Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS FOR TRANSFERRING THA CAPABILITY TO PRODUCE A NATURAL
PRODUCT INTO A SUITABLE PRODUCTION HOST.
1. FIELD OF THE INVENTION
The present invention relates to a novel approach for drug
discovery. More particularly, the invention relates to a
system for improving the process of lead optimization and
development of compounds, when these compounds are natural
products produced by microorganisms belonging to the order
Actinomycetales or chemical derivatives of these compounds:
The invention relates to a system for transferring the
capability to produce a natural product from a
microorganism belonging to the order Actinomycetales into a
defined host, where said natural product can be optimally
produced and its biosynthetic pathway suitably modified.
2. BACKGROUND ART
Natural products are complex molecules with important uses
in medicine. Examples include: antibacterial agents, such
as erythromycin, teicoplanin, tetacycline; antitumor
compounds, such as dauxorubicin; antihelmintic compounds,
such as avermectin; immunosuppressive agents, such as
cyclosporin and FK506; antifungal compounds, such as
amphotericin and nystatin; etc. Natural products are
produced as secondary metabolites by a wide range of living
organisms. Although many secondary metabolites have been
identified, there remains the need to obtain novel
structures with new activities or enhanced properties.
Current methods of obtaining such molecules include
screening of natural isolates and chemical modification of
existing ones. Random screening of natural products from
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disparate sources has resulted in the discovery of many
important drugs and is still employed for seeking for novel
activities. This process, which consists in exposing a
miniaturized biological system to tens or hundreds of
thousands of different compounds, in order to find those
few that exhibit a desired property, is designated high
throughput screening, or HTS.
One of the used sources widely in HTS is a collection
of natural products produced by small-scale fermentation of
newly isolated microorganisms. A natural product may have
one or more potential therapeutic properties, including but
not limited to antibacterial, antifungal, antiviral,
antitumor, immunomodulating or other pharmacological
properties. Natural products have long constituted a
source of interesting, structurally original and
"imaginative" molecules endowed with potent biological
activities. In addition, recent observations indicate that
only a small fraction of the microbial flora present in
environmental samples, ranging from 0.01 to 1°s according to
the estimates, is related to known species. Microorganisms
belonging to the order Actinomycetales represent thus far
the group of producers unsurpassed for chemical and
biological diversity. However, more than 15,000 natural
products produced by microorganisms have been described,
and the chances of finding new structures are relatively
small, unless efforts are directed towards those classes of
microorganisms that have been little exploited in the past.
Poorly characterized actinomycete genera can thus
constitute a useful source of novel structures. With proper
methodologies, unusual genera can be isolated from
environmental samples and some of these isolates will
produce interesting activities. These could either
represent completely new entities, or known molecules
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acting on a novel target or in a previously unreported way.
Many of these products will have original structures and
potent biological activities. However, newly discovered
secondary metabolites will be produced for the most part by
microorganisms which have been isolated for the
characteristic of being unusual and selected for their
ability to produce a given bioactivity. Consequently,
little will be known about the best conditions for growth,
productivity and storage. Often the microorganism does not
IO produce a single bioactive compound, and other, unrelated
activities must be completely removed for a meaningful
evaluation of the properties of the lead compound.
Furthermore, rarely is a secondary metabolite produced as a
single, bioactive molecule, but is often present as a
"complex" of several, closely related compounds, only some
of which may possess the desired biological or chemical
properties. Therefore, physiological conditions, such as
nutrient and cofactor supply, that allow obtaining a
"controlled" complex need to be established empirically by a
trial and error approach. Finally, the natural product may
need structural modification, and this can be achieved only
by chemical means. In essence, the scarce knowledge
available on the physiology and genetics of the producing
strain will severely hamper the lead optimization and
development processes.
Chemical modification of preexisting natural products
has been successfully employed to generate derivatives of
natural products, but it still suffers from practical
limitations to the type of compounds obtainable. Many
natural products are often structurally complex molecules,
with relatively large molecular weights. Due to their
structural complexity, total synthesis of natural products
is often prohibitive for the number of necessary steps and
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the overall yield; furthermore, selective modification of a
natural product can often be efficiently performed only on
limited portions of the molecule. This difficulty of
generating structural derivatives by conventional medicinal
chemistry slows down the process of lead optimization and
supply. Microorganisms employ intricate biosynthetic
machineries to make natural products: for example,
synthesis of the macrolide antibiotic erythromycin, a
secondary metabolite in the medium-range structural
complexity, requires the participation of over 40 different
enzymatic activities (Katz and Donadio, 1995, Macrolides,
in Genetics and Biochemistry of Antibiotic Production,
Vining and Stuttard eds., Butterworth-Heinemann, Boston CT,
p. 385-420). Biosynthetic pathways can often be redirected
through manipulation of the fermentation conditions or of
the biosynthesis genes, in order to produce desired analogs
of the original structure. The availability of genes
involved in the formation of secondary metabolites has been
exploited for the formation of derivatives of natural
products obtained after genetic manipulation of the
producing organism (Hopwood, 1997, Chem. Rev. 99:0-39).
These manipulations have resulted in novel molecules, many
of which would be extremely hard if not impossible to
produce by chemical derivatization of the parent compound.
The obvious economical and environmental benefits resulting
from the formation of the desired structure in one
fermentation step constitute an additional stimulus for the
application of pathway engineering for the rational design
of novel structures. The compounds obtained in this way are
amenable evaluation of their biological properties as well
as being substrates for further derivatization by chemical
or biological means.
In summary, the supply of a natural product produced
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by a newly discovered microorganism, the optimization of
the complex composition, and the process of lead
optimization will all benefit from a detailed knowledge of
the genetics and physiology of the producing strain. The
present invention describes a general method for
transferring the capability to produce any secondary
metabolite from the original actinomycete producer to an
established and genetically manipulatable production host.
The general concept of the invention is illustrated in Fig.
1. Conditions for optimal growth, metabolite production and
maintenance need therefore to be developed for one host. In
addition, the availability of the cloned genes in a
genetically manipulatable and well characterized host
allows the utilization of all the genetic tools developed
for these strains for the creation of novel derivatives of
the natural product after genetic intervention.
3. SUi~IARY OF THE INVENTION
The present invention provides a system for producing and
manipulating natural products produced by a large group of
bacteria for the purpose of drug discovery, development~and
production. The method of the invention transfers the
ability to produce a secondary metabolite from an
actinomycete that is the original producer of the natural
product, to another production host that has desirable
characteristics.
In one embodiment, the invention involves the
construction of a library from a donor organism, the
producer of a natural product, in an Artificial Chromosome
that can be shuttled between a convenient, neutral cloning
host, such as the bacterium Escherichia coli, and a
production host, such as the actinomycetes Streptomyces
Iividans or Streptomyces coelicolor. The clones directing
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the synthesis of the natural product are identified in said
library, transferred into the production host where said
natural product is synthesized.
In another embodiment, the invention involves the
reconstruction of a large segment that directs the
synthesis of a natural product, starting from smaller DNA
fragments cloned from the genome of a donor organism. This
reconstruction occurs in an Artificial Chromosome that can
be maintained in a convenient neutral host, such as the
bacterium Escherichia coli, and subsequently transferred
into an actinomycete production host. The reconstructed
genomic segment in the Artificial Chromosome is transferred
into the production host where said natural product is
synthesized.
The present invention also relates to Escherichia
coli-Streptomyces Artificial Chromosomes, recombinant DNA
vectors useful for shuttling the genetic information
necessary to synthesize a given natural product between a
donor actinomycete producer and a production host.
3.1 DEFINITIONS
As used herein, the following terms will have the meaning
indicated.
An "Escherichia coli-Streptomyces Artificial
Chromosome", or ESAC, is a recombinant DNA construct that
can maintain very large DNA inserts in an Escherichia coli
host and that can be introduced and maintained in an
actinomycete production host.
An "Escherichia coli-Streptomyces Artificial
0 Chromosome" library, or ESAC library, is a library of
different recombinant constructs carrying very large DNA
inserts that can be maintained in an Escherichia coli host
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and introduced and maintained in an actinomycete production
host.
A pESAC is a vector used to construct an "Escherichia
coli-Streptorriyces Artificial Chromosome" or an ESAC library:
S A "natural product" is a secondary metabolite made by a
microorganism through a series of biosynthetic steps. This
natural product may or may not have any useful biological
activity.
A "complex" is the mixture of related natural products
with similar properties and biological activity that are
often produced by the same biosynthetic pathway.
A "donor organism" is the original producer of a
natural product, where the synthesis of said compound is
governed by a defined number of genetic elements.
A "gene cluster", a "cluster", a "biosynthesis cluster"
all designate a contiguous segment of the donor organism's
genome that contains all the genes required for the
synthesis of a natural product.
A "production host" is a microorganism where the
formation of a natural product is directed by a gene
cluster derived from a donor organism.
As used in the present invention, the following
abbreviations are employed: °C (Celsius degree): h (hour);
min (minute); kb (kilobase); ~,.1 (microliter); ml
(milliliter); mm (millimeter); mg (milligram); ~.g
(microgram); ng (nanogram); M (molar); Mb (megabase); UV
(ultraviolet); kV (kilovolt); S2 (Ohm); mFa (millifaraday).
In addition, the following abbreviations are used: Ab,
antibiotic; Ap, ampicillin; attB, chromosomal attachment
site; attP, phage or plasmid attachment site; bp, base
pair; ca., circa (i.e. "about"); Cm, chloramphenicol; E.,
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Escherichia; ESAC, E. coli-Streptomyces Artificial
Chromosome; GC, guanosine + cytosine; HTS, high throughput
screening; Km, kanamycin; int , integrase encoding gene;
LB, Luria Broth; LMP, low melting point; P., Planobispora;
S PCR, polymerase chain reaction; PFGE, Pulsed Field Gel
Electrophoresis; R, resistance; rpm, rounds per minute; S.,
Streptomyces; S, sensitive; Sac., Saccharopolyspora; sacB,
gene conferring sensitivity to sucrose; SDS, sodium dodecyl
sulfate; Tc, tetacycline; TE, TrisHCl EDTA buffer; tet,
tetacycline resistance gene; Th, thiostrepton; ts,
temperature sensitive; tsr , thiostrepton resistance gene;
U, units; vol, volume; wt, weight; YEME, yeast extract malt
extract medium.
4. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Scheme of the invention. The general concept
of the invention, whereby the gene cluster required for the
synthesis of a natural product in a donor organism is
established as an ESAC in an Escherichia coli host, and
then transferred into a desired production host, where it
integrates into the chromosome and directs production of
the secondary metabolite. The hexagon represents the
natural product, the twisted thin line the bacterial
chromosomes, and the thick line the desired gene cluster.
The pESAC episome is represented by a circle.
Figure 2. E. coli-Streptomyces Artificial Chromosome
vectors. Vectors pPAC-S1 and pPAC-S2 differ solely for the
orientation of the int-tsr cassette. Relevant features of
the vectors are illustrated. Kmr indicates resistance to
kanamycin; sacB indicates sensitivity to sucrose. Suitable
cloning sites are shown as: B, BamHI; S, ScaI; X, XbaI. The
replicating function of bacteriophage P1 are indicated by
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the thick bars.
Figure 3. General scheme of the invention, top-down
approach. High molecular weight DNA from the donor organism
is cloned into a pESAC.The resulting library in E. coli is
screened with the required probes, and the relevant ESACs
are identified. These are introduced into the desired
production host strain, where they integrate site-
specifically into the host chromosome. Symbols and
abbreviations are as in Fig. 1.
Figure 4. General scheme of the invention, bottom-up
approach. A cosmid library is prepared with DNA from the
donor organism and screened with the required probes. The
overlapping inserts from the positive cosmids, which
consitute the correct contig,are assembled into a pESAC via
homologous recombination in E. coli. The reconstructed ESAC
is introduced into the desired production host, where it
integrates site-specifically into the host chromosome.
Symbols and abbreviations are as in Fig. 1.
Figure 5. Scheme. of assemblage. The figure illustrates
a hypothetical genomic segment from a donor organism that
is covered by the inserts from three overlapping clones.
The relevant fragments A and D, which denote the ends of
the segment, and B and C, which represent regions of
overlap, are indicated with their relative orientation
(thick side on the fragment rectangle). The bottom part
illustrates the reconstructed ESAC.
Figure 6. Constructs required for cluster assemblage.
The plasmids indicated are generated by routine in vitro
DNA manipulations. Fragments A, B, C and D are as in Fig.
5. Fragment pairs are in this example separated by a
marker, indicated as AbR for antibiotic resistance.
Selective markers present on the two compatible replicons
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are, as an example: CmR and KmR.
Figure 7. Interplasmid insert exchange. Each of the CmR
derivatives, as of Fig. 6, is introduced in the same E.
coli cell as the cognate clone of Fig. 5 (for example a
5 cosmid that carries a KmR marker). Formation and then
resolution of the cointegrate leads to the transfer of the
cosmid's insert, indicated here by a looping line, in the
CmR replicon .
Figure 8. Sequel of assembling steps. A series of
10 interplasmid cointegration and resolution events is
conducted. Only the growing ESAC is indicated. The starting
construct (Fig. 6) is recombined with plasmid pAB2 (Fig.
7), leading to the insertion of the segment flanked by
fragments A and B. Next, the AbR marker from pBC1 (Fig. 6)
is introduced between fragments B and C, and subsequently
replaced by the insert from pBC2 (Fig. 7). Finally, the AbR
marker from pCD1 (Fig. 6) is introduced between fragments C
and D, and subsequently replaced by the insert from pCD2
(Fig. 7) .
Figure 9. A gene cluster from Planobispora rosea. The
extension of a gene cluster from P. rosea ATCC 53733 is
reported, together with the cosmids pRPl6, pRP31 and pRP58.
The fragments A, B, C and D used for assemblage are
highlighted. Restriction sites are abbreviated as: M, SmaI;
P, PstI; S, Sstl.
Figure 10. Site-specific integration of an ESAC. PFGE
analysis of S. lividans ZX7 transformed with ESAC-70. Lanes
1 and 2: S. coelicolor M145; lane 3: S. lividans ZX7 DNA;
lane 4: ZX7 attB::ESAC-70 DNA, colony 1; lane 5: ZX7
attB::ESAC-70 DNA, colony 2; lane 6: 50-kb ladder, size
marker. All DNAs in lanes 1-5 are digested with DraI.
Conditions for PFGE are: 200 Volts, 70 s switching for 7 15
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h, 120 s switching for 11 h.
Figure 11. Characterization of S. lividans
transformants. Southern hybridization of S. lividans
attB::PAD6, grown with (lane 1) or without (lane 2)
thiostrepton. P, rosea DNA is shown as control (lane 3).
Lane 4 contains 1-kb ladder. All DNAs are digested with
BamHI and probed with labeled PAD6.
5. DETAILED DESCRIPTION OF THE INVENTION
In its broadest sense, the present invention entails a
general procedure for constructing a Streptomyces host
producing any natural product after selective transfer of
the relevant genes from the original actinomycete producer,
the donor strain. This general procedure is outlined in
Fig. 1. The present invention can be applied with only
limited information on the structure of the natural product
and very little knowledge of the original producer's
genetics. The present invention has a substantial impact on
the process of drug discovery involving natural products or
their structural derivatives. The transfer of the producing
capability to a well characterized host can substantially
improve several portions of the process of lead
optimization and development: the titer of the natural
product in the producing strain can be more effectively
increased; the purification of the natural product can be
carried out in a known background of possible interfering
activities; the composition of the complex can be more
effectively controlled; altered derivatives of the natural
product can be more effectively produced through
manipulation of the fermentation conditions or by pathway
engineering. In order to better understand the value of the
present invention, a brief description is reported below of
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the current methods for optimizing the productivity of the
producing strain, for purifying a natural product, for
controlling the composition of a complex, and for producing
derivatives of a natural product.
The production of a natural product is controlled by
several mechanisms, few of which have been established in
detail. Generally, the level of production of a natural
product depends on the composition of the growth medium; on
the presence of appropriate precursors or on the absence of
specific inhibitors; on the expression level and timing of
genes controlling the biosynthetic pathway and competing
routes; and on the level and specific activity of key
enzymes in the pathway. Because of this complexity, the
productivity of the original strain is usually increased by
an empirical process, which may include, among other
things, one or more of the following steps: strain
purification, selection of phenotypic variants arising
spontaneously or after mutagenic treatment of the strain,
variation in the fermentation medium or in the fermentation
parameters; genetic engineering of the producing strain.
