The identification of enzyme targets for the optimization of a valine producing Corynebacterium glutamicum strain using a kinetic model

Abstract

The enzyme targets for the rational optimization of a Corynebacterium glutamicum strain constructed for valine production are identified by analyzing the control of flux in the valine/leucine pathway. The control analysis is based on measurements of the intracellular metabolite concentrations and on a kinetic model of the reactions in the investigated pathway. Data-driven and model-based methods are used and evaluated against each other. The approach taken gives a quantitative evaluation of the flux control and it is demonstrated how the understanding of flux control is used to reach specific recommendations for strain optimization. The flux control coefficients (FCCs) with respect to the valine excretion rate were calculated, and it was found that the control is distributed mainly between the acetohydroxyacid synthase enzyme (FCC = 0.32), the branched chain amino acid transaminase (FCC = 0.27), and the exporting translocase (FCC = 0.43). The availability of the precursor pyruvate has substantial influence on the valine flux, whereas the cometabolites are less important as demonstrated by the calculation of the respective response coefficients. The model is further used to make in-silico predictions of the change in valine flux following a change in enzyme level. A doubling of the enzyme level of valine translocase will result in an increase in valine flux of 31%. By optimizing the enzyme levels with respect to valine flux it was found that the valine flux can be increased by a factor 2.5 when the optimal enzyme levels are implemented.

Analysing overexpression of l-valine biosynthesis genes in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum

“…However, the ilvD gene encoding DHAD may be omitted, as shown by the comparatively low intra-and extracellular KIV concentrations after ilvE overexpression. This conclusion was also drawn by Magnus et al [28] when determining the flux control coefficients for the L-valine pathway. The main flux control for L-valine formation was through AHAS, transamination and L-valine export.…”

Model-based metabolism design: constraints for kinetic and stoichiometric models

“…The steady state of metabolism (concentrations of internal metabolites are constant while at least one flux has non-zero value) is frequently requested in kinetic modelling and optimization looking for biotechnologically applicable strain designs: organisms should be able to sustain the designed process and not just reach it for a moment in a transition phase. Another issue is the stability of the steady state that can be set as a constraint: eigenvalues of the Jacobian matrix can have only negative real parts [ 15 ]. At the same time, the duration of this transition process to a steady state might be very slow.…”

Total enzyme activity constraint and homeostatic constraint impact on the optimization potential of a kinetic model

“…This idea is developed later (Heinrich and Schuster, 1996) and used as total enzyme activity constraint that limits the overexpression of enzymes. Two variants of this constraint are common: i) the total enzyme quantity is fixed at initial or some other value (Magnus et al, 2009;Nikolaev, 2010) or ii) some total quantity can not be exceeded (Klipp et al, 2002;Mauch et al, 2001;Schmid et al, 2004;Stalidzans et al, 2017a).…”

“…This constraint limits large changes in metabolite concentrations in the model to avoid their potential impact on other reactions not included in the kinetic model but present in the living organism and to avoid cytotoxicity (Kell and Mendes, 2000). The constraint has variations in its application: i) limitation of metabolite pool total concentration increase (Magnus et al, 2009;Mauch et al, 2001;Nikolaev, 2010;Schmid et al, 2004) without constraining each metabolite individually, ii) limitation of changes of individual metabolites (Rodrıǵuez-Acosta et al, 1999;Stalidzans et al, 2017a;Villaverde et al, 2016) and iii) combination of both (Visser et al, 2004).…”