Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.     

Sugar sensing in higher plants.

Sugar repression of photosynthetic genes is likely a central control mechanism mediating energy homeostasis in a wide range of algae and higher plants. It overrides light activation and is coupled to developmental and environmental regulations. How sugar signals are sensed and transduced to the nucleus remains unclear. To elucidate sugar-sensing mechanisms, we monitored the effects of a variety of sugars, glucose analogs, and metabolic intermediates on photosynthetic fusion genes in a sensitive and versatile maize protoplast transient expression system. The results show that sugars that are the substrates of hexokinase (HK) cause repression at a low concentration (1 to 10 mM), indicating a low degree of specificity and the irrelevance of osmotic change. Studies with various glucose analogs suggest that glucose transport across the plasma membrane is necessary but not sufficient to trigger repression, whereas subsequent phosphorylation by HK may be required. The effectiveness of 2-deoxyglucose, a nonmetabolizable glucose analog, and the ineffectiveness of various metabolic intermediates in eliciting repression eliminate the involvement of glycolysis and other metabolic pathways. Replenishing intracellular phosphate and ATP diminished by hexoses does not overcome repression. Because mannoheptulose, a specific HK inhibitor, blocks the severe repression triggered by 2-deoxyglucose and yet the phosphorylated products per se do not act as repression signals, we propose that HK may have dual functions and may act as a key sensor and signal transmitter of sugar repression in higher plants.     

Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells

We present evidence that the activity of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.     

The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, ¹⁴C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[¹⁴C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m^?3 U-[¹⁴C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose-phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the ¹⁴C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6–8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.     

The regulation of sugar uptake and accumulation in bean pod tissue

The identity, localization and physiological significance of enzymes involved in sugar uptake and accumulation were determined for endocarp tissue of pods of Kentucky Wonder pole beans (Phaseolus vulgaris). An intracellular, alkaline invertase (pH optimum, 8) was assayed in extracted protein, as well as enzymes involved in sucrose synthesis, namely, uridinediphosphate (UDP-glucose pyrophosphorylase and UDP-glucose-fructose transglucosylase). Indirect evidence indicated the presence also of hexokinase, phosphohexoseisomerase and phosphoglucomutase. The data suggested that sucrose synthesis occurred in the cytoplasm, and that both sugar storage and an alkaline invertase occurred in the vacuole. The latter functions to hydrolyze accumulated sucrose. An outer space invertase (pH optimum, 4.0) was detected, but was variable in occurrence. Although its activity at the cell surface enhanced sucrose uptake, sucrose may be taken up unaltered.

Over a wide range of concentrations of exogenous glucose the sucrose/reducing sugar ratio of accumulated sugars remained unchanged at about 20. Synthesis of sucrose appears to be requisite to initial accumulation from glucose or fructose, as free hexoses do not increase at the apparent saturating concentration for uptake. Sucrose accumulation from exogenous hexose represents a steady-state value, in which sucrose is transported across the tonoplast into the vacuole at a rate equivalent to its rate of synthesis. Evidence indicates that this component of the accumulation process involves active transport of sucrose against a concentration gradient. The ratio of sucrose/reducing sugars in the accumulated sugars immediately after a period of uptake was inversely related to the level of inner space invertase. Within 16 hours after a period of accumulation, practically all of the sugar occurs as glucose and fructose.

The absence of competition among hexoses and sucrose indicated that a common carrier was not involved in their uptake. From a series of studies on the kinetics of uptake of glucose and fructose, including competition studies, the effects of inhibitors, radioactive assay of accumulated sugars and the distribution of label in accumulated sucrose it appeared that rate limitation for glucose or fructose uptake resides in the sequence of reactions leading to sucrose synthesis, rather than in a process mediated by a carrier protein.

