Besides proteins and nucleic acids, carbohydrates are also ubiquitous building blocks of living systems. Approximately 70% of mammalian proteins are glycosylated. Glycans not only provide structural support for living systems but also act as crucial regulators of cellular functions. As a result, they are considered essential pieces of the life science puzzle. However, research on glycans has lagged far behind that on proteins and nucleic acids. The main reason is that glycans are not direct products of gene coding, and their synthesis is nontemplated. In addition, the diversity of monosaccharide species and their linkage patterns contribute to the complexity of the glycan structures, which is the molecular basis for their diverse functions. Research in glycobiology is extremely challenging, especially for the in situ elucidation of glycan structures and functions. There is an urgent need to develop highly specific glycan labeling tools and imaging methods and devise glycan editing strategies. This Perspective focuses on the challenges of in situ analysis of glycans in living systems at three spatial levels (i.e., cell, tissue, and in vivo) and highlights recent advances and directions in glycan labeling, imaging, and editing tools. We believe that examining the current development landscape and the existing bottlenecks can drive the evolution of in situ glycan analysis and intervention strategies and provide glycan-based insights for clinical diagnosis and therapeutics. 

In Situ Glycan Analysis and Editing in Living Systems

Sugar esters (SEs) are non-ionic amphiphilic surfactants that possess excellent emulsifying properties, and consequently have potential applications in the food, pharmaceutical, and other related industries. In the food sector, SEs are particularly attractive, due to their reported effective usage in dessert processing, fruit and vegetable preservation, and as an antibacterial agent. In this review, the chemical and enzymatic methods for the synthesis of SEs were highlighted, and the efficiency of enzymatic synthesis was explored based on solvent tolerance. While traditional chemical synthesis has been simplified and made more efficient, it still necessitates the use of high temperature and pressure, resulting in low selectivity and potential environmental impact. The enzymatic synthesis of SEs has become increasingly popular due to its high regional selectivity and sustainable reaction conditions. Nonetheless, this method's further implementation in the food industry is hindered by the lower solvent tolerance of most lipases in organic solvents. The strategies for solvent-tolerance engineering of lipase based on computational assistance methods were discussed from insights of hydration-shell, salt bridges, interactions (van der Waals, hydrogen bonds, and hydrophobic interactions), active sites, and solvent tunnels. Finally, several practical and commonly used strategies were summarized and compared, which provides some guidance and theoretical basis for further applications of enzymatic methods in the synthesis of SEs. 

Computer-aided engineering of lipases solvent tolerance enhanced their applications in sugar esters synthesis: State of the art

Oxidases are of interest to chemical and pharmaceutical industries because they catalyze highly selective oxidations. However, oxidases found in nature often need to be re-engineered for synthetic applications. Herein, we developed the versatile and robust flow cytometry-based screening platform "FlOxi" for directed oxidase evolution. FlOxi utilizes hydrogen peroxide produced by oxidases expressed in E. coli to oxidize Fe2+ to Fe3+ (Fenton reaction). Fe3+ mediates the immobilization of a His6-tagged eGFP (eGFPHis) on the E. coli cell surface, ensuring the identification of beneficial oxidase variants by flow cytometry. FlOxi was validated with two oxidases-a galactose oxidase (GalOx) and a D-amino acid oxidase (D-AAO)--yielding a GalOx variant (T521A) with a 4.4-fold lower Km value and a D-AAO variant (L86M/G14/A48/T205) with a 4.2-fold higher kcat than their wildtypes. Thus, FlOxi can be used for the evolution of hydrogen peroxide-producing oxidases and applied for non-fluorescence substrates.

https://onlinelibrary.wiley.com/doi/pdfdirect/10.1002/anie.202214999

A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.

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A Flow Cytometry-Based Ultrahigh-Throughput Screening Method for Directed Evolution of Oxidases.