Development of a genetic system for hyperthermophilic Archaea: expression of a moderate thermophilic bacterial alcohol dehydrogenase gene in Sulfolobus solfataricus.

Abstract

The Escherichia coli/Sulfolobus solfataricus shuttle vector pEXSs was used as a cloning vehicle for the gene transfer and expression of two bacterial genes in Sulfolobus solfataricus. The alcohol dehydrogenase (adh) from the moderate thermophilic Bacillus stearothermophilus (strain LLDR) and a mutagenised version encoding a less thermostable ADH enzyme were the selected genes. S. solfataricus adh promoter and aspartate aminotransferase terminator were used to drive the heterologous gene expression and to guarantee the correct termination of the transcripts, respectively. The constructed vectors were found to be able to carry these 'passenger' genes without undergoing any rearrangements. The active transcription of bacillar mRNAs was ascertained in vivo by RT-PCR. Transformed S. solfataricus expressed functional exogenous ADHs that showed unaffected kinetic and chemical-physical features.