Coupling reprogrammed translation with enzyme engineering

Samenvatting

Enzymes are remarkable catalysts. Harnessing the prowess of enzyme catalysis for sustainable, industrial processes is a formidable challenge as biocatalysts are seldom optimal for abiological tasks. With biocatalysts displaying low or no activities for many synthetically useful reactions, enzyme engineering is crucial for potential applications.
Mimicking the Darwinian algorithm in the laboratory has become useful to tailor enzyme properties. However, the directed evolution of biocatalysts is often cumbersome, as efficient engineering protocols for many enzymes of interest remain elusive. To meet this challenge, this research proposal describes a novel, high-throughput engineering platform that is operationally simple yet broadly applicable.
Toward this end, I will take advantage of the ever-growing number of non-canonical amino acids that can efficiently be incorporated into proteins by reprogramming translation. More specifically, I aim to couple an enzyme’s activity to produce an unnatural amino acid to the subsequent incorporation of this artificial building block into a fluorescent protein (=readout). To highlight the versatility of this approach, the three objectives describe the evolution of both biotechnologically useful enzymes and biocatalysts with new-to-nature activities. The first two objectives aim to engineer enzymes for the production of tyrosine derivatives, such as L-DOPA, a psychoactive blockbuster drug. The third objective will focus on tailoring the promiscuous activity of cytochrome P450s to convert azides to ketones, a reaction not found in nature’s repertoire. Overall, the proposed research could pave the way toward a directed evolution platform that allows for fine-tuning activities of a diverse set of enzymes.
At the Stratingh Institute, I will have access to all the necessary equipment and state-of-the-art infrastructure to achieve my ambitious research goals and the University of Groningen is an ideal place to foster collaborations. This project will kick-start my independent research career and is crucial for establishing my research group in the near future.

Kenmerken

Projectnummer

722.017.007

Hoofdaanvrager

Dr. C. Mayer

Verbonden aan

Rijksuniversiteit Groningen, Faculty of Science and Engineering (FSE), Stratingh Institute for Chemistry

Looptijd

01/10/2017 tot 30/09/2020