Developing novel yeast strains for the consolidated bioprocessing of starchy substrates into bioethanol

Bioethanol belongs to the large family of renewable fuels that can be used as alternatives to fossil oil, with hundreds of billions of liters produced in the US and Brazil from maize and sugarcane. However, criticism due to the rise of food prices and food shortage is impeding the global implementation of bioethanol production from crops. Second-generation bioethanol could be made from non-food feedstocks rich in starch and lignocellulose, consisting of food waste or processing residues. The conventional first-generation bioethanol production from starch is a well-established technology, based on a multiple-step process that is hardly sustainable: high energy demand is firstly required to pretreat the material at high temperatures, then expensive commercial enzymes are needed to breakdown the granules in order to obtain free glucose for the final fermentation step. In this scenario, large cost reductions can be achieved through process integration (consolidated bioprocessing, CBP) by using a new amylolytic and fermenting microbe able to directly convert starchy biomass into fuel in a single bioreactor. To date, no natural CBP microorganism has been described. Saccharomyces cerevisiae is the most common fermenting yeast, traditionally used in the food industry. The high fermenting activity, the Generally Regarded As Safe (GRAS) status and industrial robustness are desirable characteristics for large scale bioethanol production. Unfortunately, S. cerevisiae lacks hydrolytic enzymes and cannot use starch as a carbon source. A new S. cerevisiae strain genetically engineered with fungal amylases can significantly contribute to improve the feasibility of granular starch hydrolysis. This study aimed at searching for novel yeast strains with high fermenting abilities on starchy by-products to be used as host strains for the development of new and efficient CBP yeast. A cluster of twenty-one novel S. cerevisiae isolates was screened for their fermenting potential and the industrial Ethanol RedTM was used as the benchmark. The fermenting performances were assessed on starchy substrates, broken rice, and raw corn starch, at high loading (20% w/v) in simultaneous saccharification and fermentation (SSF) set up at 30° C. The starch hydrolysis was carried out with the commercial amylases cocktail STARGENTM 002. The novel S. cerevisiae L20 strain outperformed the industrial benchmark and was selected as the host strain for the development of novel starch-to-ethanol CBP strains through the engineering of two efficient starch-hydrolyzing genes from Aspergillus tubigensis: the alpha-amylase AmyA and the glucoamylase GlaA. To this aim, both -integration and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technologies are being currently applied. The transformants strains obtained so far have been assessed for enzymatic activity, resulting in excellent performances, and will be further employed for fermentation of starchy substrates.