Enzymes from (hyper)thermophiles “Thermozymes” offer a great potential for biotechnological applications. Thermophilic adaptation does not only provide stability toward high temperature but is also often accompanied by a higher resistance to other harsh physicochemical conditions, which are also frequently employed in industrial processes, such as the presence of, e.g., denaturing agents as well as low or high pH of the medium. In order to find new thermostable, xylan degrading hydrolases with potential for biotechnological application we used an in situ enrichment strategy incubating Hungate tubes with xylan as the energy substrate in a hot vent located in the tidal zone of Kunashir Island (Kuril archipelago). Using this approach a hyperthermophilic euryarchaeon, designated Thermococcus sp. strain 2319x1, growing on xylan as sole energy and carbon source was isolated. The organism grows optimally at 85°C and pH 7.0 on a variety of natural polysaccharides including xylan, carboxymethyl cellulose (CMC), amorphous cellulose (AMC), xyloglucan, and chitin. The protein fraction extracted from the cells surface with Tween 80 exhibited endoxylanase, endoglucanase and xyloglucanase activities. The genome of Thermococcus sp. strain 2319x1 was sequenced and assembled into one circular chromosome. Within the newly sequenced genome, a gene, encoding a novel type of glycosidase (143 kDa) with a unique five-domain structure, was identified. It consists of three glycoside hydrolase (GH) domains and two carbohydrate-binding modules (CBM) with the domain order GH5-12-12-CBM2-2 (N- to C-terminal direction). The full length protein, as well as truncated versions, were heterologously expressed in Escherichia coli and their activity was analyzed. The full length multidomain glycosidase (MDG) was able to hydrolyze various polysaccharides, with the highest activity for barley β-glucan (β- 1,3/1,4-glucoside), followed by that for CMC (β-1,4-glucoside), cellooligosaccharides and galactomannan. The results reported here indicate that the modular MDG structure with multiple glycosidase and carbohydrate-binding domains not only extends the substrate spectrum, but also seems to allow the degradation of partially soluble and insoluble polymers in a processive manner. This report highlights the great potential in a multi-pronged approach consisting of a combined in situ enrichment, (comparative) genomics, and biochemistry strategy for the screening for novel enzymes of biotechnological relevance.

The Crenarchaeon Sulfolobus acidocaldarius has been described to synthesize trehalose via the maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ) pathway and the trehalose glycosyltransferring synthase (TreT) pathway has been predicted. Deletion mutant analysis of single and double deletion strains of ΔtreY and ΔtreT in S. acidocaldarius revealed that next to these two pathways a third, novel trehalose biosynthesis pathway is operative in vivo: the trehalose-6-phosphate (T6P) synthase/T6P phosphatase (TPS/TPP) pathway. In contrast to known TPS proteins, which belong to the GT20 family, the S. acidocaldarius TPS belongs to the GT4 family establishing a new function within this group of enzymes. This novel GT4-like TPS turned out to be mainly present in the Sulfolobales. The ΔtreY/ΔtreT/Δtps triple mutant of S. acidocaldarius lacking the ability to synthesize trehalose showed no altered phenotype under standard conditions or heat stress, but was unable to grow under salt stress. Accordingly, in the wild type strain a significant increase of intracellular trehalose formation was observed under salt stress. Quantitative real-time PCR showed a salt stress mediated induction of all three trehalose synthesizing pathways. This demonstrates in Archaea that trehalose plays an essential role for growth under high salt conditions. Importance The metabolism and function of trehalose as compatible solute was not well understood in Archaea. This combined genetic and enzymatic approach at the interface of microbiology, physiology and microbial ecology gives important insights into survival under stress, adaptation to extreme environments, and the role of compatible solutes in Archaea. Here we unravelled the complexity of trehalose metabolism and present a comprehensive study on trehalose function in stress response in S. acidocaldarius. This sheds light on the general microbiology and the fascinating metabolic repertoire of Archaea involving many novel biocatalysts such as glycosyltransferases with great potential in biotechnology.