The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of insulin, adrenaline and some metabolites in vitro

1. Adipose tissues from rats fed a balanced diet were incubated in the presence of glucose (20mm) with the following additions: insulin, anti-insulin serum, insulin+acetate, insulin+pyruvate, insulin+lactate, insulin+phenazine methosulphate, insulin+oleate+albumin, insulin+adrenaline+albumin, insulin+6-N-2'-O-dibutyryl 3':5'-cyclic AMP+albumin. 2. Measurements were made of the whole tissue concentrations of adenine nucleotides, hexose phosphates, triose phosphates, glycerol 1-phosphate, 3 phosphoglycerate, 6-phosphogluconate, long-chain fatty acyl-CoA, acid-soluble CoA, citrate, isocitrate, malate and 2-oxoglutarate, and of the release into the incubation medium of lactate, pyruvate and glycerol after 1h of incubation. 3. Fluxes of [(14)C]glucose carbon through the major pathways of glucose metabolism were calculated from the yields of (14)C in various products after 2h of incubation. Fluxes of [(14)C]acetate, [(14)C]pyruvate or [(14)C]lactate carbon in the presence of glucose were also determined. 4. Measurements were also made of the whole-tissue concentrations of metabolites in tissues taken directly from Nembutal-anaesthetized rats. 5. Whole tissue mass-action ratios for phosphofructokinase, phosphoglucose isomerase and the combined (aldolasextriose phosphate isomerase) reaction were similar in vivo and in vitro. The reactants of phosphofructokinase appeared to be far from mass-action equilibrium. In vitro, the reactants of hexokinase also appeared to be far from mass-action equilibrium. 6. Correlation of observed changes in glycolytic flux with changes in fructose 6-phosphate concentration suggested that phosphofructokinase may show regulatory behaviour. The enzyme appeared to be activated in the presence of oleate or adrenaline and to be inhibited in the presence of lactate or pyruvate. 7. Evidence is presented that the reactants of lactate dehydrogenase and glycerol 1-phosphate dehydrogenase may be near to mass-action equilibrium in the cytoplasm. 8. No satisfactory correlations could be drawn between the whole-tissue concentrations of long-chain fatty acyl-CoA, citrate and glycerol 1-phosphate and the observed rates of triglyceride and fatty acid synthesis. Under the conditions employed, the concentration of glycerol 1-phosphate appeared to depend mainly on the cytoplasmic [NAD(+)]/[NADH] ratios. 9. Calculated hexose monophosphate pathway flux rates roughly correlated with fatty acid synthesis rates and with whole tissue [6-phosphogluconate]/[glucose 6-phosphate] ratios. The relative rates of production of NADPH for fatty acid synthesis by the hexose monophosphate pathway and by the ;malic enzyme' are discussed. It is suggested that all NADH produced in the cytoplasm may be used in that compartment for reductive synthesis of fatty acids, lactate or glycerol 1-phosphate.     

Formation of glycerol from glucose in rat brain and cultured brain cells. Augmentation with kainate or ischemia

An increase in the concentration of glycerol in the ischemic brain is assumed to reflect degradation of phospholipids of plasma membranes. However, glycerol could, theoretically, be formed from glucose, which after glycolytic conversion to dihydroxyacetone phosphate, could be converted to glycerol-3-phosphate and hence to glycerol. We show here that (13)C-labeled glycerol accumulate in incubation media of cultured cerebellar granule cells and astrocytes incubated with [(13)C]glucose, 3 mmol/L, demonstrating the formation of glycerol from glucose. Co-incubation of cerebellar granule cells with kainate, 50 micromol/L, led to increased glucose metabolism and increased accumulation of [(13)C]glycerol. Accumulation of [(13)C]glycerol and its precursor, [(13)C]glycerol-3-phosphate, was evident in brain, but not in serum, of kainate-treated rats that received [U-(13)C]glucose, 5 micromol/g bodyweight, intravenously and survived for 5 min. Global ischemia induced by decapitation also caused accumulation of [(13)C]glycerol and [(13)C]glycerol-3-phosphate. These results show that glycerol can be formed from glucose in brain; they also demonstrate the existence of a cerebral glycerol-3-phosphatase activity. Ischemia-induced increases in brain glycerol may, in part, reflect an altered metabolism of glucose, in which glycerol formation, like lactate formation, acts as a redox sink.     

