Johannes P. van Dijken

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: Current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden–Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient␣anaerobic fermentation of this pentose. l-Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under ‘academic’ conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

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.

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, 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.

The role of redox balances in the anaerobic fermentation of xylose by yeasts

SummaryThe kinetics of glucose and xylose utilization by batch cultures of Candida utilis were studied under aerobic and anaerobic conditions during growth in complex media. Rapid ethanol formation occurred during growth on glucose when aerobic cultures were shifted to anaerobic conditions. However, with xylose as a substrate, transfer to anaerobiosis resulted in an immediate cessation of metabolic activity, as evidenced by the absence of both ethanol formation and xylose utilization. The inability of the yeast to ferment xylose anaerobically was not due to the absence of key enzymes of the fermentation pathway, since the addition of glucose to such cultures resulted in the immediate conversion of glucose to ethanol. Furthermore, when the enzyme xylose isomerase was added to an anaerobic xylose culture, immediate conversion of xylose to ethanol was observed. This indicates that the inability of the yeast to form ethanol from xylose under anaerobic conditions is caused by metabolic events associated with the conversion of xylose to xylulose. A hypothesis is put forward which explains that ethanol production from xylose by yeast under anaerobic conditions is negligible. It is suggested that the failure to ferment xylose anaerobically is due to a discrepancy between production and consumption of NADH in the overall conversion of xylose to ethanol. When a hydrogen acceptor (i.e. acetoin) was added to anaerobic cultures of C. utilis, xylose utilization resumed, and ethanol and acetate were produced with the concomitant stoicheiometric reduction of acetoin to 2,3-butanediol.

Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild‐type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate‐decarboxylase‐negative (Pdc−) mutant lacking all three PDC genes exhibited a three‐fold lower growth rate in complex medium with glucose than the isogenic wild‐type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc− strains. Furthermore, in ethanol‐limited chemostat cultures, the biomass yield of Pdc− and wild‐type S. cerevisiae were identical. However, Pdc− S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol‐limited chemostat cultures (D = 0·10 h−1) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed‐substrate cultures were switched to a feed containing glucose as the sole carbon source, wash‐out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl‐CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose‐limited chemostat cultures.

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.

The Saccharomyces cerevisiae NDE1 and NDE2 genes encode separate mitochondrial NADH dehydrogenases catalyzing the oxidation of cytosolic NADH.

In Saccharomyces cerevisiae, theNDI1 gene encodes a mitochondrial NADH dehydrogenase, the catalytic side of which projects to the matrix side of the inner mitochondrial membrane. In addition to this NADH dehydrogenase,S. cerevisiae exhibits another mitochondrial NADH-dehydrogenase activity, which oxidizes NADH at the cytosolic side of the inner membrane. To investigate whether open reading framesYMR145c/NDE1 and YDL 085w/NDE2, which exhibit sequence similarity with NDI1, encode the latter enzyme, NADH-dependent mitochondrial respiration was assayed in wild-type S. cerevisiae and nde deletion mutants. Mitochondria were isolated from aerobic, glucose-limited chemostat cultures grown at a dilution rate (D) of 0.10 h−1, in which reoxidation of cytosolic NADH by wild-type cells occurred exclusively by respiration. Compared with the wild type, rates of mitochondrial NADH oxidation were about 3-fold reduced in annde1Δ mutant and unaffected in an nde2Δmutant. NADH-dependent mitochondrial respiration was completely abolished in an nde1Δ nde2Δ double mutant. Mitochondrial respiration of substrates other than NADH was not affected in nde mutants. In shake flasks, an nde1Δ nde2Δ mutant exhibited reduced specific growth rates on ethanol and galactose but not on glucose. Glucose metabolism in aerobic, glucose-limited chemostat cultures (D = 0.10 h−1) of an nde1Δ nde2Δ mutant was essentially respiratory. Apparently, under these conditions alternative systems for reoxidation of cytosolic NADH could replace the role of Nde1p and Nde2p in S. cerevisiae.

Directed Evolution of Pyruvate Decarboxylase-Negative Saccharomyces cerevisiae, Yielding a C2-Independent, Glucose-Tolerant, and Pyruvate-Hyperproducing Yeast

ABSTRACT 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 C2 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 C2 independence. Further selection for glucose tolerance in shake flasks resulted in a Pdc−S. cerevisiae mutant (TAM) that could grow in batch cultures (μmax = 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.

The Mitochondrial Alcohol Dehydrogenase Adh3p Is Involved in a Redox Shuttle in Saccharomyces cerevisiae

NDI1 is the unique gene encoding the internal mitochondrial NADH dehydrogenase of Saccharomyces cerevisiae. The enzyme catalyzes the transfer of electrons from intramitochondrial NADH to ubiquinone. Surprisingly, NDI1 is not essential for respiratory growth. Here we demonstrate that this is due to in vivo activity of an ethanol-acetaldehyde redox shuttle, which transfers the redox equivalents from the mitochondria to the cytosol. Cytosolic NADH can be oxidized by the external NADH dehydrogenases. Deletion of ADH3, encoding mitochondrial alcohol dehydrogenase, did not affect respiratory growth in aerobic, glucose-limited chemostat cultures. Also, an ndi1Delta mutant was capable of respiratory growth under these conditions. However, when both ADH3 and NDI1 were deleted, metabolism became respirofermentative, indicating that the ethanol-acetaldehyde shuttle is essential for respiratory growth of the ndi1 delta mutant. In anaerobic batch cultures, the maximum specific growth rate of the adh3 delta mutant (0.22 h(-1)) was substantially reduced compared to that of the wild-type strain (0.33 h(-1)). This is consistent with the hypothesis that the ethanol-acetaldehyde shuttle is also involved in maintenance of the mitochondrial redox balance under anaerobic conditions. Finally, it is shown that another mitochondrial alcohol dehydrogenase is active in the adh3 delta ndi1 delta mutant, contributing to residual redox-shuttle activity in this strain.

Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359

Kluyveromyces lactis is an important industrial yeast, as well as a popular laboratory model. There is currently no consensus in the literature on the physiology of this yeast, in particular with respect to aerobic alcoholic fermentation (‘Crabtree effect’). This study deals with regulation of alcoholic fermentation in K. lactis CBS 2359, a proposed reference strain for molecular studies. In aerobic, glucose‐limited chemostat cultures (D=0·05–0·40 h−1) growth was entirely respiratory, without significant accumulation of ethanol or other metabolites. Alcoholic fermentation occurred in glucose‐grown shake‐flask cultures, but was absent during batch cultivation on glucose in fermenters under strictly aerobic conditions. This indicated that ethanol formation in the shake‐flask cultures resulted from oxygen limitation. Indeed, when the oxygen feed to steady‐state chemostat cultures (D=0·10 h−1) was lowered, a mixed respirofermentative metabolism only occurred at very low dissolved oxygen concentrations (less than 1% of air saturation). The onset of respirofermentative metabolism as a result of oxygen limitation was accompanied by an increase of the levels of pyruvate decarboxylase and alcohol dehydrogenase. When aerobic, glucose‐limited chemostat cultures (D=0·10 h−1) were pulsed with excess glucose, ethanol production did not occur during the first 40 min after the pulse. However, a slow aerobic ethanol formation was invariably observed after this period. Since alcoholic fermentation did not occur in aerobic batch cultures this is probably a transient response, caused by an imbalanced adjustment of enzyme levels during the transition from steady‐state growth at μ=0·10 h−1 to growth at μmax. It is concluded that in K. lactis, as in other Crabtree‐negative yeasts, the primary environmental trigger for occurrence of alcoholic fermentation is oxygen limitation.