Zhou Li-Bang, Zeng An-Ping
Institute of Bioprocess and Biosystems Engineering, Hamburg University of Technology Denickestrasse 15, D-21073 Hamburg, Germany.
ACS Synth Biol. 2015 Jun 19;4(6):729-34. doi: 10.1021/sb500332c. Epub 2015 Jan 16.
Riboswitch, a regulatory part of an mRNA molecule that can specifically bind a metabolite and regulate gene expression, is attractive for engineering biological systems, especially for the control of metabolic fluxes in industrial microorganisms. Here, we demonstrate the use of lysine riboswitch and intracellular l-lysine as a signal to control the competing but essential metabolic by-pathways of lysine biosynthesis. To this end, we first examined the natural lysine riboswitches of Eschericia coli (ECRS) and Bacillus subtilis (BSRS) to control the expression of citrate synthase (gltA) and thus the metabolic flux in the tricarboxylic acid (TCA) cycle in E. coli. ECRS and BSRS were then successfully used to control the gltA gene and TCA cycle activity in a lysine producing strain Corynebacterium glutamicum LP917, respectively. Compared with the strain LP917, the growth of both lysine riboswitch-gltA mutants was slower, suggesting a reduced TCA cycle activity. The lysine production was 63% higher in the mutant ECRS-gltA and 38% higher in the mutant BSRS-gltA, indicating a higher metabolic flux into the lysine synthesis pathway. This is the first report on using an amino acid riboswitch for improvement of lysine biosynthesis. The lysine riboswitches can be easily adapted to dynamically control other essential but competing metabolic pathways or even be engineered as an "on-switch" to enhance the metabolic fluxes of desired metabolic pathways.
核糖开关是mRNA分子的一个调控部分，能够特异性结合一种代谢物并调节基因表达，在工程生物系统中具有吸引力，特别是在控制工业微生物的代谢通量方面。在此，我们展示了利用赖氨酸核糖开关和细胞内L-赖氨酸作为信号来控制赖氨酸生物合成中相互竞争但又必不可少的代谢旁路。为此，我们首先研究了大肠杆菌（ECRS）和枯草芽孢杆菌（BSRS）的天然赖氨酸核糖开关，以控制柠檬酸合酶（gltA）的表达，从而控制大肠杆菌三羧酸（TCA）循环中的代谢通量。然后，ECRS和BSRS分别成功用于控制产赖氨酸菌株谷氨酸棒杆菌LP917中的gltA基因和TCA循环活性。与LP917菌株相比，两个赖氨酸核糖开关-gltA突变体的生长都较慢，表明TCA循环活性降低。突变体ECRS-gltA的赖氨酸产量提高了63%，突变体BSRS-gltA的赖氨酸产量提高了38%，这表明进入赖氨酸合成途径的代谢通量更高。这是关于利用氨基酸核糖开关改善赖氨酸生物合成的首次报道。赖氨酸核糖开关可以很容易地用于动态控制其他必不可少但相互竞争的代谢途径，甚至可以设计成一个“开启开关”来增强所需代谢途径的代谢通量。
