Department of Chemistry, North Carolina State University, Raleigh, North Carolina, United States.
ACS Chem Biol. 2013 Jan 18;8(1):200-8. doi: 10.1021/cb3003489. Epub 2012 Oct 29.
Polyketide synthases construct polyketides with diverse structures and biological activities via the condensation of extender units and acyl thioesters. Although a growing body of evidence suggests that polyketide synthases might be tolerant to non-natural extender units, in vitro and in vivo studies aimed at probing and utilizing polyketide synthase specificity are severely limited to only a small number of extender units, owing to the lack of synthetic routes to a broad variety of acyl-CoA extender units. Here, we report the construction of promiscuous malonyl-CoA synthetase variants that can be used to synthesize a broad range of malonyl-CoA extender units substituted at the C2-position, several of which contain handles for chemoselective ligation and are not found in natural biosynthetic systems. We highlighted utility of these enzymes by probing the acyl-CoA specificity of several trans-acyltransferases, leading to the unprecedented discovery of poly specificity toward non-natural extender units, several of which are not found in naturally occurring biosynthetic pathways. These results reveal that polyketide biosynthetic machinery might be more tolerant to non-natural substrates than previously established, and that mutant synthetases are valuable tools for probing the specificity of biosynthetic machinery. Our data suggest new synthetic biology strategies for harnessing this promiscuity and enabling the regioselective modification of polyketides.
聚酮合酶通过 extender 单元和酰基辅酶 A 的缩合作用构建具有多种结构和生物活性的聚酮。尽管越来越多的证据表明聚酮合酶可能对非天然 extender 单元具有耐受性，但由于缺乏合成各种酰基辅酶 A extender 单元的途径，旨在探测和利用聚酮合酶特异性的体外和体内研究严重局限于少数 extender 单元。在这里，我们报告了构建混杂的丙二酰辅酶 A 合成酶变体，可用于合成广泛的 C2 位取代的丙二酰辅酶 A extender 单元，其中一些含有用于化学选择性连接的接头，并且在天然生物合成系统中不存在。我们通过探测几种反酰基转移酶的酰基辅酶 A 特异性来突出这些酶的实用性，从而发现了对非天然 extender 单元的前所未有的多特异性，其中一些在天然生物合成途径中不存在。这些结果表明，聚酮生物合成机制可能比以前建立的机制对非天然底物更具耐受性，并且突变合成酶是探测生物合成机制特异性的有价值的工具。我们的数据表明了一种新的合成生物学策略，可以利用这种混杂性并实现聚酮的区域选择性修饰。
