The GAR1 gene, encoding D-galacturonate reductase in Cryptococcus diffluens, was isolated, and the GAR1-expression plasmid was constructed by insertion of GAR1 downstream of the yeast constitutive promoter in the yeast-integrating vector. Recombinant Saccharomyces cerevisiae expressing C. diffluens D-galacturonate reductase from a genome integrated copy of the gene was cultured for use the conversion of D-galacturonic acid to L-galactonic acid. The optimum conditions for L-galactonic acid production were determined in terms of the initial concentration of D-galacturonic acid, fermentation pH, and mixed sugars. The following conditions yielded high efficiency in the conversion of D-galacturonic acid to L-galactonic acid in large-scale cultures: 0.1% initial D-galacturonic acid concentration, pH 3.5, and glucose as additional sugar. The aerobic condition was necessary for the conversion of D-galacturonic acid. Subculture of that recombinant was not showing to decrease of the D-galacturonic acid conversion rate even though it was repeated in ten generations. Culturing in scale-up, the conversion rate of D-galacturonic acid to L-galactonic acid was increased. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.