Manipulation of malic enzyme in Saccharomyces cerevisiae for increasing NADPH production capacity aerobically in different cellular compartments

The yeast Saccharomyces cerevisiae is an attractive cell factory, but in many cases there are constraints related with balancing the formation and consumption of redox cofactors. In this work, we studied the effect of having an additional source of NADPH in the cell. In order to do this, two strains were engineered by overexpression of malic enzyme. In one of them, malic enzyme was overexpressed as its wild-type mitochondrial form, and in the other strain a short form lacking the mitochondrial targeting sequence was overexpressed. The recombinant strains were analyzed in aerobic batch and continuous cultivations, and the basic growth characteristics were generally not affected to a great extent, even though pleiotropic effects of the manipulations could be seen by the altered in vitro activities of selected enzymes of the central metabolism. Moreover, the decreased pentose-phosphate pathway flux and the ratios of redox cofactors showed that a net transhydrogenase effect was obtained, which can be directed to the cytosol or the mitochondria. This may find application in redirecting fluxes for improving specific biotechnological applications.     

Sequential inactivation of ammonium and glucose transport in Saccharomyces cerevisiae during fermentation

Abstract Ethanol at concentrations above 12% (v/v) in mineral medium with glucose and with ammonium as the only nitrogen source induced rapid inactivation of the ammonium transport system in the strain IGC 3507 of Saccharomyces cerevisiae terminating protein synthesis. Subsequently, when glucose was present, the glucose transport system was irreversibly inactivated. This two-step mechanism may play a decisive role when ethanol stops fermentation by S. cerevisiae , before all the fermentable sugar has been consumed.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

The pathways of ammonium assimilation in Rhizobium meliloti

Two pathways of ammonium assimilation are known in bacteria, one mediated by glutamate dehydrogenase, the other by glutamine synthetase and glutamate synthase. The activities of these three enzymes were measured in crude extracts from four Rhizobium meliloti wild-type strains, 2011, M15S, 444 and 12. All the strains had active glutamine synthetase and NADP-linked glutamate synthase. Assimilatory glutamate dehydrogenase activity was present in strains 2011, M15S, 444, but not in strain 12. Three glutamate synthase deficient mutants were isolated from strain 2011. They were unable to use 1 mM ammonium as a sole nitrogen source. However, increased ammonium concentration allowed these mutants to assimilate ammonium via glutamate dehydrogenase. It was found that the sole mode of ammonium assimilation in strain 12 is the glutamine synthetase-glutamate synthase route; whereas the two pathways are functional in strain 2011.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamate dehydrogenase     

Involvement of oxidative stress in the regulation of H(2)S production during ultradian metabolic oscillation of Saccharomyces cerevisiae

Periodic evolution of H(2)S during aerobic chemostat culture of Saccharomyces cerevisiae resulted in ultradian metabolic oscillation via periodic inhibition of respiratory activity. To understand the nature of periodic H(2)S evolution, we investigated whether oxidative stress is associated with H(2)S production. The cellular oxidative states represented by intracellular level of lipid peroxides oscillated out of phase with the oscillation of dissolved O(2). Pulse addition of antioxidant, oxidative agent or inhibitor of antioxidation enzymes perturbed metabolic oscillation producing changes in H(2)S evolution. Analysis of H(2)S production profiles during perturbation of oscillation revealed that the amount of H(2)S production is closely linked with cellular oxidative states. Based on these results and our previous reports, we suggest that oxidative stresses result in periodic depletion of glutathione and cysteine, which in turn causes stimulation of the sulfate assimilation pathway and H(2)S production.     

