Development of a colourimetric assay for glycosynthases

Hayes, Marc R.; Bochinsky, Kevin; Seibt, Lisa; Elling, Lothar; Pietruszka, Jörg

doi:10.1016/j.jbiotec.2017.02.005

Cited by 9 publications

(5 citation statements)

References 32 publications

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“…20 However, there are limited universal HTS options available for rapid screening of GSs. 23,24,29,51 There are currently limited HTS methods available that facilitate ultrahigh-throughput cell-based screening and directed evolution of GSs using azido sugars as glycosyl donors. 17 One of the advantages of using azido sugars as substrates for GS reactions is that the azido moiety can be selectively conjugated to alkyne-based fluorophore groups using a modified Staudinger ligation or copper-free click-chemistry under reaction conditions compatible with the in vivo environment.…”

Section: ■ Discussionmentioning

confidence: 99%

“…Azido sugars can be readily chemically synthesized using one-pot reactions from unprotected sugar monomers, as well as produced enzymatically at high yields, unlike other activated donor sugars. ,, Furthermore, with CAZymes being rapidly discovered from diverse genomic sources, , GHs offer a large selection of enzymes that can be exploited for developing better GSs using directed evolution-based approaches . However, there are limited universal HTS options available for rapid screening of GSs. ,,, There are currently limited HTS methods available that facilitate ultrahigh-throughput cell-based screening and directed evolution of GSs using azido sugars as glycosyl donors …”

Section: Discussionmentioning

confidence: 99%

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Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity

et al. 2021

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Engineering of carbohydrate-active enzymes such as glycosynthases to enable chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable ultrahigh-throughput screening methods capable of robustly detecting either starting substrates or end-products of the glycosidic bond formation reaction. Currently, there are limited screening methods available for rapid and highly sensitive single-cell-based screening of glycosynthase enzymes employing azido sugars as activated donor glycosyl substrates. Here, we report a fluorescence-based approach employing click-chemistry for the selective detection of glycosyl azides as substrates versus free inorganic azides as reaction products that facilitated an ultrahigh-throughput in vivo single-cell-based assay of glycosynthase activity. This assay was developed based on the distinct differences observed in relative fluorescence intensity of the triazole-containing fluorophore product formed during the click-chemistry reaction of organic glycosyl azides versus inorganic azides. This discovery formed the basis for proof of concept validation of a directed evolution methodology for screening and sorting glycosynthase mutants capable of synthesis of targeted fucosylated oligosaccharides. Our screening approach facilitated fluorescence-activated cell sorting of an error-prone polymerase chain reaction-based mutant library of fucosynthases expressed in Escherichia coli to identify several novel mutants that showed increased activity for β-fucosyl azide-activated donor sugars toward desired acceptor sugars (e.g., pNP-xylose and lactose). Finally, we discuss avenues for improving this proof of concept in vivo assay method to identify better glycosynthase mutants and further demonstrate the broader applicability of this screening methodology for synthesis of bespoke glycans.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity

et al. 2021

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“…21 However, currently there are very limited options available for universal HTS methods for rational engineering or cell-based directed evolution of GSs. 24,25,30,45 Specifically, other than this report, there are currently no HTS methods available that facilitate ultrahigh-throughput cell-based screening and directed evolution of GSs using azido sugars as glycosyl donors. 18 Here, we have now developed, validated, and applied a novel SPAAC or click-chemistry enabled uHTS method for screening large GSs libraries.…”

Section: Discussionmentioning

confidence: 99%

Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

Agrawal

Bandi

Burgin

et al. 2020

Preprint

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Engineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of evolved glycosynthase enzymes employing azido sugars as substrates. Here, we report a fluorescencebased approach employing click-chemistry for the selective detection of glycosyl azides (versus free inorganic azides) that facilitated ultrahigh-throughput in-vivo single cell-based assay of glycosynthase activity. This discovery has led to the development of a directed evolution methodology for screening and sorting glycosynthase mutants for synthesis of desired fucosylated oligosaccharides. Our screening technique facilitated rapid fluorescence activated cell sorting of a large library of glycosynthase variants (>10 6 mutants) expressed in E. coli to identify several novel mutants with increased activity for b-fucosyl-azide activated donor sugars towards desired acceptor sugars, demonstrating the broader applicability of this methodology.

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“…Also progress in methods for identification and characterization of glycosynthases from mutant libraries will be critical to produce enzymes with new acceptor or donor scopes. Many advances have already been made such as biological and biochemical assays utilizing chemical complementation, fluorescence and photometric methods [ 50 , 88 , 89 , 90 ]. Subsequent analysis of structural and mechanistic details will help to identify structural requirements, which can be transferred to new glycosynthases by genetic engineering and rational design [ 28 , 91 ].…”

Section: Discussionmentioning

confidence: 99%

Synthesis of Glycosides by Glycosynthases

Hayes

Pietruszka

2017

Molecules

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The many advances in glycoscience have more and more brought to light the crucial role of glycosides and glycoconjugates in biological processes. Their major influence on the functionality and stability of peptides, cell recognition, health and immunity and many other processes throughout biology has increased the demand for simple synthetic methods allowing the defined syntheses of target glycosides. Additional interest in glycoside synthesis has arisen with the prospect of producing sustainable materials from these abundant polymers. Enzymatic synthesis has proven itself to be a promising alternative to the laborious chemical synthesis of glycosides by avoiding the necessity of numerous protecting group strategies. Among the biocatalytic strategies, glycosynthases, genetically engineered glycosidases void of hydrolytic activity, have gained much interest in recent years, enabling not only the selective synthesis of small glycosides and glycoconjugates, but also the production of highly functionalized polysaccharides. This review provides a detailed overview over the glycosylation possibilities of the variety of glycosynthases produced until now, focusing on the transfer of the most common glucosyl-, galactosyl-, xylosyl-, mannosyl-, fucosyl-residues and of whole glycan blocks by the different glycosynthase enzyme variants.

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Development of a colourimetric assay for glycosynthases

Cited by 9 publications

References 32 publications

Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity

Click-Chemistry-Based Free Azide versus Azido Sugar Detection Enables Rapid In Vivo Screening of Glycosynthase Activity

Click-chemistry enabled directed evolution of glycosynthases for bespoke glycans synthesis

Synthesis of Glycosides by Glycosynthases

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