Affiliation:
1. Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan
Abstract
ABSTRACT
We have recently developed a gene disruption system for the hyperthermophilic archaeon
Thermococcus kodakaraensis
by utilizing a
pyrF
-deficient mutant, KU25, as a host strain and the
pyrF
gene as a selectable marker. To achieve multiple genetic manipulations for more advanced functional analyses of genes in vivo, it is necessary to establish multiple host-marker systems or to develop a system in which repeated utilization of one marker gene is possible. In this study, we first constructed a new host strain, KU216 (Δ
pyrF
), by specific and almost complete deletion of endogenous
pyrF
through homologous recombination. In this refined host, there is no need to consider unknown mutations caused by random mutagenesis, and unlike in the previous host, KU25, there is little, if any, possibility that unintended recombination between the marker gene and the chromosomal allele occurs. Furthermore, a new host-marker combination of a
trpE
deletant, KW128 (Δ
pyrF
Δ
trpE
::
pyrF
), and the
trpE
gene was developed. This system made it possible to isolate transformants through a more simple selection procedure as well as to deduce the transformation efficiency, overcoming practical disadvantages of the first system. The effects of the transformation conditions were also investigated using this system. Finally, we have also established a system in which repeated utilization of the counterselectable
pyrF
marker is possible through its excision by pop-out recombination. Both endogenous and exogenous sequences could be applied as tandem repeats flanking the marker
pyrF
for pop-out recombination. A double deletion mutant, KUW1 (Δ
pyrF
Δ
trpE
), constructed with the pop-out strategy, was demonstrated to be a useful host for the dual markers
pyrF
and
trpE
. Likewise, a triple deletion mutant, KUWH1 (Δ
pyrF
Δ
trpE
Δ
hisD
), could also be constructed. The transformation systems developed here now provide the means for extensive genetic studies in this hyperthermophilic archaeon.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
193 articles.
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