Malic acid production by Saccharomyces cerevisiae: engineering of pyruvate carboxylation, oxaloacetate reduction, and malate export.

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.

Development of efficient xylose fermentation in Saccharomyces cerevisiae: xylose isomerase as a key component.

Metabolic engineering of Saccharomyces cerevisiae for ethanol production from D-xylose, an abundant sugar in plant biomass hydrolysates, has been pursued vigorously for the past 15 years. Whereas wild-type S. cerevisiae cannot ferment D-xylose, the keto-isomer D-xylulose can be metabolised slowly. Conversion of D-xylose into D-xylulose is therefore crucial in metabolic engineering of xylose fermentation by S. cerevisiae. Expression of heterologous xylose reductase and xylitol dehydrogenase does enable D-xylose utilisation, but intrinsic redox constraints of this pathway result in undesirable byproduct formation in the absence of oxygen. In contrast, expression of xylose isomerase (XI, EC 5.3.1.5), which directly interconverts D-xylose and D-xylulose, does not have these constraints. However, several problems with the functional expression of various bacterial and Archaeal XI genes have precluded successful use of XI in yeast metabolic engineering. This changed with the discovery of a fungal XI gene in Piromyces sp. E2, expression of which led to high XI activities in S. cerevisiae. When combined with over-expression of the genes of the non-oxidative pentose phosphate pathway of S. cerevisiae, the resulting strain grew anaerobically on D-xylose with a doubling time of ca. 8 h, with the same ethanol yield as on glucose. Additional evolutionary engineering was used to improve the fermentation kinetics of mixed-substrate utilisation, resulting in efficient D-xylose utilisation in synthetic media. Although industrial pilot experiments have already demonstrated high ethanol yields from the D-xylose present in plant biomass hydrolysates, strain robustness, especially with respect to tolerance to inhibitors present in hydrolysates, can still be further improved.

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose.

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.

Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain.

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.

Metabolic engineering of a xylose-isomerase-expressing Saccharomyces cerevisiae strain for rapid anaerobic xylose fermentation.

After an extensive selection procedure, Saccharomyces cerevisiae strains that express the xylose isomerase gene from the fungus Piromyces sp. E2 can grow anaerobically on xylose with a mu(max) of 0.03 h(-1). In order to investigate whether reactions downstream of the isomerase control the rate of xylose consumption, we overexpressed structural genes for all enzymes involved in the conversion of xylulose to glycolytic intermediates, in a xylose-isomerase-expressing S. cerevisiae strain. The overexpressed enzymes were xylulokinase (EC 2.7.1.17), ribulose 5-phosphate isomerase (EC 5.3.1.6), ribulose 5-phosphate epimerase (EC 5.3.1.1), transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2). In addition, the GRE3 gene encoding aldose reductase was deleted to further minimise xylitol production. Surprisingly the resulting strain grew anaerobically on xylose in synthetic media with a mu(max) as high as 0.09 h(-1) without any non-defined mutagenesis or selection. During growth on xylose, xylulose formation was absent and xylitol production was negligible. The specific xylose consumption rate in anaerobic xylose cultures was 1.1 g xylose (g biomass)(-1) h(-1). Mixtures of glucose and xylose were sequentially but completely consumed by anaerobic batch cultures, with glucose as the preferred substrate.

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export.

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.

Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle.

When xylose metabolism in yeasts proceeds exclusively via NADPH-specific xylose reductase and NAD-specific xylitol dehydrogenase, anaerobic conversion of the pentose to ethanol is intrinsically impossible. When xylose reductase has a dual specificity for both NADPH and NADH, anaerobic alcoholic fermentation is feasible but requires the formation of large amounts of polyols (e.g., xylitol) to maintain a closed redox balance. As a result, the ethanol yield on xylose will be sub-optimal. This paper demonstrates that anaerobic conversion of xylose to ethanol, without substantial by-product formation, is possible in Saccharomyces cerevisiae when a heterologous xylose isomerase (EC 5.3.1.5) is functionally expressed. Transformants expressing the XylA gene from the anaerobic fungus Piromyces sp. E2 (ATCC 76762) grew in synthetic medium in shake-flask cultures on xylose with a specific growth rate of 0.005 h(-1). After prolonged cultivation on xylose, a mutant strain was obtained that grew aerobically and anaerobically on xylose, at specific growth rates of 0.18 and 0.03 h(-1), respectively. The anaerobic ethanol yield was 0.42 g ethanol x g xylose(-1) and also by-product formation was comparable to that of glucose-grown anaerobic cultures. These results illustrate that only minimal genetic engineering is required to recruit a functional xylose metabolic pathway in Saccharomyces cerevisiae. Activities and/or regulatory properties of native S. cerevisiae gene products can subsequently be optimised via evolutionary engineering. These results provide a gateway towards commercially viable ethanol production from xylose with S. cerevisiae.

Directed evolution of pyruvate decarboxylase-negative Saccharomyces cerevisiae, yielding a C2-independent, glucose-tolerant, and pyruvate-hyperproducing yeast.

The absence of alcoholic fermentation makes pyruvate decarboxylase-negative (Pdc(-)) strains of Saccharomyces cerevisiae an interesting platform for further metabolic engineering of central metabolism. However, Pdc(-) S. cerevisiae strains have two growth defects: (i) growth on synthetic medium in glucose-limited chemostat cultures requires the addition of small amounts of ethanol or acetate and (ii) even in the presence of a C(2) compound, these strains cannot grow in batch cultures on synthetic medium with glucose. We used two subsequent phenotypic selection strategies to obtain a Pdc(-) strain without these growth defects. An acetate-independent Pdc(-) mutant was obtained via (otherwise) glucose-limited chemostat cultivation by progressively lowering the acetate content in the feed. Transcriptome analysis did not reveal the mechanisms behind the C(2) independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc(-) S. cerevisiae mutant (TAM) that could grow in batch cultures ( micro (max) = 0.20 h(-1)) on synthetic medium, with glucose as the sole carbon source. Although the exact molecular mechanisms underlying the glucose-tolerant phenotype were not resolved, transcriptome analysis of the TAM strain revealed increased transcript levels of many glucose-repressible genes relative to the isogenic wild type in nitrogen-limited chemostat cultures with excess glucose. In pH-controlled aerobic batch cultures, the TAM strain produced large amounts of pyruvate. By repeated glucose feeding, a pyruvate concentration of 135 g liter(-1) was obtained, with a specific pyruvate production rate of 6 to 7 mmol g of biomass(-1) h(-1) during the exponential-growth phase and an overall yield of 0.54 g of pyruvate g of glucose(-1).

High-level functional expression of a fungal xylose isomerase: the key to efficient ethanolic fermentation of xylose by Saccharomyces cerevisiae?

Evidence is presented that xylose metabolism in the anaerobic cellulolytic fungus Piromyces sp. E2 proceeds via a xylose isomerase rather than via the xylose reductase/xylitol-dehydrogenase pathway found in xylose-metabolising yeasts. The XylA gene encoding the Piromyces xylose isomerase was functionally expressed in Saccharomyces cerevisiae. Heterologous isomerase activities in cell extracts, assayed at 30 degrees C, were 0.3-1.1 micromol min(-1) (mg protein)(-1), with a Km for xylose of 20 mM. The engineered S. cerevisiae strain grew very slowly on xylose. It co-consumed xylose in aerobic and anaerobic glucose-limited chemostat cultures at rates of 0.33 and 0.73 mmol (g biomass)(-1) h(-1), respectively.

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.