Genetic biosensor enables in vivo glycosyltransferase screening
September 15-19, 2019
Glycosylation of natural products can alter their solubility and bioavailability, among other properties, which makes glycosyltransferases useful tools for increasing the production and/or generating novel compounds in microbial cell factories. However, the discovery and screening of new enzymes and engineered variants is often a low-throughput endeavor due to the need for over-expression and purification prior to in vitro experiments, which do not necessarily represent the in vivo activities of the enzyme. Therefore, a genetic biosensor controlling GFP expression was developed based on the flavonoid responsive transcriptional-repressor QdoR and expressed in E. coli. Due to the induced fluorescent response upon feeding the flavonoids Quercetin and Kaempferol, but not to their glucosides, the activity of UDP-dependent glycosyltransferases (UGTs) could be screened in vivo. Furthermore, a variant of QdoR was generated by directed evolution that showed greater dose-responsiveness and proved to allow greater discrimination of cellular populations and was thus more useful for in vivo UGT screening. The designed biosensor-based method will greatly increase the throughput of glycosyltransferase discovery and engineering.
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Evan K. Chaberski, Michael K. Jensen, and Ditte H. Welner, "Genetic biosensor enables in vivo glycosyltransferase screening" in "Enzyme Engineering XXV", Huimin Zhao, University of Illinois at Urbana-Champaign, USA John Wong, Pfizer, USA Eds, ECI Symposium Series, (2019). https://dc.engconfintl.org/enzyme_xxv/79