U.S. patent application number 10/423231 was filed with the patent office on 2005-02-10 for screening for novel bioactivities.
Invention is credited to Short, Jay M..
Application Number | 20050032054 10/423231 |
Document ID | / |
Family ID | 25440325 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032054 |
Kind Code |
A1 |
Short, Jay M. |
February 10, 2005 |
Screening for novel bioactivities
Abstract
Disclosed is a process for identifying clones having a specified
activity of interest, which process comprises (i) generating one or
more expression libraries derived from nucleic acid directly
isolated from the environment; and (ii) screening said libraries
utilizing an assay system. More particularly, this is a process for
identifying clones having a specified activity of interest by (i)
generating one or more expression libraries derived from nucleic
acid directly or indirectly isolated from the environment; (ii)
exposing said libraries to a particular substrate or substrates of
interest; and (iii) screening said exposed libraries utilizing a
fluorescence activated cell sorter to identify clones which react
with the substrate or substrates. Also provided is a process for
identifying clones having a specified activity of interest by (i)
generating one or more expression libraries derived from nucleic
acid directly or indirectly isolated from the environment; and (ii)
screening said exposed libraries utilizing an assay requiring a
binding event or the covalent modification of a target, and a
fluorescence activated cell sorter to identify positive clones.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
DIVERSA CORPORATION
4955 DIRECTORS PLACE
SAN DIEGO
CA
92121
US
|
Family ID: |
25440325 |
Appl. No.: |
10/423231 |
Filed: |
April 25, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10423231 |
Apr 25, 2003 |
|
|
|
09561597 |
Apr 27, 2000 |
|
|
|
6555315 |
|
|
|
|
09561597 |
Apr 27, 2000 |
|
|
|
08918406 |
Aug 26, 1997 |
|
|
|
6057103 |
|
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/471; 435/6.1 |
Current CPC
Class: |
C12N 15/1086 20130101;
C12Q 1/00 20130101; C12N 15/1034 20130101; C12N 15/52 20130101;
C12N 15/1093 20130101 |
Class at
Publication: |
435/006 ;
435/471 |
International
Class: |
C12Q 001/68; C12N
015/74 |
Claims
What is claimed is:
1. A method for identifying a desired activity encoded by a genomic
DNA population comprising: (a) obtaining a single-stranded genomic
DNA population; (b) contacting the single-stranded DNA population
of (a) with a DNA probe bound to a ligand under conditions and for
sufficient time to allow hybridization and to produce a
double-stranded complex of probe and members of the genomic DNA
population which hybridize thereto; (c) contacting the
double-stranded complex of (b) with a solid phase specific binding
partner for said ligand so as to produce a solid phase complex; (d)
separating the solid phase complex from the single-stranded DNA
population of (b); (e) releasing from the probe the members of the
genomic population which had bound to the solid phase bound probe;
(f) forming double-stranded DNA from the members of the genomic
population of (e); (g) introducing the double-stranded DNA of (f)
into a suitable host cell to produce an expression library
containing a plurality of clones containing the selected DNA; and
(h) screening the expression library for the desired activity.
2. The method of claim 1, wherein the genomic DNA population is
derived from uncultivated or cultivated microorganisms.
3. The method of claim 2, wherein the uncultivated or cultivated
microorganisms are isolated from an environmental sample.
4. The method of claim 3, wherein the microorganisms isolated from
an environmental sample are extremophiles.
5. The method of claim 4, wherein the extremophiles are selected
from the group consisting of thermophiles, hyperthermophiles,
psychrophiles, halophiles, acidophiles, barophiles and
psychrotrophs.
6. The method of claim 1, wherein the genomic DNA, or fragments
thereof, comprise one or more operons, or portions thereof.
7. The method of claim 6, wherein the operons, or portions thereof,
encodes a complete or partial metabolic pathway.
8. The method of claim 7, wherein the operons or portions thereof
encoding a complete or partial metabolic pathway encodes polyketide
synthases.
9. The method of claim 1, wherein the expression library containing
a plurality of clones is selected from the group consisting of
phage, plasmids, phagemids, cosmids, phosmids, viral vectors and
artificial chromosomes.
10. The method of claim 1, wherein the a suitable host cell is
selected from the group consisting of a bacterium, fungus, plant
cell, insect cell and animal cell.
11. The method of claim 1, wherein the DNA probe bound to a ligand
is comprised of at least a portion of the coding region sequence of
DNA for a known bioactivity.
12. The method of claim 1, wherein the ligand is selected from the
group consisting of antigens or haptens, biotin or iminobiotin,
sugars, enzymes, apoenzymes homopolymeric oligonucleotides and
hormones.
13. The method of claim 1, wherein the binding partner for said
ligand is selected from the group consisting of antibodies or
specific binding fragments thereof, avidin or streptavidin,
lectins, enzyme inhibitors, apoenzyme cofactors, homopolymeric
oligonucleotides and hormone receptors.
14. The method of claim 1, wherein a solid phase is selected from
the group consisting of a glass or polymeric surface, a packed
column of polymeric beads or magnetic or paramagnetic
particles.
15. The method of claim 1, further comprising producing an extract
of the expression library.
16. The method of claim 15, further comprising combining the
expression library extract with an enzyme extract from a
metabolically rich host organism.
17. The method of claim 16, wherein the host organism is
Streptomyces.
18. The method of claim 16, wherein the host organism is
Bacillus.
19. A method for preselecting a desired DNA from a genomic DNA
population comprising: (a) obtaining a single-stranded genomic DNA
population; (b) contacting the single-stranded DNA population of
(a) with a ligand-bound oligonucleotide probe that is complementary
to a secretion signal sequence unique to a given class of proteins
under conditions permissive of hybridization to form a
double-stranded complex; (c) contacting the double-stranded complex
of (a) with a solid phase specific binding partner for said ligand
so as to produce a solid phase complex; (d) separating the solid
phase complex from the single-stranded DNA population of (a); (e)
releasing the members of the genomic population which had bound to
said solid phase bound probe; (f) separating the solid phase bound
probe from the members of the genomic population which had bound
thereto; (g) forming double-stranded DNA from the members of the
genomic population of (e); (h) introducing the double-stranded DNA
of (g) into a suitable host cell to form an expression library
containing a plurality of clones containing the selected DNA; and
(i) screening the expression library for the desired activity.
20. The method of claim 19, wherein the genomic DNA population is
derived from uncultivated or cultivated microorganisms.
21. The method of claim 20, wherein the uncultivated or cultivated
microorganisms are isolated from an environmental sample.
22. The method of claim 21, wherein the microorganisms isolated
from an environmental sample are extremophiles.
23. The method of claim 22, wherein the extremophiles are selected
from the group consisting of thermophiles, hyperthermophiles,
psychrophiles, halophiles, acidophiles, barophiles and
psychrotrophs.
24. The method of claim 19, wherein the genomic DNA, or fragments
thereof, comprise one or more operons, or portions thereof.
25. The method of claim 24, wherein the operons, or portions
thereof, encodes a complete or partial metabolic pathway.
26. The method of claim 25, wherein the operons or portions thereof
encoding a complete or partial metabolic pathway encodes polyketide
synthases.
27. The method of claim 19, wherein the expression library
containing a plurality of clones is selected from the group
consisting of phage, plasmids, phagemids, cosmids, phosmids, viral
vectors and artificial chromosomes.
28. The method of claim 19, wherein the a suitable host cell is
selected from the group consisting of a bacterium, fungus, plant
cell, insect cell and animal cell.
29. The method of claim 19, wherein the DNA probe bound to a ligand
is comprised of at least a portion of the coding region sequence of
DNA for a known bioactivity.
30. The method of claim 19, wherein the ligand is selected from the
group consisting of antigens or haptens, biotin or iminobiotin,
sugars, enzymes, apoenzymes homopolymeric oligonucleotides and
hormones.
31. The method of claim 19, wherein the binding partner for said
ligand is selected from the group consisting of antibodies or
specific binding fragments thereof, avidin or streptavidin,
lectins, enzyme inhibitors, apoenzyme cofactors, homopolymeric
oligonucleotides and hormone receptors.
32. The method of claim 19, wherein a solid phase is selected from
the group consisting of a glass or polymeric surface, a packed
column of polymeric beads or magnetic or paramagnetic
particles.
33. The method of claim 19, further comprising producing an extract
of the expression library.
34. The method of claim 33, further comprising combining the
expression library extract with an enzyme extract from a
metabolically rich host organism.
35. The method of claim 34, wherein the host organism is
Streptomyces.
36. The method of claim 34, wherein the host organism is Bacillus.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the discovery of new
bio-active molecules, such as antibiotics, anti-virals, anti-tumor
agents and regulatory proteins. More particularly, the invention
relates to a system for capturing genes potentially encoding novel
biochemical pathways of interest in prokaryotic systems, and
screening for these pathways utilizing high, throughput screening
assays.
BACKGROUND OF THE INVENTION
[0002] Within the last decade there has been a dramatic increase in
the need for bioactive compounds with novel activities. This demand
has arisen largely from changes in worldwide demographics coupled
with the clear and increasing trend in the number of pathogenic
organisms that are resistant to currently available antibiotics.
For example, while there has been a surge in demand for
antibacterial drugs in emerging nations with young populations,
countries with aging populations, such as the US, require a growing
repertoire of drugs against cancer, diabetes, arthritis and other
debilitating conditions. The death rate from infectious diseases
has increased 58% between 1980 and 1992 and it has been estimated
that the emergence of antibiotic resistant microbes has added in
excess of $30 billion annually to the cost of health care in the US
alone. (Adams et al., Chemical and Engineering News, 1995; Amann et
al., Microbiological Reviews, 59, 1995). As a response to this
trend pharmaceutical companies have significantly increased their
screening of microbial diversity for compounds with unique
activities or specificities.
[0003] There are several common sources of lead compounds (drug
candidates), including natural product collections, synthetic
chemical collections, and synthetic combinatorial chemical
libraries, such as nucleotides, peptides, or other polymeric
molecules. Each of these sources has advantages and disadvantages.
The success of programs to screen these candidates depends largely
on the number of compounds entering the programs, and
pharmaceutical companies have to date screened hundred of thousands
of synthetic and natural compounds in search of lead compounds.
Unfortunately, the ratio of novel to previously-discovered
compounds has diminished with time. The discovery rate of novel
lead compounds has not kept pace with demand despite the best
efforts of pharmaceutical companies. There exists a strong need for
accessing new sources of potential drug candidates.
[0004] The majority of bioactive compounds currently in use are
derived from soil microorganisms. Many microbes inhabiting soils
and other complex ecological communities produce a variety of
compounds that increase their ability to survive and proliferate.
These compounds are generally thought to be nonessential for growth
of the organism and are synthesized with the aid of genes involved
in intermediary metabolism hence their name--secondary metabolites.
Secondary metabolites that influence the growth or survival of
other organisms are known as "bioactive" compounds and serve as key
components of the chemical defense arsenal of both micro- and
macroorganisms. Humans have exploited these compounds for use as
antibiotics, antiinfectives and other bioactive compounds with
activity against a broad range of prokaryotic and eukaryotic
pathogens. Approximately 6,000 bioactive compounds of microbial
origin have been characterized, with more than 60% produced by the
gram positive soil bacteria of the genus Streptomyces. (Barnes et
al., Proc. Nat. Acad. Sci. U.S.A., 91, 1994). Of these, at least 70
are currently used for biomedical and agricultural applications.
The largest class of bioactive compounds, the polyketides, include
a broad range of antibiotics, immunosuppressants and anticancer
agents which together account for sales of over $5 billion per
year.