Fundamental knowledge about the physiology of the producing
strain and the variables affecting titer must be achieved
for an effective improvement of productivity. This
knowledge is very scant in a newly identified producer
strain.
During the discovery and development phase, sufficient
quantities of a natural product must be available for an
evaluation of its properties and/or for the generation of
analogs. Because of its uniqueness, a specific purification
process must be developed for each natural product.
However, it is highly desirable to have the natural product
as free as possible of compounds that may interfere with
the biological activity of the molecule. Contaminating
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impurities must be characterized analytically and
biologically. In a poorly characterized producer, little
information is available on the relevance of contaminating
impurities.
S A natural product may be produced by a microorganism
as a complex of a few or tens of molecules with minor
structural differences, designated congeners. Although most
of the congeners are usually biologically active, only one
or a few may represent the desired product: for example,
one congener may be substantially more active than the
others; it may possess better physico-chemical properties;
or it may be a better substrate for chemical modification.
The composition of a complex can be somehow controlled by
intervening on the fermentation parameters. However, the
most effective way is usually the altered expression of
selected genes by genetic engineering (e. g. Sezonov et al.,
1997, Nature Biotechnol. 15:349-353).
Chemical modification of natural products represents
the most commonly used means of obtaining novel structures.
This approach has been successfully employed, but it still
suffers from practical limitations to the number and type
of compounds obtainable. The structural complexity of many
natural products makes their total synthesis often too
lengthy and expensive to be of any practical use. This same
structural complexity, with either the presence of several
closely related functional groups or their absence, limits
modification of a natural product to selected portions of
the molecule. Methods of combinatorial synthesis need an
initial scaffold as the starting building block, and this
can be often generated only through a low yield degradation
of the natural product. However, derivatives of natural
products that would be very hard if not impossible to
produce by chemical means have been obtained after genetic
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alteration of the biosynthetic pathway. Examples include
the introduction of additional genetic information (Epp et
al., 1989, Gene 85:293-301), the targeted inactivation of
selected genes or portion thereof (Donadio et al., 1993;
Proc. Natl. Acad. Sci. USA 90:7119-7123), the "mixing and
matching" of genes or portions thereof from different
pathways (McDaniel et al., 1994, Nature 375:549-554).
All the above activities are important for the process
of lead optimization and for the development of selected
lead structures. They can all benefit, to different extent,
from a detailed knowledge of the physiology of the
producing strain, and from the possibility of genetically
manipulating it. The process by which a given organism is
genetically manipulated in order to alter the type, quality
or quantity of a natural product is referred to as pathway
engineering. The ability to perform pathway engineering in
a newly isolated microorganism producing a bioactive
molecule with promising characteristics can therefore
considerably expedite the optimization of a lead structure
and the development process. Pathway engineering can be
schematized as a sequel of three steps: a) isolation of the
genes of interest; b) performing on selected genes) the
manipulations required by the specific objective; and c)
introduction of the modified genes) i.n suitable form in an
appropriate host.
Isolation of the genes of interest from most
actinomycetes can be achieved quite easily. The genes for
primary metabolism are usually well conserved, and they can
be easily accessed in any microorganism by using suitable
hybridization probes or by the PCR. The genetic elements
governing the biosynthesis of the major classes of
secondary metabolites have been also described, and many
genes can similarly be identified. Since natural product
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biosynthesis is governed by clusters, one needs to identify
just a few genes in order to have them all. However,
synthesis of the vast majority of natural products requires
a considerable extent of genetic information. For examples;
5 biosynthesis of the natural products erythromycin (an
antibiotic), avermectin (an antihelmintic agent) and
rapamycin (an immunosuppressant) requires 55, 90 and 95 kb,
respectively, of genetic information (Katz and Donadio,
1993, Annu. Rev. Microbiol. 47:875-912; MacNeil, 1995,
10 Avermectins, in Genetics and Biochemistry of Antibiotic
Production, Vining and Stuttard eds., Butterworth-
Heinemann, Boston CT, p.421-442; Schwecke et al., 1995,
Proc. Natl. Acad. Sci. USA 92:7839-7843). Other natural
products may require even larger extent of genetic
15 information. Therefore, in order to isolate an entire
cluster in a single piece, cloning vectors capable of
accepting and maintaining large DNA segments are necessary.
The manipulation of the isolated genes is generally
best performed in a convenient cloning host, such as E.
coli. Manipulations relevant to pathway engineering can
include some or all of the following: site directed
mutagenesis, gene inactivation, gene fusions, modification
of regulatory sequences, etc. Techniques for the in vitro
manipulation of DNA and for the propagation of the mutated
alleles in E. coli are well developed and can be applied to
DNA from virtually any source (Sambrook et al., 1989, In
Molecular Cloning: A laboratory Manual, 2nd edn, Cold
Spring Harbor, New York: Cold Spring Harbor Laboratory
Press).
The final step in pathway engineering requires the
introduction of modified or heterologous gene(s), in
suitable form, in a strain where these genes can be
appropriately expressed. This strain is often the strain
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producing the natural product whose quantity, quality or
type one wants to alter. The genes of interest must be
carried on appropriate vectors: according to the particular
objective of pathway engineering, one may need, among
others, vectors that can be stably maintained as single or
multicopy episomes; that can insert into the host
chromosome at a fixed location; that allow replacement of
an endogenous gene with an in vitro modified allele; that
allow deletion of selected genes from the host chromosome.
In addition, for each strain one must have means for
introducing heterologous DNA and selecting for its
presence. Therefore, in order to genetically manipulate a
given producer, one must establish conditions for rendering
the bacterial cell capable of receiving incoming DNA; for
selecting the incoming DNA; and develop vectors and
methodologies for the various types of manipulations
exemplified above. Low- and high copy-number, integrative,
non-replicating vectors must be developed with appropriate
selection markers. Thus, for each producing strain,
specific gene transfer tools and conditions must be
developed, starting in most cases from extremely poor
knowledge about the microorganism. In addition, techniques
developed for one species do not necessarily apply to a new
species from the same genus, and often not even to a new
strain. It is then no wonder that, among the thousands of
strains described as producers of interesting natural
products, gene transfer systems have been developed only
for a limited number of species, which serve either as
model organisms for genetic and physiological studies, or
produce a commercially important molecule. The present
invention provides tools for the general manipulation of
any secondary metabolite pathway, and overcomes the
difficulties of developing ad hoc conditions for a new
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producer.
Naive hosts have been shown to produce the appropriate
natural product or its intermediates) when the relevant
DNA was introduced into them (Malpartida and Hopwood, 1984;
Nature 309:462-464; Hong et al., 1997, J. Bacteriol.
179:470-476; Kao et al., 1994, Science 265:509-512; McGowan
et al., 1996, Mol. Microbiol. 22:415-426; Kealey et al.,
1998; Proc. Natl. Acad. Sci. USA 95:505-509). However, the
examples reported thus far represent special cases. Indeed,
they include the introduction of relatively small DNA
segments into a production host; or the transfer of gene
clusters within members of the same bacterial genus; or
they have required the careful engineering of specific
biosynthesis genes under the control of appropriate genetic
elements that direct their expression. Furthermore, the
Streptomyces vectors currently available have an upper
limit of ca. 40 kb (Hopwood et al., 1987, Methods Enzymol.
153:116-167).
Until now, it was not established that DNA fragments
exceeding 100 kb, derived from the high GC genome of
actinomycetes, could be cloned and stably maintained in an
E. coli host. Nor was any report of the introduction of
large DNA segments into a Streptomyces host. The unexpected
finding described herein is that these cloning tasks can be
achieved according to the principles and methodologies of
the present invention. Furthermore, the genetic elements
required for the synthesis of a natural product in the
original producer are genetically stable in a heterologous
host, where they can direct the synthesis of the desired
molecule. It was also unexpected and unprecedented that
this heterologous stability and expression can occur when
the donor organism and the production host belong to
different bacterial genera.
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The present invention rests on the fact that the genes
required for the formation of a natural product are found
as gene clusters of a defined size; that these gene
clusters can be conveniently isolated, manipulated and
transferred among different actinomycete strains; that they
are expressed in a heterologous host; and on the fact that
all the primary metabolite precursors required for the
formation of a particular natural product are either
produced by selected enzymes encoded by cluster-specific
genes, or are present and available in the heterologous
host at the time of formation of the natural product. The
present invention addresses also the crucial aspect of
natural product formation in actinomycetes: i.e. synthesis
of many natural products may require over 100 kb of genetic
information. To be generally applicable, transferring all
the genes necessary for the production of any natural
product requires cloning vectors capable of accommodating
fragments as large as 150 kb, and possibly more. An object
of the present invention is therefore represented by
vectors capable of accommodating such large fragments which
are also capable of being stably maintained in a suitable
microbial host, such as a Streptomyces host.
Examples of these vectors are designated with the
generic name pESAC. They are derived from bacterial
artificial chromosomes (Shizuya et al., 1992, Proc. Natl.
Acad. Sci. USA 89:8794-8797; Ioannou et al., 1994, Nature
Genet. 6:84-89}and can carry inserts up to 300 kb, or more.
As a general example of the broad applicability of the
principles and methodologies described in the present
invention, the Examples reported below describe how a
convenient Streptomyces host can be engineered to carry a
large gene cluster in order to produce a desired natural
product through the use of an appropriate ESAC. The
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exemplary organism chosen as the donor organism is the
actinomycete P. rosea, belonging to one of the lesser
characterized genera of actinomycetes (Goodfellow, 1992, In
The Prokaryotes, 2nd edn., Balows, Trueper, Dworkin, Harder
and Schleifer eds, Springer-Verlag, New York, NY, USA).
This organism produces the natural product GE2270 (Selva et
al., 1991, J. Antibiotics 44:693-701), an antibacterial
agent. This particular case therefore describes the general
applicability of the present invention, since very little
information is available on the donor organism, on its
genetics and physiology, and on the gene clusters present
in its genome. Further examples described herein illustrate
the application of the principles and methodologies of the
present invention to other gene clusters described in the
literature.
The present invention, relating to a general method
for transferring the capability to produce any natural
product from the original actinomycete to an established
and genetically manipulatable Streptomyces host, can be
schematized in a series of passages summarized as: 1)
design of suitable vectors; 2) construction of a large-
insert library in said vectors; 3} selection of the desired
clones with appropriate probes; 4) insertion of the
selected clones into a convenient Streptomyces host; and 5)
growth of the recombinant strain under appropriate
conditions to produce the natural product.
Actinomycetes produce a large number of natural
products with important applications. However, other
important classes of microbial producers are known, and
newer ones are likely to be discovered in the upcoming
years, as more microbial sources are screened for potential
new drugs. Important classes of microbial producers
include, among others, filamentous fungi, bacilli,
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mixobacteria, pseudomonas and cyanobacteria. The series of
passages described above can therefore be applied to other
important classes of microbial producers, provided that two
requisites are met: the synthesis of the desired natural
5 product is governed by a gene cluster; suitable production
hosts) exist; and appropriate selective markers) and
maintenance functions) are introduced into the Artificial
Chromosome.
Furthermore, the series of passages summarized above
10 and described in detail in the Examples, involve the use of
a neutral cloning host. This host, as described in the
present invention, is the bacterium Escherichia coli. In a
preferred example of such a host, a high cloning efficiency
can be obtained, and many of the analyses of the ESACs can
15 be quickly performed. However, it is evident to one of
ordinary skill in this art that any other host that allows
high cloning efficiency can be used as neutral cloning
host. Additionally, the use of such a host is not a
conditio sine qua non for the applicability of the present
20 invention. In fact, when it is possible to establish
directly a library in a production host, there is no need
for an intermediate neutral cloning host.
In summary, the present invention consists of a method
for transferring the production of a natural product from
an actinomycete donor organism that is the original
producer of said natural product to a different
actinomycete host, where this transfer is achieved by means
of an E, coli-Streptomyces Artificial Chromosome that
carries a gene cluster governing the biosynthesis of said
natural product derived from said donor organism. This
method comprises the steps of .
(a) isolating large fragments of chromosomal DNA of
the actinomycete donor organism of a size which encompasses
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the gene cluster that directs the biosynthesis of the
natural product;
(b) constructing a suitable vector capable of
accomodating said large fragments of chromosomal DNA and of
introducing and stably maintaining said large fragments of
DNA into an E. coli host;
(c) constructing an E. coli-Streptomyces Artificial
Chromosome by inserting said large fragments of chromosomal
DNA of step (a}into the above said vector of step (b) and
selecting the E. coli-Streptomyces Artificial Chromosome
comprising the entire gene cluster construct that directs
the biosynthesis of the above said natural product;
(d) transforming an actinomycete host different from
the donor actinomycete host with the E. coli-Streptomyces
Artificial Chromosome of step (c) that carries the gene
cluster governing the biosynthesis of said natural product
wherein the actinomycete host carries a region which is
specific for the integration of the E. coli-Streptomyces
Artificial Chromosome.
6. GENERAL METSODS
Plasmids, Bacterial Strains and Growth Conditions
Plasmids pUCBM20, pUCBM21, pBR322 and pUCl8 are obtained
from Boheringer Mannheim; plasmid pIJ:39 and ~C31 DNA have
been described (Hopwood et al., 1985, Genetic Manipulation
of Streptomyces: A Laboratory Manual, The John Innes
Foundation, Norwich, UK) and are available from prof. David
Hopwood, The John Innes Centre, Norwich, UK; plasmid
pCYPAC2 has been described (Ioannou et al., 1994, Nature
Genetics 6:84-89) and is available from prof. Pieter de
Jong, Roswell Park Cancer Institute, Buffalo, NY, USA;
plasmid pMAK705 has been described (Hamilton, et al., 1989,
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J. Bacteriol, 171:4617) and is available from prof. Sidney
Kushner, University of Georgia, Athens, USA; cosmid Lorist6
has been described (Gibson et al., 1987, Gene, 53:283-286)
and is from prof. Stewart Cole, Pasteur Institute, Paris',
France. E. coli strains are obtained from commercial
sources: DHSa (Life Technologies), DHlOB (Life
Technologies), C600 (E. coli Genetic Stock Center), DH1
(Life Technologies} and XLlblue (Stratagene}. S. coelicolor
M145 and S. l.ividans ZX7 have been described (Hopwood et
al., 1985, Genetic Manipulation of Streptomyces: A
.Laboratory Manual, The John Innes Foundation, Norwich, UK)
and are available from prof . David Hopwood, The John Innes
Institute, Norwich, UK. Planobispora rosea ATCC 53733,
Streptomyces hygroscopicus ATCC 29253, Amycolatopsis
mediterranei ATCC 13685 and Saccharopolyspora erythraea
NRRL2338 are from the ATCC culture collection. All other
materials are from commercial sources. Media for
cultivation of E. coli (Sambrook et al., 1989, In Molecular
Cloning: A laboratory Manual, 2nd edn, Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press) and
Streptomyces (Hopwood et al., 1985, Genetic Manipulation of
Streptomyces: A Laboratory Manual., The John Innes
Foundation, Norwich, UK) have been described. The JM medium
for S. coelicolor has been described (Puglia et al., 1995,
Mol. Microbiol. 17:737-746).
DNA Manipulations DNA manipulations are performed
following described procedures, using the appropriate E.
coli strains as cloning hosts (Sambrook et al., 1989, In
Molecular Cloning: A laboratory Manual, 2nd edn, Cold
Spring Harbor, New York: Cold Spring Harbor Laboratory
Press). Genomic DNA from actinomycetes is prepared as
described (Hopwood et al., 1985, Genetic Manipulation of
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Streptomyces: A Laboratory Manual, The John Innes
Foundation, Norwich, UK). A cosmid library of P. rosea DNA
is constructed in the cosmid vector Lorist6 following
published procedures (Sambrook et al., 1989, In Molecular
Cloning: A laboratory Manual, 2nd edn, Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press).
Amplification by the PCR are performed following published
guidelines (Innis, Gelfand, Sninsky and White, eds., 1990,
PCR Protocols: A guide to Methods and Applications,
Academic Press, San Diego, CA, USA).
Hybridizations Probes Pep6 and Pep8 are derived from
conserved motifs in peptide synthetase gene sequences
(Turgay and Marahiel, 1994, Pept. Res. 7:238-241).
