Gal operon of streptomyces

BACKGROUND OF THE INVENTION 
This invention relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon. 
Hodgson, J. Gen. Micro., 128, 2417-2430 (1982), report that Streptomyces 
coelicolor A3(2) has a glucose repression system which allows repression 
at the level of transcription of the arabinose uptake system, one of the 
glycerol uptake systems, and also repression of the galactose uptake 
system in wild type strains. There is no report in Hodgson of actual 
galactose metabolism by S. coelicolor A3(2). 
Okeda et a. Mol. Gen. Genet., 196, 501-507 (1984), report that glucose 
kinase activity, 2-deoxyglu-cose-sensitivity, glucose utilization and 
glucose repression were all restored to S. coelicolor A3(2) glk (glucose 
kinase) mutants transformed by a 3.5 kb DNA fragment which contained the 
glk gene cloned from S. coelicolor into a phage vector. 
Seno et al., Mol. Gen. Genet., 193, 119-128 (1984), report the glycerol 
(gyl) operon of Streptomyces coelicolor, and state such operon is 
substrate-inducible and catabolite-repressible. 
Debouck, et al., Nuc. Acids, Res., 13(6), 1841-1853 (1985), report that 
the gal operon of E. coli consists of three structurally contiguous genes 
which specify the enzymes required for the metabolism of galactose, i.e., 
galE (uridine diphosphogalactose-4-epimerase), galT (galactose-1-phosphate 
uridyltransferase) and galK (galactokinase) that such genes are expressed 
from a polycistronic mRNA in the order E, T, K; that the expression of the 
promoter distal gene of the operon, galK, is known to be coupled 
translationally to the galT gene immediately preceding it; that such 
translational coupling results from a structural overlap between the end 
of the galT coding sequence and the ribosome binding region of galK; and 
that the translational coupling of galT and galK ensures the coordinate 
expression of these genes during the metabolism of galactose. 
SUMMARY OF THE INVENTION 
This invention relates to a recombinant DNA molecule comprising a 
Streptomyces gal operon galK gene; galE gene; galT gene; P2 promoter 
expression unit, or P2 promoter or any functional derivative thereof as 
well as a recombinant DNA molecule comprising a Streptomyces gal operon P1 
promoter, P1 promoter regulated region or the entire gal operon or any 
regulatable and functional derivative thereof. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon or any regulatable and functional derivative 
thereof; a recombinant DNA molecule comprising a Streptomyces lividans or 
Streptomyces coelicolor gal operon containing a wild-type gal operon P1 
promoter or any regulatable and functional P1 promoter derivative, a 
functional DNA molecule operatively linked to such operon; a recombinant 
DNA vector comprising and such DNA molecule, and, optionally, additionally 
comprising a replicon; a method of preparing a host cell transformed with 
such vector; the transformed host prepared by such method; a method of 
expressing such functional DNA sequence which comprises cultivating such 
transformed host under suitable conditions such that the functional DNA 
sequence is expressed; and to a method of regulating the expression of 
such functional DNA sequence which comprises cultivating such transformed 
host under conditions which regulate such expression. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P2 promoter expression unit or any functional 
derivative thereof and a functional DNA molecule operatively linked to 
such unit; a recombinant DNA vector comprising such DNA molecule, and, 
optionally, additionally comprising a replicon; a method of preparing a 
host cell transformed with such vector; the transformed host prepared by 
such method; and to a method of expressing such functional DNA sequence 
which comprises cultivating such transformed host under suitable 
conditions such that the functional DNA sequence is expressed. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P1 promoter regulated region or any regulatable 
and functional derivative thereof and a functional DNA molecule 
operatively linked to such region; a recombinant DNA vector comprising 
such DNA molecule, and, optionally,, additionally comprising a replicon; a 
method of preparing a host cell transformed with such vector, the 
transformed host prepared by such method; a method of expressing such 
functional DNA sequence which comprises cultivating such transformed host 
under suitable conditions such that the functional DNA sequence is 
expressed; and to a method of regulating the expression of such functional 
DNA sequence which comprises cultivating such transformed host under 
conditions which regulate such expression. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P1 promoter or any regulator and functional 
derivative thereof and a foreign functional DNA molecule operatively 
linked to such region; a recombinant DNA vector comprising such DNA 
molecule, and, optionally, additionally comprising a replicon; a method of 
preparing a host cell transformed with such vector; the transformed host 
prepared by such method; a method of expressing such functional DNA 
sequence which comprises cultivating such transformed host under suitable 
conditions such that the functional DNA sequence is expressed; and to a 
method of regulating the expression of such functional DNA sequence which 
comprises cultivating such transformed host under conditions which 
regulate such expression. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P2 promoter or any functional derivative thereof 
and a foreign functional DNA molecule operatively linked to such region; a 
recombinant DNA vector comprising such DNA molecule, and, optionally, 
additionally comprising a replicon; a method of preparing a host cell 
transformed with such vector; the transformed host prepared by such 
method; and to a method of expressing such functional DNA sequence which 
comprises cultivating such transformed host under suitable conditions such 
that the functional DNA sequence is expressed. 
This invention also relates to a method of enabling a non-galactose 
utilizing host microorganism or cell to utilize galactose which comprises 
transforming such host with a recombinant DNA molecule comprising a 
Streptomyces gal operon or any portion of the Streptomyces gal operon, or 
any functional derivative thereof, which is adequate to enable such 
transformed host to utilize galactose. This invention relates to the 
recombinant DNA vector employed in such method and to the host prepared by 
such method.

DETAILED DESCRIPTION OF THE INVENTION 
It has now been discovered that the Streptomyces genome contains an operon 
for the metabolism of galactose (i.e., a gal operon) which comprises three 
structural genes (galT, galE and galK) and two promoters (P1 and P2). The 
galT gene product is known as galactose-1-phosphate uridyltransferase 
(transferase), the galE gene product is known as uridine 
diphosphogalactose-4-epimerase (epimerase), and the galK gene product is 
known as galactose-1-kinase (galactokinase). The function of the gene 
products of galT, galE and galK in galactose metabolism in Streptomyces is 
explained by the following diagram: 
______________________________________ 
1. galactose + ATP galactokinase 
galactose-1-phosphate + ADP 
2. galactose-1-phosphate + UDP-glucose transferase 
UDP-galactose + glucose-1-phosphate 
3. UDP-galactose epimerase UDP-glucose 
______________________________________ 
By the term "promoter" is meant any region upstream of a structural gene 
which permits binding of RNA polymerase and transcription to occur. 
By the term "structural gene" is meant a coding sequence for a polypeptide 
which serves to be the template for the synthesis of mRNA. 
By the term "operon" is meant a group of closely linked genes responsible 
for the synthesis of one or a group of enzymes which are functionally 
related as members of one enzyme system. An operon comprises an operator 
gene, a number of structural genes (equivalent to the number of enzymes in 
the system) and a regulator gene. By "operator" or "operator gene" is 
meant a DNA sequence which controls the biosynthesis of the contiguous 
structural gene(s) within an operon. By "regulator gene" is meant a gene 
which controls the operator gene in an operon through the production of a 
repressor which can be either active (enzyme induction) or inactive 
(enzyme repression). The transcription of the structural gene(s) in an 
operon is switched on or off by the operator gene which is itself 
controlled in one or more of three ways: 1) in inducible enzyme systems, 
the operator is switched off by a repressor produced by the regulator gene 
and which can be inactivated by some metabolite or signal substance (an 
inducer) coming from elsewhere in the cell or outside the cell, so that 
the presence of the inducer results in the operon becoming active; or 2) 
in repressed enzyme systems, the operator is switched off by a 
repressor-corepressor complex which is a combination of an inactive 
repressor produced by the regulator gene with a corepressor from 
elsewhere, so that the presence of the corepressor renders the operon 
inactive; or 3) in activated gene systems, the promoter is switched on by 
an activator produced by a regulator gene which can be activated by some 
metabolic or signal substance. 
The Streptomyces gal operon is naturally present in the Streptomyces 
genome. 
By the term "Streptomyces gal operon" is meant that region of the 
Streptomyces genome which comprises the P1 promoter, P2 promoter, galT, 
galE and galK structural genes and any other regulatory regions required 
for transcription and translation of such structural genes. 
By the term "regulatory region" is meant a DNA sequence, such as a promoter 
or operator, which regulates transcription of a structural gene. 
The following model is suggested for gene expression within the 
Streptomyces gal operon. The P1 promoter is a galactose inducible promoter 
(i.e., it is induced in the presence of galactose and repressed in the 
presence of glucose). According to S1 data, the P2 promoter is 
constitutive, i.e., it is "turned on" regardless of the presence or 
absence of galactose or any other carbon source. 
A cosmid library was constructed for Streptomyces lividans 1326 DNA by 
using cosmid pJW357 (which encodes the ability to replicate in both 
Streptomyces and E. coli. 
