A strategy for increasing an <i>in vivo</i> flux by genetic manipulations. The tryptophan system of yeast

Niederberger, Peter; Prasad, Rajendra; Miozzari, Giuseppe; Kacser, H.

doi:10.1042/bj2870473

Cited by 165 publications

(102 citation statements)

References 31 publications

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“…This finding accords with similar studies on the overexpression of PFK-1 in other systems [7][8][9], and the finding that, in order to obtain a significant increase in flux in a metabolic pathway, the activity of several enzymes must be increased [10,45]. This is also consistent with the expectation that flux will not increase unless there is an increased demand for glycolytically derived ATP or for glycolytic intermediates.…”

Section: Discussionsupporting

confidence: 91%

“…In order to effect large increases in pathway flux, the activities of several enzymes need to be increased simultaneously. This was demonstrated experimentally in S. cere isiae, where a large increase in tryptophan synthesis was achieved only by simultaneous overexpression of five enzymes, when increased expression of between one and four enzymes produced relatively poor results [45]. This requirement for increasing the activity of several enzymes in order to increase pathway flux is echoed in the long-term response of the glycolytic pathway to tissue hypoxia.…”

Section: Discussionmentioning

confidence: 94%

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Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

Urbano

Gillham

Groner

et al. 2000

Biochemical Journal

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Overexpression of the liver subunit of 6-phosphofructo-1-kinase in Chinese hamster ovary K1 cells was shown to increase the steady-state level of the enzyme's product, fructose 1,6-bisphosphate, and to produce a small but significant decrease in the concentration of fructose 2,6-bisphosphate, which is an allosteric activator of the enzyme. However, overexpression of the enzyme had no effect on glycolytic flux under a variety of different substrate conditions. This latter observation is consistent with similar studies in fungi and in potato tubers which indicate that 6-phosphofructo-1-kinase has very little control over flux in glycolysis.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 94%

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

Urbano

Gillham

Groner

et al. 2000

Biochemical Journal

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“…r > 1, 'expected' x-,,, values based on either of these hypotheses will underestimate the true value given by Eqn (24) or Eqn (27) (see e.g. [53]). …”

Section: Predictions Of Changes In Fluxmentioning

confidence: 99%

Responses of metabolic systems to large changes in enzyme activities and effectors

Small

Kacser

1993

European Journal of Biochemistry

156

124

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This first paper in a series investigates the problem of predicting and analysing the effects of large changes in enzyme activities or external nutrients/effectors on metabolic fluxes. We introduce the concept of a deviation index, D, which gives a measure of the relative change in a metabolic variable (e.g. flux) due to a large (non-infinitesimal) relative change in a parameter (e.g. enzyme). Using simplifying kinetic assumptions we have found, for an unbranched metabolic chain, a direct relationship between deviation indices and flux control coefficients. This relationship provides a method to estimate flux control coefficients using a single large change in enzyme activity. We also provide a method of predicting the effects of, for example, DNA manipulation or other techniques for enzyme activity/concentration changes on metabolic fluxes. Up-modulations of single enzymes rarely produce significant changes in fluxes. We show that combined changes of activity of a group of enzymes will produce a more than 'additive' response. We provide a method of predicting the effects of these combined changes, given either the flux control coefficients of the group of enzymes or the effects on the flux of changing the enzymes individually. A similar analysis is carried out for large changes in external nutrients or effectors. These amplification factors, f, give experimentally accessible estimates of the expected changes in metabolic variables. We provide three 'case studies' to illustrate our results.The investigation of metabolism has had two principal aims : to understand how organisms work and to change them in desired directions. An understanding will be achieved if the physiological responses of the organism can be described in terms of the many thousand of 'unit' parts; the enzymecatalysed reactions. These responses happen as a result of naturally occurring changes in external conditions or due to the experimenter's manipulations. The desire to change organisms, including man, is testified by the long history of medicine and agriculture: to change sickness into health, to change low yields into high yields.The well-known complexity of metabolic structures along with many experimental explorations has made it evident that simple predictions of the outcome of external, or internal, manipulations are rarely possible. This is so in spite of the considerable progress in the enzymology and molecular biology of individual steps in the system. It is now accepted that a solution must come by taking a systemic view, rather than the study of isolated parts.The development of genetic manipulation technology has high-lighted the possibility of altering the metabolic properties of organisms in specific ways. In particular, the possible use of DNA manipulation in order to increase the concenCorrespondence to H. Kacser,

