Use of the Red- Recombineering Method for Genetic Engineering - Genetic Engineering: Recent Developments in Applications - page 28

Although the frequency of selection of the desired mutation was not high, in
principle, λ Beta-dependent integration of ssDNAs allowed the construction of
the unmarked mutations in the P. ananatis.
discussion
It is currently well established that high-frequency recombination between short
homologies can be catalyzed in E. coli cells by the λ Red functions [4-7,43-45].
Unfortunately, up to the present, the range of bacteria for which this system has
been utilized is limited. The main goal of the present study was to widen use of
the λ Red-recombineering technology to P. ananatis, a bacterium of interest in
the field of metabolic engineering. A broad-host-range λ Red-expressing plasmid
useful for P. ananatis was constructed. The observed general toxicity of simulta-
neous expression of the λ gam, bet, and exo genes for P. ananatis SC17 cells was
overcome by selection of the special recipient, SC17(0).
It is known that expression of λ Red functions in E. coli cells can lead to toxic
effects [46,47]. Certainly, expression of λ Red genes has been investigated for
E. coli in detail. In addition to its direct influence on traditional recombination
pathways due to inhibition of RecBCD and SbcCD nucleases by λ Gam, the ac-
tivity of λ Red proteins can interfere with the processes of replication and repair
(see, [48,49] for reviews). For example, prolonged expression of gam gene could
lead to formation of linear multimers of high, medium and low copy-number
plasmids, and, even, of minichromosomes [50-53]. Expression of exo gene in
addition to gam enhances this effect [50,52]. The plasmid linear multimers, also,
may interfere with λ Red-recombination [18]. Murphy and co-workers showed
that extended expression of λ Red-recombination functions could significantly
induce a spontaneous mutagenesis, probably caused by interfering with mismatch
repair UvrD-dependent pathway of E. coli [18].
Toxicity of expression of λ Red genes for the bacteria closely related to E. coli,
but differing in the enzymes of replication, recombination and reparation, could
not be predicted in advanced, as in the case of P. ananatis SC17. It is difficult, as
well, to give an univocal explanation of how λ Red-mediated toxicity could be
overcome. It seems that the decrease of this toxicity could be based on (i) lower
intracellular level of λ Red proteins in the mutant cell, caused by reduced level
in the synthesis of λ Red proteins or by increased efficiency of the specific prote-
olysis of λ Red proteins, (ii) decreased affinity of specific targets for interaction
with λ Red proteins or increased level of biosynthesis of these targets. As for the P.
ananatis mutant strain obtained, both variants of the explanation may be possible.
As mentioned in the Results section, it is not very probable that SC17(0) strain