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.

FIG. 1.

Four possible pathways for malate production in S. cerevisiae, using oxaloacetate and/or acetyl-CoA as precursors. (I) Direct reduction of oxaloacetate. (II) Oxidation of citrate via the TCA cycle. (III) Formation from acetyl-CoA via the cyclic glyoxylate route. (IV) Formation from acetyl-CoA and oxaloacetate via the noncyclic glyoxylate route. OAA, oxaloacetate; MAL, malate; CIT, citrate; ICI, isocitrate; AKG, α-ketoglutarate; SUCC, succinyl-CoA; SUC, succinate; FUM, fumarate; C2, acetyl-CoA; Y_sp^max, maximum theoretical yield (in mol malate per mol glucose).

FIG. 2.

Malate concentrations obtained in shake flask cultivations of different strains of S. cerevisiae overexpressing combinations of pyruvate carboxylase, malate dehydrogenase, or malate permease. Concentrations were determined after 96 h of cultivation when glucose was depleted (100 ml synthetic medium in 500-ml shake flasks, 100 g liter⁻¹ glucose, 5 g CaCO₃). Each bar represents a strain. A plus sign (+) below the bar indicates that the gene shown at the end of the row (PYC2, MDH3ΔSKL, or SpMAE1) is overexpressed in that particular strain. If a minus sign (−) is listed, the gene was not overexpressed. Error bars indicate standard deviations (each strain was tested at least twice).

FIG. 3.

Extracellular metabolite concentrations during a representative shake flask fermentation of RWB525 (100 ml synthetic medium in a 500-ml shake flask, 188 g liter⁻¹ initial glucose, 15 g CaCO₃, 1 g liter⁻¹ initial biomass). Glucose (▪), malate (□), glycerol (•), succinate (○), pyruvate (▴), fumarate (▵), and pH (⧫; dashed line) are shown. End point determinations of two additional experiments yielded quantitatively similar results. Standard deviations for these experiments are indicated in the text.

FIG. 4.

Flux estimates for the ¹³C-based metabolic flux analysis of RWB525 under malate-producing conditions in shake flasks. Fluxes (in moles) are normalized for a glucose uptake of 100 mol. Arrows indicate the direction of the (net) flux. In the case of reversible reactions, the exchange flux is included in parentheses. Some arrows are dashed to improve readability. Metabolites present in both compartments are differentiated by the indicators “cyt” (cytosol) and “mit” (mitochondrion).

FIG. 5.

Sensitivity analysis showing the covariance-weighted sum of squared residuals (the flux-fit error) between measured and fitted ¹³C-enrichment data over a range of malic enzyme and net cytosolic malate dehydrogenase fluxes. Optimizations were run at various fixed malic enzyme fluxes. For each malic enzyme flux, the net cytosolic malate dehydrogenase was forced stepwise from the free fit value to lower values. Flux distributions of the square data points are shown in detail in Fig 4 and 6: the top right point represents the best fit (Fig. 4), the lower left, top left, and lower right are, respectively, the upper, middle, and lower values in Fig. 6. The black surface in the lower left of the figure contains errors higher than 150.

FIG. 6.

Flux profiles for three highlighted situations from the sensitivity analysis of Fig. 5. Fluxes (in moles) are normalized for a glucose uptake of 100 mol. Arrows indicate the direction of the (net) flux. In case of reversible reactions, the exchange flux is included in parentheses. Some arrows are dashed to improve readability. Metabolites present in both compartments are differentiated by the indicators “cyt” (cytosol) and “mit” (mitochondrion). Upper numbers in each set of three, malic enzyme and net cytosolic malate dehydrogenase fluxes forced to 0; middle numbers, malic enzyme flux forced to 0; lower numbers, malic enzyme flux forced to the value obtained for the best fit and net cytosolic malate dehydrogenase flux forced to 0.