[0005] Despite the seemingly large number of available bioactive
compounds, it is clear that one of the greatest challenges facing
modern biomedical science is the proliferation of antibiotic
resistant pathogens. Because of their short generation time and
ability to readily exchange genetic information, pathogenic
microbes have rapidly evolved and disseminated resistance
mechanisms against virtually all classes of antibiotic compounds.
For example, there are virulent strains of the human pathogens
Staphylococcus and Streptococcus that can now be treated with but a
single antibiotic, vancomycin, and resistance to this compound will
require only the transfer of a single gene, vanA, from resistant
Enterococcus species for this to occur. (Bateson et al., System.
Appl. Microbiol, 12, 1989). When this crucial need for novel
antibacterial compounds is superimposed on the growing demand for
enzyme inhibitors, immunosuppressants and anti-cancer agents it
becomes readily apparent why pharmaceutical companies have stepped
up their screening of microbial diversity for bioactive compounds
with novel properties.
[0006] The approach currently used to screen microbes for new
bioactive compounds has been largely unchanged since the inception
of the field. New isolates of bacteria, particularly gram positive
strains from soil environments, are collected and their metabolites
tested for pharmacological activity. A more recent approach has
been to use recombinant techniques to synthesize hybrid antibiotic
pathways by combining gene subunits from previously characterized
pathways. This approach, called "combinatorial biosynthesis" has
focused primarily on the polyketide antibiotics and has resulted in
a number of structurally unique compounds which have displayed
activity. (Betz et al., Cytometry, 5, 1984; Davey et al.,
Microbiological Reviews, 60, 1989). However, compounds with novel
antibiotic activities have not yet been reported; an observation
that may be do to the fact that the pathway subunits are derived
from those encoding previously characterized compounds. Dramatic
success in using recombinant approaches due to small molecule
synthesis has been recently reported in the engineering of
biosynthetic pathways to increase the production of desirable
antibiotics. (Diaper et al., Appl. Bacteriol., 77, 1994; Enzyme
Nomenclature, Academic Press: NY, 1992).
[0007] There is still tremendous biodiversity that remains untapped
as the source of lead compounds. However, the currently available
methods for screening and producing lead compounds cannot be
applied efficiently to these under-explored resources. For
instance, it is estimated that at least 99% of marine bacteria
species do not survive on laboratory media, and commercially
available fermentation equipment is not optimal for use in the
conditions under which these species will grow, hence these
organisms are difficult or impossible to culture for screening or
re-supply. Recollection, growth, strain improvement, media
improvement and scale-up production of the drug-producing organisms
often pose problems for synthesis and development of lead
compounds. Furthermore, the need for the interaction of specific
organisms to synthesize some compounds makes their use in discovery
extremely difficult. New methods to harness the genetic resources
and chemical diversity of these untapped sources of compounds for
use in drug discovery are very valuable. The present invention
provides a path to access this untapped biodiversity and to rapidly
screen for activities of interest utilizing recombinant DNA
technology. This invention combines the benefits associated with
the ability to rapidly screen natural compounds with the
flexibility and reproducibility afforded with working with the
genetic material of organisms.
[0008] The present invention allows one to identify genes encoding
bioactivities of interest from complex environmental gene
expression libraries, and to manipulate cloned pathways to evolve
recombinant small molecules with unique activities. Bacteria and
many eukaryotes have a coordinated mechanism for regulating genes
whose products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. The gene cluster,
the promoter, and additional sequences that function in regulation
altogether are referred to as an "operon" and can include up to 20
or more genes, usually from 2 to 6 genes. Thus, a gene cluster is a
group of adjacent genes that are either identical or related,
usually as to their function. Gene clusters are of interest in drug
discovery processes since product(s) of gene clusters include, for
example, antibiotics, antivirals, antitumor agents and regulatory
proteins.
[0009] Some gene families consist of one or more identical members.
Clustering is a prerequisite for maintaining identity between
genes, although clustered genes are not necessarily identical. Gene
clusters range from extremes where a duplication is generated of
adjacent related genes to cases where hundreds of identical genes
lie in a tandem array. Sometimes no significance is discernable in
a repetition of a particular gene. A principal example of this is
the expressed duplicate insulin genes in some species, whereas a
single insulin gene is adequate in other mammalian species.
[0010] Gene clusters undergo continual reorganization and, thus,
the ability to create heterogeneous libraries of gene clusters
from, for example, bacterial or other prokaryote sources is
valuable in determining sources of novel bioactivities, including
enzymes such as, for example, the polyketide synthases that are
responsible for the synthesis of polyketides having a vast array of
useful activities.
[0011] Polyketides are molecules which are an extremely rich source
of bioactivities, including antibiotics (such as tetracyclines and
erythromycin), anti-cancer agents (daunomycin), immunosuppressants
(FK506 and rapamycin), and veterinary products (monensin). Many
polyketides (produced by polyketide synthases) are valuable as
therapeutic agents. Polyketide synthases (PKSs) are multifunctional
enzymes that catalyze the biosynthesis of a huge variety of carbon
chains differing in length and patterns of functionality and
cyclization. Despite their apparent structural diversity, they are
synthesized by a common pathway in which units derived from acetate
or propionate are condensed onto the growing chain in a process
resembling fatty acid biosynthesis. The intermediates remain bound
to the polyketide synthase during multiple cycles of chain
extension and (to a variable extent) reduction of the
(.beta.-ketone group formed in e ach condensation. The structural
variation between naturally occurring polyketides arises largely
from the way in which each PKS controls the number and type of
units added, and from the extent and stereochemistry of reduction
at each cycle. Still greater diversity is produced by the action of
regiospecific glycosylases, methyltransferases and oxidative
enzymes on the product of the PKS.
[0012] Polyketide synthase genes fall into gene clusters. At least
one type (designated type I) of polyketide synthases have large
size genes and encoded enzymes, complicating genetic manipulation
and in vitro studies of these genes/proteins. Progress in
understanding the enzymology of such type I systems have previously
been frustrated by the lack of cell-free systems to study
polyketide chain synthesis by any of these multienzymes, although
several partial reactions of certain pathways have been
successfully assayed in vitro. Cell-free enzymatic synthesis of
complex polyketides has proved unsuccessful, despite more than 30
years of intense efforts, presumably because of the difficulties in
isolating fully active forms of these large, poorly expressed
multifunctional proteins from naturally occurring producer
organisms, and because of the relative lability of intermediates
formed during the course of polyketide biosynthesis. In an attempt
to overcome some of these limitations, modular PKS subunits have
been expressed in heterologous hosts such as Escherichia coli and
Streptomyces coelicolor. Whereas the proteins expressed in E. coli
are not fully active, heterologous expression of certain PKSs in S.
coelicolor resulted in the production of active protein. Cell-free
enzymatic synthesis of polyketides from PKSs with substantially
fewer active sites, such as the 6-methylsalicylate synthase,
chalcone synthase, tetracenomycin synthase, and the PKS responsible
for the polyketide component of cyclosporin, have been
reported.
[0013] Hence, studies have indicated that in vitro synthesis of
polyketides is possible, however, synthesis was always performed
with purified enzymes. Heterologous expression of genes encoding
PKS modular subunits have allowed synthesis of functional
polyketides in vivo, however, there are several challenges
presented by this approach, which had to be overcome. The large
sizes of modular PKS gene clusters (>30 kb) make their
manipulation on plasmids difficult. Modular PKSs also often utilize
substrates that may be absent in a heterologous host. Finally,
proper folding, assembly, and posttranslational modification of
very large foreign polypeptides are not guaranteed.
[0014] Novel systems to clone and screen for bioactivities of
interest in vitro are desirable. The method(s) of the present
invention allow the cloning and discovery of novel bioactive
molecules in vitro, and in particular novel bioactive molecules
derived from uncultivated samples. Large size gene clusters can be
cloned and screened using the method(s) of the present invention.
Unlike previous strategies, the method(s) of the present invention
allow one to clone utilizing well-known genetic systems, and to
screen in vitro with crude (impure) preparations.
SUMMARY OF THE INVENTION
[0015] The present invention allows one to clone genes potentially
encoding novel biochemical pathways of interest in prokaryotic
systems, and screen for these pathways utilizing a novel process.
Sources of the genes may be isolated, individual organisms
("isolates"), collections of organisms that have been grown in
defined media ("enrichment cultures"), or, most preferably,
uncultivated organisms ("environmental samples"). The use of a
culture-independent approach to directly clone genes encoding novel
bioactivities from environmental samples is most preferable since
it allows one to access untapped resources of biodiversity.
[0016] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample that may be
under-represented by several orders of magnitude compared to the
dominant species.
[0017] In the evaluation of complex environmental expression
libraries, a rate-limiting step occurs at the level of discovery of
bioactivities. The present invention allows the rapid screening of
complex environmental expression libraries, containing, for
example, thousands of different organisms.
[0018] In the present invention, for example, gene libraries
generated from one or more uncultivated microorganisms are screened
for an activity of interest. Potential pathways encoding bioactive
molecules of interest are first captured in prokaryotic cells in
the form of gene expression libraries; crude or partially purified
extracts, or pure proteins from metabolically rich cell lines are
then combined with the gene expression libraries to create
potentially active molecules; and the combination is screened for
an activity of interest. Common approaches to drug discovery
involve screening assays in which disease targets (macromolecules
implicated in causing a disease) are exposed to potential drug
candidates that are tested for therapeutic activity. In other
approaches, whole cells or organisms that are representative of the
causative agent of the disease, such as bacteria or tumor cell
lines, are exposed to the potential candidates for screening
purposes. Any of these approaches can be employed with the present
invention.
[0019] The present invention also allows for the transfer of cloned
pathways derived from uncultivated samples into metabolically rich
hosts for heterologous expression and downstream screening for
bioactive compounds of interest using a variety of screening
approaches briefly described above.
[0020] Accordingly, in one aspect, the present invention provides a
process for identifying clones encoding a specified activity of
interest, which process comprises (i) generating one or more
expression libraries derived from nucleic acid directly isolated
from the environment; and (ii) combining the expression libraries
with crude or partially purified extracts, or pure proteins from
metabolically rich cell lines; and (iii) screening said libraries
utilizing any of a variety of screening assays to identify said
clones.
[0021] In another aspect, the present invention provides a process
for identifying clones encoding a specified activity of interest,
which process comprises (i) generating one or more expression
libraries derived from nucleic acid directly isolated from the
environment; and (ii) transferring the clones into a metabolically
rich cell line; and (iii) screening said cell line utilizing any of
a variety of screening assays to identify said clones.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] FIG. 1 shows a scheme to capture, clone and archive large
genome fragments from uncultivated microbes from natural
environments. The cloning vectors used in this process can archive
from 40 kbp (fosmids) to greater than 100 kbp (BACs).
[0023] FIG. 2 shows the nucleotide alignment of a region of the
ketosynthase I gene of polyketide pathways from a variety of
Streptomyces species. These regions are aligned with a homologous
region encoding a fatty acid synthase from E. coli. Observed
sequence differences were used to construct probes that hybridize
to cloned polyketide sequences but not to fatty acid sequences
carried by the E. coli host strain.
[0024] FIG. 3 shows an example of a high density filter array of
environmental fosmid clones probed with a labeled oligonucleotide
probe. The 2400 arrayed clones contain approximately 96 million
base pairs of DNA cloned from a naturally occurring microbial
community.
