Regulation of alcohol oxidase of a recombinant Pichia pastoris Mut+ strain in transient continuous cultures

In the methylotrophic yeast Pichia pastoris, alcohol oxidase (AOX) is a key enzyme involved in the dissimilation of methanol. Heterologous proteins are usually expressed under the control of the AOX1 promoter, which drives the expression of alcohol oxidase 1 in the wild-type strain. This study investigates the regulation of the alcohol oxidase enzyme of a recombinant P. pastoris Mut+ strain in cultures on glycerol and methanol as sole carbon sources and in mixed substrate cultures on both substrates. The aim was to have a better insight in the transition from growth on glycerol to growth on methanol, which is a key step in standard high cell density P. pastoris cultures for the production of foreign proteins. Nutrient shifts in chemostat cultures showed that after growth on glycerol use of mixed feeds of glycerol and methanol allowed faster induction of alcohol oxidase and faster adaptation of cellular metabolism than with a feed containing methanol as sole carbon source. The results of this study showed also how critical it is to avoid transient methanol accumulation during P. pastoris cultures operated at low residual methanol concentrations. Indeed, pulse experiments during chemostat cultures showed that sudden increase in methanol concentrations in cultures performed under methanol-limited or dual methanol and glycerol-limited growth conditions leads to wash-out of the culture because of too high consumption rate of methanol, which leads to excretion of toxic intermediates. High rate of methanol consumption was due to high specific AOX activities observed at low residual methanol concentrations.     

The potential of a sucrose ester coating material for improving the storage and shelf-life qualities of Cox's Orange Pippin apples

A sucrose-ester coating material was tested for its potential as a storage technique and as an extender of the shelf life of apple (cv. Cox's Orange Pippin). Apples treated with 1·25% sucrose ester formulation were stored in air at 3·5°C for up to 5 months. Sucrose ester treatment did not reduce detrimental changes in terms of fruit firmness, yellowing and weight loss but did increase core flush incidence. When applied after storage, the sucrose ester reduced yellowing and loss of firmness and markedly increased internal carbon dioxide levels during a 21 day simulated marketing period. Effects were enhanced with increasing sucrose ester concentrations between 1% and 4%. Sucrose ester did not markedly reduce weight loss in the fruit, did not cause accumulation of alcohol or induce any internal physiological disorders during the simulated marketing period. Treatment of fruit with an external atmosphere containing 8% carbon dioxide, a level similar to that found in fruit treated with 3% sucrose ester, did not have the same effects as 3% sucrose ester on firmness or ground colour changes, suggesting that the effects of the sucrose ester are not solely the result of the raised carbon dioxide level.     

Polysaccharide production in liquid cell suspension cultures of Phleum pratense L.

The production of carbohydrates by cell suspension cultures of Phleum pratense (timothy grass) is described. Extracellular polysaccharides similar in monosaccharide composition to native cell wall polymers were accumulated, together with polymers of fructose (fructans). The fructans had similar properties to the intracellular reserve polymers found in intact plants, and were found in both cells and media of young, slow-growing cultures.Production of extracellular polysaccharides differed in cultures grown on sucrose or equimolar glucose/fructose as carbon source. These differences were observed only when autoclaved media were used, and were not related to changes in either pH or osmolarity. Autoclaving medium containing radioactive glucose and fructose produced a novel, unidentified labelled compound which was absent in medium containing labelled sucrose.     

Degradation of tetrahydrofurfuryl alcohol by Ralstonia eutropha is initiated by an inducible pyrroloquinoline quinone-dependent alcohol dehydrogenase.

An organism tentatively identified as Ralstonia eutropha was isolated from enrichment cultures containing tetrahydrofurfuryl alcohol (THFA) as the sole source of carbon and energy. The strain was able to tolerate up to 200 mM THFA in mineral salt medium. The degradation was initiated by an inducible ferricyanide-dependent alcohol dehydrogenase (ADH) which was detected in the soluble fraction of cell extracts. The enzyme catalyzed the oxidation of THFA to the corresponding tetrahydrofuran-2-carboxylic acid. Studies with n-pentanol as the substrate revealed that the corresponding aldehyde was released as a free intermediate. The enzyme was purified 211-fold to apparent homogeneity and could be identified as a quinohemoprotein containing one pyrroloquinoline quinone and one covalently bound heme c per monomer. It was a monomer of 73 kDa and had an isoelectric point of 9.1. A broad substrate spectrum was obtained for the enzyme, which converted different primary alcohols, starting from C2 compounds, secondary alcohols, diols, polyethylene glycol 6000, and aldehydes, including formaldehyde. A sequence identity of 65% with a quinohemoprotein ADH from Comamonas testosteroni was found by comparing 36 N-terminal amino acids. The ferricyanide-dependent ADH activity was induced during growth on different alcohols except ethanol. In addition to this activity, an NAD-dependent ADH was present depending on the alcohol used as the carbon source.     