Oligonucleotide probe Pep6 consists of an equimolar mixture
of 5'-GCSTACATCATCTACACSTCSGGSACSACS-GGSAAGCCSAAGGG-
3'(SEQID N°1) and 5'-
GGSTACATCATCTACACSAGCGGSACSACSGGSAAGCCSAAGGG-3'(SEQID N°2).
Oligonucleotide probe Pep8 consists of an equimolar mixture
of 5'-AKGCTGTCSCCSCCSAGSNNGAAG-AAGTYGTCGTCGATSCC-3'(SEQID
N°3) and 5'-AKGGAGTCSCCSCCSAGSNNGAAGAAGTYGTCGTCGATSCC-
3'(SEQID N°4). [S indicates G or C; K indicates G or T; Y,
C or T; and N, any base]. Hybridizations are performed with
a hybridization stringency set at 2xSSC, 55 °C, and a final
wash set at the same stringency.
Preparation of high molecular weight DNA Procedures for
the preparation of high molecular weight DNA from
actinomycetes for PFGE have been described (Dyson, 1993,
Trends Genet. 9:72; Kieser et al., 1992, J. Bacteriol.
174:5496-5507). They are modified for constructing
libraries as described in the Examples.
7. EXAMPLES
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The present invention consists in a series of passages,
involving the design of suitable vectors; the introduction
of large DNA inserts in said vectors employing genomic DNA
from the donor organism; the selection of clones carrying
the cluster specifying the synthesis of the desired natural
product; the introduction of selected clones) into the
appropriate production host; and the growth of the
recombinant strain under appropriate conditions for
metabolite production. These passages are described in
detail in the Examples reported herein. These Examples
outline the steps necessary to accomplish each passage, for
the overall purpose of the present invention: the
production of a natural product in a different host. They
serve to illustrate the principles and methodologies of the
present invention, and are not meant to restrict its scope
to the Examples specified herein.
7.1 Cloning vectors
Bacterial Artificial Chromosomes are circular plasmids that
can be easily propagated in and prepared from E. coli cells
by standard miniprep methods (Shizuya et al., 1992, Proc.
Natl. Acad. Sci. USA 89:8794-8797; Ioannou et al., 1994,
Nature Genet. 6:84-89). In order to adapt Bacterial
Artificial Chromosomes to a Streptomyces host, they need to
be endowed with a selectable marker and maintenance
functions. Site-specific integration, mediated by the
action of an integrase encoded by the int gene, allows the
stable incorporation of episomal elements into the host
genome, at a defined locus designated attB. The episomal
element needs to carry the cognate attP site and it may
lack replicative functions. In addition, int-mediated
excision of the integrated element from the chromosome via
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reversal of the integration event can be prevented through
selection of the resistance marker carried by the
integrated episome; or, if necessary, after site-specific
integration has occurred, the int gene on the integrated
5 episome can be inactivated. Site-specific integration
therefore allows the introduction of foreign DNA in single
copy at a defined genetic locus. Several systems capable of
directing site-specific integration of incoming circular
DNA into the chromosome of a Streptomyces host have been
10 described. A convenient system that can be used in the
present invention is for istance the int-attP system
derived from the temperate bacteriophage ~C31 (Kuhstoss and
Rao, 1991, J. Mol. Biol. 222:897-908), which directs,
during lysogen formation, integration of the 41-kb phage
15 genome at the attB site, located in a stable segment of the
S. coelicolor chromosome (Redenbach et al., 1996, Mol.
Microbiol. 21:77-96). Several selectable markers have been
described that can be used for Streptomyces (Hopwood et
al., 1985, Genetic Manipulation of Streptomyces: A
20 Laboratory Manual, The John Innes Foundation, Norwich, UK).
The tsr gene, conferring resistance to the antibiotic
thiostrepton (Thompson et al., 1982, Gene 20:51-62), is
used in the present invention. The pESAC vectors, pPAC-S1
and pPAC-S2, described in the present invention, are
25 depicted in Fig. 2. Their relevant features are: ability to
accommodate DNA inserts up to 300 kb; low copy number in E.
coli for increased stability; ease of propagation in E.
coli because of the pUCl9 stuffer segment; BamHI, XbaI or
ScaI cloning sites, with positive selection of inserts for
resistance to sucrose; T7 and SP6 promoters flanking the
cloning site; KmR or ThR for selection in E. coli or
actinomycetes, respectively; site-specific integration at
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the ~C31 attB site into the Streptomyces genome. Vectors
pPAC-S1 and pPAC-S2 are 22 kb in size and differ solely for
the orientation of the int-tsr cassette. After release of
the stuffer pUCl9 segment, the vector size is reduced to
19.7 kb. When cloning in the BamHI site, the vector can be
released by digestion with DraI, resulting in vector
fragments of 7.4, 4.2 and 0.6 kb. The additional 7.5 kb of
vector DNA will be associated with the insert. DraI rarely
cuts in the high-GC genome of actinomycetes, so that the
insert size can be easily calculated.
Example 1
Isolation of the int region from ~C31
Two pairs of PCR primers, 5'-TTTTTGGTACCTGACGTCCCGAAGG
CGTG-3'(SEQID N°5) and 5'-CAGCTTGTCCATGGCGGA-3' (SEQID
N°6); and 5'-TCTGTCCGCCATGGACAAGC-3' (SEQID N°7) and 5'
TTTTTGGATCCGGCTAACTAACTAAACCGAGA-3' (SEQID N°8), are used
to amplify the int-containing fragments of 1.3 and 0.9 kb,
respectively. The template is c~C31 DNA. The amplified
fragments are digested with KpnI + NcoI and NcoI + BamHI,
respectively, and recovered from an agarose gel.
Example 2
Construction of plasmid pINT
The 1.3 and 0.9 kb fragment, prepared as described in
Example 1, are ligated to pUCMB21, digested with KpnI +
BamHI. The resulting mixture contains the desired plasmid
pINT.
Example 3
Construction of E. coli K12 DHSa/pINT
Approximately 10 ng of plasmid pINT, prepared as described
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in Example 2, are used to transform E. coli DHSa and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pINT, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.0 and
0.9 kb after digestion of the plasmid with NcoI + BamHI.
Example 4
Construction of plasmids pUIT1
The 1.8 kb BamHI fragment containing the tsr gene is
isolated from pIJ39 and ligated to pINT, prepared as
described in Example 3 and previously digested with BamHI.
The resulting mixture contains the desired plasmids pUITl.
Example 5
Construction of E. coli K12 DHSa/pUITl
Approximately 10 ng of plasmid pUITl, prepared as described
in Example 4, are used to transform E. coli DHSa and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUITl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.9 and
1.8 kb after BamHI digestion of the plasmid.
Example 6
Construction of plasmid pUIT3
The 3.7 kb ApaI fragment, containing the int-tsr cassette,
is isolated from plasmid pUITl, prepared as described in
Example 5, and ligated to pUCBM21 digested with ApaI. The
resulting mixture contains the desired plasmid pUIT3.
Example 7
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Construction of E. coli K12 DHSa/pUIT3
Approximately 10 ng of plasmid pUIT3, prepared as described
in Example 6, are used to transform E. coli DHSa and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUIT3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of of 4.2
and 2.2 kb after BamHI digestion of the plasmid.
Example 8
Construction of plasmid pUIT4
The BamHI site present in the int-tsr cassette of plasmid
pUIT3 is eliminated as follows. Plasmid pUIT3, prepared as
described in Example 7, is partially digested with BamHI,
followed by filling-in of the resulting ends, and treated
with DNA ligase. The resulting mixture contains the desired
plasmid pUIT4.
Example 9
Construction of E. coli K12 DHSa/pUIT4
Approximately 10 ng of plasmid pUIT4, prepared as described
in Example 8, are used to transform E. coli DHSa and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUIT4, as verified by the observation,
upon agarose gel-electrophoresis, of a 6.4 kb fragment
after BamHI digestion of the plasmid.
Example 10
Construction of plasmid pPAC-S1 and pPAC-S2
The 3.7 kb ApaI fragment from pUIT4, prepared as described
in Example 9, is mixed with pCYPAC2, previously digested
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with NheI. After filling-in of the ends, DNA ligase is
added. The resulting mixture contains the desired plasmids
pPAC-S1 and pPAC-S2.
Example 11
Construction of E. coli K12 DH10B/pPAC-S1 and DH10B/pPAC-S2
Approximately 10 ng of plasmids pPAC-S1 and pPAC-S2,
prepared as described in Example 10, are used to transform
E. coli DH10B and a few of the resulting KmR colonies that
appear on the LB-agar plates are analyzed for their plasmid
content. One colony is found to carry pPAC-S1, as verified
by the observation, upon agarose gel-electrophoresis, of
fragments of 8.1, 4.8, 4.6, 2.2, 2.2, 0.5 and 0.1 kb after
EcoRI digestion of the plasmid. Another colony is found to
carry pPAC-S2, as verified by the observation, upon agarose
gel-electrophoresis, of fragments of 8.1, 7.8, 2.2, 2.2,
1.5, 0.5 and 0.1 kb after EcoRI + BamHI digestion of the
plasmid.
Although the present invention is described in the
Examples listed above in terms of preferred embodiments,
they are not to be regarded as limiting the scope of the
invention. The above Examples serve to illustrate the
principles and methodologies for constructing Bacterial
Artificial Chromosomes that can be introduced in a
Streptomyces host. It will occur to those skilled in the
art that selectable markers different from the tsr gene can
be employed for selection in Streptomyces. Other useful
markers are described in detail in laboratory manuals
(Hopwood et al., 1985, Genetic Manipulation of
Streptomyces: A Laboratory Manual, The John Innes
Foundation, Norwich, UK) and include but are not limited
to: genes conferring resistance to apramycin, kanamycin,
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erythromycin, hygromycin, viomycin. It will also occur to
those skilled in the art that functions other than those
specified by ~C31 can be used for directing site-specific
integration in the Streptomyces chromosome. These functions
5 are described in recent literature (Hopwood and Kieser,
1991, Methods Enzymol. 204:430- 458) and include but are
not limited to those derived from pSAM2, SLP1, IS117.
Bacterial Artificial Chromosomes derived from the E. coli F
plasmid have been described (Shizuya et al., 1992, Proc.
10 Natl. Acad. Sci. USA 89:8794-8797). It will occur to those
skilled in the art that, using the principles and
methodologies described above, the int-tsr cassette from
pUIT4, prepared as described in Example 9, could be
inserted into a unique site of pBAC108L (Shizuya et al.,
15 1992, Proc. Natl. Acad. Sci. USA 89:8794-8797) or of
suitable derivatives of this vector, leading to the
formation of a BAC-based series of pESAC. It will occur to
those skilled in the art that other pESACs differing, for
example, in their size, in the E. coli replicon they carry,
20 in the selectable marker for E. coli, in the cloning sites,
can also be used in the present invention. Other
differences and variations in the technical aspects of the
present invention could be employed. These include but are
not limited to: different methods and sources for
25 obtaining selectable markers and integrative functions;
different cloning sites and methodologies; different
orientation of the insert; different E. coli hosts for
amplifying the recombinant constructs. All these variations
fall within the scope of the present invention.
7.2 Construction of large inserts in pESAC
Two distinct methodologies for introducing large DNA
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fragments into the vectors described in Section 7.1 fall
within the scope of the present invention. The first
methodology can be referred to as the top-down approach and
is depicted in Fig. 3. It consists of directly cloning the
desired gene cluster into a pESAC through the construction
of a genomic library of DNA fragments of average size of
100 kb, or more. The library is then screened with suitable
probes (Section 7.3) in order to :identify the desired
cluster. The second methodology can be considered a bottom-
up approach and is illustrated in Fig. 4. It consists of
assembling the desired gene cluster from pre-existing
smaller segments of cloned, overlapping DNA, through the
iterative use of homologous recombination in E. coli. The
desired overlapping clones encompass the desired gene
cluster and are identified as described in Section 7.3.
Both methodologies fall within the scope of this invention.
Depending on factors such as previous knowledge about the
biosynthesis cluster, the extent of characterization of the
producing strain, the existence of other natural products
of interest produced by the original microorganism, one
methodology may be preferred over the other. However, the
two methodologies are not mutually exclusive.
7.2.1 Preparation of a large insert library
In order to prepare a large-insert library, particular care
must be taken in the preparation of genomic DNA from the
actinomycete strain of choice. Although several procedures
have been described for the isolation of genomic DNA, few
are suitable for obtaining sufficient yields of high
molecular weight DNA. The strain of choice is grown in a
medium that allows dispersed growth to facilitate lysis of
the cells. Examples of suitable growth media for different
genera of actinomycetes can be found in the literature
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(Balows, Trueper, Dworkin, Harder and Schleifer eds., 1992,
The Prokaryotes, 2nd edn., Springer-Verlag, New York, NY,
USA). The growth time should allow formation of a
sufficient quantity of biomass; however, long incubation
times should be avoided, since mycelia are generally more
resistant to lysis as they age. The mycelium is pelleted,
washed and embedded in agarose for the subsequent lytic
steps. Lysis of the cells is achieved by a combination of
enzymatic (e. g., incubation with lysozyme and/or
achromopeptidase) and mild physical treatments (e. g., SDS).
The concentrations of reagents and the incubation times
need to be optimized for each strain. A good starting point
is represented by the conditions described in Example 12.
The quality of the DNA preparation is checked by PFGE under
appropriate conditions. Once a suitable preparation is
obtained, the DNA can be digested as described in Example
13. The exact incubation time and the units of restriction
endonuclease are adjusted to the particular DNA preparation
fox optimizing the size and yield of the bulk of digested
DNA, which should exceed 150 kb. The partially digested DNA
is size-fractionated on a PFGE gel, without exposure to
ethidium bromide or UV light, in order to avoid damage to
the DNA. The gel slice containing the desired DNA fraction
is localized by staining the marker-containing portion of
the gel and cut. All subsequent manipulations are performed
with great care (Birren and Lai, 1993, Pulsed Field Gel
Electrophoresis: A Practical Guide, Academic Press, New
York, NY). The size-selected DNA is ligated to an
appropriately prepared pESAC (see Example 14) employing a
high molar excess of vector to insert (ca. 10:1) in order
to minimize the formation of chimeric clones (i.e. those
constituted by the religation of two uncontiguous inserts).
Subsequent steps are performed using published procedures
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for the cloning in Bacterial Artificial Chromosomes, as
described in Examples 16 and 17.
The genome size of actinomycetes is around 8 Mb.
Consequently, a 10-genome equivalents library consisting of
800 clones with an average insert size of 100 kb has >99.9~
probability of containing the desired clone (Sambrook et
al., 1989, In Molecular Cloning: A laboratory Manual, 2nd
edn, Cold Spring Harbor, New York: Cold Spring Harbor
Laboratory Press). Therefore, the average clone in the
library will have a 10-kb segment (8, 000 kb divided by 800
clones = 10 kb/clone) of unique DNA , i.e. DNA not found in
any other clone. Consequently, a 90 kb cluster will have a
high chance of being exactly contained within one or two
100-kb clones in a 800-clone library. The number of clones
to be screened and the average insert size to be looked for
in the ESAC library depends on the expected size of the
biosynthesis gene cluster. The larger the difference
between the average insert size and the expected size of
the gene cluster, the smaller the number of clones to
screen in order to identify an entire gene cluster in a
single clone. ESAC DNA is prepared from a representative
number of clones obtained after electroporation of a
ligation mixture and analyzed for determining the frequency
of insert-carrying clones and their average size. If
necessary, all insert containing clones can be analyzed by
miniprep procedure (Birren and Lai, 1993, Pulsed Field Gel
Electrophoresis: A Practical Guide, Academic Press, New
York, NY, USA) and clones carrying inserts below a certain
threshold can be discarded. Alternatively, the number of
clones carrying insert of the appropriate size can be
estimated after analysis of a representative number of
ESACs. The quality of the library can be evaluated by
probing with cloned genes from the strain (if available),
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or from highly conserved "housekeeping" genes from a strain
with a similar GC content, such as S. coelicolor.
