Ortholog Analysis and Transformation of Glycoside Hydrolase Genes in Hyperthermophilic Archaeal Thermococcus Species

Archaea thrive in extreme environments, exhibiting unique traits with significant biotechnological potential. In this study, we investigated whether <i>Thermococcus onnurineus</i> NA1 could stably integrate a large glycoside hydrolase (GH) gene cluster from <i>T. pacificus</i> P-4, enhancing β-linked polysaccharides degradation for hydrogen production. Among 35 <i>Thermococcus</i> genomes examined via OrthoFinder2 and OrthoVenn3, and selecting <i>Tpa</i>-GH gene clusters as the target, we cloned and integrated <i>Tpa</i>-GH into <i>T. onnurineus</i> NA1 using a fosmid-based system, creating the GH03 mutant. Cultivation in a modified MM1 medium supplemented with laminarin revealed significantly higher growth and hydrogen production in <i>T. onnurineus</i> GH03 than in the wild-type strain. Our findings demonstrate the feasibility of stable, large-fragment DNA integration in hyperthermophilic archaea and underscore the promise of <i>T. onnurineus</i> GH03 as a strain for high-temperature biomass conversion.