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.
Laboratoire de Génétique Microbienne, Institut de Biotechnologie, INRA, Jouy en Josas, France.
M A
MA
Petit
G
G
Joliff
J M
JM
Mesas
A
A
Klier
G
G
Rapoport
S D
SD
Ehrlich
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