Ambri Francesca, Snoek Tim, Skjoedt Mette L, Jensen Michael K, Keasling Jay D
The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kongens Lyngby, Denmark.
Joint BioEnergy Institute, Emeryville, CA, USA.
Methods Mol Biol. 2018;1671:269-290. doi: 10.1007/978-1-4939-7295-1_17.
In cell factory development, screening procedures, often relying on low-throughput analytical methods, are lagging far behind diversity generation methods. This renders the identification and selection of the best cell factory designs tiresome and costly, conclusively hindering the manufacturing process. In the yeast Saccharomyces cerevisiae, implementation of allosterically regulated transcription factors from prokaryotes as metabolite biosensors has proven a valuable strategy to alleviate this screening bottleneck. Here, we present a protocol to select and incorporate prokaryotic transcriptional activators as metabolite biosensors in S. cerevisiae. As an example, we outline the engineering and characterization of the LysR-type transcriptional regulator (LTTR) family member BenM from Acetinobacter sp. ADP1 for monitoring accumulation of cis,cis-muconic acid, a bioplast precursor, in yeast by means of flow cytometry.
在细胞工厂的开发中，筛选程序通常依赖低通量分析方法，远远落后于多样性生成方法。这使得识别和选择最佳细胞工厂设计既繁琐又昂贵，最终阻碍了制造过程。在酿酒酵母中，将来自原核生物的变构调节转录因子用作代谢物生物传感器已被证明是缓解这一筛选瓶颈的有效策略。在此，我们提出了一种在酿酒酵母中选择并整合原核转录激活因子作为代谢物生物传感器的方案。例如，我们概述了醋酸杆菌属ADP1菌株的LysR型转录调节因子（LTTR）家族成员BenM的工程改造和表征，以通过流式细胞术监测酵母中生物塑料前体顺，顺-粘康酸的积累。