This library was then transfected into E. coli K21 which is a derivative of 
the E. coli strain MM294 which contained a bacteriophage P1 transduced 
galactokinase (galK) mutation. Transfected cells were plated under media 
conditions which select for both the presence of the cosmid and the 
presence of an active galK gene. Weakly positive colonies were isolated 
and the cosmid DNA derived from these colonies was transformed into the 
K21 strain. These transformations yielded two cosmids which consistently 
produced positive growth with galactose as the only carbon source. These 
galK.sup.+ cosmids were then transformed into a Streptomyces host (i.e., 
Streptomyces lividans 1326-12K) which had been isolated by the inventors 
of the subject invention as unable to grow on medium in which galactose 
was the only carbon source by using 2-deoxygalactose selection [see, 
Brawner et al., Gene, 40, 191 (1985), in press]. Under conditions which 
differentiate strains able and unable to produce galactokinase, only one 
of the cosmids caused the Streptomyces lividans 1326-12K host to become 
galK.sup.+. Further studies have demonstrated that this cosmid encodes a 
gene with galactokinase activity. Additional studies, including DNA 
sequence analysis and protein studies demonstrate that the Streptomyces 
gene shares homology with the E. coli and yeast galactokinase genes. 
Regulation studies indicate that the cosmid encoded galactokinase gene 
regulated in the same manner as the chromosome encoded gene. 
A S. lividans gal operon was originally isolated from a ca. 9 kilobase (Kb) 
region of Streptomyces lividans 1326. The ca. 9 Kb region of Streptomyces 
lividans 1326 containing the Streptomyces gal operon has been mapped 
substantially as follows in Table A. By "substantially" is meant (i) that 
the relative positions of the restriction sites are approximate, (ii) that 
one or more restriction sites can be lost or gained by mutations not 
otherwise significantly affecting the operon, and (iii) that additional 
sites for the indicated enzymes and, especially for enzymes not tested, 
may exist. The restriction enzymes used herein are commercially available. 
All are described by Roberts, Nuc. Acids. Res., 10(5);p117 (1982). 
TABLE A 
______________________________________ 
Map Position 
Restriction Enzyme 
Location (kb) 
______________________________________ 
1 HindIII -.40 
1a NruI 0 
2 BglII .75 
3 EcoRI 1.05 
4 PvuII 1.15 
5 MlUI 2.30 
6 PvuII 2.80 
7 EcoRI 4.00 
8 PvuII 4.10 
8a SacI 4.25 
9 PvuII 5.00 
10 XhoI 5.50 
11 BamHI 5.80 
12 BamHI 6.50 
13 MluI 6.90 
13a PvuII 7.20 
14 MluI 7.80 
15 BamHI 8.00 
16 SphI 8.30 
______________________________________ 
FIG. 1 represents a restriction endonuclease map of the Streptomyces 
lividans 1326 gal operon and indicates locations for structural genes 
(galT, galE and galK) and promoters (P1 and P2) comprised within the 
operon. 
Referring to Table A and FIG. 1, the location of the promoters and 
structural genes of the Streptomyces lividans 1326 gal operon are mapped 
substantially as follows in Table B: 
TABLE B 
______________________________________ 
Location (Kb) 
______________________________________ 
P1 transcription start site 
.10 
galT translation initiation codon 
.15 
P2 transcription start site 
1.25 
galE translation initiation codon 
1.50 
galK translation initiation codon 
2.40 
3' end of galK message 
3.60 
______________________________________ 
Microorganisms of the genus have historically been used as a source of 
antibiotics for the pharmaceutical industry. Consequently, the technical 
skills necessary to scale-up the production of biological products using 
Streptomyces as the vehicle for the production of such products are 
presently available. However, before Streptomyces can be used as a vehicle 
for the production of bioactive molecules using the new recombinant DNA 
technologies, there is a need to define regulatory elements in 
Streptomyces analogous to those which have proved useful in E. coli. These 
regulatory elements include ribosomal binding sites and regulated 
transcriptional elements. 
The existence of a galE, galT or galK gene or gene product or gal operon in 
Streptomyces has not been previously reported. The instant invention, 
i.e., the cloning of the Streptomyces gal operon, enables construction of 
regulatable expression/cloning vectors in Streptomyces, other 
actinomycetes, and other host organisms. Furthermore, the instant 
invention led to the discovery that the Streptomyces gal operon is 
polycistronic. Perhaps the most important feature of the cloning of the 
Streptomyces gal operon is the observation that there are sequences 
essential for regulation of the Streptomyces galK gene. Direct analogy to 
the initial use of the lac promoter from E. coli as an expression system 
can be made. In fact, Brosius et al., Proc. Natl. Acad. Sci. USA, 81 
6929-6933 (1984), utilized the regulatory elements of the E. coli lac 
promoter to regulate the exceptionally strong E. coli ribosomal promoters. 
Because it is likely that the Streptomyces gal operon ribosomal promoters 
are also exceptionally strong, such promoters enable the construction of 
regulatable expression vectors which will be very useful in Streptomyces, 
other actinomycetes, and other host organisms. The instant invention also 
enabled the unexpected discovery that the 2-deoxygalactose selection which 
has been used in E. coli to select for galK mutants also operates in 
Streptomyces to select for galK mutants [see, Brawner et al., Gene 40, 191 
(1985), in press]. This observation, combined with the ability to clone 
the Streptomyces galK gene and the promoter and regulatory regions 
required for its transcription and translation on a cosmid, as described 
herein, allows the direct insertion of any structural gene into the 
chromosomally located galK gene of Streptomyces by homologous 
recombination. This manipulation will allow molecular biologists to stably 
insert DNA fragments of interest into the Streptomyces chromosome. Such an 
approach will allow researchers to tag or mark a Streptomyces strain of 
interest or to insert expression cassettes into the organism without the 
need of maintaining an antibiotic selection such as that presently 
required by most Streptomyces expression vectors. 
This invention relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon on any regulatable and functional derivative 
thereof. By "regulatable and functional derivative" is meant any 
derivative of the Streptomyces gal operon which functions in substantially 
the same way as the naturally occurring Streptomyces gal operon in terms 
of regulatable production of the galT, galE and galK gene products. Such 
derivatives include partial sequences of the gal operon, as well as 
derivatives produced by modification of the gal operon coding sequence. 
Techniques for modifying the gal operon which are known in the art 
include, for example, treatment with chemical mutagens, irradiation or 
direct genetic engineering, such as by inserting, deleting or substituting 
nucleic acids by the use of enzymes or recombination techniques. The 
naturally occurring Streptomyces gal operon can be isolated from any 
galactose utilizing Streptomyces strain by employing the techniques 
described herein. 
Numerous strains of various Streptomyces species are publicly available 
from many sources. For example, the American Type Culture Collection, 
Rockville, Md., U.S.A. has approximately 400 different species of 
Streptomyces available to the public. The ability of a particular strain 
of Streptomyces to utilize galactose can be readily determined by 
conventional techniques, such as by growing such strain on a medium 
containing galactose as the sole carbon source. The preferred Streptomyces 
species from which to isolate a gal operon include S. lividans, S. 
coelicolor, S. azuraeus and S. albus, S. carzinostaticus, S. 
antibibrinolyticus and S. longisporus, S. lividans is most preferred. The 
Streptomyces gal operon, and smaller portions thereof, is useful as a 
nucleic acid probe to obtain homologous sequences from other cells and 
organisms. The Streptomyces gal operon is also useful as a selection 
marker in an appropriate host mutant, and for providing regulatory 
elements. By "appropriate host mutant" is meant a host which does not 
utilize galactose because it (a) does not contain a gal operon or (b) 
contains a nonfunctional gal operon, or (c) contains a defect within a 
homologous structural gene or regulatory region comprised by the 
Streptomyces gal operon such as a defective P1 promoter, P2 promoter, galT 
gene, galK gene and/or galE gene. Thus, a recombinant DNA molecule 
(comprising the Streptomyces gal operon and a foreign functional DNA 
sequence operatively linked thereto), which can be prepared by 
conventional techniques, can be transformed into an appropriate host 
mutant by conventional techniques for incorporation into the host genome 
by homologous recombination to enable regulatable expression of the 
foreign functional DNA sequence without the need of maintaining an 
expensive antibiotic selection. Such operon may therefore also be 
incorporated on recombinant DNA expression vectors for regulatable 
expression of a foreign functional DNA sequence operatively linked to such 
operon in an appropriate host mutant transformed with such vector without 
the need of maintaining an expensive antibiotic selection. Such operon is 
also useful for transforming those cells, viruses and microorganisms, such 
as strains of Streptomyces, other actinomycetes, and other prokaryotic 
organisms, such as gal.sup.- E. coli strains, which do not utilize 
galactose into galactose utilizing strains. Such transformation may have 
pleiotrophic effects on the transformed host. By the term "functional DNA 
sequence" is meant any discrete region of DNA derived directly or 
indirectly form Streptomyces or any other source which functions in a host 
organism transformed therewith as a gene expression unit, structural gene, 
promoter or a regulatory region. Preferred functional DNA sequences 
include those coding for polypeptides of pharmaceutical importance, such 
as, but not limited to, insulin, growth hormone, tissue plasminogen 
activator, alpha-1-anti-trypsin or antigens used in vaccine production. By 
the term "foreign functional DNA sequence" is meant a functional DNA 
sequence not derived from the Streptomyces gal operon coding region. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P2 promoter expression unit or any functional 
derivative thereof. By the term "P2 promoter expression unit" is means 
that region of the Streptomyces gal operon comprising the Streptomyces gal 
operon P2 promoter, galE and galK structural genes and any other 
regulatory regions required for transcription and translation of such 
structural genes. By "functional derivative" is meany any derivative of 
the Streptomyces gal operon P2 promoter expression unit which functions in 
substantially the same was as the naturally occurring region in terms of 
production of the Streptomyces gal operon galE and galK gene products. 