Example 12
Preparation of high molecular weight chromosomal DNA
S. coelicolor strain M145 is grown in YEME medium
containing 0.5°s (wt/vol) glycine for 40 h at 30°C on an
orbital shaker (ca. 200 rpm). The mycelium is pelleted by
centrifugation, washed with 10.3 sucrose and the
chromosomal DNA is extracted from the mycelium embedded in
0.750 LMP agarose by treatment with 1 mg/ml lysozyme and
with 1 mg/ml proteinase K in O.lo SDS for 40 h at 50°C.
Example 13
Preparation of partially digested chromosomal DNA
S. coelicolor M145 chromosomal DNA, prepared as described
in Example 12 and embedded in LMP agarose plugs, is
partially digested by limiting the magnesium concentration
for 20 min with 4 U of Sau3AI. The resulting DNA fragments
are resolved by PFGE and the size-selected genomic DNA
fraction (larger than 100 kb) is recovered and released
from the agarose gel by digestion with gelase.
Example 14
Preparation of pPAC-S1 for library construction
The vector pPAC-S1, prepared as described in Example 11, is
cut with ScaI and then treated with calf intestinal
phosphatase. The recovered DNA is then digested with BamHI
and treated with an excess of calf intestinal phosphatase.
The short ScaI-BamHI linker fragments are removed by spin
dialysis.
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Example 15
Construction of the ESAC library
Size selected genomic DNA, prepared as described in Example
13, is ligated to pPAC-S1, prepared as described in Example
5 14, employing 300 Molecular Biology Units of T4 DNA ligase
in a 50 ~,1 final volume and using a ca. 10:1 molar ratio of
vector to insert. The resulting ligation mixture contains
the desired ESAC library, consisting of fragments S.
coelicolor DNA inserted into the pPAC-S1 vector.
Example 16
Introduction of the library into E. coli K12 DH10B
The ligation mixture, prepared as described in Example 15,
is drop-dialyzed against 0.5 X TE for 2 h using 0.025 mm
type VS membranes (Millipore) and a few ~1 are used to
electroporate 40 ~,l of electrocompetent E. coli DH10B
cells. The electroporation conditions are: 2.5 kV, 100 SZ
and 25 mFa employing the Biorad Gene Pulser II. The cells
are plated on LB-agar plates containing 25 ~tg/ml Km and 5°s
sucrose to select for recombinant cells harboring insert-
carrying pPAC-S1. Individual colonies are picked into 0.1
ml of LB broth containing 25 ~g/ml Km in 96-well microtiter
plates, where they are stored at -80 °C after overnight
incubation and addition of glycerol to 200 (v/v).
Example 17
Preparation of recombinant ESACs
Individual colonies, prepared as described in Example 16,
are inoculated into 5 ml of LB broth containing 25 ~tg/ml Km
and grown overnight. ESAC DNA is isolated using the
alkaline extraction procedure (Sambrook et al., 1989, In
Molecular Cloning: A laboratory Manual, 2nd edn, Cold
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Spring Harbor, New York: Cold Spring Harbor Laboratory
Press)) without the phenol extraction step. The DNA is
analyzed, after digestion with DraI, by PFGE. Three bands
of 7.4, 4.2 and 0.6 kb are common to all clones and
represent vector DNA.
The examples described above illustrate the principles
and methodologies of constructing a large-insert library of
S. coelicolor DNA in a pESAC . Although the present
invention is described in the Examples listed above in
terms of preferred embodiments, they are not to be regarded
as limiting the scope of the invention. The above
descriptions serve to illustrate the principles and
methodologies for constructing a large-insert DNA library
in a pESAC. It will occur to those skilled in the art that
other Streptomyces strains can be used as a source of DNA
for constructing the library. For example, an ESAC library
of the rapamycin producer Streptomyces hygroscopicus ATCC
29253 can be constructed, employing the procedures reported
for PFGE analysis, (Ruan et al., 1997, Gene 203:1-9) and
applying the principles and methodologies described in
Examples 12 through 17.
It will also occur to those skilled in the art that
strains from actinomycete genera other than Streptomyces
can be used as a source of DNA for constructing an ESAC
library. These strains can belong to any genus of the order
Actinomycetales, which include but are not limited to the
genera reported in Table 1. As another example, an ESAC
library of the erythromycin producer Saccharopolyspora
erythraea can be constructed, employing the procedures
reported for PFGE analysis (Reeves et al., 1998,
Microbiology 144:2151-2159) and applying the principles and
methodologies described in Examples 12 through 17. Those
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skilled in the art understand that bacterial taxonomy is a
rapidly evolving field and new genera may be described
while old genera may be reclassified. Therefore, the list
of bacteria genera related to actinomycetes is very likely
to change. Nonetheless, the principles and methodologies of
the present invention can be applied to any donor organism
related to the actinomycetes.
It will also occur to those skilled in the art that
different actinomycete strains will require growth media
different from those reported in Example 12. Furthermore,
alternative media and conditions for growth can be employed
for obtaining mycelia for DNA preparation; that alternative
methods of lysis of mycelia can be utilized; that
restriction endonucleases other than Sau3AI can be equally
effective for constructing a library; that other methods
for fragmenting DNA can be employed. In addition, it will
occur to those skilled in the art that pESAC other than
pPAC-S1, which include but are not limited to the possible
vectors described in Section 7.1, can be used for
constructing a library. Alternative methods for ligating
DNA, for introducing the library in E. coli cells, and
hosts other than DH10B are well described in the literature
and can be employed in the present invention. All the above
variations in strains, reagents and methodologies that can
be employed for preparing a large-insert library of
actinomycete DNA into a pESAC fall within the scope of the
present invention.
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Table 1
List of exemplary genera of Actinomycetales
Acidothermus Cellulomonas Kineococcus
Actinobispora Chainia Kineosporia
Actinocorallia Clavibacter Kitasatoa
Actinokineospora Coriobacterium Kitasatosporia
Actinomadura Corynebacterium Kocuria
Actinomyces Couchioplanes Kutzneria
Actinoplanes Cryobacteriurn Kytococcus
Actinopolyspora Curtobacterium Lentzea
Actinopycnidium Dactylosporangium Luteococcus
Actinosporangim Demetria Microbacterium
Actinosynnema Dermabacter Microbispora
Aeromicrobium Dermacoccus Micrococcus
Agrococcus Dermatophilus Microellobosporia
Agromyces Dietzia Microlunatus
Ampullariella Elytrosporangium Micromonospora
Amycolata Excellospora Microsphaera
Amycolatopsis Exiguobacterium Micro raspora
Arcanobacterium Frankia Microthrix
Arthrobacter Friedmanniella Mobiluncus
Atopobium Gardnerella Mycobacterium
Aureobacterium Geodermatophilus Nesterenkonia
Bifidobacterium Glycomyces Nocardia
Blastococcus Gordona Nocardioides
Bogoriella Herbidospora Nocardiopsis
Brachybacterium Intrasporangium Oerskovia
Brevibacterium Janibacter Pelczaria
Catellatospora Jonesia Phenylobacterium
Catenuloplanes Kibdelosporangium Pilimelia
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Pimelobacter Rhodococcus Streptomyces
Planobispora Rothia Streptosporangium
Planomonospora Rubrobacter Streptoverticillium
Planopolyspora Saccharomonospora Terrabacter
Planotetaspora Saccharopolyspora Terracoccus
Prauseria Saccharothrix Thermoactinomyces
Promicromonospora Sanguibacter Thermocrispum
Propionibacterium Skermania Thermomonospora
Propioniferax Spiri11ip1anes Tropheryma
Pseudonocardia Spirillospora Tsukamurella
Rarobacter Sporichthya Turicella
Rathayibacter Stomatococcus
Renibacterium Streptoalloteichus
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7.2.2 Assemblage by homologous recombination
The bottom--up strategy of assembling large fragments from a
set of pre-existing smaller segments of partially
overlapping DNA cloned from the genome of the actinomycete
5 donor organism, is described in this section. This
methodology makes use of the same pESAC described in the
present invention under Section 7.1. The desired cluster is
assembled from existing partially overlapping clones by the
iterative use of homologous recombination in E. coli. In
10 the example of Fig. 5, three overlapping clones, designated
1, 2 and 3, and derived from the genome of a donor
organism, encompass the desired biosynthesis cluster. These
clones include leftward fragment "A" unique to clone 1;
fragment "B" common to clones 1 and 2; fragment "C" common to
15 clones 2 and 3; and rightward fragment "D" unique to clone
3. These fragments can range from a few hundred by to a few
kb, and are thus amenable to routine in vitro DNA
manipulations. The number of overlapping clones
encompassing the cluster may vary. However, if n is the
20 number of overlapping clones that cover the desired genomic
segment, the number of fragments to consider will be equal
to n + I. In the example illustrated in Fig. 5, four
fragments are required. The cluster of Fig. 5 is
reconstructed into a pESAC through the use of successive
25 rounds of homologous recombination in E. coli. Fragments A
and B are cloned in a is vector, as shown in Fig. 6, which
carries a selectable marker, CmR as exemplified in Fig. 6.
The same is done with fragment pairs B-C and C-D (Fig. 6).
The relative orientation of each fragment pair in the is
30 vector must be the same as in the gene cluster. The
fragments in each pair may be separated by a selectable
marker, designated AbR in Fig. 6, to monitor interplasmid
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insert exchange. Therefore, three constructs in the is
vector, designated pABl, pAB2 and pAB3, are required. The
A-B-C-D four-fragment cassette is cloned in a pESAC (Fig.
6). The relative orientation of the four fragments in the
pESAC must be the same as in the gene cluster. Again, a
selectable marker may separate any of two fragments to
monitor interplasmid insert exchange. The original clone
(for example, a cosmid, which carries a selectable marker,
KmR as exemplified in Fig. 7) containing part of the cluster
and the cognate is construct (Fig. 7) are introduced into
the same E. coli cell. The interplasmid cointegrate between
the original clone and the is construct is selected at the
non-permissive temperature for the is replicon. This occurs
through a single, reciprocal homologous recombination
mediated by either one of the two fragments in the A-B, B-C
or C-D pairs. The cointegrate is then resolved at the
permissive temperature, leading to insert exchange between
the two replicons (Fig. 7). The presence in the is replicon
of the genomic segment comprised between fragments A and B
can be monitored by the appearance of CmR Abs colonies. This
is done for clone 1 and pABl, resulting in pAB2; for clone
2 and pBCl, resulting in pBC2; and for clone 3 and pCDl,
resulting in pCD2. Each insert from the original
overlapping clones (Fig. 5) is thus transferred into the is
replicon, as outlined in Fig. 7. Subsequently, the inserts
from clone 1, now present in the is plasmid pAB2, is
introduced into the pESAC construct carrying the entire A-
B-C-D cassette. This is done by selecting for the
interplasmid cointegrate between pAB2 and the pESAC
construct at the non-permissive temperature, and then
resolving the cointegrate at the permissive temperature,
selecting for KmR Abs colonies. This leads to insert
exchange between the two replicons (as shown in Fig. 8).
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Next, a selectable marker is introduced in the growing ESAC
between the next fragment pair, again through the use of
two rounds of single, reciprocal homologous recombination
mediated by plasmid pBCl, leading to the appearance of KmR
AbR colonies. Subsequently, the interplasmid exchange with
pBC2 leads to the introduction of the genomic segment
comprised between fragments B and C. Finally, the use of
pCDl first and subsequently of pCD2 leads to the
reconstruction of the genomic segment into the pESAC.
Therefore, through the use of alternating steps, the AbR
marker first and the genomic segment later are introduced
between any fragment pair, as schematized in Fig. 8. This
iterative procedure results in the reconstruction of the
original chromosomal region in the pESAC.
A series of examples described herein illustrate how a
90-kb gene segment from the actinomycete P. rosea is
assembled from three pre-existing cosmids via homologous
recombination. The cosmids, designated pRPl6, pRP31 and
pRP58, are identified in a cosmid library constructed in
the vector Lorist6 by the use of selective hybridization
probes. The relevant information about the cluster is
reported in Fig. 9. The reconstruction of the cluster
results in the formation of the intermediate derivatives
pPADl, PAD2, PAD4 and PAD6, carrying inserts of 10, 39, 68
and 89 kb, respectively. The examples reported herein serve
to illustrate the principles and methodologies of the
present invention and are not meant to restrict its scope.
Example 18
Isolation of cosmid clones pRPl6, pRP31 and pRP58
A cosmid library of P, rosea DNA prepared in the vector
Lorist6 is screened with oligonucleotide probes Pep6 and
Pep8, according to the conditions described under Section
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6. Among the positive colonies identified, several cosmids
were found to span the ca. 90 kb segment of the P. rosea
chromosome reported in Fig. 9. Signature sequences close to
the left and right ends of this segment are reported as
SEQID N°9 and SEQID N°10, respectively. Three cosmids are
chosen for further studies. Cosmids pRPl6, pRP31 and pRP58
exhibits, after digestion with BamHI and resolution by
agarose gel-electrophoresis, fragments of 7.5, 7.2, 5.6,
5.2, 2.7, 2.0, 1.9, 1.9, 1.8, 1.6, 1.4, 0.9 and 0.7 kb; of
10.5, 6.2, 3.1, 2.8, 2.6, 2.5, 2.1, 1.9, 1.9, 1.5, 1.4,
1.2, 1.0, 1.0, 0.9, 0.9, 0.7, 0.6, 0.5, 0.5, 0.1 and 0.1
kb; and of 10.0, 7.6, 6.7, 6.2, 3.4, 3.0, 2.8, 2.1, 1.0,
1.0, 0.9, 0.9, 0.7, 0.6, 0.5, 0.5, 0.3 and 0.1 kb;
respectively.
Example 19
Construction of plasmid pUA1
The 0.9 kb SmaI-SstI fragment, comprised between map
coordinates 2.0-2.9 kb of Fig. 9, is obtained from cosmid
pRPl6, prepared as described in Example 18, and ligated to
pUCl8 previously digested with SstI and SmaI. The resulting
mixture contains the desired plasmid pUAl.
Example 20
Construction of E. coli K12 XLlblue/pUAl
Approximately 10 ng of plasmid pUAl, prepared as described
in Example 19, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
~is found to carry pUAl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
0.9 kb after digestion of the plasmid with BamHI + SstI.
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Example 21
Construction of plasmid pUA2
The 0.9 kb BamHI-SstI fragment from pUAl, prepared as
described in Example 20, is ligated to pUCBM20 previously
digested with BamHI and SstI. The resulting mixture
contains the desired plasmid pUA2.
Example 22
Construction of E. coli K12 XLlblue/pUA2
Approximately 10 ng of plasmid pUA2, prepared as described
in Example 21, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUA2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
0.9 kb after digestion of the plasmid with EcoRI + Sstl.
Example 23
Construction of plasmid pUBl
The 1.8 kb SstI-BamHI fragment, comprised between map
coordinates 33.4-35.2 of Fig. 9, is obtained from cosmid
pRPl6, prepared as described in Example 18, and ligated to
pUCl8 previously digested with SstI + BamHI. The ligation
mixture contains the desired plasmid pUBl.
Example 24
Construction of E. coli K12 XLlblue/pUBl
Approximately 10 ng of plasmid pUBl, prepared as described
in Example 23, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to contain plasmid pUBl as verified by the
observation, upon agarose gel electrophoresis, of fragments
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2.7 and 1.8 kb after digestion with SstI + XbaI.
Example 25
Construction of plasmid pUC1
5 The 6.2 kb BamHI fragment, comprised between map
coordinates 54.2-60.4 kb of Fig. 9, is obtained from cosmid
pRP58, prepared as described in Example 18, and ligated to
pUCl8 previously digested with BamHI. The ligation mixture
contains the desired plasmid pUCl.
Example 26
Construction of E. coli K12 XLlblue/pUCl
Approximately 10 ng of plasmid pUCl, prepared as described
in Example 25, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUCl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.9 and
4.0 kb after digestion of the plasmid with PstI.
Example 27
Construction of plasmid pUDl
Synthetic oligonucleotides 5'-GATCTAAGCTTGGGGG-3' (SEQID
N°11) and 5'-CCCCCAAGCTTA-3' (SEQID N°12) are annealed and
ligated to the 1.5 kb PstI-BamHI fragment, comprised
between map coordinates 89.5-91.0 kb of Fig. 9 and obtained
from cosmid pRP58, prepared as described in Example 18. The
ligation mixture is digested with H.indIII and ligated to
pUCl8 previously digested with PstI + HindIII. The
resulting mixture contains the desired plasmid pUDl.