[0025] FIG. 4 shows the results of mixed extract experiment
measuring conferral of bioactivity on recombinant backbones
heterologously expressed in E. coli. A. Organic extracts from 3
oxytetracylin clones (1-3) and 3 gramicidin clones (4-6) were
incubated with a protein extract from Streptomyces lividans strain
TK24. After incubation the mixture was reextracted with methyl
ethyl ketone, spotted on to filter disks, allowed to dry, then
placed on a lawn of an E. coli test strain. Distinct zones of
clearing can be seen around disks 2, 3 and 5. Extracts from 2 and 3
were subsequently separated by thin layer chromatography that
showed UV fluorescent spots with similar Rf and appearance to
authentic oxytetracylin. B. Filters corresponding to those in A but
without incubation with protein extract from Streptomyces. The
Streptomyces extract alone also showed no bioactivity.
[0026] FIG. 5 shows a strategy for FACS screening for recombinant
bioactive molecules in Streptomyces venezuelae.
[0027] FIG. 6 shows a micrograph of pMF4 oxytetracyclin clone
expressed in S. lividans strain TK24. The red fluorescence near the
end of the mycelia suggests that recombinant expression of
oxytetracyclin may be induced at the onset sporulation as is the
activity of the endogenous actinorhodin pathway.
[0028] FIG. 7 shows an approach to screen for small molecules that
enhance or inhibit transcription factor initiation. Both the small
molecule pathway and the GFP reporter construct are co-expressed.
Clones altered in GFP expression can then be sorted by FACS and the
pathway clone isolated for characterization.
[0029] FIG. 8 shows the gene replacement vector pLL25 designed to
inactivate the actinorhodin pathway in Streptomyces lividans strain
TK24.
[0030] FIG. 9 shows the possible recombination events and predicted
phenotypes from replacement of the actinorhodin gene cluster in S.
lividans by the spectinomycin gene resident on pLL25.
[0031] FIG. 10 shows a tandem duplication of a pMF3 clone into the
S. lividans chromosome. Duplicated clones will contain cos sites at
the appropriate spacing for lambda packaging.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0032] Sample Source/Collection
[0033] The method of the present invention begins with the
construction of gene libraries that represent the collective
genomes of naturally occurring organisms archived in cloning
vectors that can be propagated in suitable prokaryotic hosts.
[0034] The microorganisms from which the libraries may be prepared
include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria, and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Libraries may be produced from
environmental samples in which case DNA may be recovered without
culturing of an organism or the DNA may be recovered from one or
more cultured organisms. Such microorganisms may be extremophiles,
such as hyperthermophiles, psychrophiles, psychrotrophs,
halophiles, barophiles, acidophiles, etc.
[0035] The microorganisms from which the libraries may be prepared
may be collected using a variety of techniques known in the art.
Samples may also be collected using the methods detailed in the
example provided below. Briefly, the example below provides a
method of selective in situ enrichment of bacterial and archaeal
species while at the same time inhibiting the proliferation of
eukaryotic members of the population. In situ enrichments can to
increase the likelihood of recovering rare species and previously
uncultivated members of a microbial population. If one desires to
obtain bacterial and archaeal species, nucleic acids from
eukaryotes in an environmental sample can seriously complicate DNA
library construction and decrease the number of desired bacterial
species by grazing. The method described below employs selective
agents, such as antifungal agents, to inhibit the growth of
eukaryotic species.
[0036] In situ enrichment is achieved by using a microbial
containment device composed of growth substrates and nutritional
amendments with the intent to lure, selectively, members of the
surrounding environmental matrix. Choice of substrates (carbon
sources) and nutritional amendments (i.e., nitrogen, phosphorous,
etc.) is dependent upon the members of the community for which one
desires to enrich. Selective agents against eukaryotic members are
also added to the trap. Again, the exact composition depends upon
which members of the community one desires to enrich and which
members of the community one desires to inhibit. Some of the
enrichment "media" which may be useful in pulling out particular
members of the community is described in the example provided
herein.
[0037] Sources of microorganism DNA as a starting material library
from which target DNA is obtained are particularly contemplated to
include environmental samples, such as microbial samples obtained
from Arctic and Antarctic ice, water or permafrost sources,
materials of volcanic origin, materials from soil or plant sources
in tropical areas, etc. Thus, for example, genomic DNA may be
recovered from either a culturable or non-culturable organism and
employed to produce an appropriate recombinant expression library
for subsequent determination of a biological activity.
[0038] DNA Isolation
[0039] The preparation of DNA from the sample is an important step
in the generation DNA libraries from environmental samples composed
of uncultivated organisms, or for the generation of libraries from
cultivated organisms. DNA can be isolated from samples using
various techniques well known in the art (Nucleic Acids in the
Environment Methods & Applications, J. T. Trevors, D. D. van
Elsas, Springer Laboratory, 1995). Preferably, DNA obtained will be
of large size and free of enzyme inhibitors or other contaminants.
DNA can be isolated directly from an environmental sample (direct
lysis), or cells may be harvested from the sample prior to DNA
recovery (cell separation). Direct lysis procedures have several
advantages over protocols based on cell separation. The direct
lysis technique provides more DNA with a generally higher
representation of the microbial community; however, it is sometimes
smaller in size and more likely to contain enzyme inhibitors than
DNA recovered using the cell separation technique. Very useful
direct lysis techniques have been described which provide DNA of
high molecular weight and high purity (Barns, 1994; Holben, 1994).
If inhibitors are present, there are several protocols that utilize
cell isolation that can be employed (Holben, 1994). Additionally, a
fractionation technique, such as the bis-benzimide separation
(cesium chloride isolation) described herein, can be used to
enhance the purity of the DNA.
[0040] Isolation of total genomic DNA from extreme environmental
samples varies depending on the source and quantity of material.
Uncontaminated, good quality (>20 kbp) DNA is required for the
construction of a representative library for the present invention.
A successful general DNA isolation protocol is the standard
cetyl-trimethyl-ammonium-bromide (CTAB) precipitation technique. A
biomass pellet is lysed and proteins digested by the nonspecific
protease, proteinase K, in the presence of the detergent SDS. At
elevated temperatures and high salt concentrations, CTAB forms
insoluble complexes with denatured protein, polysaccharides and
cell debris. Chloroform extractions are performed until the white
interface containing the CTAB complexes is reduced substantially.
The nucleic acids in the supernatant are precipitated with
isopropanol and resuspended in TE buffer.
[0041] For cells that are recalcitrant to lysis, a combination of
chemical and mechanical methods with cocktails of various
cell-lysing enzymes may be employed. Isolated nucleic acid may then
further be purified using small cesium gradients.
[0042] A further example of an isolation strategy is detailed in an
example below. This type of isolation strategy is optimal for
obtaining good quality, large size DNA fragments for cloning.
[0043] Normalization
[0044] The present invention can further optimize methods for
isolation of activities of interest from a variety of sources,
including consortias of microorganisms, primary enrichments, and
environmental "uncultivated" samples. Libraries which have been
"normalized" in their representation of the genome populations in
the original samples are possible with the present invention. These
libraries can then be screened utilizing the methods of the present
invention, for enzyme and other bioactivities of interest.
[0045] Libraries with equivalent representation of genomes from
microbes that can differ vastly in abundance in natural populations
are generated and screened. This "normalization" approach reduces
the redundancy of clones from abundant species and increases the
representation of clones from rare species. These normalized
libraries allow for greater screening efficiency resulting in the
identification of cells encoding novel biological catalysts.
[0046] In one embodiment, viable or non-viable cells isolated from
the environment are, prior to the isolation of nucleic acid for
generation of the expression gene library, FACS sorted to separate
cells from the sample based on, for instance, DNA or AT/GC content
of the cells. Various dyes or stains well known in the art, for
example those described in "Practical Flow Cytometry", 1995
Wiley-Liss, Inc., Howard M. Shapiro, M. D., are used to intercalate
or associate with nucleic acid of cells, and cells are separated on
the FACS based on relative DNA content or AT/GC DNA content in the
cells. Other criteria can be used to separate cells from the
sample, as well. DNA is then isolated from the cells and used for
the generation of expression gene libraries, which are then
screened for activities of interest.
[0047] Alternatively, the nucleic acid is isolated directly from
the environment and is, prior to generation of the gene library,
sorted based on DNA or AT/GC content. DNA isolated directly from
the environment, is used intact, randomly sheared or digested to
general fragmented DNA. The DNA is then bound to an intercalating
agent as described above, and separated on the analyzer based on
relative base content to isolate DNA of interest. Sorted DNA is
then used for the generation of gene libraries, which are then
screened for activities of interest.
[0048] As indicated, one embodiment for forming a normalized
library from an environmental sample begins with the isolation of
nucleic acid from the sample. This nucleic acid can then be
fractionated prior to normalization to increase the chances of
cloning DNA from minor species from the pool of organisms sampled.
DNA can be fractionated using a density centrifugation technique,
such as a cesium-chloride gradient. When an intercalating agent,
such as bis-benzimide is employed to change the buoyant density of
the nucleic acid, gradients will fractionate the DNA based on
relative base content. Nucleic acid from multiple organisms can be
separated in this manner, and this technique can be used to
fractionate complex mixtures of genomes. This can be of particular
value when working with complex environmental samples.
Alternatively, the DNA does not have to be fractionated prior to
normalization. Samples are recovered from the fractionated DNA, and
the strands of nucleic acid are then melted and allowed to
selectively reanneal under fixed conditions (C.sub.ot driven
hybridization). When a mixture of nucleic acid fragments is melted
and allowed to reanneal under stringent conditions, the common
sequences find their complementary strands faster than the rare
sequences. After an optional single-stranded nucleic acid isolation
step, single-stranded nucleic acid representing an enrichment of
rare sequences is amplified using techniques well known in the art,
such as a polymerase chain reaction (Barnes, 1994), and used to
generate gene libraries. This procedure leads to the amplification
of rare or low abundance nucleic acid molecules, which are then
used to generate a gene library that can be screened for a desired
bioactivity. While DNA will be recovered, the identification of the
organism(s) originally containing the DNA may be lost. This method
offers the ability to recover DNA from "unclonable" sources. This
method is further detailed in the example below.
[0049] Hence, one embodiment for forming a normalized library from
environmental sample(s) is by (a) isolating nucleic acid from the
environmental sample(s); (b) optionally fractionating the nucleic
acid and recovering desired fractions; (c) normalizing the
representation of the DNA within the population so as to form a
normalized expression library from the DNA of the environmental
sample(s). The normalization process is described and exemplified
in detail in co-pending, commonly assigned U.S. Ser. No.
08/665,565, filed Jun. 18, 1996.
[0050] Gene Libraries
[0051] Gene libraries can be generated by inserting the normalized
or non-normalized DNA isolated or derived from a sample into a
vector or a plasmid. Such vectors or plasmids are preferably those
containing expression regulatory sequences, including promoters,
enhancers and the like. Such polynucleotides can be part of a
vector and/or a composition and still be isolated, in that such
vector or composition is not part of its natural environment.
Particularly preferred phage or plasmids and methods for
introduction and packaging into them are described herein.
[0052] The examples below detail procedures for producing libraries
from both cultured and non-cultured organisms.
[0053] Cloning of DNA fragments prepared by random cleavage of the
target DNA can also be used to generate a representative library.
DNA dissolved in TE buffer is vigorously passed through a 25-gauge
double-hubbed needle until the sheared fragments are in the desired
size range. The DNA ends are "polished" or blunted with Mung Bean
Nuclease, and EcoRI restriction sites in the target DNA are
protected with EcoRI Methylase. EcoRI linkers (GGAATTCC) are
ligated to the blunted/protected DNA using a very high molar ratio
of linkers to target DNA. This lowers the probability of two DNA
molecules ligating together to create a chimeric clone. The linkers
are cut back with EcoRI restriction endonuclease, and the DNA is
size fractionated. The removal of sub-optimal DNA fragments and the
small linkers is critical because ligation to the vector will
result in recombinant molecules that are unpackageable, or the
construction of a library containing only linkers as inserts.
