Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, USA.
McKetta Department of Chemical Engineering, University of Texas at Austin, Austin, TX, USA.
Nat Chem Biol. 2022 Sep;18(9):981-989. doi: 10.1038/s41589-022-01072-w. Epub 2022 Jul 7.
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 (EC) < 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.
在利用微生物生产治疗性植物代谢物的过程中，一个关键的瓶颈是确定能够提高产量的酶。易于识别的遗传编码生物传感器可以克服这一限制，并成为工程规模化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，可以快速改进通用转录因子的亲和力和特异性；以 RamR 为起点，我们进化出了高度特异性（>100 倍偏好）和灵敏（半最大有效浓度 (EC) < 30 μM）的生物传感器，用于检测生物碱四氢罂粟碱、罂粟碱、白屈菜碱、阿朴啡碱和北美黄连碱。高分辨率结构揭示了效应物结合位点的多种进化途径，并为不同化学部分创造了新的口袋。这些传感器进一步使四氢罂粟碱的简化途径得以进化，四氢罂粟碱是四种现代药物的前体，将多个甲基化步骤简化为单个进化酶。我们用于进化生物传感器的方法可以快速设计治疗性生物碱的途径。
