Recognition sequences and substrate evolution in cyanobactin biosynthesis.
Recognition sequences and substrate evolution in cyanobactin biosynthesis.
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DOI：
10.1021/sb500019b
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发表时间：
2015-02-20
影响因子：
4.7
通讯作者：
Schmidt, Eric W.
中科院分区：
文献类型：
--
作者：
Sardar, Debosmita;Pierce, Elizabeth;McIntosh, John A.;Schmidt, Eric W.
关键词：
核糖体合成和后修饰肽（RiPP）天然产物由于其内在的生物活性和合成生物学的潜力而受到广泛关注。RiPP氰氧化菌素途径pat和tru已被实验证明对突变具有极强的耐受性。在自然界中，这些途径表现出“底物进化”，其中酶保持恒定，而这些酶的底物是高度可变的并且容易进化。在这里，我们试图确定这种滥交背后的机制。分析了一系列不同的酶-底物组合从五个不同的氰氧化菌素基因簇，除了工程基板，使我们能够定义短离散识别元件基板内负责指导酶。我们发现，这些识别序列（RS）是便携式的，可以互换，以控制哪些官能团被添加到最终的天然产物。除了先前指定的N-和C-末端蛋白水解RS之外，这里我们指定RS用于杂环化修饰。我们表明，底物元素可以在体内交换，从而成功地生产天然产物在E。杆菌这些元件的可交换性在合成生物学方法中有望在体内和体外定制肽产品。
Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit “substrate evolution”, where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme–substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.
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