Note: Descriptions are shown in the official language in which they were submitted.
~203~5
TITL~
CLONED STREPTOMYCETE GENE
FIELD OF THE INVEMTION
This invention relates to the field of
biotechnology, specifically to genetic engineering. More
particularly, the invention relates to cloning of genes
coding for ~-galactosidase and for another excreted
protein from a Streptomyces`species and a promotor
therefor onto suitable vectors, expression of such cloned
genes in other microorganisms, the method of detecting and
identifying such ~-galac~osidase gene by monitoring
B-galactosidase which i~ excreted into the growth medium
and the use of such cloned genes ~or various genetic
engineering purposes.
BACKGROUND INFORMATION
Although the Actinomycetales produce more than
half of the known antibiotics having valuable clinical and
other applications as secondary metabolites and, thus/ are
recognized as a key target for application of gene
manipulation techniques, many problems remain to be
overcome before specific useful genes are successfully
.~
3i~
- 2 -
1 identified and cloned E "Molecular Breeding and Genetics of
Applied Microorganisms", Sakaguchi and Okanishi, eds.,
Academic Press (New York) Kodansha Ltd. (Toyko) 1980, pgs.
130-131]. Until the present work, clo~ing of a
~-galactosidase gene from a Streptomyces species onto a
suitable vector followed by introduction and expression of
such vector has not been reported. Prior work has
concerned development of other cloning systems or vectors
for Streptomycetes lBibb et al. (197R), Nature
274:398-400; Hayakawa et al. (1979), J. Antibiot.
XXXII(12):1348-1350; Okanishi et al. (1980), J. Antibiot.
XXXIII(1):88-91; Bibb et al. (1980), Nature 284:526-531;
Thompson et al. (1980), Nature 286 525-5~7; Suarez et al.
(1980), Nature 286:527-529; Bibb et al. (1981), Mol. Gen.
Genet. 184:230-2~0]; Bibb ~1981), qMicrobiology-1981",
Schlessinger, ed., American Society for ~icrobiology,
(Washington, D. C.) 1981, pgs. 367-370 and Hopwood et al.
(1981)~ "Microbiology-1981", suPra~ pgs. 376-379], cloning
and expression in Streptomyces ~e. of genes derived from
Escherichia coli lSchottel et al. ~1981), JO Bacteriol.
146:360-368] and cloning of genes ~rom Streptomycetes in
Escherichia coli ["Molecular Breeding and Genetics of
Applied Microorganisms", supra; pgs. 130-137]. Chater et
al. (1982), Current Topics in Microbiol. and Immunol.
96:69-95, review gene cloning in Streptomyces.
Work with various ~-galactosidase genes, their
expression and application of such expression as an assay
or detectio~ method has been reported by Rose et al.
(19811, Proc. Natl. Acad. Sci. USA 78(4):2460-2464, for
expression in yeast of yeast genes fused to
~-galactosidase genes from Escherichia coli; by Casadaban
et al. (1980), J. Mol. ~iol. 138:179-207, for fu~ion of
~-galactosidase genes to promotors in Escherichia coli and
.~,
1~31~
1 assay following transformation; and by Talmadge et al.
(1981), Nature 294:176-178, for construction of
Escherichia coli containing a plasmid encoding a
~-galactosidase-preproinsulin fusion protein.
In general, the activity of promotors can be
assayed by measuring the amount of gene product which is
formed as a consequence o~ transcription starting from a
specific promotor. The amount of gene product formed is
determined by using a specific property of that gene
product, such as enzymatic activity. In studying gene
expression or in constructing high expression vectors
which rely on highly efficient promotorsl the gene which
is na~urally expressed from such a promotor is replaced by
the structural gene whose product can be more easily
monitored. The lacZ gene from Escherichia coli lCasadaban
et al. ~1980), ~ .] is frequently used for ~his
purpose.