Such derivatives include partial sequences of the Streptomyces gal operon 
P2 promoter expression unit coding sequence. Techniques for effecting such 
modification are known in the art, and some have been outlined above. The 
naturally occurring Streptomyces gal operon P2 promoter expression unit 
can be isolated from the naturally occurring Streptomyces gal operon by 
conventional techniques. The Streptomyces gal operon P2 expression unit 
is useful as a selection marker in an appropriate host mutant and for 
providing regulatory elements. By "appropriate host mutant" is meant a 
host which does not utilize galactose because it contains a defect within 
a homologous structural gene or regulatory region comprised by the 
Streptomyces P2 promoter expression unit such as a defective P2 promoter, 
galE gene and/or galK gene. Thus, a recombinant DNA molecule (comprising 
the Streptomyces gal operon P2 promoter expression unit and a foreign 
functional DNA sequence operatively linked thereto), which can be prepared 
by conventional techniques, can be transformed into an appropriate host 
mutant by conventional techniques for incorporation into the host genome 
by homologous recombination to enable constitutive expression of the 
foreign functional DNA sequence without the need of maintaining an 
expensive antibiotic selection. Such expression unit may also be 
incorporated on recombinant DNA expression vectors for constitutive 
expression of foreign functional DNA sequences. The Streptomyces gal 
operon P2 promoter expression unit is also useful for complementation of 
an appropriate host mutant which can then be used for constitutive 
expression of a foreign functional DNA sequence operatively linked to such 
expression unit in an appropriate host mutant transformed with such vector 
without the need of maintaining an expensive antibiotic selection. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P1 promoter regulated region or any regulatable 
and functional derivative thereof. By the term "P1 promoter regulated 
region" is meant that region of the Streptomyces gal operon comprising the 
Streptomyces gal operon P1 promoter, galT, galE and galK structural genes 
and any other regulatory regions required for transcription and 
translation of such structural genes. By "regulatable and functional 
derivative" is meant any derivative of the Streptomyces gal operon P1 
promoter regulated region which functions in substantially the same way as 
the naturally occurring region in terms of regulatable production of the 
Streptomyces gal operon galT, galE and galK gene products. Such 
derivatives include partial sequences of the Streptomyces gal operon P1 
promoter regulated region, as well as derivatives produced by modification 
of the Streptomyces gal operon P1 promoter required region coding 
sequence. Techniques for effecting such modifications are known in the 
art, and some have been outlined above. The naturally occurring 
Streptomyces gal operon P1 promotor regulated region can be isolated from 
the naturally occurring Streptomyces gal operon by conventional 
techniques, such as by excising the P2 promoter from the naturally 
occurring Streptomyces gal operon or inactivating the P2 promoter by a 
point mutation or by inserting a foreign DNA sequence within the promoter. 
The Streptomyces gal operon P1 promoter regulated region is useful for the 
utilities outlined above for the Streptomyces gal operon. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P2 promoter or any functional derivative thereof. 
By "functional derivative" is meant any derivative of the Streptomyces gal 
operon P2 promoter which functions in substantially the same was as the 
naturally occurring P2 promoter in terms of enabling the binding of RNA 
polymerase thereto and transcription of a functional DNA sequence 
operatively linked to such promoter. Such derivatives include partial 
sequences of the Streptomyces gal operon P2 promoter, as well as 
derivatives produced by modification of the gal operon P2 promoter coding 
sequence. Techniques for effecting such modification are known in the art, 
and some have been outlined above. The naturally occurring Streptomyces 
gal operon P2 promoter can be isolated from the naturally occurring 
Streptomyces gal operon by conventional techniques. A recombinant DNA 
molecule (comprising the Streptomyces gal operon P2 promoter and a foreign 
functional DNA sequence operatively linked thereto), which can be prepared 
by conventional techniques, can be transformed into an appropriate host 
mutant by conventional techniques for incorporation into the host genome 
by homologous recombination to enable constitutive expression of the 
foreign functional DNA sequence. The Streptomyces gal operon P2 promoter 
is also useful for incorporation into recombinant DNA expression vectors 
for constitutive expression of a foreign functional DNA sequence 
operatively linked thereto in viruses and eukaroyotic or prokaryotic cells 
or organisms, especially in Streptomyces or other actinomycetes, 
transformed with such vector. 
This invention also relates to a recombinant DNA molecule comprising the 
Streptomyces gal operon P1 promoter or any regulatable and functional 
derivative thereof. By "regulatable and functional derivative" is meant 
any derivative of the Streptomyces gal operon P1 promoter which functions 
in substantially the same way as the naturally occurring P1 promoter in 
terms of enabling the binding of RNA polymerase thereto and regulating the 
transcription of a functional DNA sequence operatively linked to such 
promoter. Such derivatives include partial sequences of the Streptomyces 
gal operon P1 promoter, as well as derivatives produced by modification of 
the gal operon P1 promoter coding sequence. Techniques for effecting such 
modification are known in the art, and some have been outlined above. The 
naturally occurring Streptomyces gal operon P1 promoter can be isolated 
from the naturally occurring Streptomyces gal operon by conventional 
techniques. A recombinant DNA molecule (comprising the Streptomyces gal 
operon P1 promoter and a foreign functional DNA sequence operatively 
linked thereto), which can be prepared by conventional techniques, can be 
transformed into an appropriate host mutant by conventional techniques for 
incorporation into the host genome by homologous recombination to enable 
regulatable expression of the foreign functional DNA sequence. The 
Streptomyces gal operon P1 promoter is also useful for incorporation into 
recombinant DNA expression vectors for regulatable expression of a foreign 
functional DNA sequence operatively linked thereto in viruses and 
eukaroyotic or prokaryotic cells or organisms, especially Streptomyces or 
other actinomycetes, transformed with such vector. 
This invention also related to a recombinant DNA molecule comprising the 
Streptomyces gal operon galE, galT or galK gene, or any functional 
derivative thereof. By "functional derivative" is meant any derivative of 
the Streptomyces gal operon galE, galT or galK gene which functions in 
substantially the same way as the naturally occurring gene in terms of 
production of an active galE, galT, or galK type gene product. Such 
derivatives include partial sequences of the Streptomyces gal operon galE, 
galT, or galK gene, as well as derivatives produced by modification of the 
gal operon sequence. Techniques for effecting such modifications are known 
in the art, and some have been outlined above. The naturally occurring 
Streptomyces gal operon galE, galT and/or galK gene can be isolated from 
the naturally occurring Streptomyces gal operon by conventional 
techniques. The Streptomyces gal operon galE, galT and/or galK gene can be 
used as a selection marker in an appropriate host mutant. By "appropriate 
host mutant is meant a host which does not utilize galactose because it 
contains a defect within a homologous galE, galT and/or galK gene. Thus, a 
recombinant DNA molecule (comprising the Streptomyces gal operon galE, 
galT and/or galK gene and a foreign functional DNA sequence, both of which 
are operatively linked to appropriate regulatory region). which can be 
prepared by conventional techniques can be transformed into an appropriate 
host mutant by conventional techniques for incorporation into the host 
genome by homologous recombination to enable detection of transformants 
without the need of maintaining an expensive antibiotic selection. 
Likewise, a recombinant DNA vector comprising the Streptomyces gal operon 
galE, galT and/or galK gene and a foreign functional DNA sequence, both of 
which are operatively linked to appropriate regulatory regions, as well as 
a replicon, can be transformed into an appropriate host mutant by 
conventional techniques to enable detection of transformants without the 
need of maintaining an expensive antibiotic selection. The Streptomyces 
gal operon galE, galK and/or galT gene is also useful for complementation 
of an appropriate host mutant. 
The Streptomyces gal operon galE gene is also useful for providing a 
ribosome binding site and initiation codon which can be fused to a foreign 
functional DNA sequence to enable the expression of such coding sequence 
when incorporated into an appropriate expression vector and transformed 
into an appropriate host. If such foreign functional DNA sequence is fused 
to the galE gene ribosome binding site and initiation codon in a 
recombinant DNA expression vector comprising the Streptomyces gal operon 
P2 promoter expression unit, or the entire gal operon, such DNA sequence 
will be constitutively expressed when such vector is transformed into an 
appropriate host organism. If such DNA sequence is fused to the galE gene 
ribosome binding site and initiation codon in a recombinant DNA expression 
vector comprising the Streptomyces gal operon P2 promoter regulated 
region, expression of such DNA sequence can be regulated when such vector 
is transformed into an appropriate host organism by controlling the 
presence or absence of galactose or glucose. 
The Streptomyces gal operon galT gene is also useful for providing a 
ribosome binding site and initiation codon which can be fused to a foreign 
functional DNA sequence to enable the expression of such coding sequence 
when incorporated onto an appropriate expression vector and transformed 
into an appropriate host. If such DNA sequence is fused to the galT gene 
ribosome binding site and initiation codon in a recombinant DNA expression 
vector comprising the Streptomyces gal operon P1 promoter regulated 
region, or the entire gal operon, expression of such coding sequence can 
be regulated in a host transformed with such vector as outlined above. 
