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Metabolic Flux Analysis using 13C Isotopes: III. Significance for Systems Biology and Metabolic Engineering

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Abstract

At present, 13C-MFA is a primary method for quantitatively characterizing intracellular carbon fluxes in cells in vivo under steady-state conditions. The method has been successfully used to investigate both the fundamental characteristics of prokaryotic and eukaryotic cell metabolism and to improve producer strains for more than twenty years. This publication is the last in a set of reviews that describe various aspects of the method. Here, the authors highlight recent achievements that involved using 13C-MFA to elucidate bacterial metabolism. Analyses of well-characterized bacterial model strains revealed that central metabolism robustness is provided by a set of alternative metabolic pathways; these analyses also helped develop a better understanding of the physiological significance of these pathways and identified previously unknown functions of well-studied metabolic pathways. Several examples of 13C-MFA-based fundamental investigations of poorly characterized bacteria are also analyzed. In applied investigations, flux analysis of strains that produce amino acids, vitamins and antibiotics indicated targets for modifications, suggested unconventional metabolic engineering approaches, and, most importantly, confirmed their utility. In the last section of this article, 13C-MFA prospects, including the monitoring of the dynamics of metabolic flux distribution during culture growth, are discussed.

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Abbreviations

CM:

central metabolism

AC:

acetate

ACALD:

acetaldehyde

ACCOA:

acetyl-coenzyme A

AKG:

2-oxoglutarate

CoA:

coenzyme A

C2/C3:

two-carbon and tri-carbon fragments involved in the enzymatic reactions catalyzed by transaldolase and transketolase of the non-oxidizing branch of the PP pathway according to the **-pong mechanism

CIT:

citrate

13C-MFA (13C-based metabolic flux analysis):

metabolic flux analysis in experiments using substrates containing 13C isotopes of carbon

E4P:

erythrose 4-phosphate

ED pathway:

Entner-Doudoroff pathway

EMP pathway:

Embden–Meyerhof–Parnas pathway

DHAP:

dihydroxyacetone phosphate

ICIT:

isocitate

F6P:

fructose 6-phosphate

FDP:

fructose 1,6-diphosphate

FUM:

fumarate

G6P:

glucose 6-phosphate

G3P:

glyceraldehyde 3-phosphate

GC–MS:

Gas chromatography–mass spectrometry

GLC:

glucose

GLX:

glyoxylate

KDPG:

2-keto-3-deoxy-phosphogluconate

LC:

liquid chromatography

LL-DAP:

L,L-diaminopimelic acid

MAL:

malate

MS/MS:

tandem mass spectrometry

MTHF:

methyltetrahydrofolate

NAD(H):

oxidized (reduced) nicotinamide dinucleotide

m so-DAP:

meso-diaminopimelic acid

OAA:

oxaloacetate

PP pathway:

pentose phosphate pathway

PRPP:

5-phosphoribosyl-1-pyrophosphate

PTS:

phosphoenolpyruvate-dependent phosphotransferase system

Pyr:

pyruvate

P5P:

phosphorylated pentasaccharide pool (R5P, Ru5P, and Xu5P)

PEP:

phosphoenolpyruvate

3PG:

3-phosphoglycerate

R5P:

ribose 5-phosphate

Ru5P:

ribulose 5-phosphate

S7P:

sedoheptulose 7-phosphate

SAKP:

N-succinyl- [alpha]amino-oxopimelate

SDAP:

succinyl-diaminopimelic acid

SUC:

succinate

SUCCOA:

succinyl-coenzyme A

TCA:

Tricarboxylic acid cycle

THDP:

tetrahydrodipicolinate

Xu5P:

xylulose 5-phosphate

6PG:

gluconate 6-phosphate

13DPG:

1,3-diphosphoglycerate. Amino acids are designated according to the three-letter code.

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Authors and Affiliations

  1. The Closed Joint-Stock Company A**omoto-Genetika, Moscow, 117545, Russia

    L. I. Golubeva, M. S. Shupletsov & S. V. Mashko

  2. The Faculty of Computational Mathematics and Cybernetics, Lomonosov Moscow State University, Moscow, 119991, Russia

    M. S. Shupletsov

  3. The Faculty of Biology, Lomonosov Moscow State University, Moscow, 119991, Russia

    S. V. Mashko

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Original Russian Text © L.I. Golubeva, M.S. Shupletsov, S.V. Mashko, 2017, published in Biotekhnologiya, 2017, Vol. 33, No. 2, pp. 9–28.

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Golubeva, L.I., Shupletsov, M.S. & Mashko, S.V. Metabolic Flux Analysis using 13C Isotopes: III. Significance for Systems Biology and Metabolic Engineering. Appl Biochem Microbiol 53, 827–841 (2017). https://doi.org/10.1134/S0003683817090058

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