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Applied and Environmental Microbiology, June 2002, p. 2814-2821, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2814-2821.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Metabolic Engineering of Glycerol Production in Saccharomyces cerevisiae

Karin M. Overkamp,1 Barbara M. Bakker,2 Peter Kötter,3 Marijke A. H. Luttik,1 Johannes P. van Dijken,1 and Jack T. Pronk1*

Kluyver Laboratory of Biotechnology, Delft University of Technology, NL-2628 BC Delft,1 Molecular Cell Physiology, Free University Amsterdam, NL-1081 HV Amsterdam, The Netherlands,2 Institut für Mikrobiologie, J. W. Goethe Universität Frankfurt, 60439 Frankfurt, Germany3

Received 22 January 2002/ Accepted 1 April 2002

Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1{Delta} nde1{Delta} nde2{Delta} gut2{Delta} quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h-1 at 100 g of glucose · liter-1). In aerated batch cultures grown on 400 g of glucose · liter-1, this engineered S. cerevisiae strain produced over 200 g of glycerol · liter-1, corresponding to a molar yield of glycerol on glucose close to unity.

* Corresponding author. Mailing address: Kluyver Laboratory 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, June 2002, p. 2814-2821, Vol. 68, No. 6
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.6.2814-2821.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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