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Archaeal Expression System (Sulfolobus shuttle vector)

Sulfolobus spp., a thermoacidophilic crenarchaeote, can be easily grown in

the laboratory and is interesting from a biotechnological point of view. Due to their growth conditions (75-80°C and pH 3-3.5), enzymes from this organism are thermostable and, if secreted, also acid-resistant and may have potential for industrial applications. Furthermore, Sulfolobus is developing into an important archaeal

model organism that is increasingly being used in biochemical and molecular biology studies. So far, in vivo experiments in Sulfolobus were hampered by the poor reproducibility of the few described genetic systems. This technical hurdle could be overcome with the developed Sulfolobus- Escherichia coli shuttle vector. Our construct meets the requirements for a small, episomal, easy-to-handle multicopy vector. The different components are an E. coli replicon with a selectable marker for E. coli, a Sulfolobus replicon, and a selectable marker for Sulfolobus. The plasmid pRN1 was chosen as Sulfolobus replicon because of its relatively small size of 5.4 kb. Our shuttle vector was found to be stable in E. coli and to be suitable for cloning of additional DNA sequences. Furthermore, the suitability of the vector constructs for reporter gene experiments and for the expression of proteins in Sulfolobus was demonstrated.

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