5’-untranslated region sequences enhance plasmid-based protein production in Sulfolobus acidocaldarius

Sulfolobus acidocaldarius , a thermoacidophilic archaeon of the phylum Thermoproteota (former Crenarchaeota), is a widely used model organism for gene deletion studies and recombinant protein production. Previous research has demonstrated the efficacy of the saci_2122 promoter (P _ara ), providing low basal activity and high pentose-dependent induction. However, available expression vectors lack a 5’-terminal untranslated region (5’-UTR), which is a typical element in bacterial expression vectors, usually significantly enhancing protein production in bacteria. To establish S. acidocaldarius as a production strain in biotechnology in the long-term, it is intrinsically relevant to optimize its tools and capacities to increase production efficiencies. Here we show that protein production is increased by the integration of S. acidocaldarius 5’-UTRs into P _ara expression plasmids. Using the esterase Saci_1116 as a reporter protein, we observed a fourfold increase in soluble and active protein yield upon insertion of the saci_1322 ( alba ) 5’-UTR. Screening of four additional 5’-UTRs from other highly abundant proteins ( thα , slaA , slaB, saci_0330 ) revealed a consistent enhancement in target protein production. Additionally, site-directed mutagenesis of the Shine-Dalgarno (SD) motif within the alba 5’-UTR revealed its significance for protein synthesis. Ultimately, the alba 5’-UTR optimized expression vector demonstrated successful applicability in expressing various proteins, exemplified by its utilization for archaeal glycosyltransferases. Our results demonstrate that the integration of SD-motif containing 5’-UTRs significantly boosted plasmid-based protein production in S. acidocaldarius . This advancement in recombinant expression not only broadens the utility of S. acidocaldarius as an archaeal expression platform but also marks a significant step toward potential biotechnological applications.