Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification.
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.
Petit
M A
MA
Laboratoire de Génétique Microbienne, Institut de Biotechnologie, INRA, Jouy en Josas, France.
Joliff
G
G
Mesas
J M
JM
Klier
A
A
Rapoport
G
G
Ehrlich
S D
SD
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism; D003013:Clostridium / Q000235:genetics*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000378:metabolism*; D005784:Gene Amplification; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism
D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D002480:Cellulase