Heterologous production, assembly, and secretion of a minicellulosome by Clostridium acetobutylicum ATCC 824.
The gene man5K encoding the mannanase Man5K from Clostridium cellulolyticum was cloned alone or as an operon with the gene cipC1 encoding a truncated scaffoldin (miniCipC1) of the same origin in the solventogenic Clostridium acetobutylicum. The expression of the heterologous gene(s) was under the control of a weakened thiolase promoter Pthl. The recombinant strains of the solventogenic bacterium were both found to secrete active Man5K in the range of milligrams per liter. In the case of the strain expressing only man5K, a large fraction of the recombinant enzyme was truncated and lost the N-terminal dockerin domain, but it remained active towards galactomannan. When man5K was coexpressed with cipC1 in C. acetobutylicum, the recombinant strain secreted almost exclusively full-length mannanase, which bound to the scaffoldin miniCipC1, thus showing that complexation to the scaffoldin stabilized the enzyme. The secreted heterologous complex was found to be functional: it binds to crystalline cellulose via the carbohydrate binding module of the miniscaffoldin, and the complexed mannanase is active towards galactomannan. Taken together, these data show that C. acetobutylicum is a suitable host for the production, assembly, and secretion of heterologous minicellulosomes.
Mingardon
Florence
F
BIP-CNRS, 31, Chemin Joseph-Aiguier, F-13402 Marseille Cedex 20, France.
Perret
Stéphanie
S
Bélaïch
Anne
A
Tardif
Chantal
C
Bélaïch
Jean-Pierre
JP
Fierobe
Henri-Pierre
HP
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics; D002480:Cellulase / Q000096:biosynthesis* / Q000235:genetics; D002482:Cellulose / Q000378:metabolism; D003001:Cloning, Molecular; D046969:Clostridium acetobutylicum / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005798:Genes, Bacterial; D008361:Mannosidases / Q000096:biosynthesis / Q000235:genetics; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D009876:Operon; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C473132:cellulosome, Clostridium; C074074:cellulose binding protein A, Clostridium cellulovorans; D002482:Cellulose; D008361:Mannosidases; D002480:Cellulase