This invention also relates to a recombinant DNA vector comprising a 
replicon, Streptomyces gal operon, or a functional and regulatable 
derivative thereof, and a foreign functional DNA sequence operatively 
linked to such operon. Such vector can be prepared by conventional 
techniques. The replicon employed should be one known for its ability to 
stably and extrachromosomally, maintain a vector in the host organism 
which is to be the host transformed with the vector. 
This invention also relates to a transformed host microorganism comprising 
a recombinant DNA vector wherein said vector contains a replicon, the 
Streptomyces gal operon, or a functional and regulatable derivative 
thereof, and a foreign functional DNA sequence operatively linked to such 
operon; and to the method of preparing such host which comprises 
transforming an appropriate host microorganism with such vector. 
Appropriate host microorganisms which may be employed in the method of 
this invention include viruses, and eukaroyotic and prokarylotic cells or 
organisms, especially actinomycetes, such as those of the genes 
Streptomyces. The most preferred host microorganisms belong to the genus 
Streptomyces. Preferred species of Streptomyces include Streptomyces 
lividans, S. coelicolor, S. azuraeus and S. albus. Transformation of such 
host microorganism with such vector can be accomplished using conventional 
techniques such as the method of Chater et al., Curr. Top. Micro. Imm., 
96, 69-95 (1982). This invention also related to a method of expressing 
the functional DNA sequence contained by such transformed host of this 
invention which comprises cultivating such transformed host under suitable 
conditions such that the functional DNA sequence is expressed. By 
"suitable conditions" is meant those conditions which will allow the host 
to grow and which enable the expression of the functional DNA sequence. 
Such suitable conditions can be determined by one of skill in the art 
using conventional techniques and will depend on various factors, such as 
the host organism employed and the functional DNA sequence to be 
expressed. This invention is also related to a method of regulating the 
expression of the functional DNA sequence contained by such transformed 
host which comprises cultivating a transformed host containing such 
functional DNA sequence under appropriate conditions such that its 
expression is regulatable. By "appropriate conditions" is meant those 
conditions which enable the Streptomyces gal operon (and thus the foreign 
functional DNA sequence) to be regulatable. By "regulatable" is meant 
responsive to the presence of galactose or its metabolites and the 
presence f glucose or its metabolites in the growth media of the 
transformed host cell. Such regulation can be carried out by addition or 
deletion of galactose or glucose to the transformed host's culture medium. 
The optimal elvels of galactose and/or glucose for up or down-regulation 
of the expression of the foreign functional DNA coding sequence by the 
transformed host of this invention can be readily determined by one of 
skill in the art using conventional techniques. 
This invention also related to a recombinant DNA vector comprising a 
replicon, a Streptomyces gal operon P2 promoter expression unit, or a 
functional derivative thereof, and a foreign functional DNA sequence 
operatively linked to such unit. Such a vector can be prepared by 
conventional techniques. The replicon employed should be one known for its 
ability to stably, and extra-chromosomally, maintain a vector in the host 
organism which is to be transformed with the vector. 
This invention also relates to a transformed host microorganism comprising 
a recombinant DNA vector wherein said vector contains a replicon, the 
Streptomyces gal operon P2 promoter expression unit, or a functional 
derivative thereof, and a foreign functional DNA sequence operatively 
linked to such unit and to the method of preparing such host which 
comprises transforming an appropriate host microorganism with such vector. 
By the term "operatively linked" is meant that a functional DNA sequence 
is transcriptionally or translationally linked to an expression control 
sequence (i.e., the Streptomyces gal operon P2 promoter expression unit, 
P1 promoter regulated region, P1 promoter or P2 promoter) in such a way so 
that the expression of the functional DNA sequence can be 
transcriptionally or translationally linked to the Streptomyces gal operon 
by inserting such operon within the Streptomyces gal operon P1 or P2 
promoter transcript. By the term "replicon" is meant that region of DNA on 
a plasmid which functions to maintain, extrachromosomally, such plasmid is 
a host microorganism or cell transformed therewith. It has also been 
discovered that the Streptomyces gal operon, and smaller portions thereof, 
is useful as a nucleic acid probe to obtain homologous sequences from 
other cells and organisms. Appropriate host microorganisms which may be 
employed in the method of this invention include any virus or eukaroyotic 
or prokaryltic cell or organism, especially any actinomycetes such as 
those of the genes Streptomyces. The most preferred host microorganisms 
belong to the genus Streptomyces. Preferred species of Streptomyces 
include Streptomyces lividans, S. coelicolor, S. azuraeus and S. albus. 
Transformation of such host microorganism with such vector can be 
accomplished using conventional techniques such as the method of Chater et 
al., Curr. Top. Micro. Imm., 96, 69-95 (1982). This invention relates to a 
method of expressing the functional DNA sequence contained by such 
transformed host of this invention which comprises cultivating such 
transformed host under suitable conditions such that the functional DNA 
sequence is expressed. By "suitable conditions" is meant those conditions 
which will allow the host to grow and which enable the expression of the 
functional DNA sequence. Such suitable conditions can be determined by one 
of skill in the art using conventional techniques and will depend on 
various factors, such as the host organism employed and the functional DNA 
sequence to be expressed. 
This invention also related to a recombinant DNA vector comprising a 
replicon, a Streptomyces gal operon P1 promoter regulated region, or a 
functional and regulatable derivative thereof, and a foreign functional 
DNA sequence operatively linked to such region. Such a vector can be 
prepared by conventional techniques. The replicon employed should be one 
known for its ability to stable and extrachromosomally maintain a vector 
in the host or organism which is to be the host transformed with the 
vector. 
This invention also related to a transformed host microorganism comprising 
a recombinant DNA vector wherein said vector contains a replicon, a 
Streptomyces gal operon P1 promoter regulated region, or a functional and 
regulatable derivative thereof, and a foreign functional DNA sequence 
operatively linked to such region; and to the method of preparing such 
host which comprises transforming an appropriate host microorganism with 
such vector. Appropriate host microorganisms which may be employed include 
any virus or eukaroyotic or prokaryotic cell or organism especially 
actinomycetes such as those of the microorganisms belong to the genus 
Streptomyces. Preferred species of Streptomyces include Streptomyces 
lividans, S. coelicolor, S. azuraeus and S. albus. Transformation of such 
host microorganisms with such vector can be accomplished using 
conventional techniques such as the method of Chater et al., Curr. Top. 
Micro. Imm., 96, 69-95 (1982). This invention also related to a method of 
expressing the foreign functional DNA sequence contained by such 
transformed host of this invention which comprises cultivating such 
transformed host under suitable conditions such that the functional DNA 
sequence is expressed. By "suitable conditions" is meant those conditions 
which will allow the host to grow and which enable the expression of the 
function DNA sequence. Such suitable conditions can be determined by one 
of skill in the art using conventional techniques and will depend on 
various factors, such as the host organism employed and the functional DNA 
sequence to be expressed. This invention also related to a method of 
regulating the expression of the functional DNA sequence contained by such 
transformed host which comprises cultivating a transformed host containing 
such functional DNA sequence under appropriate conditions such that its 
expression is regulatable. By "appropriate conditions" is meant those 
conditions which enable the Streptomyces gal operon P1 promoter regulated 
region (and thus the foreign functional DNA sequence) to be regulatable. 
By "regulatable" is meant responsive to the presence or absence of 
galactose or its metabolites and the presence or absence of glucose or its 
metabolites in the growth media or the transformed host cell. Such 
regulation can be carried out by addition or deletion of galactose or 
glucose to the transformed host's culture medium. 
The invention also relates to a recombinant DNA vector comprising a 
replicon, a Streptomyces gal operon P2 promoter, or a functional 
derivative thereof, and a foreign functional DNA sequence operatively 
linked to such promoter. Such a vector can be prepared by conventional 
techniques. The replicon employed should be one known for its ability to 
stably and extrachromosomally maintain a vector in the host organism which 
is to be the host transformed with the vector. 
This invention also relates to a transformed host microorganism comprising 
a recombinant DNA vector wherein said vector contains a replicon, a 
Streptomyces gal operon P2 promoter, or a functional derivative thereof, 
and a foreign functional DNA sequence operatively linked to such region; 
and to the method of preparing such host which comprises transforming an 
appropriate host microorganism with such vector. Appropriate host 
microorganisms which may be employed include actinomycetes such as those 
of the genus Streptomyces. The most preferred host microorganisms belong 
to the genus Streptomyces. Preferred species of Streptomyces include 
Streptomyces lividans, S. coelicolor, S. azuraeus and S. albus. 
Transformation of such host microorganism with such vector can be 
accomplished using conventional techniques such as the method of Chater el 
al., Curr. Top. Micro. Imm., 96, 69-95 (1982). This invention also relates 
to a method of expressing the foreign functional DNA sequence contained by 
such transformed host of this invention which comprises cultivating such 
transformed host under suitable conditions such that the functional DNA 
sequence is expressed. By "suitable conditions" is meant those conditions 
which will allow the host to grow and which enable the expression of the 
functional DNA sequence. Such suitable conditions can be determined by one 
of skill in the art using conventional techniques and will depend on 
various factors, such as the host organism employed and the functional DNA 
sequence to be expressed. 
This invention also relates to a recombinant DNA vector comprising a 
replicon, Streptomyces gal operon P1 promoter, or any regulatable and 
functional derivative, therefore, and a foreign functional DNA sequence 
operatively linked to such region. Such a vector can be prepared by 
conventional techniques. The replicon employed should be one known for its 
ability to stably and extrachromosomally maintain a vector in the host 
organism which is to be the host transformed with the vector. 
