CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
Metab Eng. 2017 Mar;40:115-123. doi: 10.1016/j.ymben.2017.01.006. Epub 2017 Jan 19.
Overexpressing key enzymes of biosynthetic pathways for overproduction of value-added products usually imposes metabolic burdens on cells, which can be circumvented by improving the key enzyme activities. p-Coumarate: CoA ligase (4CL) is a critical enzyme in the phenylpropanoid pathway that synthesizes various natural products. To screen for 4CL with improved activity, a biosensor of resveratrol whose biosynthetic pathway involves 4CL was designed by engineering the TtgR regulatory protein. The biosensor exhibited good specificity and robustness, allowing rapid and sensitive selection of resveratrol hyper-producers. A 4CL variant with improved activity was selected from a 4CL mutagenesis library constructed in the resveratrol biosynthetic pathway in Escherichia coli. This mutant led to increased production of not only resveratrol but also the flavonoid naringenin, when introduced in their corresponding biosynthetic pathways. These findings demonstrate the feasibility of improving key enzyme activities in important biosynthetic pathways with the aid of designed biosensors of pathway products.
过量表达生物合成途径中的关键酶通常会给细胞带来代谢负担，而通过提高关键酶的活性可以避免这种负担。对香豆酸：辅酶 A 连接酶（4CL）是苯丙烷途径中合成各种天然产物的关键酶。为了筛选活性提高的 4CL，通过工程改造 TtgR 调节蛋白，设计了白藜芦醇生物传感器，其生物合成途径涉及 4CL。该生物传感器具有良好的特异性和稳健性，能够快速灵敏地选择白藜芦醇高产菌。从大肠杆菌中构建的白藜芦醇生物合成途径中的 4CL 突变文库中筛选到一种活性提高的 4CL 变体。当该突变体被引入相应的生物合成途径时，不仅增加了白藜芦醇的产量，还增加了类黄酮柚皮素的产量。这些发现证明了利用设计的产物途径生物传感器来提高重要生物合成途径中关键酶活性的可行性。
