Streptomyces secretion vector

Cloning vectors are disclosed to obtain secretion of a desired protein from a host Streptomyces when a structural gene coding for the protein is inserted into the vector. The vector has: (1) regulatory DNA that includes a promoter sequence effective to start transcription in the host Streptomyces and DNA that encodes a ribosome-binding site; (2) a DNA sequence that codes for a signal sequence that occurs naturally in a Streptomyces strain or that derives from such a DNA sequence; and (3) at least one engineered restriction endonuclease recognition site positioned for the insertion of a structural gene, the DNA that encodes a signal sequence and attached structural gene being transcribed and translated together under the control of the regulatory DNA. Expression vectors are disclosed which include a structural gene coding for a desired protein so positioned. Streptomyces cells containing the vector and methods of using them to produce the desired protein are disclosed. Finally, an alkaline phosphatase gene is inserted in secretion vectors for use in a method of evaluating the ability of those vectors to cause secretion in host gram positive bacteria; the method may be used to evaluate either a potential host or a potential secretion vector.

BACKGROUND OF THE INVENTION 
This invention relates to genetically engineered strains of bacteria of the 
genus Streptomyces. As used in this application, the term "Streptomyces 
bacteria" or "Streptomyces" means any bacterial strain that is a member of 
the genus Streptomyces as classified in Buchanan et al., The Shorter 
Bergey's Manual For Determinative Bacteriology (Williams & Wilkins 1982). 
The invention also relates to vectors for engineering Streptomyces and 
methods of producing desired compounds with the resulting engineered 
organisms. 
For some time now, the pharmaceutical industry has used strains of 
Streptomyces to produce desired compounds, particularly antibiotics. 
Vectors carrying various genes that are expressed in Streptomyces have been 
reported. Katz et al. (1983) J. General Microbiol. 129:2703-2714 report 
cloning a DNA fragment coding for tyrosinase from S. antibioticus DNA into 
two plasmids. The resulting hybrid plasmids are inserted in a strain of S. 
lividans. They report that most of the tyrosinase activity of S. 
antibioticus is secreted; in contrast, most of the activity remains 
intracellular in S. lividans clones. There is no indication of the 
mechanism for transport of the activity through the cell wall into the 
medium. 
Thompson et al. (1980) Nature 286:525-527 report cloning the tsr gene from 
S. azureus which is expressed under its own promoter on various vectors. 
Thompson et al. (1983) PNAS (USA) 80:5190-5194 report sequencing the S. 
fradiae aph (neomycin resistance) gene together with its promoter region. 
They also report that the aph gene has been incorporated into high 
copy-number, broad host-range vectors. 
Burnett et al. (1984) Abstracts of the Annual Meeting of the American 
Society of Microbiology H98, p. 107, report cloning a 10-kb fragment from 
S. lividans containing a transcription start site and two structural genes 
(beta-galactosidase and Bgl protein). They have determined the sequence of 
that promoter region as well as of a ribosome-binding site. 
Robbins et al. (1981) J. Biol. Chem. 256:10,640-10,644 report cloning the 
gene coding for the enzyme endo-N-acetylglucosaminidase (endo H) from S. 
plicatus into E. coli plasmid pBR322. The enzyme is expressed in E. coli 
and is found in the cytoplasmic, periplasmic, and supernatant fractions. 
There have been reports of vectors that include a leader sequence which 
enables secretion of an expressed protein in other bacterial species. For 
example, see Gilbert U.S. Pat. Nos. 4,338,397 and 4,411,994 [E. coli]; and 
Palva et al. (1983) Gene 22:229-235 [Bacillus subtilis]. 
SUMMARY OF THE INVENTION 
One aspect of the invention features, in general, a cloning vector 
engineered to receive a structural gene coding for a desired protein to 
cause expression of the gene in, and secretion of the protein from, a host 
Streptomyces bacterial strain. The vector comprises: (1) regulatory DNA 
which includes a promoter sequence effective to start transcription in the 
host Streptomyces and DNA that encodes a ribosome-binding site; (2) a DNA 
sequence that codes for a signal sequence, the DNA sequence deriving from 
DNA that occurs naturally in a Streptomyces strain and (3) at least one 
engineered restriction endonuclease recognition site positioned for the 
insertion of a structural gene, the DNA encoding a signal sequence and the 
inserted structural gene being transcribed and translated together under 
the control of the regulatory DNA. For convenience, we will use the term 
"protein" to include proteins or smaller peptides. By a sequence that 
"derives from" another sequence, we mean a sequence that is identical to 
that other sequence or is a naturally occurring or engineered variant or 
chemically synthesized copy or variant thereof that preserves the desired 
function. By a "signal sequence" we mean a portion of a protein that 
causes the protein or a fragment of it to be transported through a cell 
membrane. 