Sucrose gradient fractionation is used since it is extremely easy,
rapid and reliable. Although the sucrose gradients do not provide
the resolution of agarose gel isolations, they do produce DNA that
is relatively free of inhibiting contaminants. The prepared target
DNA is ligated to the lambda vector, packaged using in vitro
packaging extracts and grown on the appropriate E. coli.
[0054] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g. vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus, yeast, etc.) Thus, for example, the DNA may
be included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used as long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention.
[0055] A preferred type of vector for use in the present invention
contains an f-factor origin replication. The f-factor (or fertility
factor) in E. coli is a plasmid which effects high frequency
transfer of itself during conjugation and less frequent transfer of
the bacterial chromosome itself. A particularly preferred
embodiment is to use cloning vectors, referred to as "fosmids" or
bacterial artificial chromosome (BAC) vectors. These are derived
from E. coli f-factor which is able to stably integrate large
segments of genomic DNA. When integrated with DNA from a mixed
uncultured environmental sample, this makes it possible to achieve
large genomic fragments in the form of a stable "environmental DNA
library."
[0056] Another preferred type of vector for use in the present
invention is a cosmid vector. Cosmid vectors were originally
designed to clone and propagate large segments of genomic DNA.
Cloning into cosmid vectors is described in detail in Sambrook, et
al., Molecular Cloning A Laboratory Manual, Second Edition, Cold
Spring Harbor Laboratory Press, 1989.
[0057] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers.
[0058] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0059] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium.
[0060] The cloning strategy permits expression via both vector
driven and endogenous promoters; vector promotion may be important
with expression of genes whose endogenous promoter will not
function in E. coli.
[0061] The DNA derived from a microorganism(s) may be inserted into
the vector by a variety of procedures. In general, the DNA sequence
is inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0062] The DNA selected and isolated as hereinabove described is
introduced into a suitable host to prepare a library that is
screened for the desired activity. The selected DNA is preferably
already in a vector which includes appropriate control sequences
whereby selected DNA which encodes for a bio-activity may be
expressed, for detection of the desired activity. The host cell is
a prokaryotic cell, such as a bacterial cell. Particularly
preferred host cells are E. coli. Introduction of the construct
into the host cell can be effected by calcium phosphate
transfection, DEAE-Dextran mediated transfection, or
electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods
in Molecular Biology, (1986)). The selection of an appropriate host
is deemed to be within the scope of those skilled in the art from
the teachings herein.
[0063] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors. The engineered host
cells can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying genes. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to the ordinarily skilled
artisan.
[0064] Since it appears that many bioactive compounds of bacterial
origin are encoded in contiguous multigene pathways varying from 15
to 100 kbp, cloning large genome fragments is preferred with the
present invention, in order to express novel pathways from natural
assemblages of microorganisms. Capturing and replicating DNA
fragments of 40 to 100 kbp in surrogate hosts such as E. coli,
Bacillus or Streptomyces is in effect "propagating" uncultivated
microbes, albeit in the form of large DNA fragments each
representing from 2 to 5% of a typical eubacterial genome.
[0065] Two hurdles that must be overcome to successfully capture
large genome fragments from naturally occurring microbes and to
express multigene pathways from subsequent clones are 1) the low
cloning efficiency of environmental DNA and 2) the inherent
instability of large clones. To overcome these hurdles, high
quality large molecular weight DNA is extracted directly from soil
and other environments and vectors such as the F factor based
Bacterial Artificial Chromosome (BAC) vectors are used to
efficiently clone and propagate large genome fragments. The
environmental library approach (FIG. 1) will process such samples
with an aim to archive and replicate with a high degree of fidelity
the collective genomes in the mixed microbial assemblage. The basis
of this approach is the application of modified Bacterial
Artificial Chromosome (BAC) vectors to stably propagate 100-200 kbp
genome fragments. The BAC vector and its derivative the fosmid (for
F factor based cosmid) use the f-origin of replication to maintain
copy number at one or two per cell. This feature has been shown to
be a crucial factor in maintaining stability of large cloned
fragments. High fidelity replication is especially important in
propagating libraries comprised of high GC organisms such as the
Streptomyces from which clones maybe prone to rearrangement and
deletion of duplicate sequences.
[0066] Because the fosmid vector uses the highly efficient lambda
packaging system, comprehensive libraries can be assembled with a
minimal amount of starting DNA. Environmental fosmid libraries of
4.times.10.sup.7 clones of the present invention can be generated,
each containing approximately 40 kbp of cloned DNA, from 100 ng of
purified DNA collected from samples, including, for example, from
the microbial containment device described herein.
[0067] A potential problem with constructing libraries for the
expression of bioactive compounds in E. coli is that this
gram-negative bacterium may not have the appropriate genetic
background to express the compounds in their active form. One
aspect of the present invention allows the efficient cloning of
fragments in E. coli and the subsequent transfer to a different
suitable host for expression and screening. Shuttle vectors, which
allow propagation in two different types of hosts, can be utilized
in the present invention to clone and propagate in bacterial hosts,
such as E. coli, and transfer to alternative hosts for expression
of active molecules. Such alternative hosts may include but are not
limited to, for example, Streptomyces or Bacillus, or other
metabolically rich hosts such as Cyanobacteria, Myxobacteria, etc.
Streptomyces lividans, for example, may be used as the expression
host for the cloned pathways. This strain is routinely used in the
recombinant expression of heterologous antibiotic pathways because
it recognized a large number of promoters and appears to lack a
restriction system (Guseck, T. W. & Kinsella, J. E., (1992)
Crit. Rev. Microbiol. 18, 247-260).
[0068] In the present invention, the example below describes a
shuttle vector that can be utilized. The vector is an E.
coli-Streptomyces shuttle vector. This system allows one to stably
clone and express large inserts (40 kbp genome fragments).
Chromosomally integrated recombinants can be recovered as the
original fosmid to facilitate sequence characterization and further
manipulation of positive clones. Replicons which allow regulation
of the clone copy number in hosts can be utilized. For instance,
the SPC2 replicon, a 32 kb fertility plasmid that is present at one
copy p er c ell in Streptomyces coelicolor, can be utilized. This
replicon can be "tuned" by truncation to replicate at various copy
number in Streptomyces hosts. For instance, replicative versions of
integrative shuttle vectors may be designed containing the full
length and truncated SCP2 replicon which will regulate the clone
copy number in the Streptomyces host from 1 to 10 copies per cell.
In order to ensure that the bioactivity of the clones containing
the putative polyketide or other clustered genes is not due to the
activation of any resident gene cluster, the resident gene
sequences can be removed from the host strain by gene replacement
or deletion. An example is presented below.
[0069] Biopanning
[0070] After the expression libraries have been generated one can
include the additional step of "biopanning" such libraries prior to
transfer to a second host for expression screening. The
"biopanning" procedure refers to a process for identifying clones
having a specified biological activity by screening for sequence
homology in a library of clones prepared by (i) selectively
isolating target DNA, from DNA derived from at least one
microorganism, by use of at least one probe DNA comprising at least
a portion of a DNA sequence encoding an biological having the
specified biological activity; and (ii) transforming a host with
isolated target DNA to produce a library of clones which are then
processed for screening for the specified biological activity.
[0071] The probe DNA used for selectively isolating the target DNA
of interest from the DNA derived from at least one microorganism
can be a full-length coding region sequence or a partial coding
region sequence of DNA for a known bioactivity. The original DNA
library can be preferably probed using mixtures of probes
comprising at least a portion of the DNA sequence encoding a known
bioactivity having a desired activity. These probes or probe
libraries are preferably single-stranded and the microbial DNA
which is probed has preferably been converted into single-stranded
form. The probes that are particularly suitable are those derived
from DNA encoding bioactivities having an activity similar or
identical to the specified bioactivity that is to be screened.
[0072] The probe DNA should be at least about 10 bases and
preferably at least 15 bases. In one embodiment, an entire coding
region of one part of a pathway may be employed as a probe.
Conditions for the hybridization in which target DNA is selectively
isolated by the use of at least one DNA probe will be designed to
provide a hybridization stringency of at least about 50% sequence
identity, more particularly a stringency providing for a sequence
identity of at least about 70%.
[0073] Hybridization techniques for probing a microbial DNA library
to isolate target DNA of potential interest are well known in the
art and any of those which are described in the literature are
suitable for use herein, particularly those which use a solid
phase-bound, directly or indirectly bound, probe DNA for ease in
separation from the remainder of the DNA derived from the
microorganisms.
[0074] Preferably the probe DNA is "labeled" with one partner of a
specific binding pair (i.e., a ligand) and the other partner of the
pair is bound to a solid matrix to provide ease of separation of
target from its source. The ligand and specific binding partner can
be selected from, in either orientation, the following: (1) an
antigen or hapten and an antibody or specific binding fragment
thereof; (2) biotin or iminobiotin and avidin or streptavidin; (3)
a sugar and a lectin specific therefor; (4) a n enzyme and an
inhibitor therefor; (5) an apoenzyme and cofactor; (6)
complementary homopolymeric oligonucleotides; and (7) a hormone and
a receptor therefor. The solid phase is preferably selected from:
(1) a glass or polymeric surface; (2) a packed column of polymeric
beads; and (3) magnetic or paramagnetic particles.
[0075] Further, it is optional but desirable to perform an
amplification of the target DNA that has been isolated. In this
embodiment the target DNA is separated from the probe DNA after
isolation. It is then amplified before being used to transform
hosts. Long PCR (Barnes, W M, Proc. Natl. Acad. Sci, USA, (1994)
Mar. 15) can be used to amplify large DNA fragments (e.g., 35 kb).
The double stranded DNA selected to include as at least a portion
thereof a predetermined DNA sequence can be rendered single
stranded, subjected to amplification and reannealed to provide
amplified numbers of selected double stranded DNA. Numerous
amplification methodologies are now well known in the art.
[0076] The selected DNA is then used for preparing a library for
further processing and screening by transforming a suitable
organism. Hosts, particularly those specifically identified herein
as preferred, are transformed by artificial introduction of the
vectors containing the target DNA by inoculation under conditions
conducive for such transformation.
[0077] The resultant libraries of transformed clones are then
processed for screening for clones which display an activity of
interest. Clones can be shuttled in alternative hosts for
expression of active compounds, or screened using methods described
herein.
[0078] Having prepared a multiplicity of clones from DNA
selectively isolated from an organism, such clones are screened for
a specific activity and to identify the clones having the specified
characteristics.
[0079] The screening for activity may be effected on individual
expression clones or may be initially effected on a mixture of
expression clones to ascertain whether or not the mixture has one
or more specified activities. If the mixture has a specified
activity, then the individual clones may be rescreened for such
activity or for a more specific activity. Alternatively,
encapsulation techniques such as gel microdroplets, may be employed
to localize multiple clones in one location to be screened on a
FACS machine for positive expressing clones within the group of
clones which can then be broken out into individual clones to be
screened again on a FACS machine to identify positive individual
clones. Screening in this manner using a FACS machine is fully
described in patent application Ser. No. 08/876,276 filed Jun. 16,
1997. Thus, for example, if a clone mixture has a desirable
activity, then the individual clones may be recovered and
rescreened utilizing a FACS machine to determine which of such
clones has the specified desirable activity.
