Lab evolution platform for engineering novel enzymatic functions in brewing yeast

Overview

This research program offers a lab evolution approach for developing novel enzymatic functions in brewing yeast (S. cerevisiae). The platform exploits underground metabolism, the hidden catalytic potential of existing enzymes toward non-native substrates, by applying strong in-vitro selection pressure to evolve new catabolic activities. A proof of concept has already been demonstrated in E. coli, which was evolved to utilize oxalate as a sole carbon and energy source in under 400 generations. The same strategy is now being applied to brewing yeast to target specific molecules relevant to the food, cosmetics, cleaning, and wellness (FC CCW) industries.

The value proposition for industry partners is access to non-GMO evolved yeast strains and their purified enzymes capable of degrading or transforming target molecules of commercial interest. This enables sustainable, biology-based alternatives to chemical processing in food and consumer product manufacturing.

Technical specifications

Core approach:

• Uses adaptive laboratory evolution under carbon-limiting conditions with a target molecule and a helper carbon source to select for new enzymatic activities
• Relies on promiscuous activities of native enzymes that can be activated and refined through targeted mutation
• Identifies causative mutations via whole genome sequencing, followed by enzyme purification and in-vitro characterization
• Produces non-GMO strains suitable for applications where regulatory or consumer acceptance of genetically modified organisms is limited

Proof of concept demonstrated:

• E. coli evolved to grow on oxalate as sole carbon and energy source in fewer than 400 generations
• Repeated mutations identified in two genes (adhE and eutE) responsible for the new catabolic activity
• Mutated enzymes confirmed to catalyze oxalate degradation in-vitro

Adaptation to brewing yeast:

• Growth of S. cerevisiae in carbon-limiting medium containing a FC CCW target molecule plus a helper carbon source
• Continuous monitoring of optical density to detect emergence of target molecule utilization
• Whole genome sequencing to identify the underlying mutated enzyme
• Enzyme purification and testing on FC CCW substrates, analogous to lactase used in lactose-free dairy production

Technology readiness level

The platform has reached proof-of-concept validation in E. coli for oxalate degradation, with the responsible mutations identified and enzymes characterized in-vitro. The current stage focuses on extending the approach to S. cerevisiae and validating enzymatic activity against specific FC CCW target molecules. Future work includes scaling evolved strains and enzymes for industrial application and further characterization of substrate specificity and catalytic efficiency. The technology is at an early-to-mid stage of development, with demonstrated feasibility but pending validation in the target organism and target substrates.