Carbon catabolite repression of invertase during batch cultivations of Saccharomyces cerevisiae: the role of glucose, fructose, and mannose

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized first, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose. Received: 5 May 1998 / Received revision: 3 August 1998 / Accepted: 8 August 1998     

Effect of some disaccharides, hexoses and pentoses on nitrate reductase, glutamine synthetase and glutamate dehydrogenase in excised pea roots

The effects of exogenous sucrose, lactose, d -glucose, d (-)fructose, d -galactose, d -mannose, l -sorbose, l -arabinose and d -xylose on nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH) levels, on anaerobic nitrite production and on respiratory O₂ consumption were studied in excised roots of pea (Pisum sativum L. cv. Raman). Sucrose, glucose and fructose increase NR and GS levels and decrease GDH level (when compared with roots cultures without any sugar) at all concentrations used, but the extent of this effect varies. NR induction is enhanced by all sugars within the concentration range studied. Precultivation of roots with mannose and galactose results in an increase in anaerobic nitrite production in a medium consisting of phosphate buffer and KNO₃. GS reaches its maximum at lower sugar concentrations, this fact being especially clear-cut with galactose. The decrease in GS level observed in roots cultured without sucrose is enhanced by higher sorbose concentrations. The increase in GDH level occurring in roots cultured without sucrose is depressed by low galactose and mannose concentrations but enhanced by high galactose, mannose, xylose and a wide range of sorbose concentrations. Lactose exerts only slight influence on the enzymes. The effects of sugars are in no case consistent with their effect on respiratory O₂ consumption which is most pronounced with NR. The above results show that the effects of sugars on NR, GS and GDH are not mediated by one universal mechanism.     

The impact of supplemental carbon sources on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> growth,chlorophyll content and anthocyanin accumulation

This research explores the impacts of a broad range of supplemental carbon sources on growth and development of Arabidopsis thaliana. Parameters measured include dark-germinated hypocotyl length, light-germinated root growth, rosette growth, chlorophyll concentration and anthocyanin content. Treatment sugars include sucrose, maltose, d-glucose, d-fructose, l-arabinose, l-fucose, d-galactose, d-mannose, l-rhamnose and d-xylose each supplied at 4, 20 or 100 mM. This comparison of the effect of different carbon sources on multiple parameters and under identical conditions showed that every carbon source had unique qualitative and quantitative effects on Arabidopsis growth and development. Root growth was particularly sensitive to supplemental carbon source. Growth on 100 mM sucrose, maltose, glucose or xylose stimulated root growth by ~100%. Growth on arabinose, fucose, galactose, mannose or rhamnose inhibited root growth by 50% or more. Several sugars that strongly inhibited root growth had either no effect (galactose and fucose) or a positive effect (arabinose) on hypocotyl elongation and rosette growth. Rhamnose was the only carbon source that inhibited hypocotyl elongation across all concentrations. Sucrose, maltose, glucose, fructose, arabinose or xylose stimulated rosette growth by ~50%. Chlorophyll content was strongly reduced by mannose while sucrose, glucose, galactose and rhamnose caused smaller reductions. Anthocyanin accumulation was strongly induced by both galactose and mannose. Only mannose impacted all parameters across all concentrations. Based on these data it can be concluded that the effect of each carbon source on Arabidopsis growth and development is specific in terms of both magnitude and the parameters impacted.     

Candida glycerinogenes不同碳源代谢的初步研究

研究了不同碳源对Candidaglycerinogenes的菌体生长、发酵液pH值及代谢产物的影响,结果发现以葡萄糖、果糖等单糖为碳源时茵体生长较快,最终生物量比以蔗糖、麦芽糖等二糖为碳源时高20％～30％;导致发酵前12h发酵液pH值明显下降的主要因素是乳酸;与葡萄糖为碳源转化为甘油相比,果糖为碳源时更易累积乙醇;以蔗糖、麦芽糖为碳源时,用于转化生成甘油的碳源明显降低,碳源主要用于茵体自身生物合成及HMP途径,以蔗糖为碳源时,用于乳酸、丙酸及柠檬酸生物合成的碳源较麦芽糖明显提高,TCA途径代谢较为活跃。     

