A novel, genetically encoded whole-cell biosensor for directed evolution of myrcene synthase in Escherichia coli
A novel, genetically encoded whole-cell biosensor for directed evolution of myrcene synthase in Escherichia coli
复制标题
DOI：
10.1016/j.bios.2023.115176
复制
发表时间：
2023-03-11
影响因子：
12.6
通讯作者：
Jiang, Hui
中科院分区：
文献类型：
--
作者：
Chen, Chang;Liu, Jiajia;Jiang, Hui
β-月桂烯是一种高价值的无环单萜。月桂烯合成酶活性低，生物合成效价低。生物传感器是一种很有前途的酶导向进化工具。本工作以假单胞菌MyrR调节子为基础，建立了一种新型的对月桂烯反应的基因编码生物传感器。通过检测启动子的特性和工程，开发出具有良好的特异性和动态范围的生物传感器，并将其应用于月桂烯合成酶的定向进化。通过对月桂烯合成酶随机突变文库的高通量筛选，获得了最佳突变体R89G/N152S/D517N。其催化效率是母体的1.47倍。根据突变株，月桂烯的最终产量达到了510.38 mg/L，是迄今为止报道的最高的月桂烯效价。这项工作展示了全细胞生物传感器在提高酶活性和目标代谢物产生方面的巨大潜力。
beta-myrcene is a high-value acyclic monoterpene. The low activity of myrcene synthase resulted to low biosynthetic titer of it. Biosensor is a promising tool applied for enzyme directed evolution. In this work, a novel genetically encoded biosensor responding to myrcene was established based on the MyrR regulator from Pseudomonas sp. Through sensing promoter characterization and engineering, the biosensor exhibiting excellent specificity and dynamic range was developed, and applied for directed evolution of myrcene synthase. After high-throughput screening of the myrcene synthase random mutation library, the best mutant R89G/N152S/ D517N was obtained. Its catalytic efficiency was 1.47-fold than that of parent. Based on the mutants, the final production of myrcene reached 510.38 mg/L, which is the highest myrcene titer reported to date. This work demonstrates the great potential of whole-cell biosensor for improving enzymatic activity and the production of target metabolite.