Metabolic engineering of muconic acid production in Saccharomyces cerevisiae |
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| Affiliation: | 1. Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA;2. Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA;1. Department of Chemical Engineering, The University of Texas at Austin, 200 E Dean Keeton St. Stop C0400, Austin, TX 78712, USA;2. Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, TX 78712, USA;1. Department of Chemical and Biological Engineering, Iowa State University, 618 Bissell Road, Ames, Iowa 50011, United States;2. Department of Chemical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802, United States;3. NSF Engineering Research Center for Biorenewable Chemicals, 617 Bissell Road, Ames, Iowa 50011, United States;4. Interdepartmental Microbiology Program, Iowa State University, 2237 Osborn Dr., Ames, Iowa 50011, United States;5. US Department of Energy Ames Laboratory, 2408 Pammel Drive, Ames, Iowa 50011, United States;1. School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, United States;2. Renewable Bioproducts Institute, Georgia Institute of Technology, Atlanta, GA 30332, United States;3. School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, United States;1. Faculty of Agriculture and Life Science, Hirosaki University, Bunkyo-cho, Hirosaki 036-8561, Aomori, Japan;2. Forestry and Forest Products Research Institute, Matsuzato, Tsukuba 305-8687, Ibaraki, Japan;3. Virginia Agricultural Experiment Station, College of Agriculture and Life Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA;4. Department of Sustainable Biomaterials, Virginia Polytechnic Institute and State University, Blacksburg, VA 24060, USA |
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| 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 141 mg/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. |
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