In a second aspect, the invention features an expression vector for 
expression of a desired protein in a host from a first Streptomyces 
species. The vector includes the promoter DNA and the DNA encoding a 
signal sequence as described above and a structural gene coding for a 
desired protein. The DNA that encodes a signal sequence derives from DNA 
that occurs naturally in a second species of Streptomyces, and the vector 
is capable of expressing a preproduct in the Streptomyces host; the 
preproduct includes a signal sequence and the desired protein. The signal 
sequence permits secretion of a compound comprising the desired protein. 
In a third aspect, the invention features an expression vector as described 
above except that the host and the source from which the DNA encoding a 
signal sequence derives are not necessarily different species of 
Streptomyces; and the structural gene is one that is not naturally 
transcribed and translated with the DNA encoding a signal sequence. 
In a fourth aspect, the invention features a Streptomyces host cell that 
includes one of the above-described expression vectors. 
In a fifth aspect, the invention features a method of making a desired 
protein by growing a Streptomyces host carrying one of the expression 
vectors, in a suitable medium and under suitable conditions, and 
recovering the compound comprising the protein from the medium. 
In a sixth aspect, the invention features a method of evaluating a 
potential secretion vector or potential host gram positive bacterial 
strains using alkaline phosphatase. The secretion vector includes 
regulatory DNA as described above and DNA encoding a signal sequence. The 
method comprises providing a test vector comprising the secretion vector 
and DNA encoding for alkaline phosphatase positioned therein for 
transcription and translation of the DNA encoding a signal sequence and 
the alkaline phosphatase-coding DNA under control of the regulatory DNA. 
After transformation of gram positive host strain cells with the test 
vector, strain cells are cultured in a medium comprising an indicator 
responsive to extracellular alkaline phosphatase. 
In preferred embodiments, the DNA encoding a signal sequence, the promoter 
sequence, or the ribosome-binding site, or all three, derive from the 
respective components associated with a Streptomyces endo H gene, most 
preferably that of S. plicatus. Alternatively, the promoter may derive 
from the promoter sequence from the neomycin resistance (aph) gene of S. 
fradiae. The cloning vector comprises a sequence enabling replication in 
Streptomyces, for example from pIJ702, and, in one embodiment, a sequence 
enabling replication in E. coli, for example from pBR322. The cloning 
vector also comprises a DNA sequence capable of conferring antibiotic 
resistance, for example to thiostrepton, on Streptomyces. Preferably, an 
antibiotic resistance gene effective in E. coli is also included, for 
example, one of the resistance genes of pBR322. 
In preferred embodiments of the expression vectors, the structural gene is 
the endo H gene of S. plicatus or the alkaline phosphatase gene of E. 
coli. 
In preferred embodiments of the cell and the method of production, the 
expression host may be one of the following species, without limitation: 
S. lividans, S. coelicolor, S. fradiae, S. griseofuscus, S. reticuli, S. 
rimosus, S. albus, S. parvulus, S. ambofaciens, S. aurofaciens, S. 
plicatus, S. espinosus, S. lincolnensis, S. erythresus, S. antibioticus, 
S. griseus, or S. glaucens. Most preferably, the host is S. lividans or S. 
coelicolor. 
In preferred embodiments of the method of evaluating secretion vectors, 
after it is determined that alkaline phosphatase is secreted into the 
medium, a structural gene coding for a desired protein or peptide is 
inserted in a restriction endonuclease recognition site positioned in the 
secretion vector for transcription and translation of the DNA encoding a 
signal sequence and structural gene under control of the regulatory DNA. 
The preferred gram positive bacteria are members of the genus Streptomyces 
or the genus Bacillus (as defined in Bergey's manual, cited above). 
The invention provides a versatile and generally useful cloning vector for 
Streptomyces that can be used to engineer an expression vector that 
includes any of a broad range of structural genes to be expressed with the 
DNA encoding a signal sequence and secreted into the medium surrounding 
the recombinant cells. 
