Improvement of Lactic Acid Production in Saccharomyces cerevisiae by Cell Sorting for High Intracellular pH

Abstract

ABSTRACT Yeast strains expressing heterologous l -lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pH i ) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae , CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pH i . During batch culture, we observed in both strains a reduction of the mean pH i and the appearance of a subpopulation of cells with low pH i . Simultaneous analysis of pH i and viability proved that the cells with low pH i were dead. Based on the observation that the better lactic-acid-producing strain had a higher pH i and that the cells with low pH i were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pH i range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pH i range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.