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

## TL;DR

This study shows that integrating a gene cluster from one archaeal species into another can enhance its ability to break down complex sugars and produce hydrogen at high temperatures.

## Contribution

The study demonstrates stable integration of a large GH gene cluster in hyperthermophilic archaea for enhanced biomass conversion.

## Key findings

- T. onnurineus GH03 showed significantly higher growth when cultivated with laminarin.
- The GH03 mutant produced more hydrogen compared to the wild-type strain.
- Large-fragment DNA integration is feasible in hyperthermophilic archaea.

## Abstract

Archaea thrive in extreme environments, exhibiting unique traits with significant biotechnological potential. In this study, we investigated whether Thermococcus onnurineus NA1 could stably integrate a large glycoside hydrolase (GH) gene cluster from T. pacificus P-4, enhancing β-linked polysaccharides degradation for hydrogen production. Among 35 Thermococcus genomes examined via OrthoFinder2 and OrthoVenn3, and selecting Tpa-GH gene clusters as the target, we cloned and integrated Tpa-GH into T. onnurineus 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 T. onnurineus 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 T. onnurineus GH03 as a strain for high-temperature biomass conversion.