Novel human recombinant N-acetylgalactosamine-6-sulfate sulfatase produced in a glyco-engineered Escherichia coli strain.

Mucopolysaccharidosis IVA (MPS IVA) is a lysosomal storage disease caused by mutations in the gene encoding the lysosomal enzyme N-acetylgalactosamine-6-sulfate sulfatase (GALNS), resulting in the accumulation of keratan sulfate (KS) and chondroitin-6-sulfate (C6S). Previously, it was reported the production of an active human recombinant GALNS (rGALNS) in E. coli BL21(DE3). However, this recombinant enzyme was not taken up by HEK293 cells or MPS IVA skin fibroblasts. Here, we leveraged a glyco-engineered E. coli strain to produce a recombinant human GALNS bearing the eukaryotic trimannosyl core N-glycan, Man3GlcNAc2 (rGALNSoptGly). The N-glycosylated GALNS was produced at 100 mL and 1.65 L scales, purified and characterized with respect to pH stability, enzyme kinetic parameters, cell uptake, and KS clearance. The results showed that the addition of trimannosyl core N-glycans enhanced both protein stability and substrate affinity. rGALNSoptGly was capture through a mannose receptor-mediated process. This enzyme was delivered to the lysosome, where it reduced KS storage in human MPS IVA fibroblasts. This study demonstrates the potential of a glyco-engineered E. coli for producing a fully functional GALNS enzyme. It may offer an economic approach for the biosynthesis of a therapeutic glycoprotein that could prove useful for MPS IVA treatment. This strategy could be extended to other lysosomal enzymes that rely on the presence of mannose N-glycans for cell uptake.

Direct observation of glycans bonded to proteins and lipids at the single-molecule level.

Proteins and lipids decorated with glycans are found throughout biological entities, playing roles in biological functions and dysfunctions. Current analytical strategies for these glycan-decorated biomolecules, termed glycoconjugates, rely on ensemble-averaged methods that do not provide a full view of positions and structures of glycans attached at individual sites in a given molecule, especially for glycoproteins. We show single-molecule analysis of glycoconjugates by direct imaging of individual glycoconjugate molecules using low-temperature scanning tunneling microscopy. Intact glycoconjugate ions from electrospray are soft-landed on a surface for their direct single-molecule imaging. The submolecular imaging resolution corroborated by quantum mechanical modeling unveils whole structures and attachment sites of glycans in glycopeptides, glycolipids, N-glycoproteins, and O-glycoproteins densely decorated with glycans.

Rapid biosynthesis of glycoprotein therapeutics and vaccines from freeze-dried bacterial cell lysates.

The advent of distributed biomanufacturing platforms promises to increase agility in biologic production and expand access by reducing reliance on refrigerated supply chains. However, such platforms are not capable of robustly producing glycoproteins, which represent the majority of biologics approved or in development. To address this limitation, we developed cell-free technologies that enable rapid, modular production of glycoprotein therapeutics and vaccines from freeze-dried Escherichia coli cell lysates. Here, we describe a protocol for generation of cell-free lysates and freeze-dried reactions for on-demand synthesis of desired glycoproteins. The protocol includes construction and culture of the bacterial chassis strain, cell-free lysate production, assembly of freeze-dried reactions, cell-free glycoprotein synthesis, and glycoprotein characterization, all of which can be completed in one week or less. We anticipate that cell-free technologies, along with this comprehensive user manual, will help accelerate development and distribution of glycoprotein therapeutics and vaccines.

Isolation of full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of Escherichia coli.

Here we describe a facile and robust genetic selection for isolating full-length IgG antibodies from combinatorial libraries expressed in the cytoplasm of redox-engineered Escherichia coli cells. The method is based on the transport of a bifunctional substrate comprised of an antigen fused to chloramphenicol acetyltransferase, which allows positive selection of bacterial cells co-expressing cytoplasmic IgGs called cyclonals that specifically capture the chimeric antigen and sequester the antibiotic resistance marker in the cytoplasm. The utility of this approach is first demonstrated by isolating affinity-matured cyclonal variants that specifically bind their cognate antigen, the leucine zipper domain of a yeast transcriptional activator, with subnanomolar affinities, which represent a ~20-fold improvement over the parental IgG. We then use the genetic assay to discover antigen-specific cyclonals from a naïve human antibody repertoire, leading to the identification of lead IgG candidates with affinity and specificity for an influenza hemagglutinin-derived peptide antigen.