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.     

Normal Myelin Composition and Phospholipid Synthesis in Adrenalectomized Rats with Reduced Brain Myelin and Glycerol 3-Phosphate Dehydrogenase Activity

The composition of CNS myelin was investigated in rats adrenalectomized at day 14 and killed 7 days later, previously shown to result in a 25% reduction in the amount of bulk-isolated myelin and a 40% decrease in brain glycerol 3-phosphate dehydrogenase activity. The proportions of the major myelin proteins, as well as the specific activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase, were the same in the myelin from both adrenalectomized and control animals. The amount of total phospholipid and the proportions of individual phospholipids were also normal in myelin from the adrenalectomized animals. The amount of nonmyelin phospholipid in whole brain was unchanged by adrenalectomy. Labeling studies carried out 4 days after adrenalectomy of 14-day-old animals showed no change in the synthesis rates of the major myelin phospholipids as compared with the synthesis rate of nonmyelin phospholipids. Furthermore, incorporation of [1,(3)-14C]glycerol into the glycerol moiety of ethanolamine plasmalogen, which requires glycerol 3-phosphate dehydrogenase, was also normal, showing that the reduced oligodendroglial glycerol 3-phosphate dehydrogenase activity following adrenalectomy was not rate-limiting for myelin phospholipid synthesis.     

Kinetics of metabolic pathways. A system in vitro to study the control of flux.

A method for determining Control Coefficients is proposed for systems studied in vitro and applied to a model pathway. Rat liver extract, which converts glucose into glycerol 3-phosphate, was used with the addition to the incubation mixture of fructose-bisphosphate aldolase, triose-phosphate isomerase and glycerol-3-phosphate dehydrogenase as 'auxiliary' enzymes, which leaves all the control on the first three enzymes. The flux of the metabolic pathway was recorded by assaying NADH decay. Flux Control Coefficients (CJE) of hexokinase, glucose-6-phosphate isomerase and phosphofructokinase were calculated by titration of the system with increasing quantities of extraneous enzymes. It is shown that the summation property is fulfilled. The applicability of this procedure to study the control in any metabolic pathway is discussed. Possible relevance of the method to conditions in vivo and its limitations are considered.     

Implications of glycerol metabolism for lipid production

Triacylglycerol (TAG) is an important product in oil-producing organisms. Biosynthesis of TAG can be completed through either esterification of fatty acids to glycerol backbone, or through esterification of 2-monoacylglycerol. This review will focus on the former pathway in which two precursors, fatty acid and glycerol-3-phosphate (G3P), are required for TAG formation. Tremendous progress has been made about the enzymes or genes that regulate the biosynthetic pathway of TAG. However, much attention has been paid to the fatty acid provision and the esterification process, while the possible role of G3P is largely neglected. Glycerol is extensively studied on its usage as carbon source for value-added products, but the modification of glycerol metabolism, which is directly associated with G3P synthesis, is seldom recognized in lipid investigations. The relevance among glycerol metabolism, G3P synthesis and lipid production is described, and the role of G3P in glycerol metabolism and lipid production are discussed in detail with an emphasis on how G3P affects lipid production through the modulation of glycerol metabolism. Observations of lipid metabolic changes due to glycerol related disruption in mammals, plants, and microorganisms are introduced. Altering glycerol metabolism results in the changes of final lipid content. Possible regulatory mechanisms concerning the relationship between glycerol metabolism and lipid production are summarized.     

