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 synthetic biology tools and design principles to better understand and engineer glycosylation. Specifically, we focus on how the biosynthetic and analytical tools of synthetic biology have been used to redesign glycosylation systems to obtain defined glycosylation structures on proteins for diverse applications in medicine, materials, and diagnostics. We review the key biological parts available to synthetic biologists interested in engineering glycoproteins to solve compelling problems in glycoscience, describe recent efforts to construct synthetic glycoprotein synthesis systems, and outline exemplary applications as well as new opportunities in this emerging space.
Highlights
We review the exciting area of synthetic glycobiology with a focus on useful abstractions, tools, and methods regularly employed by the synthetic biology community at large
This review focuses primarily on the synthesis of glycoproteins, knowledge of lectin specificities has often been leveraged in the field of synthetic glycobiology to design glycanbased selection schemes,[58−60] develop new approaches to fight infectious and autoimmune disease,[61−63] produce functional biomaterials,[64−66] and to understand and manipulate protein trafficking within the human body.[67−71] Key resources for the identification of relevant enzymes, glycans, and glycan-binding proteins include the Carbohydrate-active enzyme (CAZY) database,[72] the GlyCosmos Portal, and the GlycoGene Database
Because the host organisms in which these glycosylation pathways are constructed strongly affect their challenges, advantages, and applications, we describe examples of synthetic glycosylation pathways developed in mammalian, insect, plant, yeast, and bacterial cells, cell-free, and chemoenzymatic backgrounds
Summary
Synthetic biology has made great strides in engineering living systems for desired purposes and in creating novel biological processes with compositions and properties not found in nature.[1−4] While the field is historically rooted in the development of methods to better read, write, edit, and design. There is no O-GalNAc glycosylation in plants and N-glycans generally terminate with N-linked Man3GlcNAc2 that may be modified with bianntenary GlcNAc residues.[195] These simplified pathways and the apparent tolerance of plants for heterologous glycosylation pathways offer excellent opportunities for de novo construction of desired glycosylation systems with a freedom of design and homogeneity that may be more difficult to achieve in mammalian systems.[195] far, glycoprotein engineering in plants (reviewed thoroughly here195,213) has focused on (i) ensuring homogeneous expression of N-linked GlcNAcylated trimannose by removal of β-hexosaminidases;[214] (ii) the removal of nonhuman sugar linkages including β1−2 Xylose, α1−3 Fucose,[215] arabinosylated hydroxyproline,[216] and Lewis A structures;[217] and (iii) the addition of metabolic machinery and human GTs to obtain human-like, sialylated N- and Oglycans.[218−222] to the GlycoDelete strategy in mammalian cells, plants were recently engineered to generate a minimal trisaccharide.[223] The end result of these works is the ability to produce glycoprotein therapeutics in a number of model plant and plant cell systems (such as Nicotiana bethamiana, Arabidopsis thaliana, and Nicotiana tabacum) with highly similar glycosylation to mammalian systems.[195] Key remaining challenges lie in the optimization of homogeneity and production levels without affecting plant fitness and control of potentially immunogenic nonhuman hydroxylproline modifications.[190,195].
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