Engineering Enzyme Systems by Recombination Thesis by Devin Lee Trudeau In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California 2014 (Defended December 11, 2013) 2014 Devin L. Trudeau All Rights Reserved iii ACKNOWLEDGEMENTS I’d like to thank my advisor Frances Arnold for seeing me through four years of failure and success. Frances has continually challenged my ideas and work, which has taught me how to be a better scientist, thinker, and bioengineer. In her lab I’ve had the freedom to pursue many different ideas, and interact with people of diverse personalities and research interests. The Arnold lab has been my home these last few years. I’d like to thank Florence Mingardon and Yousong Ding for being my two mentors as I was learning the art of protein engineering. Toni Lee has been a great collaborator on our endoglucanase project, and I’m happy that our efforts were as synergistic as our enzymes. Matthew Smith and Indira Wu have both been invaluable sources of advice on cellulase engineering. Sabrina Sun was a pleasure to work with over the course of two summers and a fourth-year project. Joseph Meyerowitz was my collaborator on the aldehyde tolerance project. Sabine Bastian kept the lab running smoothly, and has provided excellent advice on directed evolution methodology. Jackson Cahn, Ardemis Boghossian, Phil Romero, Ryan Lauchli, John McIntosh, Martin Engqvist, Chris Farwell, and Jane Wang have been great feedback for my (mostly crazy) ideas— and good friends. I’d like to thank Frank Chang and the rest of my biofuels collaborators in the Ho and Chao labs at Academia Sinica. I’d like to thank my committee members Professors David Tirrell, Thomas F. Miller III, Mikhail Shapiro, Steve Mayo, and Rob Phillips for their time and guidance during my candidacy and defense. Lastly I would like to thank my parents and brothers for their support and keeping me sane. iv ABSTRACT Homologous recombination is a source of diversity in both natural and directed evolution. Standing genetic variation that has passed the test of natural selection is combined in new ways, generating functional and sometimes unexpected changes. In this work we evaluate the utility of homologous recombination as a protein engineering tool, both in comparison with and combined with other protein engineering techniques, and apply it to an industrially important enzyme: Hypocrea jecorina Cel5a. Chapter 1 reviews work over the last five years on protein engineering by recombination. Chapter 2 describes the recombination of Hypocrea jecorina Cel5a endoglucanase with homologous enzymes in order to improve its activity at high temperatures. A chimeric Cel5a that is 10.1 °C more stable than wild-type and hydrolyzes 25% more cellulose at elevated temperatures is reported. Chapter 3 describes an investigation into the synergy of thermostable cellulases that have been engineered by recombination and other methods. An engineered endoglucanase and two engineered cellobiohydrolases synergistically hydrolyzed cellulose at high temperatures, releasing over 200% more reducing sugars over 60 h at their optimal mixture relative to the best mixture of wild-type enzymes. These results provide a framework for engineering cellulolytic enzyme mixtures for the industrial conditions of high temperatures and long incubation times. In addition to this work on recombination, we explored three other problems in protein engineering. Chapter 4 describes an investigation into replacing enzymes with complex cofactors with simple cofactors, using an E. coli enolase as a model system. Chapter 5 describes engineering broad-spectrum aldehyde resistance in Saccharomyces cerevisiae by evolving an alcohol dehydrogenase simultaneously for activity and promiscuity. Chapter 6 describes an attempt to engineer gene-targeted hypermutagenesis into E. coli to facilitate continuous in vivo selection systems. v TABLE OF CONTENTS Acknowledgements ........................................................................................................ iii Abstract ........................................................................................................................... iv Table of Contents ............................................................................................................ v List of Figures ................................................................................................................. ix List of Tables .................................................................................................................. xi Nomenclature ................................................................................................................ xii Chapter 1: Innovation by homologous recombination ................................................... 1 1.1 Abstract ............................................................................................................... 1 1.2 Introduction ......................................................................................................... 1 1.3 Structural and sequence information facilitates recombination ........................ 4 1.4 Exploring the limits of homologous recombination .......................................... 6 1.5 Recombination promotes innovation ................................................................. 9 1.5.1 Stability ..................................................................................................... 9 1.5.2 Enzyme substrate specificity .................................................................. 11 1.5.3 Optogenetic properties ............................................................................ 12 1.6 Modeling and predicting desired chimeras ...................................................... 13 1.7 Conclusions....................................................................................................... 15 Chapter 2: Recombination-based thermostabilization of a fungal endoglucanase II reveals protein fold dependence of contact epistasis ....................... 16 2.1 Abstract ............................................................................................................. 16 2.2 Introduction ....................................................................................................... 17 2.3 Non-contiguous recombination of fungal endoglucanase II ........................... 18 vi 2.4 Adapting linear regression to account for non-additive block interactions .... 20 2.5 Regression modeling predicts a highly stable chimera ................................... 24 2.6 Contact penalties among SCHEMA libraries .................................................. 24 2.7 A highly thermostable fungal endoglucanase II chimera ................................ 27 2.8 Non-additive block interactions can constrain thermostabilization by recombination ........................................................................................................ 29 2.9 Methods ............................................................................................................ 32 2.10 Supplementary information ............................................................................ 35 Chapter 3: A synergistic set of engineered thermostable fungal cellulases accelerates high-temperature cellulose hydrolysis ....................................................... 41 3.1 Abstract ............................................................................................................. 41 3.2 The utility of thermostable cellulase mixtures ................................................. 42 3.3 Engineering the most stable known fungal endoglucanase ............................. 43 3.4 Evaluating the synergy of engineered thermostable cellulases ....................... 47 3.5 An optimized mixture of engineered cellulases accelerates cellulose hydrolysis ............................................................................................................... 49 3.6 Discussion ......................................................................................................... 51 3.7 Methods ............................................................................................................ 53 3.8 Supplementary information .............................................................................. 55 Chapter 4: Directed evolution of an enolase for next-generation biofuels and chemicals ...................................................................................................................... 56 4.1 Abstract ............................................................................................................. 56 4.2 Natural enzymes limit production of biofuels and biochemical ..................... 57 4.3 Assessment of enolase candidates for engineering.......................................... 60 4.4 Directed evolution of an enolase– Targeted mutagenesis .............................. 62 vii 4.5 Directed evolution of an enolase– Substrate walking ..................................... 64 4.6 Directed evolution of an enolase– Growth selections ..................................... 65 4.7 Directed evolution of an enolase– Rational design ......................................... 65 4.8 Discussion ......................................................................................................... 68 4.9 Methods ...........................................................................................................