Identification of the aldo-keto reductase responsible for d-galacturonic acid conversion to l-galactonate in saccharomyces cerevisiae

Abstract

D-galacturonic acid (D-GalUA) is the main constituent of pectin, a complex polysaccharide abundant in several agro-industrial by-products such as sugar beet pulp or citrus peel. During several attempts to valorise D-GalUA by engineering the popular cell factory Saccharomyces cerevisiae, it became obvious that D-GalUA is, to a certain degree, converted to L-galactonate (L-GalA) by an endogenous enzymatic activity. The goal of the current work was to clarify the identity of the responsible enzyme(s). A protein homology search identified three NADPH-dependent unspecific aldo-keto reductases in baker’s yeast (encoded by GCY1, YPR1 and GRE3) that show sequence similarities to known D-GalUA reductases from filamentous fungi. Characterization of the respective deletion mutants and an in vitro enzyme assay with a Gcy1 overproducing strain verified that Gcy1 is mainly responsible for the detectable reduction of D-GalUA to L-GalA.