The rosettazyme: a synthetic cellulosome.
Cellulose is an attractive feedstock for biofuel production because of its abundance, but the cellulose polymer is extremely stable and its constituent sugars are difficult to access. In nature, extracellular multi-enzyme complexes known as cellulosomes are among the most effective ways to transform cellulose to useable sugars. Cellulosomes consist of a diversity of secreted cellulases and other plant cell-wall degrading enzymes bound to a protein scaffold. These scaffold proteins have cohesin modules that bind conserved dockerin modules on the enzymes. It is thought that the localization of these diverse enzymes on the scaffold allows them to function synergistically. In order to understand and harness this synergy smaller, simplified cellulosomes have been constructed, expressed, and reconstituted using truncated cohesin-containing scaffolds. Here we show that an 18-subunit protein complex called a rosettasome can be genetically engineered to bind dockerin-containing enzymes and function like a cellulosome. Rosettasomes are thermostable, group II chaperonins from the hyperthermo-acidophilic archaeon Sulfolobus shibatae, which in the presence of ATP/Mg(2+) assemble into 18-subunit, double-ring structures. We fused a cohesin module from Clostridium thermocellum to a circular permutant of a rosettasome subunit, and we demonstrate that the cohesin-rosettasomes: (1) bind dockerin-containing endo- and exo-gluconases, (2) the bound enzymes have increased cellulose-degrading activity compared to their activity free in solution, and (3) this increased activity depends on the number and ratio of the bound glucanases. We call these engineered multi-enzyme structures rosettazymes.
Mitsuzawa
Shigenobu
S
Biomolecular Engineering Department, University of California, Santa Cruz, CA 95064, USA.
Kagawa
Hiromi
H
Li
Yifen
Y
Chan
Suzanne L
SL
Paavola
Chad D
CD
Trent
Jonathan D
JD
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001674:Bioelectric Energy Sources; D044602:Cellulases / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D002482:Cellulose / Q000378:metabolism; D044603:Cellulosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D018833:Chaperonins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D016944:Sulfolobus / Q000201:enzymology / Q000235:genetics
D001426:Bacterial Proteins; C080369:CipA protein, Clostridium; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D002482:Cellulose; D044602:Cellulases; D018833:Chaperonins