Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

Jasmine M Hershewe,Katherine F Warfel,Michael C Jewett,Justin A Peruzzi,Neha P Kamat,Matthew P Delisa,Eric W Roth,Claretta J Sullivan,Shaelyn M Iyer

doi:10.1038/s41467-021-22329-3

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.

Highlights

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology
We aimed to characterize and engineer membrane vesicles in E. coli CFE extracts (Fig. 1)
We: (i) used nanocharacterization techniques to determine the sizes and quantities of membrane vesicles in E. coli extracts; (ii) determined how extract processing can control the enrichment of vesicles in extracts; (iii) enriched several heterologous, membrane-bound components in extracts via vesicles; and (iv) demonstrated that increasing enrichment of membranebound components significantly improves cell-free glycoprotein synthesis (CFGpS) systems for Nand O-linked glycosylation

Summary

Introduction

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. We apply our methods to enrich membranebound 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 (CFE) systems activate transcription and translation using crude cellular extracts instead of living, intact cells[1]. Despite the absence of intact cellular membranes, membrane structures are present in crude extract-based CFE systems They form upon fragmentation and rearrangement of cell membranes during cell lysis and extract preparation and have been studied and characterized for decades[51,54,55,56,57]. In eukaryotic-derived crude extract-based CFE systems, endoplasmic reticulum (ER)derived microsomes enhance functionality, enabling the synthesis of membrane proteins and proteins with disulfide bonds, among others[5,26,60,61,62,63,64,65]

Objectives

Methods

Results