Abstract
The UDP-glucosyltransferase UGT76G1 from Stevia rebaudiana converts stevioside to rebaudioside A via a one-step glycosylation reaction, which increases the amount of sweet-tasting rebaudioside A and decreases the amount of stevioside that has a bitter aftertaste. This enzyme could, therefore, conceivably be used to improve the organoleptic properties of steviol glycosides and offer a cost-effective preparation of high-purity rebaudioside A. Producing soluble enzymes by overexpression is a prerequisite for large-scale biocatalysis. However, most of the UGT76G1 overexpressed in Escherichia coli is in inclusion bodies. In this study, three N-terminal fusion partners, 3'-phosphoadenosine-5'-phosphatase (CysQ), 2-keto-3-deoxy-6-phosphogluconate aldolase (EDA) and N-utilisation substance A (NusA), were tested to improve UGT76G1 expression and solubility in E. coli. Compared with the fusion-free protein, the solubility of UGT76G1 was increased 40% by fusion with CysQ, and the glucosyltransferase activity of the crude extract was increased 82%. This successful CysQ fusion strategy could be applied to enhance the expression and solubility of other plant-derived glucosyltransferases and presumably other unrelated proteins in the popular, convenient and cost-effective E. coli host.
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