Department of Chemical and Biological Engineering, Northwestern University, Technological Institute E136, Evanston, IL, 60208, USA.
Chemistry of Life Processes Institute, Northwestern University, Evanston, IL, 60208, USA.
Nat Commun. 2021 Apr 22;12(1):2363. doi: 10.1038/s41467-021-22329-3.
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.
无细胞基因表达 (CFE) 系统源自粗细胞提取物，因其在生物制造和合成生物学方面的应用而备受关注。然而，在细菌 CFE 系统中激活源自细胞的囊泡的膜依赖性功能一直受到限制。在这里，我们通过对基于大肠杆菌的 CFE 提取物中的天然膜囊泡进行表征，并描述了富集具有异源膜结合机制的囊泡的方法来解决这一限制。作为模型，我们专注于细菌糖基工程。我们首先使用多种正交技术对囊泡进行表征，并展示了如何使用提取物处理方法来增加 CFE 系统中膜囊泡的浓度。然后，我们表明，囊泡数量丰富的提取物还显示出异源膜蛋白货物的浓度增加。最后，我们应用我们的方法来富集膜结合的寡糖基转移酶和脂连接的寡糖，以提高无细胞 N 连接和 O 连接糖蛋白的合成。我们预计这些方法将促进按需糖蛋白生产，并为具有膜相关活性的新型 CFE 系统提供支持。
