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Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16

  • Applied Genetics and Molecular Biotechnology
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Abstract

Five kinds of promoters were evaluated as tools for regulated gene expression in the PHA-producing bacterium Cupriavidus necator. Several broad-host-range expression vectors were constructed by which expression of a reporter gene gfp was controlled by P lac , P tac , or P BAD derived from Escherichia coli, or promoter regions of phaC1 (P phaC ) or phaP1 (P phaP ) derived from C. necator. Then, the gfp-expression profiles were determined in C. necator strains harboring the constructed vectors when the cells were grown on fructose or soybean oil. P lac , P tac , P phaC , and P phaP mediated constitutive gene expression, among which P tac was the strongest promoter. lacI-P tac was not thoroughly functional even after addition of isopropyl-β-d-thiogalactopyranoside (IPTG), probably due to inability of C. necator to uptake IPTG. Gene expression by araC-P BAD could be regulated by varying l-arabinose concentration in the medium, although P(3HB) production rate was slightly decreased in the recombinant. phaR-P phaP exhibited an expression profile tightly coupled with P(3HB) accumulation, suggesting application of the vector harboring phaR-P phaP for gene expression specific at the PHA-biosynthesis phase. The properties of these promoters were expected to be useful for effective engineering of PHA biosynthesis in C. necator.

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Acknowledgements

This study was supported by Industrial Technology Research Grant Program in 2005 from New Energy and Industrial Technology Development Organization (NEDO), KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas Applied Genomics from the Ministry of Education, Culture, Sports, Science and Technology (MEXT).

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Authors and Affiliations

  1. Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, B-37 4259 Nagatsuta, Midori-ku, Yokohama, 226-8501, Japan

    Toshiaki Fukui, Kei Ohsawa, Jun Mifune, Izumi Orita & Satoshi Nakamura

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  1. Toshiaki Fukui
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  2. Kei Ohsawa
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  3. Jun Mifune
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  4. Izumi Orita
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  5. Satoshi Nakamura
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Correspondence to Toshiaki Fukui.

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Fukui, T., Ohsawa, K., Mifune, J. et al. Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16. Appl Microbiol Biotechnol 89, 1527–1536 (2011). https://doi.org/10.1007/s00253-011-3100-2

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  • DOI: https://doi.org/10.1007/s00253-011-3100-2

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