Development and characterisation of a promoter library for Sulfolobus acidocaldarius

The hyperthermoacidophilic archaeon Sulfolobus acidocaldarius is a promising production host for industrial biotechnology applications due to its ability to thrive in extreme conditions. However, the lack of well-characterised genetic parts and tools, particularly promoters, limits its potential for metabolic engineering. In this study, we developed the first promoter library for S. acidocaldarius by randomising specific regions of the core promoter sequence of P _{Saci 2137} , a promoter known to function in both S. acidocaldarius and Escherichia coli . The library was initially screened in E. coli using mKate2, a red fluorescent reporter protein, and seven promoters were selected for characterisation in S. acidocaldarius using the thermostable β-galactosidase reporter LacS. The resulting promoter collection exhibited a 5-fold range of expression levels in S. acidocaldarius , spanning from low to high constitutive expression when compared to S. acidocaldarius promoters P _sac7d and P _malE . This study demonstrates a successful workflow for generating and characterising S. acidocaldarius promoters, providing a valuable toolkit for fine-tuning gene expression and optimising metabolic pathways in this extremophilic archaeon. The design principles established here can be extended to other archaeal systems with similar promoter architectures.