Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance.
Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme-Substrate Tolerance.
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评价Lasso肽形成和酶-底物耐受性的无细胞生物合成。
DOI：
10.1021/jacs.1c01452
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发表时间：
2021-04-21
影响因子：
15
通讯作者：
Mitchell DA
中科院分区：
文献类型：
--
作者：
Si Y;Kretsch AM;Daigh LM;Burk MJ;Mitchell DA
Lasso 肽是核糖体合成和翻译后修饰肽 (RiPP) 天然产物，具有独特的套索状螺纹构象。由于具有锁定的三维结构，套索肽对于热和蛋白水解降解具有异常稳定的稳定性。一些套索肽已被证明可以结合人类细胞表面受体并表现出抗癌特性，而其他套索肽则表现出抗菌或抗病毒活性。所有已知的套索肽都是由细菌产生的，基因组挖掘研究表明套索肽是一类相对普遍的 RiPP；然而，套索肽的发现、分离和表征由于缺乏有效的生产系统而受到限制。在这项研究中，我们采用无细胞生物合成（CFB）策略来解决与套索肽生产相关的长期挑战。我们报告了成功地使用 CFB 形成了一系列序列多样化的套索肽，其中包括已知的例子以及来自 Thermobifida halotolerans 的新预测套索肽。我们进一步证明了 CFB 在快速生成和表征多位点前体肽变体以评估生物合成途径的底物耐受性方面的实用性。通过评估 1,000 多个随机选择的变体，我们表明来自 Fusilassin 途径的套索形成环化酶能够通过 CFB 产生数百万个序列多样化的套索肽。这些数据为使用 CFB 创建大型套索肽库来识别具有独特特性的新变体奠定了坚实的基础。
Lasso peptides are ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that display a unique lariat-like, threaded conformation. Owing a locked three-dimensional structure, lasso peptides can be unusually stable towards heat and proteolytic degradation. Some lasso peptides have been shown to bind human cell-surface receptors and exhibit anticancer properties, while others display antibacterial or antiviral activities. All known lasso peptides are produced by bacteria and genome-mining studies indicate that lasso peptides are a relatively prevalent class of RiPPs; however, the discovery, isolation, and characterization of lasso peptides are constrained by the lack of an efficient production system. In this study, we employ a cell-free biosynthesis (CFB) strategy to address longstanding challenges associated with lasso peptide production. We report the successful use of CFB for the formation of an array of sequence-diverse lasso peptides that include known examples as well as a new predicted lasso peptide from Thermobifida halotolerans. We further demonstrate the utility of CFB to rapidly generate and characterize multisite precursor peptide variants to evaluate the substrate tolerance of the biosynthetic pathway. By evaluating more than 1,000 randomly chosen variants, we show that the lasso-forming cyclase from the fusilassin pathway is capable of producing millions of sequence-diverse lasso peptides via CFB. These data lay a firm foundation for the creation of large lasso peptide libraries using CFB to identify new variants with unique properties.
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影响因子：
--
作者：
Gavrish E;Sit CS;Cao S;Kandror O;Spoering A;Peoples A;Ling L;Fetterman A;Hughes D;Bissell A;Torrey H;Akopian T;Mueller A;Epstein S;Goldberg A;Clardy J;Lewis K
通讯作者：
Lewis K
影响因子：
18.2
作者：
Burkhart BJ;Kakkar N;Hudson GA;van der Donk WA;Mitchell DA
通讯作者：
Mitchell DA
影响因子：
4.3
作者：
Fouque, Kevin Jeanne Dit;Bisram, Vikash;Fernandez-Lima, Francisco
通讯作者：
Fernandez-Lima, Francisco
影响因子：
--
作者：
Knappe, Thomas A.;Linne, Uwe;Marahiel, Mohamed A.
通讯作者：
Marahiel, Mohamed A.
影响因子：
3.2
作者：
Al Toma, Rashed S.;Kuthning, Anja;Suessmuth, Roderich D.
通讯作者：
Suessmuth, Roderich D.