Toward a global database for the molecular typing of Saccharomyces cerevisiae strains

Most of the yeast strains used in fermented beverages and foods are classified as Saccharomyces cerevisiae. However, different strains are suitable for different fermentation processes. The purpose of this work is the proposal of a standardized methodology for the molecular genotyping of S. cerevisiae strains based on polymorphisms at microsatellite loci and/or single nucleotide polymorphisms (SNPs). Single nucleotide variants in the coding region of FLO8, a key regulator of flocculation and pseudohyphae formation, were analyzed in a subset of Uruguayan wine strains. Polymorphism analysis at nine microsatellite loci (selected from 33 loci tested) was performed in a collection of 120 strains, mostly wine strains, from different origins. From a total of 184 different alleles scored, 50 were exclusive alleles that could identify 29 strains. Four selected microsatellite loci are located within or near genes of putative enological interest. The Uruguayan strains are highly diverse and evenly distributed in the phylogenetic reconstructions, suggesting an evolutionary history previous to human use. The Saccharomyces cerevisiae Microsatellites and SNPs Genotyping Database is presented (www.pasteur.edu.uy/yeast). Comparison of standardized results from strains coming from different settings (industrial, clinical, environmental) will provide a reliable and growing source of information on the molecular biodiversity of S. cerevisiae strains.     

Metabolic engineering of Saccharomyces cerevisiae for increased bioconversion of lignocellulose to ethanol

The absence of pentose-utilizing enzymes in Saccharomyces cerevisiae is an obstacle for efficiently converting lignocellulosic materials to ethanol. In the present study, the genes coding xylose reductase (XYL1) and xylitol dehydrogenase (XYL2) from Pichia stipitis were successfully engineered into S. cerevisae. As compared to the control transformant, engineering of XYL1 and XYL2 into yeasts significantly increased the microbial biomass (8.1 vs. 3.4 g/L), xylose consumption rate (0.15 vs. 0.02 g/h) and ethanol yield (6.8 vs. 3.5 g/L) after 72 h fermentation using a xylose-based medium. Interestingly, engineering of XYL1 and XYL2 into yeasts also elevated the ethanol yield from sugarcane bagasse hydrolysate (SUBH). This study not only provides an effective approach to increase the xylose utilization by yeasts, but the results also suggest that production of ethanol by this recombinant yeasts using unconventional nutrient sources, such as components in SUBH deserves further attention in the future.     

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.     

Epistatic interactions of spontaneous mutations in haploid strains of the yeast Saccharomyces cerevisiae

Several important biological phenomena, including genetic recombination and sexual reproduction, could have evolved to counteract genome contamination by deleterious mutations. This postulate would be especially relevant if it were shown that deleterious mutations interact in such a way that their individual negative effects are reinforced by each other. The hypothesis of synergism can be tested experimentally by crossing organisms bearing deleterious mutations and comparing the fitness of the parents and their progeny. The present study used laboratory strains of the budding yeast burdened with mutations resulting from absence of a major DNA mismatch repair function. Only in one, or possibly two, crosses out of eight did fitness of the progeny deviate from that of their parents in a direction indicating synergism. Furthermore, the distributions of progeny fitness were not skewed as would be expected if strong interactions were present. The choice of experimental material ensured that genetic recombination was extensive, all four meiotic products were available for fitness assays, and that the mutations were probably numerous. Despite this generally favourable experimental setting, synergism did not appear to be a dominating force shaping fitness of yeast containing randomly generated mutations.     

Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

Abstract Malolactic fermentation, a crucial step in winemaking, results mostly in degradation by lactic acid bacteria of L-malic acid into L-lactic acid. This direct decarboxylation is catalysed by the malolactic enzyme. Recently we, and others, have cloned the mleS gene of Lactococcus lactis encoding malolactic enzyme. Heterologous expression of mleS in Saccha-romyces cerevisiae was tested to perform simultaneously alcoholic and malolactic fermentations by yeast. mleS gene was cloned in a yeast multicopy vector under a strong promoter. Malolactic activity was present in crude extracts of recombinant yeasts. Malic acid degradation was tested during alcoholic fermentation in synthetic media and must. Yeasts expressing the mleS gene actually produced L-lactate from L-malate; nevertheless malate degradation was far from complete.     

