Development of consolidated-bioprocessing yeasts for the second-generation bioethanol production from agricultural residues

Today, the fossil materials currently represent the major share of the fuel market. In order to reduce the environmental impact resulting from the massive use of these non-renewable sources, particularly associated with the transport sector, bioethanol represents one of the most favorable, sustainable and ecological alternatives. However, the second-generation bioethanol production from waste plant biomass requires an expensive multi-step process and large dosages of commercial hydrolytic enzymes. The consolidated bioprocessing (CBP) performed by a single fermenting microbe could provide significant energy savings as well as being more cost-effective. Nevertheless, to date no naturally occurring CBP microbe has been described yet. In this study, a collection of newly isolated Saccharomyces cerevisiae strains was screened with the aim of selecting a wild type yeast with superior fermentative traits than the industrial S. cerevisiae Ethanol Red®, which is currently used at industrial scale for firstgeneration bioethanol production. The collection has been evaluated for the conversion of starchy substrates into ethanol by a simultaneous saccharification and fermentation (SSF) configuration. The S. cerevisiae L20 strain, which demonstrated the highest fermentation rate and ethanol production, was selected for a genetic engineering program in order to obtain an amylolytic yeast, for an efficient conversion of starch into ethanol. S. cerevisiae L20 and Ethanol Red® were engineered for the constitutive expression of two genes, encoding the α-amylase AmyA and the glucoamylase GlaA from Aspergillus tubingensis T8.4, in order to develop a stable recombinant strain. The well-established d-integration strategy was used to obtain recombinants at d- sequences by using homologous cassettes for AmyA and GlaA. Alongside, the innovative CRISPR / Cas9 knock-in system was used for the site-specific integration of the same genes in two selected genomic loci, namely mk114 and AD7. Both approaches were evaluated in terms of strain stability and enzymatic activity. The recombinant strains were verified for correct integration and examined for the effective secretion of amylases on agar plates containing starch. The nzymatic activity of the strains presenting the largest hydrolysis halos was quantified, and their recombinant proteins characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The performances of the new CBP strains were then demonstrated on starchy substrates. The most promising recombinant yeast was found to be L20 dT8, co-expressing both AmyA and GlaA. This study demonstrated the superior fermenting abilities of S. cerevisiae L20 compared to Ethanol Red®, confirming its promise as a starting point for the development of a CBP yeast. Genetic editing technologies have both proven to be effective, although further efforts are needed.