Example 28
Construction of E. coli K12 XLlblue/pUDl
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Approximately 10 ng of plasmid pUDl, prepared as described
in Example 27, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to contain plasmid pUDl as verified by the
observation, upon agarose gel-electrophoresis, of fragments
of 2.7 and 1.5 kb after digestion with PstI + HindIII.
Example 29
Construction of plasmid pUABl
The 0.9 kb EcoRI-SstI fragment from plasmid pUA2, prepared
as described in Example 22, and the 1.8 kb SstI-BamHI
fragment from pUBl, prepared as described in Example 24,
are ligated to pUCl8 previously digested with EcoRI +
BamHI. The ligation mixture contains the desired plasmid
pUABl.
Example 30
Construction of E. coli K12 XLlblue/pUABl
Approximately 10 ng of plasmid pUABl, prepared as described
in Example 29, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUABl, as verified by the observation,
upon agarose gel-electrophoresis, of two fragments of 2.7
kb after digestion of the plasmid with EcoRI + XbaI.
Example 31
Isolation of the tet fragment
The 1.6 kb fragment containing the tet gene is isolated
after PCR amplification of pBR322 DNA using
oligonucleotides 5'-GAGCTCTCATGTTTGACAGCT-3'(SEQID N°13)
and 5'-GAGCTCTGACTTCCGCGTTTCCAG-3'(SEQID N°14) as primers,
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followed by digestion with SstI.
Example 32
Construction of plasmid pUAB2
Plasmid pUABl, prepared as described in Example 30, is
digested with SstI and ligated to the tet fragment prepared
as described in Example 31. The ligation mixture contains
the desired plasmid pUAB2.
Example 33
Construction of E. coli K12 DHSa/pUAB2
Approximately 10 ng of plasmid pUAB2, prepared as described
in Example 32, are used to transform E. coli DHSa, and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUAB2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.3 and
2.7 kb after digestion of the plasmid with EcoRI + XbaI.
Example 34
Construction of plasmid pUBCl
The 1.8 kb SstI-XbaI fragment obtained from plasmid pUBl,
prepared as described in Example 24, and the 4.0 kb XbaI-
PstI fragment obtained from plasmid pUCl, prepared as
described in Example 26, are ligated to pUCl8 previously
digested with SstI + PstI. The ligation mixture contains
the desired plasmid pUBCl.
Example 35
Construction of E. coli K12 XLlblue/pUBCl
Approximately 10 ng of plasmid pUBCl, prepared as described
in Example 34, are used to transform E. co.Ii XLlblue and a
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few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUBCl,- as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 5.8 and
2.7 kb after digestion of the plasmid with EcoRI + HindIII.
Example 36
Construction of plasmid pUBC2
Plasmid pUBCl, prepared as described in Example 35 and
previously digested with XbaI, and the tet fragment,
prepared as described in Example 31, are treated with T4
DNA polymerase and T4 DNA ligase. The ligation mixture
contains the desired plasmid pUBC2.
Example 37
Construction of E. coli K12 DHSa/pUBC2
Approximately 10 ng of plasmid pUBC2, prepared as described
in Example 36, are used to transform E. coli DHSa and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUBC2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 5.6 and
9.5 kb after digestion of the plasmid with HindIII.
Example 38
Construction of plasmid pUCDl
The 4.0 kb XbaI-PstI fragment obtained from plasmid pUCl,
prepared as described in Example 26, and the 1.5 kb PstI-
HindIII fragment isolated from plasmid pUDl, prepared as
described in Example 28, are ligated to pUCl8 previously
digested with XbaI and HindIII. The mixture contains the
desired plasmid pUCDl.
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Example 39
Construction of E. co.ti K12 XLlblue/pUCDl
Approximately 10 ng of plasmid pUCDl, prepared as described
S in Example 38, are used to transform E. coli XLlblue and a
few of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUCDl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 5.5 and
2.7 kb after digestion of the plasmid with XbaI + HindIII.
Example 40
Construction of plasmid pUCD2
Plasmid pUCDl, prepared as described in Example 39 and
previously digested with PstI, and the tet fragment
prepared as described in Example 31, are treated with T4
DNA polymerase and T4 DNA ligase. The ligation mixture
contains the desired plasmid pUCD2.
Example 41
Construction of E. coli K12 DHSa/.-pUCD2
Approximately 10 ng of plasmid pUCD2, prepared as described
in Example 40, are used to transform E. coli DHSa and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUCD2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 6.7 and
3.1 kb after digestion of the plasmid with HindIII.
Example 42
Construction of plasmid pUADl
The 4.3 kb EcoRI-XbaI fragment obtained from plasmid pUAB2,
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prepared as described in Example 33, and the 5.5 XbaI
HindIII fragment from plasmid pUCDl, prepared as described
in Example 39, are ligated to pUCl8, previously digested
with EcoRI + HindIII. The ligation mixture contains the
5 desired plasmid pUADI.
Example 43
Construction of E. coli K12 DHSa/-pUAD1
Approximately 10 ng of plasmid pUADl, prepared as described
10 in Example 42, are used to transform E. coli DHSa and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUADl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 8.9 and
15 3.6 kb after digestion of the plasmid with HindIII.
Example 44
Construction of plasmid pMABl
The 4.3 kb EcoRI-XbaI fragment obtained from plasmid pUAB2,
20 prepared as described in Example 33, is treated with T4 DNA
Polymerase and ligated to pMAK705 previously digested with
HincII. The ligation mixture contains the desired plasmid
pMABl.
25 Example 45
Construction of E. coli K12 C600/pMAB1
Approximately 10 ng of plasmid pMABl, prepared as described
in Example 44, are used to transform E. coli C600 and a few
of the resulting CmRTcR colonies that appear on the LB-agar
30 plates are analyzed for their plasmid content. One colony
is found to carry pMABl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.1, 3.4,
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1.4 and 0.9 kb after digestion of the plasmid with HindIII
+ EcoRI.
Example 46
S Construction of plasmid pMBC1
The 7.1 kb fragment from plasmid pUBC2, prepared as
described in Example 37, is obtained after partial
digestion with PstI, treated with T4 DNA polymerase and
ligated to pMAK?05 previously digested with HincII. The
ligation mixture contains the desired plasmid pMBCl.
Example 47
Construction of E. coli K12 C600/pMBCl
Approximately 10 ng of plasmid pMBCl, prepared as described
in Example 46, are used to transform E. coli C600 and a few
of the resulting CmRTcR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pMBCl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 9.5, 1.5,
1.3 and 0.3 kb after digestion of the plasmid with BamHI.
Example 48
Construction of plasmid pMCDl
The 7.1 kb fragment from plasmid pUCD2, prepared as
described in Example 41, is obtained by complete digestion
with XbaI and partial digestion with HindIII, treated with
T4 DNA polymerase and ligated to pMAK705, previously
digested with HincII. The ligation mixture contains the
desired plasmid pMCDl.
Example 49
Construction of E. coli K12 C600/pMCDl
Approximately 10 ng of plasmid pMCDl, prepared as described
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in Example 48, are used to transform E. coli C600 and a few
of the resulting CmRTcR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pMCDl, as verified by the observation;
upon agarose gel-electrophoresis, of fragments of 8.6 and
4.3 kb after digestion of the plasmid with BamHI.
Example 50
Construction of plasmid pPADl
The 10.0 kb EcoRI-NdeI fragment from plasmid pUADl,
prepared as described in Example 43, is ligated to pPAC-S1,
prepared as described in Example 11 and previously digested
with ScaI. The ligation mixture contains the desired
plasmid pPADl.
Example 51
Construction of E. coli K12 C600/pPADl
Approximately 10 ng of plasmid pPADl, prepared as described
in Example 50, are used to transform E'. coli C600 and a few
of the resulting KmRTck colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pPADl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 19.7,
5.8, 3.1 and 1.2 kb after digestion of the plasmid with
BamHI. After digestion with DraI and resolution by PFGE,
pPADl yields fragments of 17.4, 7.4, 4.2 and 0.6 kb.
Example 52
Construction of E. coli K12 C600/pMABI::pRPl6
E. coli C600/pMABl, prepared as described in Example 45, is
transformed with ca. 50 ng of pRPl6, prepared as described
in Example 18. The CmRKmR colonies that appear at 30 °C on
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the LB-agar plates are grown at 30°C in LB broth containing
Km and Cm, aliquots are withdrawn at various times and
appropriate dilutions plated. Few of the CmRKmR colonies
that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
Cm for 16 h at 44°C and analyzed for their plasmid content.
One colony is found to carry pMABI::pRPl6, as verified by
the observation, upon agarose gel-electrophoresis, of
fragments of 34, 10.7, 1.7, 1.6, 1.5, 1.2 and 0.6 kb after
digestion of the plasmid with EcoRI.
Example 53
Construction of E. coli K12 C600/pMAB2
Several colonies of E. coli C600/pMABI::pRPl6, prepared as
described in Example 52, are grown individually in LB broth
containing Cm for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h. Appropriate dilutions are plated. Few of
the resulting CmRKmSTcs colonies that appear at 30°C are
analyzed for their plasmid content. One colony is found to
carry pMAB2, as verified by the observation, upon agarose
gel-electrophoresis, of fragments of 37 and 1.5 kb after
digestion of the plasmid with EcoRI.
Example 54
Construction of E. coli K12 DH1/pMBCI::pRP31
Approximately 50 ng of pRP3l, prepared as described in
Example 18, are used to transform E. coli DH1 cells.
Competent cells are prepared from one of the resulting KmR
colonies and transformed with ca. 10 ng of plasmid pMBCl,
prepared as described in Example 47. The CmRKmR colonies
that appear at 30°C on the LB-agar plates are grown at 30°C
in LB broth containing Km and Cm, aliquots are withdrawn at
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various times and appropriate dilutions plated. Few of the
CmRKmR colonies that appear on the LB-agar plates after
overnight incubation at 44°C are grown in LB broth
containing Km and Cm for 16 h at 44°C and analyzed for their
plasmid content. One colony is found to carry pMBCI::pRP3l,
as verified by the observation, upon agarose gel-
electrophoresis, of fragments of 22.2, 14.1, 14.0 and 6.0
kb after digestion of the plasmid with EcoRV.
Example 55
Construction of E. coli K12 DHl/pMBC2
Several colonies of E. coli DH1/pMBCI::pRP3l, prepared as
described in Example 54, are grown individually in LB broth
containing Cm for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h. Appropriate dilutions are plated. Few of
the resulting CmRKmSTcS colonies that appear at 30°C are
analyzed for their plasmid content. One colony is found to
carry pMBC2, as verified by the observation, upon agarose
gel-electrophoresis, of fragments of 14.4, 14.1 and 1.5 kb
after digestion of the plasmid with EcoRI.
Example 56
Construction of E. coli K12 DH1/pMCDl::pRP58
Approximately 50 ng of pRP58, prepared as described in
Example 18, are used to transform E. coli DH1 cells.
Competent cells are prepared from one of the resulting KmR
colonies and transformed with ca. 10 ng of plasmid pMCDl,
prepared as described in Example 48. The CmRKmR colonies
that appear at 30°C on the LB-agar plates are grown at 30°C
in LB broth containing Km and Cm, aliquots are withdrawn at
various times and appropriate dilutions plated. Few of the
CmRKmR colonies that appear on the LB-agar plates after
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overnight incubation at 44°C are grown in LB broth
containing Km and Cm for 16 h at 44°C and analyzed for their
plasmid content. One colony is found to carry pMCDI::pRP58,
as verified by the observation, upon agarose gel-
s electrophoresis, of fragments of 39, 16, 1.7, 1.6, 1.5, 1.2
and 0.6 kb after digestion of the plasmid with EcoRI.
Example 57
Construction of E.coli K12 DH1/pMCD2
10 Several colonies of E. coli DH1/pMCDI::pRP58, prepared as
described in Example 56, are grown individually in LB broth
containing Cm for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h. Appropriate dilutions are plated: Few of
the resulting CmRKmSTcs colonies that. appear at 30°C are
15 analyzed for their plasmid content. One colony is found to
carry pMCD2, as verified by the observation, upon agarose
gel-electrophoresis, of fragments of 42 and 1.5 kb after
digestion of the plasmid with EcoRI.
20 Example 58
Construction of E. coli K12 C600/pMAB2::pPAD1
E. coli C600/pMAB2, prepared as described in Example 53, is
transformed with ca. 50 ng of plasmid pPADl, prepared as
described in Example 51. The CmRKmR colonies that appear at
25 30°C on the LB-agar plates are grown at 30°C in LB broth
containing Km and Cm, aliquots are withdrawn at various
times and appropriate dilutions plated. Few of the CmRKmR
colonies that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
30 Cm for 16 h at 44°C and analyzed for their plasmid content.
One colony is found to carry pMAB2::pPADl, as verified by
the observation, upon agarose gel-electrophoresis, of
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fragments of 19.7, 7.2, 5.6, 5.6, 5.5, 5.2, 3.1, 2.7, 1.9,
1.9, 1.8, 1.8, 1.6, 1.9, 1.2, 0.9, 0.9 and 0.7 kb after
digestion of the plasmid with BamHI.
Example 59
Construction of E.coli K12 C600/PAD2
Several colonies of E. coli C600/pMAB2::pPADl, prepared as
described in Example 58, are grown individually in LB
containing Km for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h at 44°C. Appropriate dilutions are plated.
Few of the resulting KmRCmSTcs colonies that appear at 37°C
are analyzed for their plasmid content. One colony is found
to carry PAD2, as verified by the observation, upon agarose
gel-electrophoresis, of fragments of 19.7, 7.2, 5.6, 5.5,
5.2, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 0.9 and 0.7 kb
after digestion of the plasmid with BamHI. After DraI
digestion and resolution by PFGE, PAD2 yields fragments of
45, 7.4, 4.2 and 0.6 kb.
Example 60
Construction of plasmid pMCD3
The 1.4 kb KpnI-XhoII fragment obtained from plasmid
pCYPAC2 after digestion with XhoII, treatment with T4 DNA
polymerase and digestion with KpnI, and the 7.1 kb XbaI-
HindIII fragment from pUCD2, prepared as described in
Example 40 and obtained after partial digestion with
HindIII, complete digestion with XbaI and treatment with T4
DNA polymerase, are ligated to pMAK705, previously digested
with KpnI + HincII. The ligation mixture contains the
desired plasmid pMCD3.
Example 61
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Construction of E. coli K12 C600/pMCD3
Approximately 10 ng of plasmid pMCD3, prepared as described
in Example 60, are used to transform E, coli C600 and a few
of the resulting CmRTcR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pMCD3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 9.8 and
4.3 kb after digestion of the plasmid with BamHI.
Example 62
Construction of E. coli K12 C600/PAD2::pMCD3
E. coli C600/PAD2, prepared as described in Example 59, is
transformed with ca. 10 ng of plasmid pMCD3, prepared as
described in Example 61. The CmRKmR colonies that appear at
30°C on the LB-agar plates are grown at 30°C in LB broth
containing Km and Cm, aliquots are withdrawn at various
times and appropriate dilutions plated. Few of the CmRKmR
colonies that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
Cm for 16 h at 44°C and analyzed for their plasmid content.
One colony is found to carry PAD2::pMCD3, as verified by
the observation, upon agarose gel-electrophoresis, of
fragments of 19.7, 9.8, 7.2, 5.6, 5.5, 5.2, 4.3, 2.7, 1.9,
1.9, 1.8, 1.8, 1.6, 1.4, 0.9 and 0.7 kb after digestion of
the plasmid with BamHI.
Example 63
Construction of E. co~i K12 C600/PAD3
Several colonies of E. coli C600/PAD2::pMCD3, prepared as
described in Example 62, are grown individually in LB broth
containing Km for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h at 44°C. Appropriate dilutions are plated.
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Few of the resulting KmRCmSTcR colonies are analyzed for
their plasmid content. One colony is found to carry PAD3,
as verified by the observation, upon agarose gel-
electrophoresis, of fragments of 21.5, 7.2, 5.6, 5.2, 4.3-,
2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 0.9 and 0.7 kb after
digestion of the plasmid with BamHI.
