Metabolic Control Analysis of Glycerol Synthesis in Saccharomyces cerevisiae

Glycerol, a major by-product of ethanol fermentation by Saccharomyces cerevisiae, is of significant importance to the wine, beer, and ethanol production industries. To gain a clearer understanding of and to quantify the extent to which parameters of the pathway affect glycerol flux in S. cerevisiae, a kinetic model of the glycerol synthesis pathway has been constructed. Kinetic parameters were collected from published values. Maximal enzyme activities and intracellular effector concentrations were determined experimentally. The model was validated by comparing experimental results on the rate of glycerol production to the rate calculated by the model. Values calculated by the model agreed well with those measured in independent experiments. The model also mimics the changes in the rate of glycerol synthesis at different phases of growth. Metabolic control analysis values calculated by the model indicate that the NAD⁺-dependent glycerol 3-phosphate dehydrogenase-catalyzed reaction has a flux control coefficient (Cv1J) of approximately 0.85 and exercises the majority of the control of flux through the pathway. Response coefficients of parameter metabolites indicate that flux through the pathway is most responsive to dihydroxyacetone phosphate concentration (RDHAPJ = 0.48 to 0.69), followed by ATP concentration (RATPJ = −0.21 to −0.50). Interestingly, the pathway responds weakly to NADH concentration (RNADHJ = 0.03 to 0.08). The model indicates that the best strategy to increase flux through the pathway is not to increase enzyme activity, substrate concentration, or coenzyme concentration alone but to increase all of these parameters in conjunction with each other.     

Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation

Ethanol is still one of the most important products originating from the biotechnological industry with respect to both value and amount. In addition to ethanol, a number of byproducts are formed during an anaerobic fermentation of Saccharomyces cerevisiae. One of the most important of these compounds, glycerol, is produced by yeast to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD+. The purpose of this study was to evaluate whether a reduced formation of surplus NADH and an increased consumption of ATP in biosynthesis would result in a decreased glycerol yield and an increased ethanol yield in anaerobic cultivations of S. cerevisiae. A yeast strain was constructed in which GLN1, encoding glutamine synthetase, and GLT1, encoding glutamate synthase, were overexpressed, and GDH1, encoding the NADPH-dependent glutamate dehydrogenase, was deleted. Hereby the normal NADPH-consuming synthesis of glutamate from ammonium and 2-oxoglutarate was substituted by a new pathway in which ATP and NADH were consumed. The resulting strain TN19 (gdh1-A1 PGK1p-GLT1 PGK1p-GLN1) had a 10% higher ethanol yield and a 38% lower glycerol yield compared to the wild type in anaerobic batch fermentations. The maximum specific growth rate of strain TN19 was slightly lower than the wild-type value, but earlier results suggest that this can be circumvented by increasing the specific activities of Gln1p and Glt1p even more. Thus, the results verify the proposed concept of increasing the ethanol yield in S. cerevisiae by metabolic engineering of pathways involved in biomass synthesis.     

In vivo dynamics of galactose metabolism in Saccharomyces cerevisiae: metabolic fluxes and metabolite levels

The dynamics of galactose metabolism in Saccharomyces cerevisiae was studied by analyzing the metabolic response of the CEN.PK 113-7D wild-type strain when exposed to a galactose pulse during aerobic growth in a galactose-limited steady-state cultivation at a dilution rate of 0.097 h(-1). A fast sampling technique and subsequent methanol-chloroform/solid phase extractions were applied for in vivo measurements of the dynamic changes of the AMP, ADP, ATP levels and the sugar phosphates of the Leloir pathway. The ATP level was found to be significantly lower for yeast growing under galactose limitation (0.37 +/- 0.05 micromol/g CDW) than what has been reported for growth under glucose limitation. The galactose pulse of 5.58 mM was consumed within 40 min (t = 40) and 7 min after the pulse was added cell growth stopped. Subsequently, the cells started to grow and at t = 30 the specific growth rate had recovered to half the steady-state growth rate (0.047 h(-1)). To evaluate the change in flux distribution at steady state and during the galactose transient, a stoichiometric model describing the aerobic metabolism of S. cerevisiae was set up for quantification of the metabolic fluxes. At t = 7 the flux entering the TCA cycle was low and acetate and ethanol started to be excreted to the extracellular medium. During recovery of cell growth the flux entering the TCA cycle increased again, and at t = 30 this flux exceeded the corresponding steady-state flux. During the pulse an enhanced level of Gal-1P was measured, which may be responsible for a toxic metabolic response in S. cerevisiae. The increase in the Gal-1P concentration is intensified by the low affinity of Gal7 towards Gal-1P and, hence, under the physiological conditions examined Gal7 seems to exert control over flux through the Leloir pathway.     