A variety of chromogenic substr.ates, such as
S-bromo-4-chloro-3-indolyl-~-D-galactoside (referred to as
"X-gal") or o-nitrophenyl-~-D-galactoside (referred to as
"ONPG") can be used to monitor enzymatic activity as
described by Miller (1972), "Experiments in Molecular
Genetics", Cold Spring Harbor Laboratories (Cold Spring
Harbor, New York). These substrates are advantageous
since the efficiency of a promotor fused to a aene coding
for an enzyme which can react with the substrate, such as
the lacZ gene, can be monitored by growing the organism on
a solid agar medium containing the substrate and observing
for enzyme-substrate reaction. In this manner r several
hundred individual colonies can be scored at one time for
their ability to express the gene~ Thus, relatively rare
events such as the occurrence of a highly efficient
promotor can be detected. ~-Galactosidase expression can
be used in such a procedure to assay gene transcription
and to detect and isolate mutants which over-produce a
1; :03~S
1 particularly desired protein, such as an enzyme involved
in antibio-" c production.
In order to effectively use such a powerful
approach as described above, it is crucial that the chosen
substrate has the opportunity to react with the enzyme.
If, as in the case of B-galactosidase produced by
Escherichia coli, the enzyme is intraceLlular, the
substrate must enter the cell in order for the
enzyme-substrate reaction to occur. With Actinomycetes,
however, the commonly used substrates, X-gal and ONPG,
enter the cell only poorly as verified by comparing the
intracellular B-galactosidase activity of a suitable
organism with dye formation on plates. For example, we
have found that although intracellular activity as
measured with cell extracts and with ~NPG as substrate was
very high (300 nmoles/mg protein/min), no significant
color reaction with whole cells and with either ONPG or
X-gal was found. Furthermore, Actinomycetes differ from
many other microorganisms by the formation of an aerial
mycelium which separates the cells physically ~ro~ the
substrate, thus further restricting access of the
substrate to the cells [Kalakouts~ii et al. (1976~,
Bacteriol. Rev. 40(2~:469 524].
SUMMARY OF THE INVENTIOM
The invention lies in the discovery that certain
strains of Streptomyces s~. prod~ce an excretable
B-galactosidase. The enzyme is useul for degradation of
certain B-galactosides, such as lactose, and can be used
as a diagnostic or laboratory reagent.
Another embodiment of the invention is a DNA
fragment comprising a gene naturally present in
Streptomyces sp. which can code fof an excretable
B-galactosidase. The gene can be readily expressed in a
variety of Streptomycetes by inserting it onto a suitable
vector. The DNA fragment contains at least the nucleotide
~2~)3il~5
-- 5 --
1 sequence which codes for excretable ~-galactosidase.
Other nucleotides, including, for example, promotor
sequences, from the donor organism or other sources, can
also be present.
Another embodiment of the invention is a DNA
fragment from Streptomyces sp. comprising a gene which can
code for an excretable protein, the Bgl protein, said gene
being naturally present in Streptomyces ~. located
immediately upstream of the Streptomyces fl-galactosidase
gene.
Another embodiment of the invention is a DNA
fragment comprising the Streptomyces ~-galactosidase
promotor.
Another embodiment of the invention is a
procedure for assaying promotion of gene expression
comprising transforming a microorganism with the
~-galactosidase gene fused to a promotor and assaying
excreted B-galactosidase by use of a chromogenic substrate
not readily taken up in the host cells. ~s the
~-galactosidase of the invention is excreted b~ the
microorganisms, detection and assay of the enzyme are
significantly advantageous and useful over, for example,
detection and assay of non-excretable ~-galactosidases.
Since a variety of chromogenic substrates are available
for ~-galactosidase which may be used on plates, assay,
identification and obtention of high expression cloning
vectors and potent promotors are facilitated. The cloned
D~A fragment, which is readily expressed in a variety of
Streptomyces sp. and other microorganisms, can be easily
used to construct high expression vectors.
Another embodiment of the invention is a vector,
that is, a recombinant DNA molecule, containing one or
more of the DNA fragments described above. The invention
also comprises microorganisms transformed ~ith the DNA
fragments of the invention, including such vectors.
