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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. The ability to produce homogeneous glycoproteins is expected to advance fundamental understanding in glycoscience, but current in vivo-based production systems have several limitations. Here, the authors develop an E. coli extract-based one-pot system for customized production of N -linked glycoproteins.

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. Antigen display on outer membrane vesicles (OMVs) can be difficult to control and highly variable. Here, the authors describe a universal approach called AvidVax for linking biotinylated antigens to the exterior of OMVs and enabling rapid vaccine assembly.

Glycosylation plays important roles in cellular function and endows protein therapeutics with beneficial properties. However, constructing biosynthetic pathways to study and engineer precise glycan structures on proteins remains a bottleneck. Here, we report a modular, versatile cell-free platform for glyco sylation p athway assembly by r apid i n vitro m ixing and e xpression (GlycoPRIME). In GlycoPRIME, glycosylation pathways are assembled by mixing-and-matching cell-free synthesized glycosyltransferases that can elaborate a glucose primer installed onto protein targets by an N -glycosyltransferase. We demonstrate GlycoPRIME by constructing 37 putative protein glycosylation pathways, creating 23 unique glycan motifs, 18 of which have not yet been synthesized on proteins. We use selected pathways to synthesize a protein vaccine candidate with an α-galactose adjuvant motif in a one-pot cell-free system and human antibody constant regions with minimal sialic acid motifs in glycoengineered Escherichia coli . We anticipate that these methods and pathways will facilitate glycoscience and make possible new glycoengineering applications. Constructing biosynthetic pathways to study and engineer glycoprotein structures is difficult. Here, the authors use GlycoPRIME, a cell-free workflow for mixing-and-matching glycosylation enzymes, to evaluate 37 putative glycosylation pathways and discover routes to 18 new glycoprotein structures

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli- based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N- linked and O -linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities. Cell-free gene expression systems are an attractive platform for biomanufacturing and synthetic biology. Here the authors characterize native membrane vesicles in E. coli extracts for improved glycoengineering.

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. Access to glycoenzymes for basic and applied research is limited by difficulties with their recombinant expression. Here, the authors describe a universal strategy for converting membrane-bound glycosyltransferases into water-soluble biocatalysts, which are expressed at high levels with retention of activity.

As new disease threats arise and existing pathogens grow resistant to conventional interventions, attention increasingly focuses on the development of vaccines to induce protective immune responses. Given their admirable safety records, protein subunit vaccines are attractive for widespread immunization, but their disadvantages include poor immunogenicity and expensive manufacture. We show here that engineered Escherichia coli outer membrane vesicles (OMVs) are an easily purified vaccine-delivery system capable of greatly enhancing the immunogenicity of a low-immunogenicity protein antigen without added adjuvants. Using green-fluorescent protein (GFP) as the model subunit antigen, genetic fusion of GFP with the bacterial hemolysin ClyA resulted in a chimeric protein that elicited strong anti-GFP antibody titers in immunized mice, whereas immunization with GFP alone did not elicit such titers. Harnessing the specific secretion of ClyA to OMVs, the ClyA-GFP fusion was found localized in OMVs, resulting in engineered recombinant OMVs. The anti-GFP humoral response in mice immunized with the engineered OMV formulations was indistinguishable from the response to the purified ClyA-GFP fusion protein alone and equal to purified proteins absorbed to aluminum hydroxide, a standard adjuvant. In a major improvement over current practice, engineered OMVs containing ClyA-GFP were easily isolated by ultracentrifugation, effectively eliminating the need for laborious antigen purification from cell-culture expression systems. With the diverse collection of heterologous proteins that can be functionally localized with OMVs when fused with ClyA, this work signals the possibility of OMVs as a robust and tunable technology platform for a new generation of prophylactic and therapeutic vaccines.

