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  • 1
    Electronic Resource
    Electronic Resource
    Pathway analysis, engineering, and physiological considerations for redirecting central metabolism (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 52 (1996), S. 129-140 
    Details
    ISSN: 0006-3592
    Keywords: pathway engineering ; central metabolism ; phosphoenolpyruvate synthase ; phosphoenolpyruvate carboxykinase ; aromatic amino acid ; Escherichia coli ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The rate and yield of producing a metabolite is ultimately limited by the ability to channel metabolic fluxes from central metabolism to the desired biosynthesis pathway. Redirection of central metabolism thus is essential to high-efficiency production of biochemicals. This task begins with pathway analysis, which considers only the stoichiometry of the reaction networks but not the regulatory mechanisms. An approach extended from convex analysis is used to determine the basic reaction modes, which allows the determination of optimal and suboptimal flux distributions, yield, and the dispensable sets of reactions. Genes responsible for reactions in the same dispensable set can be deleted simultaneously. This analysis serves as an initial guideline for pathway engineering. Using this analysis, we successfully constructed an Escherichia coli strain that can channel the metabolic flow from carbohydrate to the aromatic pathway with theoretical yield. This analysis also predicts a novel cycle involving phosphoenolpyruvate (PEP) carboxykinase (Pck) and the glyoxylate shunt, which can substitute the tricarboxylic acid cycle with only slightly less efficiency. However, the full cycle could not be confirmed in vivo, possibly because of the regulatory mechanism not considered in the pathway analysis.In addition to the kinetic regulation, we have obtained evidence suggesting that central metabolites are involved in specific regulons in E. coli. Overexpression of PEP-forming enzymes (phosphoenolpyruvate synthase [Pps] and Pck) stimulates the glucose consumption rate, represses the heat shock response, and negatively regulates the Ntr regulon. These results suggest that some glycolytic intermediates may serve as a signal in the regulation of the phosphotransferase system, heat shock response, and nitrogen regulation. However, the role of central metabolites in these regulations has not been determined conclusively. © 1996 John Wiley & Sons, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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    Paper (German National Licenses)
  • 2
    Electronic Resource
    Electronic Resource
    Metabolic engineering and control analysis for production of aromatics: Role of transaldolase (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 53 (1997), S. 132-138 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; metabolic control analysis ; transaldolase ; aromatics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Aromatic metabolites in Escherichia coli and other microorganisms are derived from two common precursors: phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P). During growth on glucose, the levels of both E4P and PEP are insufficient for high throughput of aromatics because of the low carbon flux through the pentose pathway and the use of PEP in the phosphotransferase system. It has been shown that transketolase and PEP synthase are effective in relieving this limitation and promoting high throughput of aromatics. To determine whether transaldolase, another E4P-producing enzyme, is also a limiting factor in directing carbon flux to the aromatic pathway, E. coli transaldolase gene (tal) was cloned and overexpressed in an aroB strain which excretes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), the first intermediate in the aromatic pathway. We found that overexpression of transaldolase did significantly increase the production of DAHP from glucose. This result further supports the contention that the supply of E4P is limiting when glucose is the carbon source. However, overexpression of transaldolase in strains which already overexpress transketolase did not show a further increase in production of aromatics. This result was attributed to the saturation of E4P supply when TktA was overexpressed. The flux control of DAHP production was discussed on the basis of Metabolic Control Analysis. © 1997 John Wiley & Sons, Inc.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Pathway engineering for production of aromatics in Escherichia coli: Confirmation of stoichiometric analysis by independent modulation of AroG, TktA, and Pps activities (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 46 (1995), S. 361-370 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The synthesis of 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) is the first commitment of resources toward aromatics production in Escherichia coli. DAHP is produced during a condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) catalyzed by DAHP synthases (coded by aroF, aroG, and aroH). Stoichiometric analysis has shown a severe PEP limitation in the theoretical yield of DAHP production from glucose due to the phosphotransferase system (PTS) for sugar uptake. This limitation can be relieved by (i) the recycling of pyruvate from PEP using PEP synthase (Pps) or (ii) use of non-PTS sugars such as xylose. Previous studies have shown the usefulness of overexpressing tktA (encoding transketolase), aroG, and