Deletion of the Cel48S cellulase from Clostridium thermocellum.
Clostridium thermocellum is a thermophilic anaerobic bacterium that rapidly solubilizes cellulose with the aid of a multienzyme cellulosome complex. Creation of knockout mutants for Cel48S (also known as CelS, S(S), and S8), the most abundant cellulosome subunit, was undertaken to gain insight into its role in enzymatic and microbial cellulose solubilization. Cultures of the Cel48S deletion mutant (S mutant) were able to completely solubilize 10 g/L crystalline cellulose. The cellulose hydrolysis rate of the S mutant strain was 60% lower than the parent strain, with the S mutant strain also exhibiting a 40% reduction in cell yield. The cellulosome produced by the S mutant strain was purified by affinity digestion, characterized enzymatically, and found to have a 35% lower specific activity on Avicel. The composition of the purified cellulosome was analyzed by tandem mass spectrometry with APEX quantification and no significant changes in abundance were observed in any of the major (>1% of cellulosomal protein) enzymatic subunits. Although most cellulolytic bacteria have one family 48 cellulase, C. thermocellum has two, Cel48S and Cel48Y. Cellulose solubilization by a Cel48S and Cel48Y double knockout was essentially the same as that of the Cel48S single knockout. Our results indicate that solubilization of crystalline cellulose by C. thermocellum can proceed to completion without expression of a family 48 cellulase.
Olson
Daniel G
DG
Mascoma Corporation, Lebanon, NH 03766, USA.
Tripathi
Shital A
SA
Giannone
Richard J
RJ
Lo
Jonathan
J
Caiazza
Nicky C
NC
Hogsett
David A
DA
Hettich
Robert L
RL
Guss
Adam M
AM
Dubrovsky
Genia
G
Lynd
Lee R
LR
D001483:Base Sequence; D002480:Cellulase / Q000235:genetics* / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D048013:Acetivibrio thermocellus / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D055786:Gene Knockout Techniques; D006868:Hydrolysis; D008969:Molecular Sequence Data; D040901:Proteomics; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
D002482:Cellulose; D002480:Cellulase