Streptomyces are particularly useful hosts for protein production for 
several reasons. In Streptomyces, antigenic lipopolysaccharide 
contaminants are not produced; a single membrane permeability barrier 
allows protein export into the culture medium; and secretion of a variety 
of proteins occurs in the regular course of growth. As with other gram 
positive bacteria, Streptomyces cells are capable of protein secretion 
because they lack a gram-negative outer membrane. Streptomyces generally 
do not produce toxic materials that must be separated from the desired 
product. Furthermore, the pharmaceutical industry has acquired extensive 
experience with large scale industrial growth and fermentation of 
Streptomyces strains, thus providing standard fermentation protocols. The 
life cycle of Streptomyces enables long time courses of high level 
production of desired proteins during the secondary stage of metabolism. 
High copy-number Streptomyces plasmids have been characterized, and 
genetically engineered derivatives are available for cloning purposes. The 
transcriptional and translational regulatory components of Streptomyces 
biosynthesis apparently do not limit expression of foreign genes. 
Since the product is secreted, it is possible to obtain quantities that 
might be lethal if localized within the cell. Moreover, recovery of the 
product is simplified by the absence of intracellular contaminants and the 
avoidance of product purification therefrom. In addition, various 
cell-immobilization techniques are made more useful when the product is 
secreted into extracellular medium. 
The method of evaluating potential secretion vectors and potential gram 
positive secretion hosts is a simple technique using an indicator that can 
be easily evaluated. Once promising candidates are isolated, a cloned gene 
coding for a desired protein can be inserted in the vector to enable 
expression of the protein in, and secretion of it from, the gram positive 
bacteria. 
Other features and advantages of the invention will be apparent from the 
following description of the preferred embodiments thereof and from the 
claims.

The primary components of the expression and cloning vector are illustrated 
by the construction and structure of expression vectors pGH202 and pGH205, 
and by derivatives of pNH224, a vector shown in FIG. 4. pGH202 and pGH205, 
have been deposited with the American Type Culture Collection and they 
bear the following accession numbers, respectively: ATCC 39896 and ATCC 
39895. Applicants' assignee, BioTechnica International, Inc. acknowledges 
its responsibility to replace these cultures should they die before the 
end of the term of a patent issued hereon, and its responsibility to 
notify the ATCC of the issuance of such a patent, at which time the 
deposits will be made available to the public. Until that time the 
deposits will be made available to the Commissioner of Patents under the 
terms of 37 CFR .sctn.1.14 and 35 USC .sctn.112. 
The Vectors 
FIG. 3 shows maps of expression vectors pGH202 and pGH205 for expressing 
the secreted endo H. pGH202 and pGH205 contain the entire endo H gene from 
S. plicatus including a 600-bp region containing the transcriptional and 
translational regulatory controls of that gene. Of particular interest is 
the sequence of DNA that is about 120-bp long and that includes the DNA 
that encodes the signal sequence of that gene. The DNA sequence and 
resulting amino-acid sequence that functions as a signal sequence for endo 
H are included in the following sequences: 
EQU ATG TTC ACT CCG GTT CGC AGA AGG GTG CGG ACG GCT GCG CTC Met Phe Thr Pro Val 
Arg Arg Arg Val Arg Thr Ala Ala Leu 
EQU GCG CTC TCG GCC GCC GCG GCC CTC GTC CTC GGT TCC ACC GCC Ala Leu Ser Ala Ala 
Ala Ala Leu Val Leu Gly Ser Thr Ala 
EQU GCG AGC GGC GCG TCA GCG ACC CCC TCA CCC GCT CCG GCC CCG Ala Ser Gly Ala Ser 
Ala Thr Pro Ser Pro Ala Pro Ala Pro. 
While it may be preferable to include in the expression vector the entire 
DNA sequence set out above, the secretion signaling ability does not 
require that entire sequence in every host. For example, primary cleavage 
may occur at one or more sites within one peptide residue of the 29th 
amino acid (Ala) of the above listed sequence, and the resulting 28-30 
amino acids may be sufficient to enable secretion. 
Other genetic components of pGH202 and pGH205 include: 
1. pIJ702, including the promoter-proximal part of the mel gene from S. 
antibioticus, the tsr gene from S. azureus, the pIJ101 replicon, and the 
promoter-distal portion of the mel gene; and 
2. the pBR322 fragment including the promoter distal portion of the gene 
for tetracycline-resistance, the pBR322 origin of replication, and the 
gene for ampicillin resistance. 
pGH205 contains all of the genes known to be carried by pGH202, and, in 
addition, it includes the promoter of the aminoglycoside 
phosphotransferase (aph) gene coding for resistance to neomycin. 