pps (PEP synthase) for DAHP production in an aroB strain unable to utilize DAHP further. In the present study we confirm the predictions of the stoichiometric analysis by introducing pps, tktA, and aroG into vectors under independently controlled promoters. In glucose medium, although TktA has some positive effect on the final DAHP concentration, it has no effect on the yield (percent conversion). With Pps overexpression, the DAHP concentration produced from glucose is increased almost twofold and the yield is approaching the theoretical maximum, as predicted by the stoichiometric analysis. However, this Pps effect is observed only in the presence of both increased AroG and TktA. In xylose mimimal medium, the final DAHP concentration and the yield are completely determined by the AroG activity. TktA and Pps play no or insignificant roles, and the yield can reach the theoretical maximum without overexpression of these two enzymes. The results shown here are important for both rational design of metabolic pathways and industrial production of aromatics such as tryptophan, phenylalanine, indigo, quinic acid, and catechol.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260460409
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  • 4
    Electronic Resource
    Electronic Resource
    Gas phase composition effects on suspension cultures of Taxus cuspidata (1995)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 48 (1995), S. 123-132 
    Details
    ISSN: 0006-3592
    Keywords: secondary metabolite ; taxol ; ethylene ; carbon dioxide ; oxygen ; taxus cuspidata ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The effect of different concentrations and combinations of oxygen, carbon dioxide, and ethylene on cell growth and taxol production in suspension cultures of Taxus cuspidata was investigated using several factorial design experiments. Low head space oxygen concentration (10% v/v) promoted early production oftaxol. High carbon dioxide concentration (10% v/v) inhibited taxol production. The most effective gas mixture composition in terms of taxol production was 10% (v/v) oxygen, 0.5% (v/v) carbon dioxide, and 5 ppm ethylene. Cultures grown underambient concentration of oxygen had a delayed uptake of glucose and fructose compared to cultures grown under 10% (v/v) oxygen. Average calcium uptake rates into the cultured cells decreased and average phosphate uptake rates increased as ethylene was increased from 0 to 10 ppm. These results may indicate that gas composition alters partitioning of nutrients, which in turn affects secondary metabolite production. © 1995 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260480206
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  • 5
    Electronic Resource
    Electronic Resource
    Control of packed column fouling in the continuous fermentation and stripping of ethanol (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 51 (1996), S. 33-39 
    Details
    ISSN: 0006-3592
    Keywords: yeast ; fuel ethanol ; flocculation ; glucose conversion ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: By recycling the contents of a 14 L fermentor through a stripping column to continuously remove ethanol and reduce product inhibition, continuous complete conversion of nutrient feed containing 600 g/L glucose was achieved in a small pilot plant. Ethanol was recovered from the carbon dioxide stripping gas in a refrigerated condenser, and the gas was reheated with steam and recycled by a blower. Productivity of ethanol in the fermentor as high as 15.8 g/L/h and condensate production of up to 10 L/day of almost 50% by volume ethanol were maintained for up to 60 days of continuous operation. Weekly washing of the column packing in situ was required to prevent loss of performance caused by attached growth of yeast cells, which restricts the gas flow rate through the stripping column. © 1996 John Wiley & Sons, Inc.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    DNA profiling and plant variety registration: 1. The use of random amplified DNA polymorphisms to discriminate between varieties of oilseed rape (1996)
    Weinheim : Wiley-Blackwell
    Electrophoresis 17 (1996), S. 261-265 
    Details
    ISSN: 0173-0835
    Keywords: Random amplified polymorphic DNA ; Oilseed rape ; Variety identification ; Brassica napus ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Before they can be marketed in the UK, newly bred varieties of crop species have to undergo a process of statutory testing, part of which involves the examination of the distinctness, uniformity and stability (DUS) of the variety. DUS testing is also used as the basis for the award of Plant Breeders' Rights. This paper examines the potential of DNA polymorphisms, amplified using arbitrary primers (RAPDs) for use in DUS testing of varieties of oilseed rape. RAPDs using suitable primers can produce high levels of discrimination (〉 95%) between varieties, although there are certain problems in gel ‘scoring’ that are only partially resolved by computerised gel scanning/evaluation techniques. Varieties of oilseed rape are also heterogeneous in their RAPD profiles using certain primers, which could cause problems in the DUS testing context. DNA profiling with RAPDs could be used for discrimination between and identification of oilseed rape varieties, but its use for DUS testing needs to be considered carefully.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/elps.1150170145
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