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     

Analysis of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in continuous culture on glucose and fructose

The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l⁻¹ exopolysaccharides with glucose and 30 mg l⁻¹ with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced on glucose showed the presence of two fractions with relative molecular masses (M _r) of 1.7 × 10⁶ and 4 × 10⁴ in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M _r of 4 × 10⁴. The high-M _r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose and rhamnose in the molar ratio of 5:1:1, whereas the low-M _r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose, 1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production of the high-M _r fractions appeared to be dependent on the carbohydrate source, whereas the low-M _r fractions were produced more continuously. Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997     

Intra- and extracellular concentrations of glutamate, lactate and acetate during growth of Corynebacterium glutamicum on different media

Corynebacterium glutamicum ATCC 17965 was cultivated in a 4-L batch aerated fermentor with glucose, fructose and mixtures of these two sugars in various proportions as carbon sources and with different concentrations of minerals and vitamins. A multilayer centrifugation technique was devised to obtain cell extracts in order to assess intracellular production of glutamate and partitioning between intracellular and extracellular spaces for lactate and acetate, the main by-products produced during the growth phase. Glutamate production increased with the proportion of glucose in the carbon source. The average value for the intracellular concentration of glutamate obtained with basic glucose medium was increased three-fold when initial concentrations of vitamins and minerals were increased four-fold. In this case, overall production of glutamate (16.3 mM) reached the highest value obtained. Production of acetate was weak on all media types (< 1.6 mm). it was the same for lactate synthesis in media where glucose remained the major carbon source (< 2.3 mm). production of lactate was significantly higher on media where fructose was the main carbon source (> 10 mM to 60 mM). The increase in lactate production and the decrease in glutamate production were correlated to a modification of carbon flux distribution between the metabolic pathways as the fructose proportion was increased. An increase in the concentration of minerals favoured production of glutamate during growth. This was correlated with an increase in the NADPH,H⁺ production rate. Received 16 January 1996/ Accepted in revised form 14 January 1997     

The diversion of lactose carbon through the tagatose pathway reduces the intracellular fructose 1,6-bisphosphate and growth rate of Streptococcus bovis

Twenty strains of Streptococcus bovis grew more slowly on lactose (1.21 ± 0.12 h⁻¹) than on glucose (1.67 ± 0.12 h⁻¹), and repeated transfers or prolonged growth in continuous culture (more than 200 generations each) did not enhance the growth rate on lactose. Lactose transport activity was poorly correlated with growth rate, and slow growth could not be explained by the ATP production rate (catabolic rate). Batch cultures growing on lactose always had less␣intracellular fructose 1,6-bisphosphate (Fru1,6P ₂) than cells growing on glucose (6.6 mM compared to 16.7 mM), and this difference could be explained by the pathway of carbon metabolism. Glucose and the glucose moiety of lactose were metabolized by the Embden-Meyerhoff-Parnas (EMP) pathway, but the galactose moiety of lactose was catabolized by the tagatose pathway, a scheme that by-passed Fru1,6P ₂. A mutant capable of co-metabolizing lactose and glucose grew more rapidly when glucose was added, even though the total rate of hexose fermentation did not change. Wild-type S. bovis grew rapidly with galactose and melibiose, but these galactose-containing sugars were activated by galactokinase and catabolized via EMP. On the basis of these results, rapid glycolytic flux through the EMP pathway is needed for the rapid growth (more than 1.2 h⁻¹) of S.␣bovis. Received: 3 June 1997 / Received revision: 10 September 1997 / Accepted: 6 January 1998     

Glucose, and not sucrose, is transported from wheat to wheat powdery mildew

The main host carbon energy source transferred from wheat leaves (Triticum aestivum L.) to wheat powdery mildew (Erysiphe graminis f.sp. tritici) has been investigated in three ways. When the uptake of sugars by isolated mycelial suspensions was examined, the uptake rate for glucose was considerably higher than that for a range of other solutes. Analysis by high-performance liquid chromatography of leaf and mycelial extracts following uptake of sugars into infected leaf pieces confirmed that sucrose was rapidly hydrolyzed in the leaf; no sucrose or fructose could be detected in mycelial extracts. Furthermore, studies of the uptake of asymmetrically labelled sucrose indicated that this sugar is cleaved prior to uptake by the pathogen. Thus several lines of evidence show that glucose, and not sucrose, is the major carbon energy source transferred from host to fungal mycelium. Received: 11 November 1998 / Accepted: 18 January 1999     