     

Phenotypic consequences resulting from a methionine-to-valine substitution at position 48 in the HPr protein of Streptococcus salivarius

In gram-positive bacteria, the HPr protein of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) can be phosphorylated on a histidine residue at position 15 (His(15)) by enzyme I (EI) of the PTS and on a serine residue at position 46 (Ser(46)) by an ATP-dependent protein kinase (His approximately P and Ser-P, respectively). We have isolated from Streptococcus salivarius ATCC 25975, by independent selection from separate cultures, two spontaneous mutants (Ga3.78 and Ga3.14) that possess a missense mutation in ptsH (the gene encoding HPr) replacing the methionine at position 48 by a valine. The mutation did not prevent the phosphorylation of HPr at His(15) by EI nor the phosphorylation at Ser(46) by the ATP-dependent HPr kinase. The levels of HPr(Ser-P) in glucose-grown cells of the parental and mutant Ga3.78 were virtually the same. However, mutant cells growing on glucose produced two- to threefold less HPr(Ser-P)(His approximately P) than the wild-type strain, while the levels of free HPr and HPr(His approximately P) were increased 18- and 3-fold, respectively. The mutants grew as well as the wild-type strain on PTS sugars (glucose, fructose, and mannose) and on the non-PTS sugars lactose and melibiose. However, the growth rate of both mutants on galactose, also a non-PTS sugar, decreased rapidly with time. The M48V substitution had only a minor effect on the repression of alpha-galactosidase, beta-galactosidase, and galactokinase by glucose, but this mutation abolished diauxie by rendering cells unable to prevent the catabolism of a non-PTS sugar (lactose, galactose, and melibiose) when glucose was available. The results suggested that the capacity of the wild-type cells to preferentially metabolize glucose over non-PTS sugars resulted mainly from inhibition of the catabolism of these secondary energy sources via a HPr-dependent mechanism. This mechanism was activated following glucose but not lactose metabolism, and it did not involve HPr(Ser-P) as the only regulatory molecule.     

Mycoplasma phosphoenolpyruvate-dependent sugar phosphotransferase system: glucose-negative mutant and regulation of intracellular cyclic AMP.

Glucose-negative mutants of Mycoplasma capricolum were selected for growth on fructose in the presence of the toxic glucose analog alpha-methyl-D-glucopyranoside. The mutants are defective in the phosphoenolpyruvate:sugar phosphotransferase system for glucose. One mutant, pts-4, was studied in detail. It lacks the glucose-specific, membrane-bound enzyme II, IIGlc, as well as the general, low-molecular-weight, phosphocarrier protein, HPr. In place of the latter, however, it has a fructose-specific protein, HPrFru. Consistent with these changes, the mutant lost the ability to grow on glucosamine and maltose but retained its ability to grow on sucrose. In the glucose-negative mutant, glucose did not regulate the intracellular concentration of cyclic AMP. The intracellular concentration of cyclic AMP in M. capricolum is regulated by the presence of metabolizable sugars. In the wild-type, both glucose and fructose reduced the intracellular concentration of cyclic AMP; however, in the glucose-negative mutant, glucose no longer regulated the intracellular level of cyclic AMP.     

Cellular levels, excretion, and synthesis rates of cyclic AMP in Escherichia coli grown in continuous culture.

Changes in dilution rate did not elicit large and systematic changes in cellular cyclic AMP levels in Escherichia coli grown in a chemostat under carbon or phosphate limitation. However, the technical difficulties of measuring low levels of cellular cyclic AMP in the presence of a large background of extracellular cyclic AMP precluded firm conclusions in this point. The net rate of cyclic AMP synthesis increased exponentially with increasing dilution rate through either the entire range of dilution rates examined (phosphate limitation) or a substantial part of the range (lactose and glucose limitations). Thus, it is probable that growth rate regulates the synthesis of adenylate cyclase. The maximum rate of net cyclic AMP synthesis was greater under lactose than under glucose limitation, which is consistent with the notion that the uptake of phosphotransferase sugars is more inhibitory to adenylate cyclase than the uptake of other carbon substrates. Phosphate-limited cultures exhibited the lowest rate of net cyclic AMP synthesis, which could be due to the role of phosphorylated metabolites in the regulation of adenylate cyclase activity. Under all growth conditions examined, greater than 99.9% of the cyclic AMP synthesized was found in the culture medium. The function of this excretion, which consumed up to 9% of the total energy available to the cell and which evidently resulted from elaborate regulatory mechanisms, remains entirely unknown.     

