Building Carbohydrates with Glycoside Phosphorylases

Abstract: Glycoside phosphorylases (GPs) have recently been recognized as potentially useful biocatalysts for the synthesis and biotransformation of glycans. These enzymes ordinarily carry out phosphorolysis of the glycosidic linkage by transferring a glycosyl moiety from the non-reducing end of a di- or polysaccharide substrate onto inorganic phosphate, thereby cleaving the glycosidic bond and generating a sugar-1-phosphate. GPs distinguish themselves from most carbohydrate-active enzymes in that the hydrolytic free energy associated with the ester-linkage of the sugar-1-phosphate product is roughly equivalent to that of the glycosidic linkage in the glycan substrate. Therefore, the equilibrium position can be tipped in favour of glycoside synthesis by manipulation of reaction conditions. GPs thus have considerable potential for the assembly of glycans, especially since their reversibility would allow the use of one GP to degrade an inexpensive glycan to produce a pool of sugar-1-phosphates, while a second GP could be deployed to use those sugar-1-phosphates as donors to synthesize a different, more valuable target glycan. The bottleneck in this approach, however, is the limited range of GPs available, which restricts the classes of glycan that can be assembled. To help increase the spectrum of known GPs available, we have turned to metagenomics as a means to discover new enzymes belonging to this class. We have developed two complementary function-based high-throughput screening approaches to identify  expression of novel GPs from bacterial genome fragments recovered directly from the environment. Our initial screens have revealed a total of 9 GPs, including the first phosphorylase activity reported in the GH3 CAZy family and another belonging to the newly discovered GH149 family. Characterization of these new GPs are currently underway to determine their substrate specificity and potential application towards carbohydrate synthesis. Moving forward we are planning to cast a wider net by screening new metagenomic libraries from diverse environments with a wider range of substrates.