Example 64
Construction of E. coli K12 C600/PAD3::pMCD2
E. coli C600/PAD3, prepared as described in Example 63, is
transformed with ca. 50 ng of plasmi.d pMCD2, prepared as
described in Example 57. The CmRKmR colonies that appear at
30°C on the LB-agar plates are grown at 30°C in LB broth
containing Km and Cm, aliquots are withdrawn at various
times and appropriate dilutions plated. Few of the CmRKmR
colonies that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
Cm for 16 h at 44°C and analyzed for their plasmid content.
One colony is found to carry PAD3::pMCD2, as verified by
the observation, upon agarose gel-electrophoresis, of
fragments of 21.5, 10, 9,0, 7.6, 7.2, 6.2, 5.6, 5.2, 4.3,
3.1, 2.8, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 1.0, 0.9, 0.9,
0.9, 0.7, 0.5, 0.3 and 0.1 kb after digestion of the
plasmid with BamHI.
Example 65
Construction of E. coli K12 C600/PAD4
Several colonies of E. coli C600/PAD3::pMCD2, prepared as
described in Example 64, are grown individually in LB broth
containing Km for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h at 44°C. Appropriate dilutions are plated.
Few of the resulting KmRCmSTcs colonies are analyzed for
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their plasmid content. One colony is found to carry PAD4,
as verified by the observation, upon agarose gel-
electrophoresis, of fragments of 22, 10, 7.6, 7.2, 6.2,
5.6, 5.2, 3.1, 2.8, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 1.0;
0.9, 0.9, 0.9, 0.7, 0.5, 0.3 and 0.1 kb after digestion of
the plasmid with BamHI. After DraI digestion and resolution
by PFGE, PAD4 yields fragments of 79, 4.2 and 0.6 kb.
Example 66
Construction of E. coli K12 C600/PAD4::pMBC1
E. coli C600/PAD4, prepared as described in Example 65, is
transformed with ca. 10 ng of plasmid pMBCl, prepared as
described in Example 47. The CmRKmR colonies that appear at
30°C on the LB-agar plates are grown at 30°C in LB broth
containing Km and Cm, aliquots are withdrawn at various
times and appropriate dilutions plated. Few of the CmRKmR
colonies that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
Cm for 16 h at 44°C and analyzed for their plasmid content.
One colony is found to carry PAD4::pMBCl, as verified by
the observation, upon agarose gel-electrophoresis, of
fragments of 22, 10, 9.6, 7.6, 7.2, 6.2, 5.6, 5.2, 3.1,
2.8, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.5, 1.4, 1.3, 1.0, 0.9,
0.9, 0.9, 0.7, 0.5, 0.3, 0.3 and 0.1 kb after digestion of
the plasmid with BamHI.
Example 67
Construction of E. coli K12 C600/PAD5
Several colonies of E. coli C600/PAD4::pMBCl, prepared as
described in Example 66, are grown individually in LB broth
containing Km for 24 h at 30°C, diluted 1:100 and incubated
for further 8 h at 44°C. Appropriate dilutions are plated.
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Few of the resulting KmR Cms TcR colonies are analyzed for
their plasmid content. One colony is found to carry PAD5,
as verified by the observation, upon agarose gel-
electrophoresis, of fragments of 22, 10, 7.6, 7.2, 6.2;
5 5.6, 5.2, 3.1, 2.8, 2.7, 1.9, 1.9, 1.8, 1.8, 1.6, 1.4, 1.3,
1.0, 0.9, 0.9, 0.9, 0.7, 0.5, 0.3, 0.3 and 0.1 kb after
digestion of the plasmid with BamHI.
Example 68
10 Construction of E. coli K12 C600/PAD5::pMBC2
E. coli C600/PAD5, prepared as described in Example 67, is
transformed with ca. 50 ng of plasmid pMBC2, prepared as
described in Example 55. The CmRKmR colonies that appear at
30°C on the LB-agar plates are grown at 30°C in LB broth
15 containing Km and Cm, aliquots are withdrawn at various
times and appropriate dilutions plated. Few of the CmRKmR
colonies that appear on the LB-agar plates after overnight
incubation at 44°C are grown in LB broth containing Km and
Cm for 16 h at 44°C and analyzed for their plasmid content.
20 One colony is found to carry PADS::pMBC2, as verified by
the observation, upon agarose gel-electrophoresis, of
fragments of 65, 33, 5.6, 4.7, 3.4, 2.8, 2.1, 1.2, 1.2, 1.0
and 0.4 kb after digestion of the plasmid with HindIII.
25 Example 69
Construction of E. coli K12 C600/PAD6
Several colonies of E. coli C600/PAD5::pMBC2, prepared as
described in Example 68, are grown individually in LB broth
containing Km for 24 h at 30°C, diluted 1:100 and incubated
30 for further 8 h at 44°C. Appropriate dilutions are plated.
Few of the resulting KmRCmSTcS colonies are analyzed for
their plasmid content. One colony is found to carry the
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correct ESAC, designated PAD6, as verified by the
observation, upon agarose gel-electrophoresis, of fragments
of 47, 46, 8.1, 4.6, 2.2, 0.5 and 0.1 kb after digestion of
the plasmid with EcoRI. After digestion with DraI arid
resolution by PFGE, PAD6 yields fragments of 102, 4.2 and
0.6 kb.
Although the present invention is described in the
Examples listed above in terms of preferred embodiments,
they are not to be regarded as limiting the scope of the
invention. The above Examples serve to illustrate the
principles and methodologies for assembling pre-existing
overlapping segments of DNA into pESAC.
It will occur to those skilled in the art that the
cluster of Fig. 9 can be assembled using A-B-C-D fragments
other than those specified in the Examples. Any A fragment,
such that no useful genes are present to its left (using
the orientation of Fig. 9) can be used for assembling the
cluster. Similarly, any D fragment, such that no useful
genes are present to its right (using the orientation of
Fig. 9) can also be used. Furthermore, any fragment common
to pRPl6 and pRP3l, or to pRP31 and pRP58, can be used in
place of the fragments B and C, respectively, described
above. It will also occur to those skilled in the art that
other methods for obtaining these fragments, such as use of
different segments from the cluster of Fig. 9, of different
restriction endonucleases, or of the PCR, can be used for
achieving equivalent results. In addition, intermediate
vectors, other than the pUC- series used in the above
Examples, can be used for subcloning fragments A through D,
and the use of these intermediate vectors is merely
instrumental to the transfer of the fragment pairs into the
is vector. Some or all of the fragment pairs could
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therefore be cloned directly into a is vector.
It will also occur to those skilled in the art that
cosmids other than pRPl6, pRP31 and pRP58 can be used to
achieve equivalent results, provided that they encompass
the entire gene cluster and they have overlapping segments.
It will also occur to those skilled in the art that
pMAK705, Lorist6 and pPAC-S1, are merely examples of ts,
cosmid and pESAC, respectively. Any of the several cosmid
vectors described in the literature, other is replicons
derived from pMAK705 or other sources, and any of the pESAC
other than pPAC-Sl, which include the possible vectors
described in Section 7.1, can be used for obtaining
equivalent results.
Those skilled in the art understand that the purpose
of a is replicon is to select for interplasmid cointegrates
at the non-permissive temperature. However, cointegrate
formation can occur between any two replicons, and
cointegrate can be isolated after transformation of
suitable hosts with a plasmid preparation made from an E.
coli cell harboring both replicons. Selection for the
antibiotic resistance markers carried by both replicons can
lead to the isolation of cointegrates from the resulting
transformants.
Furthermore, it will occur to those skilled in the art
that the inclusion of the tet marker between the A-B, B-C
and C-D fragment pairs serves solely the scope of
recognizing insert exchange after resolution of the
interplasmid cointegrate. Selectable markers other than tet
can be equally effective, as long as they are not present
in the vectors. Those skilled in the art understand that
the presence of a selectable marker within the fragment
cassettes is not absolutely necessary, as insert exchange
can be observed by other methods, such as selective
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hybridization or PCR. Similarly, different E. coli hosts
other than those used in the above Examples can be also
employed.
It will also occur to those skilled in the art that,
as described in Examples 58 through 69, interplasmid insert
exchange can be obtained in a sequel independent of the
order of the overlapping cosmid clones in the genomic
contig. Indeed, the schematic of Fig. 8 illustrates the
sequel of interplasmid exchanges A-B, followed by B-C and
then by C-D, while Examples 58 through 69 describe the
sequel A-B, C-D and last B-C. Furthermore, technical
variations on the methodologies employed here can produced
equivalent results. All these variations fall within the
scope of the present invention.
It will occur to those skilled in the art that the
principles and methodologies described in Sections 7.2.1
and 7.2.2 are not mutually exclusive. For example, a
construct equivalent to PAD6 can be directly isolated by
subjecting the producer strain P. rosea to the principles
and methodologies described in Section 7.2.1. Similarly,
selected cosmids from the described S, coelicolor library
(Redenbach et al., 1996, Mol. Microbiol. 21:77-96) can be
used for assembling a large chromosomal segment into pPAC-
S1, following the principles and methodologies described in
Section 7.2.2. Furthermore, it will occur to those skilled
in the art that the principles and methodologies of Section
7.2.1 and 7.2.2 can complement each other. For example,
after constructing an ESAC library of P. rosea DNA, inserts
from individual ESACs may be enlarged by applying the
principles and methodologies of Section 7.2.2, using, for
example, cosmids overlapping the cognate ESACs.
Those skilled in the art understand that the
principles and methodologies described in Section 7.2.2 and
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illustrated in schematic form in Fig. 4 are general enough
that they can be applied to other strains and clusters.
Methods for preparing high molecular weight DNA, for
constructing and propagating in E. co~i an ESAC library can
be developed from the principles and methodologies
described in Examples 12 through 17. Methods for preparing
the appropriate combinations of fragment pairs to yield the
starting plasmids described in Fig. 6, can be developed for
other clusters following the principles and methodologies
described in Examples 19 through 51; methods for assembling
an entire cluster into a pESAC can be developed following
the principles and methodologies described in Examples 52
through 69. In order to illustrate how the principles and
methodologies described in Section 7.2 can be extended to
other actinomycete strains producing different natural
products, the constructions of ESACs carrying large gene
clusters from different producer strains are reported
herein. The Examples describe, for each cluster, the
selection of the appropriate fragments A, B, C and D; and
the construction of the starting plasmids, equivalent to
those reported in Fig. 6. These plasmids can then be used
to to reassemble each cluster according to the scheme of
Fig. 8.
The rapamycin gene cluster from S. hygroscopicus is
contained within three overlapping cosmids designated
cos58, cos25 and cost (Schwecke et al., 1995, Proc. Natl.
Acad. Sci. USA 92:7839-7843). The Examples described herein
report the preparation of the appropriate fragments A, B, C
and D; the construction of the plasmids containing the A-B,
B-C and C-D cassettes; and the cloning approach to obtain
constrcuts equivalent to those reported in Fig. 6.
Example 70
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Preparation of the rapamycin fragments A, B, C and D
Primers 5'-TTTTTGAATTCGGTACCAGCCGACGGCGA-3' (SEQID N°15)
and 5'-TTTTTGGATCCCTGTTCCACCAGCGCACC-3'(SEQID 16) are used
to amplify a 1.2 kb fragment from cos58; primers 5'-
5 TTTTTTCTAGACCGTCGTCGGTGGTTCTC-3'(SEQID N° 17) and 5'-
TTTTTGGATCCAGGAAGCCCTGTGCTGTC-3'(SEQID N°18) a 1.2 kb
fragment from cos58; primers 5'-
TTTTTTGTAGAGGTCAAGATCCGGGGCAT-3' (SEQID N°19) and 5'-
TTTTTCTGCAGGACAGCGCCCTTGAGGTG-3' (SEQID N°20) a 1.2 kb
10 fragment from cos25; and primers 5'-TTT-
TTCTGCAGGCGACGAAGAGGGGC-3' (SEQID N°21) and 5'-
TTTTTAAGCTTAGCGCGACCGGGGCGGT-3'(SEQID N°22) a 0.9 kb
fragment from cost. Fragment A, B, C and D are then
digested with EcoRI + BamHI, BamHI + XbaI, XbaI + PstI, and
15 PstI + HindIII, respectively.
Example 71
Construction of plasmid pURl
Fragments A and B, prepared as described in Example 70, are
20 ligated to pUCl8 digested with EcoRI + XbaI. The resulting
mixture contains the desired plasmid pURl.
Example 72
Construction of E. coli K12 DH1/pURl
25 Approximately 10 ng of plasmid pURl, prepared as described
in Example 71, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pURl, as verified by the observation,
30 upon agarose gel-electrophoresis, of fragments of 2.7 and
2.4 kb after digestion of the plasmid with Ecol~,I + XbaI.
Example 73
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Construction of plasmid pUR2
Fragments B and C, prepared as described in Example 70, are
ligated to pUCl8 digested with BamHI + PstI. The resulting
mixture contains the desired plasmid ptJR2.
Example 74
Construction of E. coli K12 DH1/pUR2
Approximately 10 ng of plasmid pUR2, prepared as described
in Example 73, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUR2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
2.4 kb after digestion of the plasmid with BamHI + Pstl.
Example 75
Construction of plasmid pUR3
Fragments C and D, prepared as described in Example 70, are
ligated to pUCl8 digested with XbaI + HindIII. The
resulting mixture contains the desired plasmid pUR3.
Example 76
Construction of E. coli K12 DH1/pUR3
Approximately 10 ng of plasmid pUR3, prepared as described
in Example 75, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUR3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
2.1 kb after digestion of the plasmid with EcoRI + HindIII.
Example 77
Construction of plasmid pURll
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Plasmid pURl, prepared as described in Example 72 and
previously digested with BamHI, and the tet fragment,
prepared as described in Example 31, are treated with T4
DNA Polymerase and DNA ligase. The ligation mixture
contains the desired plasmid pURll.
Example 78
Construction of E. coli K12 DHl/pURll
Approximately 10 ng of plasmid pURll, prepared as described
in Example 77, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pURll, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.9 and
2.8 kb after digestion of the plasmid with HindIII.
Example 79
Construction of the plasmid pUR21
Plasmid pUR2, prepared as described in Example 74 and
previously digested with XbaI, and the tet fragment,
prepared as described in Example 31, are treated with T4
DNA Polymerase and DNA ligase. The ligation mixture
contains the desired plasmid pUR2l.
Example 80
Construction of E. coli K12 DH1/pUR21
Approximately 10 ng of plasmid pUR2l, prepared as described
in Example 79, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUR2l, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.9 and
2.8 kb after digestion of the plasmid with HindIII.
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Example 81
Construction of the plasmid pUR31
Plasmid pUR3, prepared as described in Example 76 and
digested with PstI, and the tet fragment, prepared as
described in Example 31, are treated with T4 DNA Polymerase
and DNA ligase. The ligation mixture contains the desired
plasmid pUR3l.
Example 82
Construction of E. coli K12 DH1/pUR31
Approximately 10 ng of plasmid pUR3l, prepared as described
in Example 81, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUR3l, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.9 and
2.5 kb after digestion of the plasmid with HindIII.
Example 83
Construction of plasmid pURl3
The 4.0 kb EcoRI-XbaI fragment obtained from plasmid pURll,
prepared as described in Example 78, and the 2.1 kb XbaI-
HindIII fragment obtained from plasmid pUR3, prepared as
described in Example 76, are ligated to pUCl8 digested with
EcoRI + HindIII. The ligation mixture contains the desired
plasmid pURl3.
Example 84
Construction of E. coli K12 DH1/pURl3
Approximately 10 ng of plasmid pURl3, prepared as described
in Example 83, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
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plates are analyzed for their plasmid content. One colony
is found to carry pURl3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.9 and
3.9 kb after digestion of the plasmid with HindIII.
Those of ordinary skill in the art understand that the
plasmids constructed above can be used for transfering the
two-fragment cassettes present in pURll, pUR21 and pUR31
into a is vector. This can be achieved by recovering the
4.0 kb insert from pURll, the 4.0 kb ~_nsert from pUR2l, and
the 3.7 kb insert from pUR3l, after digestion with EcoRI +
XbaI, EcoRI + PstI, and EcoRI + NdeI, respectively.
Similarly, those of ordinary skill in the art understand
that the 6.1 kb four-fragment cassette present in plasmid
pURl3 can be easily transfered into pPAC-S1 after digestion
with EcoRI + NdeI. These subcloning experiments lead to the
formation of plasmids equivalent to those reported in Fig.
6.