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia.

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Factors influencing the formation and stability of D-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens.

D-glucoside 3-dehydrogenase specific activity in Agrobacterium tumefaciens was maximal towards the end of the exponential growth phase of batch cultures; over 90% of the activity disappeared within the next 15 h. Manganese ions, although essential for growth of the organism, strongly repressed D-glucoside 3-dehydrogenase synthesis in sucrose medium but had little effect when the carbon source was methyl alpha-D-glucoside. D-Glucoside 3-dehydrogenase activity increased linearly with increasing specific growth rate in chemostat cultures limited by carbon, nitrogen, phosphate or manganese when methyl alpha-D-glucoside was the carbon source. High enzyme activity was found with sucrose as carbon source only when the growth medium was manganese-limited. D-Glucoside 3-dehydrogenase activity disappeared from A. tumefaciens incubated in carbon- and nitrogen-free medium or in nitrogen-free medium containing succinate, but on continued incubation the activity returned and was then stable. The recovery of activity could be prevented by chloramphenicol or erythromycin. Bacteria containing the recovered dehydrogenase activity could not convert sucrose to 3-ketosucrose when oxygen acted as the terminal electron acceptor, but produced 3-ketosucrose at the normal rate in the presence of ferricyanide. D-Glucoside 3-dehydrogenase activity disappeared irreversibly from bacteria incubated in nitrogen-free medium containing sucrose. Loss of activity followed first order kinetics in bacteria taken from nitrogen-, phosphate- or manganese-limited chemostat steady states; an accelerating rate of decay occurred in cells grown under carbon-limitation. 8-Hydroxyquinoline, chloramphenicol, erythromycin, 2,4-dinitrophenol and manganese ions could reduce the rate of decay.     

Stimulation of fermentation and yeast-like morphogenesis in Mucor rouxii by phenethyl alcohol

The germination of fungal spores into hyphae was inhibited by concentrations of phenethyl alcohol (PEA) from 0.05 to 0.3%. Spores of Mucor formed budding spherical cells instead of filaments. These cells were abundant in cultures of Mucor rouxii at 0.22% PEA, provided that the carbon source was a hexose at 2 to 5%. Morphology was filamentous with xylose, maltose, sucrose, or a mixture of amino acids. Removal of PEA resulted in the conversion of yeast-like cells into hyphae. PEA did not inhibit biosynthesis of cytochromes or oxygen uptake, but it stimulated CO₂ and ethyl alcohol production. PEA had no effect on the rate of oxygen uptake, but it inhibited the oxidative-phosphorylation activity of mitochondria. These results suggested that growth inhibition by PEA could result from uncoupling of oxidative phosphorylation and that, in Mucor, yeast-like morphology and fermentation were linked.     

Generalization of specific taste aversions to alcohol in the rat

Two experiments were conducted to examine the taste qualitiesof alcohol in the rat. In Experiment 1, rats were trained toavoid sucrose, quinine or a sucrose + quinine mixture. In Experiment2, rats were trained to avoid sucrose, hydrochloric acid ora sucrose + hydrochloric acid mixture. In both experiments ratswere then tested for generalization to 3, 6 and 9% (v/v) alcoholsolutions. Following the alcohol tests rats were tested withthe taste solutions used during training. Results showed thatrats trained to avoid sucrose (Experiments 1 and 2), quinineor the sucrose + quinine mixture generalized that aversion tothe 6% alcohol solution. No generalization was found to the3% alcohol solution. No generalization was displayed by ratstrained to avoid either the hydrochloric acid solution or thesucrose + hydrochloric acid solution. Following the alcoholtests, all trained rats exhibited strong aversions to the solutionthey were trained to avoid. In addition, rats trained to avoida single solution generalized that aversion to the mixture andrats trained to avoid a mixture generalized to the single components.     

