DataSheet1_A ubiquitous amino acid source for prokaryotic and eukaryotic cell-free transcription-translation systems.pdf

Cell-free gene expression (CFE) systems are an attractive tool for engineering within synthetic biology and for industrial production of high-value recombinant proteins. CFE reactions require a cell extract, energy system, amino acids, and DNA, to catalyse mRNA transcription and protein synthesis. To provide an amino acid source, CFE systems typically use a commercial standard, which is often proprietary. Herein we show that a range of common microbiology rich media (i.e., tryptone, peptone, yeast extract and casamino acids) unexpectedly provide an effective and low-cost amino acid source. We show that this approach is generalisable, by comparing batch variability and protein production in the following range of CFE systems: Escherichia coli (Rosetta™ 2 (DE3), BL21(DE3)), Streptomyces venezuelae and Pichia pastoris. In all CFE systems, we show equivalent or increased protein synthesis capacity upon replacement of the commercial amino acid source. In conclusion, we suggest rich microbiology media provides a new amino acid source for CFE systems with potential broad use in synthetic biology and industrial biotechnology applications.

无细胞基因表达（Cell-free gene expression, CFE）系统是合成生物学工程化研究与工业生产高价值重组蛋白的极具吸引力的工具。CFE反应体系需要细胞提取物、能量系统、氨基酸与DNA，以催化mRNA转录及蛋白质合成过程。为提供氨基酸来源，CFE系统通常采用商业化标准试剂，而这类试剂往往具有专利性。本研究证实，一系列常见的微生物富集培养基（即胰蛋白胨、蛋白胨、酵母提取物与酪蛋白氨基酸）可意外地作为高效且低成本的氨基酸来源。通过对以下数种CFE系统的批次间差异与蛋白表达水平进行对比，我们证实该方法具备普适性：大肠杆菌（Escherichia coli）Rosetta™ 2 (DE3)、BL21(DE3)，委内瑞拉链霉菌（Streptomyces venezuelae）以及巴斯德毕赤酵母（Pichia pastoris）。在所有测试的CFE系统中，替换商业化氨基酸来源后，蛋白合成能力均达到或超越原有水平。综上，我们认为微生物富集培养基可作为CFE系统的新型氨基酸来源，在合成生物学与工业生物技术领域具备广阔的应用潜力。