Unlocking nature’s glycosylation potential : characterization and engineering of novel sucrose/trehalose synthases

Abstract

Glycosylation - the addition of a sugar molecule onto an acceptor substrate - is a promising strategy to improve the activity, solubility, stability, flavor and/or pharmacokinetic behavior of chemicals such as pharmaceuticals, neutraceuticals or cosmetics. Glycosylation can efficiently be performed in an aqueous environment under mild reaction conditions by enzymes called GlycosylTransferases (GTs). However, the industrial application of these enzymes in vitro is mainly hampered by their need for nucleotide-activated sugars (e.g. UDP-glucose) as donor substrates, which are highly expensive and rarely available in large quantities. In this doctoral thesis, two strategies to make in vitro reactions with GTs more cost-efficient were evaluated: the use of Sucrose Synthase (SuSy) as intermediate enzyme to produce UDP-glucose from the cheap substrate sucrose and the engineering of GTs to alter the sugar donor specificity towards cheaper glycosyl-phosphates. To this end, several new bacterial SuSy enzymes were cloned, expressed, purified and characterized. In addition, one of them was subjected to extensive mutagenesis to improve or change properties such as substrate affinity, substrate specificity and stability. The enzyme Trehalose glycosylTransferring synthase, on the other hand, was used as a test-case to scrutinize the possibility of changing the donor specificity of GTs from nucleotide sugars towards glycosyl-phosphates through mutagenesis