title: Assembly of minicellulosomes on the surface of Bacillus subtilis.
abstract: To cost-efficiently produce biofuels, new methods are needed to convert lignocellulosic biomass into fermentable sugars. One promising approach is to degrade biomass using cellulosomes, which are surface-displayed multicellulase-containing complexes present in cellulolytic Clostridium and Ruminococcus species. In this study we created cellulolytic strains of Bacillus subtilis that display one or more cellulase enzymes. Proteins containing the appropriate cell wall sorting signal are covalently anchored to the peptidoglycan by coexpressing them with the Bacillus anthracis sortase A (SrtA) transpeptidase. This approach was used to covalently attach the Cel8A endoglucanase from Clostridium thermocellum to the cell wall. In addition, a Cel8A-dockerin fusion protein was anchored on the surface of B. subtilis via noncovalent interactions with a cell wall-attached cohesin module. We also demonstrate that it is possible to assemble multienzyme complexes on the cell surface. A three-enzyme-containing minicellulosome was displayed on the cell surface; it consisted of a cell wall-attached scaffoldin protein noncovalently bound to three cellulase-dockerin fusion proteins that were produced in Escherichia coli. B. subtilis has a robust genetic system and is currently used in a wide range of industrial processes. Thus, grafting larger, more elaborate minicellulosomes onto the surface of B. subtilis may yield cellulolytic bacteria with increased potency that can be used to degrade biomass.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles, 611 Charles E. Young Drive, Los Angeles, CA 90095-1570, USA.
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Anderson
authors.forename: Scott A
authors.initials: SA
authors.lastname: Robson
authors.forename: Xiao Wen
authors.initials: XW
authors.lastname: Jiang
authors.forename: G Reza
authors.initials: GR
authors.lastname: Malmirchegini
authors.forename: Henri-Pierre
authors.initials: HP
authors.lastname: Fierobe
authors.forename: Beth A
authors.initials: BA
authors.lastname: Lazazzera
authors.forename: Robert T
authors.initials: RT
authors.lastname: Clubb
mesh_terms: D019881:Aminoacyltransferases / Q000096:biosynthesis / Q000235:genetics; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D057005:Bioengineering; D018797:Cell Cycle Proteins; D002473:Cell Wall / Q000378:metabolism*; D002480:Cellulase / Q000378:metabolism; D044603:Cellulosomes / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone; D048013:Acetivibrio thermocellus / Q000201:enzymology; D003546:Cysteine Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D015151:Immunoblotting; D008031:Lignin / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000097722:Cohesins
chemical_list: D019881:Aminoacyltransferases; D001426:Bacterial Proteins; D018797:Cell Cycle Proteins; D002480:Cellulase; D002868:Chromosomal Proteins, Non-Histone; D003546:Cysteine Endopeptidases; D008031:Lignin; D008565:Membrane Proteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D000097722:Cohesins; C036909:lignocellulose; C120542:sortase A