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Study on aerobic biosynthesis of 4-hydroxybutyric acid by Escherichia coli cells upon heterologous expression of the 2-ketoglutarate decarboxylase gene

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Abstract

The Mycobacterium tuberculosis Rv1248c (kgd) gene has been expressed in the recombinant Escherichia coli strain with the inactivated pathways of mixed-acid fermentation and anaerobic generation of acetyl-CoA, and also with modified system of glucose transport and phosphorylation, and altered regulation of ydfG gene encoding NADPH-dependent dehydrogenase of hydroxy carboxylic acids. It was found that with the intensive 2-ketoglutarate formation during aerobic glucose utilization, 4-hydroxybutyrate synthesis could be resulted not only from the direct conversion of 2-ketoglutarate to succinate semialdehyde by the heterologous enzymatic activity, but also from the involvement of respective tricarboxylic acid cycle intermediate in a cascade of native biochemical reactions. Induced expression of the 2-ketoglutarate decarboxylase gene in the recombinant strain provided an efficient conversion of 2-ketoglutarate to succinate semialdehyde derivatives, while the concentration of synthesized 4-hydroxybutyric acid reached 0.3 mM and has apparently been limited by the activity of the enzyme responsible for the terminal stage of precursor reduction.

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Abbreviations

GHB:

4-hydroxybutyric acid

HPLC:

high-performance liquid chromatography

GBL:

γ-butyrolactone

IPTG:

isopropyl β-D-l-thiogalactopyranoside

PCR:

polymerase chain reaction

SSA:

succinate semialdehyde

TCA:

tricarboxylic acid cycle

NADPH:

reduced form of nicotinamide adenine dinucleotide phosphate

References

  1. Zhu, Y.L., Yang, J., Dong, G.Q., Zheng, H.Y., Zhang, H.H., Xiang, H.W., and Li, Y.W., An environmentally benign route to gamma-butyrolactone through the coupling of hydrogenation and dehydrogenation, Appl. Catal., 2005, Vol. 57, pp. 183–190.

    Article  CAS  Google Scholar 

  2. Yim, H., Haselbeck, R., Niu, W., Pujol-Baxley, C., Burgard, A., Boldt, J., Khandurina, J., Trawick, J.D., Osterhout, R.E., Stephen, R., Estadilla, J., Teisan, S., Schreyer, H.B., Andrae, S., Yang, T.H., Lee, S.Y., Burk, M.J., and Van Dien, S., Metabolic engineering of escherichia coli for direct production of 1,4-butanediol, Nat. Chem. Biol., 2011, Vol. 7, pp. 445–452.

    Article  CAS  PubMed  Google Scholar 

  3. Choi, S., Kim, H.U., Kim, T.Y., Kim, W.J., Lee, M.H., and Lee, S.Y., Production of 4-hydroxybutyric acid by metabolically engineered Mannheimia succiniciproducens and its conversion to ?-butyrolactone by acid treatment, Metab. Eng., 2013, Vol. 20, pp. 73–83.

    Article  CAS  PubMed  Google Scholar 

  4. Efe, C., Straathof, A.J., and van der Wielen, L.A., Options for biochemical production of 4-hydroxybutyrate and its lactone as a substitute for petrochemical production, Biotechnol. Bioeng., 2008, Vol. 99, pp. 1392–1406.

    Article  CAS  PubMed  Google Scholar 

  5. Morzhakova, A.A., Skorokhodova, A.Yu., Gulevich, A.Yu., and Debabov, V.G., Recombinant Escherichia coli strains deficient in mixed acid fermentation pathways and capable of rapid aerobic growth on glucose with a reduced crabtree effect, Appl. Biochem. Microbiol., 2013, Vol. 49, No. 2, pp. 113–119.

    Article  CAS  Google Scholar 

  6. Sambrook, J., Fritsch, E., and Maniatis, T., Molecular Cloning: a Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Lab. Press, 1989.

    Google Scholar 

  7. Datsenko, K.A. and Wanner, B.L., One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc. Natl. Acad. Sci. USA, 2000, Vol. 99, pp. 6640–6645.