[0080] As described with respect to one of the above aspects, the
invention provides a process for activity screening of clones
containing selected DNA derived from a microorganism which process
comprises:
[0081] (a) screening a library for specified bioactivity, said
library including a plurality of clones, said clones having been
prepared by recovering from genomic DNA of a microorganism selected
DNA, which DNA is selected by hybridization to at least one DNA
sequence which is all or a portion of a DNA sequence encoding a
bioactivity having a desirable activity; and
[0082] (b) transforming a host with the selected DNA to produce
clones which are further processed and/or screened for the
specified bioactivity.
[0083] In one embodiment, a DNA library derived from a
microorganism is subjected to a selection procedure to select
therefrom DNA which hybridizes to one or more probe DNA sequences
which is all or a portion of a DNA sequence encoding an activity
having a desirable activity by:
[0084] (a) rendering the double-stranded genomic DNA population
into a single-stranded DNA population;
[0085] (b) contacting the single-stranded DNA population of (a)
with the DNA probe bound to a ligand under conditions permissive of
hybridization so as to produce a double-stranded complex of probe
and members of the genomic DNA population which hybridize
thereto;
[0086] (c) contacting the double-stranded complex of (b) with a
solid phase specific binding partner for said ligand so as to
produce a solid phase complex;
[0087] (d) separating the solid phase complex from the
single-stranded DNA population of (b);
[0088] (e) releasing from the probe the members of the genomic
population which had bound to the solid phase bound probe;
[0089] (f) forming double-stranded DNA from the members of the
genomic population of (e);
[0090] (g) introducing the double-stranded DNA of (f) into a
suitable host to form a library containing a plurality of clones
containing the selected DNA; and
[0091] (h) screening the library for the desired activity.
[0092] In another aspect, the process includes a preselection to
recover DNA including signal or secretion sequences. In this manner
it is possible to select from the genomic DNA population by
hybridization as hereinabove described only DNA which includes a
signal or secretion sequence. The following paragraphs describe the
protocol for this embodiment of the invention, the nature and
function of secretion signal sequences in general and a specific
exemplary application of such sequences to an assay or selection
process.
[0093] A particularly preferred embodiment of this aspect further
comprises, after (a) but before (b) above, the steps of:
[0094] (a i) contacting the single-stranded DNA population of (a)
with a ligand-bound oligonucleotide probe that is complementary to
a secretion signal sequence unique to a given class of proteins
under conditions permissive of hybridization to form a
double-stranded complex;
[0095] (a ii) contacting the double-stranded complex of (a i) with
a solid phase specific binding partner for said ligand so as to
produce a solid phase complex;
[0096] (a iii) separating the solid phase complex from the
single-stranded DNA population of (a);
[0097] (a iv) releasing the members of the genomic population which
had bound to said solid phase bound probe; and
[0098] (a v) separating the solid phase bound probe from the
members of the genomic population which had bound thereto.
[0099] The DNA which has been selected and isolated to include a
signal sequence is then subjected to the selection procedure
hereinabove described to select and isolate therefrom DNA which
binds to one or more probe DNA sequences derived from DNA encoding
a bioactivity having a desirable bioactivity.
[0100] This procedure of "biopanning" is described and exemplified
in U.S. Ser. No. 08/692,002, filed Aug. 2, 1996.
[0101] Further, it is possible to combine all the above embodiments
such that a normalization step is performed prior to generation of
the expression library, the expression library is then generated,
the expression library so generated is then biopanned, and the
biopanned expression library is then screened using a high
throughput cell sorting and screening instrument. Thus there are a
variety of options: i.e. (i) one can just generate the library and
then screen it; (ii) normalize the target DNA, generate the
expression library and screen it; (iii) normalize, generate the
library, biopan and screen; or (iv) generate, biopan and screen the
library.
[0102] The clones which are identified as having the specified
activity may then be sequenced to identify the DNA sequence
encoding a bioactivity having the specified activity. Thus, in
accordance with the present invention it is possible to isolate and
identify: (i) DNA encoding a bioactivity having a specified
activity, (ii) bioactivities having such activity (including the
amino acid sequence thereof) and (iii) produce recombinant
molecules having such activity.
[0103] Screening
[0104] The present invention offers the ability to screen for many
types of bioactivities. For instance, the ability to select and
combine desired components from a library of polyketides and
postpolyketide biosynthesis genes for generation of novel
polyketides for study is appealing. The method(s) of the present
invention make it possible to and facilitate the cloning of novel
polyketide synthases, and other relevant pathways or genes encoding
commercially relevant secondary metabolites, since one can generate
gene banks with clones containing large inserts (especially when
using vectors which can accept large inserts, such as the f-factor
based vectors), which facilitates cloning of gene clusters.
[0105] Preferably, the gene cluster or pathway DNA is ligated into
a vector, particularly wherein a vector further comprises
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. As previously indicated, this
f-factor of E. coli is a plasmid which affect high-frequency
transfer of itself during conjugation and is ideal to achieve and
stably propagate large DNA fragments, such as gene clusters from
mixed microbial samples. Other examples of vectors include cosmids,
bacterial artificial chromosome vectors (BAC vectors), and P1
vectors.
[0106] Gene expression libraries of the present invention,
capturing potential pathways encoding bioactive molecules of
interest can first be induced in prokaryotic cells to express
desirable precursors (e.g. backbone molecules which will be capable
of being modified) which can then be screened in another host
system which allows the expression of active molecules.
Particularly preferred prokaryotic cells are E. coli cells.
Alternatively, crude or partially purified extracts, or pure
proteins from metabolically rich cell lines can be combined with
the original gene expression libraries to create potentially active
molecules, which can then be screened for an activity of
interest.
[0107] For example, gene libraries can be generated in E. coli as a
host, and a shuttle vector as the vector, according to the examples
provided herein. These libraries may then be screened using
"hybridization screening". "Hybridization screening" is an approach
used to detect pathways encoding compounds related to previously
characterized small molecules that relies on the hybridization of
probes to conserved genes within the pathway. This approach appears
effective for the polyketide class of molecules that have highly
conserved regions within the polyketide synthase genes in the
pathway. Because of the highly conserved nature of these genes,
hybridization of probes to high density filter arrays of clones
from low complexity libraries is an effective approach to identify
clones carrying potential full length pathways. Alternatively,
multiplex PCR using primers designed against the conserved pathway
genes can be used on DNA pools from clones arrayed in microtiter
dish format.
[0108] Libraries made from complex communities require an
enrichment procedure to increase the likelihood of identifying by
hybridization any clones carrying homologous sequences. For
example, the .about.100 million base pairs of DNA immobilized on
the filter shown in FIG. 3 represents approximately 5-fold coverage
of 3 typical Streptomyces genomes. However, a gram of soil can
contain approximately 10.sup.6 bacterial cells representing over
10.sup.4 species. Screening a library made from such a sample would
require over 3,000 filters.
[0109] The biopanning approach described above can be used to
create libraries enriched with clones carrying sequences homologous
to a given probe sequence. Using this approach libraries containing
clones with inserts of up to 40 kbp can be enriched approximately
1,000 fold after each round of panning. This enables one to reduce
the above 3,000-filter fosmid library to 3 filters after 1 round of
biopanning enrichment. This approach can be applied to create
libraries enriched for clones carrying polyketide sequences.
[0110] Hybridization screening using high density filters or
biopanning has proven an efficient approach to detect homologues of
pathways containing conserved genes. To discover novel bioactive
molecules that may have no known counterparts, however, other
approaches are necessary. Another approach of the present invention
is to screen in E. coli for the expression of small molecule ring
structures or "backbones". Because the genes encoding these
polycyclic structures can often be expressed in E. coli the small
molecule backbone can be manufactured albeit in an inactive form.
Bioactivity is conferred upon transferring the molecule or pathway
to an appropriate host that expresses the requisite glycosylation
and methylation genes that can modify or "decorate" the structure
to its active form. Thus, inactive ring compounds, recombinantly
expressed in E. coli are detected to identify clones which are then
shuttled to a metabolically rich host, such as Streptomyces, for
subsequent production of the bioactive molecule. The use of high
throughput robotic systems allows the screening of hundreds of
thousands of clones in multiplexed arrays in microtiter dishes.
[0111] One approach to detect and enrich for clones carrying these
structures is to use FACS screening, a procedure described and
exemplified in U.S. Ser. No. 08/876,276, filed Jun. 16, 1997.
Polycyclic ring compounds typically have characteristic fluorescent
spectra when excited by ultraviolet light. Thus clones expressing
these structures can be distinguished from background using a
sufficiently sensitive detection method. High throughput FACS
screening can be utilized to screen for small molecule backbones in
E. coli libraries. Commercially available FACS machines are capable
of screening up to 100,000 clones per second for UV active
molecules. These clones can be sorted for further FACS screening or
the resident plasmids can be extracted and shuttled to Streptomyces
for activity screening.
[0112] In an alternate screening approach, after shuttling to
Streptomyces hosts, organic extracts from candidate clones can be
tested for bioactivity by susceptibility screening against test
organisms such as Staphylococcus aureus, E. coli, or Saccharomyces
cervisiae. FACS screening can be used in this approach by
co-encapsulating clones with the test organism (FIG. 5).
[0113] An alternative to the abovementioned screening methods
provided by the present invention is an approach termed "mixed
extract" screening. The "mixed extract" screening approach takes
advantage of the fact that the accessory genes needed to confer
activity upon the polycyclic backbones are expressed in
metabolically rich hosts, such as Streptomyces, and that the
enzymes can be extracted and combined with the backbones extracted
from E. coli clones to produce the bioactive compound in vitro.
Enzyme extract preparations from metabolically rich hosts, such as
Streptomyces strains, at various growth stages are combined with
pools of organic extracts from E. coli libraries and then evaluated
for bioactivity. A description of this is provided in the examples
below.
[0114] Another approach to detect activity in the E. coli clones is
to screen for genes that can convert bioactive compounds to
different forms. For example, a recombinant enzyme was recently
discovered that can convert the low value daunomycin to the higher
value doxorubicin. Similar enzyme pathways are being sought to
convert penicillins to cephalosporins.
[0115] FACS screening can also be used to detect expression of V
fluorescent molecules in metabolically rich hosts, such as
Streptomyces. Recombinant oxytetracylin retains its diagnostic red
fluorescence when produced heterologously in S. lividans TK24 (FIG.
6). Pathway clones, which can be sorted by FACS, can thus be
screened for polycyclic molecules in a high throughput fashion.
[0116] Recombinant bioactive compounds can also be screened in vivo
using "two-hybrid" systems, which can detect enhancers and
inhibitors of protein-protein or other interactions such as those
between transcription factors and their activators, or receptors
and their cognate targets. FIG. 7 depicts an approach to screen for
small molecules that enhance or inhibit transcription factor
initiation. Both the small molecule pathway and the GFP reporter
construct are co-expressed. Clones altered in GFP expression can
then be sorted by FACS and the pathway clone isolated for
characterization.
[0117] As indicated, common approaches to drug discovery involve
screening assays in which disease targets (macromolecules
implicated in causing a disease) are exposed to potential drug
candidates which are tested for therapeutic activity. In other
approaches, whole cells or organisms that are representative of the
causative agent of the disease, such as bacteria or tumor cell
lines, are exposed to the potential candidates for screening
purposes. Any of these approaches can be employed with the present
invention.
[0118] The present invention also allows for the transfer of cloned
pathways derived from uncultivated samples into metabolically rich
hosts for heterologous expression and downstream screening for
bioactive compounds of interest using a variety of screening
approaches briefly described above.
