title: Expression of three Trichoderma reesei cellulase genes in Saccharomyces pastorianus for the development of a two-step process of hydrolysis and fermentation of cellulose.
abstract: OBJECTIVE
  To compare the production of recombinant cellulase enzymes in two Saccharomyces species so as to ascertain the most suitable heterologous host for the degradation of cellulose-based biomass and its conversion into bioethanol.
  
  
  RESULTS
  cDNA copies of genes representing the three major classes of cellulases (Endoglucanases, Cellobiohydrolases and β-glucosidases) from Trichoderma reesei were expressed in Saccharomyces pastorianus and Saccharomyces cerevisiae. The recombinant enzymes were secreted by the yeast hosts into the medium and were shown to act in synergy to hydrolyse cellulose. The conditions required to achieve maximum release of glucose from cellulose by the recombinant enzymes were defined and the activity of the recombinant enzymes was compared to a commercial cocktail of T. reesei cellulases.
  
  
  CONCLUSIONS
  We demonstrate that significantly higher levels of cellulase activity were achieved by expression of the genes in S. pastorianus compared to S. cerevisiae. Hydrolysis of cellulose by the combined activity of the recombinant enzymes was significantly better at 50°C than at 30°C, the temperature used for mesophilic yeast fermentations, reflecting the known temperature profiles of the native enzymes.
  
  
  CONCLUSIONS
  The results demonstrate that host choice is important for the heterologous production of cellulases. On the basis of the low activity of the T. reesei recombinant enzymes at fermentation temperatures, we propose a two-step process for the hydrolysis of cellulose and its fermentation into alcohol using cellulases produced in situ.
authors.affiliation: School of Genetics and Microbiology, Moyne Institute, Trinity College Dublin, College Green, Dublin 2, Ireland.
authors.forename: J
authors.initials: J
authors.lastname: Fitzpatrick
authors.forename: W
authors.initials: W
authors.lastname: Kricka
authors.forename: T C
authors.initials: TC
authors.lastname: James
authors.forename: U
authors.initials: U
authors.lastname: Bond
mesh_terms: D018533:Biomass; D044602:Cellulases / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006868:Hydrolysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012440:Saccharomyces / Q000201:enzymology / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013045:Species Specificity; D014242:Trichoderma / Q000201:enzymology / Q000235:genetics*
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D000431:Ethanol; D002482:Cellulose; D044602:Cellulases; D005947:Glucose
keywords: Saccharomyces pastorianus; T. reesei; cellulase genes; cellulose hydrolysis; fermentation