Successes in engineering glucansucrases to enhance glycodiversification
Carbohydrates are biomolecules that have an essential role in every form of life. The reservoir of naturally occurring glyco-structures is incredibly large and involves a tremendous number of carbohydrate-active enzymes (more than 280,000 released modules in the Carbohydrate Active enZymes database) for their synthesis and degradation. Nevertheless, natural enzymes do not necessarily present all the requested properties in terms of efficiency, specificity or stability when considering their usage for carbohydrate or glyco-derivative manufacturing. In addition, if existing, the identification of an enzyme perfectly adapted to a specific function from the natural diversity may be critical due to the lack of available biochemical data and may necessitate intensive screening efforts. To circumvent such limitations and provide optimized solutions, protein engineering has been considered. Leloir-type glycosyltransferases, for example, are mainly involved in the biosynthesis of glycoconjugates in Nature and they have been widely studied and engineered for this purpose. However, these enzymes are often found as membrane-bound proteins, what renders difficult their isolation and purification. In addition, their need of low-abundant activated sugars as glycosyl donors also impairs their usage. Alternatively, enzymes that use more abundant glycosyl donor directly issued from agro-ressources have been considered to access to new glyco-derivatives. This has promoted the use of glucansucrases (GS) that catalyze transglycosylation reactions from sucrose substrate. These enzymes are of particular interest for synthetic purpose and have found industrial interest for pharmaceutical and fine chemical applications. To diversify their applications, various approaches of engineering have been exploited to improve expression level, stability, or change substrate or product specificity of these enzymes. In particular, the range of molecules recognized and the osidic linkages formed by GS is broad but yet limited. Therefore, protein engineering methods have been applied to further increase the diversity of glycosylation reactions catalyzed by these enzymes. Sequence analysis and mutagenesis experiments have enabled the identification of key amino acid residues of glucansucrases either involved in catalysis or substrate specificity. Moreover, the determination of three-dimensional structures of glucansucrases from both families 13 and 70 of Glycoside-Hydrolases (GH) have provided powerful information for understanding the sequence-structure-function relationships and guiding structure-based rational and semi-rational engineering of these proteins. To assist these efforts, high-throughput screening and biomolecular methods have been developed for the directed evolution of these enzymes. Here are reported some of the successes in the bioengineering of glucansucrases from precursor work to latest results, as well as the methods developed for screening and developing efficient variant libraries. The major progresses and breakthroughs in the field will be highlighted and further prospects will be considered and discussed.