Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains
Engineering of Chimeric Polyketide Synthases Using SYNZIP Docking Domains
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DOI：
10.1021/acschembio.8b01060
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发表时间：
2019-03-01
影响因子：
4
通讯作者：
Grininger, Martin
中科院分区：
文献类型：
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作者：
Klaus, Maja;D'Souza, Alicia D.;Grininger, Martin
20多年来，设计流水线聚酮合成酶(PKSS)以生产新型生物活性化合物一直是人们追求的目标。PKSS的明显模块化激发了许多工程尝试，在这些尝试中，整个模块或单个域被交换。近年来，很明显，某些结构域的相互作用在进化上得到了优化，如果中断，会导致嵌合PKS的总周转率下降。在本研究中，我们对不同类型的嵌合PKSS进行了比较，以确定最小侵袭界面，并扩展PKS工程工具箱。我们产生了双模嵌合PKS，在其中整个模块被交换，同时在异源模块之间保留共价连接物或引入非共价对接结构域，或SYNZIP结构域，介导的界面。这些嵌合系统在模块间聚酮链易位过程中表现出非自然的结构域-结构域相互作用。将它们与其他等价的双模PKS进行比较，在双模PKS中，在模块的缩合和加工部分之间引入了非共价界面，导致在其催化循环的延伸单元酰化和聚酮链延长步骤中产生非天然结构域相互作用。我们证明了SYNZIP结构域可以有效地替代天然的PKS对接结构域，并且新引入的模块冷凝和加工部分之间的非共价界面可以被用于PKS工程。此外，我们建立了SYNZIP结构域作为一种新的工具，通过在不干扰PKS活动的情况下有效地桥接非本地接口来设计PKS。
Engineering of assembly line polyketide synthases (PKSs) to produce novel bioactive compounds has been a goal for over 20 years. The apparent modularity of PKSs has inspired many engineering attempts in which entire modules or single domains were exchanged. In recent years, it has become evident that certain domain domain interactions are evolutionarily optimized and, if disrupted, cause a decrease of the overall turnover rate of the chimeric PKS. In this study, we compared different types of chimeric PKSs in order to define the least invasive interface and to expand the toolbox for PKS engineering. We generated bimodular chimeric PKSs in which entire modules were exchanged, while either retaining a covalent linker between heterologous modules or introducing a noncovalent docking domain, or SYNZIP domain, mediated interface. These chimeric systems exhibited non-native domain-domain interactions during intermodular polyketide chain translocation. They were compared to otherwise equivalent bimodular PKSs in which a noncovalent interface was introduced between the condensing and processing parts of a module, resulting in non-native domain interactions during the extender unit acylation and polyketide chain elongation steps of their catalytic cycles. We show that the natural PKS docking domains can be efficiently substituted with SYNZIP domains and that the newly introduced noncovalent interface between the condensing and processing parts of a module can be harnessed for PKS engineering. Additionally, we established SYNZIP domains as a new tool for engineering PKSs by efficiently bridging non-native interfaces without perturbing PKS activity.