Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using a micro-organism to make a protein or polypeptide
Reexamination Certificate
1999-09-10
2001-07-10
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using a micro-organism to make a protein or polypeptide
C435S252100, C435S320100, C435S440000, C536S023100, C536S024100
Reexamination Certificate
active
06258565
ABSTRACT:
The present invention relates to novel expression vectors for expressing desired genes within a range of bacterial hosts and, in particular, to expression vectors based on the RK2 replicon and the TOL plasmid regulatory functions.
The cloning and expression of genes is a central tool in biotechnology. Traditionally, genes have been cloned and expressed in enteric bacteria, most notably
E. coli
, which for a long time was regarded as the most useful host for gene cloning. However, the inability of
E. coli
to express some biological properties, for example certain metabolic activities, or to carry out appropriate modifications and processing of certain gene products, has encouraged the development of alternative host-vector systems, in particular for different hosts. The use of non-enteric bacteria for basic and applied molecular research has extended the need for well characterised vector systems for such organisms. Thus, vector systems have been designed which are specific for the bacterial species of interest, e.g. soil bacteria. However, a more useful approach would be to design vectors which may be used across a broad range of microbial hosts, and work in recent years has been directed to this.
In addition, expression of foreign genes, and indeed over expression of native genes, can significantly perturb the physiology of the host cell and constitute a strong selective pressure for elimination or inactivation of the cloned genes. Vectors in which the expression of cloned genes can be regulated and controlled have therefore become increasingly important.
The present invention is directed towards meeting this continuing need for new and improved expression vectors for the controlled expression of genes in a wide range of hosts. In particular, it has been found that efficient and controlled expression of cloned genes in a broad range of hosts may be achieved by constructing expression vectors which combine the replicon from the RK2 plasmid family with the expression regulatory functions of the TOL plasmids.
In its broadest aspect, the present invention thus provides an expression vector comprising an RK2 minimum replicon together with an expression cassette comprising the regulatory functions of a TOL plasmid.
As used herein the term “expression cassette” refers to a nucleotide sequence encoding or comprising the various functions required to express a DNA sequence, notably the promoter-operator functions and the associated regulatory sequences required for expression from that promoter, e.g translational and transcriptional control elements and/or sequences encoding regulatory proteins, which may act to regulate expression, for example at the level of the promoter.
RK2 is a well-characterised naturally occurring 60 Kb self-transmissible plasmid of the IncP incompatibility group well known for its ability to replicate in a wide range of gram-negative bacteria (Thomas and Helinski, 1989, in Promiscous Plasmids in Gram-negative bacteria (Thomas, C. M., Ed.) Chapter 1, pp 1-25, Academic Press Inc (London) Ltd, London). It has been determined that the minimal replicating unit of RK2 consists of two genetic elements, the origin of vegetative replication (oriV), and a gene (trfA) encoding an essential initiator protein (TrfA) that binds to short repeated sequences (iterons) in oriV (Schmidhauser and Helinski, 1985, J. Bacteriol. 164, 446-455; Perri et al., 1991, J. Biol. Chem; 266, 12536-12543). This minimal replicating unit is termed the so-called “RK2 minimum replicon”, and has been extensively characterised and studied in the literature. A wide range of replicons (termed “mini-RK2 replicons”) and cloning vectors based on the RK2 minimum replicon or on derivatives of the RK2 plasmid have been prepared and described in the literature (see, for example, Li et al., 1995, J. Bacteriol. 177, 6866-6873; Morris et al., J. Bacteriol., 177, 6825-6831; Franklin and Spooner, in Promiscous Plasmids in Gram-negative bacteria (Thomas, C. M., ed) Ch. 10, pp 247-267, Academic Press Inc. (London) Ltd., London; Haugan et al., 1992, J. Bacteriol 174:7026-7032; and Valla et al. 1., 1991, Plasmid, 25, 131-136).
The TOL plasmids are another series of well-characterised naturally occurring plasmids and their derivatives, which occur in Pseudomonas sp. and which encode the enzymes required for the catabolism of toluene and xylenes (for a review see Assinder and Williams 1990, Adv. Microb. Physiol., 31, 1-69).
The catabolic genes of TOL plasmids are organised in two operons, an upper pathway operon (OP1) encoding genes and regulatory sequences required for the oxidation of aromatic hydrocarbons to aromatic carboxylic acids, and a lower, or meta pathway operon (OP2) necessary for the oxidation and ring clearage of the aromatic nucleus of aromatic carboxylic acids, giving rise to intermediates which are channelled into the intermediary metabolism. The expression of the two operons is controlled by two positive regulatory proteins XylR and XylS, in the presence of the corresponding substrate ligands toluene/xylene and benzoate/toluate respectively. Activated XylR stimulates transcription from the Promoter Pu of the upper pathway operon, whereas activated XylS induces the meta pathway operon from the promoter Pm. XylR may also induce the promoter Ps of the xylS gene (see Assinder and Williams, Supra). A regulatory cassette based on the xylR gene and Pu promoter has been described and used to prepare expression vectors which enable regulated gene expression induced by aromatic hydrocarbons (Keil and Keil, 1992, Plasmid, 27, 191-199). However, it has not previously been proposed to combine the TOL regulatory functions Pu/xylR or Pm/xylS with an RK2-based replicon within an expression vector construct.
Viewed from a further aspect, the present invention thus provides an expression vector comprising a RK2 minimum replicon together with a promoter Pm and/or Pu and a corresponding regulatory gene xylS and/or xylR as derived from a TOL plasmid.
In such expression vectors of the invention the catabolic genes of the TOL plasmids, encoding the enzymes of the metabolic pathway, are generally absent. Especially, the full complement of catabolic structural genes, in any one, or both, of the operons, are absent.
The novel vectors of the invention allow the regulated expression of cloned genes in a wide range of host cells.
As mentioned above, the RK2 replicon has been well studied and its complete nucleotide sequence is reported (Pansegrau et al., 1994, J. Mol. Biol., 239, 623-633). Thus, sources for the RK2 minimum replicon are well established and readily available. Hence, for example, the RK2 minimum replicon may be derived from the parental plasmid RK2 or from any of the vast number of derivatives or mini RK2 plasmids described and available from the literature (see e.g. Li et al; Morris et al., Franklin and Spooner; Haugen et al; and Valla et al., Supra). As exemplary of a suitable source plasmid for the minimum RK2 replicon may be mentioned plasmid pFF1 (Durland et al., 1990, J. Bacteriol, 172, 3859-3867), but many other source plasmids are available and could be used. The separate elements of the minimum replicon, oriV and the trfA gene may be isolated from the same source together or separately or from separate sources.
Likewise, any of the TOL plasmids and their derivatives widely known and described in the literature could be used as the source of the TOL regulatory functions (see e.g. Assinder and Williams, Keil and Keil, Supra and Mermod at al., 1986, J. Bacteriol., 167, 447-454). Indeed, a number of plasmids are known in the literature which have TOL genes inserted, and any of these could be used as the source of the TOL regulatory functions for the present invention. The regulatory genes xylR and/or xylS may be inserted together with the Pu and/or Pm promoter from the same source or the promoter and regulatory gene may be derived independently from separate sources. Thus, for example a Pm promoter may be derived from plasmid pERD21, (a RSF1010-based replicon, Ramos at al., 1988, Febs Letters, 226, 241-246
Blatny Janet M.
Karunakaran Ponniah
Valla Svein
Leiv Eiriksson Nyfotek AS
Sandals William
Schwartzman Robert A.
Schwegman Lundberg Woessner & Kluth P.A.
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