DeoxyBioCat: redesign of enzyme cascades for deoxy sugars

Carbohydrates (sugars) are the structurally most diverse class of biomolecules. Many key processes of biological recognition, like cell-to-cell or protein-to-protein interactions, involve carbohydrates as the main elements of specificity. The broad diversity of natural carbohydrates results from structural variation of the building blocks (monosaccharides) and from the way how the building blocks are linked one to another. An important strategy of cellular biosynthesis to enhance building block structural diversity is to deoxygenate precursor monosaccharides. Chemically, deoxygenation is the substitution of a sugar hydroxy group by a hydrogen. The structural modification confers unique chemical properties to the monosaccharide and enables new biological functions. Many biologically important sugars are deoxygenated, like the L-fucose which is prevalent in human glycoproteins or various sugars present in secondary metabolites that have antibiotic activity. To exploit deoxy-sugars more broadly for different applications in medicine and nutrition but also for possible industrial uses, efficient ways of their synthesis as chemically defined structures are required. Considering that a sugar substrate contains multiple hydroxy groups of which only one should be replaced, a synthesis that builds upon the exquisite selectivity of enzymes is highly desired. Deoxy-sugar synthesis in nature happens on a universal precursor form, referred to as sugar nucleotide. Deoxygenation is then achieved by enzymes that are called dehydratases. The dehydratase reaction is mechanistically challenging for it involves multiple catalytic steps (oxidation, elimination of water, reduction) that must be precisely coordinated by the enzyme. The dehydratase reactions are made practical for synthesis by their use in multistep enzyme cascades. The idea of the cascade is to combine multiple enzymes in a one-pot transformation to obtain the desired deoxy-sugar from expedient and cost-efficient substrates. The goals of the current project are to advance the mechanistic understanding of sugar nucleotide dehydratases in order to provide a solid basis for rational enzyme engineering; to identify and develop new enzymes for synthesis of various deoxy-sugars in reactions that may be new to nature; and integrate dehydratases into enzyme cascade transformations for efficient synthesis. Among others, L-fucose is selected as an example of high interest. The project combines molecular enzymology with synthetic biology to establish multienzyme cascades for deoxy-sugar synthesis.