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Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z

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Abstract

A number of vectors were constructed based on the plasmid from the broad range of pMHA200 hosts. Also, the expression of some key genes of the haloalkalitolerant methanotroph Methylomicrobium alcaliphilum 20Z was studied. The activities of the promoter regions of genes for hexulose phosphate synthase, glutamine synthetase, and glucokinase, as well as the promoter of the ectABC-ask operon, which encodes enzymes for osmoprotectant ectoine biosynthesis, were evaluated with the use of the gfp gene; the evaluation was proven to be ineffective. Conversely, glucokinase and a heterologous enzyme of chloramphenicol acetyltransferase were useful for the evaluation of promoter activity. In M. alcaliphilum 20Z cells, the expression level of chloramphenicol acetyltransferase transcribed from the methanol dehydrogenase promoter was higher as compared with that of glucokinase. This seems to be due to a regulatory mechanism for homologous protein expression. The introduction of a synthetic nucleotide sequence forming the secondary structure in the 5′ untranslated region of the glucokinase mRNA resulted in an increase of this enzyme level. This is the first attempt to use M. alcaliphilum 20Z for homo- and heterologous protein expression.

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Authors and Affiliations

  1. G. K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290, Russia

    I. I. Mustakhimov, S. Y. But, A. S. Reshetnikov, V. N. Khmelenina & Y. A. Trotsenko

  2. Pushchino State Institute of Natural Science, Pushchino, Moscow oblast, 142290, Russia

    I. I. Mustakhimov & Y. A. Trotsenko

Authors
  1. I. I. Mustakhimov
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  2. S. Y. But
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  3. A. S. Reshetnikov
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  4. V. N. Khmelenina
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  5. Y. A. Trotsenko
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Corresponding author

Correspondence to Y. A. Trotsenko.

Additional information

Original Russian Text © I.I. Mustakhimov, S.Y. But, A.S. Reshetnikov, V.N. Khmelenina, Y.A. Trotsenko, 2016, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2016, Vol. 52, No. 3, pp. 279–286.

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Mustakhimov, I.I., But, S.Y., Reshetnikov, A.S. et al. Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z. Appl Biochem Microbiol 52, 263–268 (2016). https://doi.org/10.1134/S0003683816030157

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  • DOI: https://doi.org/10.1134/S0003683816030157

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