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  • 1
    Electronic Resource
    Electronic Resource
    Metabolic engineering of yeast: the perils of auxotrophic hosts (1999)
    Springer
    Biotechnology letters 21 (1999), S. 611-616 
    Details
    ISSN: 1573-6776
    Keywords: auxotrophy ; growth physiology ; leucine ; metabolic engineering ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Auxotrophic mutants may have physiological alterations and sensitivities which are not generally recognized. Such features are shown here by observations that final cell densities attained by several leucine-auxotrophic Saccharomyces cerevisiae strains depend differently on the initial leucine concentration in the medium. Furthermore, complementing such auxotrophic strains with the plasmid-based LEU2 selection marker resulted in different final cell densities than chromosomal expression of LEU2 in the otherwise isogenic, prototrophic strains. These results warn that auxotrophic host-related physiological influences overlay any metabolic effect of a cloned gene expressed in such a host, clearly complicating interpretation of the effect of that gene's product in scientific or metabolic engineering research.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005576004215
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    Paper (German National Licenses)
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  • 2
    Electronic Resource
    Electronic Resource
    Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 273-279 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; CHO cell ; E2F-1 ; serum-free cell culture ; two-dimensional electrophoresis of proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation. © 1996 John Wiley & Sons, Inc.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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    Paper (German National Licenses)
  • 3
    Electronic Resource
    Electronic Resource
    Metabolic capacity of Bacillus subtilis for the production of purine nucleosides, riboflavin, and folic acid (1998)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 59 (1998), S. 227-238 
    Details
    ISSN: 0006-3592
    Keywords: Bacillus subtilis ; folic acid ; metabolic engineering ; metabolic fluxes ; purine nucleosides ; riboflavin ; stoichiometric model ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: We developed a stoichiometric model of Bacillus subtilis metabolism for quantitative analysis of theoretical growth and biochemicals production capacity. This work concentrated on biochemicals that are derived from the purine biosynthesis pathway; inosine, guanosine, riboflavin, and folic acid. These are examples of commercially relevant biochemicals for which Bacillus species are commonly used production hosts. Two previously unrecognized, but highly desirable properties of good producers of purine pathway-related biochemicals have been identified for optimally engineered product biosynthesis; high capacity for reoxidation of NADPH and high bioenergetic efficiency. Reoxidation of NADPH, through the transhydrogenase or otherwise, appears to be particularly important for growth on glucose, as deduced from the corresponding optimal carbon flux distribution. The importance of cellular energetics on optimal performance was quantitatively assessed by including a bioenergetic efficiency parameter as an unrestricted, ATP dissipating flux in the simulations. An estimate for the bioenergetic efficiency was generated by fitting the model to experimentally determined growth yields. The results show that the maximum theoretical yields of all products studied are limited by pathway stoichiometry at high bioenergetic efficiencies. Simulations with the estimated bioenergetic efficiency of B. subtilis, growing under glucose-limiting conditions, indicate that the yield of these biochemicals is primarily limited by energy and thus is very sensitive to the process conditions. The maximum yields that can reasonably be expected with B. subtilis on glucose were estimated to be 0.343, 0.160, and 0.161 (mol product/mol glucose) for purine nucleosides, riboflavin, and folic acid, respectively. Potential strategies for improving these maximum yields are discussed. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 227-238, 1998.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Two-dimensional electrophoresis of proteins as a tool in the metabolic engineering of cell cycle regulation (1996)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 50 (1996), S. 336-340 
    Details
    ISSN: 0006-3592
    Keywords: cell cycle ; metabolic engineering ; two-dimensional electrophoresis of proteins ; CHO cell ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic engineering of cell cycle regulation addresses important biotechnological problems about serum removal from animal cell culture systems. Chinese hamster ovary cells stimulated to grow by fetal calf serum, insulin, or basic fibroblast growth factor were studied by two-dimensional electrophoresis (2DE) and the resulting protein expression patterns were analyzed. Detailed 2DE protocols are provided and at least 24 gene products are identified which may play an important role in growth factor signaling. Moreover, a correlation between the expression of three proteins (cyclin D1, cyclin E, and E2F-1) and mitogenic strength was found. © 1996 John Wiley & Sons, Inc.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
