg Ile His Asp His Thr Val Pro Gly Gly Val Trp
325 330 335Ile Asn Ser Asp Val Cys Gly Pro Asp Asp Pro Arg Arg Gln
Val Leu 340 345 350Leu Arg Leu Ser Thr Asp Asp Gly Asp Asn Pro Ala
Ala Pro Arg Pro 355 360 365Asp Leu Ala Glu Leu Thr Ser Ala Glu Val
Arg Arg Tyr Val Gly Gly 370 375 380Leu Ser Thr Arg Ala Arg Leu Asp
Gln Phe Ala Val Asp Phe Ala Phe385 390 395 400Asp Phe Asp Tyr Glu
Pro Leu Pro Asp Gly Ala Val Arg Leu Thr Leu 405 410 415Gly Ala Ala
Met Asp Tyr Leu Thr Arg Lys Asp Tyr Thr Asp Asn Trp 420 425 430Leu
Ser Glu Thr Gln Glu Gln Phe Cys Gly Leu Ser Phe Ala Asp Trp 435 440
445Thr Asp Leu Leu Thr Glu Ala Gly Phe Glu Ile Gly Pro Ala Ser Ala
450 455 460Pro Val Arg Asn Glu Trp Val Ile Asp Asn Arg Ile Ala Pro
Val Ala465 470 475 480Ser Leu Thr Asp Leu Asp Gly Arg Pro Leu Asp
Trp Pro Thr Thr His 485 490 495Val Leu Thr Val Ala His Arg Pro Arg
Asn Gln 500 505851524DNAMicromonospora sp. strain 046-ECO11
85atgaccgacg cgccggcccg cttcgtgctc ttcccggggc ggcaccacct gctgacccgg
60ttccaggccg actacctgcg gcggctggcc ggggacgacg ccacagtggt ctgggcggtg
120acgtcggcca accacgagaa caccaggcgc aacccggtgc cctaccaccg
gcgggaggcc 180gcgatcgaac gattcagcgt gctgagcggg ctgcgctcgg
tggtggtgcc gatcttcgac 240accgcgtaca ccgacgcgtt cgccgaggtg
acgctgaagt ccatcgcggt ggccaccggg 300ctcgaactca cccccgccga
caccgtgctg gcctgctcca cgccggaggt cgcgaagctg 360tacgagcagc
tcggcttttc gatcgcgccg gtcgaggcgg acccggacct gcccgagccg
420cccgaacggc cgtgggacgt gctgctgcgc ctggccgccg gggacgagac
ctggcgcgcg 480ctcacccacc cggccaccat cgacgtgttc gagcgctacc
gcctggtcga gtcgatccgg 540tcggtggtga acgacccgct cgtcggcgac
gagggcggtc tcacagtgac ccgcgactac 600cggacctacg tcgaggcgtt
cgccacggcc gcgcagcgca agtgggactc ggtacgccgg 660tacgtgcagc
ccggccgcat cgtggacatc ggctgcggcg cgggcgccgt cctggaactc
720gccgaccggg aggccgcgct gcgtgagagc gacctgatcg gcgtggaggt
cgcccgccac 780ctctaccagg agtgcctgca caagaaggcg cagggcgtgt
tccgcaacgc caacgtctac 840ttcttccacc gcaacgtcct cggcggcgcg
gtgttcaagg accgctcggt cgacaccacg 900ctcacgttcg cgctgaccca
cgagatctgg tcgtacgggc ggcggcggga gtcgctgctg 960cagttcgccc
gccgcatcca cgaccacacg gtgcccggcg gcgtctggat caacagcgac
1020gtgtgcggtc cggacgaccc ccggcggcag gtgctcctgc gactgtccac
cgacgacggc 1080gacaacccgg ccgcgccccg ccccgacctc gccgagctga
cctcggcgga ggtccggcgt 1140tacgtcggcg ggctgtcgac gcgggcgcgg
ctggaccagt tcgccgtcga cttcgcgttc 1200gacttcgact acgagccgct
ccccgacggc gcggtacgcc tgacgctggg cgccgcgatg 1260gactacctga
cccgcaagga ctacacggac aactggctgt cggagacgca ggagcagttc
1320tgcggcctga gcttcgccga ctggacggac ctgctcaccg aggcggggtt
cgagatcggc 1380ccggcgtcgg cgccggtgcg caacgagtgg gtgatcgaca
accggatcgc gccagtcgcg 1440tccctcaccg acctcgacgg ccggccgctg
gactggccga ccacccacgt cctcaccgtc 1500gcccaccgcc cccgcaacca gtga
152486232PRTMicromonospora sp. strain 046-ECO11 86Val Ser Asp Ile
Gln Ile Ile Ser Phe Val Ala Ala Ser Leu Leu Ile1 5 10 15Ile Ile Val
Pro Gly Val Asp Phe Ala Leu Val Thr Arg Gln Thr Val 20 25 30Arg Tyr
