Yeast display for directed evolution of amadoriase, an enzyme capable of cleaving protein glycation products - MINIATURA 8 (2024/08/X/NZ1/01781) (dataset)

Abstract

This dataset was generated as part of the Miniatura 8 project, which focused on amadoriases. These FAD-dependent enzymes catalyze the breakdown of glycated amino acids, producing a free amino acid, glucosone, and hydrogen peroxide. Naturally occurring in fungi and bacteria, they are of considerable practical importance due to their applications in medical diagnostics and their potential therapeutic use, particularly in the treatment of diseases such as diabetes. Previous attempts to improve amadoriase I from Aspergillus through targeted amino acid substitutions increased the enzyme's thermostability but did not enhance its catalytic activity. Similarly, directed evolution of the related enzyme amadoriase II resulted in only marginal improvements. The aim of this project was to develop methods for the in vitro evolution of amadoriases to increase their activity using a non-directed, ultra-high-throughput approach based on yeast surface display. This technique enables the generation of random enzyme variants displayed on the surface of yeast cells, which can then be screened for catalytic activity. A biotin- and azide-modified fructoselysine analogue, designed to mimic a glycated substrate, was successfully synthesized and immobilized on the yeast surface to serve as a substrate for activity screening. Upon catalysis by an amadoriase, the substrate was expected to lose its fluorescence, allowing fluorescence-activated cell sorting (FACS) to isolate yeast cells displaying enzyme variants with higher catalytic activity. A previously engineered variant of amadoriase II from Aspergillus fumigatus (AmaII_SII_82) was used as the starting point for the evolution experiments. Unfortunately, because the initial enzyme exhibited only negligible activity toward the biotin-modified substrate, the variants obtained after three rounds of sorting also failed to show the expected improvement in activity. Nevertheless, the techniques developed during this research provide a strong foundation for future studies aimed at improving other classes of deglycating enzymes, such as FN3K and FrlB/FrlD.