Conidiogenesis and secondary metabolism in Penicillium urticae.

Submerged cultures of Penicillium urticae (NRRL 2159A) produced the antibiotics patulin and griseofulvin when grown in a glucose-nitrate medium. A high concentration of calcium (i.e., 68 mM) inhibited the production of both antibiotics while stimulating conidiogenesis. Conidial mutants that were defective in an early stage of conidiogenesis produced markedly less patulin, even under growth conditions that favored secondary metabolism. A mutant which lacked the ability to produce the patulin pathway metabolites m-cresol, toluquinol, m-hydroxybenzyl-alcohol, m-hydroxybenzaldehyde, gentisaldehyde, gentisyl alcohol, gentisic acid and patulin, as well as the pathway enzyme m-hydroxybenzyl-alcohol dehydrogenase, still produced yields of conidia that were equivalent to or greater than those of the parent strain. Other mutants which were blocked at later steps of the patulin pathway also produced conidia. These results indicate that patulin and the other related secondary metabolites noted above are not a prerequisite to conidiogenesis in P. urticae. Environmental and developmental factors such as calcium levels and conidiogenesis do, however, indirectly affect the production of patulin pathway metabolites.     

Patulin biosynthesis: the metabolism of m-hydroxybenzyl alcohol and m-hydroxybenzaldehyde by particulate preparations from Penicillium patulum.

The ring hydroxylation of m-hydroxybenzyl alcohol to gentisyl alcohol by a particulate preparation from Penicillium patulum has been characterised. The activity was shown to be closely associated with, but not necessarily identical to, m-cresol 2-hydroxylase activity of the 105 000 X g microsomal fraction. As with both the m-cresol hydroxylases of this system, m-hydroxybenzyl alcohol hydroxylase required oxygen and NADPH for activity. A Km value for m-hydroxybenzyl alcohol of 15 muM was measured. Inhibition of the hydroxylase activity and its reversal by light, as well as the action of cytochrome c, KCN and other effectors suggested a mixed-function oxidase reaction of the cytochrome P-450, NADPH-cytochrome reductase type. m-Hydroxybenzaldehyde was not ring hydroxylated by any preparation from P. patulum. Apart from the previously described conversion to m-hydroxybenzyl alcohol by a predominantly soluble dehydrogenase, m-hydroxybenzaldehyde was metabolized to m-hydroxybenzoic acid by a particulate fraction. This activity required NADPH. It was concluded that the main biosynthetic pathway to patulin must be through m-hydroxybenzyl alcohol, gentisyl alcohol and gentisaldehyde.     

Spore inoculum optimization to maximize cyclosporin A production in Tolypocladium niveum

The cyclic undecapeptide, cyclosporin A (CyA), is one of the most commonly prescribed immunosuppressive drugs. It is generated nonribosomally from a multifunctional cyclosporin synthetase enzyme complex by the filamentous fungus Tolypocladium niveum. In order to maximize the production of CyA by wild-type T. niveum (ATCC 34921), each of three culture stages (sporulation culture, growth culture, and production culture) were sequentially optimized. Among the three potential sporulation media, the SSMA medium generated the highest numbers of T. niveum spores. The SSM and SM media were then selected as the optimal growth and production culture media, respectively. The addition of valine and fructose to the SM production medium was also determined to be crucial for CyA biosynthesis. In this optimized three-stage culture system, 3% of the spore inoculum generated the highest level of CyA productivity in a 15-day T. niveum production culture, thereby implying that the determination of an appropriate size of T. niveum spore inoculum plays a critical role in the maximization of CyA production.     

Enzyme Pattern and Aerobic Growth of Saccharomyces cerevisiae Under Various Degrees of Glucose Limitation

The enzyme pattern of Saccharomyces cerevisiae was followed during batch growth and in continuous culture in a synthetic medium limited for glucose under aerobic conditions. Seven enzymes were measured: succinate-cytochrome c oxidoreductase, malate dehydrogenase, nicotinamide adenine dinucleotide-linked glutamate dehydrogenase, malate synthase, isocitrate lyase, aldolase, and nicotinamide adenine dinucleotide phosphate (NADP(+))-linked glutamate dehydrogenase. During fermentation of glucose and high growth rate (mu) during the first log phase in batch experiments, the first five enzymes (group I) were repressed, and aldolase and NADP(+)-linked glutamate dehydrogenase (group II) were derepressed. During growth on the accumulated ethyl alcohol and lower mu, the group I enzymes were preferentially formed and the other two were repressed. A sequence of derepression of the group I enzymes was found during the shift from glucose to ethyl alcohol metabolism, which can be correlated with a strong increase in the percentage of single (nonbudding) cells in the population. A correlation between the state of cells in the budding cycle and enzyme repression and derepression is suggested. In continuous culture, the enzyme pattern was shown to be related to the growth rate. The group I enzymes were repressed at high growth rates, while the group II enzymes were derepressed. Each enzyme exhibits a different dependence. The enzyme pattern is shown to depend on the rate of substrate consumption as well as on the type of metabolism and to be correlated with the budding cycle. The enzyme pattern is considered to be controlled by changes of intracellular catabolic or metabolic conditions inherent in the division cycle.     

