The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.     

Balance of production and consumption of ATP in ammonium-starved Saccharomyces cerevisiae

To establish a balance between the ATP produced in catabolism and the ATP consumed in net biosynthesis of cellular components the energy metabolism of Saccharomyces cerevisiae utilizing glucose in the absence of a nitrogen source (resting cells) was studied. The following results were obtained. (i) Cell number and biomass increased 2- and 2.5-fold, respectively, during the first 8 h of ammonium starvation. After this period, both values remained constant. (ii) The rate of sugar consumption and ATP production decreased with the duration of starvation to about 20% of the original in 24 h. (iii) About 60% of the sugar consumed was fermented to ethanol and about 10% assimilated as cellular material. Of the assimilated sugar, as much as 80% was accumulated as carbohydrate. (iv) Only 15% of the total ATP produced in catabolism seems to be consumed in net biosynthesis and maintenance of intracellular pH. The fate of the remaining 85% is unknown.     

Composition and synthesis of cellular lipids in Neurospora crassa during cellular differentiation.

The synthesis of cellular lipids of Neurospora crassa was measured during growth on low (2% sucrose)- and high (15% glucose)-carbohydrate supplementation. The amount of lipid per dry weight of cells does not change during the germination and early logarithmic growth periods, but the percentage of phospholipid in the lipid does increase, reaching a maximal value of 90% at 4 to 5 h after inoculation, at which time the phospholipid content of the cells is approximately 60 mumol/g (dry weight). The content of the anionic phospholipids, as a percentage of the lipid fraction, is relatively constant during the growth period, but the contents of the zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine change in a reciprocal fashion. During the first 8 h of growth, phosphatidylcholine falls from 53% of the phospholipid to 43%, whereas phosphatidylethanolamine rises from 29 to 38%. The total of these two phospholipids is approximately 83% during the growth period studied. The synthesis of cellular phospholipids, measured either by [32P]H3PO4 or [14C]glucose incorporation, reached maximal levels between 3 and 5 h of growth. The effect of the high-carbohydrate supplement on cellular lipids was minimal. Inclusion of 15% glucose decreased the labeling of phospholipid by [32P]H3PO4, but did not affect lipid composition. This observation is in contrast to the effects of high glucose on mitochondrial phospholipid synthesis.     

Culture fluorescence studies on aerobic continuous cultures ofSaccharomyces cerevisiae

Summary Fluorometric measurements were performed in continuous aerobic cultures ofSaccharomyces cerevisiae in order to study the effect of substrate concentration and residence time on the intracellular NADH-level. A modified Beyelermicrofluorometer probe (Beyeler et al. 1981) was used for the experiments. It was possible to use this sensor continuously up to five weeks without problems. The relative NADH-values obtained by the on-line monitoring of the NADH-dependent culture fluorescence were compared to the enzymatically determined NADH-content. Biomass estimation from fluorescence data was performed. During oxidative-reductive catabolism the deviation between calculated and measured data were below 5%. The differences between oxidative and oxidative-reductive catabolism were studied regarding glucose addition, dilution rate increase and aerobic-anaerobic transition. For synchronized continuous cultures, changes in dilution rate resulted in changes of the oscillating behaviour. Flow cytometric studies in comparison with fluorometric studies showed changes in budding behaviour during the oscillations.     

Tryptophan catabolism during sporulation in Bacillus cereus

1. Two intermediates of tryptophan catabolism were isolated from a sporulating culture of Bacillus cereus and identified as anthranilic acid and kynurenine by their spectral properties. 2. During sporulation the rate of formation of anthranilic acid and kynurenine by whole cells increased and reached a maximum at the pre-spore stage. 3. The specific activities of tryptophan pyrrolase and formylase also increased during sporulation and exhibited a maximal activity at the pre-spore stage. 4. Kynureninase activity reached a maximum during early stages of sporulation and then started to decline. 5. There was a net increase in the activity of tryptophan pyrrolase when cells were grown in the presence of l-tryptophan or dl-kynurenine. 6. The cultures exhibited the maximal activity of kynureninase 2h earlier in the presence of dl-kynurenine whereas l-tryptophan delayed the appearance of the maximal activity by 2h. 7. The omission of glucose from the medium had no effect on the pattern of development of tryptophan pyrrolase during growth and sporulation. 8. On the addition of tryptophan to a chemically defined medium no significant change in the pattern of development of tryptophan pyrrolase was observed.     