Gly Arg Arg Ala Gly Phe Val Val Leu Ala Gly Leu Phe Val 35 40 45Ala
Ala Leu Val His Ala Ser Phe Ala Thr Ala Gly Leu Ser Ala Leu 50 55
60Leu Val Ser Ser Pro Thr Leu Tyr Thr Val Leu Arg Val Ala Gly Ala65
70 75 80Leu Tyr Leu Leu Tyr Leu Gly Gly Thr Ile Leu Trp Ala Thr Arg
Pro 85 90 95Arg Arg Thr Val Pro Ala Ala Gln Pro Val Thr Val Gly Ala
Gly Gly 100 105 110Ala Gly Pro Asp Thr Asp Thr Gly Pro Ala Pro Val
Pro Asp Thr Pro 115 120 125Ala Ala Asp Glu Pro His Val Ala Arg Arg
Ser Phe Val Met Gly Val 130 135 140Thr Ser Gln Leu Leu Asn Val Lys
Val Val Val Phe Tyr Val Ser Phe145 150 155 160Val Pro Gln Phe Val
Lys Pro Gly Glu Gly Ala Ala Ala Arg Thr Ala 165 170 175Val Leu Ala
Ala Thr Phe Ile Gly Leu Ala Val Leu Trp Trp Ala Cys 180 185 190Tyr
Ile Met Leu Ile Asp Arg Leu Gln Pro Trp Leu Thr Arg Pro Ser 195 200
205Val Leu Leu Val Ile Glu Arg Leu Thr Gly Leu Ile Leu Ile Val Leu
210 215 220Ala Ile Arg Ile Ala Leu Ser Arg225
23087699DNAMicromonospora sp. strain 046-ECO11 87gtgtctgaca
tccagatcat cagtttcgtc gccgccagcc tgctcatcat catcgtgccg 60ggcgtcgact
tcgcgctcgt cacccggcag accgtcaggt acggccggcg ggccgggttc
120gtggtgctgg ccgggctgtt cgtcgccgcg ctggtgcacg cgtcgttcgc
gaccgccggc 180ctgtccgccc tgctggtctc ctcgccgacg ctctacacgg
tgctgcgcgt cgccggcgcg 240ctgtacctgc tctacctggg cggcacgatc
ctctgggcga cccggccgcg ccggacggtc 300ccggcggcgc agccggtcac
tgtcggcgcg ggcggcgccg ggccggacac ggacaccggc 360cccgcgccgg
tgccggacac cccggccgcc gacgagccgc acgtggcccg ccgctcgttc
420gtcatgggcg tcaccagcca gctgctgaac gtcaaggtgg tcgtcttcta
cgtctcgttc 480gtgccgcagt tcgtcaagcc cggcgagggg gcggcggccc
gtacggcggt gctcgccgcc 540acgttcatcg gcctcgcggt gctctggtgg
gcctgctaca tcatgctcat cgacaggttg 600cagccctggc tgacccggcc
gtccgtgctg ctggtgatcg aacggctgac cgggctcatc 660ctgatcgtcc
tggcgatccg gatcgcgctg agccggtga 69988132PRTMicromonospora sp.
strain 046-ECO11 88Val Gly Val Ser Ala Met Thr Thr Phe Asp Tyr Asp
Gly Arg Val Phe1 5 10 15Val Ser Val Asp His Asp Ala Gly Asp Gly Ala
Glu Pro Leu Arg Gly 20 25 30His Tyr His Gln Arg Gly Asp Leu Val Trp
Ala Glu Ile Thr Gly Gly 35 40 45Pro Val Arg His Gly Arg Leu Ala Gly
Thr Cys Asp Ala Gln Gly Val 50 55 60Val Arg Phe Ala Tyr Leu Glu Val
Leu Thr Asp Gly Thr Ile Val Ile65 70 75 80Gly Glu Cys Glu Ser Arg
Pro Glu Arg Leu Pro Asp Gly Arg Ile Arg 85 90 95Leu Arg Glu Gln Trp
Arg Arg His Gly Pro Arg Gln Asp Ser Gly Val 100 105 110Ser Val Ile
Glu Glu Ala Val Pro Ala Leu Ala Gly Gly Gln Glu Ser 115 120 125Arg
Arg Arg Val 13089399DNAMicromonospora sp. strain 046-ECO11
89gtgggcgtga gcgcgatgac gacattcgac tacgacggcc gcgtcttcgt ctcggtggac
60cacgacgccg gtgacggcgc cgagccgctg cgggggcact accaccagcg tggcgacctg
120gtctgggcgg agatcaccgg cggcccggtc cggcacggcc ggctggccgg
cacctgcgac 180gcgcagggcg tcgtgcgctt cgcctacctg gaggtgctca
ccgacggcac catagtcatc 240ggcgagtgcg agtcccggcc cgaacggctg
ccggacggcc ggatccggct gcgggaacag 300tggcgccggc acggaccacg
ccaggacagc ggcgtctccg tcatcgagga ggcagtgccg 360gcgctcgccg
gaggacagga gagccggcgt cgtgtctga 399
* * * * *