SusChEM: Engineering and Evolution of Cytochrome P450 Enzymes for Non-Natural Chemistry

1403077阿诺德，弗朗西丝h .一个可持续和环境友好的未来化学工业将越来越多地利用生物过程将可再生资源和化石资源转化为有用的化学品。迄今为止，利用工程微生物生产燃料和化学品依赖于将现有的酶重新组装成生物合成途径。不幸的是，许多期望的产物超出了已知的相当有限的酶催化转化的范围，或者可以使用合成化学更有效地制造。因此，代谢工程可能产生几乎所有社会所依赖的有机分子的未来仍然很遥远。生物生产的进步将取决于我们基因编码新催化剂的能力。本研究的目的是通过对一种天然酶细菌细胞色素P450进行工程改造，创造新的环萘化催化剂。初步结果表明这是可能的——新发现的环丙烷化反应将利用现代蛋白质工程技术进行优化和扩展。自然界的催化性创新是罕见的事件，很难观察到。在实验室中，通过模拟自然的进化方法来发现全新的活动也是很困难的。然而最近，PI在加州理工学院的实验室利用化学直觉启动了这一过程，并发现细胞色素P450-BM3的变体在提供适当的合成试剂时是形式碳转移的有效催化剂。基于对P450结构和催化机制的了解，利用半理性诱变技术可以显著提高这种非天然活性。需要更多的研究途径来继续改进这些催化剂并发现新的催化剂。设计了一个三层策略来测试关于改进的特定假设，同时也允许进化创新。具体来说，(i)血红素氧化还原电位对非自然反应性的影响将被研究，期望达到更高的氧化还原电位将增加反应性；（ii）关键保守残基将通过确定突变对动力学参数和总体周转水平的影响来检查。(3)采用一种真正的进化策略，利用新型高通量环丙烷筛选来优化非自然活性，并鉴定和表征有益的突变。这项研究将探索和解释这种重要的新酶功能的进化潜力。该奖项由CBET部门的生物技术和生化工程项目以及分子和细胞生物学部门的系统和合成生物学项目共同资助。

1403077Arnold, Frances H.A future chemicals industry that is sustainable and environmentally friendly will make increasing use of biological processes to convert both renewable and fossil resources to useful chemicals. To date, the use of engineered microbes to produce fuels and chemicals has depended on reassembling existing enzymes into biosynthetic pathways. Unfortunately, many desired products fall outside the reach of the rather limited set of known enzyme-catalyzed transformations or can be made more efficiently using synthetic chemistry. Thus, a future where metabolic engineering might produce nearly all of the organic molecules upon which society depends is still a ways off. Progress in biological production will depend on our ability to genetically encode new catalysts. The goal of this research is to create new cyclopranation catalysts by engineering of a natural enzyme, a bacterial cytochrome P450. Preliminary results indicate that this is possible - the newly-discovered cyclopropanation reaction will be optimized and expanded using modern protein engineering techniques.Catalytic innovations in nature are rare events and difficult to observe. It is also difficult to discover entirely new activities in the laboratory by evolutionary methods designed to mimic nature. Recently, however, the PI's lab at Caltech used chemical intuition to jumpstart the process and discover that variants of cytochrome P450-BM3 are efficient catalysts of formal carbene transfers when provided with appropriate synthetic reagents. This highly desirable non-natural activity could be increased significantly using semi-rational mutagenesis based on knowledge of the P450 structure and catalytic mechanism. Additional avenues of investigation are needed to continue improving these catalysts and to discover new ones. A three-tiered strategy has been designed to test specific hypotheses regarding improvements while also allowing for evolutionary innovation. Specifically, (i) the effect of heme redox potential on non-natural reactivity will be investigated, with the expectation that achieving more oxidizing redox potentials will increase reactivity; (ii) key conserved residues will be examined by determining the effect of mutations on kinetic parameters and overall turnover levels. (3) a true evolutionary strategy will be employed to optimize non-natural activity using a novel high-throughput cyclopropanation screen and identify and characterize beneficial mutations. This research will both explore and explain the evolutionary potential of this important new enzyme function.This award is co-funded by the Biotechnology and Biochemical Engineering Program of the CBET Division and by the Systems and Synthetic Biology Program of the Division of Molecular and Cellular Biology.