Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.
Identification of a Thioesterase Bottleneck in the Pikromycin Pathway through Full-Module Processing of Unnatural Pentaketides.
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DOI：
10.1021/jacs.7b06432
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发表时间：
2017-09-27
影响因子：
15
通讯作者：
Sherman DH
中科院分区：
文献类型：
--
作者：
Hansen DA;Koch AA;Sherman DH
二十多年来，聚酮生物合成途径一直被设计成产生天然产物类似物。然而，操纵模块化的I型聚酮合成酶(PKS)来产生非天然代谢物通常会导致产量下降或完全失去活性途径，而由于限速或无活性区域(S)的存在，导致产量下降的机制很少被阐明。因此，我们合成并检测了一系列模拟早期通路工程的修饰吡克罗霉素(Pikromycin，Pik)五肽，以探讨PikAIII-TE模块的体外底物耐受性。截短的五酮类化合物的处理效率与相应的大内酯不同，而具有异构化手性中心的五酮类化合物对PikAIII-TE的处理效果较差，无法生成12元环产品。对延长的但过早卸载的分流产物的分离和鉴定表明，Pik硫代酯酶(TE)结构域具有有限的底物灵活性，并在非天然底物的加工中起到把关的作用。合成具有异构化亲核羟基的类似六酮，可以直接评估被切除的TE结构域的底物立体选择性。异构化的六酮未发生环化反应，被排他性地水解，证实TE结构域是关键的催化瓶颈。在随附的一篇论文中，我们改造了独立的Pik硫代酯酶(TE)，以产生硫代酯酶(TES148C)和模块(PikAIII-TES148C)，该模块显示了具有倒置羟基的底物的功能增益处理。
Polyketide biosynthetic pathways have been engineered to generate natural product analogs for over two decades. However, manipulation of modular type I polyketide synthases (PKSs) to make unnatural metabolites commonly results in attenuated yields or entirely inactive pathways, and the mechanistic basis for compromised production is rarely elucidated since rate limiting or inactive domain(s) remain unidentified. Accordingly, we synthesized and assayed a series of modified pikromycin (Pik) pentaketides that mimic early pathway engineering to probe the substrate tolerance of the PikAIII-TE module in vitro. Truncated pentaketides were processed with varying efficiencies to corresponding macrolactones, while pentaketides with epimerized chiral centers were poorly processed by PikAIII-TE and failed to generate 12-membered ring products. Isolation and identification of extended but prematurely offloaded shunt products suggested that the Pik thioesterase (TE) domain has limited substrate flexibility and functions as a gatekeeper in the processing of unnatural substrates. Synthesis of an analogous hexaketide with an epimerized nucleophilic hydroxyl group allowed for direct evaluation of the substrate stereoselectivity of the excised TE domain. The epimerized hexaketide failed to undergo cyclization and was exclusively hydrolyzed, confirming the TE domain as a key catalytic bottle-neck. In an accompanying paper, we engineer the standalone Pik thioesterase (TE) to yield a thioesterase (TES148C) and module (PikAIII-TES148C) that display gain-of-function processing of substrates with inverted hydroxyl groups.
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