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Expanding the Scope of Metalloprotein Families and Substrate Classes in New-To- Nature Reactions

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Expanding the Scope of Metalloprotein Families and Substrate Classes in New-To- Nature Reactions EXPANDING THE SCOPE OF METALLOPROTEIN FAMILIES AND SUBSTRATE CLASSES IN NEW-TO- NATURE REACTIONS Thesis by Anders Matthew Knight In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2020 (Defended June 9, 2020) ii © 2020 Anders Matthew Knight ORCID: 0000-0001-9665-8197 iii ACKNOWLEDGEMENTS I would first like to thank my advisor, Prof. Frances Arnold, for her help and guidance during my thesis work. When I spoke with Frances at my Caltech interview, she told me that if I wanted to learn how to engineer proteins, the Arnold lab was the best place in the world to do it. As usual, Frances was correct. Thank you for challenging me to come up with interesting, novel, and useful research questions, and for giving me the freedom to apply myself in answering those questions. I am thankful to my committee members, Prof. Sarah Reisman, Prof. William Clemons, Prof. Mikhail Shapiro, and Prof. Justin Bois for their insights and guidance on my PhD research. I would also like to thank Justin for his pivotal role in my graduate education, having taught most of the courses I took at Caltech and teaching me most of the programming I know. His skill and enthusiasm for educating junior scientists is immediately apparent. The Arnold laboratory is full of incredibly talented scientists. Dr. Sabine Brinkmann-Chen has been there from the beginning for research, writing, and life advice. I am indebted to Dr. Andrew Buller and Dr. David Romney for mentoring me during my rotation and beyond. Dr. Rusty Lewis, Dr. Stephan Hammer, Dr. Oliver Brandenberg, Dr. Tina Boville, Dr. Nicholas Porter, and Dr. Xiongyi Huang all provided excellent feedback on my new project ideas and helped me decipher data. I would like to thank Dr. Jennifer Kan in particular for her constant mentorship, advice, and support throughout my PhD – my time at Caltech would have been very different without your guidance. I have learned so much from collaborations and discussions with Lucas Schaus, Ruijie Zhang, Patrick Almhjell, Ella Watkins, Bruce Wittmann, Nicholas Porter, and Nat Goldberg. Thank you all for making me a better scientist, and, together with Austin Dulaney and Brad Boville, for making my time outside of work enjoyable. I am richer for having known you, friends. I would not have gotten to Caltech in the first place if it were not for the many role models and advisors on my way. My high-school chemistry teacher, Michael Smits, inspired me to study chemistry and chemical engineering in college. My undergraduate research advisor, Prof. Silvia Cavagnero, took a chance in hiring an enthusiastic but inexperienced freshman with no background on proteins to do research in her group. At the time I thought I would be a “traditional” chemical engineer, but I was quickly hooked on researching proteins. The foundation of both technical skills iv and the research process that I learned in your laboratory have been invaluable. Prof. Randall Goldsmith, on top of being a fantastic teacher, helped me navigate the process of deciding to pursue a PhD and which program to choose. Prof. Uwe Bornscheuer guided me through my first forays into protein engineering and helped lay the groundwork for me to hit the ground running when I started at Caltech. Mental health is a common topic when speaking about graduate school, and I am not alone in my struggles over the last five years. I am grateful to Dr. Lee Coleman for helping me to persevere, and to be kind to myself along the way. I am thankful to have an incredibly supportive family. I know that my parents, Matthew and Carolyn Knight, and sister, Freya Ludeman, are always there for me. I look forward to celebrating in person when we can. Lastly, thank you to my partner, Silken Jones, for being with me through the ups and downs of grad school over the last four years. I cannot wait to start the next chapter of our adventure together. v ABSTRACT Heme proteins, in particular cytochromes P450, have been extensively used in biocatalytic applications due to their high degree of regio-, chemo-, and stereoselectivity in oxene-transfer reactions. In 2013, it was shown for the first time that engineered heme proteins can also catalyze analogous carbene- and nitrene-transfer reactions. Research in this field has since grown dramatically, with emphasis on developing new heme protein variants to increase the scope of biotransformations accessible through these new transfer reactions. This thesis details the expansion of these new-to-nature carbene and nitrene-transfer reactions to include new substrate classes previously unexplored with iron-porphyrin proteins, the use of non-heme metalloproteins