[0119] Recovering Desirable Bioactivities
[0120] After viable or non-viable cells, each containing a
different expression clone from the gene library are screened, and
positive clones are recovered, DNA is isolated from positive clones
utilizing techniques well known in the art. The DNA can then be
amplified either in vivo or in vitro by utilizing any of the
various amplification techniques known in the art. In vivo
amplification would include transformation of the clone(s) or
subclone(s) of the clones into a viable host, followed by growth of
the host. In vitro amplification can be performed using techniques
such as the polymerase chain reaction.
[0121] Evolution
[0122] One advantage afforded by a recombinant approach to the
discovery of novel bioactive compounds is the ability to manipulate
pathway subunits to generate and select for variants with altered
specificity. Pathway subunits can be substituted or individual
subunits can be evolved utilizing methods described below, to
select for resultant bioactive molecules with different
activities.
[0123] Clones found to have the bioactivity for which the screen
was performed can be subjected to directed mutagenesis to develop
new bioactivities with desired properties or to develop modified
bioactivities with particularly desired properties that are absent
or less pronounced in the wild-type activity, such as stability to
heat or organic solvents. Any of the known techniques for directed
mutagenesis are applicable to the invention. For example,
particularly preferred mutagenesis techniques for use in accordance
with the invention include those described below.
[0124] The term "error-prone PCR" refers to a process for
performing PCR under conditions where the copying fidelity of the
DNA polymerase is low, such that a high rate of point mutations is
obtained along the entire length of the PCR product. Leung, D. W.,
et al., Technique, 1:11-15 (1989) and Caldwell, R. C. & Joyce G
F., PCR Methods Applic., 2:28-33 (1992).
[0125] The term "oligonucleotide directed mutagenesis" refers to a
process which allows for the generation of site-specific mutations
in any cloned DNA segment of interest. Reidhaar-Olson, J. F. &
Sauer, R. T., et al., Science, 241:53-57 (1988).
[0126] The term "assembly PCR" refers to a process which involves
the assembly of a PCR product from a mixture of small DNA
fragments. A large number of different PCR reactions occur in
parallel in the same vial, with the products of one reaction
priming the products of another reaction.
[0127] The term "sexual PCR mutagenesis" (also known as "DNA
shuffling") refers to forced homologous recombination between DNA
molecules of different but highly related DNA sequence in vitro,
caused by random fragmentation of the DNA molecule based on
sequence homology, followed by fixation of the crossover by primer
extension in a PCR reaction. Stemmer, W. P., PNAS, USA,
91:10747-10751 (1994).
[0128] The term "in vivo mutagenesis" refers to a process of
generating random mutations in any cloned DNA of interest which
involves the propagation of the DNA in a strain of E. coli that
carries mutations in one or more of the DNA repair pathways. These
"mutator" strains have a higher random mutation rate than that of a
wild-type parent. Propagating the DNA in one of these strains will
eventually generate random mutations within the DNA.
[0129] The term "cassette mutagenesis" refers to any process for
replacing a small region of a double stranded DNA molecule with a
synthetic oligonucleotide "cassette" that differs from the native
sequence. The oligonucleotide often contains completely and/or
partially randomized native sequence.
[0130] The term "recursive ensemble mutagenesis" refers to an
algorithm for protein engineering (protein mutagenesis) developed
to produce diverse populations of phenotypically related mutants
whose members differ in amino acid sequence. This method uses a
feedback mechanism to control successive rounds of combinatorial
cassette mutagenesis. Arkin, A. P. and Youvan, D. C., PNAS, USA,
89:7811-7815 (1992).
[0131] The term "exponential ensemble mutagenesis" refers to a
process for generating combinatorial libraries with a high
percentage of unique and functional mutants, wherein small groups
of residues are randomized in parallel to identify, at each altered
position, amino acids which lead to functional proteins, Delegrave,
S. and Youvan, D. C., Biotechnology Research, 11:1548-1552 (1993);
and random and site-directed mutagenesis, Arnold, F. H., Current
Opinion in Biotechnology, 4:450-455 (1993).
[0132] All of the references mentioned above are hereby
incorporated by reference in their entirety. Each of these
techniques is described in detail in the references mentioned. DNA
can be mutagenized, or "evolved", utilizing any one or more of
these techniques, and rescreened to identify more desirable clones.
The invention will now be illustrated by the following working
examples, which are in no way a limitation thereof.
EXAMPLE 1
Sample Collection Using A Microbial Containment Device
[0133] Sample to be utilized for downstream nucleic acid isolation
for library generation may be collected according to the following
example:
[0134] The following represents a method of selective in situ
enrichment of bacterial and archaeal species while at the same time
inhibiting the proliferation of eukaryotic members of the
population.
[0135] In situ enrichment is achieved by using "traps" composed of
growth substrates and nutritional amendments with the intent to
lure, selectively, members of the surrounding environmental matrix,
coated onto surfaces. Choice of substrates (carbon sources) and
nutritional amendments (i.e., nitrogen, phosphorous, etc.) is
dependent upon the members of the community one desires to enrich.
Selective agents against eukaryotic members are also added to the
trap. Again, the exact composition will depend upon which members
of the community one desires to enrich and which members of the
community one desires to inhibit. Substrates include monomers and
polymers. Monomers of substrates, such as glucosamine, cellulose,
pentanoic or other acids, xylan, chitin, etc., can be utilized for
attraction of certain types of microbes. Polymers can also be used
to attract microbes that can degrade them. Some of the enrichment
"media" which may be useful in pulling out particular members of
the community is described below:
[0136] 1. Addition of bioactive compounds against fungi and
microscopic eukaryotes:
[0137] Proliferation of eukaryotic members of the community may be
inhibited by the use of one or more commercially available
compounds such as nystatin, cycloheximide, and/or pimaricin. These
compounds may be sprinkled as a powder or incorporated as a liquid
in the selective enrichment medium.
[0138] 2. Addition of bioactive compounds against other bacterial
species:
[0139] Compounds which inhibit the growth of some bacterial species
but not others (i.e., polymyxin, penicillin, and rifampin) maybe
incorporated into the enrichment medium. Use of the compounds is
dependent upon which members of the bacterial community one desires
to enrich. For example, while a majority of the Streptomyces are
sensitive to polymyxin, penicillin, and rifampin, these may be used
to enrich for "rare" members of the family which are resistant.
Selective agents may also be used in enrichments for archaeal
members of the community.
[0140] 3. Use of carbon sources as selective agents:
[0141] Any particular carbon source can be utilized by some members
of the community and not others. Carbon source selection thus
depends upon the members of the community one desires to enrich.
For example, members of the Streptomycetales tend to utilize
complex, polymeric substrates such as cellulose, chitin, and
lignin. These complex subtrates, while utilized by other genera,
are recalcitrant to most bacteria. These complex substrates are
utilized by fungi, which necessitates the use of anti-fungal
agents, mentioned above.
[0142] 4. Addition of nitrogen sources:
[0143] The use of additional nitrogen sources may be called for
depending upon the choice for carbon source. For example, while
chitin is balanced in its C:N ratio, cellulose is not. To enhance
utilization of cellulose (or other carbon-rich substrates), it is
often useful to add nitrogen sources such as nitrate or
ammonia.
[0144] 5. Addition of trace elements:
[0145] In general, the environmental matrix tends to be a good
source of trace elements, but in certain environments, the elements
may be limiting. Addition of trace elements may enhance growth of
some members of the community while inhibiting others.
[0146] Large surface area materials, such as glass beads or silica
aerogels can be utilized as surfaces in the present example. This
allows a high concentration of microbes to be collected in a
relatively small device holding multiple collections of
substrate-surface conjugates.
[0147] Glass beads can be derivitized with N-Acetyl
B-D-glucosamine-phenylisothiocyanate as follows:
[0148] Bead Preparation:
[0149] 30 ml glass beads (Biospec Products, Bartlesville, Okla.)
are mixed with 50 ml
[0150] APS/Toluene (10% APS) (Sigma Chemical Co.)
[0151] Reflux overnight
[0152] Decant and wash 3 times with Toluene
[0153] Wash 3 times with ethanol and dry in oven
[0154] Derivitize with N-Acetyl
B-D-glucosamine-phenylisothiocyanate as Follows:
[0155] Combine in Falcon Tube:
[0156] 25 ml prepared glass beads from above
[0157] 15 ml 0.1M NaHCO.sub.3+25 mg N-Acetyl-B-D-glucosamine-PITC
(Sigma Chemical Co.)+1 ml DMSO
[0158] Add 10 ml NaHCO.sub.3+1 ml DMSO
[0159] Pour over glass beads
[0160] Let shake in Falcon Tube overnight
[0161] Wash with 20 ml 0.1M NaHCO.sub.3
[0162] Wash with 50 ml ddH.sub.2O
[0163] Dry at 55EC for 1 hour
[0164] Beads can then be placed in mesh filter "bags" (Spectrum,
Houston, Tex.) created to allow containment of the beads, while
simultaneously allowing migration of microbes, which are then
placed in any device used as a solid support which allows
containment of the bag. Particularly preferred devices are made of
inert materials, such as plexiglass. Alternatively, beads can be
placed directly into Falcon Tubes (VWR, Fisher Scientific) which
have been punctured with holes using a needle. These "containment"
devices are then deployed in desired biotopes for a period of time
to allow attraction and growth of desirable microbes. The following
protocol details one method for generating a simple "microbial
containment device":
[0165] Puncture holes using a heated needle or other pointed device
into a 15 ml Falcon Tube (VWR, Fisher Scientific).
[0166] Place approximately 1-5 mls of the derivitized beads into a
Spectra/mesh nylon filter, such as those available from Spectrum
(Houston, Tex.) with a mesh opening of 70 .mu.m, an open area of
43%, and a thickness of 70 .mu.m. Seal the nylon filter to create a
"bag" containing the beads using, for instance, Goop, Houshold
Adhesive & Sealant.
[0167] Place the filter containing the beads into the ventilated
Falcon Tube and deploy the tube into the desired biotope for a
period of time (typically days).
EXAMPLE 2
DNA Isolation and Library Construction from Cultivated Organism
[0168] The following outlines the procedures used to generate a
gene library from an isolate, Streptomyces rimosus.
[0169] Isolate DNA.
[0170] 1. Inoculate 25 ml Trypticase Soy Broth (BBL Microbiology
Systems) in 250 ml baffled erlenmeyer flasks with spores of
Streptomyces rimosus. Incubate at 30.degree. C. at 250 rpm for 48
hours.
[0171] 2. Collect mycelin by centrifugation. Use 50 ml conical
tubes and centrifuge at 25.degree. C. at 4000 rpm for 10
minutes.
[0172] 3. Decant supernatent and wash pellet 2.times. with 10 ml
10.3% sucrose (centrifuge as above between washes).
[0173] 4. Store pellet at -20.degree. C. for future use.
[0174] 5. Resuspend pellet in 40 ml TE (10 mM Tris, 1 mM EDTA; pH
7.5) containing lysozyme (1 mg/ml; Sigma Chemical Co.) and incubate
at 37.degree. C. for 45 minutes.
[0175] 6. Add sarcosyl (N-lauroylsarcosine, sodium salt, Sigma
Chemical Co.) to final concentration of 1% and invert gently to mix
for several minutes.
[0176] 7. Transfer 20 ml of preparation to clean tube and add
proteinase K (Stratagene Cloning Systems) to a final concentration
of 1 mg/ml. Incubate overnight at 50.degree. C.
[0177] 8. Extract 2.times. with Phenol (saturated with TE).
[0178] 9. Extract 1.times. with Phenol:CH.sub.3Cl.
[0179] 10. Extract 1.times. with CH.sub.3Cl: Isoamyl alcohol.