The effect of the sugar source on citric acid production by Aspergillus niger

Summary Under otherwise identical fermentation conditions, the sugar source has been shown to have a marked effect on citric acid production by Aspergillus niger. Sucrose was the most favourable source, followed by glucose and fructose and then lactose. No citric acid was produced from galactose. Strong relationships were observed between citric acid production and the activities of certain enzymes in myccelial cell-free extracts prepared from fermentation samples. When sucrose, glucose, or fructose was the sugar source pyruvate carboxylase activity was high, but 2-oxoglutarate dehydrogenase activity was not detected. When galactose was the sugar source pyruvate carboxylase activity was low, but 2-oxoglutarate dehydrogenase activity was high. It is suggested that whereas glucose and fructose repress 2-oxoglutarate dehydrogenase, thereby causing accumulation of citric acid, galactose does not. The activity of aconitase showed a direct relationship to the citric acid production rate. Thus, the activity was highest when sucrose was the sugar source, and lowest when galactose was the source. It is suggested that when large amounts of citric acid are lost from the cell the activity of aconitase increases as a response to the diminished intracellular supply of its substrate.     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Golgi Apparatus Mediated Polysaccharide Secretion by Outer Root Cap Cells of Zea mays. III. Control by Exogenous Sugars

Sugars supplied to germinating seedlings of maize (Zea mays L.) regulate the secretion of polysaccharides by the outer cells of the root cap. The polysaccharide secreted by these cells adheres to the root tip as a droplet and the size of the droplet was used to quantitate polysaccharide secretion. The polysaccharide contains glucose, galacrose, and galacturonic acid residues with smaller quantities of mannose, arabinose, xylose, fucose and rhamnose. These sugars supplied to maize seedlings had marked effects on the rate of polysaccharide secretion by root tips. The effects on secretion were independent of the growth rates of the roots. Glucose, fucose and xylose increased droplet size 1.5–2 fold (as did sucrose, maltose, lacrose, fructose and ribose) whereas galactose, arabinose and galacturonic acid were inhibitory. Mannose increased dropler size 5–7 fold. The marked effect of mannose on polysaccharide secretion was due to an increased rate of secretion combined with a longer phase of extrusion of polysaccharide into the forming droplet. The effect of mannose was partially reversed by inorganic phosphate and other sugars (except for fucose which had no effect or promoted secretion in the presence of mannose). In contrast to sucrose, mannose stimulated secretion in a maize variety having a high sugar endosperm (high endogenous sugar). The results suggest that regulation of secretion by mannose is due to an alteration of normal sugar metabolism; whereas stimulation of secretion by sucrose and other sugars may be due to an increased availability of sugars for metabolism.     

Different effects of galactose and mannose on cell proliferation and intracellular soluble sugar levels in <Emphasis Type="Italic">Vigna angularis</Emphasis> suspension cultures

Plant cells utilize various sugars as carbon sources for growth, respiration and biosynthesis of cellular components. Suspension-cultured cells of azuki bean (Vigna angularis) proliferated actively in liquid growth medium containing 1% (w/v) sucrose, glucose, fructose, arabinose or xylose, but did not proliferate in medium containing galactose or mannose. These two latter sugars thus appeared distinct from other sugars used as growth substrates. Galactose strongly inhibited cell growth even in the presence of sucrose but mannose did not, suggesting a substantial difference in their effects on cell metabolism. Analysis of intracellular soluble-sugar fractions revealed that galactose, but not mannose, caused a conspicuous decrease in the cellular level of sucrose with no apparent effects on the levels of glucose or fructose. Such a galactose-specific decrease in sucrose levels also occurred in cells that had been cultured together with glucose in place of sucrose, suggesting that galactose inhibits the biosynthesis, rather than uptake, of sucrose in the cells. By contrast, mannose seemed to be metabolically inert in the presence of sucrose. From these results, we conclude that sucrose metabolism is important for the heterotrophic growth of cells in plant suspension-cultures.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.     

