Development of a synthetic diversification platform for targeted EVOLUTION of ENZYMES supporting a sustainable bioeconomy – EvoZyme

The ongoing climate change requires immediate actions to reduce the CO2 concentration in the atmosphere.

One such action is the transition from a fossil-based to a bio-based economy that builds on CO2-derived one-carbon (C1) feedstocks like formate. To this end, synthetic C1 assimilation routes are being engineered into biotechnological relevant microbes.

Among them, the reductive glycine pathway (rGlyP) bears enormous potential for biotechnological applications as it is short, energy efficient, and easy to engineer.

So far, optimization of growth on formate via the rGlyP was achieved by adaptive laboratory evolution, a time-consuming process that leads to incorporation of undesired mutations and does not allow targeted pathway optimization (debottlenecking).In EvoZyme, I aim to develop a Synthetic Biology platform for targeted evolution of pathway enzymes.

This platform consists of specialized Escherichia coli strains that are equipped with state-of-the-art diversification machineries allowing tunable hyperdiversification/evolution of target enzymes. Furthermore, the platform is capable of transferring the diversified enzyme libraries to various bacterial hosts.

To benchmark the platform, I aim to debottleneck the rGlyP by targeting its core enzyme, the glycine cleavage system (GCS) which catalyzes the rate-limiting step of glycine synthesis.

Diversification and optimization of the GCS will be tested in dedicated selection strains from different bacterial hosts that can only grow if a functional GCS is expressed. Ultimately, optimized GCS variants will be expressed in a formatotrophic E. coli strain lacking a GCS.

Improved formatotrophic growth will be analyzed in lab-scale bioreactors to determine techno-economic parameters required for the establishment of a formate bioeconomy.

The platform developed in this project will enable debottlenecking of rate-limiting enzymes in virtually any pathway and presents a valuable tool for Synthetic Biology.