The systematic organization of enzymes is a key feature for the efficient operation of cascade reactions in nature. Here, we demonstrate a facile method to create nanoscale enzyme cascades by using engineered bacterial outer membrane vesicles (OMVs) that are spheroid nanoparticles (roughly 50 nm in diameter) produced by Gram-negative bacteria during all phases of growth. By taking advantage of the fact that OMVs naturally contain proteins found in the outer cell membrane, we displayed a trivalent protein scaffold containing three divergent cohesin domains for the position-specific presentation of a three-enzyme cascade on OMVs through a truncated ice nucleation protein anchoring motif (INP). The positional assembly of three enzymes for cellulose hydrolysis was demonstrated. The enzyme-decorated OMVs provided synergistic cellulose hydrolysis resulting in 23-fold enhancement in glucose production than free enzymes.

Dysregulated protein degradation via the ubiquitin-proteasomal pathway can induce numerous disease phenotypes, including cancer, neurodegeneration, and diabetes. While small molecule-based targeted protein degradation (TPD) and targeted protein stabilization (TPS) platforms can address this dysregulation, they rely on structured and stable binding pockets, which do not exist to classically “undruggable” targets. Here, we expand the TPS target space by engineering “deubiquibodies” (duAbs) via fusion of computationally-designed peptide binders to the catalytic domain of the potent OTUB1 deubiquitinase. In human cells, duAbs effectively stabilize exogenous and endogenous proteins in a DUB-dependent manner. Using protein language models to generate target-binding peptides, we engineer duAbs to conformationally diverse target proteins, including key tumor suppressor proteins p53 and WEE1, and heavily-disordered fusion oncoproteins, such as PAX3::FOXO1. We further encapsulate p53-targeting duAbs as mRNA in lipid nanoparticles and demonstrate effective intracellular delivery, p53 stabilization, and apoptosis activation, motivating further in vivo translation. Dysregulated protein degradation drives diseases like cancer. Here, authors use protein language models to design target-binding peptides, which are subsequently attached to the catalytic domain of the OTUB1 deubiquitinase, generating “deubiquibodies” (duAbs). duAbs restore tumor suppressors and fusion oncoproteins, offering a programmable strategy for protein stabilization.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive. Here, we describe an alternative methodology for producing glycoconjugate vaccines whereby recombinant O-PS biosynthesis is coordinated with vesiculation in laboratory strains of Escherichia coli to yield glycosylated outer membrane vesicles (glycOMVs) decorated with pathogen-mimetic glycotopes. Using this approach, glycOMVs corresponding to eight different pathogenic bacteria were generated. For example, expression of a 17-kb O-PS gene cluster from the highly virulent Francisella tularensis subsp. tularensis (type A) strain Schu S4 in hypervesiculating E. coli cells yielded glycOMVs that displayed F. tularensis O-PS. Immunization of BALB/c mice with glycOMVs elicited significant titers of O-PS–specific serum IgG antibodies as well as vaginal and bronchoalveolar IgA antibodies. Importantly, glycOMVs significantly prolonged survival upon subsequent challenge with F. tularensis Schu S4 and provided complete protection against challenge with two different F. tularensis subsp. holarctica (type B) live vaccine strains, thereby demonstrating the vaccine potential of glycOMVs. Given the ease with which recombinant glycotopes can be expressed on OMVs, the strategy described here could be readily adapted for developing vaccines against many other bacterial pathogens.

•Glycosylation is the most prevalent post-translational protein modification.•N-Linked protein glycosylation pathways in bacteria have led to novel therapeutics, vaccines, and diagnostics.•Bacteria with recombinant N-linked glycosylation pathways improve our understanding of N-glycosylation.•Current and future prospects in glycoengineering are discussed. Glycosylation is the most prevalent post-translational modification found on proteins, occurring in all domains of life. Ever since the discovery of asparagine-linked (N-linked) protein glycosylation pathways in bacteria, major efforts have been made to harness these systems for the creation of novel therapeutics, vaccines, and diagnostics. Recent advances such as the ability to produce designer glycans in bacteria, some containing unnatural sugars, and techniques for evolving glycosylation enzymes have spawned an entirely new discipline known as bacterial glycoengineering. In addition to their biotechnological and therapeutic potential, bacteria equipped with recombinant N-linked glycosylation pathways are improving our understanding of the N-glycosylation process. This review discusses the key role played by microorganisms in glycosciences, particularly in the context of N-linked glycosylation.