Engineering industrial yeast strains for Consolidated Bioprocessing of starchy substrates and by-products to ethanol.

Introduction. Commercial bioethanol is currently obtained from starchy substrates, with a relatively mature technology for corn in the USA. However, starch-to-ethanol processes are still expensive and the development of a Consolidated Bioprocessing (CBP) through amylolytic yeast could considerably reduce commercial costs (van Zyl et al. 2012). This research project aimed to construct efficient amylolytic CBP Saccharomyces cerevisiae strains for the industrial ethanol production from starchy feedstock. Materials and methods. In the last eight years, ten fungal glucoamylase and alpha-amylase genes, native and codon-optimized, were screened in different combinations for their high activity into the laboratory strain S. cerevisiae Y294. The most proficient sequences were δ-integrated into industrial yeast strains (van Zyl et al. 2011; Favaro et al., 2012; Favaro et al. 2015). Results. This report gives an overview of the research outcomes we obtained towards the CBP of starchy materials into ethanol. So far, the most effective raw starch-hydrolyzing combination was found to be the codon-optimized glucoamylase of Thermomyces lanuginosus glucoamylase (TLG1) and α-amylase of Saccharomycopsis fibuligera (SFA1) and their gene were δ-integrated into the industrial S. cerevisiae strains M2n and MEL2. The resulting recombinant yeast displayed high activities on raw starch (up to 4461 nkat/g dry cell weight) and produced in a bioreactor about 64 g/L ethanol from 200 g/L raw corn starch, corresponding to 55% of the theoretical yield (g of ethanol/g of glucose equivalent). Their starch conversion efficiencies were even higher on sorghum and triticale (62 and 73% of the theoretical yield, respectively). Moreover, both recombinant strains were efficiently used also for the CBP of starchy by-products, such as wheat bran and rice husk, where starch content is about 10-30% of the biomass. Supplementing the CBP with recombinant cellulases was beneficial to hydrolyze also the cellulose content of the agricultural residues, thus increasing the overall ethanol yield. Discussion. This is the first report of CBP from natural starchy substrates and by-products using industrial yeast strains co-secreting glucoamylase and α-amylase. The high ethanol yields achieved at bioreactor scale pave the way for their large scale CBP applications.