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Biosynthesis of bacterial polysaccharides by novel glycosyltransferase enzymes

Time: Fri 2023-12-15 10.00

Location: Kollegiesalen, Brinellvägen 8, Stockholm

Video link: https://kth-se.zoom.us/j/61815154778

Language: English

Subject area: Biotechnology

Doctoral student: Shu-Chieh Chang , Glykovetenskap

Opponent: Professor Ute Römling, Department of Microbiology, Tumor and Cell Biology Karolinska Institutet, Stockholm, Sweden

Supervisor: Universitetslektor Yves S. Y. Hsieh, Glykovetenskap

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QC 2023-11-06

Abstract

Within the intricate microbiological realm, exopolysaccharides (EPS) emerge as a significant class of high molecular weight polysaccharides, serving pivotal roles in bacterial survival, virulence, communication, and defense against environmental adversities. The versatile nature of bacterial EPS extends beyond mere biological functions, reaching into medications, cosmetics, functional food, and sustainable industries. Although EPS is a vital and much-exploited class of polysaccharide, their biosynthesis remain less explored or understood. This thesis delves into the exploration of enzymes integral to EPS synthesis, focusing specifically on the characterization of putative membrane-bound enzymes from the so-called glycosyltransferase family 2 (GT2). The initial investigations of the research are centered on (1,3;1,4)-β-D-glucans, polymers with mixed linkage backbones that are widely distributed across various biological systems. Despite their prevalence, collective understanding of the biosynthesis of these polymers in the bacterial domain, particularly in gram-positive strains, remains limited. Through extensive research, distinct genes encoding (1,3;1,4)-β-D-glucan synthases were identified in two gram-positive bacteria: Romboutsia ilealis and Clostridium ventriculi. A gain-of-function approach was employed and provided conclusive evidence of the synthase activity of these identified genes. Subsequently, the thesis shifts focus to Chitinophaga pinensis, a gram-negative bacterium with roles in maintaining ecosystem balance through its proficiency in carbohydrate breakdown and recycling. The exploration led to the discovery of an uncommon GT2 β-glucan synthase with activity in curdlan synthesis. Several unusual features of the C. pinensis enzyme highlight the extensive diversity and nuances within the GT2 polysaccharide synthase family, particularly the fact that such catalysts sometimes have close connections with carbohydrate-degrading enzymes. Characterization of these putative GT2 proteins was verified by a variety of techniques, including gene expression in E. coli and yeast host systems, enzyme-coupled oligosaccharide profiling, and in vitro radiometric activity assays. To advance the investigation, bioinformatics tools such as protein alignment, phylogenetic analysis, and model structure analysis were employed.

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