This invention also relates to a transformed host microorganism comprising 
a recombinant DNA vector wherein said vector contains a replicon, the 
Streptomyces gal operon P1 promoter, or any regulatable and functional 
derivative thereof, and a foreign functional DNA sequence operatively 
linked to such region; and to the method of preparing such host which 
comprises transforming an appropriate host microorganism with such vector. 
Appropriate host microorganisms which may be employed include viruses or 
prokaryotic or eukaroyotic cells or organisms, especially actinomycetes 
such as those of the genus Streptomyces. The most preferred host 
microorganisms belong to the genus Streptomyces. Preferred species of 
Streptomyces include Streptomyces lividans, S. coelicolor, S. azuraeus and 
S. albus. Transformation of such host microorganism with such vector can 
be accomplished using conventional techniques such as the method of Chater 
et al., Curr, Top. Micro. Imm., 96 , 69-95 (1982). This invention also 
relates to a method of expressing the foreign functional DNA sequence 
contained by such transformed host of this invention which comprises 
cultivating such transformed host under suitable conditions such that the 
functional DNA sequence is expressed. By "suitable conditions" is meant 
those conditions which will allow the host to grow and which enable the 
expression of the functional DNA sequence. Such suitable conditions can be 
determined by one of skill in the art using conventional techniques and 
will depend on various factors, such as the host organism employed and the 
foreign functional DNA sequence to be expressed. This invention also 
relates to a method of regulating the expression of the functional DNA 
sequence contained by such transformed host which comprises cultivating a 
transformed host containing such foreign functional DNA sequence under 
appropriate conditions such that its expression is regulatable. By 
"appropriate conditions" is meant those conditions which enagle the gal 
operon P1 promoter (and thus the functional DNA sequence) to the 
regulatable. By "regulatable" is meant responsive to the presence or 
absence of galactose or its metabolites and the presence of glucose or its 
metabolites in the growth media of the transformed host cell. Such 
regulation can be carried out addition or deletion of galactose or glucose 
to the transformed host's culture medium. 
EXAMPLES 
In the following Examples, specific embodiments of the invention are more 
fully disclosed. These Examples are intended to be illustrative of the 
subject invention and should not be construed as limiting its scope. In 
all Examples, temperature is in degrees Centigrade (.degree.C.) 
By utilizing conventional methods, such as those outlined in the following 
Examples, one of skill in the art can isolate the gal operon from any 
galactose utilizing strain of Streptomyces. Furthermore, by utilizing 
techniques similar to those employed herein to isolate the Streptomyces 
gal operon, one of skill in the art can attempt to use the Streptomyces 
gal operon to isolate a gal operon from other galactose utilizing other 
strains of Streptomyces, especially S. coelicolor, S. azuraeus, S. albus 
and other S. lividans strains. 
Molecular genetic manipulations and other techniques employed in the 
following Examples are described in Hopwood et al., Genetic Manipulation 
of Streptomyces: A Laboratory Manual, John Innes Foundation, Norwich, 
England (1985). 
ABBREVIATIONS 
In the following Examples, the following abbreviations may be employed: 
LB: 10 grams (g) tryptone, 5 g yeast extract, 5 g NaCl 
MBSM (modified MBSM): See, Brawner et al., Gene, 40, 191 (1985)(in press) 
MOPS: (3)-N-morpholino-(proprane-sulfonic acid) 
YEME+MgCL.sub.2 +Glycine: [per liter(1)] 3 g yeast extract, 5 peptone, 3 g 
malt extract, 10 g glucose, 10 g MgCL.sub.2 "62H.sub.2 O, 340 g sucrose. 
SL: Mix together (NH.sub.4).sub.2 SO.sub.4 (1 g/l); L-asparagine (2 g/l); 
K.sub.2 HPO.sub.4 (9 g/l); NaH.sub.2 PI.sub.4 (1 g/l) for 0.2% agar and 
autoclave. Then mix with yeast extract (20 g/l), MgCl.sub.2 (5 g/l); 
CuCl.sub.2 (0.1 g/l); Trace elements [20 ml/l--include ZnCl.sub.2 --40 
mg/l); FeCl.sub.3 "6H.sub.2 O (200 mg/l); CuCl.sub.2 "2H.sub.2 O (10 
mg/l); NaB.sub.4 O.sub.7 "10H.sub.2 O (10 mg/l); (NH.sub.4).sub.6 MO.sub.7 
O.sub.24 "4H.sub.2 O (10 mg/l )] filter and sterilize. 
YEME (Ym base): (per liter) yeast extract (3 g); peptone (5 g); malt 
extract (3 g); MgCl.sub.2 "6H.sub.2 O (2 g) 
Ymglu: YEME+glucose (10 g) 
Ymgal: YEME+galactose (10 g) 
BACTERIAL STRAINS 
In the following Examples, the following strains of E. coli are employed; 
______________________________________ 
CGSC Strain Chromosomal 
Strain #(a) 
Designation 
Sex Markers 
______________________________________ 
4473 (galE.sup.-) 
W3109 F.sup.- 
galE9,.sup.(b) g.sup.-, 
IN(rrnD-rrnE)1 
4467 (galT.sup.-) 
W3101 F.sup.- 
galT22.sup.(b) g.sup.- ; 
IN(rrnD-rrnE)1 
4498 (galE.sup.-) 
PL-2 Hfr relAl, spoT1 
thi-1, 921E28, g.sup.-, 
______________________________________ 
.sup.(a) CGSC Strain # is the stock number designated for such strain by 
the E. coli Genetic Stock Center of the Department of Human Genetics, Yal 
University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven 
Connecticut, 06510,U.S.A. 
.sup.(b) galE9 is the old Lederberg gal9; galT22 is the old Lederberg 
gal1. 
S1 ANALYSIS 
S1 analysis is used to identify the 5' end of RNAs and the length of a RNA 
of interest. In the following Examples, S1 analysis refers to S1 
experiments carried out according to the method of Weaver et al., Nucl. 
Acids Res., 7, 1175 (1979) and Berk et al., Proc. Natl. Acad. Sci. USA, 
75, 1214 (1978). 
EXAMPLE I 
A. Cloning of a Streptomyces Lividans Galactokinase Gene 
Streptomyces lividans strain 1326 is described by Bibb et al., Mol. Gen. 
Genetics, 184, 230-240 (1981) and was obtained from D. A. Hopwood, John 
Innes Foundation, Norwich, England. Streptomyces lividans strain 1326 and 
S. lividans strain 1326 containing the pIJ6 plasmid were deposited in the 
Agricultural Research Culture Collection, See, Adams et al., Biochem. 
Biophys. Res. Comm., 89(2), 650-58 (1979)] with 30 mg/ml chloramphenicol. 
Twenty plates were spread with approximately 200 transformants per plate. 
After three days incubation at 37.degree. C., no transformants were 
detected. The minimal plates were then sprayed with nicotinic acid to 5 
ug/ml to supplement the nicotinic acid requirement of E. coli strain K21, 
and the incubation was continued for 3 more days at 37.degree. C. and for 
2 additional days at room temperature. After such incubation, the 
surviving colonies were patched to both MacConkey galactose agar (MAC-GAL) 
[See, Miller et al., cited above ] with 30 ug/ml chloramphenicol and to 
M63 minimal agar [See, Miller et al., cited above] supplemented with 0.5% 
galactose, 5 ug/ml nicotinic acid, 5 ug/ml thiamine and 30 ug/ml 
chloramphenicol. Only two colonies contained cosmid DNA that transformed 
E. coli K21 to a galK.sup.+ phenotype. Such cosmids were designated as 
psLIVGAL-1 and pSLAIVAG-2. Both colonies were light red on MAC-GAL (i.e., 
they were galK.sup.+) and also grew on the M63 medium. 
Plasmid pSLIVGAL-1 and PSLIVGAL-2 were isolated from the two galK.sup.+ 
colonies described above and were transformed, according to the method of 
Chater et al., Curr. Top. Micro. Inn., 96, 69-95 (1982), into Streptomyces 
lividans strain 1326-12 K (a galK deficient strain isolated after UV 
mutagenesis of S. lividans strain 1326, See, Brawner et al., Gene, 40, 191 
(1985), (in press). Plasmid encoded complementation of the S. lividans 
1326-12K (galK.sup.-) host was tested by observing growth of spores plated 
on MBSM-gal-thiostrepton according to the method of Brawner et al., Gene, 
40, 191 (1985) (in press). pSLIVGAL-2 showed no detectable complementation 
of the Streptomyces 1326-12K host. 
Cell extracts were prepared from cultures grown in SL medium supplemented 
with 1% glucose or galactose and 10 ug/ml thiostrepton. The extracts were 
analyzed for galactokinase production by immunoblot analysis (see, Brawner 
et al., Gene, 40, 191 (1985), in press) using rabbit antisera prepared 
against E. coli galactokinase. The protein detected by immunoblot analysis 
was the approximate size of E. coli galK. Such protein appears in 
galactose supplemented cultures of Streptomyces at levels several fold 
higher than in glucose cultures. 