The kinetics of simultaneous glucose and fructose uptake and product formation by Aspergillus niger in citric acid fermentation

The kinetics of substrate uptake and product formation in the process of citric acid accumulation by Aspergillus niger on sucrose as a sole carbon source are presented. The experiments are aimed at studying if glucose and fructose obtained from the hydrolysis of sucrose are equivalent carbon sources for A. niger and how the presence of the two different carbon substrates might influence the citric acid formation process. Beet sugar was used as a sole carbon source in the first series of experiments conducted in two types of bioreactors: stirred tank and air-lift. The fructose uptake rate was significantly lower than the glucose uptake rate in the late idiophase. A substrate utilisation breakpoint occurred when a large amount of citric acid was accumulated in the fermentation broth. A similar phenomenon was also detected in repeated fed-batch fermentation. This phenomenon was confirmed by the second series of parallel shake culture runs, in which fungal growth and citric acid accumulation by A. niger was simultaneously tested on the media containing the following carbon sources: sucrose, glucose and fructose, with and without addition of concentrated citric acid solution. Finally, it was shown that high concentration of citric acid strongly depleted fructose uptake rate.     

GROWTH OF EXCISED PEA EMBRYONIC AXES ON DIFFERENT SUGARS

Excised pea embryonic axes were cultured on mineral salts plus various carbon sources. Growth continued for at least 3 wk, as measured by increased length, fresh and dry wt, sugar content, and β-amylase activity. The optimum sucrose concentration for elongation and fresh wt accumulation was 5% (w/v), although dry wt and sugar content increased in cultures containing 10 to 20%. Comparable growth was observed for axes cultured on 2% sucrose, glucose, fructose, or maltose.     

Regulation of in vitro bud formation of date palm (Phoenix dactylifera L.) cv. Khanezi by different carbon sources

The influence of different carbon sources and concentrations on in vitro shoot multiplication of date palm cv. Khanezi were investigated. Sucrose, glucose, fructose and maltose sugars were used at the following concentrations; 0, 30, 60, 90 and 120 g l(-1). Shoot dry weight was significantly increased with increasing sugar concentrations while little growth was obtained in the absence of sugar. Concentrations of 30 and 60 g l(-1) were optimal for qualitative and quantitative shoot growth while abnormal growth was observed at 90 and 120 g l(-1) possibly due to osmotic stress. Sugar type had significant effects on most parameters measured except bud formation. Fructose produced the highest values of dry weights compared with other carbon sources. In addition, glucose, fructose and maltose were almost equivalently effective as a carbon source for culture of date palm compared with sucrose. It is also noticed in this study that the root formation was enhanced as the sugar concentration increased (60 g l(-1) and above). This rooting of buds generally reduced their ability to multiply during multiplication stage and occasionally inhibited bud formation especially in higher concentration of sugar.     

Evaluating potential sugar food sources from the olive grove agroecosystems for Prays oleae parasitoid Chelonus elaeaphilus

Chelonus elaeaphilus Silvestri (Hymenoptera: Braconidae) is a host-specific parasitoid of the olive moth, Prays oleae (Bernard), that can cause parasitism rates of up to 80% in Mediterranean olive groves. A laboratory study was carried out to assess the potential of sugars provided by wild plant species in olive grove agroecosystem to enhance the fitness of C. elaeaphilus. Insects were reared in a climate-controlled chamber at 25?±?2°C, 60?±?5% relative humidity (RH) with a photoperiod of 16:8 (L:D) h. Five naturally occurring wild plant nectar sugars (sucrose, fructose, glucose, maltose and mannose) were tested for their effect on insect longevity. The nectar sugar content of sucrose, fructose and glucose in 12 selected olive grove agroecosystem plant species was analysed and categorised on the basis of sugar ratios. Female insect longevity was increased when they were fed on both sucrose and glucose compared to either maltose or fructose, suggesting that sucrose-dominant nectars would bene?t this parasitoid. Sucrose was predominant in the nectar of five of the studied plant species (Silene gallica, Borago officinalis, Echium plantagineum, Lavandula stoechas and Lonicera hispânica). The results are discussed in terms of potential enhancement of the biological control of P. oleae.     

The Growth of Acer pseudoplatanus Cells in a Synthetic Liquid Medium: Response to the Carbohydrate, Nitrogenous and Growth Hormone Constituents