Carbon Catabolite Repression Regulates Glyoxylate Cycle Gene Expression in Cucumber

We have previously proposed that metabolic status is important in the regulation of cucumber malate synthase (MS) and isocitrate lyase (ICL) gene expression during plant development. In this article, we used a cell culture system to demonstrate that intracellular metabolic status does influence expression of both of these genes. Starvation of cucumber cell cultures resulted in the coordinate induction of the expression of MS and ICL genes, and this effect was reversed when sucrose was returned to the culture media. The induction of gene expression was closely correlated with a drop in intracellular sucrose, glucose, and fructose below threshold concentrations, but it was not correlated with a decrease in respiration rate. Glucose, fructose, or raffinose in the culture media also resulted in repression of MS and ICL. Both 2-deoxyglucose and mannose, which are phosphorylated by hexokinase but not further metabolized, specifically repressed MS and ICL gene expression relative to a third glyoxylate cycle gene, malate dehydrogenase. However, the addition of 3-methylglucose, an analog of glucose that is not phosphorylated, did not result in repression of either MS or ICL. It is proposed that the signal giving rise to a change in gene expression originates from the intracellular concentration of hexose sugars or the flux of hexose sugars into glycolysis.     

Enterotoxin A synthesis in Staphylococcus aureus: inhibition by glycerol and maltose

Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS- mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of beta-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.     

Various Hexoses and di-hexoses Differently Influence Growth,Morphology and Pigment Synthesis in Transformed Root Cultures of Red Beet (Beta vulgaris)

Red beet hairy root cultures, obtained after genetic transformation with Agrobacterium rhizogenes, are completely heterotrophic and synthesize betalaines (BNs). Upon subjecting the hairy roots to treatments containing different sugars (3% w/v) it was found that sucrose was rapidly utilized, followed by maltose, and a very limited use of glucose, but the other hexoses – fructose, lactose, xylose and galactose or glycerol totally suppressed both growth and BN synthesis. No habituation or adaptability to maltose or glucose occurred, evidenced by the lack of growth upon re-culture in respective medium. Glycerol, was not taken up alone, but was utilized to a considerable extent in the presence of low levels of sucrose for growth only but not BN synthesis. Red beet hairy root culture did not exogenously hydrolyse sucrose to hexoses, as there were only traces of reducing sugar present in the medium soon after inoculation, without an increase later, confirmed by HPLC. There was an increase in medium osmolarity in the presence of fructose indicating the exudation of certain compounds from the roots. Red beet hairy roots appear useful as a model system to study sugar metabolism/signalling due to their sensitivity to different sugars that may directly link to morphological changes and BN synthesis.     

Growth rate,sugar consumption,chlortetracycline production and anhydrotetracycline oxygenase activity inStreptomyces aureofaciens

The relationship between the activity of ATC oxygenase, CTC production and growth rate was investigated in a low-producing strain ofStreptomyces aureofaciens, closely related to the wildtype strain, and in a higher-producing variant. Different growth rates were achieved by using glucose, fructose and sucrose as carbon sources. Activity of the enzyme and CTC yield in both strains were inversely proportional to the rate of sugar utilization but in the higherproducing variant sugar utilization was slower than in the low-producing strain. The expression of ATC oxygenase was less sensitive to “catabolite repression” in the higherproduc ing strain. BT, a stimulator of CTC production, markedly inhibited growth of the higher-producing variant in a medium with slowly utilized sugars (fructose and sucrose) but had little effect on growth of the lowproducing strain. It also increased the level of ATC oxygenase in both strains under all experimental conditions. It could be established that there was no obligatory relationship between the increase of antibiotic synthesis and the increase of enzyme activity.     

Biosynthesis of growth hormone in the rat anterior pituitary gland control of biosynthesis in vitro by glucose

We have studied the effect of altering the concentration of glucose (in the incubation medium) on the ability of rat pituitary lobes, incubated in vitro, to incorporate [³H]leucine into total protein and growth hormone. Increasing the glucose concentration caused an increase in the rate of total protein synthesis, but a decrease in growth hormone synthesis.Phloridzin and 2-deoxyglucose both lowered the rate of growth hormone synthesis; they were more effective in media containing 2.8 mM glucose than in 28 mM glucose. Various alternative sugars were substituted for D-glucose in the medium; they had differing effects on the synthesis of total pituitary protein and growth hormone. None of these other sugars was metabolised as effectively as glucose at physiological concentrations, though all could be utilised to some extent.The physiological role of the effect of glucose on growth hormone synthesis is discussed, particularly with reference to what is known about the control of insulin synthesis by glucose. It is proposed that glucose interacts with two control systems in the pituitary. One is concerned with growth hormone synthesis and the other influences the rate of synthesis of other (unidentified) proteins.     

On the regulation of adenosine 3', 5'-monophosphate synthesis in bacteria. I. Effect of carbon source variation on cyclic AMP synthesis in Escherichia coli B/r.