Biotransformation of cinnamyl alcohol to rosavins by non-transformed wild type and hairy root cultures of Rhodiola kirilowii

Non-transformed wild type (NTWT) and hairy root cultures of Rhodiola kirilowii were grown in medium supplemented with 2.5 mM cinnamyl alcohol as a precursor and/or sucrose (1 %) on the day of inoculation or on the 14th day of culture. Rosarin, rosavin, and rosin were produced by NTWT root culture but only rosarin and rosavin by hairy roots. Approximately 80 and 95 % of the glycosides were released into the medium for NTWT and hairy root cultures, respectively. The highest rosavin yield, 505 ± 106 mg/l, was in hairy root culture when cinnamyl alcohol was applied on the day of inoculation with the addition of sucrose on the 14th day of culture. For rosin production, supplementation with cinnamyl alcohol alone on day 14 was more favourable with the highest amount 74 ± 10 mg/l in NTWT root culture. Only traces of rosarin were detected.     

Constitutive biosynthesis and localization of alcohol oxidase in the ethanol-insensitive catabolite repression mutant ecr1 of the yeast Pichia methanolica

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.     

Sucrose Suppression of Chlorophyll Synthesis in Tissue Culture: Changes in the Activity of the Enzymes of the Chlorophyll Biosynthetic Pathway

Chlorophyll synthesis in carrot root tissue cultures grown ona medium containing sucrose is inhibited. Examination of theactivities of the first enzymes in the chlorophyll biosyntheticpathway shows that the major effect of sucrose upon chlorophyllsynthesis occurs at the stage controlling 5-aminolevulinic acid(ALA) synthesis. The toxic nature of ALA in this tissue precluded its use toalleviate the sucrose inhibition effect but the utilizationof various other precursors for chlorophyll synthesis was consistentwith a block occurring at the level of ALA synthesis. The activities of other enzymes involved in chlorophyll synthesisdecreased in activity paralleling decreases in chlorophyll amount.These latter changes are thought to result as a consequenceof chloroplast degeneration rather than representing a primecause in the loss of greening.     

Activity and substrate specificity of the alcohol dehydrogenases of n-alkane oxidizing yeasts

The activity and substrate specificity of alcohol dehydrogenases (ADH) in the fractions of cytosol and membrane particles were compared in the yeasts Torulopsis candida, Candida lipolytica and Candida tropicalis grown in media with glucose and hexadecane. In all studied yeast cultures growing in the medium with hexadecane, NAD-dependent ADH specifically dehydrogenating only medium and higher alcohols are induced in the membrane structures of the cells. Soluble ADH are found in the cytosol of the cultures grown either on glucose or on hexadecane. These ADH oxidize all alcohols with the carbon chain length from C2 to C16. As was found by electrophoresis in polyacrylamide gel, the number of ADH molecular forms in the cytosol fraction of the cultures depends on the carbon growth substrate being used and the peculiarities of yeast culture.     

Changes in the proportion of endogenous osmotic solutes accumulated by Chlorella emersonii in the light and dark

Abstract. The type of endogenous osmotic solute accumulated by Chlorella emersonii grown at high external osmotic pressure (π_ext) depended on the light/dark conditions: proline accumulated to high concentrations in cells in the light, while sucrose accumulated to high concentrations in the dark. These findings were made during the alternating light dark cycles used to obtain synchronized cultures, i.e. cultures containing cells at only one stage of development at any one time. Similar decreases in proline and increases in sucrose in the dark were found for cells previously grown in continuous light to obtain non-synchronized cultures, i.e. cultures containing cells at all stages of development.
In cultures synchronized at 200 mol m ⁻³ NaCl (π_ext= 1.01 MPa), recently divided 'daughter cells' at the beginning of the light periods contained 60 mol m⁻³ proline and 100mol m⁻³ sucrose, while mature cells towards the end of light periods contained 130 mol m proline and 20 mol m⁻³ sucrose. The changes in proline and sucrose which occurred in synchronized cultures were due mainly to light/dark conditions and to a much lesser extent to different stages of cell development. The proportion of proline to sucrose in daughter cells collected from non-synchronized cultures in continuous light was not different from the proportion in heterogeneous populations of cells.
Results are discussed in relation to the accumulations of two, rather than one, endogenous osmotic solute and to growth reductions of C. emersonii exposed to high external osmotic pressures.     