Metabolic changes induced during adaptation of Saccharomyces cerevisiae to a water stress

When exponentially growing Saccharomyces cerevisiae was transferred from a normal high water activity growth medium (a_w 0.997) to a medium containing 8% NaCl low water activity growth medium (a_w 0.955), glycerol accumulation during the first eight hours of the adaptation was both retarded and greatly diminished in magnitude. Investigation of the underlying reasons for the slow onset of glycerol accumulation revealed that not only was overall glycerol production reduced by salt transfer, but also the rates of ethanol production and glucose consumption were reduced. Measurement of glycolytic intermediates revealed an accumulation of glucose-6-phosphate, fructose-6-phosphate, fructose 1,6 bisphosphate and phosphoenolpyruvate in S. cerevisiae 3 to 4 h after transfer to salt, suggesting that one or more glycolytic enzymes were inhibited. Potassium ions accumulated in S. cerevisiae after salt transfer and reached a maximum about 6 h after transfer, whereas the sodium ion content increased progressively during the adaptation period. The trehalose content also increased in adapting cells. It is suggested that inhibition of glycerol production during the initial period of adaptation could be due to either the inhibition of glycerol-3-phosphate dehydrogenase by increased cation content or the inhibitin of glycolysis, glycerol being produced glycolytically in S. cerevisiae. The increased accumulation of glycerol towards the end of the 8-h period suggests that the osmoregulatory response of S. cerevisiae involves complex sets of adjustments in which inhibition of glycerol-3-phosphate dehydrogenase must be relieved before glycerol functions as a major osmoregulator.     

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism.

sn-Glycerol 3-phosphorothioate was found to be bacteriocidal to strains of Escherichia coli which have a functional sn-glycerol 3-phosphate transport system. This effect was manifest in strains 7 and 8, which are constitutive mutants for the utilization and transport of sn-glycerol 3-phosphate (glpRc2). Strain E15, which is considered to be wild type for the glycerol phosphate functional units, was affected by the phosphorothioate analog only under conditions that are known to induce the transport system for sn-glycerol 3-phosphate. In addition, another strain of E. coli, strain 6, which is isogenic with strain E15 but has an impaired sn-glycerol 3-phosphate transport system (glpT13), was not affected by similar concentrations of sn-glycerol 3-phosphorothioate. Transport studies in which [3H]glycerol phosphate and its phosphorothioate analog were used demonstrated that the latter compound was taken up via the specific active transport system for sn-glycerol 3-phosphate; the Km values were 9 and 11 microM, respectively. The rates of macromolecular synthesis were found to be inhibited severely by sn-glycerol 3-phosphorothioate at a concentration at which sn-glycerol 3-phosphate had no effect (5 microM). At a lower concentration of the analog (0.5 microM), the rates of protein synthesis and RNA synthesis (52 and 58% below control values after 90 min, respectively) were more sensitive than the rates of DNA synthesis and cell wall synthesis (18% below control values after 3 h for DNA; transient decrease in the cell wall values after 90 min). The levels of the nucleoside triphosphates were not affected by the presence of the phospholipid precursor or its analog at a concentration of 5 microM. The phospholipid composition was significantly altered in the presence of bacteriocidal concentrations (5 microM) of sn-glycerol 3-phosphorothioate. The amount of phosphatidylglycerol in the membranes decreased from 13.5 to 3.5%. Concomitant with this decrease in phosphatidylglycerol content was a fourfold increase in the 32P content of cardiolipin (from 6.8 to 24.2%), whereas the phosphatidylethanolamine content showed only a minor reduction (8%) after 3 h. The rates of synthesis of all of the phospholipids decreased in the presence of 5 microM sn-glycerol 3-phosphorothioate, with the most significant effects observed for phosphatidylglycerol (63% after 3 h). Phosphatidylglycerol showed increased rates of turnover after 90 min (21%) and 3 h (11%), with concomitant increases in the levels of cardiolipin of more than twofold. Our data suggest that a considerably greater proportion of phosphatidylglycerol turnover may be recover in cardiolipin than is metabolized via other pathways (e.g., the membrane-derived oligosaccharide pathway).     

