1] Department of Biosystems Science and Engineering, ETH Zurich, Basel 4058, Switzerland [2] FGen GmbH, Basel 4057, Switzerland.
DSM Nutritional Products, Kaiseraugst 4303, Switzerland.
Nat Chem. 2015 Aug;7(8):673-8. doi: 10.1038/nchem.2301. Epub 2015 Jul 13.
Microcompartmentalization offers a high-throughput method for screening large numbers of biocatalysts generated from genetic libraries. Here we present a microcompartmentalization protocol for benchmarking the performance of whole-cell biocatalysts. Gel capsules served as nanolitre reactors (nLRs) for the cultivation and analysis of a library of Bacillus subtilis biocatalysts. The B. subtilis cells, which were co-confined with E. coli sensor cells inside the nLRs, converted the starting material cellobiose into the industrial product vitamin B2. Product formation triggered a sequence of reactions in the sensor cells: (1) conversion of B2 into flavin mononucleotide (FMN), (2) binding of FMN by a RNA riboswitch and (3) self-cleavage of RNA, which resulted in (4) the synthesis of a green fluorescent protein (GFP). The intensity of GFP fluorescence was then used to isolate B. subtilis variants that convert cellobiose into vitamin B2 with elevated efficiency. The underlying design principles of the assay are general and enable the development of similar protocols, which ultimately will speed up the optimization of whole-cell biocatalysts.
微区室化提供了一种高通量的方法，用于筛选来自遗传文库的大量生物催化剂。在这里，我们提出了一种用于基准测试全细胞生物催化剂性能的微区室化方案。凝胶胶囊用作纳升反应器（nLR），用于培养和分析枯草芽孢杆菌生物催化剂文库。枯草芽孢杆菌细胞与大肠杆菌感应细胞一起被限制在 nLR 内，将起始材料纤维二糖转化为工业产品维生素 B2。产物形成在感应细胞中引发了一系列反应：（1）B2 转化为黄素单核苷酸（FMN），（2）FMN 与 RNA 核糖开关结合，（3）RNA 自我切割，导致（4）绿色荧光蛋白（GFP）的合成。然后，使用 GFP 荧光的强度来分离将纤维二糖转化为维生素 B2 的枯草芽孢杆菌变体，其转化效率更高。该测定的基本设计原则是通用的，能够开发类似的方案，最终将加快全细胞生物催化剂的优化。
