Genetic engineering of Lactobacillus diolivorans

Authors

  • Stefan Pflügl,

    1. School of Bioengineering, FH Campus Wien – University of Applied Sciences, Vienna, Austria
    2. Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences, Vienna, Austria
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  • Hans Marx,

    Corresponding author
    1. Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences, Vienna, Austria
    • School of Bioengineering, FH Campus Wien – University of Applied Sciences, Vienna, Austria
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  • Diethard Mattanovich,

    1. Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria
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  • Michael Sauer

    1. Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences, Vienna, Austria
    2. Austrian Centre of Industrial Biotechnology (ACIB GmbH), Vienna, Austria
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Correspondence: Hans Marx, Department of Biotechnology, BOKU – VIBT University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria. Tel.: +43 1 3180078; fax: +43 1 3189900401; e-mail: hans.marx@boku.ac.at

Abstract

In this study, we developed a toolbox for genetic manipulation of Lactobacillus diolivorans, a promising production organism for 1,3-propanediol from glycerol. Two major findings play a key role for successful transformation of this organism: (1) the absence of a native plasmid, because a native plasmid is a major obstacle for transformation of L. diolivorans, and (2) the absence of DNA methylation. A suitable expression plasmid, pSHM, for homologous and heterologous protein expression in L. diolivorans was constructed. This plasmid is based on the replication origin repA of L. diolivorans. The native glyceraldehyde-3-phosphate dehydrogenase promoter is used for constitutive expression of the genes of interest. Functional expression of genes in L. diolivorans was shown with two examples: production of green fluorescent protein resulted in a 40- to 60-fold higher fluorescence of the obtained clones compared with the wild-type strain. Finally, the homologous overexpression of a putatively NADPH-dependent 1,3-propanediol oxidoreductase improved 1,3-propanediol production by 20% in batch cultures.

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