Construction of Promoter-Ribosome Binding Site Library for Manipulating Gene Expression in the Hyperthermophilic Archaeon <i>Thermococcus kodakarensis</i>

Extremely thermophilic archaea, such as <i>Thermococcus kodakarensis</i>, possess biotechnological potential as sources of either thermostable enzymes or biotransformation processes under high temperature. However, the absence of a tool for fine-tuning of gene expression impedes its advancement as a platform organism. Here, we constructed a genetic element library in <i>T. kodakarensis</i>, which includes constitutive promoters, inducible promoters, and ribosome binding site (RBS). The promoter library consisted of 76 constitutive promoters with expression strengths spanning a ∼8 × 10³-fold dynamic range and 22 inducible promoters consisting of 15 maltodextrin-inducible promoters and 7 pressure-inducible promoters with maximum induction strength achieving a ∼8-fold increase. We also generated an RBS library containing 31 different RBS sequences, with translation strengths covering an ∼5-fold dynamic range. Utilizing the characterized and identified element library, we constructed a high hydrostatic pressure-inducible toxin-antitoxin (TA) system as the toxin counterselectable cassette regulated by an antitoxin switch for genetic modifications in <i>T. kodakarensis</i> to realize markerless gene disruption directly in rich medium. Moreover, the rational control of the relative expression levels of the TA system enhanced the knockout efficiency. We then replaced the native promoters of genes associated with hydrogen production pathways with various types and strengths of promoters, resulting in a 2.68-fold increase in hydrogen yield (59.4 mmol liter^-1 vs 22.2 mmol liter^-1). Therefore, the genetic toolbox developed in this work is highly significant for advancing fundamental biological research and biotechnological engineering of hyperthermophilic <i>Thermococcales</i>.