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Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

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Genome-wide host-pathway interactions affecting cis-cis-muconic acid production in yeast.

Paul Cachera¹, Nikolaj Can Kurt², Andreas Røpke²

¹Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.

Metabolic Engineering

|March 1, 2024

Summary

We enhanced a high-throughput gene editing method (CRI-SPA) to map host:pathway interactions for cis-cis-muconic acid (CCM) production in yeast. This approach identifies novel targets and reveals a link between CCM biosynthesis and cellular redox.

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Area of Science:

Metabolic Engineering
Synthetic Biology
Yeast Systems Biology

Background:

Forward metabolic engineering requires understanding heterologous pathway behavior in hosts.
Current high-throughput methods like CRI-SPA are limited by product phenotypes.
A method to study colorless products is needed to expand pathway analysis.

Purpose of the Study:

To adapt CRI-SPA for analyzing colorless product biosynthesis.
To create a genome-wide map of host:pathway interactions for cis-cis-muconic acid (CCM).
To identify novel host factors influencing CCM production.

Main Methods:

Adapted CRI-SPA with a biosensor for CCM production.
Phenotyped over 9,000 yeast genotypes (knock-out and overexpression).
Quantified biosensor fluorescence in high-density agar arrays.

Main Results:

Identified novel metabolic targets across diverse cellular functions impacting CCM biosynthesis.
Confirmed positive effects of identified targets on CCM production.
Discovered an interplay between CCM biosynthesis and cytosolic redox via the oxidative pentose phosphate pathway.

Conclusions:

The adapted CRI-SPA enables genome-wide analysis of colorless metabolite production.
New strategies for enhancing CCM production in yeast cell factories are proposed.
Understanding host:pathway interactions is crucial for optimizing metabolic engineering.