Expanding the genetic toolbox for thermostable protein production

by
Schematic overview of the study. The plasmid library was constructed by cloning a 200-nucleotide random sequence upstream of the thermostable reporter gene, superfolder citrine fluorescent protein (IFP). Plasmid DNA library was pooled from E. coli and transformed to T. thermophilus. Approximately 1500 T. thermophilus clones, arrayed on 96-well plates, were screened under UV light, and 53 fluorescent clones were selected for further characterization. The thermostable enzymes were transcriptionally fused to the IFP CDS. Plasmid DNAs were subsequently transformed into E. coli for comparative expression analysis.

A new Open Access publication in Synthetic Biology journal presents an important step forward in enabling efficient protein production in the thermophilic bacterium Thermus thermophilus.

The study addresses current limitations in genetic tools for extremophiles by developing 53 novel artificial 5′ regulatory sequences (ARES), combining promoter and untranslated regions. These sequences were successfully characterised in both Thermus thermophilus and Escherichia coli. It demonstrates distinct host-specific expression patterns and improved control of gene expression.

Importantly, the newly developed ARES enabled high-level expression of thermostable proteins, underlining their potential for industrial biotechnology applications operating at elevated temperatures. By expanding the available genetic toolkit, this work contributes to strengthening the use of extremophilic hosts as robust and versatile platforms in synthetic biology.

🔗 Read the full publication here.