Tuesday, April 20, 2010
11-11

Metabolic engineering of malic acid production by Saccharomyces cerevisiae

R.M. Zelle, A.F. de Hulster, J.T. Pronk, and A.J.A. van Maris. Department of Biotechnology, Delft University of Technology, Julianalaan 67, Delft, Netherlands

Malic acid is a potential biomass-derivable "building block" for chemical synthesis. However, wild-type Saccharomyces cerevisiae strains produce this acid only in small amounts. Metabolic engineering proved successful in introducing a redox- and ATP-neutral pathway for malate production in a previously engineered glucose-tolerant, C2-independent pyruvate decarboxylase-negative S. cerevisiae strain. This pathway, that proceeds via carboxylation of pyruvate, followed by reduction of oxaloacetate to malate, can theoretically yield 2 mol malate (mol glucose)-1 by fixating CO2.  By (over)-expression of pyruvate carboxylase, malate dehydrogenase and a heterologous malate transporter from Schizosaccharomyces pombe, malate titers of up to 59 g liter-1 at a yield of 0.42 mol (mol glucose)-1 were obtained in calcium carbonate-buffered shake flask cultivation. Further gains were achieved in 1L batch bioreactors, with yields of up to 0.48 ± 0.01 mol (mol glucose)-1. Studies in the latter cultivation system highlighted the importance of pH, CO2 and O2 for efficient C4-dicarboxylic acid production: First, malate formation was found to correlate strongly with increasing medium pH. Secondly, moderate carbon dioxide enrichment of the sparging gas improved production of both malate and succinate. However, at CO2-concentrations above 15%, succinate titers kept increasing, reaching 0.29 mol (mol glucose)-1, whereas malate formation severely decreased. Finally, although fully aerobic conditions could easily be achieved in the bioreactors, it was found that moderate oxygen limitation benefited malate production. Both the malate and succinate titers achieved in this study are the highest reported for S. cerevisiae.

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