FWF - DeoxyBioCat - Redesign of enzyme cascades for deoxy sugars

Project: Research project

Project Details

Description

DeoxyBioCat: Unlocking Synthetic Biology of Deoxy Sugars via Redesigned Biocatalytic Cascades
Wider research context/theoretical framework. Deoxy-sugars are special carbohydrates in which at least one hydroxy group is substituted. The deoxy-sugars are important recognition sites in oligosaccharides.
They are prominently found in natural product glycosides, including antibiotics. The bioactivity of these glycosides depends on the deoxy-sugars present. Deoxy-sugars are biosynthesized from nucleotideactivated precursors. Although deoxygenation can be introduced by different chemistries depending on the sugar position, the one most frequently used in nature occurs at C6 and is catalyzed by dehydratase enzymes. The 4,6-dehydratases have evolved from more primitive oxidoreductases from the superfamily of
short-chain dehydrogenases/reductases (SDRs).
Hypotheses/research questions/objectives. The dehydratases are special among SDRs for integrating catalysis to the elimination of water from C6 into a unique catalytic cycle of C4-alcohol oxidation and C6 carbon-carbon double bond reduction post-dehydration. Natural biosynthesis of deoxy-sugars is restricted to only a few specialized pathways. Due to the high substrate specificity of the enzymes (dehydratases, epimerases, reductases), these pathways are separated biologically and cannot be interconnected or mixed
in vitro. This limits cascade bio-catalysis and synthetic biology in creating artificial pathways towards newto-nature deoxy-sugar nucleotides. Engineering the SDR-type enzymes for altered/relaxed specificity would open up important opportunities for both diversity- and target-oriented deoxy-sugar synthesis. However, relationships between structure and reactivity in these enzymes are subtle which renders their engineering a fundamental challenge. This project, therefore, aims at development of new dehydratase and related SDR
enzymes (epimerase, epimerase-reductase) that show programmable reactivity and specificity and that are more flexibly recombined in synthetic cascades than the native enzymes.
Approaches/methods and Level of originality/innovation. A comprehensive engineering approach, built on fundamentally advanced understanding of the mechanistically complex enzymes, is taken in two main directions. One is to broaden the substrate acceptance and to change the selectivity of existing enzymes.
The other is to elicit a different reactivity in an enzyme that offers the desired substrate scope. Focused mutagenesis combined with detailed biochemical and mechanistic assessment of variant enzymes are applied. As practical target, a new pathway towards the important 6-deoxy-sugar L-fucose is designed. The L-fucose, in its natural GDP-activated or alternative nucleotide-activated form (UDP-L-fucose) will be produced from sucrose in an artificial synthetic cascade based on engineered enzymes, prominently involving new dehydratase(s) as key deoxygenating catalyst(s).
StatusActive
Effective start/end date1/11/2031/10/24