S92: Metabolic Engineering of Muconic Acid Production in Saccharomyces cerevisiae

Tuesday, August 14, 2012: 3:30 PM
Meeting Room 11-12, Columbia Hall, Terrace level (Washington Hilton)
Kathleen Curran1, John Leavitt2, Ashty Karim1 and Hal Alper1, (1)Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, (2)Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX
The dicarboxylic acid muconic acid has recently garnered significant interest due to its potential use as a platform chemical for the production of several valuable consumer bio-plastics such as nylon-6,6 and polyurethane (via an adipic acid intermediate) and polyethylene terephthalate (PET) (via a terephthalic acid intermediate).  Here, we present the first heterologous production of muconic acid in the yeast Saccharomyces cerevisiae.   A three-step synthetic pathway was introduced in yeast utilizing codon-optimized genes to convert a native metabolite in the shikimate pathway, dehydroshikimate, to cis,cis-muconic acid.  This pathway contains the enzymes dehydroshikimate dehydratase from Podospora anserina, protocatechuic acid decarboxylase from Enterobacter cloacae, and catechol 1,2-dioxygenase from Candida albicans.  These genes were chosen from a selection of candidates using both in vitro and in vivo enzymatic assays and performance tests.  Once the pathway was established, genetic modifications guided by metabolic modeling 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 deletion of ZWF1 and over-expression of TKL1 increased flux of necessary precursors into the pathway.  Finally, the gene for the protocatechuic acid decarboxylase, which had lower activity than the other two heterologous enzymes, was integrated into the chromosome multiple times by targeting Ty elements as the integration sites.  Each of these modifications led to increases in overall titer and collectively demonstrates that yeast has the potential to be a platform for the bioproduction of muconic acid.