La Fleur Lindsay C, Zhang Zhongtian, McRoberts-Amador Christian, Christopher Jayani, Reed Megan, Zheng Jianting, Cropp T Ashton, J Williams Gavin
Department of Chemistry, NC State University, Raleigh, North Carolina 27695-8204, United States.
Department of Cell and Molecular Biology, Duke University, Durham, North Carolina 27708, United States.
Biochemistry. 2025 Apr 1;64(7):1560-1571. doi: 10.1021/acs.biochem.4c00795. Epub 2025 Mar 18.
Glycosylated macrolactones (macrolides) often display broad and potent biological activities and are targets for drug development and discovery. The modular genetic organization of macrolide polyketide synthases (PKSs) and various polyketide tailoring enzymes has inspired the combinatorial biosynthesis of new-to-nature macrolides. However, most engineered PKS and macrolide biosynthetic pathways are ineffective and produce reduced or negligible product titers. Directed evolution could improve the activity of engineered PKSs and associated pathways but critically requires a high-throughput screen to identify active variants from large libraries. Transcription factor-based biosensors can be used for this purpose. However, the effector specificity of the only known macrolide-sensing transcription factor MphR is limited to macrolides modified with the sugar, desosamine. The potential applications of MphR are subsequently limited, ruling out the possibility of leveraging MphR to screen libraries of pathway variants that make macrolactones that lack sugars (i.e., macrolide aglycones) such as the direct products of PKSs. In this study, we aimed to engineer the effector specificity of the MphR biosensor strain for detecting macrolide aglycones. By developing an "effector walking" strategy coupled with efflux pump deletion, the effector profile of MphR was dramatically broadened to include several erythronolide macrolactones. This work sets the stage for applying directed evolution and other high-throughput screening approaches to various PKSs. Our results suggest a broadly applicable approach to developing biosensors that detect ligands that are very different in structure from the native effector.
糖基化大环内酯类（大环内酯）通常具有广泛而强大的生物活性，是药物开发和发现的目标。大环内酯聚酮合酶（PKSs）和各种聚酮修饰酶的模块化遗传组织激发了新型大环内酯的组合生物合成。然而，大多数工程化的PKS和大环内酯生物合成途径效率低下，产物滴度降低或可忽略不计。定向进化可以提高工程化PKSs和相关途径的活性，但关键需要高通量筛选来从大型文库中鉴定活性变体。基于转录因子的生物传感器可用于此目的。然而，唯一已知的大环内酯感应转录因子MphR的效应物特异性仅限于用去氧氨基糖修饰的大环内酯。MphR的潜在应用随后受到限制，排除了利用MphR筛选产生无糖大环内酯（即大环内酯苷元）的途径变体文库的可能性，例如PKSs的直接产物。在本研究中，我们旨在改造MphR生物传感器菌株的效应物特异性以检测大环内酯苷元。通过开发一种“效应物步移”策略并结合外排泵缺失，MphR的效应物谱显著拓宽，包括几种红霉内酯大环内酯。这项工作为将定向进化和其他高通量筛选方法应用于各种PKSs奠定了基础。我们的结果表明了一种广泛适用的方法来开发检测与天然效应物结构非常不同的配体的生物传感器。
