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Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in Escherichia coli Cells Capable and Incapable of Phenylalanine Production

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Abstract

The activity of chorismate synthase, the terminal enzyme of the common aromatic pathway, is absolutely dependent on reduced flavin mononucleotide. The bifunctional chorismate synthase of Saccharomyces cerevisiae (product of the ARO2 gene) can reduce flavin in a reaction that involves NADPH, in contrast to the monofunctional chorismate synthase of Escherichia coli (product of the aro C gene). The latter enzyme does not have the capacity for flavin reduction, and its activity therefore depends on the flavin reductase function of the cell. Chemical synthesis of the structural part of the ARO2 gene that involved the substitution of rare E. coli codons was performed for an in vivo comparison of the two types of chorismate synthase. ARO2 expression was tested in the T7 system, and isogenic E. coli strains TG1Δ aro CPtac-ARO2 and TG1Δ aro CPtac- aro C were obtained. Comparative analysis of proteins from the cell extracts of these strains and in silico assessment of hybrid RBS efficiency showed that the level of AroC protein synthesis in TG1Δ aro CPtac- aro C was higher than the level of ARO2 synthesis in the TG1Δ aro CPtac-ARO2 cells. The introduction of Ptac-ARO2 and Ptac- aro C modifications led to complete recovery of the growth of the aromatic auxotroph TG1Δ aro C on minimal mineral medium supplemented with glucose and restored phenylalanine production in the E. coli strain DV1017Δ aro C, which lacked chorismate synthase activity. The similar positive effects of Ptac- aro C and Ptac-ARO2 on phenylalanine biosynthesis in the DV1017ΔtyrR strain, in which chorismate synthase played a “bottleneck” role, indicated the absence of a limiting effect of reduced flavin on monofunctional chorismate synthase overexpressed in E. coli cells.

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Abbreviations

HPLC:

high-performance liquid chromatography

IPTG:

isopropyl-β-D-1-thiogalactopyranoside

OD:

optical density

PAG:

polyacrylamide gel

PCR:

polymerase chain reaction

LB medium:

Luria-Bertani medium

FMNH2 :

reduced form of flavin mononucleotide

MALDI-TOF:

matrix-assisted laser desorption-ionization time-of-flight (mass spectrometry)

NADH:

reduced form of nicotinamide dinucleotide

NADPH:

reduced form of nicotinamide dinucleotide phosphate

RBS:

ribosome binding sites

SD sequence:

Shine-Dalgarno sequence

SDS:

sodium dodecyl sulfate.

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Authors and Affiliations

  1. ZAO Ajinomoto Genetika Research Institute (ZAO AGRI), Moscow, 117545, Russia

    A. E. Slesareva, L. G. Kuhn & V. G. Doroshenko

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  1. A. E. Slesareva
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  2. L. G. Kuhn
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  3. V. G. Doroshenko
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Correspondence to V. G. Doroshenko.

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Original Russian Text © A.E. Slesareva, L.G. Kuhn, V.G. Doroshenko, 2017, published in Biotekhnologiya, 2017, Vol. 33, No. 2, pp. 48–55.

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Slesareva, A.E., Kuhn, L.G. & Doroshenko, V.G. Comparative Analysis of Mono- and Bifunctional Chorismate Synthases in Escherichia coli Cells Capable and Incapable of Phenylalanine Production. Appl Biochem Microbiol 53, 867–873 (2017). https://doi.org/10.1134/S0003683817090071

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  • DOI: https://doi.org/10.1134/S0003683817090071

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