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 Dec 18;4(12):1335-40. doi: 10.1021/acssynbio.5b00075. Epub 2015 Sep 1.
Riboswitches are natural RNA elements that regulate gene expression by binding a ligand. Here, we demonstrate the possibility of altering a natural lysine-OFF riboswitch from Eschericia coli (ECRS) to a synthetic lysine-ON riboswitch and using it for metabolic control. To this end, a lysine-ON riboswitch library was constructed using tetA-based dual genetic selection. After screening the library, the functionality of the selected lysine-ON riboswitches was examined using a report gene, lacZ. Selected lysine-ON riboswitches were introduced into the lysE gene (encoding a lysine transport protein) of Corynebacterium glutamicum and used to achieve dynamic control of lysine transport in a recombinant lysine-producing strain, C. glutamicum LPECRS, which bears a deregulated aspartokinase and a lysine-OFF riboswitch for dynamic control of the enzyme citrate synthase. Batch fermentation results of the strains showed that the C. glutamicum LPECRS strain with an additional lysine-ON riboswitch for the control of lysE achieved a 21% increase in the yield of lysine compared to that of the C. glutamicum LPECRS strain and even a 89% increase in yield compared to that of the strain with deregulated aspartokinase. This work provides a useful approach to generate lysine-ON riboswitches for C. glutamicum metabolic engineering and demonstrates for the first time a synergetic effect of lysine-ON and -OFF riboswitches for improving lysine production in this industrially important microorganism. The approach can be used to dynamically control other genes and can be applied to other microorganisms.
核糖开关是通过结合配体来调节基因表达的天然RNA元件。在此，我们展示了将大肠杆菌天然赖氨酸关闭型核糖开关（ECRS）改造为合成赖氨酸开启型核糖开关并将其用于代谢控制的可能性。为此，利用基于tetA的双重遗传筛选构建了一个赖氨酸开启型核糖开关文库。筛选文库后，使用报告基因lacZ检测所选赖氨酸开启型核糖开关的功能。将所选的赖氨酸开启型核糖开关导入谷氨酸棒杆菌的lysE基因（编码赖氨酸转运蛋白），用于在重组赖氨酸生产菌株谷氨酸棒杆菌LPECRS中实现赖氨酸转运的动态控制，该菌株带有一个解除调控的天冬氨酸激酶和一个用于动态控制柠檬酸合酶的赖氨酸关闭型核糖开关。菌株的分批发酵结果表明，与谷氨酸棒杆菌LPECRS菌株相比，带有额外用于控制lysE的赖氨酸开启型核糖开关的谷氨酸棒杆菌LPECRS菌株赖氨酸产量提高了21%，与天冬氨酸激酶解除调控的菌株相比产量甚至提高了89%。这项工作为谷氨酸棒杆菌代谢工程生成赖氨酸开启型核糖开关提供了一种有用的方法，并首次证明了赖氨酸开启型和关闭型核糖开关在提高这种工业重要微生物赖氨酸产量方面的协同作用。该方法可用于动态控制其他基因，并可应用于其他微生物。