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens.

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

A universal glycoenzyme biosynthesis pipeline that enables efficient cell-free remodeling of glycans.

The ability to reconstitute natural glycosylation pathways or prototype entirely new ones from scratch is hampered by the limited availability of functional glycoenzymes, many of which are membrane proteins that fail to express in heterologous hosts. Here, we describe a strategy for topologically converting membrane-bound glycosyltransferases (GTs) into water soluble biocatalysts, which are expressed at high levels in the cytoplasm of living cells with retention of biological activity. We demonstrate the universality of the approach through facile production of 98 difficult-to-express GTs, predominantly of human origin, across several commonly used expression platforms. Using a subset of these water-soluble enzymes, we perform structural remodeling of both free and protein-linked glycans including those found on the monoclonal antibody therapeutic trastuzumab. Overall, our strategy for rationally redesigning GTs provides an effective and versatile biosynthetic route to large quantities of diverse, enzymatically active GTs, which should find use in structure-function studies as well as in biochemical and biomedical applications involving complex glycomolecules.

Shotgun scanning glycomutagenesis: A simple and efficient strategy for constructing and characterizing neoglycoproteins.

As a common protein modification, asparagine-linked (N-linked) glycosylation has the capacity to greatly influence the biological and biophysical properties of proteins. However, the routine use of glycosylation as a strategy for engineering proteins with advantageous properties is limited by our inability to construct and screen large collections of glycoproteins for cataloguing the consequences of glycan installation. To address this challenge, we describe a combinatorial strategy termed shotgun scanning glycomutagenesis in which DNA libraries encoding all possible glycosylation site variants of a given protein are constructed and subsequently expressed in glycosylation-competent bacteria, thereby enabling rapid determination of glycosylatable sites in the protein. The resulting neoglycoproteins can be readily subjected to available high-throughput assays, making it possible to systematically investigate the structural and functional consequences of glycan conjugation along a protein backbone. The utility of this approach was demonstrated with three different acceptor proteins, namely bacterial immunity protein Im7, bovine pancreatic ribonuclease A, and human anti-HER2 single-chain Fv antibody, all of which were found to tolerate N-glycan attachment at a large number of positions and with relatively high efficiency. The stability and activity of many glycovariants was measurably altered by N-linked glycans in a manner that critically depended on the precise location of the modification. Structural models suggested that affinity was improved by creating novel interfacial contacts with a glycan at the periphery of a protein-protein interface. Importantly, we anticipate that our glycomutagenesis workflow should provide access to unexplored regions of glycoprotein structural space and to custom-made neoglycoproteins with desirable properties.

On-demand biomanufacturing of protective conjugate vaccines.

Conjugate vaccines are among the most effective methods for preventing bacterial infections. However, existing manufacturing approaches limit access to conjugate vaccines due to centralized production and cold chain distribution requirements. To address these limitations, we developed a modular technology for in vitro conjugate vaccine expression (iVAX) in portable, freeze-dried lysates from detoxified, nonpathogenic Escherichia coli. Upon rehydration, iVAX reactions synthesize clinically relevant doses of conjugate vaccines against diverse bacterial pathogens in 1 hour. We show that iVAX-synthesized vaccines against Francisella tularensis subsp. tularensis (type A) strain Schu S4 protected mice from lethal intranasal F. tularensis challenge. The iVAX platform promises to accelerate development of new conjugate vaccines with increased access through refrigeration-independent distribution and portable production.

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

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).

Engineering orthogonal human O-linked glycoprotein biosynthesis in bacteria.