~ll;ZCi3~
l Another embodiment of the in~ention is a method
of expressing genes in microorganisms comprising fusing
the Streptomyces 13-galactosidase promotor to a gene coding
~or a protein and transforming a suitable microorganism
therewith.
Yet another embodiment of this invention is
excretable protein fusions which can be prepared by fusing
a DNA fragment carrying the gene coding for excretable
~-galactosidase or a DNA fragment carrying the gene coding
for the Bgl protein, or excret:ion signals derived rom
either, to a gene coding for another protein and using
such fused genes to transform a suitable host. By adding
the information necessary for excretion which is present
on the ~-yalactosidase gene and on the Bgl protein gene
isolated and cloned as part of this invention, other gene
products can be generated which can likewise be excreted
into the growth medium and, thus, more readily recovered
from cellular material than non-ex~reted gene products.
Still another embodiment of this invention is the use of a
DNA fragment carrying the gene coding for ~-galactosidase
or the ~ene coding for the Bgl protein as a probe for the
presence of homologous DNA sequences from any source by
DNA hydridization techniques. ~ll o~ these embodiments of
the invention, as well as others described throughout, are
readily attainable uses of this invention and are
considered as further aspects of the same invention.
The DNA fragment carrying the ~-galactosidase
gene isolated in carrying out and as part of this
invention is also useful as an easily identifiable genetic
trait for labeling plasmids and for studying plasmid
behavior such as curing procedures, stability, mechanisms
of instability, plasmid incompatibiliiy and the likeO It
is useful for various recombinant DNA experiments, for
example, as a marker for gene cloning, since DNA deletions
or insertions into the gene can be easily iden~ified.
~2~3~
1 BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a restriction endonuclease cleavage
map of an 8 kilobase DNA fragment from Streptomyces
lividans carryins the gene for excretable B-galackosidase,
which fragment was obtained in carrying out Example 1.
~igure 2 is a restriction endonuclease c}eavage
map of pSKL-l.
DISCLOSURE OF THE INVENTION
~-galactosidase produced in accordance with this
invention is a protein having B-galactosidase activity.
It has been found to be excreted by certain Streptomyces
cultures in a number of forms having differing molecular
weights depending, in some cases, on the producing strain
and can be isolated from other excreted products.
Described below are various DNA fragments of
Streptomyces origin which have been discovered to produce
an excretable ~-galactosidase. It is appreciated that
derivatives of the disclosed fragments may also produce a
~-galactosidase. Such derivatives are included within the
invention. Moreover, it is understood that fragments
similar to those disclosed herein, such as fragments
differing by the presence or absence of one or more
deoxyribonucleotides, including, perhaps, one or more
restriction enzyme sites, which differences do not
materially affect the fragments, are included within the
invention.
The DNA fragments of the invention are
recombinant DNA molecules, that is, DNA sequences~ single
or double stranded, that have been isolated from the
larger molecules in which they are naturally present, such
as chromosom~l DNA, or from their natural hosts, and which
may be fused to other DNA fragments, such as to form
expression units or cloning or expression vectors.
The B-galactosidase gene, that is, the gene which
codes for a protein having ~-galactosidase activlty~ i6
~ 3~
1 located on a 10.3 kb SDh I fragment. The fragment is
naturally present in and was originally isolated from
chromosomal DNA of S. lividans strain 1326. An 8.7 kb
I-Pst I fragment thereof (See, Fig. 1) also codes for an
excreted B-galactosidase. The SRh I-Pst I fragment has
been mapped as follows:
Restriction Location
Enzyme (kb3
ph I
Bam HI 0.50
Pvu II 0,72
Bal } 1.19
Stu I 1.37
Sal I 1.70
Bcl I 2.10
Bgl II 2047
~gl II 3.39
Pvu II 5.50
Nru I 6.25
Pvu II 7.00
Bcl 7-45
Stu I - 7.50
Pst I 8.69
This table will be used for further referPnces
herein to DNA fragments naturally present within the
~ Pst I fragment. So, for example, the 0.65 kb Pvu II
~0.72) - Stu I ~1.37) fragment will be referred to as such
whether or not there are additional deoxyribonucleotides
upstream and/or downstream thereof.