B. Mapping of the S. Lividans GalK Region Within a Cosmid 
The galK region of the pSLIVGAL-1 and pSLIVGAL2 cosmids, prepared as 
described above, was identified by cloning random fragments from the 
cosmids into a pUC18 derivative [See, Norrander et al., Gene, 26, 101-106 
(1983)] and scoring complementation of E. coli strain MM294 (galK.sup.-) 
on MAC-GAL medium. The cosmid clone was partially digested with Sau3AI 
(using conditions which maximized the yield of 2 to 4 kilobase fragments), 
and the products of this reaction were ligated into the BglII site of 
pUC18-TT6, a derivative of pUC18 constructed by insertion of the following 
synthetic DNA sequence into the BamHI site of pUC18: 
5'GATCAGATCTTGATCACTAGCTAGCTAG 3' 
3' TCTAGAACTAGTGATCGATCGATCCTAG 5' 
Twelve galK.sup.+ clones (red on MAC-GAL) were screened for size. One 
clone, designated as plasmid pSAU10, was the smallest and had an insert 
size of approximately 1.4 Kb. 
In contrast to colonies containing pSLIVGAL1, the pUC clones were very red 
on MAC-GAL medium, indicating and increased production of galactokinase. 
The most likely explanation for the increased enzyme level was that the S. 
lividans galK gene was now being transcribed by an E. coli promoter which 
was stronger than the upstream promoter on the cosmid. 
The insert of pSAU10 was isolated as an EcoRI to HindIII fragment (these 
sites flank the insert region of pUC18-TT6) for use as a probe for the S. 
lividans galK gene. The chromosomal DNA used in the cloning was restricted 
with EcoRI plus MluI and BamHI plus BglII, and then blotted according to 
the method of Southern, J. Mol. Biol., 98, 503 (1975). The pSAU10 fragment 
was nick translated and hybridized to the blot. The probe identified a 1.3 
kb EcoRI-MluI fragment and a 5 kb BamHI-BalII fragment in the chromosomal 
digests. When this data was compared to the map of the cosmid insert, the 
location of the galK gene (between map positions 5 and 7, See Table A) was 
confirmed. 
C. DNA Sequencing of the S. Lividans Gal Operon 
The Streptomyces lividans gal operon was sequenced by chain termination 
[(See, Sanger et al., Proc. Nat'l Acad. Sci., U.S.A., 65, 499 (1980)]. The 
initial sequences of galK were derived from Sau3AI and SalI fragments of 
the insert of pSAU6 (a 2.3 Kb sibling of pSAU10) shotgun cloned into the 
BamHI and SalI sites (respectively) of M13 mp 10 [See, Messing, Methods in 
Enzymology, 101, 20 (1983)]. Amino acid sequences of S. lividans galT, 
galE and galK genes were predicted by computer, and further analyzed by 
comparison with amino acid sequences of the E. coli and or S. cerevisiae 
galactokinase, gal-1-phosphate uridyltransferase and UDP-4-epimerase 
enzymes. The sequences of these proteins were predicted by computer 
analysis using the total or partial DNA sequence of the genes which encode 
the gal enzymes [See, Debouck et al., Nuc Acids. Res., 13(6), 1841-1853 
(1985), and Citron and Donelson, J. Bacteriology, 158, 269 (1984)]. Some 
homology was found between the inferred protein sequence for the S. 
lividans galK, galT, galE gene products and their respective E. coli 
and/or S. cerevisiae gene products. 
The complete DNA sequence of the S. lividans gal operon is shown in Table 
1. Includes in Table 1 are the transcription start sites for the operon's 
promoters and the predicted amino acid sequences of the galT, galE and 
galK products. 
TABLE 1 
__________________________________________________________________________ 
TRANSLATED SEQUENCE OF STREPTOMYCES LIVIDANS 
GALACTOSE OPERON 
__________________________________________________________________________ 
##STR1## 
##STR2## 
##STR3## 
##STR4## 
##STR5## 
##STR6## 
##STR7## 
##STR8## 
##STR9## 
##STR10## 
##STR11## 
##STR12## 
##STR13## 
##STR14## 
##STR15## 
##STR16## 
##STR17## 
##STR18## 
##STR19## 
##STR20## 
##STR21## 
##STR22## 
##STR23## 
##STR24## 
##STR25## 
##STR26## 
##STR27## 
##STR28## 
##STR29## 
##STR30## 
##STR31## 
##STR32## 
##STR33## 
##STR34## 
##STR35## 
##STR36## 
##STR37## 
##STR38## 
##STR39## 
##STR40## 
##STR41## 
##STR42## 
##STR43## 
##STR44## 
##STR45## 
##STR46## 
##STR47## 
##STR48## 
##STR49## 
##STR50## 
##STR51## 
##STR52## 
##STR53## 
##STR54## 
##STR55## 
##STR56## 
##STR57## 
##STR58## 
##STR59## 
##STR60## 
##STR61## 
__________________________________________________________________________ 
EXAMPLE 2 
Promoters of the S. Lividans Gal Operon 
a) P1 promoter 
(i) Summary 
This promoter is galactose inducible, glucose repressible and is the 
regulatable promoter for the entire Streptomyces gal operon. S1 data 
indicates that the Streptomyces lividans gal operon encodes a 
polycistronic transcript of approximately 3.4 kilobases (Kb). The 
transcript consists of approximately 1 Kb for galT, followed by 
approximately 1 Kb each for galE and galK. (See, FIG. 1). 
Galactose induction of P1 is mediated, at least in part, by an operator 
sequence whose 5' end is located 31 bp upstream of the transcription start 
site and a represssor protein which recognizes the operator. 
(ii) Experimental: Isolation, Localization, and Characterization of the P1 
Promoter 
The sequences upstream of the Streptomyces lividans galK ATG were screened 
for promoters using the E. coli galK promoter probe system of Brawner, et 
al., Gene, 40, 191, (1985), in press. The HindIII-MluI fragment (See, 
Table A, map positions 1-5) was restricted with Sau3AI. ligated into the 
unique BamHI site of pK21 (FIG. 2), and transformed into E. coli K21 
(galK) according to the method of Example 1. pK21 is a derivative of pSKO3 
and is an E. coli-Streptomyces shuttle vector containing the E. Coli galK 
gene (See, FIG. 2. The construction of pSKO3 is described in Rosenberg et 
al., Genetic Engineering, 8, (1986), in press. The clones which expressed 
galK, i.e., those which had promoter activity, were identified on 
MacConkey-galactose plates. Two galK.sup.+ clones (designated as pK21 MH1 
and 2) were transformed into Streptomyces 1326-12 (galK). Extracts from 
transformants were cultured in Ymglu and Ymgal, and were analyzed by 
western blot analysis using anti-E. coli galactokinase antiserum. The 
blots showed significantly higher levels of galactokinases in the extracts 
from the galactose induced cultures. 
pK21 MH1 and 2 were shown by restriction analysis to contain a 410 bp 
Sau3AI insert which is contained within the HindIII and BglII sites (see 
Table A, map positions 1-2) by Southern blot analysis according to the 
method of Southern, J. Mol. Biol., 98, 503 (1975). The cloned fragment was 
analyzed by S1 analysis using RNA isolated from Streptomyces lividans 
1326-12K and E. coli K21 cultures. The fragment yielded a 290 nucleotide 
protected fragment after S1 digestion (indicating the 5' end of an mRNS 
290 bp upstream of the Saw3AI site). Hybridization experiments (using 
single stranded M13 clones of this region) have identified the direction 
of transcription as left to right as shown in FIG. 2 (i.e., transcription 
is going toward galK). 
Conventional DNA sequence analysis and additional S1 mapping analysis were 
used to define the 5' end of the mRNA. 
The sequences responsible for regulating galactose induction of P1 were 
localized by removing sequences upstream of the transcription start site 
by nuclease Bal31. Any change in promoter function or galactose induction 
by removal of these sequences was assessed using the E. coli galK promoter 
probe plasmid used to identify P1. 
(iii) Construction of Gal Promoter Deletions 
Plasmid pHL5 was constructed by cloning a DNA fragment containing 100 bp of 
sequences downstream from the start of P1 transcription and 216 bp 
upstream from the start of P1 transcription into plasmid pUC19TT1. Plasmid 
pUC19TT1 is described in Norrander et al., Gene, 26, 101-106 (1983) and 
has the Unker as pUC18-TT6. See, Example IB. Deletions extending into the 
upstream sequence preceeding P1 were generated by linearizing pHL5 with 
HindIII and treating the ends with nuclease Bal31.The uneven ends were 
subsequently repaired with the Klenow fragment of DNA polymerase I. Bal 
31-treated pHL5 was then digested with Bam-HI and run on a 5% acrylamide 
gal. DNA fragments in the molecular weight range of 100-300 bp were eluted 
from the gel and subcloned into M13 mp 10 that had been digested with 
HindII and BamHI. [See, Messing, Methods in Enzymology, 101, 20 (1983)]. 
Individual deletions were then sequenced from the single stranded phage 
DNA the dideoxy chain termination method of Sanger, et al., cited above. 
(iv) Linking the P1 Promoter Deletions to the E. coli galK Gene 
The various mp 10 clones were digested with BamHI and HindIII, DNA 
fragments containing individual deletions were isolated from low-melting 
point agarose gels and then ligated to pK21 (see, FIG. 2) that had been 
digested with BamHI and HindIII. After transformation into E. coli MM294, 
plasmid DNA was isolated for each of the deletion derivatives and 
transformed into Streptomyces Lividans 12K. 