Phenethyl alcohol inhibition of sn-glycerol 3-phosphate acylation in Escherichia coli.

In vivo and in vitro experiments were performed to determine how phenethyl alcohol (PEA) inhibits phospholipid synthesis in Escherichia coli. This drug drastically reduced the rate of incorporation of sn-glycerol 3-phosphate into the phospholipids of an sn-glycerol 3-phosphate auxotroph. PEA also reduced the rate of fatty acid incorporation into the phospholipids of a fatty acid auxotroph. The kinetics of PEA inhibition of the rate of incorporation of sn-glycerol 3-phosphate were almost identical to those of PEA inhibition of the rate of fatty acid incorporation into phospholipids. The in vivo experiments suggested that the rate-limiting step(s) in phospholipid biosynthesis inhibited by PEA is at the level of the acylation of sn-glycerol 3-phosphate or beyond this step. PEA inhibited the sn-glycerol 3-phosphate acyltransferase with either palmitoyl coenzyme A or palmitoyl-acyl carrier protein as the acyl donor. This drug, however, had no effect on the cytidine 5'-diphosphate-diglyceride:glycerol 3-phosphate phosphatidyl transferase, cytidine 5'-diphosphate-diglyceride:L-serine phosphatidyl transferase, and acyl coenzyme A:lysophatidic acid acyltransferase. The in vitro findings suggested that PEA inhibits phospholipid synthesis primarily at the level of sn-glycerol 3-phosphate acyltransferase.     

The regulation of triglyceride synthesis and fatty acid synthesis in rat epididymal adipose tissue. Effects of altered dietary and hormonal conditions

1. Epididymal adipose tissues obtained from rats that had been previously starved, starved and refed a high fat diet for 72h, starved and refed bread for 144h or fed a normal diet were incubated in the presence of insulin+glucose or insulin+glucose+acetate. 2. Measurements were made of the whole-tissue concentrations of hexose phosphates, triose phosphates, glycerol 1-phosphate, 3-phosphoglycerate, 6-phosphogluconate, adenine nucleotides, acid-soluble CoA, long-chain fatty acyl-CoA, malate and citrate after 1h of incubation. The release of lactate, pyruvate and glycerol into the incubation medium during this period was also determined. 3. The rates of metabolism of glucose in the hexose monophosphate pathway, the glycolytic pathway, the citric acid cycle and into glyceride glycerol, fatty acids and lactate+pyruvate were also determined over a 2h period in similarly treated tissues. The metabolism of acetate to CO(2) and fatty acids in the presence of glucose was also measured. 4. The activities of acetyl-CoA carboxylase, fatty acid synthetase and isocitrate dehydrogenase were determined in adipose tissues from starved, starved and fat-refed, and alloxan-diabetic animals and also in tissues from animals that had been starved and refed bread for up to 96h. Changes in these activities were compared with the ability of similar tissues to incorporate [(14)C]glucose into fatty acids in vitro. 5. The activities of acetyl-CoA carboxylase and fatty acid synthetase roughly paralleled the ability of tissues to incorporate glucose into fatty acids. 6. Rates of triglyceride synthesis and fatty acid synthesis could not be correlated with tissue concentrations of long-chain fatty acyl-CoA, citrate or glycerol 1-phosphate. In some cases changes in phosphofructokinase flux rates could be correlated with changes in citrate concentration. 7. The main lesion in fatty acid synthesis in tissues from starved, starved and fat-refed, and alloxan-diabetic rats appeared to reside at the level of pyruvate utilization and to be related to the rate of endogenous lipolysis. 8. It is suggested that pyruvate utilization by the tissue may be regulated by the metabolism of fatty acids within the tissue. The significance of this in directing glucose utilization away from fatty acid synthesis and into glyceride-glycerol synthesis is discussed.     

