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Malic Acid Production by Saccharomyces cerevisiae: Engineering of Pyruvate Carboxylation, Oxaloacetate Reduction, and Malate Export ^,

Rintze M. Zelle,^1,4, Erik de Hulster,^1,4, Wouter A. van Winden,^1,4 Pieter de Waard,³ Cor Dijkema,³ Aaron A. Winkler,² Jan-Maarten A. Geertman,^1,4 Johannes P. van Dijken,^1,2,4 Jack T. Pronk,^1,4 and Antonius J. A. van Maris^1,4*

Department of Biotechnology, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands,¹ BIRD Engineering B.V., Westfrankelandsedijk 1, 3115 HG Schiedam, The Netherlands,² Wageningen NMR Centre, Wageningen University and Research Centre, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands,³ and Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands⁴

Received 16 November 2007/ Accepted 1 March 2008

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. Since wild-type Saccharomyces cerevisiae strains produce only low levels of malate, metabolic engineering is required to achieve efficient malate production with this yeast. A promising pathway for malate production from glucose proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate. This redox- and ATP-neutral, CO₂-fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose)^–1. A previously engineered glucose-tolerant, C₂-independent pyruvate decarboxylase-negative S. cerevisiae strain was used as the platform to evaluate the impact of individual and combined introduction of three genetic modifications: (i) overexpression of the native pyruvate carboxylase encoded by PYC2, (ii) high-level expression of an allele of the MDH3 gene, of which the encoded malate dehydrogenase was retargeted to the cytosol by deletion of the C-terminal peroxisomal targeting sequence, and (iii) functional expression of the Schizosaccharomyces pombe malate transporter gene SpMAE1. While single or double modifications improved malate production, the highest malate yields and titers were obtained with the simultaneous introduction of all three modifications. In glucose-grown batch cultures, the resulting engineered strain produced malate at titers of up to 59 g liter^–1 at a malate yield of 0.42 mol (mol glucose)^–1. Metabolic flux analysis showed that metabolite labeling patterns observed upon nuclear magnetic resonance analyses of cultures grown on ¹³C-labeled glucose were consistent with the envisaged nonoxidative, fermentative pathway for malate production. The engineered strains still produced substantial amounts of pyruvate, indicating that the pathway efficiency can be further improved.

Published ahead of print on 14 March 2008.

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R.M.Z. and E.D.H. contributed equally to this publication.