Reduced oxidative pentose phosphate pathway flux in recombinant xylose-utilizing Saccharomyces cerevisiae strains improves the ethanol yield from xylose

In recombinant, xylose-fermenting Saccharomyces cerevisiae, about 30% of the consumed xylose is converted to xylitol. Xylitol production results from a cofactor imbalance, since xylose reductase uses both NADPH and NADH, while xylitol dehydrogenase uses only NAD(+). In this study we increased the ethanol yield and decreased the xylitol yield by lowering the flux through the NADPH-producing pentose phosphate pathway. The pentose phosphate pathway was blocked either by disruption of the GND1 gene, one of the isogenes of 6-phosphogluconate dehydrogenase, or by disruption of the ZWF1 gene, which encodes glucose 6-phosphate dehydrogenase. Decreasing the phosphoglucose isomerase activity by 90% also lowered the pentose phosphate pathway flux. These modifications all resulted in lower xylitol yield and higher ethanol yield than in the control strains. TMB3255, carrying a disruption of ZWF1, gave the highest ethanol yield (0.41 g g(-1)) and the lowest xylitol yield (0.05 g g(-1)) reported for a xylose-fermenting recombinant S. cerevisiae strain, but also an 84% lower xylose consumption rate. The low xylose fermentation rate is probably due to limited NADPH-mediated xylose reduction. Metabolic flux modeling of TMB3255 confirmed that the NADPH-producing pentose phosphate pathway was blocked and that xylose reduction was mediated only by NADH, leading to a lower rate of xylose consumption. These results indicate that xylitol production is strongly connected to the flux through the oxidative part of the pentose phosphate pathway.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Fluxes of carbon, phosphorylation, and redox intermediates during growth of saccharomyces cerevisiae on different carbon sources

In the present work we develop a method for estimating anabolic fluxes when yeast are growing on various carbon substrates (glucose, glycerol, lactate, pyruvate, acetate, or ethanol) in minimal medium. Fluxes through the central amphibolic pathways were calculated from the product of the total required amount of a specified carbon intermediate times the growth rate. The required amount of each carbon intermediate was estimated from the experimentally determined macromolecular composition of cells grown in each carbon source and the monomer composition of macromolecules.Substrates sharing most metabolic pathways such as ethanol and acetate, despite changes in the macromolecular composition, namely carbohydrate content (34% +/- 1 and 21% +/- 3, respectively), did not show large variations in the overall fluxes through the main amphibolic pathways. For instance, in order to supply anabolic precursors to sustain growth rates in the range of 0.16/h to 0.205/h, similar large fluxes through Acetyl CoA synthase were required by acetate (4.2 mmol/hr g dw) or ethanol (5.2 mmol/h g dw).The V(max) activities of key enzymes of the main amphibolic pathways measured in permeabilized yeast cells allowed to confirm, qualitatively, the operation of those pathways for all substrates and were consistent on most substrates with the estimated fluxes required to sustain growth.When ATP produced from oxidation of the NADH synthesized along with the key intermediary metabolites was taken into account, higher Y(ATP) (max) values (36 with respect to 24 g dw/mol ATP) were obtained for glucose. The same result was obtained for glycerol, ethanol, and acetate. A yield index (YI) was defined as the ratio of the theoretically estimated substrate flux required to sustain a given growth rate over the experimentally measured flux of substrate consumption. Comparison of Yl between growth on various carbon sources led us to conclude that ethanol (Yl = 0.84), acetate (Yl = 0.77), and lactate (Yl = 0.77) displayed the most efficient use of substrate for biomass production. For the other substrates, the Yl decayed in the following order: pyruvate > glycerol > glucose.An improvement of the quantitative understanding of yeast metabolism, energetics, and physiology is provided by the present analysis. The methodology proposed can be applied to other eukaryotic organisms of known chemical composition. (c) 1995 John Wiley & Sons, Inc.     

Metabolic Engineering of a Phosphoketolase Pathway for Pentose Catabolism in Saccharomyces cerevisiae

Low ethanol yields on xylose hamper economically viable ethanol production from hemicellulose-rich plant material with Saccharomyces cerevisiae. A major obstacle is the limited capacity of yeast for anaerobic reoxidation of NADH. Net reoxidation of NADH could potentially be achieved by channeling carbon fluxes through a recombinant phosphoketolase pathway. By heterologous expression of phosphotransacetylase and acetaldehyde dehydrogenase in combination with the native phosphoketolase, we installed a functional phosphoketolase pathway in the xylose-fermenting Saccharomyces cerevisiae strain TMB3001c. Consequently the ethanol yield was increased by 25% because less of the by-product xylitol was formed. The flux through the recombinant phosphoketolase pathway was about 30% of the optimum flux that would be required to completely eliminate xylitol and glycerol accumulation. Further overexpression of phosphoketolase, however, increased acetate accumulation and reduced the fermentation rate. By combining the phosphoketolase pathway with the ald6 mutation, which reduced acetate formation, a strain with an ethanol yield 20% higher and a xylose fermentation rate 40% higher than those of its parent was engineered.     

