PROJECT SUMMARY 

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The successful establishment of a second-generation (2G) ethanol industry requires key technological innovations that are still awaiting effective implementation. One of them is the development of yeast strains capable of withstanding toxic compounds (i.e., inhibitors) during fermentation of lignocellulosic hydrolysates (LCHs) derived from the sugarcane biomass. This project poses the question of how tolerance to LCH inhibitors can be improved in the yeast Saccharomyces cerevisiae by the use of modern molecular genetics and synthetic biology tools. To address this important issue, we established a collaborative axis between groups belonging to the recently launched Institute for Research in Bioenergy (IPBEN, UNESP), the Brazilian Bioethanol Science and Technology Laboratory (CTBE), and key partners from the University of São Paulo (USP) and the University of Queensland, Australia. The collaborative network will take advantage of innovative experimental approaches, such as alternative adaptive laboratory evolution protocols, quantitative trait loci mapping, next generation sequencing, and flow cytometry-assisted competition and phenotyping assays, to uncover the genetic basis of yeast tolerance to inhibitor-rich LCHs from the sugarcane bagasse. The produced knowledge will be resourceful for rationally designing a yeast strain hyper-tolerant to LCHs, which will be constructed by applying modern molecular genetics tools and the CRISPR/Cas9 genome editing technology. The resulting synthetic yeast is proposed to serve as a robust "chassis" upon which further genetic modifications (such as the metabolism of pentoses) might be added to yield a reference strain suited for cellulosic ethanol production.