Purification and characterization of a glucokinase from young tomato (Lycopersicon esculentum L. Mill.) fruit

In order to clearly establish the properties of the enzymes responsible for hexose phosphorylation we have undertaken the separation and characterization of these enzymes present in tomato fruit (Martinez-Barajas and Randall 1996). This report describes the partial purification and characterization of glucokinase (EC. 2.7.1.1) from young green tomato fruit. The procedure yielded a 360-fold enrichment of glucokinase. Tomato fruit glucokinase is a monomer with a molecular mass of 53 kDa. Glucokinase activity was optimal between pH 7.5 and 8.5, preferred ATP as the phosphate donor (K _m = 0.223 mM) and exhibited low activity with GTP or UTP. The tomato fruit glucokinase showed highest affinity for glucose (K _m = 65 μM). Activity observed with glucose was 4-fold greater than with mannose and 50-fold greater than with fructose. The tomato fruit glucokinase was sensitive to product inhibition by ADP (K _i = 36 μM). Little inhibition was observed with glucose 6-phosphate (up to 15 mM) at pH 8.0; however, at pH 7.0 glucokinase activity was inhibited 30–50% by physiological concentrations of glucose 6-phosphate. Received: 4 October 1997 / Accepted: 10 January 1998     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

Improvement of ferulic acid bioconversion into vanillin by use of high-density cultures of Pycnoporus cinnabarinus

High-density cultures of Pycnoporus cinnabarinus were tested with a view to optimisation of ferulic acid bioconversion into vanillin. The dry weight was increased fourfold by using glucose, fructose or a mixture of glucose and phospholipids as carbon source instead of maltose, the carbon source previously used. 5 mmol l⁻¹ vanillin, i.e. 760 mg l⁻¹, was produced over 15 days with glucose-phospholipid medium. In contrast, formation of vanillin was lower using glucose or fructose compared to the maltose control. A bioreactor (2 l) with a glucose-phospholipid medium gave a molar yield of vanillin of 61% (4 mmol l⁻¹). An alternative strategy was to grow the fungus on a glucose or fructose medium for 3 days, then switch to maltose during the bioconversion phase: this method allowed 3.3 mmol l⁻¹ vanillin to be obtained in 10 days. Many by-products such as methoxyhydroquinone and vanillyl alcohol were also produced. Received: 19 February 1999 / Received revision: 4 June 1999 / Accepted: 4 June 1999     

Metabolic responses to different glucose and glutamine levels in baby hamster kidney cell culture

In this work, a BHK21 clone producing a recombinant antibody/cytokine fusion protein was used to study the dependence of cell metabolism on the glucose and glutamine levels in the culture medium. Results obtained indicate that both glucose and glutamine consumptions show a Michaelis-Menten dependence on glucose and glutamine concentrations respectively. A similar dependence is also observed for lactate and ammonia productions. The estimated value of the Michaelis constant for the dependence of lactate production on glucose (K ^Glc _Lac) was 1.4 ± 0.1 mM and for the dependence of ammonia production on glutamine (K ^Gln _Amm) was 0.25 ± 0.11 mM and 0.10 ± 0.03 mM, at glucose concentrations of 0.28 mM and 5.6 mM respectively. At very low glucose concentrations, the glucose to lactate yield decreased markedly, showing a metabolic shift towards lower lactate production. This␣metabolic shift was also confirmed by the significant increase in the specific oxygen consumption rate also observed at low glucose concentrations. Although it was␣highly dependent on glucose concentration, the oxygen consumption also increased with the increase in␣glutamine concentration. At very low glutamine concentrations, the glutamine to ammonia yield increased, showing a more efficient glutamine metabolism. Received: 21 August 1998 / Received revision: 11 November 1998 / Accepted: 17 January 1999     

Family-10 and Family-11 xylanases differ in their capacity to enhance the bleachability of hardwood and softwood paper pulps

