Heejin Kim, Suryang Kwak, Seungoh Seo, Jingjing Liu, and Yong-Su Jin

Department of Food Science and Human Nutrition, University of Illinois, Urbana-Champaign

Auxotrophic Saccharomyces cerevisiae, strains that are unable to synthesize particular metabolic building blocks, are widely used in yeast genetics and genomics experiments for selection purposes. Minimal growth medium, a type of defined medium composed of minimal nutrients, is commonly used for selection of auxotrophic yeast strains. As minimal essential nutrients are supplemented, minimal growth medium can impact the cellular metabolism of auxotrophic yeast strains significantly. We observed leucine auxotrophic yeast strains accumulate a significant quantity of 2-isopropylmalate (2-IPM), an intermediate of leucine biosynthesis, after leucine supplementation was depleted. 2-IPM is naturally secreted by yeast and known to chelate Al(III) ions in the environment, reducing aluminum toxicity to the yeast cell. Additionally, 2-IPM has dicarboxylic functional groups that can be industrially significant due to novel polymer synthesis.  Thus, we sought to increase the production titer of 2-IPM. First, glucose repression was relieved by using a non-repressing substrate, galactose. The 2-IPM titer was increased from 0.4 g/L to 0.7 g/L when 40 g/L of glucose or galactose was utilized, respectively. Next, nitrogen catabolite repression was relieved by deletion of a nitrogen catabolite repression transcriptional regulator, URE2. When strains that harbor a URE2-deletion were supplemented with galactose, the titer of 2-IPM was increased to 1.3 g/L. Finally, isopropylmalate synthase, encoded by LEU4, was overexpressed to maximize the conversion to 2-IPM, reaching 1.4 g/L of 2-IPM from 80 g/L of galactose supplementation. To date, this is the first report attempting to overproduce 2-IPM.