Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at Key Enzymes in Biosynthesis Pathways
Taking Control over Control: Use of Product Sensing in Single Cells to Remove Flux Control at Key Enzymes in Biosynthesis Pathways
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DOI：
10.1021/sb400059y
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发表时间：
2014-01-01
影响因子：
4.7
通讯作者：
Eggeling, Lothar
中科院分区：
文献类型：
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作者：
Schendzielorz, Georg;Dippong, Martin;Eggeling, Lothar
启动细胞结构单元生物合成的酶经常被相应途径的终产物抑制。在这里，我们提出了一种方法来快速生成套酶超越这种控制。它是基于使用基因编码的传感器在单细胞中体内检测所需的终产物。该传感器将细胞内产物浓度传输到分级光学输出中，从而通过FACS实现超高通量筛选。我们随机诱变谷氨酸棒杆菌质粒编码的ArgB，并在携带传感器pSenLys-Spc的菌株中筛选文库，该传感器可检测L-赖氨酸、L-精氨酸和L-组氨酸。六个得到的N-乙酰基-L-谷氨酸激酶蛋白进一步开发和表征，并发现是至少20倍，对L-精氨酸抑制的敏感性比野生型酶。突变蛋白ArgB-K47 H-V65 A在C.谷氨酸Δ argR导致培养基中积累34 mM L-精氨酸。我们还筛选了lysC编码的天冬氨酸激酶和hisG编码的ATP磷酸核糖基转移酶的突变体库。我们分离了11个LysC突变蛋白，使高达45 mM的L-赖氨酸积累，和13个HisG突变蛋白，使高达17 mM的L-组氨酸积累。这些结果表明，通过使用代谢物传感器在体内筛选酶文库非常适合于鉴定过量生产所需的高性能突变蛋白。
Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects L-lysine, L-arginine and L-histidine. Six of the resulting N-acetyl-L-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward L-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicum Delta argR led to the accumulation of 34 mM L-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM L-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM L-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.