Enzyme-aided bleaching of softwood and hardwood kraft pulps by glycosyl hydrolase family-10 and -11 xylanases and a family-26 mannanase was investigated. The ability to release reducing sugar from pulp xylan and to enhance bleachability is not a characteristic shared by all xylanases. Of the six enzymes tested, two xylanases belonging to family 11 were most effective at increasing bleachability and improving final paper brightness. None of the enzymes had a deleterious effect on pulp fibre integrity. The efficiency of individual xylanases as bleach enhancers was not dependent on the source microorganism, and could not be predicted solely on the basis of the quantity or nature of products released from pulp xylan. Cooperative interactions between xylanase/xylanase and xylanase/mannanase combinations, during the pretreatment of softwood and hardwood pulps, were investigated. Synergistic effects on reducing-sugar release and kappa number reduction were elicited by a combination of two family-10 xylanases. Pretreatment of kraft pulp with mannanase A from Pseudomonas fluorescens subsp. cellulosa and any one of a number of xylanases resulted in increased release of reducing sugar and a larger reduction in kappa number than obtained with the xylanases alone, confirming the beneficial effects of family-26 mannanases on enzyme-aided bleaching of paper pulp. Received: 6 January 1997 / Received revision: 10 April 1997 / Accepted: 19 April 1997     

Optimization of docosahexaenoic acid production by Schizochytrium limacinum SR21

Culture conditions of Schizochytrium limacinum SR21 for the purpose of microbial docosahexaenoic acid (DHA) production were investigated. The strain SR21 showed a wide tolerance to salinity; that is, the optimum salinity was between 50% and 200% that of sea water. Monosaccharides (glucose and fructose) and glycerol supported good cell growth and DHA yield. Di- and polysaccharides, oleic acid, and linseed oil gave low DHA yields. A high content of DHA (more than 30% of total fatty acids) was obtained from culture on glucose, fructose, and glycerol, and also the strain had simple polyunsaturated fatty acid profiles. The major polyunsaturated fatty acids other than DHA were n-6 docosapentaenoic acid only, and the contents of icosapentaenoic acid and arachidonic acid were less than 1%. Using corn steep liquor as a nitrogen source, a high total fatty acid content was obtained. The total fatty acid content in the dry cell weight increased as the concentration of the nitrogen source decreased, reached more than 50%. An increase in carbon source concentration led to a high DHA yield. A maximum DHA yield of more than 4 g/l was obtained in both glucose and glycerol media at 9% and 12% respectively. S. limacinum SR21 was thought to be a promising resource for microbial DHA production yielding a good level of productivity as well as a simple polyunsaturated fatty acid profile. Received: 26 June 1997 / Received revision: 29 August 1997  / Accepted: 19 September 1997     

Hydrolysis of sucrose within isolated vacuoles from Solanum tuberosum L. tubers

The soluble acid invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) from potato (Solanum tuberosum L. cv. Kennebec) tubers was located in the vacuoles. Although the functionality of this invertase in the vacuoles has been assumed, the activity of the enzyme has never been shown within isolated vacuoles. Vacuoles were prepared by gentle osmotic shock from free protoplasts obtained by enzymic digestion of tuber tissues. The mean volume of these vacuoles, (0.26 ± 0.05) × 10⁻² μl, was estimated by optical microscopy. Sucrose, glucose and fructose concentrations were calculated to be 100 mM, 20 mM and 40 mM, respectively, in the vacuoles. Sucrose hydrolysis and the increase in glucose and fructose concentrations within the vacuoles were measured during vacuolar incubations. An almost identical pattern of sucrose hydrolysis by invertase was found by an in-vitro assay reproducing the vacuolar conditions. In view of the determinations of internal vacuolar pH (5.2), the possibility of spontaneous hydrolysis of sucrose was disregarded. Vacuoles were shown to be free from proteinaceous inhibitors, confirming the extravacuolar location of these inhibitors. The vacuolar hydrolytic pattern of sucrose confirms the regulatory role of the reaction products previously proposed for in-vitro assays. Received: 21 July 1997 / Accepted: 31 August 1997     

Enhanced l-lysine production in threonine-limited continuous culture of Corynebacterium glutamicum by using gluconate as a secondary carbon source with glucose