(v) Functional Assessment of Bal 31-Generated Deletions in S. lividans 
For each individual promoter deletion, a single thiostrepton resistance 
transformant was grown to late log in YM base (YEME)+10 ug/ml 
thiostrepton. Cells were then pelleted, washed once in M56 media and 
resuspended in M56 media (see Miller, et al., cited above). The washed 
cells were then used to inoculate YM+01M MOPS (pH 7.2)+10 ug/ml 
thiostrepton supplemented with 1% galactose or 1% glucose. The cells were 
grown for 16 hours then assayed for galactokinase activity. 
Ten individual pK21 derivatives containing either 120, 67, 55, 34, 31, 24, 
20, 18, 10 or 8 bp of sequence upstream of the P1 transcription start site 
were analyzed for galactokinase expression. These results showed that 
substantially all the information necessary for galactose induction of P1, 
(i.e., 10-20 fold greater levels of galactokinase produced in galactose 
grown cells versus glucose grown cells) is included in the 31 bp of 
sequence upstream of P1, and that all such information is located in the 
67 bp of sequence upstream of P1. A deletion which leaves 34 bp of 
sequence upstream of P1 is partially inducible by galactose since 
galactose induced 6-fold greater amounts of galactokinase. Thus, one end 
of the operator must be situated within the sequences between the -24 and 
-31 position. The remaining deletions which leave either 20, 18, 10 or 8 
bp of upstream sequence result in a constitutive P1 promoter, that is the 
levels of galactokinase produced were equivalent when cells were grown in 
the presence of galactose or glucose. Although the promoter deletions 
which retained 8 and 10 bp of P1 were constitutive, the amount of 
galactokinase produced was reduced 10 fold in comparison to the promoter 
deletions which retained 18 to 120 bp of upstream sequence. This result 
indicates that sequences between the -10 and -18 positions of -1 are 
essential for promoter fashion. 
This data supports a model in which galactose induction of P1 is mediated, 
at lest in part, by an operator sequence. One end of this sequence is 
within the region 24 to 31 bp upstream of the P1 transcription start site. 
Removing part or all of the operator results in a promoter which is 
partially or totally derepressed. The other end of this sequence has now 
been defined to be contained within the 16 to 21 bp of sequence upstream 
of the P1 transcription start site. In addition, we cannot eliminate the 
possibility that the 3' end of the operator is also within the 100 bp 
downstream of the transcription start site since these sequences were 
contained within the smallest region needed to achieve galactose 
induction. These data also suggest that the factor which interacts with 
the operator sequence is a repressor protein. Finally, we do not have any 
evidence which eliminates the possibility that P1 may be controlled by 
factors other than a repressor (i.e., positive activator such as lambda 
phage cII protein) to modulate galactose induction promoter transcript. 
(vi) Construction of Additional P1 Promoter Mutations 
Oligonucleotide directed mutagenesis was performed as originally described 
by Kunke, et a. 1987 Methods Enzymol. 154:367 using the Mutagene Kit from 
Biorad (Cat. No. 170-3571) according to the manufacturer's instructions. 
M13mp18 containing a 196 base pair HindIII-BamHI1 fragment that includes 
the galP1 promoter from -69 to +103 with respect to the apparent 
transcription start site was used as template. Fragments containing 
mutations in a single hexamer were constructed by annealing an 
oligonucleotide (Sanger et al., 1977 Proc. Natl. Acad. Sci USA 
74:5463-5467) containing the desired base changes to wild type 
gal-P1-containing template DNA. Fragments containing mutations in more 
than one hexamer were constructed by annealing an oligonucleotide (Sanger 
et al. 1977 Proc. Natl. Acad. Sci USA 74:5463-5467) containing base 
changes in one hexamer to a template DNA that contained base changes in 
the other hexamer. The DNA sequence for each construction was confirmed by 
subjecting the various promotercontaining fragments to the dideoxy 
sequencing reactions of Sanger et al. 1977 Proc. Natl. Acad. Sci USA 
74:5463-5467 using the Sequenase kit (United States Biochemical Corp., 
Ohio) and the forward (-40) sequencing primer (P.No. 70736). 
(vii) Assay of Promoter Function Using XylE Fusions 
To assay the effect of various base changes within the galP1 promoter the 
196 bp BamHI-HindIII fragments containing the promoter mutations (prepared 
as described in Example 2avi, above) were ligated to the larger 
Bam-HI-HindIII fragment of pXe4 (Ingram, et al., 1989, J. Bacteriol. 
171:6617-6624) thereby generating transcriptional fusions between galP1 
and a promoterless copy of the xylE gene contained in pXE4. In all cases, 
plasmid DNA was isolated after transformation to verify the presence of 
the insert. Plasmid DNA was transformed into S. lividans 1326 using 
standard procedures (see Hopwood, et al., Genetic Manipulation of 
Streptomyces-A Laboratory Manual, F. Crows & Sons. Ltd., Norwich, England 
(1985)). Transformants were selected by overlaying the transformation 
plates with agar (0.4%) containing 100 mg/ml thiostrepton. Catechol 
dioxygenase activity was detected on plates and assayed as described by 
Ingram, 1989, J. Bacteriol., supra, except that the assays were performed 
at 30 degrees Centigrade. The results are indicated in the following 
Tables X, Y and Z. 
TABLE X 
______________________________________ 
Catechol dioxygenase activity 
(% fully induced wild type promoter) 
Position/Change Glucose Galactose 
______________________________________ 
wild type 7 100 
-35, G to C 5 206 
-34, G to C 1 12 
-33, G to C 7 333 
-32, G to C 8 9 
______________________________________ 
TABLE Y 
______________________________________ 
Catechol dioxygenase activity 
(% fully induced wild type promoter) 
Position/Change Glucose Galactose 
______________________________________ 
wild type 9 100 
-34, G to A 34 306 
-34, G to T 17 850 
-32, G to A 65 650 
-32, G to T 8 280 
______________________________________ 
TABLE Z 
______________________________________ 
Catechol dioxygenase activity 
(% fully induced wild type promoter) 
Hexamer/Change Glucose Galactose Glycerol 
______________________________________ 
wild type 10 100 10 
(-21 to -16)IV, TCTCAA 
76 474 75 
(-47 to -42)II, TCTCAA 
66 1996 ND 
(-53 to -48)I, TATCAA 
60 75 55 
(-7 to -2)VI, TATCAA 
37 91 21 
______________________________________ 
ND = not determined 
b) P2 promoter 
(i) Summary 
The P2 promoter of the Streptomyces gal operon is upstream of the galE gene 
and transcribes both galE and galK genes. 
P2 promoter expression is constitutive (i.e., not glucose 
repressed/galactose inducted) as shown by S1 analysis. 
(ii) Experimental: Isolation, Localization, and Characterization of the P2 
Promoter 
The existence of the Streptomyces gal operon P2 promoter became apparent 
when the BglIII-MluI fragment (see, Table A, map positions 2-5) of S. 
lividans 1326 DNA was inserted into plasmid pK21 see, FIG. 2) and 
galactokinase expression was observed in Streptomyces lividans 1326-12K 
transformed therewith. 
DNA sequence analysis and S1 analysis were used to identify the 5' end of 
the S. lividans gal operon P2. The 5' end of the P2 promoter transcript is 
within the 100 bp upstream of the predicted galE ATG. 
EXAMPLE 3 
Evidence of a Polycistonic Message in the Streptomyces Gal Operon 
S1 analysis was used to map the transcripts upstream and downstream of the 
Streptomyces lividans gal operon galK gene. In general, overlapping DNA 
fragments of 1-2 Kb were isolated from subclones, further restricted, and 
end labelled. The message was followed from the 3' end of galK to the 
upstream end at P1. 
The 3' end of the Streptomyces lividans gal operon transcript probably 
occurs within the first hundred bases downstream of galK. Fragments 3' 
labelled at sites within the galK sequence were not protected to their 
full length (S1 analysis) if they extend into this downstream region. One 
experiment showed a possible protected region that terminated 50-100 bp 
downstream of the galK translation stop. The existence of a transcription 
terminator can be confirmed by conventional techniques by using a 
terminator probe system. The gal operon transcript clearly does not extend 
to the PvuII site (see, Table A, map position 8) because no full length 
protection of 5' labelled PvuII fragments occurs from that site. 
5' end labelled fragments from two PvuII fragments, fragment I, (map 
positions 4-6, See, Table A), and fragment II, (map positions 6-8, see 
Table A), and the insert of pSau10 were used as sources of probes for S1 
walking from the 3' to 5' end of the message. All fragments through this 
region are protected, except the fragment containing the P2 promoter which 
shows partial and full protection. The complete protection from S1 digest 
indicates a polycistronic message which initiates upstream at P1 and 
continues to approximately 100 bp downstream of galK. 
The above data is indirect evidence of a polycistronic mRNA of the 
Streptomyces gal operon. S1 analysis using a long contiguous DNA fragment 
(e.g., the 4.5 kb HindIII-SacI fragment, see map position 7 of Table A) 
has been used to confirm the transcript size. 
EXAMPLE 4 
Localization of S. Lividans gal Operon GalE and GalT Genes 
(i) Summary 
The S. lividans gal operon galE gene was localized to 1.5 Kb PvuII fragment 
(mag position, 4-6 of Table A) of pLIVGAL1 (FIG. 1). 
