Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors
Optimization of a whole-cell biocatalyst by employing genetically encoded product sensors inside nanolitre reactors
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DOI：
10.1038/nchem.2301
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发表时间：
2015-08-01
期刊：
影响因子：
21.8
通讯作者：
Held, Martin
中科院分区：
文献类型：
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作者：
Meyer, Andreas;Pellaux, Rene;Held, Martin
微区室化提供了一种高通量的方法来筛选大量的生物催化剂产生的基因文库。在这里，我们提出了一个microcompartmentalization协议的基准性能的全细胞生物催化剂。凝胶胶囊作为纳升反应器（nLR）的枯草芽孢杆菌生物催化剂库的培养和分析。B。subtilis细胞，与E.在nLR内的大肠杆菌传感器细胞中，将起始材料纤维二糖转化为工业产品维生素B2。产物的形成触发了传感器细胞中的一系列反应：（1）B2转化为黄素单核苷酸（FMN），（2）FMN与RNA核糖开关结合，（3）RNA自切割，导致（4）合成绿色荧光蛋白（GFP）。然后使用GFP荧光的强度来分离B。枯草芽孢杆菌变体以提高的效率将纤维二糖转化为维生素B2。该分析的基本设计原则是通用的，并且能够开发类似的方案，这最终将加速全细胞生物催化剂的优化。
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) selfcleavage 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.