Monday, August 2, 2010
Pacific Concourse (Hyatt Regency San Francisco)
Cellulose is the world's most abundant renewable carbon source. Presently, cellulose is converted to renewable ethanol in a three-stage process: production of saccharolytic enzymes (cellulases), enzymatic hydrolysis of the cellulose into fermentable glucose, and fermentation of the glucose into ethanol. These three stages are combined in a single step in a process arrangement called consolidated bioprocessing (CBP). In CBP, saccharolytic enzyme production, hydrolysis and fermentation are carried out by a single microbe or a mixed stable culture. Our goal is to engineer CBP-competent S. cerevisiae strains for ethanol production. Towards this goal, we began by screening for heterologous endoglucanases (EGs) and cellobiohydrolases (CBHs) that could be functionally expressed in S. cerevisiae. For this, we cloned PCR amplified ORF sequences into yeast vectors that direct expression using strong constitutive promoters. Genes expressing secreted endoglucanase were identified using Congo red indicator plates. The ability of the expressed endoglucanases to hydrolyze various cellulosic substrates was investigated. Five endoglucanaes and three cellobiohydrolases were functionally expressed at levels exceeding 1µg/ml. One of the Cel5A /EG2 class endoglucanases was expressed at levels that equalled the amount of Cel5A/EG2 present in a commercial cellulase system that hydrolysed 0.5% phosphoric acid swollen cellulose (PASC) in 70 hours. In an effort to increase the amount of secreted cellulases produced by yeast, we are examining how codon usage, gene dosage, the signal peptide sequence and genetic manipulation of the yeast secretory pathway affect expression. Future efforts will also study cellulose hydrolysis by strains co-expressing genes for endoglucanases, exoglucanases and β-glucosidases.