Regulatory nucleic acid sequences and uses in actinomycetes

Novel nucleic acid sequences, vectors for expressing same, and uses of said sequences, in particular in actinomycetes fermentation methods.

The present invention relates to a new nucleic acid sequence, to vectors 
for its expression and to its use in fermentation processes in 
actinomycetes. 
Actinomycetes are branched filamentous Gram-(+) bacteria. Among 
actinomycetes, streptomycetes represent the largest family. Streptomycetes 
are spore-forming filamentous bacteria which live naturally in the soil 
under strictly aerobic conditions. 
Actinomycetes, and streptomycetes in particular, are of great importance 
from an industrial standpoint. In particular, they possess the feature of 
producing a wide variety of secondary metabolites (Demain, Biology of 
Actinomycetes 88, Okami (Eds), Tokyo, Japan scientific societies press, 
1988, p. 19-25). These metabolites can have very different structures and 
biological properties (herbicides, anticancer agents, anthelmintics, 
anabolic agents, antibiotics, and the like). The best known of these 
metabolites are antibiotics (chemical substances produced by an organism 
and having a deleterious effect on other organisms). Streptomyces are 
currently the source of 70% of industrially produced antibiotics. The 
structural diversity of the antibiotics synthesized is not to be found in 
any other bacterial genus. Thus, almost all types of structure are 
represented: .beta.-lactams (e.g. ampicillin), polypeptide antibiotics 
(e.g. streptogramins), aminoglycoside antibiotics (e.g streptomycin, 
kanamycin, and the like), macrolides (e.g. erythromycin, spiramycin, and 
the like) or alternatively cyclines (e.g. tetracycline), and the like. 
For these reason, it is especially advantageous to be able to have tools 
(vectors, promoters, and the like) at one's disposal enabling these 
microorganisms to be manipulated genetically. Such tools would make it 
possible, in effect, to modify the levels of synthesis of these 
metabolites, or to prepare synthesis intermediates or derivatives of these 
metabolites, and the like. Such tools would also make it possible to make 
these microorganisms manufacture recombinant products, in particular 
heterologous proteins, according to genetic engineering techniques. 
In this connection, Patent Application No. EP 350,341 describes vectors 
derived from plasmid pSAM2 having very advantageous properties. Thus, 
these vectors are capable of integrating in a site-specific manner in the 
genome of actinomycetes, and possess a broad host range and high 
stability. Moreover, they may be used for transferring nucleic acids into 
actinomycetes and expressing these nucleic acids therein. However, these 
vectors possess some drawbacks, which lie, in particular, in their low 
copy number per cell, and in the absence of means of controlling the copy 
number. Thus, pSAM2 and its derivatives generally integrate on the basis 
of a single copy per chromosome. 
The present invention supplies a solution to this problem, by providing 
tools capable of improving the conditions of industrial use of the vectors 
derived from pSAM2. The present invention describes, in effect, a gene 
whose expression product leads to the appearance, from integrated forms, 
of replicative free forms of plasmid pSAM2 or of vectors derived from the 
latter. This has the effect of increasing the copy number of pSAM2 or its 
derivatives, since the free forms are present in a high copy number per 
cell. 
The present invention also describes cassettes for the expression of this 
gene, vectors containing it and their use for inducing the appearance of 
free copies of pSAM2 or integrative vectors derived from the latter. 
The Applicant has, in effect, isolated, sequenced and characterized a 
region of plasmid pSAM2 capable of inducing the appearance of replicative 
free copies of pSAM2 or its derivatives. The Applicant has also shown that 
this region could be used in cis (on the same vector) or in trans (not on 
the same vector) to act on pSAM2 or its derivatives. The sequence of this 
region is presented in the sequence SEQ ID NOS:1 and 2. More specifically, 
this region and its functional role were demonstrated by studying variants 
of plasmid pSAM2: on the one hand pSAM2(B2) originating from S. 
ambofaciens ATCC 15154 for which no free form is observed, and on the 
other hand pSAM2 (B3) and pSAM2 (B4) originating from other S. ambofaciens 
strains for which the free form is observed with the integrated form. This 
study enabled two different point mutations to be characterized (those of 
pSAM2 (B3) and that of pSAM2 (B4), both localized in the promoter region 
controlling the expression of a pSAM2 gene (see SEQ ID NOS:1 and 2. These 
mutations lead to the appearance of the free form of pSAM2 (B3) and of 
pSAM2 (B4). This gene was then cloned and sequenced, and its capacity to 
cause the appearance of the free form of plasmids derived from pSAM2 from 
the integrated copy was demonstrated. 