Biosynthesis of fredericamycin A, a new antitumor antibiotic

Fredericamycin A (FM A), produced by a strain of Streptomyces griseus, represents a new structural class of antitumor antibiotics containing a spiro ring system. Studies on the producer organism showed that glucose in the fermentation medium is not utilized until late in the growth stage, just prior to synthesis of FM A. [14C]Glucose tracer experiments demonstrated that glucose is incorporated into FM A by catabolism to acetate. Biosynthetic enrichment of FM A with single- and double-labeled [13C]acetate showed that the entire carbon skeleton of the spiro ring system is derived from acetate. L-Methionine was shown to provide the only nonskeletal carbon in FM A, the methoxy carbon at position C-6. The direction of the polyketide chain and the position of the carbon lost during biosynthesis were established by using stable isotope experiments. A general model for FM A biosynthesis is proposed, and a possible scheme for the formation of the spiro carbon center is presented.     

Batch and continuous culture kinetics for production of carotenoids by beta-ionone-resistant mutant of Xanthophyllomyces dendrorhous

A beta-ionone-resistant mutant strain isolated from the red yeast Xanthophyllomyces dendrorhous KCTC 7704 was used for batch and continuous fermentation kinetic studies with glucose media in a 2.5-1 jar fermentor at 22 degrees C and pH 4.5. The kinetic pattern of growth and carotenoid concentration in the batch fermentations exhibited a so-called mixed-growth-associated product formation, possibly due to the fact that the content of intracellular carotenoids depends on the degree of physical maturation toward adulthood. To determine the maximum specific growth rate constant (microm) and Monod constant (k(s)) for the mutant, glucose-limited continuous culture studies were performed at different dilution rates within a range of 0.02-0.10 h(-1). A reciprocal plot of the steady-state data (viz., reciprocal of glucose concentration versus residence time) obtained from continuous culture experiments was used to estimate a microm of 0.15 h(-1) and k(s) of 1.19 g/l. The carotenoid content related to the residence time appeared to assume a typical form of saturation kinetics. The maximum carotenoid content (Xm) for the mutant was estimated to be 1.04 microg/mg dry cell weight, and the Lee constant (k(m)), which was tentatively defined in this work, was found to be 3.0 h.     

林肯链霉菌合成林可霉素代谢调节的研究

在摇瓶条件下研究了葡萄糖、铵盐、磷酸盐对林可霉素产生菌林肯链霉菌的生长及林可霉素生物合成的影响。发酵过程中林可霉素的合成主要发生在菌体生长期,逐渐下降。使用6%的葡萄糖未发现通常所说的“葡萄糖效应”。0.2%铵盐有利于细胞生长,但0.8%NH+4对林可霉素的生物合成具有抑制作用。发酵48h后补加0.6% NH^＋_４,对林可霉素的生成没有显著影响。0.05%～0.1%磷酸盐对林可霉素合成具有较强的抑制作用。并就磷酸盐对菌体由初级代谢转向次级代谢的作用作了初步考察。     

Aflatoxin formation,lipid synthesis,and glucose metabolism by Aspergillus parasiticus during incubation with and without agitation