[0180] 11. Precipitate DNA with 2 volumes of EtOH.
[0181] 12. Spool DNA on sealed pasteur pipet.
[0182] 13. Rinse with 70% EtOH.
[0183] 14. Dry in air.
[0184] 15. Resuspend DNA in 1 ml TE and store at 4.degree. C. to
rehydrate slowly.
[0185] 16. Check quality of DNA:
[0186] Digest 10 .mu.l DNA with EcoRI restriction enzyme
(Stratagene Cloning Systems) according to manufacturers protocol;
electrophorese DNA digest through 0.5% agarose, 20.degree.
overnight; stain gel in 1 g/ml EtBr
[0187] 17. Determine DNA concentration (A.sub.260-A.sub.280).
[0188] Restriction Digest DNA
[0189] 1. Incubate the following at 37.degree. C. for 3 hours:
[0190] 8 .mu.l DNA (.about.10 .mu.g)
[0191] 35 .mu.l H.sub.2O
[0192] 5 .mu.l 10.times. restriction enzyme buffer
[0193] 2 .mu.l EcoRI restriction enzyme (200 units)
[0194] Sucrose Gradient
[0195] 1. Prepare small sucrose gradient (Sambrook, Fritsch and
Maniatus, 1989) and run DNA at 45,000 rpm for 4 hours at 25.degree.
C.
[0196] 2. Examine 5 .mu.l of each fraction on 0.8% agarose gel.
[0197] 3. Pool relevant fractions and precipitate DNA with 2.5
volumes of EtOH for 1 hour at -70.degree. C.
[0198] 4. Collect DNA by centrifugation at 13,200 rpm for 15
minutes.
[0199] 5. Decant and wash with 1 ml of 70% EtOH.
[0200] 6. Dry, resuspend in 15 .mu.l TE.
[0201] 7. Store at 4.degree. C.
[0202] Dephosphorylate DNA
[0203] 1. Dephosphorylate DNA with shrimp alkaline phosphatase
according to manufacturers protocol (US Biochemicals).
[0204] Adaptor Ligation
[0205] 1. Ligate adaptors according to manufacturers protocol.
[0206] 2. Briefly, gently resuspend DNA in EcoR I-BamH I adaptors
(Stratagene Cloning Systems); add 10.times. ligation buffer, 10 mM
rATP, and T4 DNA ligase and incubate at room temperature for 4-6
hours.
[0207] Preparation of Fosmid Arms
[0208] 1. Fosmid arms can be prepared as described (Kim, et. al.,
Nucl. Acids Res., 20:10832-10835, 1992). Plasmid DNA can be
digested with PmeI restriction enzyme (New England Biolabs)
according to the manufacturers protocol, dephosphorylated
(Sambrook, Fritsch and Maniatus, 1989), followed by a digestion
with BamH I restriction enzyme (New England Biolabs) according to
the manufacturers protocol, and another dephosphorylation step to
generate two arms each of which contain a cos site in the proper
orientation for the cloning and packaging of ligated DNA between
35-45 kbp.
[0209] Ligation to Fosmid Arms
[0210] 1. Prepare the ligation reaction:
[0211] Add .about.50 ng each of insert and vector DNA to 1U of T4
DNA ligase (Boehringer Mannheim) and 10.times. ligase buffer as per
manufacturers instructions; add H.sub.2O if necessary, to total of
10 .mu.l.
[0212] 2. Incubate overnight at 16.degree. C.
[0213] Package and Plate
[0214] 1. Package the ligation reactions using Gigapack XL
packaging system (Stratagene Cloning Systems, Inc.) following
manufacturer's protocol.
[0215] 2. Transfect E. coli strain DH10B (Bethesda Research
Laboratories, Inc.) according to manufacturers protocol and spread
onto LB/Chloramphenicol plates (Sambrook, Fritsch and Maniatus,
1989).
EXAMPLE 3
Preparation of an Uncultivated Prokaryotic DNA Library
[0216] FIG. 1 shows an overview of the procedures used to construct
an environmental library from a mixed picoplankton sample. The goal
was to construct a stable, large insert DNA library representing
picoplankton genomic DNA.
[0217] Cell collection and preparation of DNA. Agarose plugs
containing concentrated picoplankton cells were prepared from
samples collected on an oceanographic cruise from Newport, Oreg. to
Honolulu, Hi. Seawater (30 liters) was collected in Niskin bottles,
screened through 10 .mu.m Nitex, and concentrated by hollow fiber
filtration (Amicon DC10) through 30,000 MW cutoff polysulfone
filters. The concentrated bacterioplankton cells were collected on
a 0.22 .mu.m, 47 mm Durapore filter, and resuspended in 1 ml of
2.times.STE buffer (1M NaCl, 0.1M EDTA, 10 mM Tris, pH 8.0) to a
final density of approximately 1.times.10.sup.10 cells per ml. The
cell suspension was mixed with one volume of 1% molten Seaplaque
LMP agarose (FMC) cooled to 40.degree. C., and then immediately
drawn into a 1 ml syringe. The syringe was sealed with parafilm and
placed on ice for 10 min. The cell-containing agarose plug was
extruded into 10 ml of Lysis Buffer (10 mM Tris pH 8.0, 50 mM NaCl,
0.1M EDTA, 1% Sarkosyl, 0.2% sodium deoxycholate, a mg/ml lysozyme)
and incubated at 37.degree. C. for one hour. The agarose plug was
then transferred to 40 mls of ESP Buffer (1% Sarcosyl, 1 mg/ml
proteinase-K, in 0.5M EDTA), and incubated at 55.degree. C. for 16
hours. The solution was decanted and replaced with fresh ESP
Buffer, and incubated at 55.degree. C. for an additional hour. The
agarose plugs were then placed in 50 mM EDTA and stored at
4.degree. C. shipboard for the duration of the oceanographic
cruise.
[0218] One slice of an agarose plug (72 .mu.l) prepared from a
sample collected off the Oregon coast was dialyzed overnight at
4.degree. C. against 1 ml of buffer A (100 mM NaCl, 10 mM Bis Tris
Propane-HCl, 100 g/ml acetylated BSA: pH 7.0 @ 25.degree. C.) in a
2 ml microcentrifuge tube. The solution was replaced with 250 .mu.l
of fresh buffer A containing 10 mM MgCl.sub.2 and 1 mM DTT and
incubated on a rocking platform for 1 hr at room temperature. The
solution was then changed to 250 .mu.l of the same buffer
containing 4U of Sau3A1 (NEB), equilibrated to 37.degree. C. in a
water bath, and then incubated on a rocking platform in a
37.degree. C. incubator for 45 min. The plug was transferred to a
1.5 ml microcentrifuge tube and incubated at 68.degree. C. for 30
min to inactivate the protein, e.g. enzyme, and to melt the
agarose. The agarose was digested and the DNA dephosphorylased
using Gelase and HK-phosphatase (Epicentre), respectively,
according to the manufacturer's recommendations. Protein was
removed by gentle phenol/chloroform extraction and the DNA was
ethanol precipitated, pelleted, and then washed with 70% ethanol.
This partially digested DNA was resuspended in sterile H.sub.2O to
a concentration of 2.5 ng/l for ligation to the pFOS1 vector.
[0219] PCR amplification results from several of the agarose plugs
(data not shown) indicated the presence of significant amounts of
archacal DNA. Quantitative hybridization experiments using rRNA
extracted from one sample, collected at 200 m of depth off the
Oregon Coast, indicated that planktonic archaea in (this assemblage
comprised approximately 4.7% of the total picoplankton biomass
(this sample corresponds to "PACI"-200 m in Table 1 of DeLong et
al., high abundance of Archaea in Antarctic marine picoplankton,
Nature, 371:695-698, 1994). Results from archaeal-biased rDNA PCR
amplification performed on agarose plug lysates confirmed the
presence of relatively large amounts of archaeal DNA in this
sample. Agarose plugs prepared from this picoplankton sample were
chosen for subsequent fosmid library preparation. Each 1 ml agarose
plug from this site contained approximately 7.5.times.10.sup.5
cells, therefore approximately 5.4.times.10.sup.5 cells were
present in the 72 .mu.l slice used in the preparation of the
partially digested DNA.
[0220] Vector arms are prepared from pFOS1 as described (Kim et
al., Stable propagation of cosmid sized human DNA inserts in an F
factor based vector, Nucl. Acids Res., 20:10832-10835, 1992).
Briefly, the plasmid is completely digested with AstII,
dephosphorylated with HK phosphatase, and then digested with BamHI
to generate two arms, each of which contains a cos site in the
proper orientation for cloning and packaging ligated DNA between
35-45 kbp. The partially digested picoplankton DNA is ligated
overnight to the pFOS1 arms in a 15 .mu.l ligation reaction
containing 25 ng each of vector and insert and 1U of T4 DNA ligase
(Boehringer-Mannheim). The ligated DNA in four microliters of this
reaction is in vitro packaged using the Gigapack XL packaging
system (Stratagene), the fosmid particles transfected to E. coli
strain DH10B (BRL), and the cells spread onto LB.sub.cm15 plates.
The resultant fosmid clones are picked into 96-well microliter
dishes containing LB.sub.cm15 supplemented with 7% glycerol.
Recombinant fosmids, each containing 40 kb of picoplankton DNA
insert, have yielded a library of 3,552 fosmid clones, containing
approximately 1.4.times.10.sup.8 base pairs of cloned DNA. All of
the clones examined contained inserts ranging from 38 to 42 kbp.
This library is stored frozen at -80.degree. C. for later
analysis.
EXAMPLE 4
Normalization of DNA from Environmental Samples
[0221] Prior to library generation, purified DNA from an
environmental sample can be normalized. DNA is first fractionated
according to the following protocol:
[0222] Sample composed of genomic DNA is purified on a
cesium-chloride gradient. The cesium chloride (Rf=1.3980) solution
is filtered through a 0.2 .mu.m filter and 15 ml is loaded into a
35 ml OptiSeal tube (Beckman). The DNA is added and thoroughly
mixed. Ten micrograms of bis-benzimide (Sigma; Hoechst 33258) is
added and mixed thoroughly. The tube is then filled with the
filtered cesium chloride solution and spun in a VTi50 rotor in a
Beckman L8-70 Ultracentrifuge at 33,000 rpm for 72 hours. Following
centrifugation, a syringe pump and fractionator (Brandel Model 186)
are used to drive the gradient through an ISCO UA-5 LV absorbance
detector set to 280 nm. Peaks representing the DNA from the
organisms present in an environmental sample are obtained.
[0223] Normalization is then accomplished as follows:
[0224] 1. Double-stranded DNA sample is resuspended in
hybridization buffer (0.12 M NaH.sub.2PO.sub.4, pH 6.8/0.82 M
NaCl/1 mM EDTA/0.1% SDS).
[0225] 2. Sample is overlaid with mineral oil and denatured by
boiling for 10 minutes.
[0226] 3. Sample is incubated at 68.degree. C. for 12-36 hours.
[0227] 4. Double-stranded DNA is separated from single-stranded DNA
according to standard protocols (Sambrook, 1989) on hydroxyapatite
at 60.degree. C.
[0228] 5. The single-stranded DNA fraction is desalted and
amplified by PCR.
[0229] The process is repeated for several more rounds (up to 5 or
more).
