Computational design and laboratory evolution of de novo oxidoreductase enzymes

点击翻译按钮获取中文摘要

At the core of the emerging field of Synthetic Biology lies a fundamental goal to construct new functional and bio-compatible parts and devices for incorporation into explicitly biological organisms or systems. To this end, we have previously demonstrated how manmade proteins can be processed through natural biochemical pathways in vivo to produce fully functional manmade c-type cytochromes without the need for further in vitro assembly. These evolutionarily naive proteins, maquettes, have proven capable of incorporating many engineering elements common to natural oxidoreductase proteins (e.g. reversible O2 binding, electron transport) within a robust but simple protein scaffold lacking the complexity of naturally evolved proteins. The prototype c-type cytochrome maquette (CTM) is capable not only of binding oxygen, but also of assembling nascent electron transfer chains, photoactive light harvesting dyads and engaging in interprotein electron transfer with natural cytochromes. Further engineering of this prototype has resulted in the assembly of a CTM enzyme capable of catalyzing chemistries common to natural heme-containing peroxidase enzymes with catalytic efficiencies equaling or surpassing those of the current state-of-the-art de novo enzymes and certain naturally evolved enzymes.With this proposal, we aim to capitalize on our recent successes with the CTM enzyme chassis and use a powerful combination of computational (i.e. using MD & QM/MM methodologies and the Rosetta design package) and experimental approaches to evolve and improve catalytic activity within these versatile protozymes. During the first rotation in the Anderson lab, the student will use the existing CTM chassis to create diverse CTM libraries that, while adhering to the basic principles of 4-helix bundle assembly and covalent heme incorporation, will facilitate high throughput screening and directed evolution strategies to incorporate and optimize catalytic activity. We will extensively use the BrisSynBio robotics suite to generate CTM libraries and for the high-throughput expression, purification and screening of variants in 96-well plates. The subsequent rotation in the Mulholland lab will focus on the computational analysis of the CTM catalytic pathway, identifying hotspots for creating substrate-binding sites, and indicating amino acid substitutions that will potentially enhance or alter reactivity. Following these two rotations, the student will return to the Anderson lab to continue the directed evolution of functional CTM enzymes while also continuing the computational work to aid redesign and analysis. This synthesis of computational and experimental techniques is an exceptional opportunity for postgraduate training and will be vital to the progression and success of the project.