As another application of the principles and
methodologies of the present invention, the Examples
reported below describe the preparation of the appropriate
fragments A, B, C and D from the Sac. erythraea
erythromycin gene cluster. This cluster has been described
and is contained within a series of overlapping clones
(Tuan et al . , 1990, Gene 90:21-29; Donadio et al . , 1993, In
Industrial Microorganisms: Basics and Applied Genetics,
Baltz, Hegeman and Skatrud eds., ASM, Washington, DC,
pp.257-265; Pereda et al., 1997, Gene 193:65-71). The
construction of the plasmids containing the A-B, B-C and C-
D cassettes and the cloning approach to obtain constructs
equivalent to those reported in Fig. 6 are also described.
Example 85
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Preparation of the erythromycin fragment A
Synthetic oligonucleotides 5'-CATGGGAATTCGGGGG-3' (SEQID
N°23) and 5'-CCCCCGAATTCC-3' (SEQID N°24) are annealed and
ligated to the 1.2 kb NcoI-BamHI fragment isolated from
5 cosmid p3B2. The resulting mixture is digested with EcoRI +
BamHI.
Example 86
Preparation of the erythromycin fragments B, C and D
10 Primers 5'-TTTTTGGATCCGGGGCAGCGGTTGGTTCC-3' (SEQID N°25}
and 5'-TTTTTTCTAGAAGGCAGCTCCAGATGATC-3' (SEQID N°26) are
used to amplify a 1.0 kb fragment from cosmid p3B2; primers
5'-TTTTTCTAGACCGGACTCGGCCGGCTCG-3'(SEQID N°27} and 5'-
TTTTTCTGCAGCCGCACGCCTCGGTGGTC-3' (SEQID N°28} a 1.1 kb
15 fragment from cosmid pSl; and primers 5'
TTTTTCTGCAGGGACCCTGAGTGCAGGTG-3' (SEQID N°29) and 5'
TTTTTAAGCTTCAGTAGCCGTCGCTGAGC-3'(SEQID N°30) a 1.1 kb
fragment from plasmid pEB6. Fragments B, C and D are then
digested with BamHI + XbaI, XbaI + PstI, and PstI +
20 HindIII, respectively.
Example 87
Construction of plasmid pUEl
Fragment A, prepared as described in Example 85, and
25 fragment B, prepared as described in Example 86, are
ligated to pUCl8 digested with EcoRI + XbaI. The resulting
mixture contains the desired plasmid pUEl.
Example 88
30 Construction of E. coli K12 DH1/pUEl
Approximately 10 ng of plasmid pUEl, prepared as described
in Example 87, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
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plates are analyzed for their plasmid content. One colony
is found to carry pURl, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
2.2 kb after digestion of the plasmid with EcoRI + Xbal.
Example 89
Construction of plasmid pUE2
Fragments B and C, prepared as described in Example 86, are
ligated to pUCl8 digested with BamHI + PstI. The resulting
mixture contains the desired plasmid pUE2.
Example 90
Construction of E. coli K12 DHl/pUE2
Approximately 10 ng of plasmid pUE2, prepared as described
in Example 89, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUE2, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
2.1 kb after digestion of the plasmid with BamHI + PstI.
Example 91
Construction of plasmid pUE3
Fragments C and D, prepared as described in Example 86, are
ligated to pUCl8 digested with XbaI + HindIII. The
resulting mixture contains the desired plasmid pUE3.
Example 92
Construction of E. coli K12 DH1/pUE3
Approximately 10 ng of plasmid pUE3, prepared as described
in Example 91, are used to transform E. coli DH1 and a few
of the resulting ApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
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is found to carry pUE3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 2.7 and
2.2 kb after digestion of the plasmid with EcoRI + HindIII.
Example 93
Construction of plasmid pUEl1
Plasmid pUEl, prepared as described in Example 88 and
previously digested with BamHI, and the tet fragment,
prepared as described in Example 31, are treated with T4
DNA Polymerase and DNA lipase. The ligation mixture
contains the desired plasmid pUEll.
Example 94
Construction of E. coli K12 DH1/pUEll
Approximately 10 ng of plasmid pUEll, prepared as described
in Example 93, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUEll, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.9 and
2.6 kb after digestion of the plasmid with HindIII.
Example 95
Construction of the plasmid pUE21
Plasmid pUE2, prepared as described in Example 90 and
previously digested with XbaI, and the tet fragment,
prepared as described in Example 31, are treated with T4
DNA Polymerase and DNA lipase. The ligation mixture
contains the desired plasmid pUE2l.
Example 96
Construction of E. coli K12 DH1/pUE21
Approximately 10 ng of plasmid pUE2l, prepared as described
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in Example 95, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUE2l, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.7 and
2.7 kb after digestion of the plasmid with HindIII.
Example 97
Construction of the plasmid pUE31
Plasmid pUE3, prepared as described in Example 92 and
digested with PstI, and the tet fragment, prepared as
described in Example 31, are treated with T4 DNA Polymerase
and DNA ligase. The ligation mixture contains the desired
plasmid pUE3l.
Example 98
Construction of E. coli K12 DH1/pUE31
Approximately 10 ng of plasmid pUE3l, prepared as described
in Example 97, are used to transform E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUE3l, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 3.8 and
2.7 kb after digestion of the plasmid with HindIII.
Example 99
Construction of plasmid pUEl3
The 3.8 kb EcoRI-XbaI fragment obtained from plasmid pUEll,
prepared as described in Example 94, and the 2.2 kb Xbal
HindIII fragment obtained from plasmid pUE3, prepared as
described in Example 92, are ligated to pUCl8 digested with
EcoRI + HindIII. The ligation mixture contains the desired
' plasmid pUEl3.
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Example 100
Construction of E. coli K12 DH1/pUEl3
Approximately 10 ng of plasmid pUEl3, prepared as described
in Example 99, are used to transform .E. coli DH1 and a few
of the resulting TcRApR colonies that appear on the LB-agar
plates are analyzed for their plasmid content. One colony
is found to carry pUEl3, as verified by the observation,
upon agarose gel-electrophoresis, of fragments of 4.8 and
3.9 kb after digestion of the plasmid with HindIII.
Those of ordinary skill in the art understand
that the plasmids constructed above can be used for
transfering the two-fragment cassettes present in pUEll,
pUE21 and pUE31 into a is vector. This can be achieved by
recovering the 3.8 kb insert from pUEll, the 3.7 kb insert
from pUE2l, and the 3.8 kb insert from pUE3l, after
digestion with EcoRI + X.bal, EcoRI + PstI, and EcoRI +
NdeI, respectively. Similarly, those of ordinary skill in
the art understand that the 6.0 kb four-fragment cassette
present in plasmid pUEl3 can be easily transfered into
pPAC-S1 after digestion with EcoRI + NdeI. These subcloning
experiments lead to the formation of plasmids equivalent to
those reported in Fig. 6.
The Examples reported above describe the principle and
methodologies for assembling the erythromycin gene cluster
into the pPAC-S1. It will occur to those skilled in the art
that the principles and methodologies illustrated in Fig. 7
and described in Examples 52 through 69 can be applied to
the erythromycin gene cluster, employing the pMAK705
derivatives constructed according to the principles
described above and the eryhtomycin cosmids.
As a further example, the preparation of the
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appropriate fragments A, B, C and D from the A.
mediterranei rifamycin gene cluster is illustrated below.
This cluster has been described and is contained within a
series of overlapping clones (August et al., 1998, Chem.
5 Biol. 5:69-79).
Example 101
Preparation of the rifamycin fragments A, B, C and D
Primers 5'-TTTTTGAATTCTGCAGACCGCCGAGGAAG-3' (SEQID N°31)
10 and 5'-TTTTTGGATCCGGAGTCGTAGCTGACGAC-3' (SEQID N°32); 5'-
TTTTGGATCCCGACCACGCGGGGACGTC-3' (SEQID N°33) and 5'-
TTTTTTCTAGACCAGGGAACCCGTGCTGC-3'(SEQID N°34); 5' -
TTTTTTCTAGACGGAAGCTCGCCGCGATC-3' (SEQID N°35) and 5'-
TTTTTCTGCAGGTCCGTAGCCCGGACACC-3'(SEQID N°36); and 5'-
15 TTTTTCTGCAGTTCGGGCGACAGTTCCTT-3' (SEQID N°37) and 5'-
TTTTTAAGCTTCAACAAGCCATCCGGGTC-3' (SEQID N°38), are used to
amplify fragments of 1.2, 1.2, 1.2 and 1.0 kb,
respectively, from A. mediterranei genomic DNA. Fragments
A, B, C and D are then digested with EcoRI + BamHI, BamHI +
20 XbaI, XbaI + PstI, and PstI + HindIII, respectively.
Those of ordinary skills in the art understa nd that
the fragments generated from the rifamycin gene cluster
contain the same restriction sites as those generated from
25 the rapamycin and erythromycin gene clusters, so that the
same cloning strategies for generating the pUCl8
derivatives containing the A-B, B-C and C-D cassettes,
described above in Examples 72-77 for the rapamycin cluster
and 88-93 for the erythromycin cluster, can also be applied
30 to the rifamycin cluster. In addition, the rifamycin
fragments A, B, C and D have been selected so that the same
cloning methodologies described above for inserting tet
within the A-B, B-C and C-D cassettes from the rapamycin
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and erythromycin clusters, described in Examples 78-83 and
94-99, respectively, can be applied in this instance as
well. Furthermore, the construction of the four-fragment
cassette can also make use of the same cloning strategy.
S Therefore, following the same principles and methodologies
described in detail for the rapamycin and erythromycin
clusters in Examples 72-85 and 88-101, respectively,
plasmids equivalent to those reported in Fig. 6 can be
constructed for assembling the rifamycin cluster into the
pPAC-S1. It will occur to those skilled in the art that the
principles and methodologies illustrated in Fig. 7 and
described in Examples 52-69 for the P. rosea cluster can be
applied to any gene cluster, once the appropriate pMAK705
derivatives have been constructed, employing available
overlapping clones.
Thus, as schematized in Fig. 7, interplasmid insert
exchange can be conducted between any plasmid containing
the desired region and the cognate is construct.,Plasmids
corresponding to pAB2, pBC2 and pCD2 can therefore be
derived from any cluster. Similarly, the principles and
methodologies illustrated in Fig. 8 can be applied
employing the appropriate A-B-C-D cassette and the cognate
pMAK705 derivatives prepared according to the scheme of
Fig. 7. The principles and methodologies illustrated in
Fig. 7 and Fig. 8 and described in Examples 52-69 can
therefore be extended to other clusters.
It will occur to those skilled in the art that,
although illustrated in Fig. 5 through 8 by three
overlapping clones and described in the Examples 58 through
69 by the use of five rounds of interplasmid insert
exchange, the principles and methodologies described in
this section of the present invention can be extended to a
different number of overlapping clones. If n is the number
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of overlapping clones that encompass the desired genomic
segment, n will also be the number of homologous
recombination rounds that introduce cluster DNA into the
pESAC. If an AbR marker is used to facilitate monitoring
insert exchange, the total number of rounds of homologous
recombination will be equal to 2r~ - 1. Interplasmid
homologous recombination has been described to introduce
large DNA segments into a desired vector (0'Connor et al.,
1989, Science 244:1307-1312; Kao et al., 1999, Science
265:509-512) or to target a smaller segment into a large
episome (Yang et a., 1997, Nature Biotechnol. 15:859-865).
However, it was not be anticipated that these procedures
could be applied iteratively for the precise reconstruction
of very large DNA segments.
7.3 Identification of positive clones
The principles and methodologies described in Section 7.2
for obtaining an entire gene cluster in a pESAC rely on the
identification of the desired genomic segment. When using
the principles and methodologies described in Section
7.2.1, the desired clones are identified by screening an
ESAC library with one of the possible strategies described
below. When using the principles and methodologies
described in Section 7.2.2, the desired clones are
identified in a genomic library, such as a cosmid library,
with one of the possible strategies described below, and
then assembled into pESAC. The principles and methodologies
for identifying the genes responsible for the biosynthesis
of natural products are well described in the literature
and are reported here solely to illustrate the fact that
they represent a necessary step in the overall scope of the
present invention.
The genes involved in the biosynthesis of natural
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products in actinomycetes are invariably found as gene
clusters in the chromosome of the producing organism, often
associated with one or more resistance determinants.
Consequently, identifying one gene allows ready access to
all the others. One or more genes responsible for the
biosynthesis of a natural product could have been
described, or the entire cluster could be known. Several
biosynthesis clusters from actinomycetes have been reported
and other clusters are likely to be described in the
future. Suitable probes from the cluster extremities can be
derived from published clusters, when available. Thus,
fragments A and D, described in Example 70, can be used as
probes to screen an ESAC library prepared from S.
hygroscopicus DNA. ESACs positive to both probes will
contain the rapamycin cluster. Similar strategies can be
applied to ESAC libraries prepared from Sac. erythraea and
A. mediterranei DNA, screened with fragments A and D,
prepared as described in Examples 85-86 and 101,
respectively.
If no biosynthesis genes are known, different
strategies for identifying them can be applied. These
strategies are well described in the literature and are
summarized below. One possible strategy involves the
isolation of the resistance genes) after cloning in a
heterologous host that is sensitive to that natural product
(for example, Stanzak et al., 1986, Bio/Technol. 4:229-
232). Another possible strategy is based on reverse
genetics: a particular biosynthetic enzyme is purified, and
from its partial protein sequences) the corresponding gene
is isolated via PCR or hybridization (for example, Fishrnan
et al., 1987, Proc. Natl. Acad. Sci. USA 84:8248-8252).
Another approach relies on the complementation of mutants
blocked in one or more biosynthesis steps, after
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introduction of a DNA library constructed in a suitable
vector into the wild type strain (for example, Malpartida
and Hopwood, 1984, Nature 309:462-464). Another approach
involves the construction of an expression library in a
suitable vector in an appropriate host, where the gene
product is sought after using specific antibodies or
looking for a particular enzymatic activity (for example,
Jones and Hopwood, 1984, J. Biol. Chem. 259:14151-14157}.
Another possible approach makes use of heterologous probes
derived from biosynthesis, resistance or regulatory genes.
Natural products can be broadly grouped into classes
according to their biosynthetic origin, and for many of
them suitable probes are available. For example, genes
encoding aromatic or modular polyketide synthases can be
effectively identified through the use of heterologous
hybridization probes (Malpartida et al., 1987, Nature
325:818-821; Schwecke et al., 1995, Proc. Natl. Acad. Sci.
USA 92:7839-7843); suitable probes have been reported for
peptide synthetase genes (Turgay and Marahiel, 1994, Pept.
Res. 7:238-241); for genes involved in the formation or
attachment of modified sugars (Decker et al., 1996, FEMS
Microbiol. Lett. 141:195-201). As the understanding of the
genetics of natural product biosynthesis increases, other
heterologous probes will become available.
The size of clusters can be estimated from those of
known clusters involved in the synthesis of structurally
similar natural products. For examples, synthesis of
macrolides is expected to require clusters in the 60-70 kb
range (Katz and Donadio, 1993, Annu. Rev. Microbiol.
47:875-912; Kuhstoss et al., 1996, Gene 183:231-236};
synthesis of glycopeptides, clusters in the 70 kb range
(van Wageningen et al., 1998, Chem. Biol. 5:155-162). In
instances where no clusters have been described for the
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same structural class of natural products, the size of the
relevant cluster can be estimated from considerations about
its known or likely biosynthesis route. Once the desired
cluster has been identified, its extent can be established
5 by analysis of the DNA sequence of the cloned cluster or of
parts thereof. Comparison of the DNA sequence to databases
can allow the identification of the likely borders of the
gene cluster.
Employing the above mentioned approaches, the desired
IO gene cluster can be identified in any library. If an ESAC
library is used, the identified cluster is ready for
transfer into the production host. If a smaller fragment
library is employed, the cluster can be assembled into a
pESAC.
15 Those skilled in the art understand that, when an ESAC
library from a donor organism is constructed, any ESAC can
be selected from said library and transferred into a
production host. Therefore, a single donor organism can be
utilized as the source of several biosynthesis clusters
20 that can be mobilized into a production host. Similarly, an
ESAC library needs not be constructed from a single donor
organism.
