_Applied_  
_sciences_  
_Article_  
L-Fucose Synthesis Using a Halo- and Thermophilic L-Fucose Isomerase from Polyextremophilic _Halothermothrix oretii_  
**[PERSON] 1** and [PERSON] 1,2,*(r)  
Footnote 10  
properties, such as anti-inflammatory, antitumor, immune-enhancement, skin-whitening, and skin-moisturization properties, that are beneficial to humans [7; 8; 9]. Although widely occurring in nature in the form of bacterial exopolysaccharides, brown seaweed polysaccharides (i.e., tucoidal), and human milk oligosaccharides, it does not meet the industrial demands to directly acquire l-fucose from natural sources [10; 11; 12]. Conventional chemical synthesis is also industrially incompatible as it is a time-consuming and labor-intensive multistep process [13; 14]. More importantly, the chemical method utilizes toxic reagents, which contradicts the consumer's call for the use of \"natural\" products. Alternatively, biocatalysts based on either enzymes or whole cells can provide a more efficient and eco-friendly method for l-fucose synthesis. The in vitro L-Fucl-based biosynthetic routes for l-fucose known to date are directed towards l-fuculose production as the main precursor, which is subsequently converted into l-fucose by L-Fucl [15; 16]. Although it is more expensive than l-fucose, strategies to obtain l-fuculose from cheap and common sugars using chemical or enzymatic methods have already been established [15; 16]. Thus, l-Fucl-mediated l-fucose production utilizing l-fuculose as the substrate is a key target for investigation. Previously, we identified and characterized two l-Fucls from _Raoultella_ sp. (_Rd_Fucl) [2] and _Thermanaeromonas toyohensis_ (_Tf_Fucl) [3], demonstrating the applicability of l-Fucl for l-fucose synthesis using l-fuculose.  
Extremozymes are considered favorable in bioprocesses in the sugar, food, and pharmaceutical fields, which usually accompany high temperatures and high salt concentrations because they are stable and more active under harsh conditions. Such robust enzymes are generally sourced from organisms that have adapted to extreme environments, such as extremely hot and/or saline habitats. To date, three extremo-(thermophilic) l-Fucls (_Tf_Fucl, _Cs_Fucl from _Calcidellusiruptor saccharolyticus_, and _Df_Fucl from _Dictyogolomus turgatum_) have been characterized [3; 6; 17; 5]. However, only _Tf_Fucl has been evaluated for l-fucose synthesis [3]. Therefore, it is of great importance to identify novel extremo- l-Fucls that could offer great advantages in industrial applications.  
_Halothemothrix renin_ is a true anaerobic polyextremophile isolated from the Tunisian salt lake in the Sahara Desert [18]. Owing to its ability to thrive under dual extreme conditions, that is, high temperature and high salinity, with optimal growth at 60 \({}^{\circ}\)C and 1.7 M NaCl, it is classified as a halothemophile [18; 19]. Polyextremophilic organisms are very rare and unique in nature, as they have evolved to develop clever strategies to adapt to more than two severe conditions. Accordingly, _H. orenii_ could serve as a useful source of enzymes with beneficial features for industrial applications. Indeed, the genome of _H. orenii_ revealed the presence of genes encoding enzymes of biotechnological significance, such as those associated with carbohydrate saccharification and metabolism of sugars, including l-fucose [18; 19]. In this study, we identified l-Fucl from _H. orenii_ (_Ho_Fucl) and performed its functional characterization for l-fucose synthesis under various biochemical conditions, such as NaCl, temperature, pH, and metal ions, to explore its unique molecular features. The results obtained from this study will not only offer insights into the fundamental knowledge of extremozymes, but also allow the biotechnological production of l-fucose and other valuable rare sugars using _Ho_Fucl.