Slow Growth Increases Myo-Inositol Availability for Glucaric Acid Production in S. Cerevisiae

Glucaric acid has been classified as a top value-added chemical from biomass for potential uses ranging from cancer therapies to sustainable nylons by the Department of Energy. A biosynthetic pathway was constructed in E. coli to convert glucose to glucaric acid but activity of the Mus musculus myo-inositol oxygenase (MIOX) enzyme was rate-limiting. New expression constructs enabled a one-step knock-in of the four pathway genes to S. cerevisiae. Protein sequence similarity mapping paired with simple structure-function hypotheses enabled efficient sampling of over 300 MIOX enzymes. Substituting a plant MIOX in the pathway increased glucaric acid titers detected in the YPD culture broth at 72hrs. Supplementation with 60mM myo-inositol tripled glucaric acid titers for yeast expressing either the plant MIOX (1.5 g/L) or the mouse MIOX (0.94g/L). However, myo-inositol supplementation did not affect glucaric acid titers in yeast expressing the mouse MIOX during slower growth on non-fermentable carbon sources such as glycerol or ethanol. Myo-inositol is a component of yeast membrane phospholipids and carbon-limited chemostat experiments demonstrated that slow growth rates lead to increased myo-inositol availability and glucaric acid titers. We pursued a fed-batch strategy in YPD with yeast expressing the plant MIOX and achieved final titers of 1.2g/L.