In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long-standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180" out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 pM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 pM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells.Keywords: cell-cell communication; signalling; control ; dynamics; self-organisation.It is becoming clear that the function of thc living cell extends beyond the steady state 11, 21. Of general interest are the states that derive from the non-linear properties of intracellular regulation and lead to oscillations i n important signallers such as the calcium ion (Ca") 13, 41. The oscillations become even more intriguing when they involve the dynamic interaction of individual cells such as in Dictyosteliunz differentiation [5]. Glycolytic oscillations in yeast are an example of the coupling of the dynamics of metabolism across cell boundaris [6, 7 I.Transient glycolytic oscillations can be induced by adding glucose followed by cyanide to a suspension of starved yeast [ 8-1 1 1. The oscillations last longer at higher cell densities [ 12, 131. This has been interpreted as being due to a synchronization mcchanism which prevents individually oscillating cells from becotning out of phase 1141. Under certain conditions sustained oscillations can be observed with populations of cells 16, 151.Although various substances have been considered to be the intercellular signaller, conclusive evidence for any onc of them has bccn elusive for 25 years 17, 13, 16, 171. In this report we identify and circumvent a complication precluding the measurement of acetaldehyde concentrations in the presence of cyanide. This allows us to demonstrate that the extracellular acetaldehyde concentration oscillates at the frequency of the intracellular glycolytic oscillations. The dependence of the phase shift on the acetaldehyde concentration and on the phase of acetaldehyde addition validates acetaldehyde as the elusive synchronizing agent. MATERIALS AND METHODSStrain and preparation of cells. on glucose as described in 161. At the diauxic shift, i.e. just after the glucose in the medium had been depleted, the cells were harvested by filtration, washed with 100 mM potassium phosphate, pH 6.8, resuspended and starved in the same buffer for 3 h at 30°C. The cells were then collected by filtration, resuspended in the same phosphate buffer and placed on ice until use. The protein concentration was d...
Although yeast are unicellular and comparatively simple organisms, they have a sense of time which is not related to reproduction cycles. The glycolytic pathway exhibits oscillatory behaviour, i.e. the metabolite concentrations oscillate around phosphofructokinase. The frequency of these oscillations is about 1 min when using intact cells. Also a yeast cell extract can oscillate, though with a lower frequency. With intact cells the macroscopic oscillations can only be observed when most of the cells oscillate in concert. Transient oscillations can be observed upon simultaneous induction; sustained oscillations require an active synchronisation mechanism. Such an active synchronisation mechanism, which involves acetaldehyde as a signalling compound, operates under certain conditions. How common these oscillations are in the absence of a synchronisation mechanism is an open question. Under aerobic conditions an oscillatory metabolism can also be observed, but with a much lower frequency than the glycolytic oscillations. The frequency is between one and several hours. These oscillations are partly related to the reproductive cycle, i.e. the budding index also oscillates; however, under some conditions they are unrelated to the reproductive cycle, i.e. the budding index is constant. These oscillations also have an active synchronisation mechanism, which involves hydrogen sulfide as a synchronising agent. Oscillations with a frequency of days can be observed with yeast colonies on plates. Here the oscillations have a synchronisation mechanism which uses ammonia as a synchronising agent.
Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD ؉ , which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP ؉ -dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO 2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO 2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO 2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD ؉ . The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO 2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.
D-Galacturonic acid is the main constituent of pectin, a naturally abundant compound. Pectin-rich residues accumulate when sugar is extracted from sugar beet or juices are produced from citrus fruits. It is a cheap raw material but currently mainly used as animal feed. Pectin has the potential to be an important raw material for biotechnological conversions to fuels or chemicals. In this paper, we review the microbial pathways for the catabolism of D-galacturonic acid that would be relevant for the microbial conversion to useful products.
D-Galacturonic acid can be obtained by hydrolyzing pectin, which is an abundant and low value raw material. By means of metabolic engineering, we constructed fungal strains for the conversion of D-galacturonate to meso-galactarate (mucate). Galactarate has applications in food, cosmetics, and pharmaceuticals and as a platform chemical. In fungi D- D-Galacturonate is the main component of pectin, an abundant and cheap raw material. Sugar beet pulp and citrus peel are both rich in pectin residues. At present, these residues are mainly used as cattle feed. However, since energy-consuming drying and pelletizing of the residues is required to prevent them from rotting, it is not always economical to process the residues, and it is desirable to find alternative uses.Various microbes which live on decaying plant material have the ability to catabolize D-galacturonate using various, completely different pathways (19). Eukaryotic microorganisms use a reductive pathway in which D-galacturonate is first reduced to L-galactonate by an NAD(P)H-dependent reductase (12,17). In the following steps a dehydratase, aldolase, and reductase convert the L-galactonate to pyruvate and glycerol (9,11,14).In Hypocrea jecorina (anamorph Trichoderma reesei) the gar1 gene codes for a strictly NADPH-dependent D-galacturonate reductase. In Aspergillus niger a homologue gene sequence, gar2, exists; however, a different gene, gaaA, is upregulated during growth on D-galacturonate containing medium (16). The gaaA codes for a D-galacturonate reductase with different kinetic properties than the H. jecorina enzyme, having a higher affinity toward D-galacturonate and using either NADH or NADPH as cofactor. It is not known whether gar2 codes for an active protein.Some bacteria, such as Agrobacterium tumefaciens or Pseudomonas syringae, have an oxidative pathway for D-galacturonate catabolism. In this pathway D-galacturonate is first oxidized to meso-galactarate (mucate) by an NAD-utilizing D-galacturonate dehydrogenase. Galactarate is then converted in the following steps to ␣-ketoglutarate. This route is sometimes called the ␣-ketoglutarate pathway (20). Galactarate can also be catabolized through the glycerate pathway (20). The products of this pathway are pyruvate and D-glycerate. These pathways have been described in prokaryotes, and it is not certain whether similar pathways also exist in fungi, some of which are able to metabolize galactarate.D-Galacturonate dehydrogenase (EC 1.1.1.203) has been described in Agrobacterium tumefaciens and in Pseudomonas syringae, and the enzymes from these organisms have been purified and characterized (3,6,22). Recently, the corresponding genes were also identified (4, 24). Both enzymes are specific for NAD as a cofactor but are not specific for the substrate. They oxidize D-galacturonate and D-glucuronate to meso-galactarate (mucate) and D-glucarate (saccharate), respectively. The reaction product is probably the hexaro-lactone which spontaneously hydrolyzes. The reverse reaction can only be observed at acidic ...
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