In vitro stabilization of 6-methylsalicylic acid synthetase from Penicillium urticae

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetase in vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesis in vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells of Penicillium urticae rapidly increased just before the loss of 6-methylsalicylic acid synthetase content. These in vitro stabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymes in vivo, and into possible ways of extending the life of these catalysts.     

Manganese and antibiotic biosynthesis. I. A specific manganese requirement for patulin production in Penicillium urticae

The effect of trace metal nutrition on the functioning of the patulin biosynthetic pathway in submerged cultures of Penicillium urticae (NRRL 2159A) was examined by both chromatographic and enzymological means. Comprehensive metal ion analysis showed generally low levels of contaminating metal ions in media components. Of eight metal ions examined, only manganese strongly influenced secondary metabolite production. In control cultures or cultures deficient in calcium, iron, cobalt, copper, zinc, or molybdenum, pathway metabolites appeared in the medium at about 25 h after inoculation. The first pathway-specific metabolite, 6-methylsalicylic acid, accumulated only transiently before being converted to patulin whose concentration steadily increased. In manganese-deficient cultures, however, 6-methylsalicylic acid continued to accumulate, with only minor amounts of patulin being produced. Additionally, a marker enzyme for the pathway showed only 0-20% of control activity. Clear dose responses (patulin versus manganese) were found in different media, with no effect on growth yield. Addition of manganese to depleted cultures at 18, 26, or 36 h resulted in increasing marker enzyme activity and patulin concentrations. It is concluded that manganese exerts a specific, positive effect on patulin biosynthesis and may in some way control the section of the patulin pathway occurring after 6-methylsalicylic acid.     

Purification of nucleotide-requiring enzymes by immunoaffinity chromatography.

Monospecific (affinity-purified) anti-(yeast glucose-6-phosphate dehydrogenase) IgG inhibits three different NADPH-requiring enzymes, chicken liver dihydrofolate reductase, pigeon liver fatty acid synthetase and chicken liver malic enzyme. The inhibition of all three enzymes was approx. 50% in a 2h incubation with 100 micrograms of IgG. Similarly, with several different NADH-requiring enzymes, an immunocrossreactivity was observed. Monospecific anti-(rabbit muscle glyceraldehyde-3-phosphate dehydrogenase) IgG inhibited yeast alcohol dehydrogenase and pig heart malate dehydrogenase by 39% and 55% respectively. The cross-reactivity observed was tested by affinity chromatography. Immunoaffinity columns made with each monospecific IgG were able to bind each of the enzymes it immunotitrated. Enzymes were eluted with a nondenaturing solvent with little loss of activity. The immunoaffinity column with monospecific anti-(glucose-6-phosphate dehydrogenase) IgG as the bound ligand was also used to purify partially (over 150-fold) both isocitrate dehydrogenase and dihydrofolate reductase from crude rat liver homogenate.     

Regulation of the synthesis of methanol oxidizing enzymes inKloeckera sp. 2201 andHansenula polymorpha,a comparison

A comparative study was made of the regulation of the synthesis of methanol dissimilating enzymes inkloeckera sp. 2201 andHansenula polymorpha using chemostat and batch growth conditions and methanol or glucose as carbon sources. During growth in methanol-limited chemostat cultures similar enzyme patterns for alcohol oxidase, catalase, formaldehyde dehydrogenase and formate dehydrogenase in the two yeasts were found. When growing in batch culture with glucoseH. polymorpha, but notKloeckera sp. 2201, was found to produce ethanol which might affect the synthesis of these enzymes.     

Effect of inoculum type on actinorhodin production by Streptomyces coelicolor A3(2)

Summary The production of actinorhodin by Streptomyces coelicolor in a defined medium was examined using spore and vegetative inocula. The spore inoculum yielded higher concentrations of biomass and actinorhodin as well as a higher maximum specific growth rate compared with the vegetative inoculum. Nevertheless, the productivity of the batch culture for actinorhodin formation with vegetative inoculum was higher than that with spore inoculum.     