for these transformations, and steps toward improving the robustness of the new-to-nature biocatalytic platform. Chapter 1 introduces the steps the field of biocatalysis has taken toward engineering enzymes with new catalytic functions and the process by which these activities are discovered and enhanced. Chapter 2 details the discovery and engineering of heme proteins which catalyze the stereodivergent cyclopropanation of unactivated and electron-deficient alkenes via carbene transfer, expanding the substrate classes beyond styrenyl alkenes. Chapter 3 shows the development of engineered variants of a heme protein (Rhodothermus marinus nitric oxide dioxygenase) for the diastereodivergent synthesis of cyclopropanes functionalized with a pinacolborane moiety, enabling product diversification through standard cross-coupling reactions. In Chapter 4, a collection of non- heme metalloproteins is curated, and a non-heme iron enzyme (Pseudomonas savastanoi ethylene- forming enzyme) is shown to be both amenable to directed evolution and non-native ligand substitution to enhance its nitrene-transfer activity. Chapter 5 describes the expansion of sequence space targeted for screening in the serine-ligated cytochrome P411 from Bacillus megaterium (P411BM3) biocatalytic platform to enhance the mutational robustness of these remarkable enzymes. Overall, this work provides a framework for bringing model new-to-nature reactions to their full potential in synthetic biocatalytic reactions. vi PUBLISHED CONTENT AND CONTRIBUTIONS † denotes equal contribution 1. Hammer, S. C.†; Knight, A. M. † Arnold, F. H. Design and Evolution of Enzymes for Non- Natural Chemistry. Curr. Opin. Green Sustain. Chem. 2017, 7, 23–30. DOI: 10.1016/j.cogsc.2017.06.002. A.M.K wrote the first draft of the Introduction and Non-natural activities are readily accessed from diverse starting points sections and participated in the writing and editing of the current opinion. 2. Knight, A. M.; Kan, S. B. J.; Lewis, R. D.; Brandenberg, O. F.; Chen, K.; Arnold, F. H. Diverse Engineered Heme Proteins Enable Stereodivergent Cyclopropanation of Unactivated Alkenes. ACS Cent. Sci. 2018, 4, 372–377. DOI: 10.1021/acscentsci.7b00548. Previously deposited on ChemRxiv, DOI: 10.26434/chemrxiv.5718076.v1. A.M.K. participated in the conception of the project, discovered initial enzymatic activity, performed the majority of the mutagenesis and screening, synthesized authentic standards, developed analytical methods to determine enantioselectivity, and wrote the manuscript with input from all authors. A.M.K. generated the crystallography constructs, purified, and carried out crystallography experiments. A.M.K. collected data for and refined the tagless RmaNOD Q52V crystal structure. 3. Brandenberg, O. F.; Prier, C. K.; Chen, K.; Knight, A. M.; Wu, Z.; Arnold, F. H. Stereoselective Enzymatic Synthesis of Heteroatom-Substituted Cyclopropanes. ACS Catal. 2018, 8, 2629–2634. DOI: 10.1021/acscatal.7b04423. A.M.K. participated in preparation of P411BM3 variants for enhanced stereoselectivity on substrate scope targets and participated in characterizing the stereoselectivity of enzymatic products. A.M.K. participated in editing the manuscript. 4. Wittmann, B. J. †; Knight, A. M. †; Hofstra, J. L.; Reisman, S. E.; Kan, S. B. J.; Arnold, F. H. Diversity-Oriented Synthesis of Cyclopropane Building Blocks. ACS Catal. 2020, 10, 7112–7116. DOI: 10.1021/acscatal.0c01888. A.M.K. and S.B.J.K. conceived the project. A.M.K. prepared compilation plates for the initial activity screen and determined enzymes with initial activity. A.M.K. designed, constructed, and screened the libraries for the cis-selective lineage. A.M.K. ran multi-gram scale enzymatic reactions. A.M.K. developed the column-free purification procedure via trifluoroboration methods. A.M.K. and B.J.W wrote the manuscript with input from all authors. 5. Goldberg, N. W. †; Knight, A. M. †; Zhang, R. K.; Arnold, F. H. Nitrene Transfer Catalyzed by a Non-Heme Iron Enzyme and Enhanced by Non-Native Small-Molecule Ligands. J. Am. vii Chem. Soc. 2019, 141, 19585–19588. DOI: 10.1021/jacs.9b11608. Previously deposited on ChemRxiv, DOI: 10.26434/chemrxiv.10062044.v1. A.M.K. and N.W.G. ran controls to confirm the activity to be enzymatic. A.M.K. carried out alternative ligand experiments. A.M.K. designed site-saturation mutagenesis libraries. A.M.K. and N.W.G. constructed the libraries, A.M.K. screened and analyzed the libraries, and N.W.G. performed shake-flask validation of potentially improved variants. A.M.K. ran alternative ligand screens, thermostability assays, and time courses for evolved variants. A. M. K. and N. W. G. wrote the manuscript with input from all authors. viii TABLE OF CONTENTS Acknowledgements ................................................................................................................
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