LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo- and enantioselective α,α-(1→1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, Keq, for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDP-glucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control.IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.@en