skip to main content


Title: Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles
Abstract 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.  more » « less
Award ID(s):
1936789 1936823 1716766 1844336
NSF-PAR ID:
10249257
Author(s) / Creator(s):
; ; ; ; ; ; ; ;
Date Published:
Journal Name:
Nature Communications
Volume:
12
Issue:
1
ISSN:
2041-1723
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of Escherichia coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease (RNase) activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNase activity yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell-free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNase activity. However, it is not clear which gene encodes the RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field.

    Graphical Abstract

     

    more » « less
  2. Abstract

    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-optimizedEscherichia colistrain 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.

     
    more » « less
  3. null (Ed.)
    Abstract 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. 
    more » « less
  4. Nikel, Pablo Ivan (Ed.)
    ABSTRACT The exchange of bacterial extracellular vesicles facilitates molecular exchange between cells, including the horizontal transfer of genetic material. Given the implications of such transfer events on cell physiology and adaptation, some bacterial cells have likely evolved mechanisms to regulate vesicle exchange. Past work has identified mechanisms that influence the formation of extracellular vesicles, including the production of small molecules that modulate membrane structure; however, whether these mechanisms also modulate vesicle uptake and have an overall impact on the rate of vesicle exchange is unknown. Here, we show that membrane-binding molecules produced by microbes influence both the formation and uptake of extracellular vesicles and have the overall impact of increasing the vesicle exchange rate within a bacterial coculture. In effect, production of compounds that increase vesicle exchange rates encourage gene exchange between neighboring cells. The ability of several membrane-binding compounds to increase vesicle exchange was demonstrated. Three of these compounds, nisin, colistin, and polymyxin B, are antimicrobial peptides added at sub-inhibitory concentrations. These results suggest that a potential function of exogenous compounds that bind to membranes may be the regulation of vesicle exchange between cells. IMPORTANCE The exchange of bacterial extracellular vesicles is one route of gene transfer between bacteria, although it was unclear if bacteria developed strategies to modulate the rate of gene transfer within vesicles. In eukaryotes, there are many examples of specialized molecules that have evolved to facilitate the production, loading, and uptake of vesicles. Recent work with bacteria has shown that some small molecules influence membrane curvature and induce vesicle formation. Here, we show that similar compounds facilitate vesicle uptake, thereby increasing the overall rate of vesicle exchange within bacterial populations. The addition of membrane-binding compounds, several of them antibiotics at subinhibitory concentrations, to a bacterial coculture increased the rate of horizontal gene transfer via vesicle exchange. 
    more » « less
  5. Abstract

    Engineering synthetic interfaces between membranes has potential applications in designing non‐native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane–membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane–membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell‐free expression (CFE) system. By utilizing co‐translational helix insertion, cell‐free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane–membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity‐inducing proteins. This technology may also prove useful where cell‐cell contacts and communication are recreated in a controlled manner using minimal components.

     
    more » « less