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Applied and Environmental Microbiology, May 2004, p. 2898-2905, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2898-2905.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

Antonius J. A. van Maris,1 Aaron A. Winkler,2 Danilo Porro,3 Johannes P. van Dijken,1,2 and Jack T. Pronk1*

Department of Biotechnology, Delft University of Technology, NL-2628 BC Delft,1 BIRD Engineering B.V., NL-3044 CK Rotterdam, The Netherlands,2 Department of Biotechnology and Biosciences, University of Milan—Bicocca, Milan 20126, Italy3

Received 24 November 2003/ Accepted 19 January 2004

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.

* Corresponding author. Mailing address: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands. Phone: 31 15 278 3214. Fax: 31 15 278 2355. E-mail: J.T.Pronk{at}TNW.TUDelft.NL.

Applied and Environmental Microbiology, May 2004, p. 2898-2905, Vol. 70, No. 5
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.5.2898-2905.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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