May 15, 2009 (Vol. 29, No. 10)

Tim Zeppenfeld

Red/ET Recombination Technology Opens Opportunities in Industrial Biotech

In white biotechnology, microorganisms serve as industrial bioreactors for large-scale production of fine chemicals, enzymes, active pharmaceutical ingredients, agro chemicals, and food additives. In particular E. coli is used for the production of aromatic amino acids (e.g., L-tryptophan, L-phenylalanine, L-tyrosine), carotenoids, or shikimic acid as starting material for chiral compounds.

Recent technological advances in biotransformation, fermentation, and metabolic engineering have made such manufacturing techniques much more attractive for chemical and protein production. E. coli strain development requires genetic modifications to gear metabolic processes toward the production of the required chemical compound. Such metabolic engineering can be accomplished through gene deletions, insertions, or differential gene expression.

The potential of homologous recombination for DNA engineering has been recognized for decades. Methods based on the endogenous Rec system have been developed for E. coli, but this approach has many limitations because linear DNA molecules become rapidly degraded and are, therefore, inapplicable as recombination partners. In addition, Rec-mediated recombination requires long (Ž500 bp) homology regions.

Gene Bridges’ Red/ET recombination technology, developed in A. Francis Stewart’s Laboratory at the European Molecular Biology Laboratory in Heidelberg, overcomes many of these drawbacks. Red/ET allows for rapid modifications of DNA molecules and does not depend on restriction sites or the size of the DNA molecule.

DNA Modification

While many systems for genetic engineering in E. coli allow only for modifications at specific positions (e.g., transposons, group II introns), the Red/ET system is sequence independent. Red/ET recombination is based either on the phage proteins RecE/RecT from Rac prophage or Reda/Redb from phage l and requires short flanking homologous stretches of only 50 bp (Figure 1).

Homology arms, therefore, of any given sequence can easily be introduced to a linear fragment by PCR. The required phage genes are provided by an expression plasmid that can be transferred into any E. coli strain, and a temperature-sensitive replication origin allows for a subsequent removal of the plasmid.

Figure 1. The central step in Red/ET Recombineering is the exchange of genetic information between two DNA fragments containing homology regions.

Genome-borne selection markers that are flanked by recognition sequences of a sequence-specific recombinase (FLP or Cre)—so-called FRT or loxP sequences— can be removed in a rapid and safe way by short expression of the appropriate enzyme. Thus, the removal of the selection markers allows for the successive modification of several genes, leaving only a FRT or loxP scar behind (Figure 2). Based on this strategy, up to seven genes have been modified in client projects.

Red/ET recombination enables rapid, easy, and precise modification of the E. coli genome. Foreign DNA fragments can be selectively inserted into any desired position. This technology facilitates the development of customized production strains for already existing biotechnological products.

There is evidence to suggest this technique is also applicable to other microorganisms besides E. coli, which remains the organism most widely used by molecular biologists.

Furthermore, Red/ET Recombineering is not limited to chromosomal modifications. The ability to clone and subclone large DNA molecules presents a variety of new ways to simplify conventional DNA engineering exercises. With this method changes can be directed to a chosen DNA sequence and point mutations can be introduced at any specific site of a target DNA molecule like a BAC or cosmid regardless of its size (Figure 3).

Figure 2. Application of suitable linear DNA fragments allows modifications such as gene knockout through disruption (A) or deletion (B) of an open reading frame, insertion of a foreign gene (C) or change of a promoter (D).

Figure 3. Red/ET allows every type of DNA engineering possible regardless of target size or type of modification.

Tim Zeppenfeld, Ph.D. ([email protected]), is senior scientist at Gene Bridges. Web: Red/ET Recombination is patented by Gene Bridges and was first published under the name ET cloning.

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