Constraint-based metabolic control analysis for rational strain engineering

AbstractThe advancements in genome editing techniques over the past years have rekindled interest in rational metabolic engineering strategies. While Metabolic Control Analysis (MCA) is a well-established method for quantifying the effects of metabolic engineering interventions on flows in metabolic networks and metabolic concentrations, it fails to account for the physiological limitations of the cellular environment and metabolic engineering design constraints. We report here a constraint-based framework based on MCA, Network Response Analysis (NRA), for the rational genetic strain design that incorporates biologically relevant constraints, as well as genome editing restrictions. The NRA core constraints being similar to the ones of Flux Balance Analysis, allow it to be used for a wide range of optimization criteria and with various physiological constraints. We show how the parametrization and introduction of biological constraints enhance the NRA formulation compared to the classical MCA approach, and we demonstrate its features and its ability to generate multiple alternative optimal strategies given several user-defined boundaries and objectives. In summary, NRA is a sophisticated alternative to classical MCA for rational metabolic engineering that accommodates the incorporation of physiological data at metabolic flux, metabolite concentration, and enzyme expression levels.

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Constraint-based metabolic control analysis for rational strain engineering

Metabolic Engineering ◽

10.1016/j.ymben.2021.03.003 ◽

2021 ◽

Author(s):

Sophia Tsouka ◽

Meric Ataman ◽

Tuure Hameri ◽

Ljubisa Miskovic ◽

Vassily Hatzimanikatis

Keyword(s):

Metabolic Control ◽

Strain Engineering ◽

Metabolic Control Analysis ◽

Control Analysis

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Metabolic Engineering des Methylerythritolphosphat-Wegs durch Metabolic Control Analysis

Chemie Ingenieur Technik ◽

10.1002/cite.201450410 ◽

2014 ◽

Vol 86 (9) ◽

pp. 1403-1403

Author(s):

D. Volke ◽

B. Engels ◽

L. Wright ◽

J. Gershenzon ◽

S. Jennewein

Keyword(s):

Metabolic Engineering ◽

Metabolic Control ◽

Metabolic Control Analysis ◽

Control Analysis

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Metabolic control analysis of insulin-stimulated glucose disposal in rat skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1999.277.3.e505 ◽

1999 ◽

Vol 277 (3) ◽

pp. E505-E512 ◽

Cited By ~ 13

Author(s):

Beat M. Jucker ◽

Nicole Barucci ◽

Gerald I. Shulman

Keyword(s):