Glucose utilization, growth of mold, and synthesis of aflatoxin and total lipid by Aspergillus parasiticus were studied with cultures that were incubated statically and with agitation. With both cultural conditions, maximal toxin formation occurred at 5 days which coincided with the end of rapid mold growth and rapid uptake of glucose. The toxin concentration decreased as incubation continued. The pattern for formation and depletion of total lipid was similar to that for aflatoxin. Maximal yields of toxin and of total lipid did not coincide with maximal production of mold mycelium. Incubation with agitation enhanced mold growth, consumption of glucose, and production of aflatoxin and total lipid during the first 3 days. Generally, more growht occured in agitated cultures, but maximal yields of aflatoxin and total lipid were lower than in quiescent cultures. The need for limited, but not excessive, O₂ for synthesis of aflatoxin and lipid also was demonstrated by varying the volume of medium in flasks that were incubated quiescently. Incorporation of [1-¹⁴C] glucose into aflatoxin indicated that limiting the O₂ supply and thereby favoring glucose catabolism via the Embden-Meyerhof pathway enhanced toxin formation. Aflatoxin formation also was greater when oxidative respiration of the mold was restricted by a metabolic inhibitor. Results suggest that the degree of aeration of the culture is important in controlling biosynthesis of aflatoxin.     

温度对丙酮酸生物合成动力学、能荷和氧化-还原度的影响

在光滑球拟酵母(Torulopsis glabrata620)生产丙酮酸的过程中,温度对丙酮酸生物合成有着重要的影响。考察了不同发酵温度下基质消耗、细胞生长、丙酮酸合成及能荷水平和氧化-还原度等方面的差异。在恒温发酵中,维持较高的发酵温度可以增强糖耗,促进菌体生长,加速丙酮酸积累,但前期胞内能荷水平较高,菌体消耗较多葡萄糖合成菌体,后续产酸能力不足,导致丙酮酸得率降低;维持较低的发酵温度可以在发酵后期提供稳定的产酸能力,但菌体代谢缓慢,后期胞内NADH/NAD 水平较高,丙酮酸生产强度降低。因此仅仅采取单一的温度控制策略很难达到丙酮酸高产量、高产率和高生产强度的统一。     

Relation of anabolic-catabolic glucose utilization in growth-limited cultures of Streptomyces griseus

Phenotypically different submerged mycelium conserves had been produced from a spore conserve of the HP-strain Streptomyces griseus and proofed in a product formation culture as a test system. The phenotypical characters induced on the base of the genotype proved in a cultivation cycle during 30-34 reduplications of the biomass constant. Employing the HP phenotype we investigated the possibility of economizing the substrate turnover by utilizing the anabolic potential for the synthesis of secondary substances and/or reducing the conservation catabolism during the stationary growth stage. As criteria for that served the stoichiometric turnover equation of the streptomycin biosynthesis and the quotient qO2/qGluc taking at full substrate oxidation the numerical value 6. During the stationary growth stage the relation of maintenance anabolism to maintenance catabolism in addition to the formation as secondary substances is not fixed in the tested HP phenotypes, but in a striking manner variable. The relation of by-product synthesis to secondary metabolism synthesis, too, is variable in the stationary growth stage with constant maintenance catabolism. Due to those response reactions on phenotypical manipulations an economization of the substrate turnover during the product formation stage with stationary growth is not possible in the streptomycin producer Streptomyces griseus.     

Cyclic AMP phosphodiesterase in Thermomonospora curvata.

Cyclic AMP phosphodiesterase (PDE; EC 3.1.4.17) in Thermomonospora curvata was purified and characterized. Fractionation of cell extracts by ion-exchange and size-exclusion chromatography revealed four PDE isozymes, which differed markedly in molecular weight, theophylline sensitivity, pH optima, and substrate affinity. Although the enzyme was labile after purification, total recovery of PDE activity was fivefold that of the crude extract. PDE biosynthesis appeared sensitive to the growth phase, growth rate, and carbon source. PDE levels in batch cultures peaked and declined rapidly during mid-exponential-phase growth. In continuous culture, maximal PDE and cellulase production occurred at dilution rates yielding mean cell generation times of about 5 and 17 h, respectively. The addition of glucose to cellulose-grown cells caused declines in both cyclic AMP and PDE levels, suggesting that the enzyme was subject to, rather than the agent of, catabolite repression.     

