Evolved thermostable transketolase from Geobacillus stearothermophilus for the synthesis of deoxyketoses

In recent years, mesophilic transketolases (TK) from S. cerevisiae and E. coli have been widely used for the synthesis of numerous chiral α-hydroxyketones preferentially polyhydroxylated. To improve the efficiency of these TKs, evolvability techniques have been applied but for biocatalytic applications, the stability against time, the resistance towards temperature and destabilizing mutagenesis factors are often provided by more robust and less flexible protein structures. To answer these criteria, the discovery of a thermostable TK from Geobacillus stearothermophilus (TK_gst) offers an efficient template for the construction of TK variants able to greatly extend the substrate scope while decreasing the reaction time and giving more resistance against unusual conditions. In this chapter, we describe a three-step workflow for the production of TK_gst variants designed for the synthesis of 1-deoxyketoses- or 1,2-dideoxyketoses from aliphatic α-ketoacids as donor substrates and their in situ generation by a d-amino acid oxidase coupled in one pot with the TK_gst variant. In a first step, TK_gst variant libraries are created on targeted positions identified in the active site by molecular modeling and are then submitted to site saturation mutagenesis. The second step consists in TK_gst variant library screening with qualitative and quantitative assays to select the best TK_gst variants which are used, in the third and final step, for the preparative-scale synthesis of the targeted 1-deoxyketoses or 1,2-dideoxyketoses. This approach can be applied to the synthesis of other α-hydroxyketones of biological interests by varying the donor and acceptor substrates.