The invention relates to an expression vector that is suitable for efficient screening of (meta)genome libraries, preferably in Escherichia coli. 
Only about 1-5% of all known microorganisms are at present cultivable in the laboratory with current methods. Methods have been developed in recent times which should make it possible to use the genetic resources of non-cultivable microorganisms. This field is also called “metagenomics”, with the term “metagenome” denoting the genetic information of all organisms of a particular habitat, regardless of whether these are cultivable or not.
By direct cloning of the DNA obtained from environmental samples into suitable vector systems (plasmids, cosmids, BACs, YACs) this resource becomes available for easy manipulation in the laboratory. These gene banks (metagenome libraries) can be used for example for searching for novel enzymes. Finding completely novel enzyme activities requires activity-based screening of prepared metagenome libraries. A precondition for this is a suitable detection system (agar plate assays, microtitre plate systems), which permits simultaneous screening of the largest possible number of clones (high-throughput screening). Furthermore, expression of the genes must be provided in a heterologous host. In addition to E. coli, other organisms such as Streptomyces lividans or Pseudomonas putida are also employed as host in metagenome studies.
Problems with the metagenome technique relate in particular to expression of the genes found. These include inadequate transcription, for example because promoters are not recognized, toxicity of the products to the host, missing cofactors or chaperones and therefore incorrect folding of the proteins in the heterologous host, and missing secretion systems (W. R. Streit et al., Curr Opin Microbiol. 2004, 7(5), 492-8).
Conventional (meta)genome libraries for screening in E. coli are generally constructed in artificial chromosomes (BAC), cosmid or fosmid systems or plasmids. Until now, (meta)genomic plasmid libraries have mainly been constructed using conventional cloning vectors, which generally have an individual, comparatively weak promoter (e.g. lac promoter) or are designed entirely for the use of internal promoters of the cloned DNA. This weak promoter was not originally intended for expression of the cloned DNA, but is present as promoter before the lacZ gene, which is often used as marker. In this connection, reference may be made for example to R. Ranjan et al., Biochem Biophys Res Commun., 2005, 335(1), 57-65; and A. Knietsch et al., Appl Environ Microbiol., 2003, 69(3), 1408-1416.
The relative weakness of the promoter does not have any negative consequences in sequence-based screening of the (meta)genome library. However, if the same plasmid libraries are used for screening the activity of the target proteins encoded by the library, expression of the target proteins is then often based on the weak promoter located at the plasmid. With the cosmid/fosmid systems that are often used, the functional expression of the target genes is based exclusively on recognition and reading of the non-E. coli promoters located on the inserted DNA. In this connection, reference may be made for example to K. S. Hong et al., J Microbiol Biotechnol., 2007, 17(10), 1655-60.
Owing to the weakness of the promoter or the non-recognition of non-E. coli promoters, some of the target proteins are barely expressed, or not at all, so that activity screening of the target proteins is far more difficult. These limitations make iterative activity screening of sub-libraries (cluster screening, cf. US 2008/220581=WO 2005/040376) impossible in most cases. Instead, complicated and time-consuming activity screening with individual clones, e.g. on agar plates, is necessary.
Another problem in activity screening is that when constructing (meta)genome libraries it is not possible to influence the orientation of the open reading frame (ORF) on the cloned DNA. It is also possible for two successive open reading frames to have different directions of reading. In activity screening with conventional expression vectors, a large part of the sequence information contained in the (meta)genome library is therefore often lost because the promoter used only covers one of the two possible directions of reading.
U.S. Pat. No. 6,780,405 (=WO 01/83785) discloses a regulated system for delivery of antigens. In this system, however, the DNA to be cloned into the insertion sequence is not under the control of both promoters. Instead, one of the two promoters controls the on or a gene for regulating the ori. Such a system is hardly suitable for screening metagenome libraries, as only 50% of the sequence information contained is captured.
U.S. Pat. No. 6,030,807 discloses an operon that codes for enzymes that are linked with the use of L-arabinose. The operon does not, however, have an insertion sequence located between two promoters converging towards each other. The system also does not include a vector with two different promoters converging towards each other, between which an insertion sequence is arranged, in each case downstream.
U.S. Pat. No. 6,977,165 (=WO 02/083910) discloses a method of production of a vector that includes at least one spliceable intron. The vector size is not, however, maximum 3000 bp.
Schmeisser et al., Appl. Microbiol. Biotechnol 2007, 75(5), 955-62 is a review of the subject: Metagenomics, biotechnology with non-cultivable microbes. The publication does not contain any information on expression in plasmids with two promoters converging towards one another, and inducible separately from one another, between which an insertion sequence is arranged, in each case downstream, so that the expression of a DNA sequence cloned into the insertion sequence is placed under the control of both promoters.
U.S. Pat. No. 7,005,423 (=WO 00/01846) discloses a method for identifying DNA that is responsible for a particular phenotype. However, that method does not use a vector with promoters that are inducible separately from one another, and flow towards one another. It is even a precondition of the method that both promoters are identical. The vector does not comprise at most 3000 bp.
S. Kim et al., Prot. Expr Purif. 2006, 50(1), 49-57 discloses rare codon clusters on the 5′-terminus, which have an influence on heterologous expression of archaic genes in E. coli. The publication does not, however, contain any mention of an expression vector that comprises two promoters inducible separately from one another, and converging towards each other, between which an insertion sequence is arranged, in each case downstream, so that the expression of a DNA sequence cloned into the insertion sequence is placed under the control of both promoters.
F. W. Studier, J. Mol. Biol. 1991, 219(1), 37-44 discloses the use of T7 lysozyme bacteriophage for improving an inducible T7 expression system. The system does not, however, have an expression vector that comprises two promoters inducible separately from one another, and converging towards each other, between which an insertion sequence is arranged, in each case downstream, so that the expression of a DNA sequence cloned into the insertion sequence is placed under the control of both promoters.