Control of enzyme activity levels by serum and hydrocortisone in neonatal rat heart cells cultured in serum-free medium

A serum-free, hormone-supplemented medium (SFHM) for maintaining neonatal rat heart cells in culture has been developed in this laboratory (Mohamed et al., 1983). Morphological assessment of heart cells grown in SFHM show it to be similar to commonly used serum-supplemented media. To quantitatively compare cell behavior in SFHM with serum-supplemented media, the activities of ten regulatory enzymes which represent four metabolic pathways were studied in heart cells cultured in SFHM. The enzyme activities which were measured included hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphofructokinase, pyruvate kinase, NAD+-linked sn-glycerol-3-phosphate dehydrogenase, malate dehydrogenase, NAD+-linked isocitrate dehydrogenase, NADH-cytochrome c reductase, and succinic cytochrome c reductase. Rat heart cells maintained in culture on SFHM are not only qualitatively and quantitatively similar to those maintained in serum-supplemented medium but also provide a more suitable model system for metabolic studies of neonatal cardiac tissue for several reasons: 1) many enzyme activities that may represent dedifferentiation are elevated by serum; 2) NAD-linked glycerol-3-phosphate dehydrogenase activity in cells maintained on SFHM is similar to the in vivo activity; 3) cells beat at or near the in vivo frequency and can be maintained 3 months on SFHM; 4) the SFHM is chemically defined and thus can be completely manipulated by the investigator. The effects of three concentrations of hydrocortisone (HC) (5,000 ng/ml, 50 micrograms/ml, 0 ng/ml) on heart cells cultured in SFHM supported our previous conclusion that function (beating) and growth (protein accumulation) are inversely related in cultured neonatal rat heart cells.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

Effect of carbon and nitrogen growth limitation upon nutrient uptake and metabolism in batch cultures of Catharanthus roseus (L) G. Don

Cell suspension cultures of the Madagascan Periwinkle, Catharanthus roseus (L). G. Don were grown as batch cultures in two different types of media; in one medium the limiting nutrient was inorganic nitrogen, and in the other it was carbon. The response of the cells to these growth-limiting conditions was monitored by measuring cellular fresh weight, dry weight and protein accumulation, cell viability, medium sugar and nitrate levels, and the activities of certain intracellular enzymes throughout growth in batch culture. The enzymes investigated were glucose-6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), hexokinase (EC 2.7.1.40), phosphofructokinase (EC 2.7.1.11), nitrate reductase (EC 1.6.6.1), glutamate dehydrogenase (EC 1.4.1.2) and glutamine synthetase (EC 6.3.1.2). The effect of culturing the cells under different nutritional regimes was apparent in all aspects of growth; only some enzyme activities were unaffected. Cell viability remained at a high level for several days after growth limitation in both types of culture. The possibility that protein degradation in nitrogen-limited batch cultures is under very stringent control is discussed.     

Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules

Soybean (Glycine max) nodules formed by inoculation with either an effective strain or an ineffective (noninvasive, nodule-forming) strain of Bradyrhizobium japonicum were assayed for changes in developmental patterns of carbon metabolic enzymes of the plant nodule cells. Of the enzyme activities measured, only sucrose synthase, glutamine synthetase, and alcohol dehydrogenase were altered in the ineffective nodules relative to the effective nodules. Sucrose synthase and glutamine synthetase activities were greatly reduced, whereas alcohol dehydrogenase activity was elevated. Dark-induced senescence severely affected sucrose synthase but had little, if any, effect on the other enzymes measured. The developmental patterns of the anaerobically induced enzymes, aldolase and alcohol dehydrogenase, were different from those expected, implying that their development is not regulated solely by oxygen deprivation. However, anaerobic treatment of nodules resulted in responses similar to those enzymes in maize. The developmental profiles of the carbon metabolic enzymes suggest that carbohydrates are metabolized via the sucrose synthase and pentose phosphate pathways. This route of carbon metabolism, compared to glycolysis, would reduce the requirement of ATP for carbohydrate catabolism, generate NADPH for biosynthetic reactions, and provide intermediates for plant secondary metabolism.     

