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  • 1
    ISSN: 1432-072X
    Keywords: Ethylene ; Ethylene-forming enzyme ; Fusarium oxysporum ; Penicillium digitatum ; 2-Oxoglutarate ; Dioxygenase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Liquid cultures of the deuteromycete, Fusarium oxysporum f. sp. tulipae, a tulip pathogen, produced high amounts of ethylene during stationary phase. 1-Aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene in plants, was not present in the fungus. Radioactivity from [3,4-3H]glutamate as well as [U-14C]glutamate was incorporated into ethylene, indicating that it was derived from C3 and C4 of glutamate or 2-oxoglutarate. Ferrous ions markedly stimulated the rate of ethylene formation in vivo, whereas Fe3+, Cu2+ or Zn2+ had little or no effect. Ethylene biosynthesis was strongly inhibited by the heavy metal chelator α,α′-dipyridine. The effect of α,α′-dipyridine was fully reversed by Fe2+ ions and partially by Cu2+ and Zn2+ ions but not by the supply of glutamate or 2-oxoglutarate, suggesting that a step in the ethylene biosynthetic pathway downstream of 2-oxoglutarate is dependent on Fe2+. When stationary phase cultures were supplied with arginine, ornithine, or proline, ethylene production increased dramatically while addition of glutamate or 2-oxoglutarate had little effect. Tracer studies were performed to test the possibility that an intermediate in the catabolism of arginine to glutamate was the direct precursor of ethylene. In cultures supplied with [U-14C]arginine or [U-14C]glutamate, the specific radioactivity of ethylene was closely similar to the specific radioactivity of the endogenous glutamate pool, indicating that glutamate was on the pathway between arginine and ethylene. An enzyme system converting 2-oxoglutarate to ethylene in a reaction dependent on oxygen, ferrous ions and arginine has previously been described in extracts from Penicillium digitatum (Fukuda et al. 1986). The present results suggest that a similar enzyme system catalyzes the final step of ethylene biosynthesis in F. oxysporum.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Applied microbiology and biotechnology 36 (1991), S. 400-403 
    ISSN: 1432-0614
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The blue fluorescence emitted by microbial cells irradiated with UV light at ∼ 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 44 (1994), S. 132-139 
    ISSN: 0006-3592
    Keywords: glycogen ; Escherichia coli ; cell growth ; acetate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Excessive production of acetate is a problem frequently encountered in aerobic high-cell-density fermentations of Escherichia coli. Here, we have examined genetic alterations resulting in glycogen overproduction as a possible means to direct the flux of carbon away from the acetate pool. Glycogen overaccumulation was achieved either by using a regulatory glgQ mutation or by transforming cells with a plasmid containing the glycogen biosynthesis genes glgC (encoding ADPG pyrophosphorylase) and glgA (encoding glycogen synthase) under their native promoter. Both strategies resulted in an approximately five-fold increase in glycogen levels but had no significant effect on acetate excretion. The glgC and glgA genes were then placed under the control of the isopropyl---D-thiogalactopyranoside (IPTG) inducible tac promoter, and this construct was used to stimulate glycogen production in a mutant defective in acetate biosynthesis due to deletion of the ack (acetate kinase) and pta (phosphotransacetylase) genes. If glycogen overproduction in the ack pta strain was induced during the late log phase, biomass production increased by 15 to 20% relative to uninduced controls. Glycogen overaccumulation had a significant influence on carbon partitioning: The output of carbon dioxide peaked earlier than in the control strain, and the levels of an unusual fermentation byproduct, pyruvate, were reduced. Exogenous pyruvate was metabolized more rapidly, suggesting higher activity of gluconeogenesis or the tricarboxylic acid (TCA) cycle as a result of glycogen overproduction. Potential mechanisms of the observed metabolic alterations are discussed. Our results suggest that ack pta mutants over producing glycogen may be a suitable starting point for constructing E. coli strains with improved characteristics in high-cell-density fermentations. © 1994 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 0749-503X
    Keywords: Yeast ; metallothionein ; heterologous proteins ; dominant selectable marker ; copy number control ; hirudin ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have constructed 2-μm-based yeast expression vectors containing a copy of the metallothionein (CUP1) gene of Saccharomyces cerevisiae as a semi-dominant, selectable marker. When used for the expression of the thrombin inhibitor hirudin, originally derived from the leech Hirudo medicinalis, these vectors displayed the following characteristics. (1) In the presence of copper salts, they were mitotically more stable than similarly designed control vectors lacking the CUP1 gene. In copper-sensitive host strains, the apparent plasmid stability was 100%, even in complex media and during fed-batch fermentation for an extended period of time. (2) Use of the CUP1-stabilized plasmids improved the production of hirudin by both copper-sensitive and copper-resistant hosts. The highest hirudin titers were obtained with a ΔCUP1 host. (3) Copper selection resulted in a moderate increase in average plasmid copy numbers (up to two-fold) as assessed by measuring hirudin expression from a constitutive promoter (GAPFL). This effect was most noticeable if the vector showed an asymmetric segregation pattern (i.e., high rates of plasmid loss in the absence of copper). (4) The CUP1 marker proved particularly useful in combination with a CUP1-promoter-controlled expression cassette on the same plasmid. In such a set-up, the rates of transcription of the heterologous protein and that of the selectable marker are tightly linked. Therefore, an increase in selective pressure directly provokes an increase in product yields. In a copper-sensitive host strain, this plasmid design allowed for the production of very high amounts of biologically active hirudin. Our results clearly establish the utility of the CUP1 marker in the construction of stable yeast expression vectors.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; metallothionein ; heterologous proteins ; CUP1 ; ACE1 genes ; secretion ; hirudin ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using the anticoagulant, hirudin, from the leech Hirudo medicinalis as a secreted reporter protein, the influence of physiological parameters on activity and regulation of the yeast (Saccharomyces cerevisiae) metallothionein (CUP1) promoter was studied. Induction of CUP1-directed hirudin expression from 2μ-based vectors was possible at any time point during diauxic batch growth, even in cells approaching stationary phase. The highest titers of hirudin were obtained when the CUP1 promoter was activated immediately following inoculation of the cultures. If such a pseudo-constitutive fermentation strategy was adopted, the promoter was superior to an optimized variant (GAPFL) of the strong, constitutive GAPDH promoter. This superiority was primarily due to the relative independence of CUP1 promoter activity of the physiological status of host cells: whilst the maximal strength of the CUP1 and GAPFL promoters was comparable, CUP1-directed hirudin expression was high in all phases of diauxic batch growth, whereas hirudin production from the GAPFL promoter declined in post-diauxic cultures. High activity of the CUP1 promoter was observed on both a fermentable (glucose) and a non-fermentable (ethanol) carbon source. Hirudin expression could be adjusted to different levels by varying the amount of inducer (cupric sulphate) added to cultures. The copper concentrations required for maximal promoter induction had no negative effects on host growth and interfered with neither hirudin secretion nor with the biological activity of the peptide. Overexpression of the transcriptional activator, ACE1, resulted in increased levels of hirudin mRNA. Hirudin titers increased in parallel to mRNA concentrations in cultures grown in the presence of low concentrations of copper. In contrast, at high copper doses, elevated levels of the ACE1 protein resulted in inferior hirudin production. Cells overexpressing ACE1 while harbouring a CUP1-drived hirudin expression cassette showed slow growth and poor plasmid maintenance. It was tested whether this might be the result of a block in the secretory pathway; however, measurements of intracellular hirudin did not support this hypothesis. The data rather indicated that hirudin production was limited by a metabolic constraint downstream of transcription but upstream of the secretory pathway.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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