Consolidated bioprocessing of raw starch with Saccharomyces cerevisiae strains expressing fungal alpha-amylase and glucoamylase combinations

Cost-effective consolidated bioprocessing (CBP) of raw starch for biofuel production requires recombinant Saccharomyces cerevisiae strains expressing alpha-amylases and glucoamylases. Native Aureobasidium pullulans apuA, Aspergillus terreus ateA, Cryptococcus sp. S-2 cryA and Saccharomycopsis fibuligera sfiA genes encoding raw-starch alpha-amylases were cloned and expressed in the S. cerevisiae Y294 laboratory strain. Recombinant S. cerevisiae Y294[ApuA] and Y294[AteA] strains produced the highest extracellular alpha-amylase activities (2.17 U mL(-1) and 2.98 U mL(-1), respectively). Both the ApuA and AteA alpha-amylases displayed a preference for pH 4 to 5 and retained more than 75% activity after 5 days at 30 degrees C. When ateA was co-expressed with the previously reported Aspergillus. tubingensis glucoamylase gene (glaA), the amylolytic S. cerevisiae Y294[AteA-GlaA] strain produced 45.77 g L-1 ethanol after 6 days. Ethanol production by this strain was improved with the addition of either 2.83 mu L STARGEN 002 (54.54 g L-1 ethanol and 70.44% carbon conversion) or 20 mu L commercial glucoamylase from Sigma-Aldrich (73.80 g L-1 ethanol and 90.19% carbon conversion). This is the first report of an engineered yeast strain that can replace up to 90% of the enzymes required for raw starch hydrolysis, and thus contributes to the realisation of a CBP yeast for starch-based biofuel production.