The 5.3 kb ~ II (3.39) - Pst I fragment codes
for an excretable B-galactosidase, as does the larger 6.9
kb ~ II (3.39) - Sph I (10.3) ~ragment~ The 2.20 kb B~
Bgl II (3.39~ fragment codes fQr an excreted protein ~hich
`o
1~33~35
g
1 is not a ~-galactosidase~ The start codan of the Bgl protein
is believed to be about 30 bases downstream of the Bal I site.
This protein is referred to as the Bgl protein.
It appears that the Bgl protein gene, which is
S immediately upstream of the ~-galactosidase gene, and ~he
~-galactosidase gene comprise a polycistronic message
promoted from the Sph I - Bgl II ~.47) fragment. It
further appears that the promotor is located within or
near the Pvu II (0.72) - Stu I (1.37) fragment, based on
expression from the Pvu II (7.20) site relative to
expression from the Stu I (1.37) site and from the Sph I
(O) site.
Both the ~gl protein and B-galactosidase genes,
or portions containing excretion signals therefrom, can be
fused to a protein which is not normally excreted, to
produce a fusion protein which will be excreted. Such
technique is described, for example, by Silhavy et al.,
UOS. Patent 4,336,336.
Both can also be used to test p~omotor activity.
The n-galactosidase gene is preferred for such procedure
because of the ease with which its expression can be
detected~ The ~-galactosidase gene can also be used as a
selection marker.
The B-galactosidase promotor can be used to
express genes, including heterologous genes, in
Streptomyces and in other microorganisms.
To carry out this invention, DNA from
Strep~omyces lividans strain 1326 lNational Collection of
Industrial Bacteria, Aberdeen, Scotland, number 11416;
Bib~ et al., (1981), Mol. Gen. Genetics 184:230-240;
Krasilnikov et al., "The Biology of Certain Groups of
Actinomycetes~, Krasilnikov, ed., Science Press (Moscow)
1965, pgs. 109-110, which contains a gene which codes for
~-~alactosidase which is naturally excreted in its
original strain, is collec~ed by standard te~hniques, such
,~
3~5
-- 10 --
1 as the technique described by Chater et al., su~ra. A DNA
fragment containing the gene which codes for an excretable
~-galac~osidase is isolated by treating the DNA with a
restriction endonuclease.
If the e~nzyme-substrate reaction yields a poorly
diffusible d~e, enzymatic activity can be monitored by tile
formation of a halo of colored dye around a producing
colony on an agar plate when assaying by this procedure.
The preferred chromogenic substrate is X-gal because the
product is such a poorly diffusible dye~ The sensitivity
of this procedure is such that one producing colony among
300 to 500 colonies can be identified on a single petri
dlsh (90 mm diameter).
The genes isolated as described above, and which
originated from Streptomyces lividans 1326, can be readily
expressed in other strains and species of Streptomyces
such as Streptomyces griseus, Streptomyces aureofaciens,
Streptomyces fradiae, Streptomyces niveus and others as
well as other microorganisms. Streptomyces lividans and
Streptomyces griseus are the preferred host species.
A variety of vectors are useful in this
invention, the choice of an advantageous one being within
the ken of one skilled in the relevant art. Examples of
usable vectors are pLasmids pIJ6 [Thompson et al. (1980),
Nature 2~6:525-527], pIJ101 [Chater et al. supra] and
others which are capable of replicating in the ultimate
host strain and permit facile selection for the presence
of the vector in such strain. Likewise, various standard
growth media can be employed. The plasmid, pIJ6, is the
preferred vector.
Incorporation of a plasmid vector containing the
desired DNA fragment into microorganisms can be
accomplished by usual transformation methods, although
other procedures such as transduction or conjugation may
be used with suitable hostsD Such procedures are
described in and known to the art.
~2C~3~35
1 ~he following examples are intended to provide a
detailed description of the present invention and manner
of carrying it out, but not to limit its scope,
applicability or utility.