Enzymological and radiotracer studies of lipid metabolism in the flight-capable and flightless morphs of the wing-polymorphic cricket,Gryllus firmus

The flight-capable morph of the wing-polymorphic cricket, Gryllus firmus, exhibited significantly higher activities of each of five lipogenic enzymes compared with the obligately flightless morph on a standard and a high-carbohydrate diet during early adulthood. Similarly, the rate of incorporation of [14C]-acetate into total lipid was higher in the flight-capable morph during this time. By contrast, activities of lipogenic enzymes and rates of lipid biosynthesis, in general, did not differ between morphs on a low nutrient diet during early adulthood. Differences in lipid biosynthesis account for previously documented differences in lipid reserves between morphs on some, but not all, diets. Results of the present and previous studies indicate that increased lipid biosynthesis in the flight capable morph on standard and high-carbohydrate diets constitutes an important adaptation for flight (production of lipid flight fuel). Lipid biosynthesis is negatively correlated with ovarian growth, and may be an important biochemical component of the trade-off between flight capability and ovarian growth in G. firmus. Morphs also differed in activities of three enzymes of lipid catabolism. However, the extent to which variation in activities of these enzymes between morphs results in variation in lipid catabolism is unclear. Finally, the flight-capable morph had a substantially higher activity of alanine aminotransferase in the fat body. Amino acids may be utilized for lipid biosynthesis or energy production to a greater degree in the dispersing morph compared with the oligately flightless morph. This study is the first to document differences in intermediary metabolism that underlie adaptations of morphs of a dispersal-polymorphic species for flight vs. egg production.     

Kinetics of polysaccharide B-1459 fermentation

Polysaccharide gum was made by fermentation with Xanthomonas campestris NRRL B-1459 in a medium of glucose, minerals, and distillers' solubles. The effect of distillers' solubles on growth rate can be described by the familiar saturation equation. Although a quasistoichiometric relationship was observed between nitrogen utilization and growth, total nitrogen supply was not growth limiting, nor was polymer formation growth associated. Cell growth primarily took place in the early part of the fermentation; polysaccharide biosynthesis occurred throughout the fermentation. Glucose was converted to polysaccharide at a fairly constant yield, which was 70–80% of glucose consumed, under optimum conditions. The kinetic patterns observed indicate that multistage continuous fermentation will be suitable for polysaccharide production.     

Glutamine synthesis is a regulatory signal controlling glucose catabolism in Saccharomyces cerevisiae.

The effect of glutamine biosynthesis and degradation on glucose catabolism in Saccharomyces cerevisiae was studied. A wild-type strain and mutants altered in glutamine biosynthesis and degradation were analyzed. Cells having low levels of glutamine synthetase activity showed high ATP/ADP ratios and a diminished rate of glucose metabolism. It is proposed that glutamine biosynthesis plays a role in the regulation of glucose catabolism.     

Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint-based modeling and experimental verification

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.     

The response by microorganisms to steady-state growth in controlled concentrations of oxygen and glucose. II. Saccharomyces carlsbergensis

The growth of Saccharomyces carlsbergensis in continuous culture has been studied when dissolved oxygen and glucose concentrations were held constant at a series of steady-state levels. Both oxygen and glucose controlled the degree of aerobic metabolism and of ethanolic fermentation. When the glucose uptake rate was low (between 1.2 and 2.8 mmoles per hour per gram of yeast) the relative distribution of glucose between ethanolic and aerobic fermentation was sensitive to oxygen: when dissolved oxygen was near to saturation, glucose metabolism was 0.98 aerobic; when dissolved oxygen was 0.01 saturated, 0.8 of intake glucose metabolism was by ethanolic fermentation. On the other hand when glucose intake was high (between 7.6 and 18.2 mmoles) metabolism was predominately by ethanolic fermentation even when dissolved oxygen concentration was at saturation. The extent, to which catabolism proceeded by an anaerobic or aerobic pathway, as judged by ethanol production, was controlled more by the uptake of glucose than of oxygen.     

Metabolic characterization of a L-lysine-producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.