Abstract:Protein
glycosylation, the attachment of sugars to amino acid side
chains, can endow proteins with a wide variety of properties of great
interest to the engineering biology community. However, natural glycosylation
systems are limited in the diversity of glycoproteins they can synthesize,
the scale at which they can be harnessed for biotechnology, and the
homogeneity of glycoprotein structures they can produce. Here we provide
an overview of the emerging field of synthetic glycobiology, the application
of synt… Show more
“…Studies using purified translation components, such as the protein synthesis using recombinant elements (PURE [ 124 ]) system are excluded, and systems using purified glycosylation components are excluded unless otherwise noted. More exhaustive reviews of CFPS [ 10 , 14 , 64 , 115 , 125 , 143 , 151 ], and glycoengineering can be found elsewhere [ 6 , 22 , 26 , 29 , 46 , 72 , 94 , 102 ]. Fig.…”
Section: Outline and Scopementioning
confidence: 99%
“…We order the sections from the most “natural systems” (containing endogenous enzymes naturally found in the host strain) to the most synthetic (containing heterologous and novel enzyme combinations, generating glycans that are not found in nature). This distinction illustrates how the use of synthetic biology principles in glycosylation systems has expanded their scope and provided access to new glycoforms [ 72 ].…”
Section: Outline and Scopementioning
confidence: 99%
“…OSTs mediate glycosylation by their substrate specificity, glycosylating only when specific polypeptide acceptor sequences and LLO donor structures are recognized. While the general acceptor peptide motif (or “sequon”) N-X-S/T is conserved, specific sequons and LLOs recognized by OSTs vary across organisms [ 72 , 121 ]. In eukaryotes, OSTs are multi-subunit complexes assembled around a core catalytic subunit called STT3 [ 66 , 85 ].…”
Section: Main Textmentioning
confidence: 99%
“…Unfortunately, building and testing defined glycoproteins in cells remains challenging for several reasons. These include glycoprotein heterogeneity [ 49 , 60 , 121 ], gaps in the methods and basic knowledge required to build defined glycoforms with desired pharmacological activities [ 72 , 153 ], and the high costs and lengthy durations required to generate stable cell lines in mammalian cell culture. To address these issues, diverse CFPS platforms that allow user-defined glycosylation have been developed [ 26 , 72 ].…”
Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic
N
-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.
“…Studies using purified translation components, such as the protein synthesis using recombinant elements (PURE [ 124 ]) system are excluded, and systems using purified glycosylation components are excluded unless otherwise noted. More exhaustive reviews of CFPS [ 10 , 14 , 64 , 115 , 125 , 143 , 151 ], and glycoengineering can be found elsewhere [ 6 , 22 , 26 , 29 , 46 , 72 , 94 , 102 ]. Fig.…”
Section: Outline and Scopementioning
confidence: 99%
“…We order the sections from the most “natural systems” (containing endogenous enzymes naturally found in the host strain) to the most synthetic (containing heterologous and novel enzyme combinations, generating glycans that are not found in nature). This distinction illustrates how the use of synthetic biology principles in glycosylation systems has expanded their scope and provided access to new glycoforms [ 72 ].…”
Section: Outline and Scopementioning
confidence: 99%
“…OSTs mediate glycosylation by their substrate specificity, glycosylating only when specific polypeptide acceptor sequences and LLO donor structures are recognized. While the general acceptor peptide motif (or “sequon”) N-X-S/T is conserved, specific sequons and LLOs recognized by OSTs vary across organisms [ 72 , 121 ]. In eukaryotes, OSTs are multi-subunit complexes assembled around a core catalytic subunit called STT3 [ 66 , 85 ].…”
Section: Main Textmentioning
confidence: 99%
“…Unfortunately, building and testing defined glycoproteins in cells remains challenging for several reasons. These include glycoprotein heterogeneity [ 49 , 60 , 121 ], gaps in the methods and basic knowledge required to build defined glycoforms with desired pharmacological activities [ 72 , 153 ], and the high costs and lengthy durations required to generate stable cell lines in mammalian cell culture. To address these issues, diverse CFPS platforms that allow user-defined glycosylation have been developed [ 26 , 72 ].…”
Protein glycosylation, the enzymatic modification of amino acid sidechains with sugar moieties, plays critical roles in cellular function, human health, and biotechnology. However, studying and producing defined glycoproteins remains challenging. Cell-free glycoprotein synthesis systems, in which protein synthesis and glycosylation are performed in crude cell extracts, offer new approaches to address these challenges. Here, we review versatile, state-of-the-art systems for biomanufacturing glycoproteins in prokaryotic and eukaryotic cell-free systems with natural and synthetic
N
-linked glycosylation pathways. We discuss existing challenges and future opportunities in the use of cell-free systems for the design, manufacture, and study of glycoprotein biomedicines.
“…We recently described cellfree glycoprotein synthesis (CFGpS), a platform for one-pot biomanufacturing of defined glycoproteins in extracts enriched with heterologous, membrane-bound glycosylation machinery (47). To date, CFGpS has been used to produce model glycoproteins, human glycoproteins, and conjugate vaccines (38,47,(58)(59)(60). Importantly, CFGpS reactions can be freeze-dried for shelfstability and rehydrated at the point of care to make effective vaccines (38).…”
Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for accelerating the design of cellular function, on-demand biomanufacturing, portable diagnostics, and educational kits. Many essential biological processes that could endow CFE systems with desired functions, such as protein glycosylation, rely on the activity of membrane-bound components. However, without the use of synthetic membrane mimics, activating membrane-dependent functionality in bacterial CFE systems remains largely unstudied. Here, we address this gap by characterizing native, cell-derived membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. We first use nanocharacterization techniques to show that lipid vesicles in CFE extracts are tens to hundreds of nanometers across, and on the order of ~3x1012 particles/mL. We then determine how extract processing methods, such as post-lysis centrifugation, can be used to modulate concentrations of membrane vesicles in CFE systems. By tuning these methods, we show that increasing the number of vesicle particles to ~7x1012 particles/mL can be used to increase concentrations of heterologous membrane protein cargo expressed prior to lysis. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving N-linked and O-linked glycoprotein synthesis. We anticipate that our findings will facilitate in vitro gene expression systems that require membrane-dependent activities and open new opportunities in glycoengineering.
Most of the reported work focus on the development of O‐, N‐, C‐ and S‐glycosylation methods. However, no study explores P(III)‐glycosylation reaction. Herein we describe a convenient protocol to realize P(III)‐glycosylation process. A simple β‐phosphino ester is adopted as P(III)‐transfer reagent for this new type of glycosylation via a nucleophilic substitution and release strategy. Diverse phosphine units are introduced to the anomeric center of various sugars efficiently and with excellent stereoselectivity. The value of this method is showcased by the prepared P(III)‐sugars as novel linkers in bioactive molecule conjugation, new chiral ligands in metal‐catalyzed asymmetric allylic substitutions and organocatalysts. Preliminary mechanistic studies corroborated the designed P(III)‐transfer process.
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