School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand; Centre for Biodiscovery, Victoria University of Wellington, Wellington 6140, New Zealand.
School of Biological Sciences, Victoria University of Wellington, Wellington 6140, New Zealand.
Cell Chem Biol. 2016 Nov 17;23(11):1395-1406. doi: 10.1016/j.chembiol.2016.09.014. Epub 2016 Oct 27.
Non-ribosomal peptide synthetases (NRPSs) are modular enzymatic assembly lines where substrates and intermediates undergo rounds of transformation catalyzed by adenylation (A), condensation (C), and thioesterase (TE) domains. Central to the NRPS biosynthesis are peptidyl carrier protein (PCP) domains, small, catalytically inactive domains that shuttle substrates and intermediates between the catalytic modules and govern product release from TE domains. There is strong interest in recombination of NRPS systems to generate new chemical entities. However, the intrinsic complexity of these systems has been a major challenge. Here, we employ domain substitution and random mutagenesis to recapitulate NRPS evolution, focusing on PCP domains. Using NRPS model systems that produce two different pigmented molecules, pyoverdine and indigoidine, we found that only evolutionarily specialized recombinant PCP domains could interact effectively with the native TE domain for product release. Overall, we highlight that substituted PCP domains require very minor changes to result in functional NRPSs, and infer that positive selection pressure may improve recombinant NRPS outcomes.
非核糖体肽合成酶（NRPSs）是模块化的酶组装线，其中底物和中间产物经历由腺苷酸化（A）、缩合（C）和硫酯酶（TE）结构域催化的循环转化。NRPS 生物合成的核心是肽酰载体蛋白（PCP）结构域，这是一种小型、无催化活性的结构域，可在催化模块之间穿梭底物和中间产物，并控制 TE 结构域产物的释放。人们对 NRPS 系统的重组产生新的化学实体非常感兴趣。然而，这些系统的内在复杂性一直是一个主要挑战。在这里，我们采用结构域取代和随机诱变来重现 NRPS 的进化，重点关注 PCP 结构域。我们使用产生两种不同色素分子（绿脓菌素和靛蓝素）的 NRPS 模型系统发现，只有进化上专门化的重组 PCP 结构域才能有效地与天然 TE 结构域相互作用以释放产物。总的来说，我们强调取代的 PCP 结构域只需要很小的变化就可以产生功能性的 NRPS，并推断正选择压力可能会改善重组 NRPS 的结果。