The effect of 3-methylindole on the rates of phospholipid and neutral lipid synthesis in cultured fibroblasts

The present study was designed to test the hypothesis that a pneumotoxin, 3-methylindole, alters the basic metabolic pathways involved in phospholipid and neutral lipid synthesis in cultured fibroblasts. Rat skin fibroblasts were obtained from day-old pups. Confluent monolayers were preincubated for up to 24 h with a range of concentrations (0-0.76 mM) of 3-methylindole. Following these treatments, the cell lipids were labelled by incubation for 6 h with [14C]glycerol. The lipids were extracted, separated by thin layer chromatography, and the radioactivity in each fraction was determined. 3-Methylindole had no effect on the total incorporation of [14C]glycerol into lipids, but significantly altered the distribution among lipid fractions. Incubation with 3-methylindole caused a decrease in the incorporation of [14C]glycerol into phosphatidylcholine, while radioactivity accumulated in the neutral lipid fraction. The other lipid fractions responded variably. Similarily, Flow 2000 human diploid lung fibroblasts were incubated for 24 h with 3-methylindole followed by treatment with [14C]glycerol, resulting in a 74% decrease in the incorporation of [14C]glycerol into phosphatidylcholine and a 50% increase in its accumulation in neutral lipid. The results indicate that 3-methylindole inhibits the synthesis of phosphatidylcholine from diacylglycerol precursors on the endoplasmic reticulum in cultured fibroblasts. This is an important observation as it shows that 3-methylindole affects the synthesis of phospholipids required for membrane turnover in cells that are not specialized for the production of phospholipids for surfactant.     

The fate of 14C in glucose 6-phosphate synthesized from [1-14C]Ribose 5-phosphate by enzymes of rat liver.

1. Glucose 5-phosphate was synthesized from ribose 5-phosphate by an enzyme extract prepared from an acetone-dried powder of rat liver. Three rates of ribose 5-phosphate utilization were observed during incubation for 17 h. An analysis of intermediates and products formed throughout the incubation revealed that as much as 20% of the substrate carbon could not be accounted for. 2. With [1-14C]ribose 5-phosphate as substrate, the specific radioactivity of [14C]glucose 6-phosphate formed was determined at 1, 2, 5 and 30 min and 3, 8 and 17 h. It increased rapidly to 1.9-fold the initial specific radioactivity of [1-14C]ribose 5-phosphate at 3 h and then decreased to a value approximately equal to that of the substrate at 6 h, and finally at 17 h reached a value 0.8-fold that of the initial substrate [1-14C]ribose 5-phosphate. 3. The specific radioactivity of [14C]ribose 5-phosphate decreased to approx. 50% of its inital value during the first 3 h of the incubation and thereafter remained unchanged. 4. The distribution of 14C in the six carbon atoms of [14C]glucose 6-phosphate formed from [1-14C]ribose 5-phosphate at 1, 2, 5 and 30 min and 3, 8 and 17 h was determined. The early time intervals (1--30 min) were characterized by large amounts of 14C in C-2 and in C-6 and with C-1 and C-3 being unlabelled. In contrast, the later time intervals (3--17 h) were characterized by the appearance of 14C in C-1 and C-3 and decreasing amounts of 14C in C-2 and C-6. 5. It is concluded that neither the currently accepted reaction sequence for the non-oxidative pentose phosphate pathway nor the 'defined' pentose phosphate-cycle mechanism can be reconciled with the labelling patterns observed in glucose 6-phosphate formed during the inital 3 h of the incubation.     

