Chemoenzymatic methods and reagents to access affordable (iso)globo- and(neo)lacto-glycosphingolipids

Project Summary All cells are covered with complex lipids, including carbohydrate-containing glycolipids. A subclass of glycolipids called glycosphingolipids (GSLs) are comprised of a sphingolipid of different structures and a glycan with more than 300 variations. Many GSLs contain a glycan extended from a lactose core. Despite the ubiquitous presence of GSLs in cell membranes and their critical roles in multi-cellular organisms, studies related to GSLs are limited. Among the major challenges are the limited access to structurally defined GSLs in sufficient quantities and the high prices due to complicated synthetic processes with poor scalabilities. The collaboration of Dr. Hai Yu and Dr. Xi Chen at UC Davis and IMCS has led to 400-fold cost reduction of key materials including sphingosines, lactosylsphingosine (LacβSph), and recombinant biosynthetic enzymes for synthesizing ganglio-series GSLs. By leveraging the established collaboration effort and extending this momentum to new targets, this proposal can exponentially increase the impact of the proposed and past advances. The first goal is to expand the inventory of commercially available recombinant glycosyltransferases and demonstrate their application in synthesizing 37 globo-/isoglobo- and lacto-/neolacto-series GSLs. The key glycosyltransferases will be initially expressed and improved by the UC Davis team. IMCS will leverage its automation systems to create large libraries of glycosyltransferase mutants with sequences based on the enzymes validated by the UC Davis team. The second goal is to optimize the preparative-scale synthesis of target GSLs and scale up manufacturing of two specific GSLs, Globo H and sialyl Lewis a, which are high value candidates for developing anti-cancer therapies. In addition, Gb3 with promising anti-bacteria properties will be produced readily from lyso-Gb3, an intermediate to produce Globo H. The success for large-scale production of complex GSLs is a requirement to lower costs of GSLs, and such low-priced GSLs are much needed for the broad research community and for industrial clients. The results from this project will provide the entire research community with affordable GSLs and the related reagents while expanding the understanding of recombinant glycosyltransferase engineering and their functionalities.