Abstract
Multistage construction of an E. coli strain containing no foreign genes which is capable of producing butyrate has been carried out. At the first stage, deletions in gene fadR encoding a protein repressor of an operon for fatty acid degradation and gene aceF responsible for the synthesis of pyruvate dehydrogenase were introduced in the strain MG1655 genome. Then, a mutant obtained from the above strain by induced mutagenesis and capable of growth on ethanol as a sole carbon source under aerobic conditions was selected. It was shown that growth of the mutant on ethanol is provided by two mutations. One of them (a substitution: 257G → A) is located in the regulatory region of gene adhE that controls the synthesis of alcohol-dehydrogenase; the other, containing a substitution Glu568 → Lys, affects the structural portion of the gene. As a result of the consequent mutagenesis of the obtained strain and selection on indicating media, variants capable of growing on butyrate and butanol as sole carbon sources and putatively bearing mutations in gene atoC (encoding transcriptional activator of atoDAB operon) were selected. At the last stage of the work, gene atoB, encoding the synthesis of the thiolase II enzyme, was placed under the control of a constitutive promoter P tet , and the functional allele of gene aceF was introduced. The resulting E. coli strain (ΔfadR, adhE, atoC, P tet -atoB) accumulates 800 mg/l of butyrate upon growth on glucose-containing medium under semi-anaerobic (oxygen limited) conditions. Introduction of an additional deletion in gene ldhA encoding lactate dehydrogenase in the strain genome leads to a further growth of a butyrate production up to 1.3 g/l.
Similar content being viewed by others
Abbreviations
- CL:
-
culture liquid
- PCR:
-
polymerase chain reaction
- LB:
-
medium-Luria-Bertani medium
- TAC:
-
tricarboxylic acid circle
- Amp:
-
ampicillin
- Ara:
-
arabinose
- Cm:
-
chloramphenicol
References
Lee, S.Y., Park, J.H., Jang, S.H., Nielsen, L.K., Kim, J., and Jung, K.S., Biotechnol. Bioeng., 2008, vol. 101, no. 2, pp. 209–228.
Atsumi, S., Cann, A.F., Connor, M.R., Shen, C.R., Smith, K.M., Brynildsen, M.P., Chou, K.J.Y., Hanail, T., and Liao, J.C., Metabol. Eng, 2008, vol. 10, pp. 305–311.
Inui, M., Suda, M., Kimura, S., Yasuda, K., Suzuki, H., Toda, H., Yamamoto, S., Okino, S., Suzuki, N., and Yukawa, H., Appl. Microbiol. Biotechnol., 2008, vol. 77, pp. 1305–1316.
Li, F., Hinderberger, J., Seedorf, H., Zhang, J., Buckel, W., and Thauer, R.K., J. Bacteriol., 2008, vol. 190, pp. 843–850.
Clark, D.P. and Rod, M.L., J. Mol. Evol., 1987, vol. 25, pp. 151–158.
Minaeva, N.I., Gak, E.R., Zimenkov, D.V., Skorokhodova, A.Yu., Biryukova, I.V., and Mashko, S.V., BMC Biotechnol., 2008, vol. 8, pp. 63.
Datsenko, K.A. and Wanner, B.L., Proc. Natl. Acad. Sci. USA, 2000, vol. 97, pp. 6640–6645.
Peredelchuk, M.Y. and Bennet, G.N., Gene, 1997, vol. 187, pp. 231–238.
Lutz, R. and Bujard, H., Nucleic Acids Res., 1997, vol. 25, pp. 1203–1210.
Miller, J., Experiments in Molecular Genetics, New York: Cold Spring Harbor, 1972. Published under the title Eksperimenty v molekulyarnoi genetike, Moscow: Mir, 1976.
Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Spring Harbor Lab., 1989.
Sanger, F., Nicklen, S., and Coulson, A.R., Proc. Natl. Acad. Sci. USA, 1977, vol. 74, pp. 5463–5467.
Horton, C.E. and Benett, G.N., Enzymes Microbial Technol., 2006, vol. 38, pp. 937–943.
Nunn, W.D., Giffin, K., Clark, D., and Cronan, J.E., J. Bacteriol., 1983, vol. 154, pp. 554–560.
Overath, P., Pauli, G., and Schairer, H.U., Eur. J. Biochem., 1969, vol. 7, pp. 559–574.
Salanitro, J.P. and Wegener, W.S., J. Bacteriol., 1971, vol. 108, pp. 885–892.
Vanderwinkel, E., Furmanski, P., Reeves, H.C., and Ajl, S.J., Biochem. Biophys. Res. Commun., 1968, vol. 33, pp. 902–908.
Clark, D.P. and Cronan, J.R., Jr., J. Bacteriol., 1980, vol. 141, pp. 177–183.
Kaga, N., Umitsuki, G., Clark, D.P., Nagai, K., and Wachi, M., Biochem. Biophys. Res. Commun., 2002, vol. 295, pp. 92–97.
Causey, T.B., Shanmugam, K.T., Yomano, L.P., and Ingram, L.O., Proc. Natl. Acad. Sci. USA, 2004, vol. 101, pp. 2235–2240.
Eiteman, M.A. and Altman, E., Trends Biotechnol., 2006, vol. 24, pp. 530–536.
Kim, Y., Ingram, L.O., and Shanmugam, K.T., Appl. Env. Microbiol., 2007, vol. 73, no. 6, pp. 1766–1771.
Wallace, K.K., Bao, Z.Y., Dai, H., Digate, R., Schuler, G., Speedie, M.K., and Reynolds, K.A., Eur. J. Biochem., 1995, vol. 223, pp. 954–962.
Ensign, S.A., Mol. Microbiol., 2006, vol. 61, no. 2, pp. 274–276.
Bermejo, L.L., Welkner, N.E., and Papoustakis, E.T., Appl. Env. Microbiol., 1998, vol. 64, pp..
Kidwell, J., Valentin, H.E., and Dennis, D., Appl. Environ. Microbiol., 1995, vol. 61, no. 4, pp. 1391–1398.
Sato, S., Nomura, C.T., Abe, H., Doi, Y., and Tsuge, T., J. Biosc. Bioeng., 2007, vol. 103, no. 1, pp. 38–44.
Donaldson, G.K., Huang, L.L., Mag-Gio-Hall, L.A., Nagarajan, V., Nakamura, Ch.E., and Suh, W., WO 041269, A2, 2007.
Author information
Authors and Affiliations
Corresponding author
Additional information
Original Russian Text © T.A. Seregina, R.S. Shakulov, V.G. Debabov, A.S. Mironov, 2009, published in Biotekhnologiya, 2009, No. 6, pp. 24–35.
Rights and permissions
About this article
Cite this article
Seregina, T.A., Shakulov, R.S., Debabov, V.G. et al. Construction of a butyrate-producing E. coli strain without the use of heterologous genes. Appl Biochem Microbiol 46, 745–754 (2010). https://doi.org/10.1134/S000368381008003X
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S000368381008003X