Mode of action of alpha-chlorohydrin as a male anti-fertility agent. Inhibition of the metabolism of ram spermatozoa by alpha-chlorohydrin and location of block in glycolysis

1. The effect of alpha-chlorohydrin on the metabolism of glycolytic and tricarboxylate-cycle substrates by ram spermatozoa was investigated. The utilization and oxidation of fructose and triose phosphate were much more sensitive to inhibition by alpha-chlorohydrin (0.1-1.0mm) than lactate or pyruvate. Inhibition of glycolysis by alpha-chlorohydrin is concluded to be between triose phosphate and pyruvate formation. Oxidation of glycerol was not as severely inhibited as that of the triose phosphate. This unexpected finding can be explained in terms of competition between glycerol and alpha-chlorohydrin. A second, much less sensitive site, of alpha-chlorohydrin inhibition appears to be associated with production of acetyl-CoA from exogenous and endogenous fatty acids. 2. Measurement of the glycolytic intermediates after incubation of spermatozoal suspensions with 15mm-fructose in the presence of 3mm-alpha-chlorohydrin showed a ;block' in the conversion of glyceraldehyde 3-phosphate into 3-phosphoglycerate. alpha-Chlorohydrin also caused conversion of most of the ATP in spermatozoa into AMP. After incubation with 3mm-alpha-chlorohydrin, glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase activities were decreased by approx. 90% and 80% respectively, and in some experiments aldolase was also inhibited. Other glycolytic enzymes were not affected by a low concentration (0.3mm) of alpha-chlorohydrin. Loss of motility of spermatozoa paralleled the decrease in glyceraldehyde 3-phosphate dehydrogenase activity. alpha-Chlorohydrin, however, did not inhibit glyceraldehyde 3-phosphate dehydrogenase or triose phosphate isomerase in sonicated enzyme preparations when added to the assay cuvette. 3. Measurement of intermediates and glycolytic enzymes in ejaculated spermatozoa before, during and after injection of rams with alpha-chlorohydrin (25mg/kg body wt.) confirmed a severe block in glycolysis in vivo at the site of triose phosphate conversion into 3-phosphoglycerate within 24h of the first injection. Glyceraldehyde 3-phosphate dehydrogenase activity was no longer detectable and both aldolase and triose phosphate isomerase were severely inhibited. Spermatozoal ATP decreased by 92% at this time, being quantitatively converted into AMP. At 1 month after injection of alpha-chlorohydrin glycolytic intermediate concentrations returned to normal in the spermatozoa but ATP was still only 38% of the pre-injection concentration. Motility of spermatozoa was, however, as good as during the pre-injection period. The activity of the inhibited enzymes also returned to normal during the recovery period and 26 days after injection were close to pre-injection values. 4. An unknown metabolic product of alpha-chlorohydrin is suggested to inhibit glyceraldehyde 3-phosphate dehydrogenase and triose phosphate isomerase of spermatozoa. This results in a lower ATP content, motility and fertility of the spermatozoa. Glycidol was shown not to be an active intermediate of alpha-chlorohydrin in vitro.     

Glycerol kinase activity and glycerol metabolism of rat granular pneumocytes in primary culture

Glycerol kinase activity and glycerol utilization by rat granular pneumocytes were determined in order to investigate the rate-limiting step for glycerol incorporation into lung lipids. Granular pneumocytes were isolated in primary culture following trypsinization of rat lungs. Glycerol kinase activity was 8.2 nmol/h per 10(6) cells. Incorporation of [1,3-14C]glycerol into total cell lipids was 0.29 nmol/h per 10(6) cells. In the presence of saturating glycerol concentration, production of 3H2O from [2-3H]glycerol was 13 times greater than incorporation of [14C]glycerol into lipids. Glycerol phosphate dehydrogenase activity in isolated cells was approximately 10 times glycerol kinase activity. In the presence of 5.6 mM glucose, glycerol incorporation into lipids was decreased 79% and detritiation of glycerol was decreased 34%. This effect of glucose was due to a 25% increase in cell glycerol 3-phosphate content, resulting in dilution of the precursor pool and possible inhibition of glycerol phosphorylation. These results indicate that the relatively limited incorporation of glycerol into surfactant phospholipids by lung epithelial cells reflects the relatively high rate of glycerol 3-phosphate oxidation.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Comparative analysis on the key enzymes of the glycerol cycle metabolic pathway in Dunaliella salina under osmotic stresses