A first subject of the invention hence lies in a nucleic acid sequence 
comprising all or part of the sequence SEQ ID NOS:1 and 2 or of a variant 
of the latter. 
For the purposes of the present invention, the term variant denotes any 
sequence differing from the sequence SEQ ID NOS:1 and 2 as a result of the 
degeneracy of the genetic code, as well as any sequence which hybridizes 
with these sequences or fragments of the latter and whose product 
possesses the stated activity. These variants may be obtained from SEQ ID 
No. 1 by any technique known to a person skilled in the art, in particular 
mutation, deletion, substitution, addition, hybridization and the like. 
Hybridizations may be carried out under conventional conditions of 
stringency (Maniatis et al., see General techniques of molecular biology) 
or, preferably, under conditions of high stringency. The capacity of the 
variants to induce the appearance of replicative free forms of pSAM2 or 
its derivatives may be determined on an actinomycete strain containing 
such a plasmid in integrated form (for example on the strain ATCC 15154), 
by transfecting the said variant into the strain under conditions 
permitting its expression, and verifying the appearance of the free forms 
(see examples). 
Another subject of the invention relates to any cassette for the expression 
of the sequence SEQ ID No. 1 or of a variant of the latter as defined 
above, comprising the said sequence or variant under the control of a 
constitutive or regulated promoter. 
The use of a constitutive promoter is especially advantageous when the 
cassette is used in tans to induce free forms of an integrated plasmid. In 
this case, the cells containing the integrated form of the plasmid are 
transfected with the expression cassette to induce the appearance of 
replicative free forms, making it possible, for example, to isolate and/or 
purify the plasmid. 
The use of a regulated promoter is especially advantageous when the 
cassette is used in cis (on the vector derived from pSAM2 itself), for 
example in an industrial fermentation process. In this case, the whole of 
the proliferation and growth phase of the cell is performed without 
expression of the gene of the invention, that is to say with a single copy 
of the plasmid per chromosome, and, for the production phase (of a 
recombinant product, of a gene for the biosynthesis or regulation of the 
synthesis of a metabolite, and the like), the regulated promoter is 
induced, bringing about the appearance of several free copies of the 
plasmid and thus an enhanced production activity. This mode of 
implementation is especially advantageous when the vector derived from 
pSAM2 carries heterologous sequences whose presence in several number of 
copies may be toxic to the cells and/or affect their growth. 
Among constitutive promoters which can be used in the context of the 
present invention, mention may be made more especially of any constitutive 
promoter which is functional in actinomycetes, such as, for example, the 
promoter of the ermE gene or a variant of the latter (Bibb et al., Gene 41 
(1986) E357), the p14 promoter of phage I19 of S. ghanaensis (Labes et 
al., Sixth DFGWT/AFAST, 27-30/11/92), or any fragment containing a 
promoter region of a ribosomal operon of S. ambofaciens (Pernodet et al., 
Gene 79 (1989) 33). 
Among regulable promoters which can be used in the context of the present 
invention, mention may be made more especially of any regulable promoter 
which is functional in actinomycetes. These can comprise promoters induced 
specifically by an agent introduced into to the culture medium, such as, 
for example, the thiostrepton-inducible promoter tipA (Murakami et al., J. 
Bactt., 171 (1989) 1459), or thermoinducible promoters such as that of the 
groEL genes, for example (Mazodier et al., J. Bact. 173 (1991) 7382). They 
can also comprise an actinomycetes promoter which is specifically active 
in the late phases of the proliferation cycle of actinomycetes, such as, 
for example, certain promoters of genes of the secondary metabolism (genes 
for the production of antibiotics, in particular). 