EXAMPL~ 1
Chromosornal DNA from Streptomyces lividans strain
1326 [Bibb et al. (1981), supr_.l was isolated using the
procedure described by Chater et al, ~E~. Plasmid pIJ6
(about 21 kb) isolated from Streptomyces lividans
[Thompson et al. ~1980), supra.~ was used as the cloning
vector as this plasmid carries the gene for thiostrepton
resistance, which is useful as a selective marker to
select for the plasmid in a given thiostrepton sensitive
strain such as 1326 and its derivatives. Treatment of the
chromosomal DNA and the pIJ6 DNA with SphI restriction
endonuclease or PstI restriction endonuclease yielded DNA
fragments having a protruding complementary 3' ~NA
sequence. The pIJ6 DNA was additionally treated with
alkaline phosphatase to prevent regeneration of the
2Q cloning vector without an additional DNA insert. The ~e~I
and the PstI generated DNA's ~5 ug of chromosomal DNA, 1
ug of pIJ6 DNA~ were ligated separately at 16 C for 7
days using standard procedures. The ligated DNA's were
transformed substantially according t~ the procedure
described by Chater et al., su~a., using about 2x107
protoplasts derived from Streptomyces lividans strain
1326-9,a nitrosoguanidine induced mutant of strain 1326
lacking any excreted ~-galactosidase activity. The
protoplasts were spread onto regeneration medium plates
and incubated for 18-24 hours at 28 C. The plates were
overlaid with a soft agar mixture (0.4~ agar in water)
containing 100 ug/ml of thiostrepton to select for
transformed offspring and 150 uy/ml of X-gal. The plates
were incubated for another 2 to 6 days at 28C, then
scored for ~he appearance of characteristic blue colonies.
~2~3~S
- 12 -
1 Of over 10,000 thiostrepton resistant colonies
resulting Erom the ~I clonina, 9 turned blue; from about
the same number of colonies resulting from the PstI
cloning, one turned blue. The plasmid DNA of all the blue
colonies was isolated and analyzed. Both plasmid DNA from
tAe SphI and the PstI cloning had one common 8 kilobase
DNA fragment derived from the chromosome and not
previously present on the pIJ6 plasmid.
A restri~tion endonuclease cleavage map of the 8
kilobase DNA fragment carrying the cloned gene for such
excreted ~-galactosidase, obtained in the standard manner,
is shown in Figure 1. A 29 k.ilobase plasmid derived from
the Sph I cloning, found to contain the 8.69 kb ~
Pst I fragment described above, was termed "pSKL-l".
Cleavage by the restriction endonucleases was carried out
in the standard manner. The plasmid derived from the Pst I
cloning was termed "pX". pSKL-l is represented by the
restriction endonuclease cleavage map shown in Figure 2.
The isolated plasmid DNA from pSKL-l was used to
transform Streptomyces lividans 1326-9. Over 70% of the
thiostrepton resistant offspring showed an excreted
~-galactosidase activity/ indicating the presence and
expression of the gene on the plasmid~ The enzyme levels
of cell extracts of the pSKL-1 transformed strain, strain
1326-9/pSKL-l, were increased, in some cases, 100 times,
thus showing the presence of the gene on the plasmid.
Results of one experiment are given in Table 1, below.
~203~BS
- 13 -
1 TABLE 1
STRAIN ~-GALACTOSIDASE ACTIVITY
(nmoles/mg protein/min)
s
CARBON SOURCE IN GROWTH MEDIUM
GLUCOSE LACTOSE GALACTOSE
1326 ~12 76 184
1326-9 7 24 302
1326-9/pSKL-1 372 843 1242
As indicated in Table 1, some 1326-9 cultures produced
more unexcreted ~-galactosidase in the presence of
galactose than some 1326 cultures.
Transformants harboring the pSKL-l plasmid produced
darkèr blue colonies than the original 1326 strains,
demonstrating the utility of a DNA fragment containing the
B-galactosidase~gene in the construction of high
expression vectors. Strain 1326-9/pSKL-l, grown in broth
culture, excreted the Bgl protein as shown by
polyacrylamide gel electrophoresis of a supernatant
concentrate. Strains 1326 and 1326-9 were not found to
produce the Bgl protein.