Skeletal Muscle ◽

Metabolic Control ◽

Glucose Transport ◽

Distributed Control ◽

Metabolic Flux ◽

Glycogen Synthesis ◽

Metabolic Control Analysis ◽

Glucose Disposal ◽

Control Analysis ◽

Awake Rats

Metabolic control analysis was used to calculate the distributed control of insulin-stimulated skeletal muscle glucose disposal in awake rats. Three separate hyperinsulinemic infusion protocols were performed: 1) protocol I was a euglycemic (∼6 mM)-hyperinsulinemic (10 mU ⋅ kg−1 ⋅ min−1) clamp, 2) protocol II was a hyperglycemic (∼11 mM)-hyperinsulinemic (10 mU ⋅ kg−1 ⋅ min−1) clamp, and 3) protocol III was a euglycemic (∼6 mM)-hyperinsulinemic (10 mU ⋅ kg−1 ⋅ min−1)-lipid/heparin (increased plasma free fatty acid) clamp. [1-13C]glucose was administered in all three protocols for a 3-h period, during which time [1-13C]glucose label incorporation into [1-13C]glycogen, [3-13C]lactate, and [3-13C]alanine was detected in the hindlimb of awake rats via13C-NMR. Combined steady-state and kinetic data were used to calculate rates of glycogen synthesis and glycolysis. Additionally, glucose 6-phosphate (G-6- P) was measured in the hindlimb muscles with the use of in vivo31P-NMR during the three infusion protocols. The clamped glucose infusion rates were 31.6 ± 2.9, 49.7 ± 1.0, and 24.0 ± 1.5 mg ⋅ kg−1 ⋅ min−1at 120 min in protocols I– III, respectively. Rates of glycolysis were 62.1 ± 10.3, 71.6 ± 11.8, and 19.5 ± 3.6 nmol ⋅ g−1 ⋅ min−1and rates of glycogen synthesis were 125 ± 15, 224 ± 23, and 104 ± 17 nmol ⋅ g−1 ⋅ min−1in protocols I– III, respectively. Insulin-stimulated G-6- Pconcentrations were 217 ± 8, 265 ± 12, and 251 ± 9 nmol/g in protocols I– III, respectively. A top-down approach to metabolic control analysis was used to calculate the distributed control among glucose transport/phosphorylation [GLUT-4/hexokinase (HK)], glycogen synthesis, and glycolysis from the metabolic flux and G-6- P data. The calculated values for the control coefficients ( C) of these three metabolic steps ([Formula: see text]= 0.55 ± 0.10,[Formula: see text]= 0.30 ± 0.06, and[Formula: see text] = 0.15 ± 0.02; where J is glucose disposal flux, and glycogen syn is glycogen synthesis) indicate that there is shared control of glucose disposal and that glucose transport/phosphorylation is responsible for the majority of control of insulin-stimulated glucose disposal in skeletal muscle.

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Investigation of the methylerythritol 4-phosphate pathway for microbial terpenoid production through metabolic control analysis

Microbial Cell Factories ◽

10.1186/s12934-019-1235-5 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 4

Author(s):

Daniel Christoph Volke ◽

Johann Rohwer ◽

Rainer Fischer ◽

Stefan Jennewein

Keyword(s):

Metabolic Engineering ◽

Metabolic Control ◽

Quantitative Description ◽

Normal Growth ◽

Building Blocks ◽

Metabolic Control Analysis ◽

Mep Pathway ◽

Control Analysis ◽

Significant Control ◽

Theoretical Yield

Abstract Background Terpenoids are of high interest as chemical building blocks and pharmaceuticals. In microbes, terpenoids can be synthesized via the methylerythritol phosphate (MEP) or mevalonate (MVA) pathways. Although the MEP pathway has a higher theoretical yield, metabolic engineering has met with little success because the regulation of the pathway is poorly understood. Results We applied metabolic control analysis to the MEP pathway in Escherichia coli expressing a heterologous isoprene synthase gene (ispS). The expression of ispS led to the accumulation of isopentenyl pyrophosphate (IPP)/dimethylallyl pyrophosphate (DMAPP) and severely impaired bacterial growth, but the coexpression of ispS and isopentenyl diphosphate isomerase (idi) restored normal growth and wild-type IPP/DMAPP levels. Targeted proteomics and metabolomics analysis provided a quantitative description of the pathway, which was perturbed by randomizing the ribosome binding site in the gene encoding 1-deoxyxylulose 5-phosphate synthase (Dxs). Dxs has a flux control coefficient of 0.35 (i.e., a 1% increase in Dxs activity resulted in a 0.35% increase in pathway flux) in the isoprene-producing strain and therefore exerted significant control over the flux though the MEP pathway. At higher dxs expression levels, the intracellular concentration of 2-C-methyl-d-erythritol-2,4-cyclopyrophosphate (MEcPP) increased substantially in contrast to the other MEP pathway intermediates, which were linearly dependent on the abundance of Dxs. This indicates that 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (IspG), which consumes MEcPP, became saturated and therefore limited the flux towards isoprene. The higher intracellular concentrations of MEcPP led to the efflux of this intermediate into the growth medium. Discussion These findings show the importance of Dxs, Idi and IspG and metabolite export for metabolic engineering of the MEP pathway and will facilitate further approaches for the microbial production of valuable isoprenoids.

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