The glycerol metabolic pathway is a special cycle way; glycerol-3-phosphate dehydrogenase (G3pdh), glycerol-3-phosphate phosphatase (G3pp), dihydroxyacetone reductase (Dhar), and dihydroxyacetone kinase (Dhak) are the key enzymes around the pathway. Glycerol is an important osmolyte for Dunaliella salina to resist osmotic stress. In this study, comparative activities of the four enzymes in D. salina and their activity changes under various salt stresses were investigated, from which glycerol metabolic flow direction in the glycerol metabolic pathway was estimated. Results showed that the salinity changes had different effects on the enzymes activities. NaCl could stimulate the activities of all the four enzymes in various degrees when D. salina was grown under continuous salt stress. When treated by hyperosmotic or hypoosmotic shock, only the activity of G3pdh in D. salina was significantly stimulated. It was speculated that, under osmotic stresses, the emergency response of the cycle pathway in D. salina was driven by G3pdh via its response to the osmotic stress. Subsequently, with the changes of salinity, other three enzymes started to respond to osmotic stress. Dhar played a role of balancing the cycle metabolic pathway by its forward and backward reactions. Through synergy, the four enzymes worked together for the effective flow of the cycle metabolic pathways to maintain the glycerol requirements of cells in order to adapt to osmotic stress environments.     

Novel fermentation strategy for enhancing glycerol production by Candida krusei

During the later stage of glycerol production by fermentation of Candida krusei, glycerol consumption by the strain was observed, although there was residual sugar in the medium. To enhance the final glycerol accumulation, a new fermentation strategy was developed by maintaining high activities of glycerol synthetic enzymes (i.e., glycerol-3-phosphate dehydrogenase (ctGPD) and glycerol-3-phosphatase (GPP)) for a relatively long period while conducting oxygen limitation at a later stage to inhibit the increase of another enzyme activity related to glycerol degradation (i.e., mitochondrial glycerol-3-phosphate dehydrogenase (mtGPD)). With oxygen limitation performed from 88 h, when ctGPD and GPP activities were already at a low level while mtGPD activity was increasing, the glycerol dissimilation was efficiently reduced. The final glycerol concentration reached 55.6 g/L, which was about 18% (96 h) and 30% (104 h) higher than control, and its productivity increased to 0.54 g/(L h). The proposed strategy based on cell physiology was proved useful to the glycerol fermentation process.     

产甘油假丝酵母产甘油关键酶基因在酿酒酵母中的表达

甘油是一种极其理想的耐高渗透压介质。利用PCR方法,从产甘油假丝酵母WL2002-5中扩增出了2个产甘油的关键酶基因GPD和GPP,分别编码3-磷酸甘油脱氢酶（glycerol 3-phosphate dehydrogenase, GPD）和3-磷酸甘油磷酸酶（glycerol 3-phosphate phosphatase, GPP）。利用T-Vector在Escherichia coli JM109中克隆得到大量的GPD和GPP基因,并成功构建了重组质粒pYX212-GPD和pYX212-GPP;通过LiAc转化法将重组质粒导入酿酒酵母Saccharomyces cerevisiae W303-1A。初步实验结果表明：发酵过程中pYX212-GPD/S. cerevisiae W303-1A的生物量高于pYX212-GPP/S. cerevisiae W303-1A和野生型S. cerevisiae W303-1A;发酵72h后,pYX212 GPD/S. cerevisiae W303-1A发酵液中甘油含量大约为12mmol/L,明显高于野生型S. cerevisiae W303-1A的甘油含量,而pYX212-GPP/S. cerevisiae W303-1A与野生型S. cerevisiae W303-1A在甘油含量上相差不大,均只有4mmol/L 左右。