Biosensor-Enabled Directed Evolution to Improve Muconic Acid Production in Saccharomyces cerevisiae

Area of Science:

• Biotechnology
• Metabolic Engineering
• Synthetic Biology

Background:

• Muconic acid is a valuable platform chemical for polymers like nylon and PET.
• Previous efforts biosynthesized muconate in E. coli and engineered muconic acid production in S. cerevisiae.

Purpose of the Study:

• To enhance muconic acid production in Saccharomyces cerevisiae.
• To employ a combined adaptive laboratory evolution (ALE) strategy and rational metabolic engineering.

Main Methods:

• Adapted a biosensor module responding to aromatic amino acids (AAA) as a surrogate for pathway flux.
• Utilized two rounds of ALE with an anti-metabolite feeding strategy.
• Engineered the composite muconic acid pathway, complemented ARO1 truncation, and overexpressed an aromatic decarboxylase.

Main Results:

• Achieved a threefold increase in the composite pathway titer compared to previous strains.
• Isolated strains with improved AAA pathway flux.
• Established a final strain producing 0.5 g/L muconic acid in shake flasks and 2.1 g/L in fed-batch bioreactors.

Conclusions:

• The engineered S. cerevisiae strain achieved the highest reported titer of muconic acid in this host.
• The study demonstrates the highest reported titer for a shikimate pathway derivative in S. cerevisiae.
• This work advances the potential for sustainable production of muconic acid and its derivatives.