Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1996-06-07
1998-04-21
Railey, II, Johnny F.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 4352523, 4353201, 536 231, 536 237, C12N 121, C12N 1531, C12N 1576, C12P 2100
Patent
active
057416753
DESCRIPTION:
BRIEF SUMMARY
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) originatin
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Friedmann Annick
Guerineau Michel
Hagege Juliette
Pernodet Jean-Luc
Sezonov Guennady
Railey II Johnny F.
Rhone-Poulenc Rorer S.A.
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