Oenological yeasts as a source of extracellular enzymes for future applications in bioethanol production.

Bioethanol is an attractive, sustainable and alternative energy source to conventional fuel. Current ethanol production processes using crops (sugar cane and corn) as a starting substrate are well-established. However, a cheaper feedstock such as lignocellulosic biomass could make bioethanol more competitive with fossil fuel, although its complex structure makes this material more resistant to biological degradation. Recent efforts have focused on the one-step microbial conversion of plant biomass into biofuel since simultaneous microbial hydrolysis and fermentation of lignocellulosic material could represent a strategy to allow cost-effective production of ethanol. Wild-type microorganisms having both the properties to utilise biomass polysaccharides and to produce ethanol have not been described. In this respect, yeasts isolated from oenological environments for their fermentative abilities and possessing efficient hydrolytic enzymes could be very promising. The aim of this study was to investigate on the extracellular enzymatic activity profile of yeast strains previously selected on the basis of their optimal fermentative performance, in order to start the development of a strain suitable for the one-step bioconversion of biomass into ethanol. One hundred and eighty non-Saccharomyces strains and two hundred and twenty Saccharomyces cerevisiae strains isolated from grape marcs were screened for their activity on cellulose, hemicellulose, pectin, protein and starch. Few strains showed activity on cellulose, pectin, protein and starch, while no xylanolytic strains were observed. Two non-Saccharomyces strains were found effective for the production of cellulase and starch-degrading enzymes. Thirteen strains of S. cerevisiae, potentially able to use starch as the sole carbon source, were selected (Favaro et al. , 2008). Their weak growth on starch minimal agar plates was unexpected since the common dogma (Pretorius, 1997) is that wild-type S. cerevisiae cannot grow on media where starch is the only carbon source. In addition, extensive biochemical, physiological and genetic knowledge on their potentially amylolytic enzyme(s) was performed to look into this possible new starch-hydrolytic mechanism. The pattern of starch degradation halo of the selected S. cerevisiae strains was very similar to that exhibited by S. diastaticus, which is clearly related to S. cerevisiae, except for ethanol performance and extracellular glucoamylase production. Starch utilisation in S. diastaticus depends on the expression of three unlinked genes, sta1, sta2 and sta3. The search of the above genes or genes with the same function is in progress in the selected strains of S. cerevisiae mentioned above. These observations provided the basis for a further research on the gene-regulation and enzymatic efficiency of these DNA-sequences in order to enhance and improve their starch degrading activity through molecular approaches. Since at least few strains showed good hydrolytic activities on complex polysaccharides (cellulose and starch), main components of low-cost plant biomass, this study encourages the selection of oenological microorganisms possessing interesting enzymatic profiles for future applications in bioethanol production. This approach could allow the development of one-step bioconversion process, relying on wild-type oenological yeasts with desired properties for both lignocellulose hydrolysis and fermentation. These natural yeasts could also be the base of genetic modification for the construction of more efficient recombinant strains.