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  • Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.

Metabolic engineering of muconic acid production in Saccharomyces cerevisiae.

Metabolic engineering (2012-11-21)
Kathleen A Curran, John M Leavitt, Ashty S Karim, Hal S Alper
ABSTRACT

The dicarboxylic acid muconic acid has garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics including nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate). Many process advantages (including lower pH levels) support the production of this molecule in yeast. Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae. A three-step synthetic, composite pathway comprised of the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans was imported into yeast. Further genetic modifications guided by metabolic modeling and feedback inhibition mitigation were introduced to increase precursor availability. Specifically, the knockout of ARO3 and overexpression of a feedback-resistant mutant of aro4 reduced feedback inhibition in the shikimate pathway, and the zwf1 deletion and over-expression of TKL1 increased flux of necessary precursors into the pathway. Further balancing of the heterologous enzyme levels led to a final titer of nearly 141mg/L muconic acid in a shake-flask culture, a value nearly 24-fold higher than the initial strain. Moreover, this strain has the highest titer and second highest yield of any reported shikimate and aromatic amino acid-based molecule in yeast in a simple batch condition. This work collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid and suggests an area that is ripe for future metabolic engineering efforts.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
trans,trans-Muconic acid, 95%
Sigma-Aldrich
Sorbic acid, tested according to Ph. Eur.
Sigma-Aldrich
Sorbic acid, 99.0-101.0% anhydrous basis
Sorbic acid, European Pharmacopoeia (EP) Reference Standard