Another subject of the invention relates to the use of the sequences of the 
invention as defined above, or of cassettes containing them, for inducing 
the appearance of free copies of vectors which are derived from pSAM2 and 
integrated in an actinomycete. 
As stated above, this use may be performed in cis (the gene or cassette 
being carried by the integrative vector derived from pSAM2) or in trans 
(the gene or cassette being on another vector or even introduced directly 
as such). 
The integrative vectors derived from pSAM2 as mentioned above are vectors 
comprising at least the elements of pSAM2 needed for integration, excision 
and replication. More especially, these vectors hence comprise at least 
the attP and int regions as described in Application EP 350,341, the Xis 
gene, the repSA gene and the origin of replication (ori). These different 
regions are shown on the map of plasmid pSAM2 given in FIG. 1, on which 
some restriction sites enabling these regions to be extracted also appear. 
Advantageously, the integrative vectors derived from pSAM2 also comprise a 
recombinant DNA sequence coding for a desired product. The latter can be a 
peptide, polypeptide or protein of pharmaceutical or agri-foodstuffs 
importance. In this case, the system of the invention makes it possible to 
increase the copy number of this sequence per cell, and hence to increase 
the levels of production of this product and thus to increase the yields 
of the preparation process. The desired product can also be a peptide, 
polypeptide or protein participating in the biosynthesis (synthesis, 
degradation, transport or regulation) of a metabolite by the actinomycete 
strain in question. In this case, the system of the invention makes it 
possible to increase the copy number of this sequence per cell, and hence 
to increase the levels of production of this product, and thus either to 
increase the levels of production of the metabolite, or to block the 
biosynthesis of the metabolite, or to produce derivatives of the 
metabolite. 
The sequences of the invention may thus be used in any actinomycete, in the 
genome of which pSAM2 vectors or its derivatives are capable of 
integrating. In particular, they may be used in fermentation processes 
involving strains of Streptomyces, of mycobacteria, of bacilli, and the 
like. As an example, there may be mentioned the strains S. 
pristinaespiralis (ATCC 25486), S. antibioticus (DSM 40868), S. 
bikiniensis (ATCC 11062), S. parvulus (ATCC 12434), S. glauescens (ETH 
22794), S. actuosus (ATCC 25421), S. coelicolor (A3(2)), S. ambofaciens, 
S. lividans, S. griseofuscus, S. limosus, and the like (see also Smokvina 
et al., Applications of the integrated plasmid pSAM2, GIM90, Proceedings, 
Vol. 1, p. 403-407). 
Vectors derived from pSAM2 containing the elements described above may be 
constructed by a person skilled in the art on the basis of his general 
body of knowledge and the teachings of the present application (see also 
General techniques of molecular biology), 
The sequences of the invention are most especially suitable for use in an 
industrial process for the production of antibiotics (spiramycin, 
streptogramins, .beta.-lactams, and the like, the genes for which are 
described in Applications EP 346,000 and PCT/FR93/00923, in particular. 
The present invention will be described more fully by means of the examples 
which follow, which are to be considered to be illustrative and 
non-limiting.

GENERAL TECHNIQUES OF MOLECULAR BIOLOGY 
The methods traditionally used in molecular biology, such as preparative 
extractions of plasmid DNA, centrifugation of plasmid DNA in a caesium 
chloride gradient, agarose or acrylamide gel electrophoresis, purification 
of DNA fragments by electroelution, protein extractions with phenol or 
phemnol/chloroform, ethanol or isopropanol precipitation of DNA in a 
saline medium, transformation in Escherichia coli, and the like, are well 
known to a person skilled in the art and are amply described in the 
literature Maniatis T. et al., "Molecular Cloning, a Laboratory Manual", 
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982; Ausubel F. 
M. et al. (eds), "Current Protocols in Molecular Biology", John Wiley & 
Sons, New York 1987!. 