EXAMPLE 5
Hybridization Screening of Libraries Generated in Prokaryotes and
Expression Screening in Metabolically Rich Hosts
[0230] Hybridization screening may be performed on fosmid clones
from a library generated according to the protocol described in
Example 3 above in any fosmid vector. For instance, the pMF3 vector
is a fosmid-based vector which can be used for efficient yet stable
cloning in E. coli and which can be integrated and maintained
stably in Streptomyces coelicolor or Streptomyces lividans. A pMF3
library generated according to the above protocol is first
transformed into E. coli DH10B cells. Chloramphenicol resistant
transformants containing tcm or oxy are identified by screening the
library by colony hybridization using sequences designed from
previously published sequences of oxy and tcm genes. }(27, }28)
Colony hybridization screening is described in detail in "Molecular
Cloning", A Laboratory Manual, Sambrook, et al., (1989) 1.90-1.104.
Colonies that test positive by hybridization can be purified and
their fosmid clones analyzed by restriction digestion and PCR to
confirm that they contain the complete biosynthetic pathway.
[0231] Alternatively, DNA from the abovementioned fosmid clones may
be used in an amplification reaction designed to identify clones
positive for an entire pathway. For example, the following
sequences may be employed in an amplification reaction to amplify a
pathway encoding the antibiotic gramicidin (gramicidin operon),
which resides on a 34 kbp DNA fragment potentially encoded on one
fosmid clone:
[0232] Primers:
1 SEQ ID NO:1 5'-CACACGGATCCGAGCTCATCGATAGGCATGTGTTTAACTTCT- TGTC
ATC-3' SEQ ID NO:2
5'-CTTATTGGATCCGAGCTCAATTGCTGAAGAGTTGAAGGAGAGCATC TTCC-3'
[0233] Amplification Reaction:
[0234] 1 .mu.l fosmid/insert DNA
[0235] 5 .mu.l each primer (50 .mu.g/.mu.l)
[0236] 1 .mu.l Boehringer Mannheim EXPAND Polymerase from their
EXPAND kit
[0237] 1 .mu.l dNTP=s
[0238] 5 .mu.l 10.times. Buffer #3 from Boehringer Mannheim EXPAND
kit
[0239] 30 .mu.l ddH.sub.2O
[0240] PCR Reaction Program:
[0241] 94.degree. C. 60 seconds
[0242] 20 cycles of:
[0243] 94.degree. C. 10 seconds
[0244] 65.degree. C. 30 seconds
[0245] 68.degree. C. 15 minutes
[0246] one cycle of:
[0247] 68.degree. C. 7 minutes
[0248] Store at 4.degree. C.
[0249] Fosmid DNA from clones that are shown to contain the
oxytetracycline or tetracenomycin polyketide encoding DNA sequences
are then used to transform S. lividans TK24 Dact protoplasts from
Example 6. Transformants are selected, by overlaying regeneration
plates with hygromycin (pMF5). Resistant transformants are screened
for bioactivity by overlaying transformation plates with 2 ml of
nutrient soft agar containing cells of the test organisms
Escherichia coli or Bacillus subtilis. E. coli is resistant to the
thiostrepton concentration (50 mg/ml) to be used in the overlays of
pMF3 clones but is sensitive to oxytetracylin at a concentration of
5 mg/ml}(29). The B. subtilis test strain is rendered resistant to
thiostrepton prior to screening by transforming with a thiostrepton
marker carried on pHT315} (30). Bioactivity is demonstrated by
inhibition of growth of the particular test strain around the S.
lividans colonies. To confirm bioactivity, presumptive active
clones are isolated and cultures extracted using a moderately polar
solvent, methanol. Extractions are prepared by addition of methanol
in a 1:1 ratio with the clone fermentation broth followed by
overnight shaking at 4.degree. C. Cell debris and media solids in
the aqueous phase are then be separated by centrifugation.
Recombinantly expressed compounds are recovered in the solvent
phase and may be concentrated or diluted as necessary. Extracts of
the clones are aliquoted onto 0.25-inch filter disks, the solvent
allowed to evaporate, and then placed on the surface of an overlay
containing the assay organisms. Following incubation at appropriate
temperatures, the diameter of the clearing zones is measured and
recorded. Diode array HPLC, using authentic oxytetracyclin and
tetracenomycin as standards, can be used to confirm expression of
these antibiotics from the recombinant clones.
[0250] Rescue of Chromosmally Integrated Pathways
[0251] Sequence analysis of chromosomally integrated pathways
identified by screening can be performed for confirmation of the
bioactive molecule. One approach which can be taken to rescue
fosmid DNA from S. lividans clones exhibiting bioactivity against
the test organisms is based on the observation that plasmid vectors
containing IS117, such as pMF3, are present as circular
intermediates at a frequency of 1 per 10-30 chromosomes. The
presumptive positive clones can be grown in 25 ml broth cultures
and plasmid DNA isolated by standard alkaline lysis procedures.
Plasmid DNA preps are then used to transform E. coli and
transformants are selected for Cm.sup.r by plating onto LB
containing chloramphenicol (15 mg/ml). Fosmid DNA from the E. Coli
Cm.sup.r transformants is isolated and analyzed by restriction
digestion analysis, PCR, and DNA sequencing.
EXAMPLE 6
Host Strain Construction
[0252] The following example describes modifications that can be
performed on the Streptomyces lividans strain to make it useful for
screening bioactive clones originally identified in E. coli
according to Example 5.
[0253] Streptomyces lividans is a strain that is routinely used in
the recombinant expression of heterologous antibiotic pathways
because it recognizes a large number of promoters and appears to
lack a restriction system. Although Streptomyces lividans does not
normally produce the polyketide antibiotic actinorhodin, it
contains the requisite gene sequences, and several genes have been
identified that activate its production in S. lividans. One strain
of S. lividans, TK24, can be utilized as a host for screening for
bioactive clones. This strain contains a mutation in the rpsL gene,
encoding ribosomal protein S12, which confers resistance to
streptomycin and activates the production of actinorhodin. In order
to ensure that the bioactivity of S. lividans clones containing
putative polyketide or other antibiotic genes is not due to the
activation of the resident act gene cluster, these sequences should
be removed from host strain by gene replacement. The outline for
the gene replacement scheme is shown in FIG. 8. Gene fragments
internal to actVI and actVB, which define the boundaries of the act
cluster, are amplified by PCR. The primers used for the
amplification have recognition sequences designed within them so
that they are cloned in the proper orientation respective to each
other and the act cluster. The actVB and actVI gene fragments are
cloned into pLL25 so that they flank the spectinomycin encoding
gene, generating pRBSV2. S. lividans TK24 protoplasts are
transformed with pRBSV2 using established transformation protocols
and transformants are selected for spectinomycin resistance. As
shown in FIG. 9, Spc.sup.r transformants can arise as a result of
several recombination events. Single recombination events within
actVI or actVB (events 1 and 2) result in the insertion of the
plasmid construct within the act cluster. A double crossover within
actVI and actVB (recombination event 3) results in the replacement
of the act cluster with the Spc.sup.r encoding gene. While both
types of recombinations can generate an Act.sup.- strain, the
present example focuses on the construction of a strain containing
the gene replacement. This is advantageous for two reasons: first,
it generates a stable Act.sup.- strain that cannot revert to
Act.sup.+ by recombination between repeated sequences, and second,
it decreases the amount of potential homology between cloned
sequences and the chromosome, and decreases the likelihood of
cloning partial pathways. Because the actinorhodin antibiotic is
pigmented, one is able to distinguish the different classes of
recombinants based on the pigment produced by the Spc.sup.r
transformants. Only Spc.sup.r transformants that are generated by
double recombination are non-pigmented. S. lividans TK24 clones
that have the act cluster replaced by spc are confirmed by Southern
hybridization and PCR analysis using standard techniques.
EXAMPLE 7
Screening of Large Insert Library for Compounds of Interest
[0254] Large insert libraries generated according to Examples 1 and
3 can be screened for potentially clinically valuable compounds of
interest using the following method(s):
[0255] Organic Extraction of Fosmid Library Clones (Aqueous):
[0256] Add equal volume of Methyl-Ethyl-Ketone (MEK)(Sigma Chemical
Co.) to each well of the microtiter plate from Example 3. Transfer
MEK phase to new plates. Spin plates to dry down. Resuspend
sample(s) in TN Buffer (50 mM Tris-7, 10 mM NaCl).
[0257] Protein Extraction of Streptomycine
[0258] 1. Inoculate 25 ml Trypticase Soy Broth (BBL Microbiology
Systems) in 250 ml baffled Erlenmeyer flasks with spores of
Streptomyces lividans TK24. Incubate at 30.degree. C. at 225 rpm
for 48 hours.
[0259] 2. Spin @ 4000 rpm in 50 ml conical to pellet cells (15
minutes).
[0260] 3. Pour off supernatant and reserve.
[0261] 4. Microscopically check pellet and supernatant.
[0262] 5. Sonicate pellet
[0263] 6. Pellet cell debris 4000 rpm/15 minutes (reserve).
[0264] 7. Pull off supernatant.
[0265] 8. Dialyze against 80% saturated Ammonium Sulfate solution
according to manufacturers instructions (Slide-A-Lyzer.TM. Dialysis
from Pierce.
[0266] 9. Spin prep at 2500 rpm for 15 minutes.
[0267] 10. Spin prep again at 3500 rpm for another 15 minutes.
[0268] 11. Pull of supernatant and reserve.
[0269] 12. Add 1 ml TN buffer (50 mM Tris pH 7; 100 mM NaCl)
[0270] In 1.5 ml screw caps, combine 50 .mu.l aqueous extract from
fosmid clones with 50 .mu.l protein extract of Streptomycine (1:1
ratio) in assay wells.
[0271] Use different ratios of aqueous extract:protein extract (1:1
as indicated above, 3:1, etc.), as desired.
[0272] Incubate at 30.degree. C. for 4 hours.
[0273] Bioassay
[0274] 1. Spot 20 .mu.l of sample onto filter disk.
[0275] 2. Lay filter disk on previously generated assay plate
(growth plate containing appropriate media to grow organism of
interest, with an overlay of .about.10D 600 of cells of test
organism solidified into soft agar). Grow cells overnight at the
appropriate incubation temperature for the test organism to grow.
Identify clearing zones for positive results (inhibition of
growth).
Sequence CWU 1
1
9 1 49 DNA Artificial Sequence oligonucleotide for PCR 1 cacacggatc
cgagctcatc gataggcatg tgtttaactt cttgtcatc 49 2 50 DNA Artificial
Sequence oligonucleotide for PCR 2 cttattggat ccgagctcaa ttgctgaaga
gttgaaggag agcatcttcc 50 3 60 DNA Streptomyces saccharopo 3
gccgccgaca ccccgatcac gccgatcgtg gtgtcctgct tcgacgccat caaggcgacc
60 4 59 DNA Streptomyces coelicolor 4 gccgccgaca ccccgatcac
cccgatcgtc gtcgcctgct tcgacgcgat ccgcgccac 59 5 60 DNA Streptomyces
gvenzuelae 5 tcctcggacg ccccgatctc cccgatcacg atggcctgct tcgacgccat
caaggcgacc 60 6 60 DNA Streptomyces fraidiae 6 gcggccgacg
ccccgatctc gcccatcacc gtggcctgct tcgatgcgat caaggcgacc 60 7 59 DNA
Streptomyces glaucescen 7 gccaccgacg cgccgatctc ccccatcacc
gtggcctgct tcgacgccat caaggcgac 59 8 60 DNA Streptomyces griseus 8
gcggtggacg cgccgatcac cccgctcacg atggcggcct tcgacgcgat ccgcgccacc
60 9 60 DNA Escherichia coli 9 ggcgcagaga aagccagtac gccgctgggc
gttggtggtt ttggcgcggc acgtgcatta 60
* * * * *