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

1403077 Arnold，Frances H.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组到生物合成途径中。 不幸的是，许多期望的产物落在相当有限的已知酶催化转化的范围之外，或者可以使用合成化学更有效地制备。 因此，代谢工程可能产生社会所依赖的几乎所有有机分子的未来仍然很遥远。 生物生产的进展将取决于我们对新催化剂进行基因编码的能力。本研究的目标是通过工程化天然酶，细菌细胞色素P450来创造新的环化催化剂。 初步结果表明这是可能的-新发现的环丙烷化反应将使用现代蛋白质工程技术进行优化和扩展。自然界的催化创新是罕见的事件，难以观察。 在实验室里，通过模仿自然的进化方法，也很难发现全新的活动。然而，最近，PI在加州理工学院的实验室使用化学直觉来启动这一过程，并发现细胞色素P450-BM 3的变体在提供适当的合成试剂时是正式卡宾转移的有效催化剂。 这种高度期望的非天然活性可以使用基于P450结构和催化机制的知识的半理性诱变显著增加。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。设计了一个三层战略，以测试有关改进的具体假设，同时也允许渐进式创新。具体而言，（i）将研究血红素氧化还原电位对非天然反应性的影响，预期实现更多的氧化还原电位将增加反应性;（ii）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关键保守残基。(3)将采用真正的进化策略，使用新的高通量环丙烷化筛选来优化非天然活性，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物化学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.