Figure 5

Bioconversion of D-galacturonic acid (D-GalA) to downstream products by genetically engineered yeast strains. (A) D-GalA can be converted to meso-galactaric acid (GalAA) and L-galactonate (L-GalOA) in S. cerevisiae strains heterologously expressing uronate dehydrogenase (UDH) or D-galacturonic acid reductase (GAAA), respectively, using endogenous cofactors. (B) Bioconversion yeast strains expressing GAT-1 exhibit rapid, high-affinity uptake of D-GalA (at pH 5.8 and an initial D-GalA concentration of 90 μM). (C) Intracellular products were detected by liquid chromatography coupled to tandem mass-spectrometry of chloroform:methanol:water-extracted yeast cells from the 1-h time point samples and their accumulation was found to be transporter-dependent. (D) Even at high D-GalA conditions (100 mM, pH 6.0) co-expression of GAT-1 in GAAA- or UDH-expressing yeast strains increases bioconversion product accumulation by an average 1.8- and 2.1-fold, respectively. NAD⁺, Nicotinamide adenine dinucleotide; NADPH, Nicotinamide adenine dinucleotide phosphate (reduced); GFP, green fluorescent protein.