FIG. 4 shows construction of an expression vector for expressing E. coli 
alkaline phosphatase and . containing the regulatory information discussed 
above regarding pGH202. Specifically, the vector includes the 
above-described DNA that encodes the S. plicatus endo H signal sequence 
and the structural gene for E. coli alkaline phosphatase. 
pGH202 and pGH205 are bifunctional in that they replicate both in 
Streptomyces and in E. coli strains, and both vectors confer resistance to 
an antibiotic on each of those organisms, thus making them useful for 
engineering both in E. coli and Streptomyces. While such bifunctionality 
is preferred, the ability to replicate in E. coli, and an antibiotic 
resistance marker for E. coli, are not essential to the use of the vector 
as a Streptomyces secretion vector. 
Construction of pGH202 
Standard methods of recombinant DNA technology may be used to construct 
pGH202 as shown in FIG. 1. Following cleavage of plasmid pEHBl.6 from S. 
plicatus [described in Robbins et al. (1981) J. Biol. Chem. 
256:10,640-10,644] with restriction enzyme BamHI, the plasmid is treated 
with alkaline phosphatase (calf intestinal) and ligated to plasmid pIJ702 
DNA [disclosed in Katz et al. (1983) cited above and available from John 
Innes Institute, Norwich, England] that had been digested with BglII. 
These ligated plasmids are transformed into E. coli 294 (ATCC 33,625), and 
ampicillin-resistant derivatives are selected. Plasmid DNA isolated from a 
mixed population of E. coli transformants is then used to transform 
protoplasts of S. lividans to thiostrepton resistance, a genetic marker 
carried on the Streptomyces pIJ702 replicon. Transformants are isolated 
and plasmid DNA purified and characterized with respect to marker 
restriction sites. 
Construction of pGH205 
As shown in FIG. 2, the promoter fragment of the gene for 
neomycin-resistance is isolated from the aph gene of S. fradiae carried on 
pIJ28 described by Hopwood et al. Ch. 4 Genetic Engineering Principles and 
Methods, pp. 119-145 Setlow et al. Eds. (Plenum Press, NY, 1982). 
Digestion of this plasmid with BamHI releases the aph gene as 1.1 kb 
promoter-proximal and 2.2 kb promoter-distal DNA fragments. The 1.1-kb 
fragment is purified by gel electrophoresis, digested with NcoI, and the 
resulting mixture of 900- and 200-bp pieces is subjected to treatment with 
DNA polymerase (Klenow fragment), ligation to EcoRI linkers, and digestion 
with EcoRI. The 900-bp promoter fragment with EcoRI ends is purified and 
ligated to EcoRI-digested plasmid pUC8, available from BRL. The reaction 
mixture is used to transform E. coli strain JM83, also available from BRL, 
and a strain bearing DNA with the aph fragment in the pUC8 backbone is 
selected from among JM83 cells using LB agar supplemented with ampicillin 
and X-gal (5'-bromo-4'-chloro-3'-indolyl-.beta.-D-galactoside). The 
resulting plasmid, pGH8, is used as the source of DNA for further 
constructions involving the 900-bp aph promoter fragment, described below. 
Digestion of pGH8 with EcoRI releases the aph promoter segment, which is 
mixed with pGH202 DNA that had been purified from an E. coli transformant 
and digested with EcoRI and treated with phosphatase (calf intestinal). 
This mixture is ligated and used to transform E. coli. Clones that are 
ampicillin-resistant and carry the 900-bp insert are identified and 
characterized for orientation of aph, and plasmid DNA from an appropriate 
clone is used to transform S. lividans protoplasts to thiostrepton 
resistance. 
Other Expression Vectors 
Streptomyces vectors for expressing and secreting other proteins may be 
constructed as illustrated in FIG. 4. The specific illustrative example of 
FIG. 4 includes the DNA that encodes the endo H signal sequence described 
above, linked to a cloned gene of interest such as the E. coli alkaline 
phosphatase gene (phoA). While alkaline phosphatase is a useful enzyme in 
its own right, it provides a particularly useful tool for screening 
potential secretion vectors for use in gram positive bacteria, 
particularly in Streptomyces, and for screening potential gram positive 
host strains. The potential secretion vector includes a promoter and other 
regulatory DNA that is functional in the bacteria of interest. The vector 
also includes a DNA that encodes a signal sequence whose function is to be 
evaluated with an alkaline phosphatase structural gene. Construction of 
such a vector is described below. 