A synthetic culture medium which supports a high level of growth of a scrially propagated cell suspension culture of Acer pseudoplatanus is described. The sucrose of this medium can be effectively replaced by glucose or fructose or a mixture of glucose and fructose or galactose or maltose or soluble starch. When the carbohydrate is glucose or fructose no other sugars appear in the culture medium in significant amounts. Glucose is absorbed in greater quantity than fructose from an equimolar mixture of these sugars. When sucrose is supplied both glucose and fructose appear in the medium. Glucose appears in maltose medium, and maltose and glucose in soluble starch medium. Under the standard conditions of culture, media containing 2 % sucrose or 2 % glucose become depleted of sugar before the 25th day of incubation. Enhanced yield of the cultures can be obtained by raising the initial sucrose concentration to 6 %. – A supply of nitrate is essential for maximum yield and healthy growth. Growth, in the presence of nitrate, is significantly enhanced by a supply of urea. Addition of casein hydrolysate or of a mixture of amino acids enhances growth in the presence of nitrate and urea and particularly when nitrate is omitted. – When kinetin is omitted or incorporated at the standard level (0.25 mg/I), 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0 mg/l is essential for continuation of growth at a high level. It cannot be replaced by indol-3yl-acetic acid (IAA). 1-naphthaleneacetic acid (NAA) at 10 mg/l permits of a low level of growth with abnormal aggregation. When the level of kinetin is raised to 10 mg/l a high level of growth occurs in the absence of added auxin but the cultures become brown and tend to show increasing aggregation on subculture.     

Aspergillus niger cyclic AMP levels are not influenced by manganese deficiency and do not correlate with citric acid accumulation

Summary The role of intracellular levels of cyclic AMP in the control of citic acid accumulation by Aspergillus niger has been investigated. For this purpose, A. niger was grown in media containing either high (14%, w/v) or low (2%, w/v) concentrations of sucrose, supplemented with 10 M Mn²⁺ (manganese-sufficient) or not (manganese-deficient), to obtain conditions leading to variable citrate accumulation. Citric acid accumulation was only observed in high-sugar, manganese-deficient medium. Intracellular levels of cyclic AMP were significantly higher in mycelia grown on low-sugar media, but were not significantly influenced by the absence of manganese ions. When sucrose in the high-sugar medium was substituted by other mono- or disaccharides, similar intracellular concentrations of cyclic AMP were observed. However, citric acid accumulation was only significant with sucrose, glucose and fructose. It is thus concluded that the intracellular level of cyclic AMP is not causally related to the accumulation of citric acid by the fungus, and —noteworthy — is not affected by manganese deficiency (despite adenylate cyclase reputed to be a manganese-requiring enzyme).Offprint requests to: C. P. Kubicek     

Sugars in crop plants

We review current knowledge of the most abundant sugars, sucrose, maltose, glucose and fructose, in the world's major crop plants. The sucrose‐accumulating crops, sugar beet and sugar cane, are included, but the main focus of the review is potato and the major cereal crops. The production of sucrose in photosynthesis and the inter‐relationships of sucrose, glucose, fructose and other metabolites in primary carbon metabolism are described, as well as the synthesis of starch, fructan and cell wall polysaccharides and the breakdown of starch to produce maltose. The importance of sugars as hormone‐like signalling molecules is discussed, including the role of another sugar, trehalose, and the trehalose biosynthetic pathway. The Maillard reaction, which occurs between reducing sugars and amino acids during thermal processing, is described because of its importance for colour and flavour in cooked foods. This reaction also leads to the formation of potentially harmful compounds, such as acrylamide, and is attracting increasing attention as food producers and regulators seek to reduce the levels of acrylamide in cooked food. Genetic and environmental factors affecting sugar concentrations are described.     

Parameters interacting with mannose selection employed for the production of transgenic sugar beet

The mannose selection system employs the phosphomannose isomerase (PMI) gene as selectable gene and mannose, converted to mannose-6-phosphate by endogenous hexokinase, as selective agent. The transgenic PMI-expressing cells have acquired the ability to convert mannose-6-phosphate to fructose-6-phosphate, while the non-transgenic cells accumulate mannose-6-phosphate with a concomitant consumption of the intracellular pools of phosphate and ATP. Thus, certain steps of mannose selection depend on the cells’ own metabolism which may be affected by a number of factors, some of which are studied here using Agrobacterium tumefaciens-mediated gene transfer to sugar beet cotyledonary explants. Four frequently employed saccharides (sucrose, glucose, fructose, and maltose) were tested at various concentrations and were found to interact strongly with the phytotoxic effect of mannose, glucose being able to counteract nearly 100% of an almost complete mannose-induced growth inhibition. Sucrose, maltose, and fructose also alleviated significantly the mannose-induced growth inhibition, but were 4-, 5-, and 7-fold less potent than glucose, respectively (calculated as hexose equivalents). The transformation frequencies were also dependent on the nature and concentration of the added carbohydrates, but in this respect sucrose resulted in the highest transformation frequencies, about 1.0%, while glucose and fructose gave significantly lower frequencies. The selection efficiencies were highest in the presence of maltose where no non-transgenic escapes were found over a range of concentrations. The effect of the light intensity was also investigated and the transformation frequencies were positively correlated to light intensity, although the relative impact of light on growth in the presence of mannose appeared not to be dependent on the mannose concentration. Additional phosphate in the selection media had a strong positive effect on the transformation frequencies, suggesting phosphate limitation during selection. The mannose selection system was found to be relatively genotype-independent, provided a slight optimization of the mannose concentrations during selection. Analysis of F₁-offspring showed that all studied primary transformants resulted in PMI-expressing plantlets and that the segregational patterns were in accordance with expectations in at least 50% of the transformants, confirming the stable and active inheritance of the PMI-gene.     

