Beer Ralf, Herbst Konrad, Ignatiadis Nikolaos, Kats Ilia, Adlung Lorenz, Meyer Hannah, Niopek Dominik, Christiansen Tania, Georgi Fanny, Kurzawa Nils, Meichsner Johanna, Rabe Sophie, Riedel Anja, Sachs Joshua, Schessner Julia, Schmidt Florian, Walch Philipp, Niopek Katharina, Heinemann Tim, Eils Roland, Di Ventura Barbara
Center for Quantitative Biology (Bioquant), Division of Integrative Bioinformatics and Systems Biology, University of Heidelberg, Germany.
Mol Biosyst. 2014 Jul;10(7):1709-18. doi: 10.1039/c3mb70594c.
Non-ribosomal peptide synthetases (NRPSs) are enzymes that catalyze ribosome-independent production of small peptides, most of which are bioactive. NRPSs act as peptide assembly lines where individual, often interconnected modules each incorporate a specific amino acid into the nascent chain. The modules themselves consist of several domains that function in the activation, modification and condensation of the substrate. NRPSs are evidently modular, yet experimental proof of the ability to engineer desired permutations of domains and modules is still sought. Here, we use a synthetic-biology approach to create a small library of engineered NRPSs, in which the domain responsible for carrying the activated amino acid (T domain) is exchanged with natural or synthetic T domains. As a model system, we employ the single-module NRPS IndC from Photorhabdus luminescens that produces the blue pigment indigoidine. As chassis we use Escherichia coli. We demonstrate that heterologous T domain exchange is possible, even for T domains derived from different organisms. Interestingly, substitution of the native T domain with a synthetic one enhanced indigoidine production. Moreover, we show that selection of appropriate inter-domain linker regions is critical for functionality. Taken together, our results extend the engineering avenues for NRPSs, as they point out the possibility of combining domain sequences coming from different pathways, organisms or from conservation criteria. Moreover, our data suggest that NRPSs can be rationally engineered to control the level of production of the corresponding peptides. This could have important implications for industrial and medical applications.
非核糖体肽合成酶（NRPSs）是一类催化不依赖核糖体合成小肽的酶，其中大多数小肽具有生物活性。NRPSs就像肽装配线，各个通常相互连接的模块分别将特定氨基酸掺入新生肽链中。这些模块本身由几个负责底物激活、修饰和缩合的结构域组成。NRPSs显然是模块化的，但仍在寻求对结构域和模块进行所需排列工程改造能力的实验证据。在这里，我们使用合成生物学方法创建了一个工程化NRPSs的小型文库，其中负责携带活化氨基酸的结构域（T结构域）与天然或合成的T结构域进行了交换。作为模型系统，我们采用了来自发光杆菌的单模块NRPS IndC，它能产生蓝色色素靛蓝。我们以大肠杆菌作为底盘。我们证明了异源T结构域交换是可行的，即使是来自不同生物体的T结构域。有趣的是，用合成T结构域替换天然T结构域可提高靛蓝的产量。此外，我们表明选择合适的结构域间连接区域对功能至关重要。综上所述我们的结果扩展了NRPSs的工程改造途径，因为它们指出了组合来自不同途径、生物体或保守标准的结构域序列的可能性。此外，我们的数据表明，可以对NRPSs进行合理设计以控制相应肽的产量水平。这可能对工业和医学应用具有重要意义。