7.4 Transformation of a Streptomyces host
25 Once the desired gene cluster has been introduced into a
pESAC, one or more ESACs are introduced into a suitable
Streptomyces host. This is accomplished by employing
published procedures for transformation of Streptomyces.
Only minor modifications from established procedures
30 (Hopwood et al., 1985, Genetic Manipulation of
Streptomyces: A .Laboratory Manual, The John Innes
Foundation, Norwich, UK) are required for obtaining a
sufficient number of transformants. Because transformations
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are performed with single, purified ESACs, transformation
efficiencies do not need to be particularly high. The
Examples reported below illustrate the principles and
methodologies for introducing ESACs into S. lividans. They
serve to describe the present invention and are not meant
to restrict its scope. Streptomyces transformants are
selected for ThR, specified by the tsr marker present in the
pESAC. Since the incoming DNA is incapable of self-
replication in Streptomyces, site-specific integration
occurs at the chromosomal attB site, mediated by the int-
attP function specified by the pESAC. That integration has
occurred at the proper site can be verified by Southern
hybridization or by PFGE analysis of the transformants.
Fig. 10 illustrates a PFGE separation of a S. lividans
derivative carrying an ESAC with a 70 kb insert integrated
into its chromosome.
Example 102
Introduction of ESACs into S. lividans ZX7
A few hundred ng of three individual ESACs, prepared as
described in Example 17 and carrying inserts of S.
coelicolor DNA of 70, 120 and 140 kb (designated ESAC-70,
ESAC-120, and ESAC-140, respectively), are used to
transform protoplasts of S. lividans ZX7. The colonies that
appear on the R2YE plates, after overlaying with Th, are
analyzed for their ThR by streaking them on fresh R2YE
plates.
Example 103
Cultivation and preservation of S. lividans ZX7/ESAC
Individual colonies of S. lividans ZX7 transformants with
the individual ESACs, prepared as described in Example 102,
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are grown for several passages in solid medium without and
with Th. Spore suspension, or mycelium prepared after
cultivation in JM or YEME medium with Th, are stored at -
80°C after addition of glycerol to 20$ (v/v).
Example 104
Characterization of S. lividans ZX7 attB::ESAC-70
Individual colonies of S. lividans ZX7 attB::ESAC-70,
prepared as described in Example 102, are grown in YEME and
total genomic DNA is prepared. The DNA is digested with
BamHI, resolved by agarose gel-electrophoresis, and
transferred onto a membrane. Hybridization to labeled pPAC-
S1 DNA, prepared as described in Example 11, reveals the
appearance of three bands of approximately 16, 8 and 2.7
kb. PFGE analysis of genomic DNA reveals the disappearance
of a 2.5 Mb DraI fragment present in ZX7 and the appearance
of two fragments of 1.4 and 1.1 Mb (Fig. 10).
Although the present invention is described in the
Examples listed above in terms of preferred embodiments,
they are not to be regarded as limiting the scope of the
invention. The above Examples serve to illustrate the
principles and methodologies for introducing ESACs into S.
lividans, for cultivating the resulting transformants and
for confirming their genotype. The above Examples serve to
illustrate the principles and methodologies for the
transformation of S. lividans with ESACs carrying DNA
inserts from a different species. It will occur to those
skilled in the art that additional ESACs, either containing
different inserts of S. coelicolor DNA, prepared as
described in Example 17, or carrying DNA inserts from other
actinomycetes can be used to transform S. lividans ZX7. As
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another example transfer of large DNA segments, the
transformation of S. lividans with a P. rosea gene cluster
is illustrated below. Confirmation of the correct genotype
of the resulting transformants is illustrated in Fig. 11.
Example 105
Construction S. lividans ZX7 attB::PAD6
A few hundred ng of PAD6, prepared as described in Example
69, are used to transform protoplasts of S. Iividans ZX7.
The colonies that appear on the R2YE plates, after
overlaying with Th, are analyzed for their ThR by streaking
them on fresh R2YE plates.
Example 106
Characterization of S. lividans ZX7 attB::PAD6
Individual colonies of S. lividans ZX7 attB::PAD6, prepared
as described in Example 105, are grown in YEME medium and
total DNA is prepared. The DNA is digested with BamHI,
resolved by agarose gel-electrophoresis and transferred
onto a membrane. Hybridization to labeled PAD6 il
illustrated in Fig. 11. Bands of 22, I0, 7.6, 7.2, 6.2,
5.6, 5.2, 3.1, 3.0, 2.8, 2.7, 2.6, 2.5, 2.1, 1.9, 1.9, 1.8,
1.6, 1.5, 1.4, 1.2, 1.0, 1.0, 0.9, 0.9, 0.9, 0.7, 0.6, 0.5,
0.5, 0.3 and 0.1 kb. The profile of P. rosea DNA is shown
for comparison.
Those skilled in the art understand that S. lividans
ZX7 attB::PAD6 contains the expected number and size of
bands expected from transfer of the cluster of Fig. 9 via
PAD6. In analogy to the above Examples, the rapamycin,
erythromycin and rifamycin clusters assembled in pESAC,
according to the principles and methodologies described in
Section 7.2.2, can be used to transform S. lividans. It
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will occur to those skilled in the art that other S.
lividans strains can be equally used as hosts for
transformation with ESACs. Furthermore, ~C31 can lysogenize
other Streptomyces species, in addition to S. lividans.
These include but are not limited to the species reported
in Table 2. Furthermore, a ~C31 attB site may be engineered
into Streptomyces species or other actinomycetes that are
not naturally lysogenized by phage ~C31. Therefore, any
ESAC, prepared according to the principles and
methodologies of Section 7.2, and any natural or engineered
actinomycete host, fall within the scope of the present
invention.
It will occur to those skilled in the art that
alternative methods for introducing DNA into an
actinomycete host can be employed. These include but are
not limited to electroporation (MacNeil, 1989, FEMS
Microbiol. Lett. 42:239-244) and conjugation from E. coli
(Mazodier et al., 1989, J. Bacteriol. 171:3583-3585). It
will also occur to those skilled in the art that
alternative media and growth conditions can be employed for
cultivating the transformants, and that they can be
analyzed by different methods than those described above.
Technical variations on the methodologies described above
can produced equivalent results. All these variations fall
within the scope of the present invention.
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Table 2
5 List of exemplary species of Streptomyces and other genera
of Actinomycetales allowing attP-mediated integration of
~C31 (Hopwood et al., 1985, Genetic Manipulation of
Streptomyces: A Laboratory Manual, The 3ohn Innes
Foundation, Norwich, UK; Lomovskaya et al., 1997,
10 Microbiol. 143:875-883; Kuhstoss et al., 1991, Gene 97:143-
146; Soldatova et al., 1994, Antibiot. Khimioter. 39:3-7).
Streptomyces coelicolor
15 Streptomyces lividans
Streptomyces hygroscopicus
Streptomyces bambergiensis
Streptomyces ambofaciens
Streptomyces griseofuscus
20 Streptomyces lipmanii
Streptomyces thermotolerans
Streptomyces clavuligerus
Streptomyces fradiae
Saccharopolyspora spinosa
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7.5 Growth of the recombinant Strepto~ayces and metabolite
production
When an ESAC, introduced into a production host according
to the principles and methodologies described in Section
7.4, carries the entire biosynthesis gene cluster derived
from a donor organism, the recombinant strain produces the
relevant natural product. Naive actinomycete hosts have
been shown to produce the appropriate natural product or
its intermediates) when the relevant DNA was introduced
into them (Malpartida and Hopwood, 1989, Nature 309:462-
464; Hong et al., 1997, J. Bacteriol. 179:470-476; Kao et
al., 1994, Science 265:509-512). Thus, transformants of
Streptomyces and other actinomycete species carrying the
relevant biosynthesis clusters are expected to produce the
corresponding natural product. The recombinant production
hosts are cultivated in a suitable medium and the presence
of the relevant metabolites is determined following
appropriate procedures, which may include biological
assays, chromatographic properties, MS, NMR, etc.
It will occur to those skilled in the art that ESACs,
containing the relevant biosynthesis cluster derived from
any donor actynomycete, can be used to transform S.
lividans. The resulting transformants will produce the
corresponding natural product. For example, an ESAC
carrying the rapamycin, erythromycin or rifamycin cluster,
prepared according to the principles of Section 7.2, can be
used to transform S. lividans and rapamycin, erythromycin
or rifamycin, respectively, can be produced by the
resulting recombinant strain. Furthermore, it will occur to
those skilled in the art that other Streptomyces or
actinomycete strains that naturally contain or have been
engineered to contain a phage ~C31 attB site, can be used
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as production hosts for desired natural products.
Therefore, any natural product produced after introduction
of the relevant cluster carried on ESAC into a suitable
production host, falls within the scope of the present
invention.
The present invention describes principles and
methodologies for optimizing and speeding up the process of
lead optimization and development in drug discovery. These
can be applied since the early stages of drug discovery as
briefly summarized herein. A natural product produced by a
donor organism has an interesting property, such as
antibacterial, antifungal, antitumor, antihelmintic,
herbicidal, immunosuppressive, or other pharmacological
activity. The potential is seen for increasing the
productivity of the producing organism, and/or for
improving the biological or physico-chemical properties of
said natural product after manipulating its structure. The
biosynthetic pathway for the natural product is inferred
from its chemical structure. This leads to a hypothesis on
the genes involved, including the approximate size of the
corresponding cluster. A large insert library is
constructed in the pESAC vectors described herein using
genomic DNA prepared from the donor organism. Through a
judicious choice of hybridization probes and PCR primers,
the desired cluster is identified in the library.
Alternatively, the cluster is assembled into the pESAC
vectors described herein from overlapping cosmid clones
identified by hybridization as above. The selected clones)
are transferred into S. lividans, S. coelicolor or other
suitable strain, and the resulting transformants are
evaluated for production of the natural product.
Once production is obtained, the desired genetic,
physiological and technological manipulations can be
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performed on the production host, employing well-developed
methodologies. The bioactive molecule is purified from a
known host, amid a background of known metabolites. If
necessary, ad hoc mutations can be conveniently introduced
in the production host to eliminate unwanted, interfering
products. Because of the deeper knowledge on the
physiological processes and regulatory networks for
secondary metabolism in the production host compared to the
donor organism, targeted approaches to strain improvement,
using classical and molecular techniques, can be applied.
Furthermore, well-characterized mutant strains are
available for the production host, and the desired ESAC
could be easily introduced into different genetic
backgrounds. In addition, the biosynthetic pathway can be
easily manipulated, because of the availability of the
cloned genes and the efficient genetic tools for the
production host. Finally, additional specialized genes or
even entire clusters can be introduced into the production
host, further expanding the possible applications of the
present invention.
As it is apparent from the above description, a
further object of this invention is to provide a process
for the procduction of a natural product by cultivating an
actinornycete strain capablre of producing said natural
product in the presence of a nutrient medium, isolating and
purifying said natural product, characterized in that the
actinomycete strain capable of producing said natural
product ( production host) is an actinomycete strain
modified by means of an E. coli-Streptomyces Artificial
Chromosome that carries a gene cluster governing the
biosynthesis of said natural product derived from an
actinomycete donor organism which is the original producer
of said natural product, according to the method described
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herein. Preferably, said modified actinomycete strain shall
be a Streptomyces Iividans or Streptomyces coelicolor
strain.
Finally, even in a case where the natural product may
not be made by the production host after transfer of the
relevant cluster, appropriate tools are available to remedy
that situation. Lack of production of the expected natural
product might be due to several possibilities: absence of
required gene(s); DNA, gene product or natural product
instability; inadequate levels of gene expression or of
appropriate precursors. In a well-defined production host,
each of these possible causes may be directly investigated
and remedied.
Therefore, the present invention provides significant
advantages over the existing process of drug discovery and
development, including production. It exploits the fact
that the host where the natural product will be produced is
an organism commonly used for process development and
genetic manipulation, with substantial information
available, including safety for handling it.
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SEQUENCE LISTING
<110> Biosearch Italia Spa
<120> Methods for transferring the capability to produce a
natural product into a suitable production host
<130> G67773RS/mg
<140>
<141>
<150> EP98111506.6
<151> 1998-06-23
<150> EP99107554.0
<151> 1999-04-15
<160> 3B
<170> PatentIn Ver. 2.1
<210> 1
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:oligo probe
<400> 1
gcstacatca tctacacstc sggsacsacs ggsaagccsa aggg 44
<210> 2
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: ,oligo
probe
<400> 2
ggstacatca tctacacsag cggsacsacs ggsaagccsa aggg 44
<210> 3
1
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo probe
<400> 3
akgctgtcsc csccsagsnn gaagaagtyg tcgtcgatsc c 41
<210> 4
<211> 41
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: oligo probe
<400> 4
akggagtcsc csccsagsnn gaagaagtyg tcgtcgatsc c 41
<210> 5
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 5
tttttggtac ctgacgtccc gaaggcgtg 29
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PCR primer
<400> 6
cagcttgtcc atggcgga 18
<210> 7
2
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 7
tctgtccgcc atggacaagc 20
<210> 8
<211> 32
< 212 > DN1'.
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:PCR primer
<400> 8
tttttggatc cggctaacta actaaaccga ga 32
<210> 9
<211> 200
<212> DNA
<213> Planobispora rosea
<400> 9
ggatcccgag caccgaccag ccgtgggcgg ggacgagaca cgggtctccc ggagcctccc 60
ccgacgactc cagcacggcc aggcccgcgg cctcgaccgg gaagcggtag ggcctgtcgt 120
ccacggttga gcagggtgag cagtgcccgg ccgggatggt ccgggtcagc cgaggccagc 180
gcggcggccc ggttgctcag 200
<210> 10
<211> 200
<212> DNA
<213> Planobispora rosea
<400> 10
ggatcccgag caccgaccag ccgtgggcgg ggacgagaca cgggtctccc ggagcctccc 60
ccgacgactc cagcacggcc aggcccgcgg cctcgaccgg gaagcggtag ggcctgtcgt 120
ccacggttga gcagggtgag cagtgcccgg ccgggatggt ccgggtcagc cgaggccagc 180
gcggcggccc ggttgctcag 200
<210> 11
3
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
<400> 11
gatctaagct tggggg 16
<210> 12
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: linker
<400> 12
cccccaagct to 12
<210> 13
<211> 21
<2I2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 13
gagctctcat gtttgacagc t 21
<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 14
gagctctgac ttccgcgttt crag 24
<210> 15
4
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 15
tttttgaatt cggtaccagc cgacggcga 29
<210> 16
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 16
tttttggatc cctgttccac cagcgcacc 29
<210> 17
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 17
ttttttctag accgtcgtcg gtggttctc 29
<210> 18
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 18
tttttggatc caggaagccc tgtgctgtc 29
<210> 19
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 19
ttttttgtag aggtcaagat ccggggcat 29
<210> 20
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 20
tttttctgca ggacagcgcc cttgaggtg 29
<210> 21
<211> 26
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 21
tttttctgca ggcgacgaag aggggc 26
<210> 22
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 22
tttttaagct tagcgcgacc ggggcggt 28
<210> 23
6
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 23
catgggaatt cggggg 16
<210> 24
<211> 12
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 24
cccccgaatt cc 12
<210> 25
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 25
tttttggatc cggggcagcg gttggttcc 29
<210> 26
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 26
ttttttctag aaggcagctc cagatgatc 29
<210> 27
7
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 27
tttttctaga ccggactcgg ccggctcg 28
<210> 28
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 28
tttttctgca gccgcacgcc tcggtggtc 29
<210> 29
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 29
tttttctgca gggaccctga gtgcaggtg 29
<210> 30
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 30
tttttaagct tcagtagccg tcgctgagc 29
<210> 31
8
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<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 31
tttttgaatt ctgcagaccg ccgaggaag 29
<210> 32
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 32
tttttggatc cggagtcgta gctgacgac 29
<210> 33
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 33
ttttggatcc cgaccacgcg gggacgtc 28
<210> 34
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 34
ttttttctag accagggaac ccgtgctgc 29
<210> 35
9
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<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 35
ttttttctag acggaagctc gccgcgatc 29
<210> 36
<211> 29
~2i2> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 36
tttttctgca ggtccgtagc ccggacacc 29
<210> 37
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 37
tttttctgca gttcgggcga cagttcctt 29
<210> 38
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: PCR primer
<400> 38
tttttaagct tcaacaagcc atccgggtc 29
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