Influence of sucrose concentration and phosphate limitation on citric acid production by immobilized cells of Aspergillus niger

Summary Immobilized cells of Aspergillus niger needed a lower initial sucrose concentration than free cells in order to obtain maximal yields of citric acid production. High sucrose concentrations led to reduced yields and increased polyol formation (glycerol, erythritol, arabitol). Continuous fermentation with media containing low sugar concentrations prevented the formation of polyols. The change from nitrogen-limited to phosphate-limited precultivation of immobilized spores significantly increased the productivity of the mycelium. The ratio of citric acid to residual sugar in the effluent distinctly lay in the direction of citric acid. Inside the alginate beads mainly large bulbous cells were observed.     

Variation in the Nature of Organic Acid Secretion and Mineral Phosphate Solubilization by Citrobacter sp. DHRSS in the Presence of Different Sugars

A novel phosphate solubilizing bacterium (PSB) was isolated from the rhizosphere of sugarcane and is capable of utilizing sucrose and rock phosphate as the sole carbon and phosphate source, respectively. This PSB exhibited mineral phosphate solubilizing (MPS) phenotype on sugars such as sucrose and fructose, which are not substrates for enzyme glucose dehydrogenase (GDH), along with GDH substrates, viz., glucose, xylose, and maltose, as carbon sources. PCR amplification of the rRNA gene and sequence analysis identified this bacterium as Citrobacter sp. DHRSS. On sucrose and fructose Citrobacter sp. DHRSS liberated 170 and 100 μM free phosphate from rock phosphate and secreted 49 mM (2.94 g/L) and 35 mM (2.1 g/L) acetic acid, respectively. Growth of Citrobacter sp. DHRSS on sucrose is mediated by an intracellular inducible neutral invertase. Interestingly, in the presence of GDH substrates like glucose and maltose, Citrobacter sp. DHRSS produced approximately 20 mM (4.36 g/L) gluconic acid and phosphate released was 520 and 570 μM, respectively. Citrobacter sp. DHRSS GDH activity was found when grown on GDH and non-GDH substrates, indicating that it is constitutive and could act on a wide range of aldose sugars. This study demonstrates the role of different organic acids in mineral phosphate solubilization by rhizobacteria depending on the nature of the available carbon source.     

Gustatory perception and metabolic utilization of sugars by<Emphasis Type="Italic"> Myrmica rubra</Emphasis> ant workers

The suitability of various nectar and honeydew sugars as a food source for the polyphagous ant species M. rubra (L.) was studied. The sugars used included monosaccharides (fructose, glucose, galactose, mannose, rhamnose), disaccharides (sucrose, maltose, trehalose, melibiose, lactose) and trisaccharides (melizitose, raffinose, erlose). Single-sugar solutions were tested on ant workers in a long-term laboratory bioassay in which acceptance of the solutions and ant survival were recorded. The acceptance of the sugars was confirmed in a second bioassay in which feeding time was established. Enzymatic hydrolysis of sucrose, maltose and melibiose was investigated through HPLC analyses of workers fed these disaccharides. Sugar acceptance and feeding time were related to ant survival. Considering the monosaccharide units of which the sugars are composed, fructose seems especially suitable as a short-term energy source, while glucose appears to be used both directly and for storage. The presence of a galactose unit appears to reduce sugar suitability. It is suggested that the workers possess invertase and maltase and to a lesser degree also galactosidase. The gustatory perception is correlated with the profitability of sugars in further metabolic processes.