Plasmids of the pBR322 or pUC type and phages of the M13 series are of 
commercial origin (Bethesda Research Laboratories). 
For ligation, the DNA fragments may be separated according to their size by 
agarose or acrylamide gel electrophoresis, extracted with phenol or with a 
phenol/chloroform mixture, precipitated with ethanol and then incubated in 
the presence of phage T4 DNA ligase (Biolabs) according to the supplier's 
recommendations. 
The filling-in of 5' protruding ends may be performed with the Klenow 
fragment of E. coli DNA polymerase I (Biolabs) according to the supplier's 
specifications. The destruction of 3' protruding ends is performed in the 
presence of phage T4 DNA polymerase (Biolabs) used according to the 
manufacturer's recommendations. The destruction of 5' protruding ends is 
performed by a controlled treatment with S1 nuclease. 
Mutagenesis directed in vitro by synthetic oligodeoxynucleotides may be 
performed according to the method developed by Taylor et al. Nucleic 
Acids Res. 13 (1985) 8749-8764! using the kit distributed by Amersham. 
The enzymatic amplification of DNA fragments by the so-called PCR 
Polymerase-catalyzed Chain Reaction, Saiki R. K. et al., Science 230 
(1985) 1350-1354; Mullis, K. B. and Faloona F. A., Meth. Enzym. 155 (1987) 
335-350! technique may be performed using a "DNA thermal cycler" (Perkin 
Elmer Cetus) according to the manufacturer's specifications. 
Verification of the nucleotide sequences may be performed by the method 
developed by Sanger et al. Proc. Natl. Acad. Sci. USA, 74 (1977) 
5463-5467! using the kit distributed by Amersham. 
EXAMPLES 
Example 1 
Cloning and sequencing of the sequence SEQ ID No. 1 
The free form of pSAM2 (B2) is not observed in S. lividans TK24 (Hopwood et 
al., J. Gen. Microbiol. 129 (1983) 2257), only the integrated form is 
observed. With pSAM2 (B3), the free form of the plasmid coexists with the 
integrated form. Hybrid plasmids were from pSAM2 (B3) in which different 
regions were replaced by the equivalent regions originating from pSAM2 
(B2). This enables it to be shown that the mutation enabling pSAM2 (B3) to 
exist in free form was localized in a 2-kb KpnI restriction fragment. The 
sequence of this KpnI fragment was determined for pSAM2 (B2) and pSAM2 
(B3). A single nucleotide differs between these two sequences: a G/C pair 
in pSAM2 (B2) is replaced by an A/T pair in pAM2 (B3). Sequence analysis 
showed that this mutation occurred upstream of an open reading frame which 
extended further than the KpnI site. The sequence of this open reading 
frame was determined; it is presented in the sequence SEQ ID No. 1. This 
open reading frame, designated rmf (or pra), is located between the korSA 
and traSA genes. The mutation causes the disappearance of a recognition 
site for the restriction enzyme ApaLI (recognition site: 5'GTGCAC 3'). 
This site is present in pSAM2 (B2) but absent in pSAM2 (B3). 
The sequence SEQ ID No. 1 also comprises 100 bp upstream of the coding 
region, comprising a protion of the promoter (residues 1 to 101) and the 
5' non-coding but transcribed region (residues 102 to 154) carrying, in 
particular, the ribosome binding site (RBS). 
In the mutant pSAM2 (B4, for which the free form can be observed, the ApaLI 
site is still present, indicating that the mutation is not localized at 
the same place as in pSAM2 (B3). The sequence of pSAM2 (B4) for the 
corresponding region showed that the sequences of pSAM2 (B2) and of pSAM2 
(B4) differed by one nucleotide. This mutation was localized 8 nucleotides 
upstream of the mutation detected in pSAM2 (B3). Thus, in two independent 
cases, the presence of the free form of pSAM2 (B3) and pSAM2 (B4), which 
coexists with the integrated form, is due to a point mutation upstream of 
a pSAM2 gene. These two independent mutations are both located in a region 
shown by messenger RNA analysis experiments to constitute the promoter of 
this gene. 
