RUI: Development of New Green-Chemical Catalysts Based on Rieske Dioxygenase Scaffolds Through Directed Evolution

酶可以减少加工过程对环境的影响。土壤细菌中发现的一类酶使它们能够在石油污染的环境中茁壮成长。以前未表征的双加氧酶将是该项目的重点。进化这些酶以扩大它们所作用的分子的宽度是一个关键目标，它们将被筛选出降解污染物和合成有价值的化学中间体的能力。该项目还将为本科生和硕士生提供现代教育和培训经验。Rieske双加氧酶经常用于有机合成和生物修复。 应用受限于有限的底物范围和严格的产品选择性。定向分子进化将产生新的Rieske双加氧酶，其表现出：1）对芳族化合物的扩大的反应性和2）在烷基烯烃的二羟基化中扩大的底物范围和增加的反应性。酶工程将遵循“设计-测试-构建-学习”循环，其中将使用合理工程和随机诱变策略产生大的Rieske双加氧酶变体文库，并将使用“底物多路复用”方法筛选增强的活性。这项研究的潜在贡献包括：A）新的、可持续的催化剂，在有机合成和生物修复中具有有价值的应用; B）第一个可靠的、可替代化学方法的酶促方法，用于进行烷基烯烃的不对称二羟基化;和C）该奖项反映了NSF的法定使命，并通过评估被认为值得支持使用基金会的知识价值和更广泛的影响审查标准。

Enzymes could reduce the environmental impact of processes. A class of enzymes found in soil bacteria enables them to thrive in petroleum-contaminated environments. Previously uncharacterized dioxygenases will be the focus of the project. Evolving these enzymes to expand the breadth of molecules on which they operate is a key objective, and they will be screened for the ability to degrade pollutants and synthesize valuable chemical intermediates. This project will also provide contemporary education and training experiences for undergraduate and master’s level students.Rieske dioxygenases have frequently been utilized in organic synthesis and bioremediation. Applications are restricted by limited substrate ranges and strict product selectivity. Directed molecular evolution will generate new Rieske dioxygenases exhibiting: 1) expanded reactivity for aromatic compounds and 2) expanded substrate range and increased reactivity in the dihydroxylation of alkyl olefins. Enzyme engineering will follow a “design-test-build-learn” cycle, wherein large Rieske dioxygenase variant libraries will be produced using both rational engineering and random mutagenesis strategies and will be screened for enhanced activity using a “substrate multiplexing” approach. Potential contributions of this research include A) new, sustainable catalysts with valuable applications in organic synthesis and bioremediation; B) the first reliable, enzymatic alternative to chemical methods for performing the asymmetric dihydroxylation of alkyl olefins; and C) development of a blueprint for the successful engineering of Rieske dioxygenases.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.