A major objective of synthetic glycobiology is to re-engineer existing cellular glycosylation pathways from the top down or construct non-natural ones from the bottom up for new and useful purposes. Here, we have developed a set of orthogonal pathways for eukaryotic O-linked protein glycosylation in Escherichia coli that installed the cancer-associated mucin-type glycans Tn, T, sialyl-Tn and sialyl-T onto serine residues in acceptor motifs derived from different human O-glycoproteins. These same glycoengineered bacteria were used to supply crude cell extracts enriched with glycosylation machinery that permitted cell-free construction of O-glycoproteins in a one-pot reaction. In addition, O-glycosylation-competent bacteria were able to generate an antigenically authentic Tn-MUC1 glycoform that exhibited reactivity with antibody 5E5, which specifically recognizes cancer-associated glycoforms of MUC1. We anticipate that the orthogonal glycoprotein biosynthesis pathways developed here will provide facile access to structurally diverse O-glycoforms for a range of important scientific and therapeutic applications.

Author Correction: Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery.

The original version of this Article contained an error in Figure 2, wherein the bottom right western blot panel in Figure 2a was blank. This has now been corrected in both the PDF and HTML versions of the Article.

Single-pot glycoprotein biosynthesis using a cell-free transcription-translation system enriched with glycosylation machinery.

The emerging discipline of bacterial glycoengineering has made it possible to produce designer glycans and glycoconjugates for use as vaccines and therapeutics. Unfortunately, cell-based production of homogeneous glycoproteins remains a significant challenge due to cell viability constraints and the inability to control glycosylation components at precise ratios in vivo. To address these challenges, we describe a novel cell-free glycoprotein synthesis (CFGpS) technology that seamlessly integrates protein biosynthesis with asparagine-linked protein glycosylation. This technology leverages a glyco-optimized Escherichia coli strain to source cell extracts that are selectively enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs). The resulting extracts enable a one-pot reaction scheme for efficient and site-specific glycosylation of target proteins. The CFGpS platform is highly modular, allowing the use of multiple distinct OSTs and structurally diverse LLOs. As such, we anticipate CFGpS will facilitate fundamental understanding in glycoscience and make possible applications in on demand biomanufacturing of glycoproteins.

A flow cytometric approach to engineering Escherichia coli for improved eukaryotic protein glycosylation.

A synthetic pathway for production of the eukaryotic trimannosyl chitobiose glycan (mannose3-N-acetylglucosamine2, Man3GlcNAc2) and its transfer to specific asparagine residues in target proteins was previously engineered in Escherichia coli, providing this simple microbe with the ability to perform a complex post-translational protein modification. Here, we leveraged a flow cytometric fluorescence-based assay to improve Man3GlcNAc2 glycan biosynthesis in E. coli cells. Specifically, pathway improvements were identified, including reducing pathway enzyme expression levels and overexpressing nucleotide sugar biosynthesis genes, which enhanced production of lipid-linked Man3GlcNAc2 by nearly 50-fold to 13.9 µg/L. In turn, cells producing higher levels of the Man3GlcNAc2 substrate yielded up to 10 times more glycosylated acceptor protein (to ~ 14 mg/L) than their non-optimized counterparts. These results demonstrate the use of flow cytometry screening as a powerful tool for interrogating the surfaces of glyco-engineered bacteria and identifying meaningful improvements in glycan biosynthesis. We anticipate this approach will enable further optimization of bacterial glycan biosynthesis pathways using new strain engineering tools from metabolic engineering and synthetic biology.

Metabolic engineering of glycoprotein biosynthesis in bacteria.

The demonstration more than a decade ago that glycoproteins could be produced in Escherichia coli cells equipped with the N-linked protein glycosylation machinery from Campylobacter jejuni opened the door to using simple bacteria for the expression and engineering of complex glycoproteins. Since that time, metabolic engineering has played an increasingly important role in developing and optimizing microbial cell glyco-factories for the production of diverse glycoproteins and other glycoconjugates. It is becoming clear that future progress in creating efficient glycoprotein expression platforms in bacteria will depend on the adoption of advanced strain engineering strategies such as rational design and assembly of orthogonal glycosylation pathways, genome-wide identification of metabolic engineering targets, and evolutionary engineering of pathway performance. Here, we highlight recent advances in the deployment of metabolic engineering tools and strategies to develop microbial cell glyco-factories for the production of high-value glycoprotein targets with applications in research and medicine.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 µg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

A Pipeline for Studying and Engineering Single-Subunit Oligosaccharyltransferases.