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    Paper (German National Licenses)
  • 5
    Electronic Resource
    Electronic Resource
    A mathematical model of N-linked glycoform biosynthesis (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 890-908 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; N-linked glycosylation ; mathematical model ; CHO cells ; glycoform ; oligosaccharides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Metabolic engineering of N-linked oligosaccharide biosynthesis to produce novel glycoforms or glycoform distributions of a recombinant glycoprotein can potentially lead to an improved therapeutic performance of the glycoprotein product. Effective engineering of this pathway to maximize the fractions of beneficial glycoforms within the glycoform population of a target glycoprotein can be aided by a mathematical model of the N-linked glycosylation process. A mathematical model is presented here, whose main function is to calculate the expected qualitative trends in the N-linked oligosaccharide distribution resulting from changes in the levels of one or more enzymes involved in the network of enzyme-catalyzed reactions that accomplish N-linked oligosaccharide biosynthesis. It consists of mass balances for 33 different oligosaccharide species N-linked to a specified protein that is being transported through the different compartments of the Golgi complex. Values of the model parameters describing Chinese hamster ovary (CHO) cells were estimated from literature information. A basal set of kinetic parameters for the enzyme-catalyzed reactions acting on free oligosaccharide substrates was also obtained from the literature. The solution of the system for this basal set of parameters gave a glycoform distribution consisting mainly of complex-galactosylated oligosaccharides distributed in structures with different numbers of antennae in a fashion similar to that observed for various recombinant proteins produced in CHO cells. Other simulations indicate that changes in the oligosaccharide distribution could easily result from alteration in glycoprotein productivity within the range currently attainable in industry. The overexpression of N-acetylglucosaminyltransferase III in CHO cells was simulated under different conditions to test the main function of the model. These simulations allow a comparison of different strategies, such as simultaneous overexpression of several enzymes or spatial relocation of enzymes, when trying to optimize a particular glycoform distribution. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55:890-908, 1997.
    Additional Material: 9 Ill.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Design of expression systems for metabolic engineering: Coordinated synthesis and degradation of glycogen (1997)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 55 (1997), S. 419-426 
    Details
    ISSN: 0006-3592
    Keywords: metabolic engineering ; glycogen synthesis ; glycogen degradation ; carbon metabolism ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: In metabolic engineering, systems which allow coordinated control of two metabolic pathways can be useful. We designed two expression systems and demonstrated their application by coordinating glycogen synthesis and degradation. The first expression vector pMSW2 expressed the glycogen synthesis genes in one operon and the glycogen degradation gene in a separate, coordinately regulated operon. The plasmid was designed to switch off expression of the first operon and activate expression of the second operon on addition of IPTG. As an alternative means to control glycogen synthesis and degradation pathways, we constructed expression vector pGTSD100, which contains the native Escherichia coli glycogen synthesis and degradation operon under control of the tac promoter. Both expression vectors work successfully to control the net synthesis and degradation of glycogen. In cultures of the E. coli strain TA3476 carrying the plasmid pMSW2, before the addition of IPTG, glycogen continued to accumulate in the culture. About three hours after IPTG was added, glycogen levels began to decrease. When no IPTG was added to cultures of TA3476:pMSW2, glycogen accumulated in the cells as before but the rate of degradation of glycogen was much lower. When IPTG was added to TA3476:pMSW2, the total cell protein at the end of batch cultivation was approximately 15% higher compared to cultures without IPTG addition. The extra biomass was formed during the glycogen degradation phase. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 419-426, 1997.
    Additional Material: 7 Ill.
    Type of Medium: Electronic Resource
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