title: A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina.
abstract: BACKGROUND
  One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence.
  
  
  RESULTS
  The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in 'fast' transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. 'Slow' transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity.
  
  
  CONCLUSIONS
  The elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.
authors.affiliation: National Bioenergy Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Jeffrey G
authors.initials: JG
authors.lastname: Linger
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Larry E
authors.initials: LE
authors.lastname: Taylor
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: John O
authors.initials: JO
authors.lastname: Baker
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Todd
authors.initials: T
authors.lastname: Vander Wall
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Sarah E
authors.initials: SE
authors.lastname: Hobdey
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Kara
authors.initials: K
authors.lastname: Podkaminer
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Himmel
authors.affiliation: Biosciences Center, National Renewable Energy Laboratory, 16253 Denver West Parkway, Golden, CO 80401 USA.
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Decker
keywords: Biomass hydrolysis; Cellobiohydrolase; Cellulase expression; Fungal molecular biology; Hypocrea jecorina; Trichoderma reesei