Asparagine-linked (N-linked) protein glycosylation is one of the most abundant types of posttranslational modification, occurring in all domains of life. The central enzyme in N-linked glycosylation is the oligosaccharyltransferase (OST), which catalyzes the covalent attachment of preassembled glycans to specific asparagine residues in target proteins. Whereas in higher eukaryotes the OST is comprised of eight different membrane proteins, of which the catalytic subunit is STT3, in kinetoplastids and prokaryotes the OST is a monomeric enzyme bearing homology to STT3. Given their relative simplicity, these single-subunit OSTs (ssOSTs) have emerged as important targets for mechanistic dissection of poorly understood aspects of N-glycosylation and at the same time hold great potential for the biosynthesis of custom glycoproteins. To take advantage of this utility, this chapter describes a multipronged approach for studying and engineering ssOSTs that integrates in vivo screening technology with in vitro characterization methods, thereby creating a versatile and readily adaptable pipeline for virtually any ssOST of interest.

Substitute sweeteners: diverse bacterial oligosaccharyltransferases with unique N-glycosylation site preferences.

The central enzyme in the Campylobacter jejuni asparagine-linked glycosylation pathway is the oligosaccharyltransferase (OST), PglB, which transfers preassembled glycans to specific asparagine residues in target proteins. While C. jejuni PglB (CjPglB) can transfer many diverse glycan structures, the acceptor sites that it recognizes are restricted predominantly to those having a negatively charged residue in the -2 position relative to the asparagine. Here, we investigated the acceptor-site preferences for 23 homologs with natural sequence variation compared to CjPglB. Using an ectopic trans-complementation assay for CjPglB function in glycosylation-competent Escherichia coli, we demonstrated in vivo activity for 16 of the candidate OSTs. Interestingly, the OSTs from Campylobacter coli, Campylobacter upsaliensis, Desulfovibrio desulfuricans, Desulfovibrio gigas, and Desulfovibrio vulgaris, exhibited significantly relaxed specificity towards the -2 position compared to CjPglB. These enzymes glycosylated minimal N-X-T motifs in multiple targets and each followed unique, as yet unknown, rules governing acceptor-site preferences. One notable example is D. gigas PglB, which was the only bacterial OST to glycosylate the Fc domain of human immunoglobulin G at its native 'QYNST' sequon. Overall, we find that a subset of bacterial OSTs follow their own rules for acceptor-site specificity, thereby expanding the glycoengineering toolbox with previously unavailable biocatalytic diversity.

One-pot synthesis of halogen exchanged silsesquioxanes: octakis(3-bromopropyl)octasilsesquioxane and octakis(3-iodopropyl)octasilsesquioxane.

Metal halides, solvent effects, phase transfer catalysts, alkylating agent and reaction times were found to have important roles to complete halogen exchange reactions in "one pot" synthesis, starting from octakis(3-chloropropyl)octasilsesquioxane to obtain more reactive halide compounds: octakis(3-bromopropyl)octasilsesquioxane and octakis(3-iodopropyl)octasilsesquioxane. To confirm the complete halogen exchange, the desired products were characterized by (1)H, (13)C and (29)Si NMR spectroscopy, ESI-MS, elemental analysis and single-crystal X-ray diffraction analysis.

Synthesis and reactivity of nitrogen nucleophiles-induced cage-rearrangement silsesquioxanes.

Novel phthalimide and o-sulfobenzimide-functionalized silsesquioxanes were successfully synthesized via nucleophilic substitution reactions from octakis(3-chloropropyl)octasilsesquioxane. Surprisingly, the formation of deca- and dodecasilsesquioxanes cages was discovered during substitution with phthalimide, but only octasilsesquioxane maintained a cage in the o-sulfobenzimide substitution reaction. Moreover, we report the electronic effect of nitrogen nucleophiles to promote cage-rearrangement of inorganic silsesquioxane core for the first time. Structures of products were confirmed by (1)H, (13)C, and (29)Si NMR spectroscopy, ESI-MS analysis, and single-crystal X-ray diffraction.