A design for the control of apoptosis in genetically modified Saccharomyces cerevisiae

We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.     

Anomalies in the growth kinetics of Saccharomyces cerevisiae strains in aerobic chemostat cultures

Aerobic glucose-limited chemostat cultivations were conducted with Saccharomyces cerevisiae strains NRRL Y132, ATCC 4126 and CBS 8066, using a complex medium. At low dilution rates all three strains utilised glucose oxidatively with high biomass yield coefficients, no ethanol production and very low steady-state residual glucose concentrations in the culture. Above a threshold dilution rate, respiro-fermentative (oxido-reductive) metabolism commenced, with simultaneous respiration and fermentation occurring, which is typical of Crabtree-positive yeasts. However, at high dilution rates the three strains responded differently. At high dilution rates S. cerevisiae CBS 8066 produced 7–8 g ethanol L⁻¹ from 20 g glucose L⁻¹ with concomitant low levels of residual glucose, which increased markedly only close to the wash-out dilution rate. By contrast, in the respiro-fermentative region both S. cerevisiae ATCC 4126 and NRRL Y132 produced much lower levels of ethanol (3–4 g L⁻¹) than S. cerevisiae CBS 8066, concomitant with very high residual sugar concentrations, which was a significant deviation from Monod kinetics and appeared to be associated either with high growth rates or with a fermentative (or respiro-fermentative) metabolism. Supplementation of the cultures with inorganic or organic nutrients failed to improve ethanol production or glucose assimilation. Journal of Industrial Microbiology & Biotechnology (2000) 24, 231–236. Received 09 August 1999/ Accepted in revised form 18 December 1999     

Gaseous environments modify physiology in the brewing yeast Saccharomyces cerevisiae during batch alcoholic fermentation

Aims:  To investigate the impact of different gaseous atmospheres on different physiological parameters in the brewing yeast Saccharomyces cerevisiae BRAS291 during batch fermentation.
Methods and Results:  Yeasts were cultivated on a defined medium with a continuous sparging of hydrogen, helium and oxygen or without gas, permitting to obtain three values of external redox. High differences were observed concerning viable cell number, size and metabolites produced during the cultures. The ethanol yields were diminished whereas glycerol, succinate, acetoin, acetate and acetaldehyde yields were enhanced significantly. Moreover, we observed major changes in the intracellular NADH/NAD⁺ and GSH/GSSG ratio.
Conclusions:  The use of gas led to drastic changes in the cell size, primary energy metabolism and internal redox balance and E _h . These changes were different depending on the gas applied throughout the culture.
Significance and Impact of the Study:  For the first time, our study describes the influence of various gases on the physiology of the brewing yeast S. cerevisiae . These influences concern mainly yeast growth, cell structure, carbon and redox metabolisms. This work may have important implications in alcohol-related industries, where different strategies are currently developed to control better the production of metabolites with a particular attention to glycerol and ethanol.     

Acceleration of the rate of fermentation by Saccharomyces cerevisiae in the presence of ammonium ion

The rate of fermentation of both d-glucose and maltose in a defined medium by a brewing strain of Saccharomyces cerevisiae was found to be dependent on the availability of NH₄⁺. The glycolytic rate did not correlate with intracellular NH₄⁺and activation by NH₄⁺was blocked by cycloheximide. The ability of several amino acids to activate glycolysis followed the same order as their effectiveness as sole sources of nitrogen. It therefore seems that NH₄⁺does not stimulate fermentation through direct activation of glycolytic enzymes, but through its function as a substrate for protein synthesis.     

