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Hypersecretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis by Induction of Chromosomal DNA Amplification

Bio/Technology volume 8, pages 559–563 (1990)Cite this article

Abstract

We have inserted a DNA fragment composed of (i) the promoter and the export signal of the Bacillus subtilis levansucrase gene; (ii) the sequence encoding the mature part of the Clostridium thermocellum endoglucanase A gene in a specific site of the B. subtilis chromosome. The insert was flanked by directly repeated pBR322 sequences of 3.9 kb. Plasmid pE194, which has a thermosensitive replication, was integrated adjacent to one of the repeats. When the integrated plasmid was allowed to replicate, the insert and one of the repeats was amplified up to a level of about 250 copies per chromosome. Endoglucanase A was efficiently synthesized in, and secreted from, cells containing the amplified structure, since the heterologous fusion protein was the major extracellular protein in a B. subtilis sacUh strain. The NH2 -terminal sequence of the secreted protein revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence.

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Author notes

  1. Marie-Agnès Petit: Corresponding author.

Authors and Affiliations

  1. Laboratoire de Génétique Microbienne, Institut de Biotechnologie, INRA, Domaine de Vilvert, 78350, Jouy en Josas, France

    Marie-Agnès Petit & S. Dusko Ehrlich

  2. Unité de Biochimie Microbienne, Département des Biotechnologies, URA 1300 CNRS, Institut Pasteur, 75724, Paris Cedex, 15, France

    Gwennaël Joliff, André Klier & Georges Rapoport

  3. Ingenasa, Hermanos Garcia Noblejas, 41, 28037, Madrid, Spain

    Juan M. Mesas

Authors
  1. Marie-Agnès Petit
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  3. Juan M. Mesas
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  4. André Klier
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  5. Georges Rapoport
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  6. S. Dusko Ehrlich
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Petit, MA., Joliff, G., Mesas, J. et al. Hypersecretion of a Cellulase from Clostridium thermocellum in Bacillus subtilis by Induction of Chromosomal DNA Amplification. Nat Biotechnol 8, 559–563 (1990). https://doi.org/10.1038/nbt0690-559

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