Regulation of ethanol metabolism in the rat

The purpose of these experiments was to examine the factors which regulate ethanol metabolism in vivo. Since the major pathway for ethanol removal requires flux through hepatic alcohol dehydrogenase, the activity of this enzyme was measured and found to be 2.9 mumol/(min X g liver). Ethanol disappearance was linear for over 120 min in vivo and the blood ethanol fell 0.1 mM/min; this is equivalent to removing 20 mumol ethanol/min and would require that flux through alcohol dehydrogenase be about 60% of its measured maximum velocity. To test whether ethanol metabolism was limited by the rate of removal of one of the end products (NADH) of alcohol dehydrogenase, fluoropyruvate was infused to reoxidize hepatic NADH and to prevent NADH generation via flux through pyruvate dehydrogenase. There was no change in the rate of ethanol clearance when fluoropyruvate was metabolized. Furthermore, enhancing endogenous hepatic NADH oxidation by increasing the rate of urea synthesis (converting ammonium bicarbonate to urea) did not augment the steady-state rate of ethanol oxidation. Hence, transport of cytoplasmic reducing power from NADH into the mitochondria was not rate limiting for ethanol oxidation. In contrast, ethanol oxidation at the earliest time periods could be augmented by increasing hepatic urea synthesis.     

NADH shuttle system regulates K(ATP) channel-dependent pathway and steps distal to cytosolic Ca(2+) concentration elevation in glucose-induced insulin secretion.

The NADH shuttle system is composed of the glycerol phosphate and malate-aspartate shuttles. We generated mice that lack mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), a rate-limiting enzyme of the glycerol phosphate shuttle. Application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle, to mGPDH-deficient islets demonstrated that the NADH shuttle system was essential for coupling glycolysis with activation of mitochondrial ATP generation to trigger glucose-induced insulin secretion. The present study revealed that blocking the NADH shuttle system severely suppressed closure of the ATP-sensitive potassium (K(ATP)) channel and depolarization of the plasma membrane in response to glucose in beta cells, although properties of the K(ATP) channel on the excised beta cell membrane were unaffected. In mGPDH-deficient islets treated with aminooxyacetate, Ca(2+) influx through the plasma membrane induced by a depolarizing concentration of KCl in the presence of the K(ATP) channel opener diazoxide restored insulin secretion. However, the level of the secretion was only approximately 40% of wild-type controls. Thus, glucose metabolism through the NADH shuttle system leading to efficient ATP generation is pivotal to activation of both the K(ATP) channel-dependent pathway and steps distal to an elevation of cytosolic Ca(2+) concentration in glucose-induced insulin secretion.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. II: intracellular metabolite and enzyme assays at steady state chemostat cultures

Intracellular metabolite concentration and enzyme activity measurements were made to explain the new metabolic and growth phenomena seen in the micro-aerobic, continuous yeast cultures described in Part I. The results of these assays suggested mechanisms for the observed maximum in the specific ethanol productivity as a function of the oxygen feed rate, changing ATP yields, the effects of antifoam, and the sharp changes in the biomass concentration with small changes in the oxygenation. Measured were the intracellular concentrations of ATP, NADH, glucose 6-phosphate, pyruvate, glycerol, and ethanol, and the activities of hexokinase and alcohol dehydrogenase. Rate-limiting steps were identified by the accumulation of metabolites upstream and the depletion of metabolites downstream of the step.A potential mechanism for the stimulation of fermentation with decreasing oxygenation was an activation of glucose transport by an accumulating intracellular ATP concentration. The inhibition of fermentation at yet lower oxygenation rates may have been caused by the continued accumulation of ATP to the point that the glycolytic kineses were inhibited. A mechanism for the changing ATP yields and intracellular ATP concentration proposed the existence of ATPases or ATP waste reactions stimulated by both oxygen and ATP. Antifoam had the effect of decreasing the resistance for glycerol transport out of the cell. The resulting stimulation of glycerol production and inhibition of ethanol production decreased the intracellular ATP content. Finally, intracellular ethanol was found not to accumulate to levels of higher than the extracellular concentration.     