Analysis of the energy metabolism after incubation of Saccharomyces cerevisiae with sulfite or nitrite

After addition of 5 mM sulfite or nitrite to glucose-metabolizing cells of Saccharomyces cerevisiae a rapid decrease of the ATP content and an inversely proportional increase in the level of inorganic phosphate was observed. The concentration of ADP shows only small and transient changes. Cells of the yeast mutant pet 936, lacking mitochondrial F₁ATPase, after addition of 5 mM sulfite or nitrite exhibit changes in ATP, ADP and inorganic phosphate very similar to those observed in wild type cells. They key enzyme of glucose degradation, glyceraldehyde-3-phosphate dehydrogenase was previously shown to be the most sulfiteor nitrite-sensitive enzyme of the glycolytic pathway. This enzyme shows the same sensitivity to sulfite or nitrite in cells of the mutant pet 936 as in wild type cells. It is concluded that the effects of sulfite or nitrite on ATP, ADP and inorganic phosphate are the result of inhibition of glyceraldehyde-3-phosphate dehydrogenase and not of inhibition of phosphorylation processes in the mitochondria. Levels of GTP, UTP and CTP show parallel changes to ATP. This is explained by the presence of very active nucleoside monophosphate kinases which cause a rapid exchange between the nucleoside phosphates. The effects of the sudden inhibition of glucose degradation by sulfite or nitrite on levels of ATP, ADP and inorganic phosphate are discussed in terms of the theory of Lynen (1942) on compensating phosphorylation and dephosphorylation in steady state glucose metabolizing yeast.Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - AMP adenosine monophosphate - P_i inorganic orthophosphate Dedicated to Prof. Dr. Hans Grisebach on the occasion of his sixtieth birthday     

Biosensor based on glutamate dehydrogenase immobilized in chitosan for the determination of ammonium in water samples

An optical biosensor based on glutamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of ammonium in water samples is described. The biosensor film was deposited on a glass slide via a spin-coating method. The ammonium was measured based on β-nicotinamide adenine dinucleotide (NADH) oxidation in the presence of α-ketoglutaric acid at a wavelength of 340 nm. The biosensor showed optimum activity at pH 8. The optimum chitosan concentrations and enzyme loading were found to be at 2% (w/v) and 0.08 mg, respectively. Optimum concentrations of NADH and α-ketoglutaric acid both were obtained at 0.15 mM. A linear response of the biosensor was obtained in the ammonium concentration range of 0.005 to 0.5 mM with a detection limit of 0.005 mM. The reproducibility of the biosensor was good, with an observed relative standard deviation of 5.9% (n = 8). The biosensor was found to be stable for at least 1 month when stored dry at 4 °C.     

Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates

The conversion of acetyl-CoA to malonyl-CoA by acetyl-CoA carboxylase (ACC) is the rate-limiting step in fatty acid biosynthesis. In this study, a gene coding for ACC was isolated and characterized from an oleaginous yeast, Lipomyces starkeyi. Real-time quantitative PCR (qPCR) analysis of L. starkeyi acetyl-CoA carboxylase gene (LsACC1) showed that the expression levels were upregulated with the fast accumulation of lipids. The LsACC1 was co-overexpressed with the glycerol 3-phosphate dehydrogenase gene (GPD1), which regulates lipids biosynthesis by supplying another substrates glycerol 3-phosphate for storage lipid assembly, in the non-oleaginous yeast Saccharomyces cerevisiae. Further, the S. cerevisiae acetyl-CoA carboxylase (ScACC1) was transferred with GPD1 and its function was analyzed in comparison with LsACC1. The results showed that overexpressed LsACC1 and GPD1 resulted in a 63% increase in S. cerevisiae. This study gives new data in understanding of the molecular mechanisms underlying the regulation of fatty acids and lipid biosynthesis in yeasts.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

The role of metabolic engineering in the improvement of Saccharomyces cerevisiae: utilization of industrial media

Metabolic engineering has become a very important approach to strain improvement in parallel with classical strain development. Although Saccharomyces cerevisiae has been domesticated for ethanol and bread production, there are still some fundamental problems associated with its industrial use. The industrially used carbon sources often consist of a sugar mixture, and due to glucose repression these sugars are utilized sequentially, resulting in prolonged production time. In this article we discuss the application of metabolic engineering for construction of glucose-derepressed strains and specify advantages as well as difficulties associated with this approach.