In general, a vector containing the DNA that encodes a signal sequence is 
cleaved at a site near the downstream end of the signal sequence. To 
optimize the distance between the DNA that encodes the signal sequence and 
the structural gene, the fragment terminus may be further digested and/or 
connected with a linker to permit insertion of the desired structural gene 
in position to be expressed in frame with the DNA that encodes the signal 
sequence. Cells containing such structures can be selected from the 
mixture with an appropriate assay for the desired compound, such as those 
described below for endo H and for alkaline phosphatase. A Streptomyces 
replicon should be added if it is not already present on the constructed 
vector. 
The above general procedure is illustrated (FIG. 4) by construction of 
pNH224 derivatives. 
First, pEHBl.6 lac-UV5 [Robbins et al. (1981) cited above] is treated to 
remove the SphI site from its pBR322 backbone leaving the SphI site 
downstream from the endo H signal sequence as the only SphI site on the 
vector (pEHBl.06B lac-2). This is accomplished by SalI digestion, 
insertion of a BamHI linker, BamHI digestion, and ligation. 
The resulting plasmid (pEHBl.06B lac-2) is subjected to SphI digestion and 
to Ba131 digestion in order to obtain fragments with a terminus close to 
the end of the DNA sequence that encodes the signal sequence. Linkers 
(e.g. HindIII) are added to allow insertion of the structural gene phoA on 
segments having a HindIII proximal terminus and a BamHI distal terminus. 
The cloned phoA gene can be obtained from A. Wright, Department of 
Microbiology and Molecular Biology, Tufts University School of Medicine, 
Boston, Mass., or it can be obtained as described in Inouye et al., (1981) 
J. Bacteriol. 146:668-675. 
By appropriate screening in E. coli, vector pNH224, which includes the phoA 
gene in proper reading frame and position, can be selected. Finally, 
pNH224 can be fused with DNA containing a Streptomyces replicon, such as 
pIJ702. 
Expression of the Gene and Secretion of the Protein 
The following description of the use of expression vectors pGH202, pGH205, 
and a derivative of pNH224 containing a Streptomyces replicon exemplify 
the featured expression strains and the featured method of producing a 
protein. Specifically, the production of endo H by, and use of alkaline 
phosphatase to monitor protein secretion from, S. lividans host cells :s 
described. 
Endo H is currently among the most expensive of commercially available 
enzymes. It is obtained from culture supernatants of a strain of 
Streptomyces plicatus (ATCC 27800) (previously classified as S. griseus) 
at yields of approximately 200 .mu.g per liter, hence purification is 
cumbersome and yield very low. The unique enzyme specificity, cleavage of 
the glycosidic bond between two N-acetylglucosamine residues of high 
mannose oligosaccharides of cell-surface glycoproteins, makes endo H an 
important analytical tool for research on processing of cell surface 
components. 
As indicated above, alkaline phosphatase serves as a convenient research 
tool to measure expression and secretion of proteins from Streptomyces and 
other gram positive bacteria. Convenient plate tests are available to 
monitor the secretion of alkaline phosphatase, such as assays using the 
dye X-P (5-bromo-3-chloro-indole phosphate) which turns blue upon cleavage 
by alkaline phosphatase, or assays using compounds such as 
para-nitrophenyl phosphate. 
To produce a desired compound or to test for alkaline phosphatase 
secretion, S. lividans mycelia, carrying the appropriate plasmid 
constructed as described above, is grown in an appropriate medium at 
30.degree. C. with aeration. For example, the broth may be YEME broth 
having the following constituents per liter: Difco yeast extract, 3 g; 
Difco peptone, 5 g; Oxoid malt extract, 3 g; glucose, 10 g; sucrose 340 g; 
MgCl.sub.2 added to 5mM after autoclaving. Samples of culture taken at 
various time points are centrifuged, and supernatant fractions assayed for 
the product. For example, endo H may be assayed as described by Robbins et 
al. (1981), cited above; and alkaline phosphatase may be assayed by the 
production of yellow color as described by Brickman and Beckwith (1975) J. 
Mol. Biol. 96:1-10. 
The protein of interest is translated and secreted in substantial 
quantities, permitting efficient recovery and purification from the 
extracellular growth medium. Maximum secretion is obtained after 7-10 
days. 
Other Embodiments 
Other embodiments are within the following claims. For example, and not by 
way of limitation, structural genes for other proteins such as calf 
rennin, insulin, growth hormone, interleukins, erythropoietin, tissue 
plasminogen activator, or hoof-and-mouth disease antigens may be inserted 
in place of the E. coli alkaline phosphatase gene described above. Various 
fermentation techniques that are known for Streptomyces fermentation 
processes may be used--for example, various cell immobilization techniques 
may be used.