Adaptive changes in rat liver plasma membranes after chronic alcohol administration and its subsequent withdrawal

Rat hepatic plasma membranes isolated after chronic alcohol feeding displayed a different buoyant density range with a significantly increased peak density value when spun isopycnically in a 30-50% sucrose (w/w) gradient. This change persisted up to 48 h of withdrawal from alcohol. Analysis of membrane lipids revealed certain significant alterations in the phospholipids as well as the fatty acyl composition in individual phospholipids of the experimental plasma membranes. During withdrawal of alcohol for 48 h, all the alcohol-induced changes in the phospholipids returned to normal. Most initial changes in fatty acids reverted to the control composition during this time, but new changes in fatty acyl distribution were also observed. These were interpreted to represent readaptation to the withdrawal of the alcohol. It is not established how long this readaptation period lasts.     

Thermostability of alcohol dehydrogenase in the presence of sucrose

Summary The influence of sucrose on the thermostability of pure alcohol dehydrogenase is investigated for various temperatures (50–70°C) in the presence and absence of sucrose (0, 80 wt.%). The thermal inactivation clearly exhibits nonlinear biphasic behavior. The thermal inactivation rate constants and the magnitude of the heat-stable and heat-labile fractions of the enzyme are quantified.     

Effects of anaerobiosis and nitrate on the expression of succinate dehydrogenase and enzymes associated with nitrogen metabolism in Klebsiella pneumoniae

We have shown that the low histidase activity found in anaerobic, nitrogen-limited cultures of Klebsiella pneumoniae is due to repression of the right-hand hut operon. In addition, we have examined the effects of NO3- on the aerobic and anaerobic expression of catabolite- and NH4+-repressible enzymes in this organism. NO3- permitted anaerobic growth of K. pneumoniae in minimal medium containing histidine as the sole carbon source, and histidase and succinate dehydrogenase were derepressed during anaerobic growth in histidine/NO3- medium. Use of sucrose rather than histidine as the carbon source reversed the effects of NO3- and repressed histidase and succinate dehydrogenase activities. Anaerobic growth in sucrose/NO3- medium also uncoupled the expression of urease and glutamine synthetase.     

The mechanism of the hypolipidemic effect of garlic oil extract in rats fed on high sucrose and alcohol diets

The effect of feeding garlic oil to white albino rats maintained on high sucrose and alcohol diets was studied. It is proposed that the mechanism of the hypolipidemic effect of the oil involves the active principle, diallyl disulphide, inactivating enzymes and substrates containing thiol groups in an exchange reaction; increased hydrolysis of triacylglycerols as increased lipase activity is induced by the oil; and the reduction in the biosynthesis of triacylglycerols as NADPH is made unavailable for the process by the metabolism of the oil.     

The taste of alcohol for rats as revealed by aversion generalization tests

In six experiments, naive rats were trained to avoid alcoholby pairing its presentation with lithium chloride-induced illness.Rats were then tested for aversion generalization by presentingvarious test solutions. Rats trained to avoid either 3, 6 or9% (v/v) alcohol generalized the aversion to a sucrose + quininehydrochloride solution. The five remaining binary combinationsof sucrose, sodium chloride, hydrochloric acid and quinine hydrochloridefailed to produce significant generalization in trained rats(Experiment 1). In further experiments, rats trained to avoid6% alcohol showed significant aversion generalization to a varietyof sucrose + other ‘bitter’ solutions (Experiment2) and sucrose + acid solutions (Experiment 3). Varying theconcentration of hydrochloric acid in a sucrose + acid mixtureproduced small but uniform degrees of aversion generalization(Experiment 4). Rats trained to avoid 6% alcohol did not generalizethe aversion to sucrose alone, regardless of concentration (Experiment5). Finally, in Experiment 6, rats trained to avoid 6% alcoholsuppressed consumption over a range of alcohol concentrations.These results confirm that, for rats, the taste of alcohol hasa complex set of characteristics; sweet taste in combinationwith other tastes appears to be the most similar as it is tothese solutions that rats with alcohol aversions show the mostgeneralized avoidance.