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

Abstract

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, CO 2 -fixing pathway has a theoretical maximum yield of 2 mol malate (mol glucose) ؊1 . A previously engineered glucose-tolerant, C 2 -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 13 C-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.

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

“…Additional advantages of this organism are that its genome has been fully sequenced and that it is considered by the American Food and Drug Administration (FDA) as an organism generally regarded as safe (GRAS) (6). In addition, in several recent studies, the yeast S. cerevisiae has been proposed as a cell factory for the production of bulk chemicals, such as malic, lactic, and citric acid from renewable substrates (1,30,36,42,43).…”

Anaplerotic Role for Cytosolic Malic Enzyme in Engineered Saccharomyces cerevisiae Strains

“…Hitherto, attempts to engineer S. cerevisiae for malate (and succinate) production were based on replacement of the native alcoholic fermentation pathway by a malate fermentation pathway. To this end, pyruvate carboxylase, a cytosolically retargeted malate dehydrogenase and a heterologous malate transporter were (over) expressed in a pyruvate decarboxylase-negative strain (38,39). Although this approach yielded malate titers of up to 59 g liter Ϫ1 , ATP hydrolysis by pyruvate carboxylase precludes a net synthesis of ATP via this fermentative pathway.…”

“…[38][39] or NADP ϩ (EC 1.1.1.40) as the redox cofactor. In eukaryotes, malic enzyme has been found in the cytosol and/or in mitochondria and, in plants, in the chloroplasts (20,35).…”

Systems-Level Engineering of Nonfermentative Metabolism in Yeast

“…These include lactate (van Maris et al 2004;Ishida et al 2006), malate (Zelle et al 2008), isoprenoids (Shiba et al 2007;Herrero et al 2008;Kizer et al 2008), glycerol (Geertman et al 2006;Cordier et al 2007), and ethanol (Alper et al 2006;Bro et al 2006), among others. Although nonfermentative by-products represent a class of biologically interesting and commercially attractive small molecules, efforts aimed at engineering microbes for increased production of these metabolites are comparatively infrequent.…”