Example 2 
Construction of cassettes and vectors for the portion of the sequence SEQ 
ID No. 1 
Several constructions were carried out using the following promoters and 
plasmids: 
Modified ermE promoter (ermE*). The modified ermE promoter used corresponds 
to the promoter described by Bibb et al. cited above, possessing a 
deletion of 3 nucleotides (bases 252-254, FIG. 2 of Bibb et al. cited 
above). This modified promoter gives a strong and constitutive expression. 
It was isolated in the form of a 275-bp KpnI-BamHI fragment. 
tipA promoter. This promoter (Murakami et al. cited above) is present in 
plasmid pPM927 (Smokvina et al., Gene 94 (1990) 53). This is induced 
specifically in the presence of thiostrepton. 
Plasmids pIJ486 and pIJ487. These two plasmids have been described by Ward 
et al. (Mol. Gen. Genet. 203 (1986) 468). These plasmids alone have no 
influence on the status (free or integrated) of pSAM2. 
2.1. Construction of the vector pOS531 
The vector pOS531 was constructed by cloning the coding portion of the rmf 
gene (residues 155 to 505 of the sequence SEQ ID No. 1) at the 
BamHI-HindIII sites of plasmid pIJ487. This vector hence carries the naked 
rmf gene (without expression signal or 5' non-coding region). A map of 
this vector is given in FIG. 2. 
2.2. Construction of the vector pOS532 
The vector pOS532 was constructed by cloning the 275-bp fragment carrying 
the modified ermE promoter at the EcoRI-BamHI sites of the vector pOS531. 
This vector hence carries the naked rmf gene under the control of the 
modified ermE promoter (FIG. 3). 
2.3. Construction of the vector pOS541 
The vector pOS541 was constructed in 2 steps: 
cloning of a fragment containing the coding portion of the rmf gene and the 
5' non-coding region (residues 102 to 505 of the sequence SEQ ID No. 1) at 
the BamHI-HindIII sites of plasmid pIJ487, 
addition of the 275-bp fragment carrying the modified ermE promoter at the 
EcoRI-BamHI sites of the vector obtained above. 
This vector hence carries the rmf gene provided with its 5' non-coding 
region, under the control of the modified ermE promoter (FIG. 4). 
2.4. Construction of the vector pOS544 
The vector pOS544 carries an rmf gene deleted in its 3' portion (residues 
102 to 276 of the sequence SEQ ID No. 1 are present). It was constructed 
by cloning this region in the form of a BamHI-BspHI fragment (BspHI end 
treated with the Klenow fragment of E. coli DNA polymerase I) into the 
BamHI-HindIII sites of plasmid pIJ487 (HindIII end treated with the Klenow 
fragment of E. coli DNA polymerase I). The vector pOS544 hence carries the 
5' portion of the rmf gens (5' non-coding region +5' coding region), which 
portion lacks, however, its promoter. A map of this vector is given in 
FIG. 5. 
Example 3 
Appearance of replicative free forms of integrative vectors derived from 
pSAM2due to the expression product of the rmf gene 
This example shows that the overexpression of the gens SEQ ID No. 1 can 
cause the appearance in free form of an integrated copy of pSAM2. 
3.1. Direct evidence for the role of replication activator played by rmf 
may be obtained by causing, by expression of rmf, the appearance in free 
form of an integrated copy of pSAM2(B2). The fact that pSAM2(B2) possesses 
an additional ApaLI site relative to pSAM2(B3) enables it to be verified 
simply by observation of the ApaLI digestion profile that the free plasmid 
is indeed pSAM2(B2). 
3.2. The activity of the sequence SEQ ID No. 1 was also demonstrated by 
transformation of S. lividans carrying an integrated copy of pSAM2(B2) 
with the different vectors described above, followed by a search for free 
forms. To this end, S. lividans containing plasmid pSAM2(B2) was 
transformed by the protoplast technique with the different vectors 
described in Example 2. The plasmid DNA or total cellular DNA is extracted 
from a stationary-phase culture of the transformed clones. The plasmid DNA 
is digested with the enzyme ApaLI and the digestion fragments separated by 
agarose gel electrophoresis. Observation of the restriction profile 
reveals whether or not free copies of pSAM2(B2) are present. These results 
are then confirmed by experiments involving hybridization of the total DNA 
with a pSAM2probe. 
