Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering
Generating Functional Recombinant NRPS Enzymes in the Laboratory Setting via Peptidyl Carrier Protein Engineering
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DOI：
10.1016/j.chembiol.2016.09.014
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发表时间：
2016-11-17
影响因子：
8.6
通讯作者：
Ackerley, David F.
中科院分区：
文献类型：
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作者：
Owen, Jeremy G.;Calcott, Mark J.;Ackerley, David F.
非核糖体肽合成酶（NRPS）是模块化酶组装线，其中底物和中间体经历由腺苷酸化（A）、缩合（C）和硫酯酶（TE）结构域催化的多轮转化。NRPS生物合成的中心是肽基载体蛋白（PCP）结构域，这是一个小的无催化活性的结构域，它在催化模块之间穿梭底物和中间体，并控制TE结构域的产物释放。人们对NRPS系统重组以产生新的化学实体有强烈的兴趣。然而，这些系统的内在复杂性一直是一个重大挑战。在这里，我们采用结构域取代和随机诱变重演NRPS的进化，集中在PCP结构域。使用NRPS模型系统，产生两种不同的色素分子，绿脓菌荧光素和靛蓝，我们发现，只有进化专门的重组PCP结构域可以有效地与天然TE结构域的产品释放。总之，我们强调，取代PCP结构域需要非常微小的变化，导致功能NRPS，并推断，积极的选择压力可能会改善重组NRPS的结果。
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.