B-galactosidase expression from a plasmid is less
stable in strain 1326-9 than in strain 1326. This is
believed to be due to recombination with chromosomal DNA.
:
EXAMPLE 2
3~ Plasmid pSKL-l was also transformed into
Streptomyces griseus strain BC6, ATCC No. 10137, a strain
which naturally does not possess an excreted
~-galactosidase, by the above described procedures. The
offspring of strain BC6 containing pSKI-l (strain
BC6/pSKL-l), however, produced an excreted
~-galactosidase, demonstrating the applicability and
s~ ~
~2U~
., ~
- 14 -
1 usefulness of the ~-galactosidase gene in other
Streptomycete h~sts.
EXAMPLE 3
The ~gL II 2.47-3.39 fragment was deleted from
pSKL-l to prepare pBB2B. The plasmid, pBB2B, is identical
to pSKL-l except for the dele~ion of the Bgl II fragment.
S. lividans 1326 was transformed with pBB2~ and its
~-galactosidase excretion after 14 hours and ~4 hours was
compared with that of a 1326 strain transformed with
pSKL-l. Results were as shown in Table 2, below.
Table 2
~-GALACTOSIDASE ACTIVITY
nmoles/mg cells/min)
STRAIN (CARBON SOURC:E:GAh~CTOSE)
14h 24h
1326/pSKL-1 .03 .06
1 26/pBB2B .002 .12
Supernatant from the 1326/pSKL-l culture
contained the Bgl protein which appeared on a
polyacrylamide gel following electrophoresis. Supernatant
from the 1326/pBB2B culture did not.
The above results show that the ~ 3.39) -
Sph I fragment can code for an excreted B-galactosidase
- and that the Bgl II ragmentis required for expression of
the excreted Bgl protein.
~XAMPLE 4
A DNA fragment from Streptomyces sp. con~aining a
gene which can code for an excretable ~-galactosidase was
cloned as fol1ows.
Plasmid pIJ6 (about 21 kb) was partia~ly digested
with Bam HI to delete the Bam HI fragment between the Bgl
3~S
- 15 -
1 II and Bam HI sites, as shown in Fig. 2. The resulting
plasmid, pSKT, there~ore has two rather than three Bam HI
si~es~ All other restriction sites, including the Sph I
sites, remain as in pIJ6.
The Bgl II (3.39)-Bam HI fragment carrying the
B-galactosidase gene was removed from pSKL-l and inserted
into the Bam HI sites in pSKT in both orientations. None
of the resulting plasmids, following transformation into
S. lividans strain 1326-9 and selection for thiostrepton
resistancet produced appreciable intracellular quantities
of B-galactosidase. For example, analysis of cell
extracts of two such clones having the ~ II (3.3g) - Bam
HI fragment inserted into the B HI site closest to the
~ I site and in the same orientation as in pSKL-l,
showed that after 15 hours with galactose as the sole
carbon source, the clones had produced ~-galactosidase at
the rate of 4 and 8 nmoles/mg protein/minute. A control,
a culture of 1326/pSKL-l grown for the same length of time
under the same conditions, produced ~-galactosidase~at the
rate of 213 nmoles/mg proteinjminute.
The B~l II (3.39) - Bam HI-containing clones did
not produce the Bgl protein whereas 1326/pSECL-l did, as
detected by polyacrylamide gel electrophoresis.
These results, together with results discussed
Z5 above, illustrate that the ~ II (2.47) region
can be used to promote gene expression.
Streptomyces lividans strains 1326 and 1326-9 and a
strain containing pIJ6 are publicly available from various
sources. To further ensure availability, these strains
have been deposited with the Agricultural Research Culture
Collection in Peoria, Illinois on June 1, 3982, and
assigned accession numbers 15091, 15090 and 15092,
respectively. They will be available upon issuance of a
United States patent hereon or publication of an European
~2~3~
1 or ~7apanese patent application hereon and in accordance
with the patent laws of the United States and of other
jurisdictions in which a patent application hereon is
filed.
