Overexpression of SYM1 in a gpdDelta mutant of Saccharomyces cerevisiae with modified ammonium assimilation for optimization of ethanol production

Abstract

To improve ethanol production by Saccharomyces cerevisiae, two engineered yeast strains, namely, KAM-20 and KAM-21, were constructed by modifying their genomes. First, to redirect carbon flux from the glycerol pathway toward the ethanol pathway, a two-step gene replacement method was used in gpd1Δ or gpd2Δ mutant strains to overexpress the GLN1 gene while simultaneously overexpressing the GLT1 gene. Second, concomitant with the overexpression of GLT1 and GLN1, overexpression of the SYM1 gene was achieved by increasing the gene dosage in gpdΔ mutant strains. These modifications enabled to improve fermentation performance by studying ethanol metabolism under elevated temperature. Experimental results of anaerobic batch fermentation showed that KAM-21 and KAM-20 with regulated redox balance showed 13.83 and 13.20% higher ethanol production and 39.51 and 38.33% lower glycerol production, respectively, than that of wild-type strain KAM-2. During the exponential phase, the growth rates of KAM-20 and KAM-21 were slightly decreased relative to that of KAM-2. The three strains showed similar biomass content at the end of growth. Acetate formation and pyruvate production were remarkably reduced in KAM-20 and KAM-21 when compared with those in KAM-2. Our results indicated that KAM-20 and KAM-21 showed decreased glycerol production and increased ethanol production concomitant with improved fermentation properties, suggesting that these genetic modifications have a potential for improvement in the efficiency of ethanol production.