Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH

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, 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. Copyright © 2006, American Society for Microbiology. All Rights Reserved.