Acetate-nonutilizing Mutants of Neurospora crassa II. Biochemical Deficiencies and the Roles of Certain Enzymes

The levels of Krebs cycle, glyoxylate cycle, and certain other enzymes were measured in a wild-type strain and in seven groups of acetate-nonutilizing (acu) mutants of Neurospora crassa, both after growth on a medium containing sucrose and after a subsequent 6-hr incubation in a similar medium, containing acetate as the sole source of carbon. In the wild strain, incubation in acetate medium caused a rise in the levels of isocitrate lyase, malate synthase, phosphoenolpyruvate carboxykinase, acetyl-coenzyme A synthetase, nicotinamide adenine dinucleotide phosphate-linked isocitrate dehydrogenase, citrate synthase, and fumarate hydratase. Isocitrate lyase activity was absent in acu-3 mutants; acu-5 mutants lacked acetyl-coenzyme A synthetase activity; and no oxoglutarate dehydrogenase activity (or only low levels) could be detected in acu-2 and acu-7 mutants. In acu-6 mutants, phosphoenolpyruvate carboxykinase activity was either very low or absent. No specific biochemical deficiencies could be attributed to the acu-1 and acu-4 mutations. The role of several of these enzymes during growth on acetate is discussed.     

The effects of a choline deficiency on the lipid composition and ethanol tolerance of Drosophila melanogaster

1. A reduction in the dietary concentration of choline, an essential nutrient for Drosophila melanogaster, from the optimal concentration of 80 micrograms/ml of defined medium to 8 micrograms/ml diminished the level of tissue phosphatidylcholine to less than one-third the normal level in third instar larvae without significantly altering the amount of phosphatidylethanolamine. 2. The rates of synthesis of phospholipids, triglycerides, diglycerides and monoglycerides were reduced by the choline-deficiency, and the chain length of fatty acids in lipids was shortened. 3. The activity of succinic dehydrogenase, a mitochondrial enzyme, was decreased by the deficiency, but the activities of fumarase, sn-glycerol-3-phosphate dehydrogenase, alcohol dehydrogenase, sn-glycerol-3-phosphate oxidase and fatty acid synthetase were unaffected. A choline-deficiency did not alter the ultrastructure of mitochondria of larval fat body cells. 4. Choline-deficient individuals were more susceptible to the toxic effects of ethanol during larval and pupal development, and less adept at utilizing ethanol as a substrate for adult tissue synthesis.     

Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism

Using primary cultures of adult rat hepatocytes, the regulation of the following lipogenic enzymes was studied: glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. The addition to the culture medium of either insulin or triiodothyronine produced a 2-3-fold increase in each of the individual enzyme activities whereas glucagon slightly decreased enzyme activities. The addition to the medium of 8-bromoguanosine 3,'5'-monophosphate had no effect on any of the enzyme activities unless glucose was also added to the culture medium. Glucose addition alone to the culture medium was without any effect; however, glucose enhanced the stimulation of enzyme activity due to insulin. The addition of fructose or glycerol, even in the absence of insulin, increased the activities of each of the enzymes studied 2-3-fold. The increases in enzyme activity brought about by insulin or fructose were apparently the result of de novo enzyme synthesis, as indicated by the observation that the increases were not noted in the presence of cordycepin or cycloheximide. Immunoprecipitation of ATP-citrate lyase from hepatocytes pulse-labeled with [3H]leucine indicated that the induction of this enzyme in response to the addition of fructose or glycerol to the culture medium was the result of an increase in the rate of synthesis of the enzyme. These results indicate that the activity and synthesis of individual enzymes involved in lipogenesis are increased in response to the metabolism of carbohydrate independently in part from hormonal effects.     

Enzymes of ammonium metabolism in ectendomycorrhizal and ectomycorrhizal symbionts of pine

Ammonium assimilation enzymes from several strains of ectendo- and ectomycorrhizal fungi were assayed after three weeks culture on a buffered synthetic medium containing ammonium as sole nitrogen source. Activity of NADP-dependent glutamate dehydrogenase (GDH, EC 1.4.1.4) of ectomycorrhizal strains was very low despite excellent mycelial growth. Only ectendomycorrhizal fungus MrgX isolated from roots of Pinus sylvestris showed high GDH activity. Similar results were obtained when the enzyme extracts were subjected to starch gel electrophoresis. Growth of the fungi, except ectendomycorrhizal MrgX, was arrested when inhibitors of glutamine synthetase (GS, EC 6.3.1.2) or glutamate synthase (GOGAT. EC 1.4.7.1) (methionine sulphoximine or albizine, respectively) were included in the culture medium. Glutamine synthetase activity was found in all fungi tested. The results suggest that the GS pathway for ammonium assimilation is potentially operative in ectomycorrhizal fungi and imply only a minor role for GDH in ammonium assimilation by the studied ectomycorrhizal symbionts of pine. Some physiological and ecological implications of these results are discussed.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.