1. The effect of carbon source variation in bacterial growth media on their growth rate, inducible enzyme and cyclic AMP synthesis was examined: an inverse relationship between the culture's growth rate and its differential rate of inducible enzyme (tryptophanase and beta-galactosidase), and cyclic AMP synthesis was found. 2. The effect of the culture's growth phase on its sensitivity or resistance to glucose catabolite repression was determined in the wild type and a catabolite insensitive mutant (ABDROI): the wild type's sensitivity to glucose repression was not affected, whereas the insensitivity of the mutant was found to be limited to its early logarithmic phase of growth. At late log, or stationary phase, the mutant was found to be sensitive to glucose repression. 3. Examination of the kinetics of glucose uptake by the mutant, using alpha-[1 4-C] methyl-glucoside showed evidence for two transport systems each with a different affinity to glucose. A low affinity transport system (apparent Km of 3.4-10-minus 5 M) which appears mostly at the early logarithmic phase of growth. A high affinity transport system (apparent Km of 1.2-10-minus 5 M) which appears mostly at the late log and stationary phases of growth. 4. The effect of the culture density variation on its sensitivity to glucose repression showed that sensitivity to glucose catabolic repression is primarily a reflection of the formation of an allosteric effector molecule between glucose and its specific transport molecule which in turn regulates the activity of the adenylate cyclase.     

Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena

Summary Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3′:5′-cyc lic monophosphate (cAMP) (1). To characterize further the initial steps in the control of galactokinase gene-expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2). In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected. Radiochromatographic analyses of extracts of wild-type cells incubated with [¹⁴C]-deoxyglucose reveal intracellular conversio to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglunose 1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P. The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase. These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels. Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic. Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

Correlation between growth rates, EIIACrr phosphorylation, and intracellular cyclic AMP levels in Escherichia coli K-12

In Escherichia coli K-12, components of the phosphoenolpyruvate-dependent phosphotransferase systems (PTSs) represent a signal transduction system involved in the global control of carbon catabolism through inducer exclusion mediated by phosphoenolpyruvate-dependent protein kinase enzyme IIA(Crr) (EIIA(Crr)) (= EIIA(Glc)) and catabolite repression mediated by the global regulator cyclic AMP (cAMP)-cAMP receptor protein (CRP). We measured in a systematic way the relation between cellular growth rates and the key parameters of catabolite repression, i.e., the phosphorylated EIIA(Crr) (EIIA(Crr) approximately P) level and the cAMP level, using in vitro and in vivo assays. Different growth rates were obtained by using either various carbon sources or by growing the cells with limited concentrations of glucose, sucrose, and mannitol in continuous bioreactor experiments. The ratio of EIIA(Crr) to EIIA(Crr) approximately P and the intracellular cAMP concentrations, deduced from the activity of a cAMP-CRP-dependent promoter, correlated well with specific growth rates between 0.3 h(-1) and 0.7 h(-1), corresponding to generation times of about 138 and 60 min, respectively. Below and above this range, these parameters were increasingly uncoupled from the growth rate, which perhaps indicates an increasing role executed by other global control systems, in particular the stringent-relaxed response system.     

Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Sugar utilization by yeast during fermentation

Summary When glucose and fructose are fermented separately, the uptake profiles indicate that both sugars are utilized at similar rates. However, when fermentations are conducted in media containing an equal concentration of glucose and fructose, glucose is utilized at approximately twice the rate of fructose. The preferential uptake of glucose also occurred when sucrose, which was first rapidly hydrolyzed into glucose and fructose by the action of the enzyme invertase, was employed as a substrate. Similar results were observed in the fermentation of brewer's wort and wort containing 30% sucrose and 30% glucose as adjuncts. In addition, the high levels of glucose in the wort exerted severe catabolite repression on maltose utilization in theSaccharmyces uvarum (carlsbergensis) brewing strain. Kinetic analysis of glucose and fructose uptake inSaccharomyces cerevisiae revealed aK _m of 1.6 mM for glucose and 20 mM for fructose. Thus, the yeast strain has a higher affinity for glucose than fructose. Growth on glucose or fructose had no repressible effect on the uptake of either sugar. In addition, glucose inhibited fructose uptake by 60% and likewise fructose inhibited, glucose uptake by 40%. These results indicate that glucose and fructose share the same membrane transport components.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.