PDF(Pubmed)
Abstract：
The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of \"un-natural\" natural products.
摘要：
微生物生物合成途径的战略重新设计是以潜在的环境可持续方式获得不同结构和功能的分子的引人注目的途径。这种方法的前景取决于对酰基载体蛋白（ACP）的更好理解，作为生物合成途径的中心枢纽。这些小,柔性蛋白质介导分子构建块和中间体向酶促伙伴的运输，这些酶促伙伴延伸和定制生长的天然产物。过去的组合生物合成努力由于不相容的ACP-酶配对而失败。在这里，我们报告了具有放线菌素聚酮合酶ACP（ACT）和大肠杆菌脂肪酸合酶（FAS）ACP（AcpP）特征的嵌合ACP的设计。我们评估了嵌合ACP与大肠杆菌FAS酮合酶FabF相互作用的能力，这代表了构建合酶分子输出的碳骨架所必需的相互作用。鉴于AcpP与FabF交互，但ACT不交互，我们试图与AcpP交换ACT的模块化功能，以赋予FabF功能。使用沉降速度实验对嵌合ACP与FabF的相互作用进行了研究，表面等离子体共振分析，基于机制的交联分析，和分子动力学模拟。结果表明，指导AcpP-FabF相容性和ACT-FabF不相容性的残基可能位于环I中，α-螺旋II区。这些发现可以为战略次级元素交换的发展提供信息，这些战略次级元素交换扩展了跨系统的ACP的酶相容性，因此代表了迈向“非天然”天然产品战略工程的关键一步。
