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Applied and Environmental Microbiology, August 2006, p. 5492-5499, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00683-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

Minoska Valli,1,2 Michael Sauer,1,2 Paola Branduardi,3 Nicole Borth,1 Danilo Porro,3 and Diethard Mattanovich1,2*

Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria,1 School of Bioengineering, University of Applied Sciences FH—Campus Vienna, Muthgasse 18, A-1190 Vienna, Austria,2 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, I-20126 Milano, Italy3

Received 24 March 2006/ Accepted 30 May 2006

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 (pHi) 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 pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi 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 pHi 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.

* Corresponding author. Mailing address: Institute of Applied Microbiology, University of Natural Resources and Applied Life Sciences, Muthgasse 18, A-1190 Vienna, Austria. Phone: 43 1 36006 6569. Fax: 43 1 3697615. E-mail: diethard.mattanovich{at}boku.ac.at.

Applied and Environmental Microbiology, August 2006, p. 5492-5499, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.00683-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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