Exploring the sequence and functional landscapes of natural and engineered polymerases through directed evolution

Promovendus/a: Paola Mishal Handal Marquez

Promotor(en): Prof. dr. Vitor Bernardes Pinheiro, Prof. dr. Matheus Froeyen, De heer Georgios Gkouridis

The principles of evolution—gene diversification and natural selection—are replicated in laboratories to engineer proteins with novel or optimized functions through a process called Directed Evolution. This method can bypass our knowledge gaps and enable the discovery of proteins with custom-made capabilities. While Directed Evolution has successfully produced proteins with tailored properties, the process itself remains a "black box," with limited efforts made to fully optimize and understand it.

In the field of DNA polymerases—enzymes crucial for DNA replication and repair—Directed Evolution has led to the isolation of variants capable of synthesizing synthetic nucleic acids (XNA), which have numerous biotechnological applications. However, despite decades of research, polymerases are still only partially understood, and the Directed Evolution of these enzymes is complex. This complexity hinders the discovery of efficient XNA polymerases, delaying their potential revolutionary impact on enzymatic synthesis and the development of XNA-based therapies and biotechnological tools.

To address these challenges, I developed protocols and roadmaps to explore the black box of Directed Evolution, aiming to accelerate and optimize polymerase development. I created a workflow for automating gene diversification, the crucial first step in Directed Evolution, to speed up the construction of high-quality protein libraries. Additionally, I investigated the effects of various parameters on artificial selection techniques, providing researchers with insights for making better-informed decisions when setting up Directed Evolution experiments. Through this work, I identified several polymerase variants capable of XNA synthesis, which has contributed to a deeper understanding of polymerase mechanisms.

In addition, the field of Directed Evolution mainly focuses on isolating the best mutant, with huge amounts of information produced by the experiments, that are typically ignored. To exploit the full potential of Directed Evolution, I developed methods that can convert the input and output of Directed Evolution experiments into a simple "functional map" of polymerases that can be used to make better, informed decisions in successive rounds of gene diversification and selection. These methods can be adapted for any other protein subjected to Directed Evolution.

In summary, my project has made significant contributions to various aspects of polymerase development through Directed Evolution, deepened our understanding of polymerase mechanisms, and provided a versatile roadmap to accelerate and optimize the Directed Evolution of any protein.