Dataset Information

Optimization of Metabolic Oligosaccharide Engineering with Ac₄GalNAlk and Ac₄GlcNAlk by an Engineered Pyrophosphorylase.

ABSTRACT: Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac₄GalNAlk and Ac₄GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

SUBMITTER: Cioce A

PROVIDER: S-EPMC8501146 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Publications

Optimization of Metabolic Oligosaccharide Engineering with Ac<sub>4</sub>GalNAlk and Ac<sub>4</sub>GlcNAlk by an Engineered Pyrophosphorylase.

Cioce Anna A Bineva-Todd Ganka G Agbay Anthony J AJ Choi Junwon J Wood Thomas M TM Debets Marjoke F MF Browne William M WM Douglas Holly L HL Roustan Chloe C Tastan Omur Y OY Kjaer Svend S Bush Jacob T JT Bertozzi Carolyn R CR Schumann Benjamin B

ACS chemical biology 20210409 10

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathway ...[more]

PMID: 33835779

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Project description:Metabolic engineering has recently been embraced as an effective tool for developing whole-cell biocatalysts for oligosaccharide and polysaccharide synthesis. Microbial catalysts now provide a practical means to derive many valuable oligosaccharides, previously inaccessible through other methods, in sufficient quantities to support research and clinical applications. The synthesis process based upon these microbes is scalable as it avoids expensive starting materials. Most impressive is the high product concentrations (up to 188 g/L) achieved through microbe-catalyzed synthesis. The overall cost for selected molecules has been brought to a reasonable range (estimated $30-50/g). Microbial synthesis of oligosaccharides and polysaccharides is a carbon-intensive and energy-intensive process, presenting some unique challenges in metabolic engineering. Unlike nicotinamide cofactors, the required sugar nucleotides are products of multiple interacting pathways, adding significant complexity to the metabolic engineering effort. Besides the challenge of providing the necessary mammalian-originated glycosyltransferases in active form, an adequate uptake of sugar acceptors can be an issue when another sugar is necessary as a carbon and energy source. These challenges are analyzed, and various strategies used to overcome these difficulties are reviewed in this article. Despite the impressive success of the microbial coupling strategy, there is a need to develop a single strain that can achieve at least the same efficiency. Host selection and the manner with which the synthesis interacts with the central metabolism are two important factors in the design of microbial catalysts. Additionally, unlike in vitro enzymatic synthesis, product degradation and byproduct formation are challenges of whole-cell systems that require additional engineering. A systematic approach that accounts for various and often conflicting requirements of the synthesis holds the key to deriving an efficient catalyst. Metabolic engineering strategies applied to selected polysaccharides (hyaluronan, alginate, and exopolysaccharides for food use) are reviewed in this article to highlight the recent progress in this area and similarity to challenges in oligosaccharide synthesis. Many naturally occurring microbes possess highly efficient mechanisms for polysaccharide synthesis. These mechanisms could potentially be engineered into a microbe for oligosaccharide and polysaccharide synthesis with enhanced efficiency.

Dataset Information

Optimization of Metabolic Oligosaccharide Engineering with Ac₄GalNAlk and Ac₄GlcNAlk by an Engineered Pyrophosphorylase.

Publications

Optimization of Metabolic Oligosaccharide Engineering with Ac<sub>4</sub>GalNAlk and Ac<sub>4</sub>GlcNAlk by an Engineered Pyrophosphorylase.

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Dataset Information

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Publications

Optimization of Metabolic Oligosaccharide Engineering with Ac&lt;sub&gt;4&lt;/sub&gt;GalNAlk and Ac&lt;sub&gt;4&lt;/sub&gt;GlcNAlk by an Engineered Pyrophosphorylase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Optimization of Metabolic Oligosaccharide Engineering with Ac₄GalNAlk and Ac₄GlcNAlk by an Engineered Pyrophosphorylase.

Optimization of Metabolic Oligosaccharide Engineering with Ac<sub>4</sub>GalNAlk and Ac<sub>4</sub>GlcNAlk by an Engineered Pyrophosphorylase.