Results obtained are presented in FIG. 7. 
These results show that pOS541, which contains the modified ermE promoter, 
the untranslated region upstream of rmf with the ribosome binding site and 
the rmf coding frame, causes the appearance of replicative free forms of 
pSAM2(B2). As expected, no effect is observed with a vector containing 
only the rmf coding portion (pOS531), or with a vector containing modified 
ermE and the coding portion of rmf but no ribosome binding region 
(pOS532). No effect is observed with a vector carrying a deletion in the 
3' portion of rmf (pOS544). 
The same type of effect is obtained when the cassette permitting inducible 
expression of rmf is localized on the plasmid for which it is desired to 
cause the appearance of free forms (vector pOS666, FIG. 6). 
These results collectively provide a clear demonstration that the sequence 
of the invention is capable of inducing, in cis or in trans, the 
appearance of free forms of vectors derived from pSAM2. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 2 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 812 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CCAGCAGCCGACTGACGACCGCTCAACTCCTCACAGCCCGTCGCGAGTTCTCTGTCGCGG60 
CGGGTTGACTCATGTATAGGAGTGGTGCACTCTTCTTCATGTCACTCATATACATGAGTG120 
ACGGAGTCCAGCCTCTATAGAGGAGTGATCCGCTGTGCGTCAGATCCCCGTCGACACCTC180 
CGCCGCAACCGTGATGGTCGCCAAGACTCCGGAGCCGAAGGTGAAGGACCGCCGGACCGG240 
TGAGCTGGCCGTCGACGCCGAGACCGGTGCCAAGCTCATGACCGTGAACGTGATGTTCGC300 
GGCCAACGACGAAGTCGAGATTCTGTCCGTGACCGTCCCGGAGACCGGTATCTCCGGTGA360 
ACTGGCCATGGGTACCCCGGTCGCGCTGACGGGGCTCATCGCCCGGCCGTGGGAGAACGA420 
GTTCAACGGCCAGAAGCGGCACGGCATCGCGTTCCGCGCGGTCGCGGTCACGTCGCTGAC480 
CGCTGCGGGCTCGAAGGCTGCCTGATCATGACGTGGTTCATGGTCGCTGTGGTTGTGGTC540 
GTCGCTGCTGCGGGTCTCCTGCGGTGGCGGCGCCCCGCCTGGTACTGGCTCACCTTCGGG600 
GCCCTGGTCGCGACGGTGCGGGTCCTGGTCCGTACGCCTCGGTCATGGAAGCGTGCGGGC660 
TGACGGTCCGCCCTCACGCTGGCGGCTGCTCTGGCCCGGATGGCGAATGCCGCGCCTGAG720 
TCCCGGCCGCCGCGCATCTTGCGGTTACGTCCCACTCGTACCGGCCTGGTCTGCGGCTCA780 
AGCTCCGGCCGGGACAGGATGCCTTCGACGTG812 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 116 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetArgGlnIleProValAspThrSerAlaAlaThrValMetValAla 
151015 
LysThrProGluProLysValLysAspArgArgThrGlyGluLeuAla 
202530 
ValAspAlaGluThrGlyAlaLysLeuMetThrValAsnValMetPhe 
354045 
AlaAlaAsnAspGluValGluIleLeuSerValThrValProGluThr 
505560 
GlyIleSerGlyGluLeuAlaMetGlyThrProValAlaLeuThrGly 
65707580 
LeuIleAlaArgProTrpGluAsnGluPheAsnGlyGlnLysArgHis 
859095 
GlyIleAlaPheArgAlaValAlaValThrSerLeuThrAlaAlaGly 
100105110 
SerLysAlaAla 
115 
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