Porting the synthetic D‐glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae

Abstract D‐Glucaric acid can be produced as a value‐added chemical from biomass through a de novo pathway in Escherichia coli . However, previous studies have identified pH‐mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo‐inositol oxygenase (MIOX) enzyme to be rate‐limiting. We ported this pathway to Saccaromyces cerevisiae , which is naturally acid‐tolerant and evaluate a codon‐optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene – either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon‐optimized for expression in S. cerevisiae – in order to identify the rate‐limiting steps for D‐glucaric acid production both from a fermentative and non‐fermentative carbon source. myo‐Inositol availability was found to be rate‐limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate‐limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed‐batch mode, and 1.6 g/L from glucose supplemented with myo‐inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.