Machine‐Driven Enzymatic Oligosaccharide Synthesis by Using a Peptide Synthesizer

Zhang, Jiabin; Chen, Congcong; Gadi, Madhusudhan Reddy; Gibbons, Christopher; Guo, Yuxi; Cao, Xuefeng; Edmunds, Garrett; Wang, Shuaishuai; Liu, Ding; Yu, Jin; Wen, Liuqing; Wang, Peng George

doi:10.1002/anie.201810661

Cited by 71 publications

(53 citation statements)

References 43 publications

(33 reference statements)

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“…Our study demonstrates thatt hese powerful catalysts could prove extremely useful for synthetic access to aw ide variety of complex natural and new-to-nature sialoconjugates for biological studies. [49][50][51][52]…”

Section: Discussionmentioning

confidence: 99%

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Mertsch

Dong

et al. 2020

Chemistry A European J

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Defined sialoglycoconjugates are important molecular probes for studying the role of sialylated glycans in biological systems. We show that the α2,3‐sialyltransferase from Photobacterium phosphoreum JT‐ISH‐467 (2,3SiaTpph) tolerates a very broad substrate scope for modifications in the sialic acid part, including bulky amide variation, C5/C9 substitution, and C5 stereoinversion. To reduce the enzyme's hydrolytic activity, which erodes the product yield, an extensive structure‐guided mutagenesis study identified three variants that show up to five times higher catalytic efficiency for sialyltransfer, up to ten times lower efficiency for substrate hydrolysis, and drastically reduced product hydrolysis. Variant 2,3SiaTpph (A151D) displayed the best performance overall in the synthesis of the GM3 trisaccharide (α2,3‐Neu5Ac‐Lac) from lactose in a one‐pot, two‐enzyme cascade. Our study demonstrates that several complementary solutions can be found to suppress the common problem of undesired hydrolysis activity of microbial GT80 sialyltransferases. The new enzymes are powerful catalysts for the synthesis of a wide variety of complex natural and new‐to‐nature sialoconjugates for biological studies.

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Section: Discussionmentioning

confidence: 99%

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Mertsch

Dong

et al. 2020

Chemistry A European J

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“…Several groups have developed arrays for additional classes such as sialylated structures (74 -76), plant cell-wall glycans (77), and microbial glycans (8,78,79). The synthesis of these structures currently is limited by the requirement for high expertise and customization, but automated synthesis, which was shown to be feasible selected glycans using enzyme-mediated methods (80,81), could alleviate that bottleneck.…”

Section: Fig 2 Defining Motifs and Families Of Motifs A Motif Typmentioning

confidence: 99%

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

Haab¹,

Klamer²

2020

Molecular & Cellular Proteomics

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Proteins that bind carbohydrate structures can serve as tools to quantify or localize specific glycans in biological specimens. Such proteins, including lectins and glycan-binding antibodies, are particularly valuable if accurate information is available about the glycans that a protein binds. Glycan arrays have been transformational for uncovering rich information about the nuances and complexities of glycan-binding specificity. A challenge, however, has been the analysis of the data. Because protein-glycan interactions are so complex, simplistic modes of analyzing the data and describing glycan-binding specificities have proven inadequate in many cases. This review surveys the methods for handling high-content data on protein-glycan interactions. We contrast the approaches that have been demonstrated and provide an overview of the resources that are available. We also give an outlook on the promising experimental technologies for generating new insights into protein-glycan interactions, as well as a perspective on the limitations that currently face the field.

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“…One of the main challenges of the Golgi apparatus was that it required multiple filtration-purification steps that hindered its efficiency. To address these challenges, Wang and colleagues recently combined a thermosensitive polymer with a commercially available peptide synthesizer to mediate automated glycan assembly (Zhang et al, 2018 ). Their system was able to prepare several blood group antigens and ganglioside glycans with yields ranging from 27 to 38% within 1–2 days.…”

Section: Enzyme-mediated In Vitro Technologies Formentioning

confidence: 99%

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

Jaroentomeechai

Taw

et al. 2020

Front. Chem.

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Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the “glycan code”) and its application (e.g., biomanufacturing high-value glycomolecules on demand).

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Machine‐Driven Enzymatic Oligosaccharide Synthesis by Using a Peptide Synthesizer

Cited by 71 publications

References 43 publications

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

An α2,3‐Sialyltransferase from Photobacterium phosphoreum with Broad Substrate Scope: Controlling Hydrolytic Activity by Directed Evolution

Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

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