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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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  • 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
    Experimental determination of flux control distribution in biochemical systems: In vitro model to analyze transient metabolite concentrations (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 1121-1128 
    Details
    ISSN: 0006-3592
    Keywords: flux control coefficient ; metabolic control analysis ; enzyme kinetics ; glycolysis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Determination of the control coefficients allows the identification of rate-controlling steps in a reaction system. However, the measurement of the flux control coefficients in a biochemical system is not a trivial task, except for some special cases. We have developed a theoretical basis for the direct determination of these coefficients from dynamic responses. In order to show the validity of this methodology experimentally, the dynamic approach is applied to an in vitro reconstituted partial glycolytic pathway to determine the flux control coefficients of hexokinase and phosphofructokinase. It is shown that the dynamic approach gives consistent results, which agree well with values obtained by the direct enzyme titration method. The detailed procedure and potential applications to other systems, such as immobilized enzyme or cell reactors, are discussed. © 1993 Wiley & Sons, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260411116
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  • 5
    Electronic Resource
    Electronic Resource
    Extension of metabolic control analysis to chemostat bioreactors (1996)
    Hoboken, NJ : Wiley-Blackwell
    AIChE Journal 42 (1996), S. 1454-1464 
    Details
    ISSN: 0001-1541
    Keywords: Chemistry ; Chemical Engineering
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method of determining control coefficients and pseudo-first-order kinetic parameters is presented for cell growth, substrate consumption, and product formation in a chemostat bioreactor. From the equations that relate the control coefficients to process variables, such as feed concentrations of the limiting substrate and the product of interest, a perturbation method is developed to determine control coefficients from steady-state measurements. This method combined with the transient response analysis provides a practical way for determining control coefficients and estimating kinetic properties in chemostat bioreactor systems. To determine the kinetic parameters, one measures cell mass, concentrations of the limiting substrate and the product of interest following a step change in the feed concentration until the system reaches a new steady state. The time courses of these variables are processed to obtain control coefficients, which yield the pseudo-first-order kinetic constants for cell growth, substrate consumption, and product formation. Only steady-state responses are needed in calculation, if a step change in cell concentration in the feed stream can also be introduced without significantly perturbing cell physiology. This method is useful in characterizing the kinetics of whole cell bioreactions: results from chemostat experiments can be used to design operating strategies for batch or fed-batch bioreactions. It is generally applicable to continuous-stir-tank reactors with interacting parallel reactions.
    Additional Material: 2 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/aic.690420526
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  • 6
    Electronic Resource
    Electronic Resource
    Characteristic reaction paths of biochemical reaction systems with time scale separation (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 31 (1988), S. 847-854 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A technique has been developed for characterizing the in vivo behavior of key enzymes from intermediate measurements. The technique is based on the identification of characteristic reaction paths, and it depends on the time scale separation characteristics of the systems. It is shown that useful information can be obtained from the phase plots of properly selected intermediate pairs or combinations which typically show process insensitive algebraic relations approached on time scales short compared to those of most practical interest. These characteristic reaction paths provide useful global measures of enzyme activity. The mathematical basis of reaction path analysis is investigated using linear transformation techniques. General theorems are developed predicting the existence of characteristic reaction paths as asymptotic limits whenever there is effective time scale separation. These limits are reached when fast reactions are relaxed, and available evidence suggests that these conditions will occur for the majority of reaction networks.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260310813
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  • 7
    Electronic Resource
    Electronic Resource
    Lumping analysis of biochemical reaction systems with time scale separation (1988)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 31 (1988), S. 869-879 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Due to the complexity of the systems, successful modelling of intracellular reaction networks must rely on lumping techniques which systematically reduce the number of variables and parameters. Fortunately, the time scale separation characteristics of biochemical systems provide opportunities for eliminating unnecessary details. Through the proper interpretation of eigenvalues and eigenvectors, this article presents a theoretical basis for systematic model reduction. Results are generalized as a semiheuristic basis for lumping systems without complete kinetic information. It is also illustrated that the simplified system can yield new insight which is otherwise unavailable.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260310815
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  • 8
    Electronic Resource
    Electronic Resource
    Fermentation data analysis and state estimation in the presence of incomplete mass balance (1989)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 33 (1989), S. 613-622 
    Details
    ISSN: 0006-3592
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: A method is developed for identifying measurement errors and estimating fermentation states in the presence of unidentified reactant or product. Unlike conventional approaches using elemental balances, this method employs an empirically determined basis, which can tolerate unidentified reaction species. The essence of this approach is derived from the concept of reaction subspace and the technique of singular value decomposition. It is shown that the subspace determined via singular value decomposition of multiple experimental data provides an empirical basis for identifying measurement errors. The same approach is applied to fermentation state estimation. Via the formulation of the reaction subspace, the sensitivity of state estimates to measurement errors is quantified in terms of a dimensionless quantity, maximum error gain (MEG). It is shown that using the empirically determined subspace, one can circumvent the problem of unidentified reaction species, meanwhile reducing the sensitivity of the estimates.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260330515
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  • 9
    Electronic Resource
    Electronic Resource
    Kinetic characterization of baculovirus-induced cell death in insect cell cultures (1993)
    New York, NY [u.a.] : Wiley-Blackwell
    Biotechnology and Bioengineering 41 (1993), S. 104-110 
    Details
    ISSN: 0006-3592
    Keywords: baculovirus ; insect cell culture ; cell death ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: The death process of baculovirus-infected insect cells was divided into two phases: a constant viability (or delay) phase characterized by a delay time (td) and a first-order death phase characterized by a half-life (t1/2). These two parameters were used in conjunction with the n-target theory to classify the kinetics of cell death under various conditions, including different multiplicity of infection (MOI), host cell lines, virus types, incubation volumes, cell density and extracellular L(+)-lactate and ammonium concentrations. Two groups of kinetic effects were found: one characterized by a constant number of hypothetical targets and the other by decreased numbers of hypothetical targets. The first group includes effects such as MOI, virus types, and host cell lines. The second includes the effects of environmental perturbations, such as incubation volume, cell density, and extracellular concentrations of L(+)-lactate and ammonium. Although the underlying mechanisms of these effects are as yet unknown, the death kinetics of infected cells significantly affects the recombinant protein production. In general, foreign protein production does not correlate with the cell life after infection © 1993 John Wiley & Sons, Inc.
    Additional Material: 10 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bit.260410114
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