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“…First, we can use the information that a very large increase above the wild-type level in tryptophan does not increase the rate of tryptophan incorporation into protein [24]. This means that this rate is essentially saturated with tryptophan at its wild-type level, and hence we do not have to make any manipulations in this part of metabolism to compensate for any increases in tryptophan levels.…”

Section: Scheme 4 Biosynthesis and Consumption Of Tryptophan In Yeastmentioning

confidence: 99%

“…It has been shown [24] that large increases in the amount of enzyme are achievable using multi-copy vectors. Little is know about both the kinetics of this step in Succharomyces and the in vivo concentrations of the four metabolites involved (see above).…”

Section: Scheme 4 Biosynthesis and Consumption Of Tryptophan In Yeastmentioning

confidence: 99%

A Method for Increasing the Concentration of a Specific Internal Metabolite in Steady‐State Systems

Small¹,

Kacser²

1994

European Journal of Biochemistry

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An approach is described by which it is possible to increase the concentration of any internal metabolite without affecting the concentrations of other metabolites and fluxes in the organism. This approach requires the manipulation of only a limited number of enzyme activities. The method shows which enzymes to manipulate and the extent of the manipulation required to achieve a given increase in a chosen metabolite. A case study involving tryptophan overproduction in Saccharomyces cerevisae is given as a practical example of how this method could be used.The aim of many biotechnologists is to increase the production or yield of commercially important metabolites. Recently, a method has been proposed by which the concentration of the final excreted product of a selected pathway can be increased without causing serious deleterious effects on the rest of metabolism [l]. This 'output' method requires that the activities of all the enzymes in the last section of the pathway, including the last excretory step, should be increased by the same factor. To balance all the other fluxes, the next section, starting with the first upstream branch metabolite, requires the elevation of all the enzyme activities of that section by a different, smaller, factor. This process is continued at each branch point upstream with progressively smaller factor increases required in each section.In many cases, the last excretory step will be troublesome for the 'output' method. If excretion is by diffusion alone, then there is no enzyme that can be increased for this step. A method dealing with this eventuality has, however, been devised which recognises that the last step can only be increased by elevation of the internal metabolite (Acerenza, L., unpublished results). Another possibility is that the excretory steps are catalysed by complex active-transport systems, i.e. multi-enzyme, energy-coupled systems, which may be difficult to identify and clone. Furthermore, in many bacteria and fungi, the active-transport systems, e.g. for amino acids, work in the direction of flow into the cell against a concentration gradient [2, 31. As a result, the product accumulates in the cells and very little is found in the supernatant of the culture. It may, nevertheless, be of interest to produce strains with large internal pools of the product if one can cope with the harvesting and extraction of this product. This study discusses how these problems can be solved.We shall first deal with the more general case which is to increase the accumulation, within the cell, of any intermediate metabolite in a pathway. Such higher values may be of interest if, for example, a desirable or valuable intermediate is normally present in very low concentration. Such intermediates may also be the starting material for other compounds. ----~S t~S 3~S l c~----Different principles apply to this problem than to the principles referred to above for increasing the yield of the final excreted product. However, they share with the 'output' method the desirability to leave the r...

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A strategy for increasing an in vivo flux by genetic manipulations. The tryptophan system of yeast

Cited by 165 publications

References 31 publications

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

Effects of overexpression of the liver subunit of 6-phosphofructo-1-kinase on the metabolism of a cultured mammalian cell line

Responses of metabolic systems to large changes in enzyme activities and effectors

A Method for Increasing the Concentration of a Specific Internal Metabolite in Steady‐State Systems

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