Nature Chemical Biology ( IF 14.8 ) Pub Date : 2022-07-07 , DOI: 10.1038/s41589-022-01072-w Simon d'Oelsnitz 1 , Wantae Kim 2 , Nathaniel T Burkholder 1 , Kamyab Javanmardi 1 , Ross Thyer 3 , Yan Zhang 1 , Hal S Alper 2 , Andrew D Ellington 1
A key bottleneck in the microbial production of therapeutic plant metabolites is identifying enzymes that can improve yield. The facile identification of genetically encoded biosensors can overcome this limitation and become part of a general method for engineering scaled production. We have developed a combined screening and selection approach that quickly refines the affinities and specificities of generalist transcription factors; using RamR as a starting point, we evolve highly specific (>100-fold preference) and sensitive (half-maximum effective concentration (EC50) < 30 μM) biosensors for the alkaloids tetrahydropapaverine, papaverine, glaucine, rotundine and noscapine. High-resolution structures reveal multiple evolutionary avenues for the malleable effector-binding site and the creation of new pockets for different chemical moieties. These sensors further enabled the evolution of a streamlined pathway for tetrahydropapaverine, a precursor to four modern pharmaceuticals, collapsing multiple methylation steps into a single evolved enzyme. Our methods for evolving biosensors enable the rapid engineering of pathways for therapeutic alkaloids.
中文翻译:
使用可替代生物传感器来改进生物碱生物合成
微生物生产治疗性植物代谢物的一个关键瓶颈是识别可以提高产量的酶。基因编码生物传感器的简便识别可以克服这一限制,并成为工程规模生产的通用方法的一部分。我们开发了一种组合筛选和选择方法,可以快速完善通用转录因子的亲和力和特异性;使用 RamR 作为起点,我们进化出高度特异性(>100 倍偏好)和敏感(半最大有效浓度(EC 50) < 30 μM) 用于生物碱四氢罂粟碱、罂粟碱、海亮碱、罗通定和诺斯卡品的生物传感器。高分辨率结构揭示了可延展效应子结合位点的多种进化途径以及为不同化学部分创建新的口袋。这些传感器进一步促进了四氢罂粟碱(四种现代药物的前体)的简化途径的进化,将多个甲基化步骤分解为单个进化酶。我们不断发展的生物传感器方法能够快速设计治疗性生物碱的途径。