title: High level secretion of cellobiohydrolases by Saccharomyces cerevisiae.
abstract: BACKGROUND
  The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.
  
  
  RESULTS
  We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.
  
  
  CONCLUSIONS
  Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.
authors.affiliation: VTT Technical Research Centre of Finland, Tietotie 2, Espoo, FI-02044 VTT, Finland. merja.penttila@vtt.fi.
authors.forename: Marja
authors.initials: M
authors.lastname: Ilmén
authors.forename: Riaan
authors.initials: R
authors.lastname: den Haan
authors.forename: Elena
authors.initials: E
authors.lastname: Brevnova
authors.forename: John
authors.initials: J
authors.lastname: McBride
authors.forename: Erin
authors.initials: E
authors.lastname: Wiswall
authors.forename: Allan
authors.initials: A
authors.lastname: Froehlich
authors.forename: Anu
authors.initials: A
authors.lastname: Koivula
authors.forename: Sanni P
authors.initials: SP
authors.lastname: Voutilainen
authors.forename: Matti
authors.initials: M
authors.lastname: Siika-Aho
authors.forename: Daniël C
authors.initials: DC
authors.lastname: la Grange
authors.forename: Naomi
authors.initials: N
authors.lastname: Thorngren
authors.forename: Simon
authors.initials: S
authors.lastname: Ahlgren
authors.forename: Mark
authors.initials: M
authors.lastname: Mellon
authors.forename: Kristen
authors.initials: K
authors.lastname: Deleault
authors.forename: Vineet
authors.initials: V
authors.lastname: Rajgarhia
authors.forename: Willem H
authors.initials: WH
authors.lastname: van Zyl
authors.forename: Merja
authors.initials: M
authors.lastname: Penttilä