酿酒酵母B5不对称还原制备手性药物中间体R-2′-氯-1-苯乙醇的研究

从 11株微生物中筛选出 4株具有不对称还原 2′ 氯 苯乙酮能力的酵母 ,其中酿酒酵母B5的还原产率与对映体选择性最佳。确定了酿酒酵母B5对 2′ 氯 苯乙酮还原的最佳反应时间为 2 4h ;最佳pH 8 0 ;最佳反应温度为2 5℃ ;最佳共底物为 5 % (体积比 )乙醇。同时研究了底物浓度、微生物的量、微生物的培养条件等对反应产率和立体选择性的影响。细胞浓度为 10 75mg mL(细胞干重 反应体积 )的酿酒酵母B5可将 6 47mmol L的 2′ 氯 苯乙酮10 0 %地转化为R 2′ 氯 1 苯乙醇 ,其对映体选择性为 10 0 %。酿酒酵母B5可重复利用的特点可提高产物的产量。     

Intracellular fluxes in a recombinant xylose-utilizing Saccharomyces cerevisiae cultivated anaerobically at different dilution rates and feed concentrations

A metabolic flux model was constructed for the yeast Saccharomyces cerevisiae comprising the most important reactions during anaerobic metabolism of xylose and glucose. The model was used to calculate the intracellular fluxes in a recombinant, xylose-utilizing strain of S. cerevisiae (TMB 3001) grown anaerobically in a defined medium at dilution rates of 0.03, 0.06, and 0.18 h(-1). The feed concentration was varied from 0 g/L xylose and 20 g/L glucose to a mixture of 15 g/L xylose and 5 g/L glucose, so that the total concentration of carbon source was kept at 20 g/L. The specific uptake of xylose increased with the xylose concentration in the feed and with increasing dilution rate. The excreted xylitol was less than half of the xylose consumed. With increasing xylose concentration in the feed, the fluxes in the pentose phosphate pathway increased, whereas the flux through glycolysis decreased. Under all cultivation conditions, nicotinamide adenine dinucleotide (NADH) was the preferred cofactor for xylose reductase. The model showed that the flux through the reaction from ribulose 5-phosphate to xylulose 5-phosphate was very low under all cultivation conditions.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Accelerating glycolytic flux of Torulopsis glabrata CCTCC M202019 at high oxidoreduction potential created using potassium ferricyanide

This study aimed to increase the glycolytic flux of the multivitamin auxotrophic yeast Torulopsis glabrata by redirecting NADH oxidation from oxidative phosphorylation to membrane-bound ferric reductase. We added potassium ferricyanide as electron acceptor to T. glabrata culture broth at 20% dissolved oxygen (DO) concentration, which resulted in: (1) decreases in the NADH content, NADH/NAD(+) ratio, and ATP level of 45.3%, 60.3%, and 15.2%, respectively; (2) high activities of the key glycolytic enzymes hexokinase, phosphofructokinase, and pyruvate kinase, as well as high expression levels of the genes encoding these enzymes; and (3) increases in the specific glucose consumption rate and pyruvate yield of T. glabrata was by 45.5% and 23.1%, respectively. Our results showed that membrane-bound ferric reductase offers an alternative and efficient NADH oxidation pathway at lower DO concentration, which increases the glycolytic flux of T. glabrata.     

Fine structural localization of alkaloid synthesis in endoplasmic reticulum of submergedClaviceps purpurea

Cyanide-insensitive mitochondria from Saccharomycopsis lipolytica possess an exogenous NADH dehydrogenase, located outside the inner mitochondrial membrane, and linked to coupling site II. These mitochondria are able to oxidize exogenous NADH via two pathways: (1) a cyanide- and antimycin-sensitive pathway, or cytochrome pathway, and (2) a cyanide- and antimycin-insensitive pathway, or alternative pathway. Although the oxidation of exogenous NADH through the cytochrome pathway occurs with an ATP/0 ratio tending to 2, it proceeds, per molecule of NADH oxidized, with the apparent ejection in the outer medium of only 3 protons instead of 4 protons, as is the case with glycerol 3-phosphate as control substrate, but leaves 1 hydroxyl ion in the outer medium after decay of the protonmotive force. These properties were used to demonstrate the non electrogenic function of the alternative pathway. Indeed, the oxidation of exogenous NADH via the alternative pathway proceeds without apparent ejection of protons, although this oxidation generates an electron flux in the alternative pathway as demonstrated by the net appearance in the outer medium of 1 hydroxyl ion per atom of oxygen reduced, appearance which proves sensitive to benhydroxamic acid, a specific inhibitor of the alternative pathway. The non electrogenicity of the alternative pathway is accompanied by the absence of ATP synthesis as expected from Mitchell's chemiosmotic model. The absence of energy conservation when the electron transfer proceeds via the alternative pathway is not the result of an uncoupling property of an active alternative pathway, as the oxidation of malate plus pyruvate via coupling site I and the alternative pathway occurs with an ATP/0 ratio tending to 1.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.