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

Abstract

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which recognises that the last step can only be increased by elevation of the internal metabolite (Acerenza, L., unpublished results).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...

Engineering a Living Cell to Desired Metabolite Concentrations and Fluxes: Pathways with Multifunctional Enzymes

“…Some additional restrictions can occur, because the enzyme concentrations calculated according to Eq. (3) must be positive, and for each flux the free-energy gradient (2G) should be negative, unless the flux is coupled to another process with a negative 2G (Kholodenko et al, 1998;Small and Kacser, 1994).…”

“…If one wishes to increase, e.g., the concentration x 2 while leaving the rest of metabolism unchanged, one should modulate the concentrations of the enzymes directly affected by X 2 (we use capital letters for metabolites and lowercase letters for their concentrations) (Small and Kacser, 1994). In the simplest case, X 2 only interacts with the enzyme that produces it in step 2 and with the enzyme that consumes it in step 3.…”

The elucidation of metabolic pathways and their improvements using stable optimization of large-scale kinetic models of cellular systems

“…In that work, automatic linearizations according to basic function approximations for arbitrary kinetic representations are combined with binary variables introduced to remove reactions from the linearized kinetic model. Insightful 'universal' perturbation methods were developed to increase desired concentrations and fluxes within complex networks (Kacser and Acerenza 1993;Small and Kacser 1994). Based on this universal approach, a conception of group flux and concentration control coefficients was introduced and then used for the optimal selection of small subsets of key enzymatic reactions with the maximum impact on the targeted flux (Stephanopoulos and Simpson 1997).…”

Metabolic design: How to engineer a living cell to desired metabolite concentrations and fluxes

“…Based on the rate equations and the mass-action ratios, Small and Kacser (1994) devised a method for increasing the concentration of an internal metabolite, leaving other metabolites and the reaction rates unperturbed. However, this method cannot be applied if one wishes to increase any flux, or when the concentrations of some intermediates are constrained by moiety conservation (such as in coenzyme and substrate cycles, see Kholodenko et al, 1994).…”

“…The ''universal method'' by Kacser and Acerenza (1993), and the engineering of metabolite concentrations by Small and Kacser (1994) and by Westerhoff and Kell (1996) are both special cases of the so-called perturbation method applied to metabolic networks (Kholodenko, 1988(Kholodenko, , 1990(Kholodenko, / 1991). This perturbation method shows how one can modulate the enzyme concentrations so as to obtain any predefined change in metabolic fluxes and concentrations that is compatible with maintenance of a steady state.…”