    Article  Google Scholar 

  8. Skorokhodova, A.Yu., Zimenkov, D.V., Gulevich, A.Yu., Minaeva, N.I., Biryukova, I.V., and Mashko, S.V., Introduction of symmetrical Olac-ideal into the region between “–35” and “–20” of the Ptrc/Olac hybrid promoter significantly increases the efficiency of its repression by the LacI protein, Biotekhnologiya, 2006, Vol. 3, pp. 1–4.

    Google Scholar 

  9. Katashkina, Zh.I., Skorokhodova, A.Yu., Zimenkov, D.V., Gulevich, A.Yu., Minaeva, N.I., Doroshenko, V.G., Biryukova, I.V., and Mashko, S.V., Tuning of expression level of the genes of interest located in the bacterial chromosome, Mol. Biol. (Moscow), 2005, Vol. 39, No. 5, pp. 823–831.

    Article  Google Scholar 

  10. Gulevich, A.Yu., Skorokhodova, A.Yu., Ermishev, V.Yu., Krylov, A.A., Minaeva, N.I., Polonskaya, Z.M., Zimenkov, D.V., Biryukova, I.V., and Mashko, S.V., New method of construction of artificial translational-coupled operons in bacterial chromosome, Mol. Biol. (Moscow), 2009, Vol. 43, No. 3, pp. 547–557.

    Article  Google Scholar 

  11. Park, S.J., Chao, G., and Gunsalus, R.P., Aerobic regulation of the sucABCD genes of Escherichia coli, which encode alpha-ketoglutarate dehydrogenase and succinyl coenzymeasynthetase: roles of ArcA, Fnr, and the upstream sdhCDAB promoter, J. Bacteriol., 1997, Vol. 179, pp. 4138–4142.

    PubMed Central  CAS  PubMed  Google Scholar 

  12. Chang, D.E., Shin, S., Rhee, J.S., and Pan, J.G., Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme a flux for growth and survival, J. Bacteriol., 1999, Vol. 181, pp. 6656–6663.

    PubMed Central  CAS  PubMed  Google Scholar 

  13. Vemuri, G.N., Eiteman, V.A., and Altman, E., Effects of growth mode and pyruvate carboxylase on succinic acid production by metabolically engineered strains of Escherichia coli, Appl. Environ. Microbiol., 2002, Vol. 68, pp. 1715–1727.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Fuhrer, T., Chen, L., Sauer, U., and Vitkup, D., Computational prediction and experimental verification of the gene encoding the NAD+/NADP+-dependent succinate semialdehyde dehydrogenase in Escherichia coli, J. Bacteriol., 2007, Vol. 189, pp. 8073–8078.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Sezonov, G., Joseleau-Petit, D., D’Ari, R., Escherichia coli physiology in Luria–Bertani broth, J. Bacteriol., 2007, Vol. 189, pp. 8746–8749.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Baev, M.V., Baev, D., Radek, A.J., and Campbell, J.W., Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays, Appl. Microbiol. Biotechnol., 2006, Vol. 71, pp. 317–322.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 117545, Russia

    A. Yu. Gulevich, M. S. Skonechny, A. V. Sukhozhenko, A. Yu. Skorokhodova & V. G. Debabov

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  1. A. Yu. Gulevich
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  2. M. S. Skonechny
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  3. A. V. Sukhozhenko
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  4. A. Yu. Skorokhodova
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  5. V. G. Debabov
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Correspondence to A. Yu. Gulevich.

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Original Russian Text © A.Yu. Gulevich, M.S. Skonechny, A.V. Sukhozhenko, A.Yu. Skorokhodova, V.G. Debabov, 2015, published in Biotekhnologiya, 2015, No. 2, pp. 46–54.

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Gulevich, A.Y., Skonechny, M.S., Sukhozhenko, A.V. et al. Study on aerobic biosynthesis of 4-hydroxybutyric acid by Escherichia coli cells upon heterologous expression of the 2-ketoglutarate decarboxylase gene. Appl Biochem Microbiol 51, 804–811 (2015). https://doi.org/10.1134/S0003683815080037

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  • DOI: https://doi.org/10.1134/S0003683815080037

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