Journal
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
Volume 98, Issue 19, Pages 8155-8164Publisher
SPRINGER
DOI: 10.1007/s00253-014-5895-0
Keywords
Itaconic acid; Metabolic engineering; Saccharomyces cerevisiae; Flux balance analysis; Genome-scale metabolic engineering
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Funding
- DuPont
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Renewable alternatives for petroleum-derived chemicals are achievable through biosynthetic production. Here, we utilize Saccharomyces cerevisiae to enable the synthesis of itaconic acid, a molecule with diverse applications as a petrochemical replacement. We first optimize pathway expression within S. cerevisiae through the use of a hybrid promoter. Next, we utilize sequential, in silico computational genome-scanning to identify beneficial genetic perturbations that are metabolically distant from the itaconic acid synthesis pathway. In this manner, we successfully identify three non-obvious genetic targets (a dagger ade3 a dagger bna2 a dagger tes1) that successively improve itaconic acid titer. We establish that focused manipulations of upstream pathway enzymes (localized refactoring) and enzyme re-localization to both mitochondria and cytosol fail to improve itaconic acid titers. Finally, we establish a higher cell density fermentation that ultimately achieves itaconic acid titer of 168 mg/L, a sevenfold improvement over initial conditions. This work represents an attempt to increase itaconic acid production in yeast and demonstrates the successful utilization of computationally guided genetic manipulation to increase metabolic capacity.
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