In order to improve the production rate of l-lysine, a mutant of Corynebacterium glutamicum ATCC 21513 was cultivated in complex medium with gluconate and glucose as mixed carbon sources. In a batch culture, this strain was found to consume gluconate and glucose simultaneously. In continuous culture at dilution rates ranging from 0.2 h⁻¹ to 0.25 h⁻¹, the specific l-lysine production rate increased to 0.12 g g⁻¹ h⁻¹ from 0.1 g g⁻¹ h⁻¹, the rate obtained with glucose as the sole carbon source [Lee et al. (1995) Appl Microbiol Biotechnol 43:1019–1027]. It is notable that l-lysine production was observed at higher dilution rates than 0.4 h⁻¹, which was not observed when glucose was the sole carbon source. The positive effect of gluconate was confirmed in the shift of the carbon source from glucose to gluconate. The metabolic transition, which has been characterized by decreased l-lysine production at the higher glucose uptake rates, was not observed when gluconate was added. These results demonstrate that the utilization of gluconate as a secondary carbon source improves the maximum l-lysine production rate in the threonine-limited continuous culture, probably by relieving the limiting factors in the lysine synthesis rate such as NADPH supply and/or phosphoenolpyruvate availability. Received: 16 May 1997 / Received revision: 28 August 1997 / Accepted: 29 August 1997     

Growth of Photorhabdus luminescens in batch and glucose fed-batch culture

Photorhabdus luminescens, a bacterial symbiont of entomopathogenic biocontrol nematodes, was grown in batch and glucose fed-batch culture. The cell density, bioluminescence, production of antibiotic substances, number of cells with inclusion bodies, glucose concentration and oxygen uptake rate were recorded. The addition of 12.4 g l⁻¹ glucose prolonged the growth, and the yield almost doubled, from 6.85 g l⁻¹ to 12.45 g l⁻¹ dry mass. The production of antibiotic substances increased by 140%. Bioluminescence was higher in the batch culture. A shift of P. luminescens to phase II variants was not detected. Received: 21 January 2000 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

Anaerobic leaching of covellite by Thiobacillus ferrooxidans

Thiobacillus ferrooxidans was able to grow under anaerobic conditions on copper sulphide with ferric ion as the electron acceptor. The dissolution of covellite under these conditions (68% after 35 days) was higher than values observed aerobically in cultures with similar media composition and almost as high as under aerobic conditions without iron. From these results we propose a mechanism for anaerobic bioleaching of covellite in the presence of ferric iron and speculate that it may occur in leach dumps where the oxygen concentration is, as reported elsewhere, very low. Received: 3 September 1996 / Received revision: 13 January 1997 / Accepted: 24 January 1997     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Glucose repression of anthracycline formation in Streptomyces peucetius var. caesius

The effect of glucose on growth and anthracycline production by Streptomyces peucetius var. caesius was examined in a chemically defined medium. Glucose concentrations above 100 mM inhibited anthracycline synthesis in the original strain without causing significant change in growth and final pH values. This effect was observed when the carbohydrate was added initially or after 24 h fermentation, but not when added during the stationary growth phase. When the microorganism was pregrown in 100 mM glucose and then transferred to a resting cell system with 444 mM glucose, no significant differences in antibiotic production were observed compared to the control without glucose. The negative effect of glucose on antibiotic synthesis was not observed in a mutant (2-dog^R–21) resistant to growth inhibition by 2-deoxyglucose. Glucose consumption by this mutant was approximately 30% of that utilized by the original strain. Compared to the original strain, the mutant 2-dog^R–21 exhibited a reduction of 50% in glucose transport and an 85% decrease in glucose kinase activity. The experimental evidence obtained suggests that glucose represses anthracycline formation in a transitory manner and that this effect is related to glucose transport and phosphorylation. Received: 15 January 1999 / Received revision: 7 April 1999 / Accepted: 1 May 1999     

Flow Cytometry Demonstrates Bacteriocin-Induced Injury to Listeria monocytogenes

Flow cytometry was used to study the effect of the bacteriocin leucocin B-TA11a on Listeria (L.) monocytogenes. Mixed proportions of dead and live control populations were analyzed by flow cytometry to determine detection limits of the Dead/Live Baclight Bacterial Viability Kit^TM. High correlations for flow cytometric detection of defined proportions of live or dead cells in mixtures between 10 and 100% of dead (r² = 0.97) or live (r² = 0.99) cells were obtained. However, mixtures containing less than 10% of either live or dead control cells gave correlations below 0.72. The growth of L. monocytogenes in the absence and presence of leucocin B-TA11a was analyzed by flow cytometry with Baclight, plate counts, and optical density measurements. Although leucocin B-TA11a initially inhibited listerial growth, the uptake of both Baclight dyes suggested that cells remained viable but became leaky, possibly indicating bacteriocin-induced pore formation in the target membranes. Received: 30 June 1997 / Accepted: 20 October 1997