Conferring lignocellulolytic capacity to a superior industrial yeast strain for second-generation bioethanol production.

Awareness of climate change encourages evolution of products towards more sustainable alternatives. One major driver for global warming is the extensive and increasing use of fossil fuels. To exonerate this issue, biofuels are investigated as potential fuel alternatives, of which bioethanol is one of the most promising biofuels. Second-generation bioethanol is produced from lignocellulosic biomass that originates from plant material. A major hurdle in the production of bioethanol with second-generation feedstocks is the high cost of the enzymes for saccharification of the lignocellulosic biomass into fermentable sugars. Simultaneous saccharification and fermentation with Saccharomyces cerevisiae yeast that secretes a range of lignocellulolytic enzymes might address this problem, ideally leading to consolidated bioprocessing. However, it has been unclear how many enzymes can be secreted simultaneously and what the consequences would be on the C6 and C5 sugar fermentation performance and robustness of the second-generation yeast strain. In this work, we have successfully expressed seven secreted lignocellulolytic enzymes, namely endoglucanase, β-glucosidase, cellobiohydrolase I and II, xylanase, β-xylosidase and acetylxylan esterase, in a single second-generation industrial S. cerevisiae strain. This AC14 strain was constructed by consecutive genomic integrations of the seven heterologous genes encoding these lignocellulolytic enzymes with CRISPR/Cas9. This method enabled efficient genome engineering in a polyploid, industrial S. cerevisiae strain. The resulting strain exhibited a high secreted activity on multiple polymeric substrates, reaching 94.5 FPU/g CDW in vitro and enabling direct conversion of lignocellulosic substrates into ethanol in vivo without preceding enzyme treatment. Neither glucose nor the engineered xylose fermentation were significantly affected by the enzyme secretion. This AC14 strain can therefore serve as a promising industrial platform strain for development of yeast cell factories for production of other bio-based chemicals which can significantly reduce the enzyme cost for saccharification of lignocellulosic feedstocks.