The S. lividans gal operon galE coding sequences extend through the MluI 
site (map position 5 of Table A). 
The S. lividans gal operon galT gene was localized within the 1.15 Kb 
Nru-PyuIII region (see, Table A, map positions 1a-4) of pSLIVGAL1. 
The direction of S. lividans gal operon galE and galT transcription is the 
same as galK gene. 
(ii) Experimental 
It was necessary to identify the other functions contained on pLIVGAL1; 
specifically, does this plasmid encode for the enzyme galactose epimerase 
(galE) or the enzyme galactose transferase (galT). The Streptomyces gal 
operon galK gene was identified by its ability to complement an E. coli 
galK host. Thus, identification of the Streptomyces galT and galE genes 
was tested for by complemenatation of E. coli galE or galT hosts, 
respectively. An E. coli galT.sup.- strain (CGSC strain #4467, W3101) and 
two galE.sup.- strains (CGSC strain #4473; W3109 and CGSC strain #4498; 
PL-2) were obtained to test for complementation by the pSLIVGAL1 clone. 
The ca. 9 Kb HindIII-SphI fragment (see, Table A, map positions 1-16) 
containing the Streptomyces lividans gal operon galK gene was inserted 
into pUC19. This fragment was situated within pUC19 such that 
transcription from the Plac promoter of pUC19 is in the same direction as 
the Streptomyces galK gene. pUC19 is described in Yanisch-Perrou, et al., 
Gene, 33, 103 (1983). Complementation was assayed by growth on 
MacConkey-galactose plates. Cells which can utilize galactose [galE.sup.+, 
galT.sup.+, GalK.sup.+ ] will be red to pink on this medium. E. coli 
strain PL-2 (see, Example 2) containing pUC19 with the HindIII-SphI insert 
were pink on the indicator plate indicating that the HindIII-SphI fragment 
contains the Streptomyces lividans galE gene. The galE gene was later 
mapped to within the 4.5 Kb Hind-III-SacI (the SacI site is near the 
region around map position 7-8 of Table A) fragment. If the sequences from 
the MluI site (map position 5 of Table A) to the SacI site were removed 
galE complimentation of E. coli PL-2 was not detected. The 5' end of the 
galK gene is 70 base pairs (bp) from the MluI site. Therefore it seemed 
likely that the MluI site was contained within the 5' or 3' end of the 
galE gene. To determine the direction of galE transcription, the 
HindIII-SacI fragment was inserted into pUC18. In this configuration, the 
Streptomyces lividans gal gene is in the opposite orientation with respect 
to Plac. The pUC18 HindIII-SphI clone did not complement E. coli PL-2 
indicating the galE is transcribed in the same direction as galK. In 
addition it was concluded that the MluI site is contained within the 3' 
end of the galE gene. DNA sequence analysis of the PvuII-MluI fragment 
(See, Table A, map position 4-5) has identified an open reading frame 
which encodes for a polypeptide of predicted molecular weight of 33,000 
daltons. The 5' end of this reading frame is located approximately 176 bp 
from the PvuII site (See, Table A, map position 4). Therefore, the 
sequencing results support the conclusion that the 3' end of galE 
traverses the MluI site (See, Table A, map position 5). 
Similar experiments to localize the galT gene on pSLIVGALI were attempted 
with the galT hosts. 
The region between P1 and the 5' end of galE was sequenced to identify the 
galT gene. Translation of the DNA sequence to the amino acid sequence 
identified a reading frame which encodes a protein showing a region of 
homology to the yeast transferase. 
EXAMPLE 5 
Galactose Induction of S. Lividans Gal Operon GalK Gene 
(i) Summary 
Galactokinase expression is induced within one hour after the addition of 
galactose to culture medium. 
Galactokinase expression is 10 times higher in the presence of galactose 
versus glucose or no additional carbon source within 6 hours after 
addition of the sugar. 
(ii) Experimental 
Galactose induction of the Streptomyces lividans galK gene was examined by 
assaying for galactokinase activity at 1, 3, 6 and 24 hours after the 
addition of galactose. Two liters of YM+0.1M MOPS (pH 7.2) were inoculated 
with 2.times.10.sup.7 spores of Streptomyces lividans 1326. After 21 hours 
growth, galactose or glucose were added to a final concentration of 1%. 
One, three, six and twenty four hours after the addition of sugar, cells 
were isolated and assayed for galactokinase activity. Total RNA was 
prepared by procedures described in Hopwood et al., cited above. 
An increase in galactokinase synthesis was observed one hour after the 
addition of galactose. The increase continued over time (1 to 24 hours). 
S1 analysis or RNA isolated from the induced cultures confirmed that the 
increase in GalK activity was due to increased levels of the P1 promoter 
transcript. 
The S1 data and the induction studies suggest the following model for gene 
expression with the Streptomyces gal operon. The P1 promoter is the 
galactose inducible promoter. The P1 transcript includes galT, galE and 
galK. The P2 promoter is constitutive and its transcript includes galE and 
galK. 
It is interesting to note that the E. coli gal operon also has two 
promoters, P1, P2. [See, Nusso et al., Cell, 12, 847 (1977)]. P1 is 
activated by cAMP-CRP binding whereas P2 is inhibited by cAMP-CRP. 
Translation of the E. coli gal operon galE coding sequence is more 
efficient when transcription initiates at P2 which serves to supply a 
constant source of epimerase even in the absence of galactose or the 
presence of glucose [See, Queen et al., Cell, 25, 251 (1981)]. The 
epimerase functions to convert galactose to glucose 1-phosphate during 
galactose utilization and convert UDP-glucose to UDP-galactose which is 
required for E. coli cell wall bisocynthesis. It is possible that the P2 
promoter of the Streptomyces galE operon also serves to supply epimerase 
and galactokinase in the absence of galactose or during secondary 
metabolism. 
EXAMPLE 6 
The S. Coelicolor Gal Operon 
(i) Summary 
The restriction map of a fragment containing the S. coelicolor galK gene is 
identical to the restriction map of the S. lividans gal operon. (See, FIG. 
3). 
S. coelicolor can grow on minimal media containing galactose as the sole 
carbon source. 
Galactokinase expression in S. coelicolor is induced by the addition of 
galactose to the growth media. 
A promoter analogous and most likely identical to P1 is responsible for 
galactose induction of the S. coelicolor gal operon. 
(ii) Experimental 
An approximately 14 kb partial Sau3A fragment containing the S. coelicolor 
galK gene was isolated by K. Kendall and J. Cullum at the University of 
Manchester Institute of Science and Technology, Manchester, UK 
(unpublished data; personal communication). They were able to localize the 
S. coelicolor galK gene within a 3 kb EcoRI fragment by complimentation of 
a S. coelicolor galK mutant. The position of the number of restriction 
sites within the S. lividans gal operon are identical to those found 
within, upstream and downstream of the EcoRI fragment containing the S. 
coelicolor galK gene (FIG. 3). Thus, it seems likely that the gene 
organization of the S. lividans gal operon is identical to the S. lividans 
gal operon. 
Galactose induction of the S. coelicolor galK gene was examined by 
immunoblotting. S. coelicolor was grown in YM+1% galactose or 1% glucose 
(Ymglu or Ymgal) for 20 hours at 28.degree. C. Galactokinase expression 
was detected using rabbit antisera prepared against purified E. coli 
galactokinase. The protein detected was the approximate size of the E. 
coli and S. lividans galK gene product. Galactokinase expression is 
galactose induced since it was detected only when S. coelicolor was grown 
in Ym+galactose (Ymgal). 
S1 nuclease protection studies were performed to determine if galactose 
induction of the S. coelicolor gal operon is directed by a promoter 
analogous to the S. lividan P1 promoter. RNA was isolated from S. 
coelicolor grown in Ym+1% galactose or 1% glucose (Ymgal or Ymglu). The 
hybridization probe used for S1 analysis of the RNA was 410 pb Sau3A 
fragment which contains the S. lividans P1 promoter. Its transcription 
start site and the 5' end of the galT gene. The S1 protected fragment 
detected by this analysis co-migrated with the protected fragment detected 
when the probe was hybridized to RNA isolated from S. lividans grown in 
the presence of galactose. Thus, this result shows that galactose 
induction of the S. coelicolor gal operon is directed by a sequence 
indistinguishable from the S. lividans P1 promoter. 
It should be noted that the following strains of Streptomyces have been 
observed to be able to grow on medium containing galactose as the only 
carbon source; 
S. albus J1074 (obtained from Dr. Chater, John Innes Foundation, Norwich, 
England) 
S. carzinostaticus--ATCC accession number 15944 
S. carzinostaticus--ATTC accession number 15945 
S. antifibrinolyticus--ATCC accession number 21869 
S. antifibrinolyticus--ATCC accession number 21870 
S. antifibrinolyticus--ATCC accession number 21871 
S. longisporus--ATCC accession number 23931 
The abbreviation "ATCC" stands for the American Type Culture Collection, 
Rockville, Md., U.S.A. 
While the above descriptions and Examples fully describe the invention and 
the preferred embodiments thereof, it is understood that the invention is 
not limited to the particular disclosed embodiments. Thus, the invention 
includes all embodiments coming within the scope of the following claims.