Directed evolution of Xanthomonas axonopodis pv. citri beta-galactosidase

Abstract

Primary cell walls of plants are mainly composed by cellulose and hemicellulose, having xyloglucan (XyG) as the main hemicellulosic polysaccharide. The XyG structure and composition largely varies according to plant type and tissue requiring a diverse and complex repertoire of glycosyl hydrolases to be depolymerized. The genus Xanthomonas poses as one of the most widespread pathogenic bacteria that employ an arsenal of hydrolases to invade and modulate plant response, beyond contributing to the intake of carbohydrates as primary carbon source. Through genome annotation and transcriptomics analysis, we detected an operon dedicated to xyloglucan degradation and could be exploit for biotechnological purposes. However, due to mild optimal conditions of these identified GHs, a further molecular engineering is required to match industrial conditions. Thus, in this project, we propose the use of computer-aided active pocket redesign and directed evolution to improve the characteristics of one component of this Xac operon for XyG degradation, in particular a novel promiscuous beta-galactosidase that relies on a CBM-like ancillary domain to form the active-site pocket. The strategy will be the combined use of random and site-saturation mutagenesis, DNA recombination and modelling/simulation tools to achieve superb thermostability, thermophilicity and substrate promiscuity, making the enzyme suitable for potential greener and lower-cost industrial applications. The obtained mutants will be characterized through activity and stability assays to access their optimal pH and temperature, besides their preference and affinity for multiple substrates.